Proceeding - Final Draft

Avinashilingam
Institute for Home Science and Higher Education for Women

University
(Estd. u/s 3 of UGC Act 1956)
Coimbatore 641 043, Tamil Nadu, India
(Deemed University under Category A by MHRD)
Re-Accredited with A Grade by NAAC
International Conference on
Herbal and Natural Components as the Future of Pharmacology
and
7 Annual Meet of the National Society of Ethnopharmacology
th
Editors
Dr. P.R. Padma
Dr. N. Santhi
Dr. D. Kavitha
Dr. M. Rajeswari
Sponsored by
Year of Publication February 2017
Copyrights :
Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore - 641043,
Tamil Nadu, INDIA
ISBN : 978-93-84234-04-1
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(Electronic or Mechanical including Photocopying etc.,)
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published in this compilation.
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Organizing Committee
Patron Padma Shri Dr. P.R. Krishnakumar

Chancellor
Avinashilingam Institute for Home Science and Higher Education
for Women, Coimbatore
Co-ordinator Dr. Premavathy Vijayan

Vice Chancellor
Advisory Committee
Members Dr. S. Kowsalya
Registrar
Dr. A. Parvathi
Dean, Faculty of Science
Dr. G.P. Jeyanthi

Director (Research & Consultancy)
Padma Shri Dr. P. Pushpangadan

President, National Society of Ethnopharmacology
Amity Institute of Phytomedicine and Phytochemistry
Thiruvananthapuram
Dr. V. George
Secretary, National Society of Ethnopharmacology
Amity Institute of Phytomedicne and Phytochemistry
Thiruvananthapuram
Convenor Dr. S. Annapurani

Professor & Head
Department of Biochemistry, Biotechnology & Bioinformatics
Organizing Secretary Dr. P.R. Padma

Professor
Department of Biochemistry, Biotechnology & Bioinformatics
iii
iv
CONTENTS
1. Elephantopus scaber and Sauropus androginus Herbal Supplement for 1

promoting Pituitary-Gonadal Hormones and Histopathological Study in
Pregnant Mice under Typhoid Fever
Yustika Aulia Rahma, Muhammad Sasmito Djati
2. Validation of traditional medicine through ethnopharmacological 10
investigation including intellectual property rights
Pushpangadan, P. (Padma Shri Awardee) and Ijinu, T.P.
3. Maintaining and Improving Sustainability of Jamu, a Traditional Local 29

Based Herbal of Indonesia for Its Fresh Preparation and Usage, Means or
End?
Joni Kusnadi
4. Chili Pepper as a Potential Medicinal Ingredient 33
Estri Laras Arumingtyas
5. Phytochemical Analysis of Leucophyllum frutescens Stems by 39
FT-IR and UV-Vis Spectroscopic Analysis
Divya, P. Prithiba, A. and Rajalakshmi, R.
6. Identification, Characterization and Immunomodulatory Role of Leaf Protein 44

Fraction of Tabernaemontana divaricata
Annapurani, S., Srilatha M. and Santhi, R.
7. Therapeutic Approaches of Nanotechnology in Herbal drugs for the 50
Management of Diabetes Mellitus
Gayathri Devi, S.
8. Cytotoxicity of isolated and purified bromelain from Ananas comosus 53
Chitra, E., Sumathi, S., Aiswarya, T., Poornima, A. and Padma, P.R.
9. Mechanistic approaches for the antimicrobial action of Phytochemicals 56
Kavitha, D., Padma, P.R. and Sudha, G.
10. Characterisation and Antibiofilm Activity of Eichhornia crassipes 60
Iron Oxide Nanoparticles
Mayuri. P.K., Sumathi. S., Kavithaa. K. and Padma. P.R.
11. Detoxification of lead toxicity using in vitro antioxidant properties of 66
Cynodon dactylon in lead induced goat liver homogenate
Poornima, A., Ragavan, B. and Sumathi, S.
12. Phytochemical screening, UV and FT-IR Analysis of Jacobina Leaves 71
Thilagavathi, R., Prithiba, A and Rajalakshmi, R.
13. Biodegradation of Bisphenol A by Bacillus sp. PK4 77
Gowthami, M., Rajeswari, M., Bhuvaneswari, V., Shobana, A. and Anitha Subash
14. Behaviour of Penicillium chrysogenum as Biocontrol Agent against 85
Aflatoxigenic Aspergillus parasiticus
Ramya, K. and Suganthi, B.
v
15. Influence of geographical location of plant growth on its metabolite 88
accumulation: A comparative quantification of secondary metabolites in in
vitro and in vivo root samples of Withania
Sangeetha, U. and Kalaiselvi Senthil
16. A comparative study of Enzymic Antioxidants in the selected Beetroot 95
Varieties
Raja Hepzi, F. and Sivagami Srinivasan
17. Identification of Protease - producing Bacteria from Organic Waste Soil 99
Sujatha, A., Anitha Subash, Shobana, A. and Sherin, A.
18. Effect of Glycyrrhiza glabra root extract against letrozole induced Poly cystic 105
ovarian syndrome (PCOS) using rat model
Velvizhi, S., Sowmiya, V., Annapurani, S. and Suganthi, B.
19. Simultaneous Determination of Proposed Anti-HIV Combination Comprising 110
of Elvitegravir and Quercetin in Rat Plasma Using the HPLCESI-MS/MS
Method: Drug Interaction Study
Lubna Azmi, Ila Shukla, Padam Kant and Ch. V. Rao
20. Characterization Of Alkaloids from the Stem of Toddalia Asiatica. L, 116
A Wild Woody Liana
Arunambiga, S., Praveena, A. and Suriyavathana, M.
21. Effect of Dietary Incorporation of Ksheerabala Residue on 123
Growth Performance in Wistar rats
Jasmine Rani, K., Shyama, K., Deepak Chandran, Minu Sara Jasmine,
Ally, K. and Gangadevi, P.
22. Characterization and antibacterial activity of biosynthesized 127
Emblica officinalis iron oxide nanoparticles
Banupriya. S.J.S., Kavithaa, K., Padma, P.R. and Sumathi, S.
23. Anti-inflammation and Anti-oxidant activity of Lowsonia inermis 133
Linn. extract on Carrageenan induced paw edema in rats
Swarnakala, N., Sri Kumaran and Vijayaraj, R.
24. Anti Inflammatory Assay of Herbal Extracts of Leaves of 141

Pisonia grandis with Tamarindus indica, Ficus racemosa and Pongamia pinnata
Nandhini, M., Shubashini K Sripathi and Poongothai, G.
25. Evaluation of anti-inflammatory activity of methanolic extract of 144
Foeniculum vulgare leaves in vitro and in vivo
Jagathambal, M. and Padma, P.R.
26. Hepatoprotective and antioxidant effects of Leucas indica leaf 155
extract against Acetaminophen induced liver damage in rats
Suja Rani, S., Devyani P., Prakruthi B.S. and Rehna, A.
27. Identification of natural inhibitor from marine weeds to treat 159
Porphyromonas gingivalis induced Periodontitis : An in silico and in vitro
approach
Chikoo Cherry Abraham Cherian and Jannet Vennila, J.
28. In silico docking studies of thymoquinone with cancer and apoptotic targets 164
proteins, inhibits proliferation and induces apoptosis in breast cancer cells
Sonia Raj, K., Sumathi, S. and Padma, P.R.
vi
29. Analysis of Citrus limetta and Citrus sinensis peel for their phytoconstituents 169
Iswariya, G.T., Padma, P.R. and Nirmaladevi, R.
30. Evaluation of thrombolytic potential of Basella alba L. and Talinum 174
portulacifolium
Pappa Ammal, Pushpa, A. and Lourdu Silvi Sinduja, R.
31. Genetic diversity of Muntingia calabura fruits collected from various districts 179
of Tamil Nadu
Preethi, K. and Priyanka, K.
32. Metabolic engineering of phenyl propanoid pathway in Rice (Oryza sativa) 183
Safia. Ashwini, M. and Sathishkumar, R.
33. Protein Profiling Of Honey Samples From Apis And Stingless Bee 190
AjithaNath, K.G.R., Athulya Raveendran, A., Jayakumaran Nair, V. and Sugunan, S.
34. Renoprotective capability of S-Allylcysteine against Streptozotocin- 194
Nicotinamide induced Diabetic Nephropathy in Rats
Sathibabu Uddandrao, V.V., Brahmanaidu, P. and Ganapathy Saravanan
35. Ameliorative Potentiality of Asiatic acid against the High Fat Diet induced 199
Obesity in Sprague dawley Rats
Ramesh Reddy, P., Sathibabu Uddandrao, V.V., Parim Brahmanaidu and
Ganapathy Saravanan
36. Investigation of the Polyethylene Degrading Potential of the Isolated Bacterial 205
Strain in Polyethylene Contaminated Soil by Bioaugmentation Strategy
Kavitha, R. and Bhuvaneswari, V.
37. In vitro antioxidant activity of Medicago sativa 210
Gomathi, R. and Usha, K.
38. Anticancer Activity Of Eugenol And Eugenol Rich Plant Extracts 215
(Piper Betle And Syzygium Aromaticum) On Oral Carcinoma Cell
Azhagumeena, C., Preethi, R., Archana, A.K. and Padma, P.R.
39. Hypoglycemic and Hypolipidemic Potentials of Cinnamon 220
(Cinnamomum zeylanicum) on Metabolic Syndrome of Adults
Balasasirekha, R. and Lakshmi, U.K.
40. Investigation of Hypoglycemic and Antioxidant Properties of Ethanolic 227
Extract of the Leaves of Boerhavia diffusa in Streptozotocin-Induced Rats
Vasundhara, C.C.S. and Gayathri Devi, S.
41. In vitro a-amylase Inhibitory Kinetics and Antioxidant Potentials of 232
Trigonella foenum-graecum
Renuka, R. and Jeyanthi, G.P.
42. Anticancer activities of Methanolic leaf extract of Prosopis cineraria 238
in Breast Cancer cell lines
Dharani, B., Sivaprabha, J., Padma, P.R. and Sumathi, S.
43. Role of Phytoproteins in in vitro antioxidant and in vivo antitumor 245
activity of Tabernamontana divaricata against Daltons Lymphoma Ascites
(DLA) cells bearing mice
Srilatha, M., Santhi, R. and Annapurani, S.
vii
44. Toxicity Studies of Punica granatum From Cell Line to Animal Model 251
Shalini H. Kumar and Pushpa, A.
45. Protective effect of A.malabarica leaves against hyperlipidemia and 256
oxidative stress in Streptozotocin induced diabetic rats
Peddanna Kotha, Kameswara Rao Badri and Appa Rao Chippada
46. Protective Effect of Ethanolic Extract of Fruits of Terminalia bellirica on 261
Experimentally Induced Obesity in Wistar Rats
Mary Shoba Das, C. and Gayathri Devi, S.
47. Pharmaceutical and Cost Benefit Analysis of Commercially Important 265
Pomegranate Varieties
Sree Preethy, K. and Sathishkumar, R.
48. Phytochemical Screening and Antioxidant Potential of the Selected Coconut 274
Products
Lalitha Ramaswamy, Madhan Shankar and Benoit, T.
49. Phytomedicines for the Treatment of Postmenopausal Osteoporosis A 278
Review
Anitha, S. and Gayathri Devi, S.
50. Influence of Elicitors on Major Withanolide Accumulation in in vitro Shoots of 282
Withania somnifera Dunal: Winter cherry
Parameswari, D., Estri Laras, A., Joni, K. and Kalaiselvi, K.
51. Antimycobacterial Activity of Tylophora indica against Isolates from Female 287
Infertility Cases
Anchana Devi, C.
52. Attenuation of biochemical changes by ethanolic extract of Erythrina variegata 294
L. flowers on streptozotocin induced diabetic rats
Hemmalakshmi, S., Priyanga, S. and Devaki, K.
53. Antimicrobial activity of isolated compounds from Hybanthus enneaspermus 301
.L
Muneeswari, P., Deepika, S. and Poornima, K.
54. In vitro antioxidant activity and phytochemical analysis of Citrus limetta and 307
Citrus sinensis
Kavitha, M., Iswariya, G.T., Padma, P.R. and Nirmaladevi, R.
55. Alteration in biochemical profiles by Macrotyloma uniflorum (Lam.) Verdc. 312
leaves in streptozotocin-nicotinamide induced diabetic rats
Priyanga, S., Hemmalakshmi, S. and Devaki, K.
56. Phytoconstituents from essential oil of Cyanthillium cinereum (l.) H. Rob and 318
their molecular docking studies
Dharani, J. and Ravi, S.
57. Evaluation of Sensory and Nutritional values of Carrot juice Incorporated 321
Cow Milk Paneer
Nivedha Raveendran, P., Sathibabu Uddandrao, V.V., Ganapathy Saravanan and
Vadivukkarasi Sasikumar
58. In Vitro Propagation and Phytochemical Studies of Anoectochilus elatus - A 326
Medicinally Important Terrestrial Orchid
Parimala Devi, N.
viii
59. Mechanism of in vitro inhibition of alpha amylase and alpha glucosidase 330
activites by ethanolic extract of Polyalthia longifolia bark
Gayathri, P. and Jeyanthi, G.P.
60. Bioactive Optimization for Antioxidant Activity of Whey protein hydrolysate 337
A Natural Antioxidant
Devi, K., Suriya, M., Srilekha, Sravanthi and Haripriya, S.
61 Emodin (1, 3, 8-trihydroxy-6-methylanthraquinone), a natural anthraquinone 344
derivative induces oxidative stress and apoptosis in hepatocellular carcinoma
cells
Aruljothi Subramaniam, Kiruthika Sundarraj, Azhwar Raghunath and Ekambaram
Perumal
62. Aloe vera extract mitigates arsenic induced neurotoxicity in rats 355
Sasi Priya, Gopalakrishnan, Vinothini Subramaniam, Lakshmikanthan
Panneerselvam and Ekambaram Perumal
63. Comparison on antioxidant potential of pulp and seed extracts of Elaeocarpus 362
tectorius fruits
Poorani Gurumallesh and Baskar Ramakrishnan
64. Protein interactions may influence the effectiveness of phytochemicals as 368
anticancer agents
Padma, P.R. and Sivapriyadharshini, S.
65. A Study on Basic Knowledge of Urban Community on Spices and Condiments
Salma. S. and Lalitha, R.
66. Differential Cytotoxic Effect of Majorana hortensis leaf extract on chick
embryo cells and Hep2 cell lines
Radha, P. and Padma, P.R.
67. Identification of ER agonist from Agele marmelos
Ganesh K. Prasanth, Divya Lakshmanan M., and Sadasivan, C.
68. Isolation and characterization of phytosterols having 5 reductase inhibitory
activity
Rani, P. and Anisha, A.
69. Antimicrobial efficiency of Chromolaena odorata extracts treated spun lace
viscose nonwoven fabric
Resmi, G. and Amsamani, S.
70. In vitro Inhibitory Potential of -amylase and Proximate Analysis of Selected
Medicinal Plants
Geethanjali, A., Lalitha, P. and Smina, C.
71. Reverse pharmacological validation of a clinically successful Ethnoveterinary
herbal preparation
Punniamurthy, N., Elamurugan, A. and Vijay Anand, J.
72. A novel method for protein quantification from mice and zebra fish
Surendran, L., Ranjith Santhosh kumar, D.S., Nasir hazza and Senthilkumar, P.
73. Modulation of GLUT2 expression by aqueous extract of Erythrina variegata L.

bark in streptozotocin induced diabetic rats
Devaki, K., Hemmalakshmi, S., Priyanga, S. and Sridhar, M.
ix
74. Antioxidant activity of isolated compounds from Hybanthus enneaspermus .L
Poornima, K., Muneeswari, P. and Deepika, S.
75. Anticancer Effect of Partially Purified Phenolic fraction in MCF-7 Breast
Cancer Cell Lines
Pavithra, K. and Vadivukkarasi, S.
76. Attenuation of Hyperglycaemia through Asiatic acid in High Fat Diet induced
Obese Rats
Swapna, K., Sathibabu Uddandrao, V.V., Brahmanaidu, P. and Ganapathy Saravanan
77. Anticancer Activity of the isolated compound from Alpinia purpurata using
PC-3 cell lines"
Anusooriya, P., Poornima, K. and Gopalakrishnan, V.K.
78. Native seaweeds: Role in mitigating the toxic effects of oxysterols under
in vitro conditions
Preetha, S.P., Devaraj, H. and Venkateswaran, K.V.
79. Cytoprotective effects of P.ginseng and G.biloba against amnesic shell fish
poison induced toxicity in Caco-2 cell line
Ramya, E.M., Phani Kumar, G. and Anila kumar, K.R.
80. Modulatory effect of tannin rich extract of Terminalia chebula against
picrotoxin induced anxiety
Chandrasekhar, Y., Phani Kumar, G., Ramya, E.M. and Anilakumar, K.R.
81. Extraction of Oil from Cucurbita maxima seeds and Evaluation of its Anti
Obesity potentiality in High Fat Diet induced Obese Rats
Kalaivani, A., Sathibabu Uddandrao, V.V., Ganapathy Saravanan and Vadivukkarsi,
S.
82. Protective effect of green tea extract against carbofuran induced toxicity in
Wistar rats
Binitha, P.P. and Ramadasan Kuttan
83. Study to assess the effectiveness of acupressure on menstrual pain perception
among adolescent girls with primary dysmenorrhea at selected schools in
Coimbatore
Poornima Sriram
84. Phytochemical analysis of freshwater algae Chlorella pyrenoidosa, Chlorella
vulgaris and Spirulina platensis and its antibiofilm activity
Sridevi, N.S.
85. Evaluation of Antioxidant Status and Anticancer Activity of Thespesia
populnea on Dalton's Lymphoma Ascites Induced Swiss Albino Mice
Santhi, R. and Annapurani, S.
86. In silico prediction of binding sites of certain phytoconstituents to select
human olfactory receptors (ORs) for intranasal drug designing
Nagarathnam, B.
87. Medicinal Plant Drugs for Asthma
Naveen, S., Prathap, M. and Ananthalakshmi, R.
x
88. An Ayurvedic Approach to Improve Quality of Life in Children with Duchenne
Muscular Dystrophy
Preethika, M., Lakshmi, B.R., Shalini H. Kumar, Kalpana, A. and Agila, N.
89. Effect of supplementation of Hibiscus rosasenensis leaves and flowers in Swiss
albino mice
Ramadevi, A., Shyama, K., Jasmine Rani, K., Ally, K. and Koorse, K.G.
90. Phytochemical Screening, Total Phenolic Content and Antioxidant Activity of
Ungerminated and Germinated Sunflower Seeds
Thamilovia, S.A., Uthira, L. and Malathi, M.
91. Identification of Bioactive Compounds and Synthesis of Silver Nanoparticles
From Avicennia marina (Forssk.) Vierh and Its Anti-inflammatory Properties
- In vitro Studies
Vijayaraj, R., Sri Kumaran, N. and Jayaprakashvel, M.
92. A Cross Sectional Study on Consumption of Indian Herbal Hypoglycaemic
Agents Among Type 2 Diabetics in Coimbatore District
Bhavani, S. and Jemima Beryl Mohankumar
xi
xii
Elephantopus scaber and Sauropus androginus Herbal Supplement for
promoting Pituitary-Gonadal Hormones and Histopathological Study in
Pregnant Mice under Typhoid Fever E. Scaber and S. Androgynus pro-
mote Pituitary-Gonadal Hormones
Yustika Aulia Rahma1, Muhammad Sasmito Djati1*
1
Departement of Biology, Faculty of Mathematic and Natural Sciences, Brawijaya University, Malang, Indonesia.
E-mail : msdjati@ub.ac.id
Abstract
The aim of this study was to investigate the efficacy of Elephantopusscaber and Sauropusandrogynusherbal
supplement in promoting the LH, FSH and histopathological study in Liver of pregnant mice model typhoid
fever. Twenty one pregnant mice were orally infected with S. typhimurium, at a concentration 107 CFU/mL .
Extract of E. scaber and S. androgynus were administered orally using formulation 100:0% (T3), 75:25% (T4),
50:50% (T5), 25:75% (T6), and 0:100% (T7), with initial doses of 200 mg/kg BW E. scaber and 150 mg/kg BW S.
androgynus. Pregnant BALB/c mice serum was isolated from the orbital vein at day 9, 13, 17 pregnancy.LH and
FSH level were determined using ELISA, the liver histological were examinating with (H&E) stain. The data were
analyzed using one-way ANOVA, with significance level p<0.05, followed by Tukey test. The result showed that
the optimum dose of P4 was effective to maintained the LH level at day 17 pregnancy of pregnant mice typhoid
fever. Therefore, P1 dose effective to maintained the FSH level at day 9, 13, 17 pregnancy of pregnant mice model
typhoid fever. The histopathological study showed that all treatments were significantly decreased the percentage
of necrotic cell on the liver of pregnant mice model typhoid fever.
Keywords: Elephantopus scaber, Sauropus androgynus, LH, Liver Histophatology, FSH, Salmonella
typhimurium.
INTRODUCTION
Salmonella typhimurium is a bacteriagram-negative that can infect the pregnant women. Previous studies
explain that S. typhimuriumcan lead pregnancy loss and disruption of hormonal metabolism1,2.LH and FSH are the
hormonal that affectsin response toSalmonella typhimurium infection. Herman, et al (2010) explain that
Lipopolysaccharides (LPS) in bacteriagram-negative could effect the levels of Hypothalamic-pituitary
gonadalhormones3. LPS could decreased the level of Gonadotropin hormones such as LH and FSH 3-5. The decreasing
LH and FSH woulddisturbs the level of steroid hormone, because they have functioned as maintain corpusluteum
to produce progesterone and estrogen level in pregnancy.
The mechanism of S. typhimuirumdisturbs the hormonal metabolism in pregnancy are not quite understand.
LPS on thecell membrane of S. typhimuriumhas been found to lead activation of macrophage, which isinduced
secretion some cytokine inflammation, such as IL-1, TNF-, IL-63.However, the inflammatory cytokine can activated
the HPA axis to release adrenocorticotropic hormone (ACTH), which is induce adrenal to secrete cortisol. Cortisol
is known as an inhibitor of gonadotropin hormone3. Therefore, the infection of Salmonella could reducedthe level
of FSH and LH in pregnant women.
Indonesia has a lot of herbal medicine, such as Elephantopus scaber(Tapak Liman) and Sauropus
androgynus(Katuk). Both can use asherbalsupplement for pregnant women,because they haveanti-inflammatory
effect, anti-microbial and stimulate reproduction hormone 6-8. Phytometabolite that conatains in those plants such
1
as apigenin and luteuzin flavonoid, could inhibits the LPS to relieve the production of cytokine inflammatory9,10.Based
on that, the aim of this study was to investigate the efficacy of E. scaber and S. androgynusherbal supplement to
promoting the pituitary gonadal hormon and histophatologicallystudy in liver of pregnant mice typhoid fevermodel.
MATERIALS AND METHODS

Experimental animals
The experimental animals that used were 21 females BALB/c mice with 6 week old and pathogen free.
Those mice were housed in pathogen-free chamber under standard laboratory conditions of temperature, humidity,
and light. The animals were given free access to food (standard mice pellets) and water ad libitum. The protocols
used in the present study was approved by the Ethics Committee at Brawijaya University (approval ref no.523-
KEP-UB).
Preparation of Elephantopus scaber and Sauropus androgynus leaf ethanol extract
Fresh leaves of E. scaber and S. androgynus were collected on September 2015, from Balai Materia Medica
Batu, Malang, Indonesia. E. scaber and S. androgynus fresh leaves were air-dried at room temperature then milled
into a powder. The powder was macerated in 70% of ethanol for 24 h at room temperature. The ethanol extract of
each plant was concentrated to dryness under reduced pressure at 50oC in a vacuum pump evaporator. The initial
dose of E. scaber was 200 mg/kg BW and S. androgynus was 150 mg/kg BW.
Preparation of S. typhi
The isolate of S. typhimurium was collected from Microbiology laboratory, Faculty of Medicine, Brawijaya
University, Malang, Indonesia. Experimental animal (except control) was given orally as much as 107 CFU/mL in
0.5 mL solvent at 5days pregnancy.
Treatments
All the animals were treated for 17 days and still allowed free access to food and water. Oral administration
of E. scaber and S. androgynus were given in first until the fourth day of pregnancy. In day five of preganancy, S.
typhimurium was given orally 107 CFU/mL. Administration of E. scaber and S. androgynus continued until the last
day of treatment according to the treatment group. The pregnant mice were randomly divided into seven treatments
groups with 3 mice each:
Treatment 1 (K-) : healthy mice without extract (control)

Treatment 2 (K-) : S. typhi infection without extract
Treatment 3 (P1) : S. typhi infection with 100% of E. scaber
Treatment 4 (P2) : S. typhiinfection with 75% of E. scaber and 25% of S. androgynus
Treatment 5 (P3) : S. typhi infection with 50% of E. scaber and 50% of S. androgynus
Treatment 6 (P4) : S. typhi infection with 25% of E. scaber and 75% S. androgynus
Treatment 7 (P5) : S. typhi infection with 100% of S. androgynus
Experimental Animal Dissection

Dissection for analysis of level LH and FSH were done atdays 9, 13, 17 of pregnancy, while analysis of liver
hitologically were done only at day 17 of pregnancy.
Serum isolation
Blood isolation was taken using a pipette capillary hematocrite through the veins of orbital for 1mL. The
2
blood was collected in a microtube, then incubated at 37C for 2 hours. It was centrifuged at 4000 rpm, 37 C for
5 minutes. The supernant (serum) was moved into another microtube and save in -4C.
Preparation of liver histological section

Liver was soaked in the 10% neutral buffered formalin for 24 h and dehidrated using ethanol series from
70%, 80%, 95%, continued with absolute alcohol twice. Tissues were clearing using xilol three times which each
step need 60 min, at room temperature. Tissues were embedding in paraffin and sectioned into 4-5 m thickness
using a rotary ultra microtome. The slides were stained with haemotoxyline and eosin (H & E).
ELISA Analysis
The procedures of ELISA for identification the levels of prolactin are according to the guidebook of ELISA
kit Elabscience.
Histopathology analysis
Liver histology slides were analyzed using light Olympus BX51 microscope under 400x magnification. The
number of necroticin the hepatocyte cell were counted from three liver section with five point of view.
Statistical analysis
Level LH, FSH, and the percentage of necrotic hepatocyte cellwere analyzed statistically used one-way
ANOVA analysis with the significance of p<0.05 and were followed by Tukey test. The application for statistical
analysis was SPSS version 16.0 (SPSS Inc. PASW Statistics For Windows, Chicago: SPSS Inc.) and Excel 2007
(Microsoft).
RESULT
Influence of E.scaber and S. androgynus modulated the concentration fo LH.
The concentration of LH throuhout preganancy at 9, 13 and 17 days showed in Figure 1. The LH concentration
of K+ group at 9days pregnancypost-infection were significantly (p <0.05) decreases compared to normal group
(K-), from 33.0341.225 ng/mL (K-) to 31.1240.7105 ng/mL (K+). After administraded the combination extract
E. scaber and S. androgynus, the LH concentration from P1-P5 group were given variation concentration. The LH
concentration of P1, P2, P4 group were shown not significantdifferent (p<0.05) compare to K+. P5 grouphave LH
concentration (33.7921.254 ng/mL ) were significantly increased (p<0.05) compared to K+ group (31.1240.7105
ng/mL) (Figure 1A). The LH concentrati at 13 days pregnancy of P1-P5 group were increased but not significant
(p<0.05) compared to K- and K+ group (Figure 1B). On the day 17 pregnancy, the LH concentration of P1, P2, P3,
P5 were increased, however only P4 group (30.0660.075 ng/mL) were equivalent to LH concentration of K-
group (33.0341.225 ng/mL) (Figure 1C).
Influence of E.scaber modulated the concentration fo FSH
The FSH concentration were showed in Figure 2. Salmonella typhi were decreased the FSH concentration
of pregnant mice at 9 and 13 days prgenancy. The FSH concentration of K+ group at 9 days pregnancy were
decreased but not significant different (p<0.05) compared to K- (normal group), from 3.1341.776 ng/mL (K-) to
1.6250.527 ng/mL (K+) (Figure 2A). The FSH concentration at 13 days pregnancy were significant different (p
<0,05) compared to K-, from 2.662 0.379 ng/mL (K-) to 1.7960.245 ng/mL (K+) (Figure 2B).The administration
of combination extract of E. scaber and S. androgynusgiven various FSH concentration. Figure 2A showed, only
P1 treatment showed increased the FSH concentration but not significant different compared with K- and K+ at 9
3
days pregnancy. At 13 days pregnancy, the P1, P2, and P4 treatment could increased FSH concentration significantly
(p <0.05) compared to K- (Figure 2B). At 17 days pregnancy, the P1and P3 treatment could increase the FSH level
but not significant different (p< 0.05) compared to K- and K+ (Figure 2C).
Influence of E. scaber and S. androgynus modulated decreasing necrotic hepatocyte cell
In the figure 3 showed the Salmonella typhi infection could increased (p < 0.05) the percentage of
necrotic hepatocyte cell mice compared to K-. The administrated E. scaber and S. androgynus could decreased the
percentage of necrotic hepatocyte cell. All treatment showed decreased the percentage of necrotic cell significantly
(p < 0.05) compared to K+.
DISCUSSION
In the normal mice pregnancy, level of LH in the early pregnancy was relatively low, and increased in day
5 until 14 of pregnancy. After day 14, the levels of LH was decreased untill day 17, while end pregnancy showed
variation levels on LH 11. Another research explain that the levels of LH in early mice pregnancy was lower than
midpregnancy (<10 ng/mL), at day 9 pregnancy the level of LH increased (20-30 ng/mL) 12.The increasing of LH
in the midpregnancy would effect on production of progesteron and estrogen. LH has roles to maintain the stimulation
corpus luteum in production of estrogen and progestereron during pregnancy. This role very important during
pregnancy beacuse it affect on the development of reproduction function of gonad 13.
FSH is an important ovarian hormone because they plays a key roles in development of follicle and ovulation
process. During pregnancy, FSH together with LH has role as maintain the production of progesteron and Estrogen.
In normal preganancy of mice, the level of FSH were lower in early preganancy. The increasing level of FSH was
occur in day 5-7 pregnancy. During midpregnancy the level of FSH was various, while in the end pregnancy the
level FSH was increased 12. This data were support our result, at day 9 pregnancy the level of FSH more higer than
level of FSH at day 13 and 17 pregnancy.
This study demonstrated that the infection of Salmonella typhimuirum could reduced the level ofpituitary
gonadal hormones at days 9, 13, and 17 pregnancy. LPS on the cell membrane of S. typhimuriumhas been found
to lead activation of macrophage, which is induced secretion some cytokine inflammation, such as IL-1, TNF-,
IL-63. However, the inflammatory cytokine can activated the HPA axis to release adrenocorticotropic hormone
(ACTH), which is induce adrenal to secrete cortisol. Cortisol is known as an inhibitor of gonadotropin hormone3.
In this study result was agree with the Solati, et al. (2011) and Obryan, et al (2000) study, they were explained the
administration of LPS on mice could reduced the level of LH significantly4,5. The level of FSH in infected group
(K+) was also reduced, during day 9, 13 and 17 pregnancy, but another study had different result, the administration
of LPS on mice were not affected on the level of FSH4.
The leaf extracts of E. scaber and S. androgynus are cotains antioxidants such as apigenin and luteolin
flavonoids 14,9. Both antioxidants are proven to inhibit the LPS action by inducing the inflammatory response.
According to Zhang, et al (2014) apigenin is able to inhibit the action of LPS-induced macrophages to secrete pro-
inflammatory cytokines such as IL-6, IL-1 and TNF-9. Apigenin is able to inhibit the activation of NF-B and
ERK1/2, which bothare intracellular signaling pathways to secrete proinflammatory cytokines. According Xagorary
et al (2002), luteolin also able to inhibit LPS aby reducing the activation of intracellular signaling EPK and the p38
that caused inhibition of production of TNF-10.
Histophatological study on the liver showed that the infection of Salmonella typhimurium caused heaptic
necrotic (Fig.4). This data were agreed with some researchs about Salmoenlla. Guiliano, et al (2015) report that
Salmonella enterica was caused acute hepatic necrosis in dog15. Kamath, et al (2000) report that some patient that
had hepatic failur have been infection by bacterial endotoxins such as bacillus cereus and hepatitis bacteria such
Salmonella typhi16. Salmonella has mechanism to impairing the surface of cell host. This mechanism was encode
4
ILLUSTRATIONS
Figure 1: The effects of combination ethanol extracts from E. scaber and S. androgynus leaves on LH levels at (A)
day 4, (B) day 8, and (C) day 12 post-infection. K-, control; K+, Salmonellatyphi infection without treatment; P1,
Salmonellatyphi infection with 100% E. scaber; P2, Salmonellatyphiinfection with 75% E. scaber and 25% S.
androgynus; P3, Salmonellatyphi infection with 50% E. scaber and 50% S. androgynus; P4, Salmonellatyphi
infection with 25% E. scaber and 75% S. androgynus; P5, Salmonellatyphi infection with 100% S. androgynus. *p
< 0.05 compared toK+.Values are mean SD (n = 3, p < 0.05).
5
Figure 2: The effects of combination ethanol extracts from E. scaber and S. androgynus leaves on FSH levels at
(A) day 4, (B) day 8, and (C) day 12 post-infection. K-, control; K+, Salmonellatyphi infection without treatment;
P1, Salmonellatyphi infection with 100% E. scaber; P2, Salmonellatyphiinfection with 75% E. scaber and 25% S.
< 0.05 compared toK+.Values are mean SD (n = 3, p < 0.05).
6
Figure 3: The effects of combination ethanol extracts from E. scaber and S. androgynus leaves on average
number of necrotic of hepatocyte cell. K-, control; K+, Salmonellatyphi infection without treatment; P1,
Salmonellatyphi infection with 100% E. scaber; P2, Salmonellatyphiinfection with 75% E. scaber and 25% S.
< 0.05 compared toK+.
A B
C D
E F
Figure 4: Liverhistophatology structure (400 x magnification). (A) Normal group (K-); (B) Salmonellatyphi
infec tion without trea tment (K+); (C ) Salmonellatyphi infe c tion with 100% E. scaber (P1);
(D)Salmonellatyphiinfection with 75% E. scaber and 25% S. androgynous(P2); (E)Salmonellatyphi infection with
50% E. scaber and 50% S. androgynous(P3); (F)Salmonellatyphi infection with 25% E. scaber and 75% S.
androgynous (P4); (G)Salmonellatyphi infection with 100% S. androgynous(P5). Black circle indicating normal
hepatocyte cell, red circle indicating necrotic hepatocyte cell.
7
by Salmonella phatogenicity island 1 (SPI-1). SPI-1 secretion system was shown to induced invasion of
macrophages that caused rapid apoptosis in host. SPI-1 will be activates SipB, a translocated effector, is a mediator
to form a hole in the host plasma membrane. SipB binds and activates caspase-1, which is has role as pro-apoptotic
protease17.
All teratment of this study showed suppresed the percentage of necrotic in hepatocyte cell. Both of E.
scaber and S. androgynus conatains deidzein, is an isoflavone that shown has role as anti-inflammation activity.
Chinta, et al (2013) report that the pretreatment using deidzein was showed to signfocantly suppresess LPS-
induced mRNA expression of pro-inflammatory in murine microgial cell18. Another study showed that the
administration of deidzein was induced the decreasing of TNF- level19.
In conclusion, the herbal supplement of E. scaber and S. androgynus can optimize the level of pituitary-
gonadal hormone and decrease the level of necrotic hepatocyte cell in pregnant mice under Salmonella typhimurium
infection. The optimum dose of P4 is effective to maintain the LH level on day 17 pregnancy of pregnant mice
typhoid fever. Therefore P1dose effective to maintain the FSH level on day 9, 13, 17 pregnancy of pregnant mice
typhoid fever. The histopathological study showed that all treatments were significantly decreased the percentage
of necrotic cell on the liver of pregnant mice under Salmonella typhimurium infection.
ACKNOWLEDGEMENT
The authors would like to thank all Laboratory of Animal Physiology team for their support in conducting
this research especially to Eltavia F, DwijayantiDR and Pristiwanto B for experimental help, and for Brawijaya
University for facilitating this research.
Conflict of Interest
There is no conflict of interest in this research.

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9
Validation of traditional medicine through ethnopharmacological
investigation including intellectual property rights
P. Pushpangadan (Padma Shri Awardee) and T P Ijinu
Amity Institute for Herbal and Biotech Products Development, 3 Ravi Nagar, Peroorkada P.O.,
Thiruvananthapuram-695005, Kerala, India. E-mail: palpuprakulam@yahoo.co.in
Abstract
The history of medicine in India can be traced back to several millennia to the written treatises of the Vedic and
Samhita periods. Until the beginning of the 19th century, Ayurveda, Siddha, Unani and Amchi systems of
medicines took care of the health care needs of the people. With the advent of modern medicine traditional
systems of medicines in India suffered a setback. However, with the growing disillusionment with the ill effects
of modern medicine, traditional systems of medicines are now regaining their lost glory. With the advancement
of science, there is an increasing demand for evidence based herbal medicines. The history of human civilization
is all about the management and utilization of the resources around him. From the very beginning of the human
civilization human communities living in different agroclimatic conditions have acquired unique knowledge
about their ambient biodiversity by inherent instinct, or intuition, or by accidental discovery, or by error, or
empirical reasoning or even by conscious trial and experimentation. Ethno- (Gr., culture or people) pharmacology
(Gr., drug) is about the intersection of medical ethnography and the biology of therapeutic action, i.e., a
transdisciplinary exploration that spans the biological and social sciences. Ethnopharmacology research aims at
giving a rational explanation to how a traditional medicine works and to scientifically validate the traditional
drugs using the principles of classical systems of medicine and theories and tools of modern science and
technology including Intellectual Property Rights.
Keywords: Biopiracy, Benefit Sharing, Traditional Knowledge/Medicine, Indian Systems of Medicine,

Ethnopharmacology, Intellectual Property Rights, Patent
INTRODUCTION
The history of human civilization and development of economic systems are all inherently and inveterately
interwoven with our biological resources (Pushpangadan et al., 1997). The grand challenge for the twenty-first
century is to harness knowledge of Earths biological diversity and how it shapes the global environmental systems
on which all of life depends. This knowledge is critical to science and society for managing natural resources, for
sustaining human health, for ensuring economic stability, and for improving the quality of human life. Urgent need
for this knowledge increases daily as the conversion of natural systems to human managed systems accelerates the
decline of biological diversity (Raven and Wilson, 1992; Wilson, 1998).
Economic activity of humankind continues to derive its sustenance directly or indirectly from the biological
resources. The unknown potentials of genetic diversity found in the biological organisms, particularly the plants
represent a never ending biological frontier of inestimable value. Genetic diversity will enable breeders to tailor
crops to meet the increased productivity, adapt to changing climatic conditions, disease resistance and to meet the
other essential needs and future aspirations of humankind. Bio-genetic resources are the primary source of valuable
genes, chemicals, drugs, pharmaceuticals, natural dyes, gums, resins, enzymes or proteins of great health, nutritional
and economic importance (Laird and ten Kate, 2002). Exploration of biodiversity for commercially valuable genetic
and biochemical resources is termed as bioprospecting - a concept pioneered by Thomas T. Eisner as Chemical
Prospecting (Eisner, 1989, Reid et al., 1993). The advancements in biotechnologies have further redefined the
10
overall scope and utility of bioprospecting to encompass all relevant activities related to systematic search for
genes, natural compounds, designs and whole organisms in wildlife with a potential for product development by
biological observations and biophysical, biochemical and genetic methods without disruption to Nature (Mateo et
al., 2001). In short, bioprospecting involves investigation of genetic resources or biochemicals for new commercial
leads (Laird and ten Kate, 2002) and includes three major areas such as Chemical Prospecting, Gene Prospecting
and Bionic Prospecting (Mateo et al., 2001).
Biodiversity and traditional knowledge

Biodiversity rich countries with a long history of cultural diversity have generated immensely valuable
knowledge systems on the use of biological and genetic resources. Such knowledge is known as Traditional
Knowledge (TK). Making the best use of the TK and generating new knowledge, products and technologies is the
method of generating economic wealth. TK is a community based system of knowledge that has been developed,
preserved and maintained over many generations by the local indigenous communities through their continuous
interactions, observations and experimentation with their surrounding environment. It is unique to a given culture
or society and is developed as a result of the co-evolution and co-existence of both the indigenous cultures and
their traditional practices of resource use and ecosystem management. TK is a general term, which refers to the
collective knowledge, beliefs and practices of indigenous/local people on sustainable use and management of their
ambient resources. Through years of observation and analysis, trial, error or experimentation, the traditional
communities have been able to identify useful as well as harmful elements of their ambient flora and fauna. Such
knowledge (acquired through ages) has always remained as part of their life, culture, traditions, beliefs, folklore,
arts, music, dance, etc. TK covers a broad spectrum of the local and indigenous peoples traditional life and
culture, art, music, architecture, agriculture, medicine, engineering and a host of other spheres of human activity.
TK thus can be of direct or indirect benefit to society as it is often developed, in part as an intellectual response to
the necessities of their life. Protection and maintenance of TK of local and indigenous communities is vital for their
wellbeing and sustainable development and for their intellectual and cultural vitality (WIPO, 2004; Pushpangadan et
al, 2008). The accumulated wisdom, knowledge, beliefs and practices embodied in the TK systems were handed
down to generations by an unbroken tradition and culture. This is still a living tradition in many parts of the
biodiversity rich countries of the world. A study of such knowledge system of traditional societies about the plant
world is the subject matter of the science Ethnobotany.
Ethnobotany and India

Since the beginning of human culture, humankind sought to use the local surroundings for health beneficent
purposes. Sometimes this knowledge was written in great tomes of medicinal plant lore, for the most part though
the knowledge was passed through the oral tradition and held closely by the various medicine keepers, whose
status in a society was usually esteemed as a healer. Until 1899, it was the therapeutic properties of medicinal
plants, the knowledge held and promulgated within and between diverse cultures over the millennia, which formed
the basis of global drug-related health care, irrespective of your wealth or status. The introduction of the semi-
synthetic drug Aspirin, based on a traditional medicine, changed that dynamic (Cordell, 2015).
The beginning of present day ethnobotanic enquiry can be traced back to Stephen Powers, who in 1874
used the term aboriginal botany, which included the total aboriginal dependence on plants for food, medicines,
etc. Walter Hough in 1898 defined it as study of plants in their relation to human culture including psychological
importance and mythological reference (Ford, 1978). As a term, ethnobotany was first applied to the study of
plants used by primitive and aboriginal people by the U.S. botanist John William Harshberger (University of
Pennsylvania) in 4th April, 1895, who was addressing a university archaeological association in America (to refer
the study of plants, used by the aboriginals of Australia) and later published in Botanical Gazette in 1896. The term
was broadened by Robbins et al. (1916) and they suggested that the science of ethnobotany should include the
investigation and evaluation of the knowledge of all phases of life amongst primitive societies and of the effects of
11
the vegetal environment upon the life customs, beliefs and history of these tribal people. It was then refined again
and again by various workers. Twenty-five years later, Jones (1941) advanced a more concise definition, the
study of the interrelationships of primitive men and plants. Schultes in 1967, expanded this to include the relationships
between man and his ambient vegetation.
Very little organized work in Ethnobotany had been done in India till about 40 years ago. Organized field
work and other studies in the subject were started in the Botanical Survey of India (BSI). Also there has been a
resurgence of interest developed in ethnobotanical research in various institutions. Dr E K Janaki Ammal initiated
researches on ethnobotany in BSI. She studied food plants of certain tribals of south India. When I joined Regional
Research Laboratory, Jammu now known as Indian Institute of Integrative Medicine, Jammu under Dr Janaki
Ammal she has fondly told me about the importance of doing ethnobotanical studies in South India. However, I
could do it only after 1984 when I started research in Ethnobotany (Pushpangadan and Atal, 1984, 1986). From
1960, Dr. S K Jain from BSI started intensive field work among the tribals of Central India. He devised methodology
for ethnobotany particularly in the Indian context. The publications from this group in the early sixties triggered the
ethnobotanical activity in many other centers, particularly among botanists, anthropologists and medical practitioners
in India (Bondya et al., 2006; Bora and Pandey, 1996; Borthakuar, 1981; Borthakur, 1996; Borthakur and Gogoi,
1994; Hajra, 1981; Hajra and Baishya 1997; Jain, 1987,1991,2002, 2005, 2006, 2010; Jain & Goel, 1987,2005; Jain
& Sikarwar, 1998; Jain et al., 1994, 1997; Janaki Ammal, 1956; Joshi, 1995, Joseph and Kharkongor 1981;
Manilal, 1978, 1980a,b,c, 1981, 1996, 2005, 2012; Manilal et al., 2003; Mohanty, 2003, 2010; Mohanty and Rout,
2001; Patil, 2000, 2001, Pushpangadan, 1986, 1990; Pushpangadan et al., 1995, 2012; Pushpangadan and Dan,
2011; Pushpangadan and George, 2010; Mitra, 1998a, 1998b; Singh et al., 2011; Subramoniam et al., 1997,
1998; Vartak, 1981; Vartak and Gadgil, 1980, 1981, Biswas and Das, 2011; Chettri et al., 2014; Chowdhury and
Das, 2007; Das and Pandey, 2007; Ghosh and Das, 2004). During the last four decades similar work has been
initiated at various centres such as National Botanical Research Institute (NBRI) at Lucknow, National Bureau of
plant Genetic Resources (NBPGR) at Delhi, Jawaharlal Nehru Tropical Garden and Research Institute (JNTBGRI),
Thiruvananthapuram, Central Council of Research in Unani Medicines (CCRUS), Central Council of Research in
Ayurveda and Siddha (CCRAS) and in some other Institutions.
Bioactive leads from ethnobotany
It was not until the 19th century that man began to isolate the active principles of medicinal plants and one
particular landmark was the discovery of quinine (anti-malarial drug) from Cinchona bark by the French pharmacists,
Caventou and Pelletier in 1820. Such discoveries led to an interest in plants from the New World and expeditions
scoured the almost impenetrable jungles and forests in the quest for new medicines (Phillipson, 2001). Today
ethnobotany has become an important and crucial area of research and development in resource management,
conservation of biodiversity of flora and fauna at ecosystem levels for socio-economic development of the region.
With the appearance of new vistas of ethnobotanical studies, the scope of ethnobotany has now greatly engorged,
both in terms of its theoretical contributions to an understanding of plant human relationship as well as for practical
applications and utilization of biological knowledge of tribes to their medicine systems along with multifarious
ways (Pushpangadan, 2014). The ways in which plants that are used by tribal peoples are valuable for modern
medicine are described below with examples (Gurib-Fakim, 2006).
a. Plants from the tropics are sometimes used as sources of direct therapeutic agents, e.g., the alkaloid D-
tubocurarine is extracted from the South American jungle Liana Chondrodendron tomentosum and is widely
used as a muscle relaxant in surgery. Chemists are still unable to produce this drug synthetically in a form
that has all the attributes of the natural product and therefore collection from the wild is still relied upon.
Surprisingly harvesting of medicinal plants is often less costly than artificial drug synthesis. Another good
example to illustrate this feature is reserpine, an important hypotensive agent extracted from Rauwolfia.
The synthesis of this molecule would cost three times as much as opposed to collection.
12
b. Tropical plants are also used as sources of starting points for the elaboration of semi-synthetic compounds.
e.g., saponin extracts that are chemically altered to produce sapogenins necessary for the manufacture of
steroidal drugs. Until relatively recently, 95% of all steroids were obtained from extracts of neo-tropical
Yams of the genus Dioscorea.
c. Flora from the tropics can serve as sources of substances that can be used as models for new synthetic
compounds. e.g., cocaine from Coca plants, Erythroxylum coca, has served as model for the synthesis of a
number of local anaesthetics such as procaine. New and unusual chemical substances found in plants will
continue to serve as blueprints for novel synthetic substances and will prove to be increasingly important in
the future.
d. Plants can also be used as taxonomic markers for the discovery of new compounds. From a phytochemical
standpoint, the Plant Kingdom has been investigated in a haphazard manner; some families have been relatively
well-studied while others have been almost completely overlooked. For example, many uses have been
documented for the Liliaceae and the family is known to be rich in alkaloids. Little, on the other hand is
known on the Orchidaceae. Some plants from the family have been investigated because of their close
relationship to the Liliaceae. Research has shown that they are not only rich in alkaloids but that some of the
alkaloids are unique and could prove useful in the future.
The Rosy Periwinkle (Catharanthus roseus, Apocynaceae) represents a classical example of the importance
of plants used by local people. This herbaceous plant, native to southeastern Madagascar, is the source of over 75
alkaloids, two of which are used to treat childhood leukaemia and Hodgkins disease with a very high rate of
success. This species was first investigated in the laboratory, mainly because of its use by local people as an oral
hypoglycaemic agent. Like Catharanthus, many drugs that are commonly used today (e.g., aspirin, ergometrine,
tubocurarine, digoxin, reserpine, atropine etc.) came through the use of indigenous medicine that is through the
scientific investigations of plants used by people throughout the world. Thus, it can be seen that the investigation
of plants used for medicinal purposes by unsophisticated people can provide us with new biodynamic compounds
that may have important applications in our society (Farnsworth et al., 1985; Balick and Cox, 1996; Duke et al.,
2000; Soejarto et al., 2011). Table 1 lists a few of the many examples of drugs derived through ethnobotanical
leads.
Table 1 : Some examples of drugs derived through ethnobotanical leads
Drug Medical use Plant species Family

Acetyldigitoxin Cardiotonic Digitalis lanata Scrophulariaceae
Aescin Anti-inflammatory Aesculus hippocastanum Hippocastanaceae
Agrimophol Antihelmintic Agrimonia eupatoria Rosaceae
Ajmalicine Circulatory stimulant Rauvolfia serpentina Apocynaceae
Andrographolide Antibacterial Andrographis paniculata Acanthaceae
Anisodamine Anticholinergic Anisodus tanguticus Solanaceae
Anisodine Anticholinergic Anisodus tanguticus Solanaceae
Arecoline Antithelmintic Areca catechu Arecaceae
Artemisinin Antimalarial Artemisia annua Asteraceae
Asiaticoside Vulnerary Centella asiatica Apiaceae
Aspirin Analgesic, inflammation Filipendula ulmaria Rosaceae
13
Atropine Anticholinergic Atropa belladonna Solanaceae

Berberine Antibacterial Berberis vulgaris Berberidaceae
Bergenin Antitussive Ardisia japonica Myrsinaceae
Caffeine CNS stimulant Camellia sinensis Theaceae
Camphor Rheumatic pain Cinnamomum camphora Lauraceae
(+)-Catechin Hemostatic Potentilla fragarioides Rosaceae
Cheirotoxin Cardiotonic Adonis vernalis Ranunculaceae
Codeine Analgesic, antitussive Papaver somniferum Papaveraceae
Colchicine Antitumor agent, anti-gout Colchicum autumnale Liliaceae
Convallatoxin Cardiotonic Convallaria majalis Liliaceae
Curcumin Choleretic Curcuma longa Zingiberaceae
Cynarin Choleretic Cynara scolymus Asteraceae
Deserpidine Antihypertensive, tranquilizer Rauvolfia tetraphylla Apocynaceae
Deslanoside Cardiotonic Digitalis lanata Scrophulariaceae
Digitalin Cardiotonic Digitalis purpurea Scrophulariaceae
Digitoxin Cardiotonic Digitalis purpurea Scrophulariaceae
Digoxin Cardiotonic Digitalis lanata Scrophulariaceae
Emetine Amebicide, emetic Psychotria ipecacuanha Rubiaceae
Ephedrine Sympathomimetic Ephedra sinica Ephedraceae
Esculetin Antidysentery Fraxinus rhynchophylla Oleaceae
Etoposide Antitumor agent Podophyllum peltatum Berberidaceae
Glaucarubin Amebicide Simarouba glauca Simaroubaceae
Glycyrrhizin Sweetener Glycrrhiza glabra Fabaceae
Gossypol Male contraceptive Gossypium sp. Malvaceae
Hemsleyadin C Antibacterial, Antipyretic Hemsleya amabilis Cucurbitaceae
Hydrastine Hemostatic, astringent Hydrastis canadensis Ranunculaceae
Hyoscyamine Anticholinergic Hyoscyamus niger Solanaceae
Kawaina Tranquilizer Piper methysticum Piperaceae
Khellin Bronchodilator Ammi visnaga Apiaceae
Lanatoside A Cardiotonic Digitalis lanata Scrophulariaceae
Lobeline Respiratory stimulant Lobelia inflata Campanulaceae
Metformin Anti-diabetic Galega officinalis Fabaceae
Monocrotaline Antitumor agent Crotalaria spectabilis Fabaceae
Morphine Analgesic Papaver somniferum Papaveraceae
14
Neoandrographolide Antibacterial Andrographis paniculata Acanthaceae
Noscapine Antitussive Papaver somniferum Papaveraceae
Ouabain Cardiotonic Strophanthus gratus Apocynaceae
Papain Proteolytic, mucolytic Carica papaya Caricaceae
Phyllodulcind Non-caloric sweetener Hydrangea macrophylla Saxifragaceae
Physostigmine Anticholinesterase Physostigma venenosum Fabaceae
Picrotoxin Analeptic Anamirta cocculus Menispermaceae
Pilocarpine Parasympathomimetic Pilocarpus jaborandi Rutaceae
Podophyllotoxin Escharotic Podophyllum peltatum Berberidaceae
Protoveratrine A Antihypertensive Veratrum album Liliaceae
Pseudoephedrineb Bronchodilator Ephedra sinica Ephedraceae
Pseudoephedrine Bronchodilator Ephedra sinica Ephedraceae
Quinine Antimalarial Cinchona pubescens Rubiaceae
Quisqualic acid Antihelmintic Quiqualis indica Combretaceae
Rescinnamine Antihypertensive, tranquilizer Rauvolfia serpentina Apocynaceae
Reserpine Antihypertensive, tranquilizer Rauvolfia serpentina Apocynaceae
Rorifone Antitussive Rorippa indica Brassicaceae
Rotenone Piscicide Lonchocarpus nicou Fabaceae
Rotundine Analgesic, sedative, tranquilizer Stephania sinica Menispermaceae
Rutin Capillary antihemorrhagic Citrus sp. Rutaceae
Salicin Analgesic Salix alba Salicaceae
Santonin Ascaricide Artemisia maritima Asteraceae
Scillaren A Cardiotonic Urginea maritima Liliaceae
Scopolamine Sedative Datura metel Solanaceae
Sennoside A Laxative Senna alexandrina Fabaceae
Silibinin Antihepatotoxic Silybum marianum Asteraceae
Stevioside Sweetener Stevia rebaudiana Asteraceae
Strychnine Central nervous system stimulant Strychnos nux-vomica Loganiaceae
Teniposidec Antitumor agent Podophyllum peltatum Berberidaceae
Tetrahydropalmatine Analgesic, sedative, tranquilizer Corydalis ambigua Papaveraceae
Theobromine Diuretic, vasodilator Theobroma cacao Sterculiaceae
Theophylline Diuretic, bronchodilator Camellia sinensis Theaceae
Tubocurarine Skeletal muscle relaxant Chondrodendron tomentosum Menispermaceae
Valepotriate Sedative Valeriana officinalis Valerianaceae
15
Vinblastine Anticancer Catharanthus roseus Apocynaceae
Vincamine Cerebral stimulant Vinca minor Apocynaceae
Vincristine Anticancer Catharanthus roseus Apocynaceae
Yohimbine Adrenergic blocker, aphrodisiac Pausinystalia johimbe Rubiaceae
Yuanhuacine Abortifacient Daphne genkwa Thymelaeaceae
Yuanhuadin Abortifacient Daphne genkwa Thymelaeaceae
Ethnopharmacology approach
Ethnopharmacology as a scientific term was first introduced at an International Symposium held at San
Francisco in 1967 (Efron et al., 1970). This was used while discussing the theme Traditional Psychoactive Drugs
in this Congress. But later Rivier and Bruhn (1979) made an attempt to define ethnopharmacology as a
multidisciplinary area of research concerned with observation, description and experimental investigation of
indigenous drugs and their biological activities. It was later redefined by Bruhn and Holmstedt (1981) as the
interdisciplinary scientific exploration of biologically active agents traditionally employed or observed by man.
In its entirety, pharmacology embraces the knowledge of the history, source, chemical and physical properties,
compounding, biochemica l and physiologic al effects, mechanism of ac tion, absorption, distribution,
biotransformation, excretion and therapeutic and other uses of drugs. A drug is broadly defined as any substance
(chemical agent) that affects life processes. Therefore, briefly, the main component of ethnopharmacology may be
defined as pharmacology of drugs used in ethnomedicine. Hansen et al. (1995) has suggested that the objectives of
ethnopharmacology should focus on: i) the basic research aiming at giving rational explanation as to how a traditional
medicine works, and ii) the applied research aiming at developing a traditional medicine into a modern medicine
(pharmacotherapy) or to develop its original usage by modern methods (phytotherapy).
Ethnopharmacological impulse to modern medicine can lead to many novel useful drugs. Ethnopharmacology
has become a scientific backbone in the development of active therapeutics, based upon traditional medicine of
various ethnic groups. The ultimate aim of ethnopharmacology is validation of these traditional preparations, either
through pharmacological findings or through the isolation of active substances. Harmful practices can be
discouraged, such as the use of plants containing tumor producing pyrrolizidine alkaloids. Selection of plant for
serious study depends basically on two approaches. One approach is random screening of plants for their medicinal
value. Another approach is ethnopharmacological survey of plants of a particular region or cultural group depending
on their use
The oldest record of granting such a monopolistic right to an individual for a new innovation was in Greek.
The Greek emperor in 700 BC granted one year monopoly right to the royal cook who prepared a delicious dish
using the spices particularly Ginger imported from the western coast of India (Kerala). It was something like a
patent right. But the patent rights as we know today perhaps started in 15th century in Italy it got its definite shape
after the Paris convention in 1883. The term Intellectual Property is loosely defined as a Product of the
mind. According to WIPO, one of the specialized agencies of the U.N system provided that Intellectual Property,
shall include rights related to:
a. Literary, artistic and scientific work.
b. Performance of performing artist, phonograms and broadcasts.
c. Invention in all fields of human endeavour.
d. Scientific discoveries.
16
e. Industrial design.
f. Trademarks, service marks and commercial names and designations.
g. Protection against unfair competition and all other rights resulting from intellectual activities in the industrial,
scientific, literary or artistic fields.
IPRs emerged strongly during the industrial revolution and it has been an important driving force behind
rapid industrial growth and prosperity of the Western countries during the last 3 centuries. IPRs of 18th and 19th
centuries were primarily to protect engineering or chemical innovations wherein identification of the novelty, the
inventive step and innovator is easy. Recent worldwide extension of the IPRs in the field of biology has however
attracted much controversy. The IPR regime from the very beginning has not given any importance and totally
neglected informal knowledge of the people. It was not even considered worth acknowledging. In many cases the
informal knowledge of traditional communities which directly or indirectly contributed to several inventions or
patentable products, was never acknowledged. The profit accrued from commercialization of such products was
never shared with the traditional communities. The traditional communities in many parts of the world were
voicing against this exploitation. In the first International Congress of Ethnobiology held in Belem (Brazil) in 1987,
leaders of indigenous communities mainly from Latin America, many environmentalists, scientists and activists for
the first time seriously discussed this issue and resolved to come out with a declaration known as Declaration of
Belem. This declaration recognized a basic obligation that procedures be developed to compensate native people
for the utilization of their knowledge and their biological resources. The second congress of the international
society held at Kunming, China in 1990 in which the senior author was elected the Treasurer of this International
Society, resolved to establish a global action plan, the Kunming Action Plan, calling for specific and urgent action
to stop destruction of biological and cultural diversity as mandated in the declaration of Belem. This meeting also
gave shape to the establishment of the Global Coalition for Bio-Cultural Diversity to unite the indigenous people,
scientists and environmentalists concerned with the protection of indigenous/local peoples rights. The presence of
this societys powerful spokesmen at Rio-de-Janeiro in 1992 during the Earth Summit was mainly responsible for
the incorporation of Article 8(j) and Article 10(c) in Convention on Biological Diversity to ensure legal protection
for traditional/local community rights (Pushpangadan and Nair 2005).
17
Table 2 CBD Provisions for Access, Benefit Sharing and Protection of Traditional Knowledge
(Pushpagadan, 2010).
CBD acknowledge IPR of the collective wisdom and common resources of the communities as a sovereign
property, whereas WTO-TRIPS recognize IPR as the monopolistic rights of individual or corporate innovators.
This disparity provides the developing countries like India with enormous challenges and opportunities. Several
countries are now working towards amending their existing laws or enacting few national legislations including sui
generis model to make them compatible with the CBD and TRIPS provisions. Government of India is quite sensitive
to the fast changes that are taking place and has displayed strong commitment for the conservation and sustainable
utilization of the bioresources and traditional knowledge systems of our country.
The Indian legislations [the Biological Diversity Act 2002, Plant Variety Protection and Farmers Rights Act
PVPFR 2001, Patent (Amendment) Act 2005] address these issues and suggest provisions to implement them
under the framework of relevant international laws subject to national priorities.
Biological Diversity Act (2002)

As per the provisions contained in the Biological Diversity Act , the national, state and local biodiversity
boards/committees are entrusted to oversee and implement benefit sharing mechanisms, documentation of
bioresources and traditional knowledge, material transfers, access agreements on genetic resources and technologies,
etc. The Act stipulates norms for access to biological resources and traditional knowledge based on three ways: (i)
Access to biological resources and traditional knowledge to foreign citizens, companies and NRIs based on prior
approval of NBA (Section 3 of the Act). (ii) Access to Indian citizens, companies, associations and other organizations
registered in India on the basis of prior intimation to the State Biodiversity Board concerned (Section 7 of the
Act). (iii) Exemption of prior approval or intimation for local people and communities, including growers and
cultivators of biodiversity, and Vaidyas and Hakims, who have been practicing indigenous medicines (Section 7 of
the Act) (NBA 2002, 2004).
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Transfer of research results
The Act does not permit any person to transfer the results of any research relating to biological resources
obtained from India for monetary consideration to foreign nationals, companies or NRIs without the prior approval
of the Authority.
Procedure for Prior Approval before Applying for IPR (Rule 18, Sub rules 16)
All the conditions for granting approval for transfer of research results shall be applicable to any person
desirous of applying for a patent or any other intellectual property rights, based on biological resources and
knowledge obtained from India.
Procedures for Third Party Transfer of Accessed Biological Resources or Knowledge (Rule 19, Sub rules
16)
The Act permits transfer of accessed biological resources or traditional knowledge to a third party on the
basis of the prior approval of the Authority.
Criteria for benefit sharing

The Act, subject to Section 21 and Rule 20 of the Biodiversity Rules, insists up on including appropriate
benefit sharing provisions in the access agreement and mutually agreed terms related to access and transfer of
biological resources or knowledge occurring in or obtained from India for commercial use, bio-survey, bio-utilization
or any other monetary purposes. The Authority shall develop guidelines and shall notify the specific details of
benefit sharing formula in an official gazette on a case to case basis. The suggested benefit sharing measures may
include monetary benefits such as royalty, joint ventures, technology transfer, product development, and non-
monetary benefits such as education and awareness raising activities, institutional capacity building, venture capital
fund, etc. The time frame and quantum of benefits to be shared shall be decided on case to case based on mutually
agreed terms between the applicant, authority, local bodies, and other relevant stakeholders, including local and
indigenous communities. One of the suggested mechanisms for benefit sharing includes direct payment to persons
or group of individuals through district administration, if the biological material or knowledge was accessed from
specific individuals or organizations. In cases where such individuals or organizations could not be identified, the
monetary benefits shall be paid to the National Biodiversity Fund. Five percent of the benefits shall be earmarked
for the Authority or State Biodiversity Board towards the administrative service charges.
The Protection of Plant Varieties and Farmers Rights (PPVFR) Act, (2001)
The PPVFR Act 2001 and the Rules framed under this Act, called the PPVFR Rules 2003, deal primarily
with the protection of plant breeders rights over the new varieties developed by them and the entitlement of
farmers to register new varieties and also to save, breed, use, exchange, share or sell the plant varieties, which the
latter have developed, improved, and maintained over many generations. The Act is a deviation from the 1991
UPOV Model and can be regarded as an alternate sui generis system that accord protection of the rights of the
formal innovations of a plant breeder and the informal knowledge system and traditional plant varieties of the
farmers as well. The important provisions contained in this Act relevant to ABS are those on the protection of
farmers rights and the mechanisms suggested for compensation or benefit sharing for the contributions of local
communities or farmers in the development of a new plant variety.
The Patents (Amendment) Act (2005)

This Act, which is an amendment to the Patent Act of 1970, is significant in the sense that it stipulates
disclosure of the source and geographical origin of the biological materials in the specification, when used in an
19
invention. This issue of disclosure of source and geographical origin is a contentious one that the LMMC and
other developing and least developing countries are still pushing ahead with the WTO-TRIPS. The new Patent Act
of India includes two important clauses for revocation of a patent on the grounds that: (i) complete specification
does not disclose or wrongly mentions the source of geographical origins of biological materials used for the
inventions~ (ii) invention so far as claimed in any claims of the complete specification is anticipated having regard
to the knowledge, oral or otherwise, available within any local or indigenous community in India or elsewhere.
These two provisions ensure protection of the rights of the source country of a biological material or traditional
knowledge of local or indigenous community, and thereby enabling recognition and reward of source countries and
traditional knowledge holders through appropriate benefit sharing mechanisms.
Access and benefit-sharing

Access and Benefit Sharing (ABS) has emerged as the most complex issues where the UN-CBD and WTO
came on a direct confrontation. Both TRIPs Council of WTO and the Conference of Parties (COP) to CBD have
been considering ironing out these contradictions. CBD began to address the ABS issues and their implementations
since the Fourth Meeting of the COP held in Bratislava in 1988, which finally led to the development of Bonn
Guidelines in October 2001 (CBD/COP decision VI/24, 2001). The Bonn guidelines provide the parties and
stakeholders with a framework to facilitate access to genetic resources and ensure fair and equitable sharing of
benefits through standard practices and procedures of Prior Informed Consent (PIC), Mutually Agreed Terms
(MAT) and Material Transfer Agreements (MTA) and other relevant agreements. The Guidelines provide details of
an overall strategy and essential steps, elements and principles to be adopted in developing ABS regime by parties
and stakeholders.
Benefit sharing is an important component in any ABS or technology transfer contracts involving genetic
resources and associated TK. MAT in accordance with Article 15.7 of UN-CBD should pay adequate attention to
reaching an agreement on fair and equitable sharing from the commercial or other utilization of the resources and
the TK accessed. The benefit sharing mechanisms and formula may significantly vary depending upon for the
purpose for which the genetic resources and/or TK are accessed (Pushpangadan and Nair, 2005). The monetary
benefits (e.g., license fees, royalties) need to be fixed depending upon the actual capital inputs including S&T
inputs, human resource inputs and intellectual inputs provided by the participating countries in any joint prospecting/
bio-partnership programmes. Although the Bonn Guidelines provide a conceptual as well as resources and/or
traditional knowledge is almost impractical. Therefore, the TWCs have to address the benefit sharing issues with
a balanced and flexible approach. Also there is a need for the Like Minded Mega-diverse Countries (LMMC) and
other regional groups of biodiversity rich countries to strengthen collaborative partnership among their members to
build up capacity building in all relevant areas of biodiversity, biotechnology, intellectual property, information
management, etc. Such regional cooperation would be helpful to develop national legal and policy frameworks on
ABS and to harmonize the various statutory mechanisms through conscientious discussions, besides developing a
joint strategic action plan to deal with all ABS and related issues at international forums.
Many countries from the South felt that while the Bonn Guidelines elaborated on access, they had left the
benefit-sharing aspect relatively unspecific. The voluntary nature of the Guidelines has been judged as insufficient
for implementing the ABS provisions of the CBD. In order to further implement the third objective of the CBD and
its ABS related provisions, the World Summit on Sustainable Development, held in Johannesburg, called for action
(WSSD 2002, 44o) to negotiate within the framework of the CBD an international regime to promote and safeguard
the fair and equitable sharing of benefits arising out of the utilization of genetic resources. In 2004, in response to
this call for action the COP mandated the Ad Hoc Open-ended Working Group on ABS (COP 5 decision V/26) with
the collaboration of the Working Group on Article 8(j) and related provisions (COP 4 decision IV/9), to elaborate
and negotiate an International Regime on Access to Genetic Resources and Benefit Sharing with the aim of adopting
instrument(s) to effectively implement the provisions in Article 15 and 8(j) of the Convention and the three objectives
20
of the Convention and at its ninth meeting, in 2008, in Bonn, Germany, the COP agreed on a schedule of meetings
to complete negotiations before its tenth meeting, in 2010 at Nagoya, Japan.
Nagoya protocol on ABS

The objective of the Nagoya Protocol is the fair and equitable sharing of benefits arising from the utilization
of genetic resources, with a view to contributing to the conservation of biodiversity and the sustainable use of its
components. Benefit-sharing is envisaged through appropriate access to genetic resources, the transfer of relevant
technologies, and funding. Benefit-sharing obligations also arise from the use of traditional knowledge associated
with such genetic resources and genetic resources held by indigenous and local communities. In this regard, the
Nagoya Protocol is particularly innovative: it is the first time that such obligations are triggered by the use of
traditional knowledge for research and development purposes in an international legally binding instrument. The
Protocol is also innovative in detailing measures to ensure compliance with ABS-related obligations an aspect that
was neglected under the CBD (Tsioumani, 2015).
The Protocol adopted at the 10th meeting of COP, in Nagoya, Japan, on 29 October 2010. The meeting also
decided to establish the Open-Ended Ad Hoc Intergovernmental Committee for the Nagoya Protocol on ABS, as an
interim governing body for the Nagoya Protocol, to undertake the preparations necessary for the first meeting of
the parties to the protocol. The intergovernmental committee met three times. The Nagota Protocol entered into
force on 12 October 2014. To date it has been ratified by 93 parties, which includes 92 UN member states and the
European Union.
Nagoya protocol and India

India, being the leading country in ABS legislation with mechanisms in place, developed in true sense the
system of fair and equitable sharing of benefits. On November 2014, the Ministry of Environment, Forests and
Climate Change, GoI, in exercise of the powers conferred by section 64 read with subsection (1) of section 18 and
subsection (4) of section 21 of the Biological Diversity Act 2002 (18 of 2003) and in pursuance of the Nagoya
protocol on access to genetic resources and the fair and equitable sharing of benefits of their utilisation to the CBD
and in consultation with National Biodiversity Authority issued guidlines of Access to Biologica Resources and
Associated Knowledge and Benefit Sharing Regulations, 2014, for research and operations of bio-survey and bio-
utilisation as well as procedure for access to biological resources. The other aspect covered under the notifications
includes the option of benefit sharing on sales price of the biological resources accessed for commercial utilisation
under regulation, collection of fees as well as procedure for transfer of results of research relating to biological
resources. It also included the issues related to IPR and procedure for transfer of accessed biological resources
and/or associated knowledge to the third party for research/ commercial utilisation.
According to NBA, Indias engagement with ABS issues has been progressive and noteworthy. India has
perhaps the first country in the world that has been able to tap into the magnitude of ABS, having dealt with over
1500 ABS applications by August, 2015, the NBA signed more than 100 agreements on ABS. The total royalty
collected by NBA would be about Rs.17 crores (US $2,559,662.80) as of now, out of which Rs.15 crores (US
$2,258,526.00) is obtained through the recent auction on red sanders. Part of this money will be ploughed to SBBs/
BMCs as the case may be, and the mode of distribution of the same is yet to be worked out.
Kani model or TBGRI model of benefit sharing a case study

In India we can be proud of having the distinction of the first country in experimenting a benefit sharing
model that implemented in letter and spirit Article 8(j) and Article 15.7 of CBD. The author and his team while at
Tropical Botanic Garden and Research Institute (TBGRI) demonstrated that indigenous knowledge system merits
support, recognition and fair and adequate compensation. Based on lead obtained from a Kani tribe of Kerala, the
21
author and his co-workers developed an antifatigue, immuno-enhancing herbal formulation named Jeevani. The
technology of production of this drug when transferred to a pharmaceutical company on payment of license fee
and a royalty of 2% on the ex-factory sale of product, TBGRI resolved to share the license fee and royalty with the
Kani tribe in a 1:1 ratio. Currently this model is acclaimed as a model to be emulated in similar situations elsewhere
in the world. Although this model was worked out in early 1994 in full consultation with the Kani tribe it took
almost 3-4 years to effect this model mainly because of the inherent inability of the Kani people to receive the
benefit. Finally, the majority members of Kani tribe resolved to form a Trust which was then registered and in
February, 1999 the license fee and royalty due to them was transferred to the Trust. The trust continued to receive
the license fee and the royalties accrued from the drug developed from their knowledge of a lesser known wild
plant till recently (Pushpangadan and Pradeep, 2008; Pushpangadan and George, 2010; Pushpangadan et al., 2014;
2015a,b; 2016a,b; 2017, Pushpangadan and Ijinu, 2017).
Outcome and lessons learnt

The sharing of benefits with the Kanis and the formation of the Kani Samudaya Kshema Trust fund have
started showing positive impacts in the sense that the tribal community is now becoming conscious about the
values of and rights over their knowledge system and associated biological resources. These developments also
have helped in bringing the Kani families to a single organizational framework, so that the benefits accrued from the
trust fund could be utilized for the economic well being and social development in the Kani tribal hamlets. The Kani
tribe is now a proud and dignified community. The trust has now a fully furnished 4 room building (office room,
conference hall, library cum reading room and a store room). The Kani community regularly meet at this building,
discuss their problems and take decisions for the welfare of the community. For the first time in the history of the
Kani tribe, now they feel confident and dignified. They have initiated various welfare programmes for the benefit of
their community. Looking back 15 years, it is unbelievable to see the kind of transformation of this otherwise
timid, nomadic forest dwelling tribe who used to be scared of outsiders and foresters. They now stand up with
dignity and claim their rights and privileges. The quality of their life has been tremendously improved. There are
now many Kani boys and girls who passed high school and even graduated for which the trust was instrumental.
The trust recently acquired a fine 12 seated vehicle and a Kani young man got the driving license and now drives
this vehicle which ply from their hamlets to the nearest village market and town. The Trust was able to persuade
the local authorities to build a motorable road of over 15-20 km inside the forest in to the tribal villages.
The TBGRI Model is perhaps a unique experiment ever done, wherein the benefits accrued from the
development of a product based on an ethno-botanical lead were shared with the holders of that traditional knowledge.
Considering the significant outcome of this model in community empowerment, income generation and poverty
eradication of a tribal community, Pushpangadan was awarded with the UN-Equator Initiative Prize (under individual
category) at the World Summit on Sustainable Development held in Johannesburg in August 2002. Now with the
CBD-Bonn and WIPO guidelines and our national legislation on Biodiversity in position, the TBGRI or Kani case
study could be taken as an ideal model of equitable benefit sharing involving genetic resources and associated
traditional knowledge (Pushpangadan, 2002).
After the expiry of the license period of 7 years TBGRI has extended the period for further 3 years to the
pharmaceutical company. After the expiry of the patent period , the drug Jeevani is now in public domain. TBGRI
was holding only a process patent and they have not converted this to a product patent. However, TBGRI is now
thinking to manufacture Jeevani with the association of Kerala Kani Samudaya Trust. This if effected will be
advantageous for the Kani Trust (Pushpangadan and Pradeep, 2008; Pushpangadan and George, 2010; Pushpangadan
et al., 2014; 2015a,b; 2016a,b; 2017, Pushpangadan and Ijinu, 2017).
22
CONCLUSION
Ethnobotanical research can provide a wealth of information regarding both past and present relationships
between plants and the traditional societies. Biodiversity and associated TK are important leads for bio-prospecting
and bio-industry. Judicious and harmonious use of these resources in bio-prospecting can lead to sustainable
human development in biodiversity-rich countries of the Third World. Investigations into traditional use and
management of local flora have demonstrated the existence of extensive local knowledge of not only about the
physical and chemical properties of many plant species, but also the phenological and ecological features in the
case of domesticated species. In addition to its traditional roles in economic botany and exploration of human
cognition, ethnobotanical research has been applied to current areas of study, such as biodiversity prospecting and
vegetation management.
Through recognition of indigenous and local communities as holders of IPRs in TK relating to biodiversity
and natural capital, the development of complementary legal infrastructure to support equitable benefit-sharing,
facilitating community governance and self-determination, and protecting against misappropriation, TK as a
technology of biodiversity conservation and innovation can be preserved for future generations and respectfully
shared based on mutually equitable terms in support of sustainable development. The recent spurt of activities
spearheaded by international agencies such as UNEP, FAO, WTO, and ILO etc. to study the complementarities and
possible harmonization of the various international legal instruments and policy frameworks on biodiversity, IP, TK
are expected to bring in some positive changes in near future. The entry into force of the Nagoya Protocol
represents a step in this direction. Thus, the valueaddition to bio-resources and TK and technology transfers
involving TK holders and their community organizations/institutions are key processes that will not only help
ensure the social, cultural, spiritual, economic and technological empowerment of the communities, but also to
promote further enrichment of their TK and traditional resource wealth.
ACKNOWLEDGEMENTS
The authors express their sincere thanks to Dr Ashok K Chauhan, Founder President, Ritnand Balved
Education Foundation (RBEF) and Amity Group of Institutions & Dr Atul Chauhan, Chancellor, AUUP, Noida and
President, RBEF for facilitating this work.
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28
Maintaining and Improving Sustainability of Jamu, aTraditional Local
Based Herbal of Indonesia for Its Fresh Preparation and Usage, Means
or End ?
Joni Kusnadi*
Department of Food Science and Technology, Faculty of Agricultural Technology, University of Brawijaya Jl. Veteran,
Malang, East Java, Indonesia. E-mail : joni.kusnadi@gmail.com
Abstract
Jamuis the traditional local based herbal medicine of Indonesia that has been practised for many centuries in
order to maintain good health and to treat diseases. Fresh preparation and usage of Jamu, still gained their
popularity in the last decade. Traditional fresh Jamu products are herbal which produced and widely used by
large number of people in Indonesia. Although nowadays fresh jamu often used by Indonesian as medicinal
herbs and other purposes, many factors have been reported reducing for their fresh preparation and usage.Jamu
as traditional herbal have been proven scientifically for their safety and efficacy, but concerns over practicality,
availability and consideration of modern medicines may disturb sustainability to use them. Jamu products are
not likely to become means as important alternative to modern standard medicines unless there are changes to
the easier preparation, practicality and usage. More research works should be conducted not only for their
efficacy and safety, but also for more efficient and simpler practical fresh preparation. Beside that the interest
and awareness of the people need to be increased. If such conditions are not be fulfilled, otherwise fresh
preparation and usage of jamu and other traditional herbs will end.
Keywords : sustainability,jamu, efficacy, safety, fresh preparation and usage.
INTRODUCTION
Jamu is the Indonesian traditional herbal medicine that has beenpractised formany centuries in theIndonesian
community to maintain good health and to treat diseases[1]. In spite ofthe great advances observed in modern
medicinein recent decades, medicinal plant still make animportant contribution to health care[2]. Based on its
traditional use,jamu is being developed into arational form of therapy, by herbal practitioners and in the form of
phytopharmaceuticals. Jamu has acquired a potential benefit, both economically and clinically.
At the present time, jamu is a part of living culture in Indonesia and it used not only for curing disease but
also for other uses [3].The uses of jamu are gouped into five categories, as medicines, health-care, beauty-care,
tonic beverage, and body protections or endurance[4]. Although nowadays fresh jamu often used by Indonesian
for many purposes as mentioned above, some factors have been reported reducing for their fresh preparation and
usage.
As many as traditional products face presssure against many modern medicines, thispaperaimstodiscuss
about maintaining and improving sustainibility of jamu related to their fresh preparation and usage. This manuscript
also open for discussion that In the future, how traditional medicines like Jamu, still means or even end for their
fresh preparation and usage.
What Is Jamu ?
Jamu is aword in Javanese tribal language, meaning the traditional medicine from plants. Today, jamu has
been adopted into Bahasa Indonesia with the similar meaning[3].Indonesia has the second biggest biodiversity in the
29
world expressed by a high number of indigenous medicinal plants. Based on this rich source of medicinal plants,
most of the Indonesian people especially in rural areas use traditional herbal medicines known as jamu to treat
disease[5].
Jamu segar is a kind of traditionaljamu consumed directly or sold as jamu gendong, it is produced without
a label and freshly prepared (notpreserved) from plant material in warung, the ubiquitous stalls along the streets in
Indonesia [6,7]. Jamu segaris instantly served to whom orders this jamu requested preparation. The sellers bring
the jamu from door to door. The word gendong itself means to carry something on the back of a body. The fresh
jamu is put inside the bottles and stored in bamboo or rattan baskets and they use along wide shawl called selendang
for carrying the baskets on their back[3].
Nowadays the production of jamu is also being developed on an industrial scale. The Indonesian government,
industry and academiaallrecognizethattofurther the developmentof jamu, extensiveresearchisrequiredto
establishthesafetyandefficacyofthemanytraditionaljamu preparations[1]. Other important thing is, how to maintain
and improvesustainability of jamu, for its fresh preparation and usage, since it is in some ways much more better
than the processed one.
Efficacy of Jamu
Jamu, astraditionalmedicinearisingfromexperiencesofthe past and embedded in the culture of society, can

not stand still but constantly changes and develops. Along with conventional medicine it shares issues in appropriate
and rational use. This includes qualification and licensing of the provider, proper use of good quality products,
good communication between traditional medicine providers and patients and provision of scientific information
and guidance to the public [8].
The WHO encourages the use and development of traditional medicine as an accessible and affordable
means to provide healthcare for all people [8]. Although the pharmacological effects of some jamu constituents
have been recorded, there is an apparent lack of records or written data reporting the eeffectiveness of jamu
medicine, especially jamu gendong. To assure the correct use of such products, the Indonesian government
(NADFC) has divided the medicinal plants into three categories based on the way they are prepared and based on
a judgementof proof of their efficacy; i.e.jamu, standardized herbal medicines, and fitofarmaka (phytomedicines;
regulation nr. HK.00.05.4.2411,2004). All preparations have to meet basic safety criteria. The therapeutic effects
of jamu have to be supported by empirical data[1].
The efficacy and the biologicalactivitiesofthemostcommonplantsusedinjamu as reported in the literatureare
summarized as follow: anti cancer, antiviral, antimalarial. antiparasitic, anti-inflamatory (antirheumatic, antypyretic,
analgesic), hepatoprotective, antidiabetic, antimicrobial and antifungal, gastro-protective. cardioprotective, antihyper-
tensive, antiasthma, antitussive and antiallergic, immunostimulating,central nervous system (cns) activity,
others.[1, 9, 10].
Preparation and Serving of Jamu

In the past Indonesian people seemed to sense what they should to do to keep their health by the choice
ofright food and right way of life. Not only the processed products but also they consume the freshly picked
leaves or other part to keep their health always in good condition. In case of illness or diseases, they made certain
decoctions from the part of plants, such as leaf, bark, fruit, flower, roots etc. for curing their illness or diseases.
Usually, the people in villages or rural areas collect the plant material from their home gardens or surrounding
forest.
Original jamu is prepared in the form of a decoction and is directly consumed by family or sold as jamu
gendong . The name of jamu gendong due to it normally carrying by ladies on their back[6]. Jamu segar is
produced by person in the family or cottage industries using traditional methods. Traditional jamu makers are
30
aware of issues relating to hygiene, sanitation and chemical contaminations from biological or non-biological
sources (such as bacterial and fungal toxins, heavy metals). They try to protect raw plant materials and products
from contamination, although this is unlikely to comply with international industrial standards. The methods of
preparation are often different from producer to producer, and production steps like selection of raw materials,
sorting, grating, scraping, crushing, mixing and cooking, followed by boiling of the plant material in a hygienic way
can differ significantly.
There are some methods for jamu preparation which have been practiced in traditional ways (a, b, c,and
d) and the new one (e)[3].
a. Jamu segar
Segar means fresh, so preparation of jamu using fresh materials and drink it in fresh condition. This type of
preparation is normaly applied in family, for jamu gendong and some small warung jamu (jamu stall).
b. Jamu godogan
Godog means to boil, so godogan means that the preparation of jamu using boiling method. In practical , the
jamu materials are boiled with water and the result is as decoction which directly can be drunk.
c. Jamu seduhan
Seduh means to sob, so seduhan means that the preparation of jamu using sobbing method. Usually the raw
material of this type of jamu in the form of powder produced by jamu industries. The powder form of raw
material is sobbed and the result is a ready to drink jamu suspension.
d. Jamu olesan
Oles means to rub, so olesan means that the application the jamu by rubbing it to the body for preventing or
curing specific disease. The forms of external jamu are called pilis and tapel, both are like paste and usually in
fresh or wet condition. So that, they put pilis or tapel on disorder part of the body.
e. Jamu in the form of pills, tablets and capsules
In modern era, nowadays jamu can be found in the form of pills, tablets and capsules. Jamu in the form
like these, become simple and practical to be consumed as other modern drugs and medicines.
What is meant as fresh preparation and usage in this manuscript that includes jamu segar and jamu go do
gan which are prepared freshly and then directly consumed. Today industrially produced jamu is nolonger only
freshly prepared in the form of adecoction but also in the form of a tablet, pill, powder, pastille, capsule, extract,
creamor ointment.
Future Prospect
The diversity and availibility of raw material, cheaper price of raw material and efficacy of jamu product
are among the reasons, why Indonesia people still use jamu products in their daily lifeto maintain good health and
to treat diseases[1]. Fresh preparation and usage of Jamu e.g. jamu segar and jamu godoganstill gained their
popularity in the last decade. Traditional fresh Jamu products are herbal which traditionally produced and widely
used by large number of people in Indonesia.
The WHO encourages the use and development of traditional medicine as an accessible and affordable
means to provide healthcare for all people[8]. Some study also reported that the use fresh raw material and fresh
preparation of some herbs in some cases are better than processed ones [11]. So, it is very important matter to
maintain and improve the use of jamu by fresh preparation in order to get more benefficial values and efficacies.
31
There are some advantages in using fresh preparation of jamu as preventing and curing agent for diseases
in Indonesia. The biodiversity and availability of raw material, cheaper price and the efficacy of jamu are among
the reasons, why Indonesian people still use the fresh preparation of these products in their daily life. Although
nowadays fresh jamu often used by Indonesian as medicinal herbs, many factors have been reported reducing for
their fresh preparation and usage such as impracticality in preparation, introduction and availability of so many
synthetic modern medicines, etc. On the other hand, in modern life, easier preparation, the availability and easily
to reach products often become the main reason in usage of modern medicines. Jamu astraditional local based
herbal have been proven scientifically for their safety and efficacy, but concerns over practicality, availability and
consideration of modern medicines may disturb sustainability to use these products.
For now and the near future it is not easy to maintain even to improve the use of fresh preparation of
jamu.Traditional jamu products are not likely to become means as important alternative and replace to modern
standard medicines unless there are changes in some aspects. Here are some suggestions: more research works
should be conducted not only for their efficacy and safety, but also for more efficient and simpler practical fresh
preparation, developing and creating new jamu formula which attracting many people, increasing interest and
awareness of the people by involving public figures, celebrities, etc.If such conditions are not be fulfilled, otherwise
fresh preparation and usage of jamu and other traditional herbs will end.
CONCLUSIONS
Jamu is and will remain an integral part of Indonesian health-care system and maybe it become one of
worlds alternative for maintaining health, preventing and curing any diseases of world people.
Need further research and collaboration work not only to improve quality and efficacy of jamu as
natural,inexpensive, and safe product but also increasing interest and awareness of the people to consume fresh
jamu.
ACKNOWLEDGEMENT
The Author would like to thanks toProf. Estri Laras Arumingtyas for valuable discussion.
REFERENCES
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32
Chili Pepper as a Potential Medicinal Ingredients
EstriLarasArumingtyas*
Biology Department, Faculty Mathematics and Natural Sciences, University of Brawijaya, Jl. Veteran, Malang, Indonesia.
E-mail: larasbio@gmail.com; laras@ub.ac.id
Abstract
Chili pepper (Capsicum sp) of the genus Capsicum, the Solanaceae family is an important pungent spices and
sometimes also eaten fresh to increase appetite. The main bioactive ingredient of chili pepper that cause pungency
is capsaicin. The beneficial effects of capsaicin have long been documented in experimental or small sized
population studies including lower incidence of cancer, reduce the risk of overweight and obesity, lower risk of
cardiovascular and gastrointestinal conditions, neurogenic bladder, liver cirrhosis and dermatological conditions,
reduced the risks of diabetes. Recent researches have confirmed the inverse relation between chili pepper
consumption and certain specific cause of mortality like cancer, heart diseases, and respiratory diseases in China
and US. While some research investigategeneral chili, other emphasized the research on hot red chili pepper. We
have investigated the variability of pungency and capsaicin content of chili cultivar, along with capsaicin
content between green mature chili and mature red chili. Antioxidant and antimicrobial properties of chili pepper
have also been widely explored. However investigation on efficacy and interaction between capsaicin and other
component of chilli pepper for healing specific disease that cause mortality still need to be done.
Keyword : antimicrobial, antioxidant, Chili Pepper, diseases, medicine.
INTRODUCTION
Chili peppers (Capsicumsp) is a vegetable plant of the genus Capsicum Solanaceae family that is widely
cultivated in tropical and temperate regions of the world [1]. So far the genus Capsicum is known to consist of 36
species, five species of which, C. anuum, C. frutescens, C. chinense, C. baccatumand C. pubescens commonly
used by the public. Among these species C. anuum and C. frutescens is a commercial chili majority traded on the
world market [2,3]. Chili is an important component in some processed food, especially regional specialties in
Indonesia and around the world who have a spicy taste.
The level of pungency of chili different from one another. It is associated with the stage of development of the
pepper fruit[4], species or varieties of chili[5,6] as well as external factors such as the environment (a defense
mechanism due to stress biotic and abiotic stress) [7,8]. The pungent taste due to the content of capsaicin alkaloid
compounds (secondary metabolites) in the chilies. Capsaicin was first detected in chili at 10-15 days after flowering,
increased at 20-30 days after flowering and decreased at 40 days after flowering[9].
Besides capsaicin chili also contains essential oils, namely capsicol and bioflavonoids. Chilies are excellent
source of Vitamins A, B, C and E with minerals like molybdenum, manganese, folate, potassium, thiamin, and
copper. Chili contains seven times more vitamin C than orange. Although large amounts of capsaicin irritant for
mammals, including humans, and cause a burning sensation and heat in any of the affected tissue, but capsaicin
has a high economic value in the pharmaceutical field.
Function chili or its capsaicin content in medicine has been widely reported. However some result are not
always in line, sometimes contradictory to other result. In addition there is no information of chili varieties and the
level of maturity which are more effective as medicine. This paper discussed the possibility of developing a
research on efficacy and interaction between capsaicin and other component of many varieties and level of maturity
of chili pepper for healing disease that cause specific mortality.
33
Medicinal Effect of Chili Pepper
Chillies can have a positive impact on people that are overweight or suffer from diabetes[10]. By eating chili
in their meal the need of insulin intake were reduced, however the exact way in which chilies act to reduce the need
of insulin is not fully understood yet. In India chilies have been included in Ayurvedic medicines and used as tonic
to prevent many diseases. The ingestion of red pepper was found to decrease appetite and energy intake in people
of Asian and European origin and might reduce the risk of overweight and obesity [11, 12]. Capsaicin content of
chilies has also shown beneficial roles in decreasing the risk of obesity, cardiovascular and gastrointestinal conditions,
various cancers, neurogenic bladder, and dermatological conditions[13-15]. Moreover, the capsaicin exhibit antibacterial
activity and affect gut microbiota populations, which in humans have been recently related to risks of diabetes,
cardiovascular disease, liver cirrhosis, and cancer [16-17].
Study showed that populations with a higher consumption of spices have a lower incidence of cancer[19].
A review on the ability of capsaicin as anticancer has also been reported[20]. Capsaicin, the active compound in chili
peppers, has been proven to induce apoptosis in various tumors so as to enhance the therapeutic effects of
chemotherapy in pancreatic cancer [21-23]. Several reports indicate that capsaicin showsits inhibitory effect on
proliferation of pancreatic cancer cells[24,25]. Capsaicin also helped stop the spread of prostate cancer by triggering
suicide in both primary types of prostate cancer cell lines[26], inhibit the proliferation of prostate cancer in vitro and
in vivo[27] and enhanced transcriptional activity of target genes associated with apoptosis [28].
Capsaicin induced apoptosis in human gastric cancer cells and may serve as an anti-tumorigenic agent in
human gastric cancer[29] and also induces apoptosis of human small cell lung cancer cells[30]. Capsaicin also
decreased the growth of human leukemic cells[31], gastric [32], nasopharyngeal[33], and hepatic carcinoma cells[34]in
vitro because of its ability to mediate cell cycle arrest and induce cell apoptosis. Capsaicin might prevent the
growth of human colorectal cancer cells[35]. Therapy with high-concentration of capsaicin inhibited colorectal
cancer cell proliferation in a dose-dependent manner[36], but it was also found thatlow-concentration of capsaicin
capsaicin has carcinogenic effects, such as enhancing tumor cell proliferation and migration.
Capsaicin has also been found to provide effective pain relief without the numbing effect. The ability of
capsaicin to generate central hypersensitivity has been valuable in understanding the consequences and mechanisms
behind enhanced central processing ofpain[37].
Chili Pepper Effect and Mortality
The associations between the regular consumption of spicy foods and cause specific mortality has shown
the habitual consumption of spicy foods was inversely associated with total and certain cause of specific mortality[38].
This findings were supported by previous evidence showing potential protective effects of spicy foods on human
health including anti-obesity, antioxidant, anti-inflammatory, anticancer, and antihypertensive effects, and in improving
glucose homeostasis, largely in experimental or small sized population studies [13-15]. Additionally, the antimicrobial
function of spiceshas long been recognized[39,40]and such a property may have an important effect on the gut
microbiota in humans. Recently many evidence has implicated that gut microbiota is an important metabolic factor
that affects the health of the host[41]and several studies in humans have related abundance, composition, and
metabolites of gut microbiota to risk of obesity[42-44]diabetes[16] liver cirrhosis[17] and cardiovascular disease[18,44].The
possible mechanisms might involve the bio accessibility and bioavailability of bioactive ingredients and nutrients of
spicy foods[45].
Consumption of hot red chili peppers was associated with a 13% reduction in the instantaneous hazard of
death. The consumption of hot red chili pepper was associated with reduced mortality[46] .
Chili pepper variability

Genetic diversity of some chili pepper has been reported[47]. Morphological and molecular variationwas
34
detected among local variety of chili pepper in Indonesia. This indicates that the DNA and its expression were
varies between genotypes studied. Research at Nigeria found that Yellow pepper (Capsicum chinense), obtained
from Nsukka, the South Eastern part of Nigeria is the most pungent of the peppers studied. All the peppers
analyzed in this study can be classified as very highly pungent as the Scoville Heat Unit (SHU) values exceed
80,000, except the tatase, Zaria (Capsicum annuum) which has a mean SHU value of 19,015.20. This implies that
all the pepper varieties studiedcan serve as potential sources of capsaicin. Variability of capsaicin content between
some variety of C. frutescent with different level of maturity has been reported[48]. All the cultivars showed higher
capsaicin content when the fruit already become red than the green mature fruit.However different finding was
reported by other study. The green mature Chiltepin chili samples cultivated in Lagunera Region, Mexicohas more
pungency level than the red one[49]. Other researcher found that different varieties of chili showed different amount
of capsaicinoid compound and pungency level [50].
Other study showed that ripe sweet bell pepper has higher antioxidant activity than the mature green one.
Enhanced antioxidant capacity at fully ripe stages reflects the nutritional importance of consuming the pepper fruits
at this stage[51] different varieties of chili has different antioxidant activity. The antioxidantactivities has a
significantlinear relationship with the totalphenolic contentsof nine chili peppers varieties extracts[52].
Variability of antimicrobial property among different variety of chili was reported. Different effect of
capsaicin extracted from different varieties of chili wasdetected[53]. Pure capsaicin had a strong inhibitory effect
towards Bacillussubtilis[54]. The antimicrobial effect of Capsicum extract on Salmonellatyphimurium inoculated in
minced beef[55]. Methanol extracts of red chili could substantially inhibit cholera toxin (CT) production[56]. Other
researcher determined that the methanol extract of red chili, and purified capsaicin could inhibit cholera toxin
without affecting their viability[57].
Future Research and Prospect of Chili as medicinal ingredient
Chilies provide widest range of physiological effects however results of some studies conducted to explore
the beneficial effects of chillies were positive and some were negative. Some researcher believe that it is the
antioxidant content of red chili in the form of beta-carotenoids of vitamin C and Vitamin A that can destroy
cholesterol which could cause major disease like atherosclerosis and other heart diseases and also reduces the risk
of colon cancer. However other researcher found that it is the capsacin content that has a role.
Contradictory effect between low and high concentration of capsaicin has also been reported. High-
concentration of capsaicin inhibited colorectal cancer cell proliferation but in low-concentration has carcinogenic
effects [36]. So it is necessary to investigate the effective concentration of capsaicin that has beneficial effect as
anticancer.
Variability effect among different variety of chili[53]has also reported. Variability of morphology followed by
differences in capsaicin content, antioxidant activity, antimicrobial activity, suggest that maybe different effect
was elucidated by different kind/ variety, maturity stage of chili.
So, it is still need to be studied which maturity stage that more efficient in curing or prevent disease, what
kind of chili, or which kind of chili that suitable in healing certain disease. Other than that, there is remain questioned
if is it only the capsaicin that cause/give medicinal effect or it is other chili component or combination of those
constituent. Or with combination that give more efficient effect.
Research on the effect of spicy foods and their bioactive ingredients on the composition and activity of gut
microbiota[18,44]also has to be further investigated.
CONCLUSION
The beneficial effect of chili has been proved. However, more detail study need to be conducted in order
35
to elucidate which maturity stage that more efficient in curing or prevent disease, what kind of chili, what combination
of chili ingredients that suitable in healing certain disease.
AKNOWLEDGEMENT
The author would like to thanks to the team Capsicum who has given technical assistant and Dr. Joni
Kusnadi for valuable discussion
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38
Phytochemical Analysis of Leucophyllum frutescens Stems by
FT-IR and UV-Vis Spectroscopic Analysis
P.Divyaa*, Dr. A Prithibab, Dr.R.Rajalakshmic
a
Research Scholar, b Assistant Professor, C Professor, Department of Chemistry,
Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore, India
*Corresponding author. Email: pdivya2494@gmail.com
Abstract
The qualitative and phytochemical analysis in the stems of Leucophyllum frutescens which belongs to figwort
family Scrophulariaceae was carried out. Ethyl acetate, methanol, ethanol and water were tested for the presence
of Alkaloids, Terpenoids, Tannins, Saponins. UV-Visible and FT-IR methods were also performed to determine
the characteristic peak values and their functional groups.
Keywords: Leucophyllum frutescens, Phytochemicals, qualitative, FTIR and UV-Visible
INTRODUCTION
Plants are enriched with several contain organic substances - secondary metabolites that provide a source
of medicine since ancient times. Phytochemicals are non-nutritive plant chemicals that have protective or disease
preventive properties. Phenols are one of the chief secondary metabolites present in the plant kingdom. They are
generally found in both edible and non-edible plants, and have been reported to have multiple biological effects
including anti-oxidant activity(1-2).
Flavonoids are a class of phytochemical that possesses a wide range of biological activities.
Qualitative phytochemical screening helps us to understand about the variety of chemical compounds pro-
duced by plants .
Leucophyllum frutescens is an evergreen shrub in the figwort family, Scrophulariaceae, native to the state
of Texas and the states of Coahuila, Nuevo Len, and Tamaulipas in northern Mexico. (3-4)
Leucophyllum frutescens is traditionally given for dysentery,
fever, cough, asthma, liver injury, cataracts. The flowers and leaves can
be brewed into a pleasant herbal tea which is a mild sedative and good
for treating flu and colds, the leaves of Leucophyllum frutescens have
been used for antihepatotoxicity (5). In the present work, qualitative
analysis, UV-Visible and FT-IR were carried out in the stem extract of
Leucophyllum frutescens.

Leucophyllum frutescens plant
Collection of plant sample
The stem sample of the Leucophyllum frutescens species were identified and collected from Coimbatore,
Tamil Nadu. The stems were separated, washed properly with distilled water and air dried at room temperature. The
dried stem samples were powdered finely using a electric blender and stored in a clean container for further analysis.
Plant extracts
5g of the powdered stem samples were taken and then fitted into a round bottom flask containing 150ml of
Methanol, Ethanol, Water, and Ethyl acetate. The solvent was boiled gently and the extraction was continued for
three hours and finally the extract was filtrated using whatmann filter paper.
39
PHYTOCHEMICAL SCREENING
Phytochemical tests were carried out in the aqueous condition with the help of distilled water, where
powdered stem samples were mixed with water using standard procedures to identify the constituents, present in
the stem samples. In identifying some secondary metabolites, some chemicals were also used other than distilled
water. 2g of stem powder were taken separately and dissolved in 20ml of distilled water. The mixture was filtered
with Whatmann filter paper.
PHYTOCHEMICALANALYSIS - QUALITATIVE ESTIMATION

Phytochemical Screening of the plant extracts
Phytochemical examinations were carried out using standard procedure (6)
40
Spectroscopic analysis
To detect the UV-VIS spectrum profile of Leucophyllum frutescens, the extracts were scanned in the
wavelength ranging from 200-800nm and the characteristic peaks were detected. FT-IR analysis was performed
which was used to detect the characteristic peaks and their functional groups. The peaks values of UV-VIS and
FTIR were recorded.
RESULTS AND DISCUSSION

Phyto chemical screening
In the present study the examined extracts of Leucophyllum frutescens was found to contain various
phytochemical constituents such as alkaloids, flavonoids, phenol, terpenoids, saponin and carbohydrates. These
phyto constituents seemed to have the potential to act as a source of useful drugs.
Table : 1 Qualitative analysis of Leucophyllum frutescens stem
+ Present, - Absent
41
The UV-Vis profile shows peaks at 658 nm and 530 nm with the
absorption 1.8041 and 1.8477 respectively.
This might be due to the presence of flavonoid type of
compounds amongst other phytochemicals like terpenoids and
iridoids.
Functional groups identification

FTIR spectrum was used to identify the functional groups of
the active components present in extract based on the peaks values in
the region of IR radiation. The results of FTIR analysis confirmed the Figure 1: UV-VIS Spectrum of stem
presence of phenols, Carboxylic group, amines, alkyl and aromatic of Leucophyllum frutescens
groups.
Figure 2 : FTIR Spectrum of Leucophyllum frutescens stem
Table 2: FTIR Peak Values of Leucophyllum frutescens stem
S.No Peak values(cm-1 ) Functional groups
1 681 Aromatic C-H bending
2 1030 C=O
3 2938 Alkyl C-H stretch
4 3470 Phenol O-H stretch
5 3563 (N-H ) group
CONCLUSION
Analysis of the extracts reflected the presence of several phytochemicals ascertaining the utilization of the
investigated plant extracts for several medical purposes. Further research will be needed to find out the structural
analysis of the different phytochemicals by advanced techniques.
42
REFERENCES
1. Dhar, ML., Dhar, MM., Dhawan, BN. and Ray, C. (1968). Screening of Indian plants for biological activity
Part I. Indian J. Ex. Bio. 6: 232-247.
2. Duke, JA. (1990). Promising phytomedicinals Advances in new crops Janick J and Simon JE (eds.) Timber
Press Portland 491-498.
3. Evans, WC., Trease and Evans. (1997). Pharmacognosy (14th Ed), Harcourt Brace and Company. Asia Pvt.
Ltd. Singapore 343.
4. Trease, G. E., & Evans, W. C. (1989). Pharmacognosy. (11th ed., pp. 1-795), London, England:Brailliar
Tiridel.
5. Edeoga, H. O., Okwu, D. E., & Mbaebie, B. O. (2005). Phytochemical constituents of some Nigerian medicinal
plants. Journal of Biotechnology, 4, 685-688.
6. Harborne, J. B. (1984). Methods of plant analysis (pp. 1-36). Springer Netherlands.
7. Sauren Das , Chiou-Rong Sheue , Yuen-Po Yang (2013). Leaf micromorphology and leaf glandular hair
ontogeny of Myoporum bontioides A. Gray. Feddes Repertorium 124, 5060:4.
8. Kadry Abdel Khalik (2005) Morphological studies on trichomes of Brassicaceae in Egypt and taxonomic
significance. Acta Bot. Croat. 64 (1): 5773.
43
Identification, Characterization and Immunomodulatory Role of
Leaf Protein Fraction of Tabernaemontana divaricata
Dr. S. Annapurani*, Ms. M. Srilatha** and R. Santhi***
*Professor and Head, **Ph.D Scholar, Department of Biotechnology, ***WOS-A Project staff
Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home Science and
Higher Education for Women, Coimbatore 641 043. Email ID: sajbiochem@gmail.com
Abstract
Identification of leaf protein fraction of Tabernamontana divaricata (Td) using recrystallized ammonium sulphate
fractionation and sephadex gel filtration. The selected protein fraction of Td was characterized by SDS PAGE,
FTIR, RP-HPLC and MALDI-TOF/MS. The characterized protein was evaluated for the in vitro Cytotoxicity
using intraperitoneally propagated DLA tumor cells in Swiss albino mice. The immunomodulatory effect of TdPf
(using Balb/C mice) was evaluated by Hematological parameters (total HB, WBC, RBC and DC), cytokine levels
(IL-2 and IFN-) and cellularity of lymphoid organs (Thymus and Spleen). The protein fraction was found to be
cytotoxic and Immunostimulant. Thus, it can be used as an antitumorogenic and immunostimulanting agent in
the oxidative degenerative diseases.
Keywords : Tabernaemontana divaricata, Protein, SDS-PAGE, FTIR, RP-HPLC and MALDI-TOF/MS
INTRODUCTION
Cancer is one of the major causes of death worldwide and only modest progress has been made in
reducing the morbidity and mortality of this disease1. Many of the currently available drugs are plant-based, plant
peptides and proteins that have turned out to be a critical source of biological compounds that exhibited bioactivities
which can be exploited as drug. The plant-based products including proteins and small molecular compounds have
been suggested as the favorable drugs for cancer treatment in view of the many adverse effects exerted by current
cancer treatments, namely chemotherapy and radiation therapy2. Immunomodulators are the substances which
modify the activity of the immune system. Immunomodulators have biphasic effects; some tend to stimulate
immune system which is low while others inhibit host parameters which are normal or already activated3.
Immunostimulation and immunosuppression both need to be tackled in order to regulate the normal immunological
functioning. Hence, both immunostimulating and immunosuppressing agents have their own capacity and search
for better agents exerting these activities is becoming the field of major interest all over the world4. Tabernaemontana
divaricata R. Br. is a glabrous, evergreen, dichotomously branched shrub, belonging to the family Apocynaceae. It
is a shrub very common in India and grows to a height of 68 feet. It is distributed in upper Ganjetic plain,
Garhwal, East Bengal, Assam, Karnataka, Kerala, Tamilnadu, and Burma. Many reseachers investigated that T.
divaricata are rich in chemical constituents such as alkaloids, triterpenoids, steroids, flavanoids, phenylpropanoids,
and phenolic acids5. However, the effects of protein extract on cancers have never been determined. Therefore, we
aimed to analyze the cytotoxic activity of the proteins extracted from the leaves of T. divaricata using tryphan blue
exclusion method. Furthermore, active proteins were identified using mass spectrometric analysis. We hope that
the data obtained will be useful for the future intervention of protein-based drug for cancer therapy.

Extraction of Proteins
Protein was extracted by the method with slight modification6. Fresh leaves of Tabernaemontana divaricata
were taken and homogenized with PBS buffer pH 7.2 (20%) and were centrifuged at 5000 rpm for 10 min. To the
supernatant added 10-100 per cent saturation of recrystallized ammonium sulphate and centrifuged at 10,000 rpm
44
for 10 minutes. The pellet was suspended in PBS and dialyzed using 0.01M PBS for salting out7. The selected
protein fraction was stored at minus 20C.
Purification of protein fraction

The selected protein fraction was freeze-dried and loaded onto a Sephadex G-75 column using AKTAprime
plus protein purification system. The column was eluted with 50mM sodium phosphate buffer at pH 7.2 with a
flow rate of 0.5mL/min for a total volume of 200ml elution buffer. 5mL fraction was collected and the concentration
of protein in each fraction was determined by Lowrys method8.
SDS PAGE analysis

Sodium dodecyl sulphate polyacrylamide gel electrophoresis is widely used technique to separate proteins
according to their electrophoretic mobility7. About 40l of protein sample was loaded into gel along with the protein
markers of molecular weight ranging from 20 to 250 KDa. The gel was kept under electrophoretic run for 2h at
100v and the protein bands were identified9.
Characterization
Identification of functional groups of TdPf was performed using Shimadzu FTIR10, molecular mass by
using RP-HPLC11 and MALDI-TOF/MS by mascot prediction12.
Immunomodulatory activity
The male BALB/c mice with 6-8 week old were purchased from and were kept under optimal conditions
of hygiene and received standard mouse chow and water ad libitum. The study was approved by Institutional
Animal Ethical Committee (Ref. No. AUW: IAEC.2016.BT:01). The animals were divided into five groups with 6 in
each. The cytotoxicity was assessed by Trypan Blue Exclusion Assay.
Assessment of Hematological Profile

Hematological parameters were assessed using a Hemavet 1500S (Drew Scientific, Waterbury, CT, USA)
and analysis of various parameters such as total white blood cell (WBC), total red blood cell (RBC), as well as
Hemoglobin levels were carried out.
Assessment of Lymphoid organs Cellularity

Spleen and Thymus of experimental mice were homogenated in Hanks balanced salt solution (HBSS) and
the cells were counted.
Assessement of cytokines production

In order to evaluate the effect of TdPf on the cytokines production, the enzyme-linked immunosorbent
assay (ELISA) method was followed to measure IL-2 and IFN-. ELISA kit was purchased from Biolegend.
Results were expressed as mean SD.
Statistical analysis was performed with one way analysis of variance (ANOVA).
RESULTS
Sixty per cent saturation of ammonium sulphate was purified in sephadex G-75 column and separated by
SDS.
45
Protein profile of standard markers and TdPf
Twelve per cent SDS-PAGE showed the presence of single protein band for TdPf whose molecular
weight is around 45Kda (Figure 1).
Figure 1 Protein profile of TdPf

Characterization
The FTIR spectrum detected the peaks at 1631.26cm-1 and 3353.97cm-1 showing the presence of carbonyl
and hydroxyl stretching vibration.
Figure 2 FTIR Spectral of TdPf
Single peak was obtained in Reverse phase- HPLC (Figure 3). It indicated the presence of single protein
in TdPf with the retention time of 12.8 minute which was detected at 220 nm.
Figure 3 RP-HPLC of TdPf
Each peak in the MALDI-TOF/MS spectrum as given in the figure 4, represented the peptides present in
the tryptic digestion of TdPf. The size of the peptide ions varies from m/z 816.524 to 3349.697.
46
Figure 4 MALDI-TOF/MS spectrum of TdPf
Immunomodulatory activity of TdPf

Immunomodulatory activity of TdPf was revealed by the analysis of Haematological parameters, Cellularity
of Lymphoid organs and Production of Cytokines by the administration of 62g of TdPf/kg body weight of animal.
Effect of TdPf on Haematological studies

The effect of protein fraction on WBC, RBC and Haemoglobin count in Balb/c mice are shown in the Table 1.
Administration of TdPf individually and in coadministered mice showed significant increase in the total WBC and
decrease in the RBC count and Hb when compared to Pyrogallol and DLA induced mice. The untreated control
animals maintained the normal total WBC, RBC and Hb count during the experimental time. Values are mean
SD of six animals; # One way ANOVA. WBC-White Blood Cells; RBC-Red Blood Cells; HB-Haemoglobin
Table 2 Effect of TdPf on WBC, RBC and HB in Balb/C mice
Cellularity of Lymphoid organs

Protein fraction of T. divaricata revealed immunostimulatory role on the proliferation of lymphoid organs
(Figure 5). The highest number of spleen and thymus cells were recorded in TdPf administered mice alone when
compared with pyrogallol on both the days of treatment whereas number of cells were decreased in DLA induced
mice on different treatment periods and the cell number was slightly increased in the group treated with TdPf and
DLA induced mice when compared with tumor induced mice.
Figure 5 Effect of TdPf on cellularity of lymphoid organs
47
Effect of TdPf on the production of cytokines
The concentration of cytokines IL-2 and INF- in the serum of mice treated with and without protein
fraction were measured against DLA induced mice. The Figure 3 showed significant increase in the production of
IL-2 and INF- in both the days of treatment period when compared with the Control and DLA induced mice in the
presence and absence of TdPf since this fraction is potent to stimulate the immune system.
Figure 3 Effect of TdPf on Cytokines
DISCUSSION
The modulation of cell immune response by molecules from the medicinal plants is an interesting area for
inflammation, autoimmunity and anticancer therapy10,11. In this study, we have identified, characterized and evaluated
the immunomodulatory effects of TdPf. Several studies showed that natural agents such as plant metabolites can
modulate immune activity13, 14, 15.
The isolation, identification and characterization of TdPf showed 45KDa protein. Similarly Wang isolated
an anticancer protein with a molecular weight of 13 kDa from Ginkgo biloba seeds16. Protein isolation studies
were also done on soya, wheat, corn etc., using FTIR. They found the functional groups as C=O and N-H17. The
denaturation of proteins and their interaction with other components also responsible for the variation of
hydrophobicities among fractionated proteins18. The flaxseed protein extracted using 0.1 M NaCl and subjected to
ion exchange chromatography gave at least three fractions19. The proteome profiling technique by MALDI-TOF/
MS provided a broad-base and effective approach for the identification of proteins with biological activity20,21. The
closest homology with the sequence of 1-aminocyclopropane-1-carboxylate deaminase of Pyrococcus horikoshii
was detected by MALDI-TOF/MS analysis in Borreria hispida seed protein22.
Immunostimulatory effects have been constantly associated to active plant proteins classified as lectins23.
The purified form of taro lectin was also able to induce in vitro splenocyte proliferation in a dose-dependent
manner24. The results were compared with the report who had earlier observed increase in haematological indices
of mice fed with protein fraction of Borreria hispida 25. A study reported the effects of ethanolic extract on immune
response showed that cytokines are expressed at very low concentrations 26. The increased levels of IL-2 and IFN-
in the sera of immunosuppressed mice suggested that LP3 not only induced a restoration of decreased T-cell-
dependent antibody response by the enhancement of IL-2-production and also stimulated the enhancement of IFN-
production27.
CONCLUSION
The natural source of bioactive protein isolated from TdPf showed cytotoxic effect against DLA tumor
cells and immunostimulatory role in Balb/c mice model.
48
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1. Hail N (2005) Apoptosis 10, 687-705
2. Tepkeeva I I, Moiseeva E V Chaadaeva A V, Zhavoronkova E V & Kessler Y V (2008) Bulletin of Experimen
Biol and Med 145, 464-466
3. Deharo E, Baelmans R, Gimenez A, Quenevo C & Bourdy G (2004) Phytomedicine 11, 516-22
4. Patwardhan B, Kalbag D, Patki P S & Nagsampagi B A (1990) A Rev Indian Drugs 28, 348-58
5. Surya S, Poornima K & Ravikumar G (2011) Immunol 2, 212-218
6. Stone K L LoPresti M B, Crawford J M, DeAngelis R & Williams K R (1989) Academic publishers New
York, 31-47
7. Jayaraman J (1981) Laboratory manual in Biochemistry, 1st Ed., Wiley Eastern Ltd., New Delhi, 44-45, 82-
83
8. Lowry O H, Rosenbrough N J, Farr A L & Randall R J (1951) J Biol Chem 193, 265-275.
9. Laemmli U K (1970) Nature 227, 680-685.
10. Widjanarko S B, Nugroho A & Estiasih T (2011) Afr. J. Food Sci 5, 12-21
11. Secknus R, Yamashita S & Ginanni G (1996) Journal of Laboratory and Clinical Medicine 2, 169-178
12. Obregon W D, Liggieri C S, Morcelle S R, Trejo S A, Avile F X & Priolo N S (2009) Protein and Peptide
Letters 16, 1323-1333
13. Ghafourian M Boroujerdnia ME, Azemi A A, Hemmati A Taghian A & Azadmehr (2011) Iran J Allergy Asthma
Immunol 10, 1281-288
14. Bin Hafeez B, Ahmad I, Haque R, & Raisuddin S, (2001) J of Ethnopharma. 75, 13-8.
15. Sumalatha P, Rama Bhat R, Shwetha Ballal Sadananda Acharya, (2012) J of Applied Pharma Sci 9, 98-107
16. Wang H, Ng T B, (2000) Biochem. Biophys. Res. Commun. 279, 407-411.
17. Thani W, Vallisuta O Siripong P Ruangwises N, (2010) Asian J.Trop. Med. Public Health 41, 482-489.
18. Lee H.C, Htoon A K, Uthayakumaran & Paterson J L (2007) Food Chem 102, 1199-1207
19. Madhusudhan K T & Singh N (1983) J. Agric. Food Chem, 33, 1219-1222.
20. Dinesh R & Leela S (2011) Journal of Pharmaceutical and Clinical Research 4, 119-123
21. Hao Z, Xijuan C, Chengshang W, Jianzhong Y & Hong C (2012) Molecules 17, 14778-14794
22. Rupachandra S & Sarada D V L (2014) BioMed Research International 14, 1-8
23. Ashraf M. T, & Khan R.H (2003) Medical Science Monitor 9, 265-269
24. Pereira P.R., Del Aguila E, Vericimo M, Zingali M.A. Paschoalin, R. B & Silva J. T (2014) The Protein
Journal 33, 92-99
25. Mercado-Feliciano M, Cora M C, Witt K L, Granville C A, Hejtmancik M R, Fomby L, Knostman K A,. Ryan
M J, Newbold R, Smith C, Foster P M, Vallant M K, Stout M D (2012) Toxicol Appl Pharmacol 263, 138-
147
26. Salazar-Onfray F, Lopez M N & Mendoza-Naranjo A (2007) Cytokine Growth Factor Rev 18, 171-182
27. Decker T, Mller M & Stockinger S (2005) Immunol 5, 675-687
49
Therapeutic Approaches of Nanotechnology in Herbal drugs for
the Management of Diabetes Mellitus
Dr. S. Gayathri Devi
Associate Professor, Department of Biochemistry, Biotechnology and Bioinformatics,
Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore 641 043.
Email ID: gayathridevi.adu@gmail.com, Mob No.: 9443935039
Abstract
Diabetes mellitus highlights a worldwide growing epidemic with a significant impact on health and life expectancy
of patients. Scientific and technological advancement of newer drugs with significant efficacy in reducing
hyperglycemia has witnessed the potential drug targets in treating diabetes mellitus causing adverse side
effects. Hence extensive investigation in identifying the natural products involving nanomedicine is an eye
catching and most promising. In recent years, there has been an upsurge interest involving the plant remedies.
Many herbs have the potential to compromise reactive oxygen species namely green tea, grape seed, ginseng
and Scutellaria baicalensis. Long while treatment with herbal medicines for diabetes has been in existence. The
herbal medicines and nutraceuticals, as well as their bioactive components, which exhibit anti-inflammatory and
antioxidative properties, provide a promising approach for the prevention and treatment of diabetic complications
using imperative nanoparticles that have a very great trait to carry and serve like an antioxidant, antihyperglycemic
and reactive oxygen species interfering action. Treatment of diabetes mellitus with potent nanoparticles involving
plants would have therapeutic value to create a new platform for herbal medicines in nanoscience for drug
delivery. Hence, the present review provides an overview of green synthesis of nanoparticles involving medicinal
plants for the management of diabetes mellitus.
Keywords : Nanoparticles, nutraceuticals, anti-inflammatory, antioxidative
INTRODUCTION
Diabetes mellitus is a group of metabolic disorders that is characterized by elevated levels of glucose in
blood (hyperglycemia) and insufficiency in production or action of insulin produced by the pancreas inside the
body1. Diabetes mellitus is a common lifestyle disorder in many countries with an estimated one-third of them
using some form of complementary and alternative medicine. Nanotechnology is a field making an impact on
human life because plant mediated biological synthesis of nanoparticles is gaining importance due to its simplicity
and eco-friendliness. In this study, the application of nanoparticle synthesis from herbal extracts for the management
of diabetes is focused2.
Nanotechnology can be defined as the science and engineering involved in the design, synthesis,
characterization and the application of materials and devices for smallest functional organization in at least one
dimension is on the nanometer scale. When this science is applied particularly to the problems of medicine, it is
called Nanomedicine. Nanomedicine is defined as integration of nanotechnology in medicine for the betterment of
human health care. The burgeoning new field of nanomedicine opened up by rapid advances in health care, creates
myriad new opportunities for advancing medical science and disease treatment in human health care3.
Nanoparticles in the management of diabetes mellitus
Nanomedicine utilizes components as tiny as 1/80,000th of the diameter of a human hair. At the scale of 1
nanometer (or 10 times the diameter of a hydrogen atom) materials and devices can interact with cells and biological
molecules in a unique way4. Nanotechnology is a focal point in diabetes research, where nanoparticles are showing
great promise in improving the treatment and management of the disease. Due to their ability to potentially enhance
50
drug delivery to areas where there are barriers or unfavourable environments for macromolecules, nanoparticles
are being explored as vehicles for improved oral insulin formulations. The use of nanotechnology in the development
of glucose sensors is also a prominent focus in non-invasive glucose monitoring systems5.
Delivery of insulin through nanoparticles
The development of improved oral insulin administration is very essential for the treatment of diabetes
mellitus to overcome the problem of daily pain and trauma caused by subcutaneous injections. Oral administration
is beneficial not only to alleviate the pain, but it can mimic the physiological fate of insulin as well. The nanomedicine
technologies that may be employed for oral insulin delivery includes prodrugs (insulin-polymer conjugation), micelles,
liposomes solid lipid nanoparticles (NPs) and NPs of biodegradable polymers4.
Oral insulin
The prodrug technology that is used most often for drug formulation is PEGylation (i.e., drug conjugation
to Polyethylene Glycol [PEG]) for enhanced solubility, permeability and stability. InsulinPEG prodrugs have
shown great advantages in oral delivery. Unfortunately, micelles did not seem to be ideal for delivery of hydrophilic
drugs. Instead, liposomes can have much better performance for oral insulin delivery. This liposomal delivery
system containing glycocholate as an enzyme inhibitor and permeation enhancer has been developed recently for
oral insulin delivery, which showed better protection of insulin against enzymatic degradation by pepsin, trypsin
and achymotrypsin6.
Microsphere for oral insulin production
The oral drug delivery device for insulin and to protect the sensitive drug from digestive enzymes and
proteolytic degradation in stomach and upper part of gastro intestinal tract can be overcome by the use of a
microsphere system which is inherently a combination strategy. Microspheres act both as protease inhibitors by
protecting the encapsulated insulin from enzymatic degradation within its matrix and as permeation enhancers by
effectively crossing the epithelial layer after oral administration5.
The Nanopump
The nanopump is a powerful device and has many possible applications in the medical field. The first
application of the pump, introduced by Debiotech, is Insulin delivery. The pump injects Insulin to the patients body
in a constant rate, balancing the amount of sugars in his or her blood. The pump can also administer small drug
doses over a long period of time7.
Herbal medicine in nanotechnology

With respect to herbal medicine the drug take a longer time to show the antidiabetic activity because they
are more insoluble in nature. To overcome this problem can be overcome by the use of nano herbal medicine is
needed. Herbal mediated silver nanoparticles (HMSNPs) are having less particle size, more surface area and more
solubility which results in the optimum dose of drug that can reach systemic circulation and onsite of action is very
quick. Recent studies on the use of herbal plant extracts in synthesis of HMSNPs are new and exciting area of
research with considerable potential for the development of new methods in nano medicine8.
Several experiments were carried out on the synthesis of silver nanoparticles using medicinal plants such
as Oryza sativa, Helianthus annus, Saccharum officinarum, Sorghum bicolour, Zea mays, Basella alba, Aloe vera,
Capsicum annuum, Magnolia kobus, Medicago sativa (Alfalfa), Cinamomum camphora and Geranium species in
the field of pharmaceutical applications and biological industries. Besides, green synthesis of silver nanoparticles
using a methanolic extract of Eucalyptus hybrida was also been investigated. In the recent days, silver nanoparticles
have been synthesized from the naturally occurring sources and their products like green tea (Camellia sinensis),
Neem (Azadirachta indica), various leaf broth, natural rubber, starch, Aloe vera plant extract, lemon grass leaves
extract9.
51
Biosynthesis of nanoparticles is a kind of bottom up approach where the reduction/oxidation is the main
occurring reaction. The need for biosynthesis of nanoparticles rose as the physical and chemical processes were
costly. This is not an issue when it comes to biosynthesized nanoparticles via green synthesis route. So, in the
search of economic pathways for nanoparticles synthesis, scientist used microbial enzymes and plant extracts
(phytoconstituents). Green synthesis provides advancement over chemical and physical method as it is cost effective,
environment friendly, easily scaled up for large scale synthesis8.
CONCLUSION
This article was an attempt to review some aspects of nanoparticles in relation to diabetes mellitus. Oral
insulin in particular could prove to be promising, especially since as a therapy it seems to have progressed with
nanotechnology research, allowing for several types of encapsulations to bypass the gastric acidic environment.
Hence, nanomedicine shows a great zeal for the future management in diabetes mellitus. Thus, we conclude
saying that nanomedicine in diabetes care is in initial stage, but with a rapid progress in unlocking the
technological advancement in diabetic research.
REFERENCES
1. Ullah A, Khan A & Khan I (2016) Saudi Pharmaceutical Journal 24, 547553
2. Ekezie F C, Suneetha J W, Maheswari U K & Prasad T N V K V (2015) International Journal of Science and
Nature 6, 687-692
3. Rahiman S & Tantry B A (2012) Journal of Nanomedicine & Nanotechnology 3, 1-7
4. Woldu M A & Lenjisa J L (2014) International Journal of Basic and Clinical Pharmacology 3, 277-284
5. Harsoliya M S, Patel V M, Modasiya M, Pathan J K, Chauhan A, Parihar M & Ali M (2012) International
Journal of Pharmaceutical & Biological Archives 3, 255-261
6. Niu M, Lu Y, Hovgaard L & Wu W (2011) International Journal of Nanomedicine 6, 1155-1166
7. Kumdhavalli S, Mayavathi P, Monisha S, Priya G T & Prathiba V (2016) World Journal of Pharmaceutical
Sciences 4, 64-72
8. Amarendra D D K G (2010) Colloid Surface A Biointerfaces 369, 27-33
9. Shanker Kalakotla S, Gottumukkala K M, Rani S M & Pravallika P L (2015) International Journal of
Pharmaceutical Science Review and Research 31,142-148
52
Cytotoxicity of isolated and purified bromelain from Ananas comosus
Chitra.E, Sumathi.S, Aiswarya.T, Poornima.A, and Padma.P.R
Department of Biochemistry, Biotechnology and Bioinformatics,
Avinashilingam Institute for Home Science and Higher education for Women, University, Coimbatore-641 043, TN, India
Email : chitraelangovan1995@gmail.com
Abstract
The enzyme bromelain occurs naturally in Ananas comosus and possess many properties such as an
anticoagulative, anti-inflammatory, antithrombotic and anticancer properties. Characterization of the bromelain
is very important step to evaluate the various activities of the enzyme. Various assays are carried out to identify
its biocompatibility and cytotoxicity. Bromelain was isolated from stem of Ananas Comosus and purified,
evaluated for theirin vitro cytotoxicity and biocompatibility. Bromelain enzyme was extracted from Ananas
comosus and then subjected to partial purification and fractionation by ethanol precipitation method which was
followed by dialysis and a cytotoxicity was assessed by MTT assay. Bromelain was found to be safe to normal
yeast cells and when exposed to oxidative stress, it protected the cells from apoptotic cell death. Bromelain is
not cytotoxic. With these findings, the mechanism of action of the bromelain will be analysed for the effective
pharmacological action for further studies.
INTRODUCTION
Ananas comosus, commonly known aspineapple belongs to Bromeliaceae family native of tropic and sub-
tropic countries. In folklore medicine, Ananas comosus is reputed to act as an abortifacient as a means of
inducing labor to avoid medical intervention of post-dates pregnancy (Monjiet al., 2016).Bromelain
isaprotease,whichwas belong to a group of protein digesting enzymes. It can be isolated from the stem , peel
,crown of Ananas Comosus.It is an protein extract present in all parts of fresh plant and fruit ofAnanas Comosus.It
contains mixture of enzymes such cysteine proteinases, peroxidases, acid phosphatases, glycosidases(Karunaet
al.,2016).The stem bromelain is the most abundant cystein endopepdidase in the pineapple(Ramalingam et al.,
2012). Bromelain is chemically known since 1875 and used as a phytotherapeutical agent.Although it exit in all
parts,stem is most commercial source.It has various properties such as antibrowning, anticancerous, anti-
inflammatary,antiedematous, antithrombotic or fibrinolytic, and immunomodulatory effects.It is also used in
preparation of various skincare products and cosmetics (Lourenco et al., 2016).Bromelain has high commercial
value because it has strongproteolytic activity. Due to its activity it has been used in numerous industrial
applications such as a meat tenderizer, a bread dough improver, a fruit anti-browning agent, a beer clarifier, a
tooth whitening agent, animal feed and cosmetic substance (Arshad et al.,2014). On the present study we
attenuated to isolate bromelain from Ananas comosusand partially purified and tested for cytotoxicity using eukaryotic
in vitro model organism saccharomyces cerevisae. Mechanism of cell death was identified by AO/EtBr staining.

Crude extract of the bromelain was isolated from the stem of Ananas Comosus. Partial purification of stem
bromelain was done by ethanolprecipitation method and followed by dialysis. Then the enzyme activity was
determined byazocaesin method and finally the cell viability of yeast cells exposed to the bromelain was
accessed by MTT Assay .To differentiate the live and apoptotic cells AO/EtBr staining was done.
SAMPLE PREPARATION:
Enzyme extract was derived from the stem of Ananas Comosus.Sample was homogenated bykitchen extractor
53
and filtered the homogenate by Whatt man No: 1 filter paper in order to avoid an impurities. The filtrate was
centrifuged at 10,000 rpm for 10 minutes at 4 . Supernatant was collected and referred as crude homogenate of
enzyme.
ETHANOL PRECIPITATION METHOD:
Ethanol precipitation is done by using 90% ice cold ethanol. Ethanol was added to the crude homogenatein
a drop wise manner till it reachedthe three different concentration (30%,70% and 100%).Then the solution was
centrifuged at 10,000 rpm for 20 minutes at 4. Retain the supernatant and discard the pellet or retain in phosphate
buffer for further research.
DIALYSIS:
Dialysis bag was cut into the required length and the bag was submerged into a solution of 2% Sodium
bicarbonate followed by 0.05% EDTA and boiled for10 minutes. Discard the solution and again boiled indistilled
water for another 10 minutes and sealed the bag at one end and added the solution to the dialysis bag. Placed the
bags in a large volume of phosphate buffer saline and agitated with a magnetic bar and kept in the stirrer motor in
the cold room. Finally the solution was allowedto reach the equilibrium by changing the phosphate buffer saline
every 3 hours at 40C.
DETERMINATION OF ENZYME ACTIVITY:
The enzyme activity was assayed by the azocasein method as described by (Oliveira et al., 2006) and the
concentration of protein was determined by Biuret method.
CYTOTOXICITY ASSAY:
The order to test cytotoxicity yeast were used. Four treatgroups were set up as follows.50 of MTT to100treated
yeast cells and incubatedat 37 for 3 hours. Then200 of acid- propanol was added to it after incubation and left for
overnight at dark room. The cell viability was noted at 650 nm in a microtitre reader by fixing the control group as
100% viability and the percentage of viable cells were calculated related to other treated groups.
AO/EtBr STAINING:
In order to identify the type of cell death induced by treatment with hydrogen peroxide and how the extract
protects/induces cell death was assessed.10 of AO/EtBr stain to the treated cells and spread by placing the cover
slip over it.Then the slides were incubated at room temperature for some times andthe normal cells emits green
fluorescence and apoptotic cells emits red fluorescence were viewed under the 40X magnification in fluorescent
microscope.
RESULT
The enzyme isolated was purified by dialysis and the enzyme activity was found to 16.25(U-mg-1)as
determined by azocasein method.The cell survival was quantified using MTT assay where the cells were exposed
to different dose of bromelain. From the results, it was found that assay the various concentration tested,50ofbromelain
did not induce any cytotoxic effect and this treatment group also contain large number of surviving cells. Hence, the
further staining study were carried out with the same concentration.The yeast cells were treated with bromelain in
the presence and absence of an oxidative stress inducing agent namely hydrogen peroxide. The extent survival of
saccharomyces cerevisiae cells on exposure to hydrogen peroxide and on treatment with bromelain was assessed
by MTT assay.
The results showed that the exposure of saccharomyces cerevisiaecells to hydrogen peroxide induced a
drastic reduction in the viability of cells. When it was co-administrated with bromelain the percentage viability was
54
found to be increased.When it was treated with bromelain alone the percentage viability was increased compared
to oxidative alone, stress treated group as evidenced by MT Tassay.
The morphological changes associated with apoptosis and to differentiate the normal and apoptosis and
nuclear changes associated with apoptosis were by done AO/EtBr staining. The number of apoptotic cells were also
counted. In AO/EtBr staining, co-administration of bromelain and hydrogen peroxide showed significant decrease
in the number of apoptotic cells, when compared to hydrogen peroxide alone treated group,where the number of
apoptotic cells was increased. In the case of bromelain alone treated group the number of apoptotic cells was found
to be decreased.
CONCLUSION
From our result it is clear bromelain shows very low toxicity and also protected the cells from oxidative
damage induced by hydrogen peroxide and can be further tested for other application.
REFERENCE
1. Monji,F.,Adaikan,G.,Lau,L.C.,Said,B.B.,Gong,Y.,Tan,H.M. and Choolani,M (2016)Investigation of uterotonic

properties of Ananas comosus extracts.Journal of Ethnopharmacology .1-22.
2. Karuna.A., Singhal,R.K.,and Kailasaa,S.K(2016) Colorimetric and fluorescence turn-on methods for
sensitive detection of bromelain using carbon dots functionalized gold nanoparticles as a dual probe. RSC
Avances.1-34.
3. Ramalingam.C .,Srinath.R. and Islam.N(2012).Isolation and characterization of Bromelain from pineapple
(Ananas Comosus) and comparing its anti-browning activity on apple juice with commercial Antibrowning
agents.Elixir Food Science,45,7822-7826.
4. Lourenc,C.B., Ataide.A.J., Cefali.L.C, . Novaes, P. Moriel, E. Silveira.E.,Tambourgi.E.B and
Mazzola.P.G(2016) .Evaluation of the enzymatic activity and stability of commercial bromelain incorporated
in topical formulations. International Journal of Cosmetic Science, 38, 535540.
5. Arshad, Z. M., Amid, A., Yusof, F., Jaswir, I., Ahmad, K., & Loke, S. P.(2014). Bromelain: An overview of
industrial application andpurification strategies. Springer-Verlag Berlin Heidelberg ,72837297.
6. Senthilraj.P.,Kathiresan.K(2015) In vitro cytotoxicity MTT assay in vero,HepG2 and MCF-7 cell lines study
of marine yeast. Journal of applied pharmaceutical science,7,80-84.
7. Margaretta,D.L.,Chow,A.,Dirgantax,Y.,Djamil,M.S. and Sandra.F(2015)Macerated-Pineapple Core Crude
Extract-derived Bromelain Has Low Cytotoxic Effectin NIH-3T3 Fibroblast .Indones Biomed,7,101-106.
55
Mechanistic approaches for the antimicrobial action of Phytochemicals
Kavitha, D*, Padma, P.R$. and Sudha,G#
$
*Assistant Professor, Professor, Department of Biochemistry, Biotechnology and Bioinformatics,
Avinashiligam Institute for HomeScience and Higher Education for Women, Coimbatore- 641043
# Scientist, Biocon, Bangalore
Abstract
Infectious diseases are closely dependent on the nature and complexity of human behavior. Bacterial infections
can cause some of the most deadly diseases and widespread epidemics in the world. The development of
antimicrobial agents has been undeniably one of the greatest accomplishments of modern diseases. The
mechanism of action of antimicrobial agents can be categorized further based on the structure of the bacteria or
the function that is affected by the agents. Natural products of higher plants may be a new source of antimicrobial
agents possibly with novel mechanism of action. One such valuable plant is Couroupita guianensis. Hence the
presence study was aimed to determine the mechanism of action of Couroupita guianensis bybacterial membrane
permeabilization and to understand the molecular mechanism of interaction of alkaloids of Couroupita guianensis
and isatin with the DNA of the pathogens. The results obtained from the experiments conducted in this study
analysed the bacterial time kill kinetics and mechanism of action of alkaloid fractions of Couroupita guianensis
and isatin on selected pathogens (Shigella flexneri and Staphylococcus aureus). This study has also proved
the hemolytic activity of alkaloids of Couroupita guianensis and isatin against goat red blood cells. Also, it is
clearly evident from DNA binding assay, alkaloids of Couroupita guianensis have found to damage DNA more
compared to isatin. Thus, the present study strongly iterates the medicinal value of alkaloids and isatin, the both
were found to have high antibacterial activity and effective against the selected pathogens.
Keywords: isatin, Couroupita guianensis, alkaloid, hemolysis
INTRODUCTION
A number of pathogenic microorganisms are widely disseminated, surrounding us and sometimes infecting
our body, causing a wide range of health problems, ranging from mild to life-threatening diseases. It is now widely
accepted to use a variety of molecular approaches to classify or type the causative microbial pathogens in both host
and hostile environments.
Antibacterial drug target interactions are well studied and prominently fall into three classes: inhibition of
DNA replication and repair, inhibition of protein synthesis and inhibition of cell wall turnover (Kohanski et al.,
2007).
The primary site of action of antimicrobial agents is the cytoplasmic membrane of Gram-positive bacteria,
or the inner membrane of Gram-negative bacteria. Natural products and secondary metabolites formed by living
systems, notably from plant origin, have shown great potential in treating human diseases, diabetes and infectious
diseases. (Lai et al., 2010). Medicinal plants contain some organic compounds which produce definite physiological
action on the human body and these bioactive substances include tannins, alkaloids, carbohydrates, terpenoids,
steroids and flavonoids, which can be derived from any part of the plant. They are of great importance to the health
of individuals and communities (Soni and Sosa, 2013).
One important plant that is used in traditional medicine is Couroupita guianensis, which is a tree belonging
to the family Lecythidaceae. Various part of the tree have been reported to contain oils, keto steroids, glycoids,
56
couroupitine, indirubin, isatin and phenolic substances.
The experimental procedure employed in the present study is to analyse the mechanism of action of
alkaloid fractions of Couroupita guianensis and isatin compound against Shigella flexineri and Staphylococcus
aureus.
The flowers of Couroupita guianensis were collected. Alkaloid fractions of Couroupita guianensis was
prepared by the procedure given by Harborne (1973). The bacterial strains used in the present study were the
clinical isolates, Shigella flexneri and Staphylococcus aureus obtained from PSG Hospital, Coimbatore.
Bacterial time kill kinetics
The bacterial time kill kinetics of the alkaloid fractions of Couroupita guianensis and isatin are determined
by estimating the inactivation time of Shigella flexneri and Staphylococcus aureus. The test cultures for this assay
are prepared by incubating Shigella flexneri and Staphylococcus aureus in nutrient medium to get 1.0 McFarland
standard to reach 2-4107 CFU/ml.
The mechanism of action of the alkaloid fractions of Couroupita guianensis and isatin compound against
Staphylococcus aureus and Shigella flexineri is determined by following methods.
Membrane permeability assay
Membrane permeation is a central event in many cytotoxic process. A disruption or damage to the membrane
structure of pathogens could result in leakage of important solutes essential for the cells survival. The alkaloid
fractions of Couroupita guianensis and isatin compound effect on leakage of the membrane in pathogenic bacteria
and are determined by estimating the amount of reducing sugar (Nelson 1944) and protein (Lowry 1951) in the
cultures containing pathogenic bacteria treated with varying concentration of alkaloid fractions and isatin.
A volume of 10 ml culture containing 108 CFU/ml of the two pathogenic bacteria were inoculated with
various concentration of alkaloid fractions (500 g, 1000 g and 1500 g) and isatin (600 g, 800 g, 1000 g and
1200 g) containing Muller Hinton medium and were incubated at 372C, shaken at 150 rpm for 18 hours. The
samples were centrifuged at 12,000 rpm and supernatant were collected and frozen at -30C (Bama et al., 2012).
Then the supernatant was used for estimation of sugars and proteins.
DNA binding assay
The DNA binding assay can assess any antimicrobial-DNA binding efficiency by noting the retardation of
the rate of migration of DNA bands through agarose gels. The DNA binding abilities of the alkaloid fractions of
Couroupita guianensis and isatin are examined by analysing the electrophoretic mobility of genomic DNA of
bacteria on 1% agarose gel. The genomic DNA was isolated by the method given by Sambrook et al., 1989. The
isolated genomic DNA can be treated with both the alklaoids and isatin and incubated for 1 hour followed by
agarose gel electrophoresis.

Bacterial time kill kinetics
Bacterial time kill kinetics were performed for both alkaloid fractions of Couroupita guianensis and isatin
against Shigella flexneri and Staphylococcus aureus. The results (Figures 1 and 2) of time kill kinetics revealed
that with the increasing concentration of isatin the number of CFU of Shigella flexneri were found to be decreased.
Among the different concentrations, 800 g of isatin has significantly reduced the viable cells to 87% within 30
minutes of incubation Complete death of viable cells can be seen in increased concentration of isatin. Hence
isatin was more effective against Staphylococcus aureus compared to Shigella flexneri.
57
Figure 2: Bacterial time kill kinetics of isatin on
Figure 1: Bacterial time kill kinetics of
Staphylococcus aureus
isatin on Shigella flexneri
Figure 3: Bacterial time kill kinetics of alkaloid Figure 4: Bacterial time kill kinetics of alkaloid
fractions of Couroupita guianensis on fractions of Couroupita guianensis on
Shigella flexneri Staphylococcus aureus
Figure 5: Effect of isatin by membrane permeability Figure 6: Effect of alkaloid fractions of Couroupita
potential on Shigella flexneri and guianensis by membrane permeability potential on
Staphylococcus aureus Shigella flexneri and Staphylococcus aureus
Figure 8 : Levels of protein released by membrane

Figure 7 : Levels of protein released by membrane permeability potential of alkaloid fractions of
permeability potential of isatin Couroupita guianensis
58
The leakage of protein was estimated to be high with increasing concentration of isatin for both the
bacteria. But the isatin was found to be more potent against Staphylococcus aureus when compared with Shigella
flexneri. In the alkaloid treated bacterial cell cultures, the leakage of protein was found to be high with the
increasing concentration of the alkaloid fractions. This implies that the alkaloid fractions of the Couroupita guianensis
has the capacity to induce the damage in cell membrane.
DNA binding assay

The genomic DNA was isolated from both Shigella flexneri and Staphylococcus aureus and the purity of
the isolated DNA was checked using Biospecnano (Shimadzu) at 260 nm/280 nm. The DNA from Shigella flexneri
and Staphylococcus aureus were treated with the alkaloids and isatin followed
by the agarose gel electrophoresis.
The results of electrophoretic mobility indicated that there is a damage in isatin
treated DNA of selected pathogens when compared with the untreated control
DNA, which was proved by calculating integral density value of the bands.
Lane 1 : Control DNA of Shigella flexneri

Lane 2 : Treated DNA with isatin compound
Lane 3 : Treated DNA with alkaloid fraction
Lane 5 : Control DNA of Staphylococcus aureus
Lane 6 : Treated DNA with isatin compound
Lane 7 : Treated DNA with alkaloid fraction
Effect of alkaloid fractions of Couroupita guianensis and isatin on genomic DNA of Shigella flexneri and
Staphylococcus aureus
Our study have clearly proved the mechanism of antibacterial action of alkaloid fractions of Couroupita
guianensis and isatin on Shigella flexneri and Staphylococcus aureus. The bacterial cell kill kinetics assay gave a
good understanding about the bactericidal property of the drug compounds. Also, the membrane permeability
potential demonstrated the importance of the tested compounds against the membrane, causing damage that leads
to the leakage of protein and reducing sugars. The DNA binding ability significantly indicated that the lead compounds
are able to bind to the nucleic acids, of the pathogenic microorganisms interact with the DNA and degraded the
stability that can cause the significant death of the microbial cells.
REFERENCES
1. Kohanski, A.M., Dwyer, D.J., Hayete, B., Lawrence, C.A. and Collins, J. (2007) A common mechanism of
cellular death induced by bactericidal antibiotics, Cell, 130: 797-810.
2. Lai, H.Y., Lim, Y.Y. and Kim, K.H. (2010) Blechnum orientale Linn-a fern with potential as antioxidant,
anticancer and antibacterial agent, BMC Complement Altern. Med., 10(15): 1-10.
3. Soni, A. and Sosa, S. (2013) Phytochemical analysis and free radical scavenging potential of herbal and
medicinal plant extracts, J. Pharm. Phytochem., 2(4): 22-29.
4. Nelson, N. (1944) A photometric adaptation of the Somogyi method for the determination of glucose, J.
Biol. Chem., 153: 375-380.
5. Harborne, J.B. (1973) Phytochemical methods-A guide to modern techniques of plant analysis, Chapman
and Hall Publ, 33- 56.
6. Bama, S.S., Kingsley, S.J., Sankaranarayanan, S. and Bama, P. (2012) Antibacterial activity of different
phytochemical extracts from the leaves of T. procumbens linn.: identification and mode of action of the
terpenoid compound as antibacterial, Int. J. Pharm. Pharmaco. Sci., 4: 557-564.
59
Characterisation and Antibiofilm Activity of Eichhornia Crassipes
Iron Oxide Nanoparticles
Mayuri.P.K1, Sumathi.S2, Kavithaa.K2 and Padma.P.R2
1) Vector Control Research Centre, ICMR, Puducherry
2) Department Of Biochemistry, Biotechnology And Bioinformatics,
Avinashilingam Institute For Homescience And Higher Education For Women, Coimbatore
Abstract
The iron oxide nanoparticles were bio-synthesized from Eichhornia crassipes and were characterized using UV
visible spectra, FTIR, SEM with EDAX and X-ray diffraction analysis. Then antibiofilm assay was done to
check the efficacy of iron oxide nanoparticle in biofilm formation. The outcome gave a clear justification of the
combined effect of green synthesized iron oxide nanoparticles from water hyacinth.
Keyword : Nanoparticles, water hyacinth, characterization, antibiofilm.
INTRODUCTION
Iron oxide (IO) nanoparticles have gained considerable interest due to their superparamagnetic properties
and their potential biomedical applications arising from its biocompatibility and non-toxicity.Recent developments
in the preparation of IO nanoparticles by thermal decomposition of iron carboxylate salts have improved the quality
of IO nanoparticles in terms of characteristics such as size tunability, monodispersity and crystalline structure1.
Plants phytochemicals, which have biological significance in terms of medicine development and extracts
of aqueous, methanol and ethanol are good source of antiviral, antitumor and antibacterial agents2. Eichhornia
crassipes (Mart.) Solms. commonly known as water hyacinth is a warm water aquatic plant belonging to the family
Pontideriaceae. It is native to Brazil. It can quickly grow to very high densities (over 60kg m-2); completely clogging
water bodies, which in turn may have negative effects on the environment, human health and economic development3.
In the present study, we attempted to synthesise iron oxide nanoparticles from Eichhornia crassipes and analyse it
by standard characterization techniques. Additionally we attempted to investigate the antibacterial and antibiofilm
properties of the same.Thus in the present study we green synthesized non-toxic iron oxide nanoparticle from water
hyacinth, which has immense medicinal properties and evaluated the same.

Collection of samples & extraction
The plant sample for the study i.e., Eichhornia crassipes was collected from Ukkadam pond, Coimbatore.The
collected plant sample were washed using double distilled water.The leaf portion of the same was cut and rinsed
thoroughly using double distilled water. Then it was homogenized and filtered using Whatmann No. 1 filter paper
and the extract was poured in an air tight container and stored in cold for further processes. The aqueous extract
prepared and filtered will be stable for 2-3 months under cold conditions.
Green synthesis of iron oxide nanoparticles

0.05 M FeSO4 ( Ferrous sulphate ) and 0.025M FeCl3 solution was prepared using deionized water.140ml of
Eichhornia crassipes extract was added the above solution .The magnet was dropped in the solution and kept in
hot plate at 600C for 2 hours. The formation of light green colour to orangish brown was observed indicating the
formation of iron oxide nano-particles4.The formed nanoparticle solution was centrifuged at 1500rpm to collect the
pellet .The pelleted particles were spread in a petridish and were allowed to dry over night in a hot air oven at 500C
60
(process shown in Plate 1). Then the particles adherent to the petridishes were collected and stored dry and used for
further studies.
Characterization of green synthesized iron oxide nanoparticles of Eichhornia crassipes :

1. UV-Visible spectra-
To determine the absorption spectrum of the synthesized
iron oxide nanoparticles were analysed by UV visible spectrometer
. Eichhornia crassipes extract (aqueous) was used as control to the
synthesized solutions. The spectral analysis with a scanning range
of 200-800nm was carried out.
2. Fourier Transform Infrared Spectrophotometer (FTIR):

Fourier transform infrared spectrophotometer (FTIR) is
perhaps the most powerful tool for identifying the types of chemical Plate 1: Green synthesis of Eichhornia
crassipes iron oxide nanoparticles
bonds /functional groups present in phytochemicals. The powder of
iron oxide nano-particles of Eichhornia crassipes was used for FTIR analysis. The sample was encapsulated in KBr
pellet,in order to prepare translucent sample disc.The sample was loaded in FTIR spectroscope with a scan range
from 400 to 4000 cm-1 with a resolution of 4cm-1. The analysis was done and recorded.
3. Scanning Elecron Microscopy with EDAX :

The green synthesized nanoparticles were characterized using high resolution scanning electron microscopy
[SEM]. EDAX analysis of the iron oxide nanoparticles was also done to find the percentage impurity and composition
of the same.
4. X-ray diffraction analysis (XRD):

The powder X-ray diffraction (XRD) pattern on the samples were recorded by a X-ray diffractometer
(miniflex II, desktop-X-ray diffractometer) using Cu-k radiation of wavelength = 1.54 A0 for 2 ranging from 200
to 800. The spectrum was analysed for the structural characterization of green synthesized iron oxide nanoparticles.
Antibiofilm activity:
A overnight culture of Staphylococcus aureus in trypticase soy broth was diluted in the ratio 1:100 in
respective fresh medium and allowed to grow for another hour.100ml of the diluted strains were added to 96 well
titre plate and the different concentrations of nano-particles of 25g,50g,75g and 100g per ml were added and
incubated at 370c for three days. The growth monitored every 12 hours at 595nm using ELISA reader .After the
incubation the medium was removed and 100l of 1% crystal violet solution was added and incubated at room
temperature for 30 minutes . The dye was removed after staining and wells were washed and finally incubated with
95% ethanol for 15 minutes and it was read in spectrophotometer at 595nm.Inhibition mediated reduction of bio
film formation was calculated by,
O.D in control - O.D in treatment 100
% of inhibition =
O.D. in control

1) Synthesis of iron oxide nanoparticles
Iron oxide nanoparticles were synthesized by green synthesis by thermal decomposition method using
Eichhornia crassipes. The colour change from light greenish yellow to blackish brown, confirmed the synthesis of
iron oxide nanoparticles (shown in Plate 2).
61
2) Particle size and morphology characterization of iron oxide nanoparticles
1.UV-visible spectra:
The UV- visible spectra is shown in the Plate 3. The Eichhornia crassipes
iron oxide nano-particles showed a absorption peak at 275nm*. Most of the studies
indicated that the UV Spectral peak of iron oxide nano-particles between 200-500
nm. In a study, they observed a similar peak in their study of nano-particles5.They
Plate 2 : Eichhornia crassipes
stated that the peak would be arising due to the -* transition. In the indole part of
iron oxide nanoparticles
the molecule of tryptophan and tyrosine residue of proteins ie., present in the
Eichhornia crassipes plant derived iron oxide nano-particles; (in the present study).The peak value is the strong
indication of either coordination of tryptophan (or) tyrosine
molecules with the surface of iron oxide nano-particles.
2.Fourier Transform Infrared Spectrophotometer(FTIR):

Eichhornia crassipes iron oxide nano-particles (FTIR)
analysis have revealed the e xistence of various chemical
constituents in the same. The absorption bands, the wave-number
(cm-1) were described in the graph (shown in Plate 4). The IR
spectrum reveals structural information about major and minor Plate 3 : UV- visible absorption spectrum
-1 -1
constituents. The peak at 3452cm and 3780cm are assigned to
strong O-H (hydroxyl) stretches,vibrations due to H-bonds and free stretch respectively. A peak value at 2322cm-
1
is attributed to C-N medium stretch. The peak value at 1190cm-1 and 1365cm-1 is the character of carbonyl (co)
stretch ,and a weak amide absorptive (C-N) stretch, it may be due to some carbonyl compounds in leaves of
Eichhornia crassipes6.The amide absorptions are considered sensitive to protein conformation , hence increase or
decrease ratio of intensity of bands at ~1365 and~1190 are
attributed to amide stretching .The characteristic peak at 763cm-1
signifies the aromatic C-H bending and alkylhalide(C-Cl) stretch
while 578cm-1 signifies C-Br stretch. The sharp peak at ~763
signifies and confirms the presence and strong interaction of
aromatic amino acid as the UV visible spectral data studies reveal.
Many workers have reported the FTIR spectrum as
Plate 4 : Fourier Transform Infrared
effective tool for differentiating , classifying and discriminating Spectrum
closely related plants and other organisms. Similar work was also
reported by Geethu et al in IR spectra from mid-infrared region (4000-400cm-1) for aqueous methanol ,chloroform
,ethylacetate,and petroleum ether extract of Eichhornia crassipes several unique bands that are pertained to
functional groups present chemical components are metabolic products in the plant.
3.Scanning Electron Microscopy (SEM):

High resolution scanning electron microscopy images
of Eichhornia crassipes iron oxide nano-particles were
analysed ( shown in Plate 5) . The working distance was
10.0mm. The range of analysis of particle size and shape was
1m.The iron oxide nano particles fall in the nano particle
range. The iron oxide nano particles synthesized by Khalil,2015
Plate 5 : Scanning Electron Microscopy
were in the range of 1m7.Iron oxide nano particles synthesized Images
by green synthesis method appeared flat while analyzing under
scanning electron microscopy.The HR-SEM images of conventionally synthesized nano particles usally range from
62
50 -100nm as reported by many studies. So, the scanning electron microscopy images revealed the qualitative
characteristics 1m size, spherical and flat surface of the synthesized iron oxide nano particles.
4. EDAX Spectrum of synthesized nanoparticles :

EDAX spectrum analysis is used to find the composition of the synthesized nanoparticles. The results of
EDAX analysis of our study is shown in Plate 6. The peaks are around 0.35, 1.0, 8.6 keV. It indicates the presence
of iron and oxygen and there are no impurities present in the synthesized iron oxide nanoparticles. Mahdavi et al.,
(2013) in their study reported that peaks around 0.8, 6.2 and 6.9 keV related to the binding energies of iron confirms
the presence of Fe3O4- NPs in the Brown seaweed aqueous extract without any impurities 8. The presence of iron
and oxygen elements in the synthesized nanoparticles as are seen in the table 1.
Table 1 : Showing the EDX values.
Element Weight % Atomic %
O 22.88 54.80
Fe 77.12 45.20
Total 100 100
Plate 6 : EDX Spectrum
5) X-Ray Diffraction Spectrum Analysis
X- ray Diffraction technique is used to analyse the crystalline nature of the iron oxide nanoparticles. The
peaks in the diffractogram indicates crystalline nature of the particles.The powder X-ray diffraction method was
used to analyse the green synthesized iron-nanoparticles from Eichhornia crassipes.
Plate 7 shows the X-ray diffraction patterns of green synthesized iron oxide nanoparticles. Strong diffraction
peaks are seen with 2values of 22, 31and 50 which corresponds to the crystal planes of (250), (120) and (175)
of crystalline iron oxide nanoparticles respectively.
Plate 7 : X- ray diffraction spectrum
63
Khalil (2015) reported that six peaks at 2 of 30.36, 35.74, 43.52, 53.95, 57.34 and 63.0 which
represents the corresponding indices of (220), (311), (440), (422), (511) and (440) respectively.
Mahdavi et al., (2011) have reported the similar peaks and the crystal planes at (200), (311), (440) and
(511) by the synthesis of iron oxide nanoparticles using green synthesis method.
A similar observation was done by Saha et al ., 2013 in their analysis of iron oxide nanoparticles.The
diffraction peaks of synthesized nanoparticles matches exactly with the XRD standard data of parent (bulk) magnetite
which confirms the crystal nature of the iron oxide nanoparticles. These supportive studies help us to confirm the
crystalline structure and phase identification of the iron oxide nanoparticles.
3) Anti-biofilm effect of iron oxide nano particles Eichhornia crassipes:

Anti biofilm effect of iron oxide nano particles synthesized from Eichhornia crassipes against S.aureus ,
recorded maximum biofilm inhibition 90% in 100g fraction (72 hours).The minimum inhibition was noticed in 0
hour 25g fraction 8.5%. The inhibition percentage was gradual and increased every 12 hour interval. This study
shows the immense anti biofilm (or) biofilm inhibition activity of the non-toxic iron nano particles derived from
Eichhornia crassipes.The effect was calculated using the formula mentioned before and the graph indicating the
advance of effect is in Plate 8.
Plate 8 : Antibiofilm activity (showing the time intervals and different concentrations)
CONCLUSION
The results in the present study has showed the effectiveness of unrevealed properties of plants like
Eichhornia crassipes in combination with developing field of nano technology. The properties of the particles were
immense and uncovered in the characterization. UV-Visible spectra showed interactions of the aromatic amino
acids present in Eichhornia crassipes with the iron oxide nano particle surface. The FTIR showed 7 peaks based on
active groups present in sample.The SEM with EDX analysis showed the shape,size and range of the nanoparticles.and
the composition of the bio synthesized iron oxide nanoparticles. The XRD peaks signifies the crystalline nature of
the nanoparticle.The anti-biofilm activity showing exponential properties of nano-particle was a laid forestep to
study more capabilities of the same in future.
REFERENCE
1) Saha S and Bunia A K, (2013) ; Synthesis of Fe2O3 Nanoparticless and study of its structural and optical
properties; Journal of Physical Sciences ; Vol: 17; pp: 191-195.
2) Prakash, S., Ramasubburayan, R., Ramkumar, V. S., Kannapiran, E., Palavesam, A., & Immanuel, G. (2016).
In vitroScientific evaluation on antimicrobial, antioxidant, cytotoxic properties and phytochemical
64
constituents of traditional coastal medicinal plants; Biomedicine & Pharmacotherapy, 83, 648-657.
3) Jayanthi P, Lalitha P, Sujitha R and Thamaraiselvi A, (2013); Anti inflammatory activity of the various
solvent extracts of Eichhornia crassipes (Mart). Solms; International Journal of PharmaTech Research ; Vol:
5; pp: 641-645.
4) Balamurugan MG, Mohanraj S, Kodhaiyolii S, and Pugalenthii V ,(2014) ; Ocmium sanctum leaf extract
mediated green synthesis of iron oxide nanoparticles: Spectroscopic and Microscopic studies ; Journal of
Chemical and Pharmaceutical sciences; Vol: 4 , pp: 201-2014.
5) Sanghi R, and Verma P, (2010); pH dependent fungal proteins in the green synthesis of gold nanoparticles;
Advanced Materials letters ; Vol 1(3);pp:193-199.
6) Geethu MG, Suchithra P.S, Kavitha C.H, Aswathy J M, Babu D, and Murugan K,(2014); FTIR analysis of
different solvent extracts of Water hyacinth- An alleopathic approach ; World Journal of Pharmacy and
Pharmaceutical sciences ; Vol: 3; pp : 1256-1266.
7) Khalil M, Lee R and Liu N, (2015); Hematite nanoparticles in aquathermolysis: A desulfurization study of
thiopene; Fuel ; Vol:145 ; pp : 214-220.
8) Mahdavi M, Namvar F and Ahmad M B, Mohamad R, (2013); Green synthesis and characterization of
magnetic iron oxide nanoparticles using Sea weed (Sargossum mutium); Molecules ; Vol:18 ; pp: 5954-
5964.
65
Detoxification of lead toxicity using in vitro antioxidant properties of
Cynodondactylon in lead induced goat liver homogenate
Poornima, A1., Ragavan, B 2. And Sumathi, S1.
1. Department of Biochemistry, Biotechnology and Bioinformatics,
Avinashilingam Institute for Home Science and Higher Education for Women
2. Department of Biochemistry, PSG College of Arts and Science
Email id : dhiyapoornima@gmail.com
Abstract
Lead exposure is considered to be a major public health problem. Toxicity of lead is mainly attributed to the
induction of oxidative stress by elevation of reactive oxygen species. Liver is an organ of paramount importance
as it has great capacity to detoxify toxic substances. The aim of the study was to neutralize the oxidative stress
created by lead with Cynodon dactylon extract as it is rich in antioxidant properties. The experimental goat liver
slices were divided into four groups such as control, lead induced, treated group, and acute toxicity. The
objective of the study is to estimate the enzymic and non-enzymic antioxidants such as catalase, SOD, glutathione
peroxidase, peroxidase, Vitamin A, E, C and reduced glutathione. Thiols, protein thiols, and protein were also
estimated. On the basis of our study, we speculate that lead has adverse effects on the antioxidant status of the
goat liver and that aqueous extract of Cynodon dactylon may have potential antioxidant properties that can
detoxify the lead induced toxicity.
INTRODUCTION
Lead is present everywhere in human environment, because of its excellent physico-chemical properties,
low cost and is widely used in many industrial and domestic activities. The lead dispersed through gasoline
exhausts, smelter emission, peeling paint, etc., never disappears completely from our environment. Human beings
are exposed to lead from numerous sources including air, food, dust, soil and water.[1]
Lead has been found to induce a wide range of biochemical, physiological and behavioural effects.[2] The
brain, liver, and kidneys are considered to be the target organs for the toxic effects of lead. [2] Toxicity of lead
mainly creates oxidative stress by increasing the reactive oxygen species (ROS) such as superoxide radicals,
hydroxyl radicals, hydrogen peroxide, and lipid peroxides, therefore, increased interest among phytotherapy
investigators to use medicinal plant with antioxidant activity for protection against metal especially lead toxicity has
been noted.[3]
Liver is an organ of great importance, which plays pivotal role in regulating various physiological processes
in the body such as metabolism, secretion and storage. It has great capacity to detoxify toxic substances and
syntheses useful principles. The damage to the liver caused by hepatotoxic agents is of grave consequences. In
spite of tremendous scientific advancement in the field of hepatology in recent years, liver problems are in rise.
Jaundice and hepatitis are two major hepatic disorders that account for high death rate.[4] Oxidative stress plays an
important role in many diseases including liver diseases.
Medicinal plants are part of human life to treat many diseases from the dawn of civilization. [5] Medicinal
plants have continued providing valuable therapeutic agents, both in modern and in traditional medicine.[6] With the
associated side effects of modern medicine, traditional medicine are gaining importance and are now being studied
to find scientific basis of their therapeutic actions.[7] Cynodon dactylon. (L). Pers belongs to the family of Poaceae[8]
and is said to have many medicinal properties including antihelmenthic, antidiuretic, anti-inflammatory,
hepatoprotective activity as well as treatment of urinary tract infection, prostaitis and dysentery. Traditionally, it is
66
used in diabetes,[9] jaundice, kidney problems, urinary disease, gastrointestinal disorder, constipation, and
abdominal pain. The whole plant is used for diuretic, dropsy, syphilis, wound infection and piles. Cynodon
dactylon is used as antihaemorrhagic in dysentery and nasal bleeding.[10]
Free radicals are known as major contributors to several clinical disorders such as diabetes mellitus,
cancer, liver disease, renal failure and degenerative diseases as a result of deficient natural antioxidant defence
mechanism. A systematic search for useful bioactivities from medicinal plants is now considered to be a rational
approach in pharmaceutical and drug research.
Hence the present study was undertaken to explore the key behind the use of Cynodon dactylon as a
detoxifying agent against lead induced hepatotoxicity in goat liver model.

PREPARATION OF PLANT EXTRACT:
The leaves of Cynodon dactylon were collected during the month of December, 2014 from the local areas
of Coimbatore district, Tamil Nadu, India. The leaves were identified and authenticated by Dr.N.Rajaram, Associate
Professor, Department of Botany, PSG college of Arts and Science. The leaves were freshly ground with water
and the juice was used for the studies.
PREPARATION OF GOAT LIVER HOMOGENATE:
The goat liver was collected fresh from the local slaughter house immediately after sacrifice of the animal.
The tissue was immediately washed with saline and was plunged into cold sterile phosphate buffer. The tissue was
cut with sterile scalpel into slices (10g). The tissues were divided into four groups. 10g of the tissue was soaked
in 100 mL Tris buffer and then it was homogenised. The homogenate is used for the assay.
The treatment groups are as follows. Group I consisted of groups treated with goat liver homogenate
alone which served as control. Group II consisted of groups of goat liver homogenate induced with lead (induced).
Group III consisted of groups of liver homogenate with lead and Cynodon dactylon juice (treated). Group IV
consisted of goat liver homogenate and Cynodon dactylon juice (acute toxicity). The groups were incubated for 1
hour and the assays were done.
The phytochemicals present in hydro ethanolic crude extract of Cynodon dactylon were separated using
TLC plates. Qualitative analysis of phytochemicals in the plant extracts were done according to the standard
methods.
The antioxidant status of all the four groups were estimated. Enzymic antioxidants such as catalase (CAT),
SOD, peroxidase (PX), glutathione peroxidase (GPX); nonenzymic antioxidants such as vitamin A, vitamin C, and
vitamin E, protein, thiols, protein thiols and and total reduced glutathione were estimated.
STATISTICAL ANALYSIS
Values were expressed as mean SD. Statistical difference in mean was analyzed using one way ANOVA. P< 0.05
was considered statistically significant.
RESULT AND DISCUSSION
The results of the present study showed that the activities of enzymic and non enzymic antioxidants were
significantly decreased in lead treated group. Treatment with Cynodon dactylon extract caused slight increase in
67
the antioxidant status when compared with lead alone treated groups.
TABLE 1: Effect of Cynodon dactylon extract on the antioxidant status of lead induced, treated,
and toxicity groups of goat liver homogenates (mean SD).
Parameters Group I Group II Group III Group IV

(mg/g tissue)
PROTEIN 10.300.59 9.261.72 a * 9.27 1.22 b* 10.440.81 c*

a
CAT 17.830.68 16.560.59 17.080.38 b 17.520.51 c
a
SOD 0.020.01 0.020.016 0.020.010 b 0.020.004 c
PX 2.040.44 1.260.83 a 2.020.61 b

1.970.60 c
a b c
GP X 3.280.04 3.23 0.12 3.260.12 3.270.04
a b c
Vit-A 0.87 0.21 0.81 0.07 0.810.02 0.56 0.37
a b c
Vit-C 3.15 0.72 2.84 0.60 2.950.53 2.41 0.59
a
Vit-E 11.860.48 10.890.52 11.030.61 b 11.460.42 c
a b c
Protein thiols 10.300.59 7.27 1.39 7.850.39 8.280.38
a b c
Thiols 5.28 1.01 4.06 1.17 4.58 1.71 5.99 0.86
a b c
Total reduced 2.51 0.24 1.62 0.39 2.05 0.23 2.26 0.15
glutatione
1. a - represents group I statistically significant with group II

2. b - represents group II statistically significant with group III
3. c- represents group I statistically significant with group IV
Statistical significance
*Values are significant p < 0.05
The lead induced liver homogenates (Group II) showed decreased levels of protein when compared with
control liver homogenates (Group I).The decreased level of group II may be due to lead interacting with enzyme
functional groups and high-affinity with metal-binding proteins, such as Pb-binding proteins and metallothioneins.[11]
The levels of protein in Group III treated with plant extract (10ml per 10g of tissue) was found to be slightly
increased, this may be due to Cynodon dactylon scavenging the metal from the metal binding enzymes or removal
of metal ions from the binding site or receptors. This shows the detoxification capacity of aqueous extract of
Cynodon dactylon on in vitro liver homogenate. Group IV shows non-significant result when compared with
normal group. Our study was supported by Kalivani et al., 2011 using Jaminum sambac (Linn) in mammalian liver
slice technique in vitro.
The levels of catalase, SOD, peroxidase, glutathione peroxidase were found to be decreased moderately in
Group II when compared with Group I (statistically not significant), this may be due to lead toxicity reducing the
antioxidant defense system of cells via depleting glutathione, interfering with some essential metals needed for
antioxidant enzyme activities and/or increasing susceptibility of cells to oxidative attack by altering the membrane
integrity.[12] Under normal conditions, cells possess enzymic and non-enzymic defenses such as SOD, GSH-Px,
catalase and GSH to cope with free radicals. [13] Oxidative damage, however, may occur when antioxidant potential
68
is decreased and/or when oxidative stress is increased. Some investigators had indicated that lead exposure increased
the production of ROS in the liver. [14]
In group III, i.e., group treated with plant extract (10ml per 10g of tissue), the enzymes level were found
to be slightly high when compared with Group II. This may be due to the antioxidant properties of Cynodon
dactylon. It can act as a good antioxidant in natural origin. The phytochemical and antioxidant activity were
reported by Kanimozhi and Bai, 2012. Our study was supported by Kalivani et al., 2011 using Jaminum sambac
(Linn) in mammalian liver slice technique in vitro. Group IV shows non-significant result when compared with
normal group.
The non-enzymic antioxidants namely vitamin C, E and A were estimated in the lead induced liver homogenate
with and without grass extract of Cynodon dactylon. A moderate decrease (but not statistically significant decrease)
was found in levels of vitamin C in the lead induced group. The lead exposure caused reduction in the levels of
GSH when compared with control. The depleting effect of the lead was very well counteracted by the administration
of leaf extract in the Group III.[15] This proves the in vitro non-enzymic antioxidant potential of Cynodon dactylon.
This study was supported by Radha and Padma, 2011 who studied the antioxidant activity of Majorana hortensis
leaves subjected to the oxidative stress in an invitro system.Group IV shows non-significant result when compared
with normal group.
The levels of the thiols and protein thiols were found to be reduced. This may be due to the lead causing
oxidative stress by inducing the generation of reactive oxygen species (ROS), reducing the antioxidant defense
system of cells via depleting glutathione, inhibiting sulfhydryl-dependent enzymes, interfering with some essential
metals needed for antioxidant enzyme activities, and/or increasing susceptibility of cells to oxidative attack.[11]
Toxicity of lead results from reactive metabolites following conversion in the liver which causes cellular damage
and altered metabolic activity.[16] Loss of cellular glutathione is indicative of cytotoxicity.[17] In Group III treated
with plant extract (10ml per 10g of tissue), the level was found to be slightly increased when compared with the
lead induced group (Group II). This may be due to the properties of Cynodon dactylon that has the capacity to
reduce the reactive oxygen species and possess in vitro antioxidant properties.Group IV shows non-significant
result when compared with normal group.
On the basis of the present study, we speculate that lead has adverse effects on antioxidant status of the
goat liver homogenate and that aqueous extract of Cynodon dactylon may have potential antioxidant properties that
can detoxify the lead toxicity. Further studies are needed for investigation of invivo antioxidant potential with
animal models.
ACKNOWLEDGEMENT
The author is grateful to the authorities of PSG College of Arts and Science, Coimbatore, Tamil Nadu, India
for providing the necessary support.
REFERENCES
1. Dsouza, H.S., Dsouza, S.A., Menezes, G. and Venkatesh, T., 2011. Diagnosis, evaluation, and treatment of
lead poisoning in general population. Indian Journal of Clinical Biochemistry, 26(2), pp.197-201.
2. Jackie, T., Haleagrahara, N. and Chakravarthi, S., 2011. Antioxidant effects of Etlingera elatior flower
extract against lead acetate-induced perturbations in free radical scavenging enzymes and lipid peroxidation
in rats. BMC research notes, 4(1), p.67.
3. Haleagrahara, N., Jackie, T., Chakravarthi, S., Rao, M. and Kulur, A., 2010. Protective effect of Etlingera
elatior (torch ginger) extract on lead acetate-induced hepatotoxicity in rats. The Journal of toxicological
sciences, 35(5), pp.663-671.
69
4. Nazeema, T.H. and Brindha, V., 2009. Antihepatotoxic and antioxidant defense potential of Mimosa pudica.
Int J Drug Discov, 1, pp.1-4.
5. Nayak, A., Nayak, R.N., Soumya, B., Bhat, K. and Kudalkar, M., 2011. Evaluation of antibacterial and
anticandidial efficacy of aqueous and alcoholic extract of Neem (Azadirachta indica) an in vitro study.
IJRAP, 2, pp.230-5.
6. Krentz, A.J, and Bailey C.J., 2005, Oral antibiotic agents: current role in type 2 diabetes mellitus drugs 65,
385-411.
7. Gupta, Y.K. and Briyal, S., 2004. Animal models of cerebral ischemia for evaluation of drugs. Indian J
Physiol Pharmacol, 48(4), pp.379-394..
8. Harlan, J.R., 1970. Cynodon species and their value for grazing and hay. In Herbage Abstracts (Vol. 40, pp.
233-238).
9. Singh, S.K., Rai, P.K., Mehta, S., Singh, R.K. and Watal, G., 2009. Curative effect of Cynodon dactylon
against STZ induced hepatic injury in diabetic rats. Indian Journal of Clinical Biochemistry, 24(4), p.410.
10. Satapathy, K.B., Sahu, B.B. and Jena, G.S., 2012. Crop weeds diversity and their ethnomedicinal uses in the
treatment of common ailments in Jajpur district of Odisha (India). International Journal of Medicinal and
Aromatic Plants, 2(1), pp.80-89.
11. Goering, P.L., 1992. Lead-protein interactions as a basis for lead toxicity. Neurotoxicology, 14(2-3), pp.45-
60.
12. Gurer, H. and Ercal, N., 2000. Can antioxidants be beneficial in the treatment of lead poisoning?. Free
Radical Biology and Medicine, 29(10), pp.927-945.
13. Rice-evans, C.A., Miller, N.J., Bolwell, P.G., Bramley, P.M. and Pridham, J.B., 1995. The relative antioxidant
activities of plant-derived polyphenolic flavonoids. Free radical research, 22(4), pp.375-383.
14. Pande, M. and Flora, S.J.S., 2002. Lead induced oxidative damage and its response to combined administration
of -lipoic acid and succimers in rats. Toxicology, 177(2), pp.187-196.
15. Radha, P. and Padma, P.R., 2011. Antioxidant activity of Majorana hortensis leaves subjected to oxidative
stress in an in vitro system. Int Res J Pharma, 6, pp.153-157.
16. Brahmachari, A.K. and Singha, A., 2011. The Hepatotoxicity of Mimosine in Precision Cut Goat Liver Slice
System. MEDICAL RESEARCH, p.29.
17. Docks, E.L. and Krishna, G., 1976. The role of glutathione in chloroform-induced hepatotoxicity.
Experimental and molecular pathology, 24(1), pp.13-22.
70
Phytochemical screening, UV and FT-IR Analysis of Jacobina Leaves
R. Thilgavathi1*, A.Prithiba2 and R.Rajalakshmi3
1
*Research Scholar, 2Asst. Professor, 3Professor Department of Chemistry,
Avinashilingam Institute for Home Science and Higher Education for Women,
Coimbatore-641043, Tamil Nadu, India.
Abstract
Bioactive principles present in eco-friendly products have been principally used as curative agents for several
diseases since olden days. The secondary metabolites present in the plant species can be analysed by preliminary
phytochemical screening of plants. In the present study phyto chemical constituents present in the methanol,
ethyl acetate and water extract of Jacobina leaves have been analysed. The presence of various phytochemical
constituents in the extracts were determined using standard screening tests .The tests reflected the presence of
alkaloids, terpenoids, tannins, saponins. FT-IR and UV visible spectrophotometric analysis have been carried
out for finding the active compounds present in the Jacobina leaves.
Keywords : Jacobina leaves, Phytochemical assay, Phenolic and Flavonoids
INTRODUCTION
Herbal plants are extensively used to treat several medical disorders. Justicia belonging to the family
Acanthaceae is a small herb distributed in the Peninsular and Deccan region of India. Justicia have been used in
the traditional system for the treatment of fever 1, pain2, inflammation3, diabetes4, diarrhoea5 and liver disease6.
They also possess antitumoral7, antiviral8, analgesic and anti-inflammatory9 activities. Juices of the leaves act as a
cooling agent and aperients and also given to children in small pox. Phytochemicals present in these plant can act
as a agent for several disorder like Ischemia, Asthma, Arthritis, Cancer, Ageing, atherosclerosis, reperfusion injury
of many tissues 10,11.
These phytochemicals naturally possess antioxidants. Medically antioxidants help the cells from prevention
of oxidative damage and degenerative diseases12,13,14. Medicinal plants can protect against harmful effects of ionizing
radiation. Natural plant extracts or pure compounds are safe ingredients, which do not have any toxic effects. The
aim of this paper is to determine the possible phytochemical components by qualitative analysis.

Collection of plant sample
The leaf samples were collected from Ooty, Niligari district and was authentified by Botanical Survey of
India, Coimbatore, Tamilnadu and a voucher specimen has been deposited in our university for further reference.
The leaves were separated, washed properly with distilled water and air dried at room temperature. The dried
leaves were powdered finely using an electric blender and stored for further analysis.
Preparation of Extract for phytochemical screening:
Collected plants were dried at room temperature and ground to make fine powder. 5gm of plant powder
was well dissolved in 100ml of solvents (Methanol, Ethyl acetate, Ethanol and water) (ratio 1:5). The suspension
was filtered and extracts were collected for further use.
71
Phytochemical tests:
The phytochemical test of these extracts was performed using the method adopted by Harborne15 and Sofowora16.
Test for carbohydrates (Molischs test):

To 2ml of plant extract, 1ml of Molischs reagent and a few drops of concentrated sulfuric acid were added.
Presence of purple or reddish color indicates the presence of carbohydrates.
Test for tannins (Ferric chloride test):

To 1ml of plant extract, 2ml of 5% ferric chloride was added. Formation of dark blue or greenish black indicates the
presence of tannins.
Test for saponins (Frothes test):

To 2ml of plant extract, 2ml of distilled water was added and shaken in a graduated cylinder for 15minutes lengthwise.
Formation of a 1cm layer of foam indicates the presence of saponins.
Test for flavonoids (Shinoda test):

To 2ml of plant extract, 1ml of 2N sodium hydroxide was added. Presence of yellow color
indicates the presence of flavonoids.
Test for alkaloids (Mayers test):

To 2ml of plant extract, 2ml of concentrated hydrochloric acid was added. Then a few drops of Mayers reagent
were added. The presence of green color or white precipitate indicates the presence of alkaloids.
Test for quinines:

To 1ml of extract, 1ml of concentrated sulfuric acid was added. Formation of red color indicates presence of
Quinones.
Test for glycosides (Molischs test):

To 2ml of plant extract, 3ml of chloroforms and 10% ammonia solution was added. Formation of pink color indicates
presence of glycosides.
Test for cardiac glycosides (Keller Kiliani test):

To 0.5ml of extract, 2ml of glacial acetic acid and a few drops of 5% ferric chloride were added. This was under
layered with 1ml of concentrated sulfuric acid. The formation of brown ring at the interface indicates presence of
cardiac glycosides.
Test for terpenoids (Salkowski test):

To 0.5ml of extract, 2ml of chloroform was added and concentrated sulfuric acid is added carefully. Formation of
red brown color at the interface indicates presence of terpenoids.
Test for triterpenoids:

To 1.5ml of extract, 1ml of Libemann Buchard Reagent (aecticanhydride+ concentrated sulphuric acid) was added.
Formation of blue green color indicates presence of triterpenoids.
Test for phenols (Ferric chloride test):

To 1ml of the extract, 2ml of distilled water followed by a few drops of 10% ferric chloride was added. Formation
of blue or green color indicates presence of phenols.
72
Test for coumarins
To 1 ml of extract, 1ml of 10% NaOH was added. Formation of yellow color indicates presence of coumarins.
Steroids and phytosteroids (Libermann- Buchard test)

To 1ml of plant extract equal volume of chloroform is added and subjected with a few drops of concentrated
sulfuric acid appearance of brown ring indicates the presence of steroids and appearance of the bluish brown ring
indicates the presence of phytosteroids.
Phlobatannins
To 1ml of plant extract few drops of 2% HCL was added appearance of red colour precipitate indicates the presence
of phlobatannins.
Anthraquinones (Borntragers test):

To 1ml of plant extract few drops of 10% ammonia solution were added, appearance pink color precipitate indicates
the presence of anthraquinones.
Spectroscopic analysis
To detect the UV-VIS spectrum profile of Jacobina leaf, the methanolic extract was scanned in the wavelength
ranging from 200-800 nm by using UV spectrophotometer and the characteristic peaks were detected17,18.FT-IR
analysis was performed using Perkin Elmer spectrophotometer system which was used to detect the characteristic
peaks and their functional groups. The peaks values of the UV-VIS and FT-IR were recorded.

Phytochemical analysis
The preliminary phytochemical screening of Justicia leaves showed the presence of plant components such as
carbohydrates, tannins, flavonoids, Alkaloids, quinones, cardiac glycosides, terpenoids, phenols, coumarins,
glycosides and steroids in methanol extract and carbohydrates, flavonoids, cardiac glycosides, terpenoids, quinones
phenols, steriods and coumarins in ethyl acetate extract, carbohydrates, tannin, quinones, cardiac glycosides,
terpenoids, phenols, coumarins and steroids in water, carbohydrates, tannin, flavonoids, alkaloids, cardiac glycosides
terpenoids, phenols present in ethanol. (Table 1).
Table 1
Qualitative Analysis
S.No Phytochemical Tests Test performed Ethyl Acetate Methanol Ethanol Water
1. Carbohydrates Molischs test + + + +
2. Tannin Ferric chloride test - + + +
3. Saponin Frothes test - - - -
4. Flavonoids Shinoda test + + + -
5. Alkaloids Mayers test + + + -
6. Quinones + + - +
7. Glycosides Molischs test - + - -
73
8. Cardiac glycosides Keller Kiliani test + + + +
9. Terpenoids Salkowski test + + + +
10 Phenols Ferric chloride Test + + + +
11. Coumarins + + - +
12. Steroids Libermann Buchard test + + - +
13. Phlobotanins - - - -
14. Anthraquinones Borntragers test - - - -
Present : + Absent : -
UV-visible spectroscopy
The UV analysis showed wavelength range from 220nm-800 nm. The UV is very useful method for
identification of unsaturated bonds present in plant component, which can be used to distinguish between conjugated
and non-conjugated system19,20. The result of the UV analysis of the fraction gave absorption peaks at 567nm.
It can be inferred that the compounds present in the fractions have chromophores and hence, absorption
takes place to allow transition.
Fig.1UV-vis spectra of Jacobina leaves
FT-IR analysis
The FT-IR spectrum was used to identify the functional groups of the active components present in plant
based on the peaks values in the region of IR radiation. When the plant extract was passed into the FTIR, the
functional groups of the components were separated based on its peaks ratio. The results of FT-IR analysis confirmed
the presence of alcohol, phenol, alkanes, aldehyde, aromatic compound, secondary alcohol, carboxylic acid and
halogen compounds.(Fig.2 Table.2)
74
Fig. 2 FT-IR spectrum of Jacobina leaves
Table.2 FT-IR peak values of Jacobina leaves
S.No. Peak values (cm-1) Interpretation

1. 3312 Alcohol
2. 2980 Alkanes
3. 2768 Carboxylic acid
4. 619 Aromatic compounds
CONCLUSION
In this study, methanol and ethyl acetate extract of Jacobina leaves shows significant amount of phenolics
and flavonoids content. The presence of phytochemicals like flavanoids, tannins, glycosides, etc were observed in
the leaf extracts of Jacobina leaves. Future works could also be made possible to investigate the specific
phytoconstituents present in the above mentioned Jacobina leaves. Phytochemicals can serve as a valuable source
of information and provide appropriate standards to establish the quality of these plant materials in medicinal and
pharmaceutical studies.
REFERENCE
1. Chen CC, Hsin WC, Ko FN, Huang YL, Ou JC, Teng CM. J Nat Prod. 1996; 59:1149.
2. Bhattarai NK. Fitoterapia 1993; 64: 483.
3. Panthong A, Kanjanapothi D. J Ethnopharmacol. 1986; 18: 213.
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Ethanopharmacol. 1992; 36: 81.
6. Yangfg LL, Yen KY, Kiso Y, Kikino HJ. Ethanopharmacol. 1987; 19: 103.
7. Fukamiya N, Lu KH. J Nat Prod. 1986; 49: 348.
8. Asano J, Chiba K, Tad M, Yoshi T. Phytochemistry. 1996; 42: 713.
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9. Lino CS, Taviera ML, Viana GSB, Matos FJA. Phytotherapy Res. 1997; 11: 211.
10. Cook, NC and Samman, S. Flavonoids chemistry metabolism cardio protective effects and dietary sources.
Nutritional biochemistry.19967; 66-76.
11. Kumpulainen, JT and Salonen, J.T. Natural Antioxidants and Anticarcinogens in Nutrition, Health and Disease,
(The Royal society of chemistry, UK) 1997; 178-187.
12. Parr, A. and Bolwell, G.P. Phenols in the plant and in man: The potential for possible nutritional enhancement
of the diet by modifying the phenols content or profile. J.sci. Food Agric., 2008; 80: 985-1012.
13. Jaleel, CA., et al. Antioxidant potentials and ajmalicine accumulation in catharathusroseus after treatment
with giberellic acid. Colloids and surfaces B:Biointerfaces, 2007; 60: (2), 195-200
14. Pourmorad, F. Antioxidant activity, Phenol and Flavonoid contents of some selected Iranian medicinal plants.
African journal of biotechnology, 2009; 5 (11), 1142-1145.
15. Harborne JB(1973). Phytochemical methods: A guide to modern techniques of plant analysis. Chapman and
Hall, New York, pp. 279, 3rd Edition.
16. Sofowora A. Medicinal plants and Traditional medicinal in Africa. 2nd edition. Sunshine house, Ibadan,
Nigeria: Spectrum books Ltd; 1993. Screening plants for bioactive agents; pp. 134 156.
17. Hashimoto A, Kameoka T. Applications of infrared spec-troscopy to biochemical, food, and agricultural
processes. Applied Spectroscopy Reviews. 2008; 43:41651.
18 Hussain K, Ismail Z, Sadikun A, Ibrahim P. Evaluation of Metabolic Changes in Fruit of Piper armentosum
in vari-ous Seasons by Metabolomics using Fourier Transform Infrared (FTIR) Spectroscopy. International
Journal of Pharmaceutical and Clinical Research. 2009; 1(2):6871.
19. Srinivasan PA, Arokiaraj A, Vijayarajan D. Isolation, spec-tral and optical analysis of fistulic acid A
phytochemical constituent of Cassia fistula Linn. Indian Journal of Science and Technology. 2011; 4(4):422
4.
20. Omodara NB, Amoko JS, Obijole OA, Ojo BM. Infrared and ultraviolet spectroscopic analysis of methanol
extract of Phyllanthus muellerianus root . Greener J. physical Sci.2013;3(4):159-164
76
Biodegradation of Bisphenol A by Bacillus sp. PK4
Gowthami, M., Rajeswari, M., Bhuvaneswari, V., Shobana, A and Anitha subash
Department of Biochemistry, Biotechnology and Bioinformaics
Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore 641043
Abstract
Bisphenol A (BPA) is known to be an endocrine-disrupting compound that has to be removed from drinking
water due to its estrogenic activity and toxicity to human bodies. However, conventional processes for drinking
water treatment may be of inefficiency for BPA elimination. Microbial communities have the most powerful
biodegradative potential. The present study was carried out to assess the potential of Bacillus sp. PK4 isolated
from paper mill effluent to degrade the BPA. The isolated Bacillus sp. PK4 was found to tolerate 5mg/L of BPA,
when the concentration was increased from 5 to 25 mg/L the growth of bacteria was inhibited. The addition of
glucose increased the growth of the bacteria however; it decreased the rate of degradation of bisphenol A. Both
the intracellular and extracellular laccase activity was found to be highest at 72 h whereas the extracellular
manganese peroxidase activity reached maximum at 48 hour. HPTLC analysis also revealed that bisphenol A was
degraded by bacteria rather than bioaccumulation.
INTRODUCTION
Bisphenol A (BPA) is an organic compound which is used as primary raw material for the production of
polycarbonates and epoxy resins (Hang et al., 2012). Polycarbonate plastics and epoxy resins are used in household
appliances and food storage containers respectively (Muncke et al., 2009 and Sultania et al.., 2011). Because of
the wide usage, BPA became a ubiquitous pollutant and it was identified as an Endocrine Disrupting Chemical
(EDC) by US Environmental Protection Agency (Mohapatra et al., 2010). BPA also has the potential to bioaccumulate
in fat and muscle cell and it is hazardous to animals even at low concentration (Lindholst et al., 2001).
The degradation of BPA has been extensively studied. Many treatment methods have been used which
includes photodegradation, oxidation and photoelectric oxidation (Zhang et al., 2013). However these technologies
present several important disadvantage since they are highly costly and lead to toxic residues (Michalowicz, 2012).
Many BPA degrading bacteria have been isolated from river water, sea water, rhizosphere and activated
sludge (Kang et al., 2006 and Zhang et al., 2013). Recently enzymatic methods have gained considerable attention
for the treatment of BPA and other EDCs. Numerous types of oxidoreductase: laccases, tyrosinases, manganese
peroxidase, lignin peroxidase and polyphenol oxidase have exhibited their potential to degrade EDC (Husain and
Qayyam, 2012).
Among these oxidative enzymes, Laccases have attracted considerable interest in many fields of industry
and environmental processes due to their broad substrate specificity and ability to oxidize high redox potential
substrates in the presence of certain low molecular weight compounds called as mediators (Diwaniyan et al.,
2012). Many authors also reported that laccase activity was enhanced during the degradation of 4n-nonlyphenol
(Moujin et al., 2002), BPA and diclofenac (Yang etal.,2013 and Nguyen etal., 2014).
In this paper, the potential of laccase producing bacteria Bacillus sp. PK4 isolated from paper mill effluent
in the degradation of BPA was studied.
MATERIALS AND METHOD

Source of the bacteria
The Bacillus sp. PK4 used for the present study was isolated from the paper mill effluent and maintained
in the Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home Science
77
and Higher Education for Women, Coimbatore. The strain was sub cultured in Luria Bertani (LB) agar and maintained
at 4 C.
Preparation of the inoculum

The loop full of Bacillus PK4 strain was inoculated into 25 ml Luria Bertani (LB) Broth and incubated at 37
C for overnight by agitation at 100 rpm. From this overnight grown culture, 10% inoculum was inoculated into
the production medium.
Preparation of basal medium
The basal media (g/L) used for the enzyme production had the following composition: Yeast extract - 6.0
g, (NH4)2 SO4 - 0.1, Mg SO4 0.1, CaCo3 0.02, CuSO4 0.001 and pH 7.0. The basal media was inoculated
with overnight grown culture (OD 600 nm~ 0.5 to 0.7) and incubated at 37 C with 100 rpm. Under each experiment
set up the growth of the bacterial culture was estimated in terms of biomass by measuring the optical density at 600
nm and the residual BPA was determined using High Performance Liquid Chromatography (HPLC). The peak area
was used to calculate the remaining amount of bisphenol A as a percentage of the initial value.
High Performance liquid chromatography
The residual non degraded BPA was quantitatively estimated in 20 l of culture supernatant taken at
different interval and filtered with 0.45m syringe filter. The residual BPA was quantitatively estimated using (LC-
6AD Schimadzu liquid chromatography) HPLC. HPLC analysis was performed on water symmetric reverse phase
C18 column, dimension (2504.6mm,5 microns) using isocratic solvent system with 85% methanol and 15%
water at the flow rate of 0.8ml/minute for 10 minutes (Saito et al., 2004).
Effect of different concentrations of Bisphenol A
Bacillus sp. PK4 was grown in different concentrations of BPA ranging from 5 to 25 mg /L as sole carbon
source. This experiment was performed in order to determine the optical concentration of BPA for the growth and
degradation by Bacillus PK4. The bacterial growth and residual BPA was analyzed at different time intervals namely
24 and 48 hours.
Effect of additional co-carbon source
In order to study the effect of additional co-carbon sources on BPA degradation by the
Bacillus sp. PK4, glucose was added at the concentration of 1% to the basal media. The basal media without
carbon source served as control. The bacterial growth and residual BPA was analyzed over the time interval of 0 -
96 hours.
Role of enzymes in Bisphenol A degradation

Manganese peroxidase and laccase are the two main groups of lignolytic enzymes which were reported to
play a major role in the degradation of BPA (Lee et al., 2005). Therefore in the present study, laccase and manganese
peroxidase were estimated in both extracellular and intracellular enzyme extract.
Extraction of extracellular enzyme
The culture medium was centrifuged at 10000 x g for 15 min at 4C after fermentation at different
experimental conditions described below and the supernatant was used as the crude enzyme extract.
Extraction of intracellular enzyme
The cells obtained as pellet were washed twice with 100mM phosphate buffer, pH 7.0. It was preserved
in ice and resuspended in 0.5 ml of phosphate buffer. The protein extraction was carried out by sonication for 5
78
min at 40 % amplitude and 0.7 seconds / cycle with the sonicator. Then it was centrifuged at 13,000 rpm for 20
minutes at 4C to pull down the cell fragment and the supernatant was taken carefully in the cool tube. The
supernatant obtained was used as intracellular enzyme.
Laccase activity assay

Laccase activity was performed at room temperature for 10 minutes in 100 mM phosphate buffer using
2,6 dimethoxy phenol as a substrate. The increase in absorbance was monitored at 470 nm in UV visible
spectrophotometric (5= 14,800 M-1 cm -1). Laccase activity was expressed as U/ml where one enzyme unit is
M of product formed per minute.
Manganese peroxidase assay

Manganese peroxidase activity was measured spectrophotometrically in the 2 ml reaction mixture containing
10mM MnSO4, sodium tartarate buffer at pH 6.4 and the enzyme solution in a final volume of 200l. The reaction
was started by adding hydrogen peroxide at final concentration of 0.4 mM and increase in absorbance was monitored
at 270nm (Wariishi et al., 1992).

Effect of different concentration of BPA
The tolerance of Bacillus PK4 strain for the BPA up to 24hours and 48 hours was studied using the various
concentrations of BPA ranging from 5mg to 25mg /L. The basal media without BPA served as control. The results
obtained are shown in Figure 1.
Figure 1 : Growth of Bacillus sp. PK4 at different concentrations of BPA

It is evident from Figure 1, that the BPA concentration at 5mg /L had no inhibitory effect on the growth of
bacteria when compared with control. However, the BPA concentration of 10mg/L, 15mg /L, 20mg /L and 25mg
/L had inhibited the growth bacteria. Hence the strain
Bacillus PK4 was found to tolerate the toxicity of BPA at the concentration of 5mg /L upto 48 hours.
Ji et al. (2014) reported that algae C.mexicana tolerated the BPA concentration of 25 mg/L. Kamaraj et al.
(2012) stated that Aspergillus niger isolated from tannery effluent showed the highest removal of 77% in 20 ppm
BPA concentration.
The residual concentration of BPA in the media was quantitatively determined using HPLC and the results
are shown in Figure 2. The residual BPA was determined using HPLC as percent of the initial value.
79
Figure 2 : Percentage of residual BPA after bacterial degradation at different time interval
From figure 2, it can be confirmed that only 77% of BPA was remaining after 24 hours and 52% after 48
hour for 5mg/ L concentration of BPA. In case of 10mg/ L, 69% of BPA was present after 48 hours of biodegradation.
In case of 15-25mg/ L about 90% of BPA was present in the medium after 24 hours. The effect of different
concentrations of BPA revealed that optimal concentration of BPA for the degradation study for the Bacillus PK4 is
less than 5mg/L. Hence for the further studies 5mg/ L BPA concentration was used.
Zhang et al. (2007) reported that the degradation of BPA by the isolated species was completed on 3rd day
at the initial concentration of 3mg/L. Soominlee et al. (2005) affirmed that both fungi H- insulare and S.hirsutum
removed 68 to 77% of BPA respectively after 3 days. Moreover after 7 day only trace amount of BPA remained in
the liquid culture and there was no residual BPA at after 14 days.
Effect of additional co carbon source

The effect of additional co carbon source glucose on the growth and BPA degradation was studied. The
effects of additional carbon source glucose (1%) on the growth of the bacteria with 5mg/ L BPA were evaluated
after 24, 48, 72 and 96 hours.
Figure 3 : Growth of Bacillus sp. PK4 in presence of additional co carbon source glucose
From Figure 3 it can be concluded that addition of glucose increased the growth of bacteria compared to the
80
medium containing only BPA as sole carbon source at all time point. The residual BPA in the media with and without
co carbon glucose was quantitatively analyzed using HPLC and it was given in Figure 4.
Figure 4 : Percentage of residual BPA in presence of additional co carbon source glucose
The Figure 4 protrayed that in presence of glucose as co carbon the degradation of BPA by bacteria was gradually
decreased from 0th minute to 96 hours and 41% of residual BPA was remaining in the media at the end of 96 hours.
In case of media without additional carbon source 95% of BPA was degraded within 96 hours. These results
demonstrated that BPA was used as carbon source by the bacteria when there was no glucose in the medium. Our
results are in par with the Fischer et al. (2010) reported that C.basilensis JF1 degraded the BPA efficiently when it
was used as sole carbon source.
Role of enzymes in biodegradation of BPA
Laccase activity assay:
In order to understand the role of enzyme in the degradation of BPA by Bacillus PK4, laccase activity was
determined every 24 hour for a period of 4 days at 5mg/ L of BPA as sole carbon source and the results as given in
Figure 5.
Figure 5 : Laccase Activity at Different Time Interval
From the figure it is evident that both extracellular and intracellular laccase activity were found to increase gradually
up to 72 hours and decreased at the end of 96 hours. At the end of 96 hours there was 100% degradation of BPA.
This might be the reason for the decrease in both intracellular and extracellular activity. The extracellular activity
81
and intracellular activity at 72 h were found to be 0.1 U/ml and 0.18 U/ml respectively. These results further
confirmed the role of laccase in BPA degradation. Sarto et al.(2004) reported that laccase from fungus chaetomiaccae
oxidized the BPA and nonyphenol efficiently within 24 hours.
Manganese peroxidase assay

Intracellular and extracellular manganese peroxidase activity at different time interval were calculated and
the results obtained are presented in Figure 6.
From figure 6, it is evident that extracellular manganese peroxidase activity was higher at 48 h (0.16 U/ml)
and 0.09 U/ml of intracellular laccase activity at 72 h. The enzyme activity in both intracellular and extracellular
declined at 96 h. Camarero (1999) stated that manganese peroxidase plays a major role in the degradation of BPA
by Pleurotus eryngii. Hirano et al. (2000) also reported that manganese peroxidase plays a major in the degradation
of BPA by Pleurotous ostreatus.
HPLC analysis of residual BPA

The residual concentration of BPA at different time interval 0th minute, 48 h and 96 h were performed and
overlapped chromatographic profile is shown in Figure 7.
Figure 7 : High Performance Liquid Chromatogram of Residual BPA at different time interval
Green color line 0th minute, blue color line 48 h and pink color line 96 h
82
Figure 7 further confirmed the degradation of bisphenol A by Bacillus PK4 as the peak area at the retention time of
3.4 minutes is drastically reduced at 48 h and 96 h. Jadhav et al., (2010) reported as the decolorization progressed
the emergence of additional peak were observed duo to degradation of parent dye with two minor peaks at retention
time 1.458, 1.746 min and two major peaks at retention time 2.288 min, 2.491 min.
HPTLC analysis of degraded product:

The degraded product of BPA was analyzed qualitatively using HPTLC. The HPTLC Chromatogram is
shown in Figure 8 .
Figure 8 : High Performance Thin Layer Chromatogram of BPA degradation by Bacillus sp. PK4
Track 1 to 3 - Standard BPA

Track 4 - Culture without BPA
Track 5 - Culture with BPA at 24 h
Track 6 - Culture with BPA at 96 h
From the chromatogram (figure 8), it is evident that there is complete absence of BPA at the end of
incubation of 96 h compared to 24 h. This further indicated the degradation of BPA by Bacillus PK4. Hirano et al.,
(2001) affirmed that degraded products of BPA as phenol, 4- isopropenyl phenol, 4- isopropyl phenol and hexestrol.
Laccase catalyse the oxidation of phenolic substance to form phenoxy radicals which initates the formation of
polymers (Duran and Eposito, 2000). Cabana et al. (2007) reported that BPA oxidized by laccase extract of
Coriolopsis polyzona also initiates the formation of polymerization. Uchida et al. (2001) Identified the polymerized
product of BPA by T. villosa laccase as 5,50-bis[1-(4-hydroxy-phenyl)-1-methyl-ethyl]-biphenyl-2,20-diol
CONCLUSION
The Bacillus sp. PK4 isolated from paper mill effluent has the potential to degrade bisphenol A and tolerate
upto 5 mg/L of bisphenol A.
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84
Behaviour Of Penicilliumchrysogenum As Biocontrol Agent Against
Aflatoxigenic Aspergillusparasiticus 2797
Ramya, K and Suganthi, B
Department of Biochemistry, Biotechnology and Bioinformaics
e-mail: ramya_ek@yahoo.com
Abstract
The capacity of P. chrysogenum to inhibit the aflatoxin production in synthetic media and in corn kernels was
ascertained during co-culture with Aspergillusparasiticus MTCC 2797. A reduction in radial growth of A.
parasiticus was evidenced in all the four types of media namely CYA, PDA, RBCA and SDA, with the highest
reduction percentage of 80% in CYAmedium. Reductions of 97% in aflatoxin levels were observed in simultaneous
co-culture with lowering of pH and disturbances in spore development. The mycelial dry weight also showed
reductions in the co-culture. A 95% reduction in aflatoxin production was obtained when the co-culture was
carried out in corn kernels. Pronounced reductions were not evidenced during delayed culture which indicates
that, P. chrysogenum does not have the capacity to degrade the preformed toxin of Aspergillusparasiticus
MTCC 2797. The reduced levels of aflatoxin in simultaneous co-culture are indicative of the potential of
Penicilliumchrysogenum to be further developed and used as a biocontrol agent.
Keywords : PenicilliumchrysogenumAspergillusparasiticus, aflatoxin, biocontrol, co-culture
INTRODUCTION
Aflatoxins are the most important mycotoxins and are produced predominantly by two fungi,
Aspergillusflavus and Aspergillusparasiticus (Hontanaya et al, 2015). Aflatoxin group comprises of aflatoxin B1,
B2, G1 an G2 and Aspergillusparasiticus produces all the four types (Grace et a l, 2015). Aflatoxins are polyketide
derivatives and are considered the most carcinogenic, mutagenic and teratogenic substances found naturally in
foods and feeds. Invasion of these fungi and resulting contamination with aflatoxins often makes the commodities
unfit for consumption (Kouchesfahaniet a l, 2015). Biological control is a promising approach for reducing both
pre-harvest and post-harvest aflatoxin contamination (Zanon et al, 2013). This involves the biocontrol strain to
effectivelycompete with aflatoxigenic strains under conditions favourable for aflatoxin contamination. Gluconic
acid accounts for 99% inhibition of aflatoxin production by Aspergillusflavus when cultured along with
Aspergillusniger (Shantha et al, 1990).Further, Penicilliumchrysogenum has a greater ability to synthesize gluconic
acid than other Penicillium strains. Moreover, gluconic acid producing bacteria or fungi have not been reported to
be biocontrol strains (Wang et al, 2014). The present study was performed to assess the behavior of
Penicilliumchrysogenum as a possible biocontrol agent against the aflatoxigenic Aspergillusparasiticus MTCC 2797.

The study was carried out in three phases. The first phase comprised of isolation of P. chrysogenum from
infected corn kernels (COH 1 variety), collection of A. parasiticusMTCC2797 and discerning the radial growth
reduction of the fungi in various solid media. The solid media used were Rose Bengal Chloramphenicol agar
(RBCA), Potato Dextrose agar (PDA), Saborauds Dextrose agar (SDA) and Czapek Yeast extract agar (CYA).
The second phase was performed to ascertain the reduction levels in aflatoxin production during simultaneous
and delayed co-cultures and to establish a relation between organic acid production of P. chrysogenum with aflatoxin
production levels. The simultaneous co-culture was done by inoculating equal amount of spores of both fungi in
85
Czapek Yeast extract broth and determining the aflatoxin concentration, pH of the medium and mycelial dry
weight at each 24 hr interval of the 7 day culture period. These were compared with control cultures of A.
parasiticus. The delayed co-culture was performed to elucidate if the P.chrysogenum was able to degrade the
preformed toxin. This involved inoculating spores of A. parasiticus first, followed by the inoculation of equal
amount of spores ofP.chrysogenum after 24 hr. This was done for 48h, 72h and 96h delayed cultures also, where
the inoculation of P. chrysogenum was done after 48h, 72h and 96h grown culture of A.parasiticus respectively.
The flasks were incubated for a further period of 96h. The third phase was carried to determine the results of co-
culture in a biological substrate. Corn kernels of the hybrid variety (COH 1) were mixed with the spores of both P.
chrysogenum and A. parasiticus and the toxin levels were monitored at every alternate day during a 14 day culture
period.
RESULTS
A reduction in radial growth of A. parasiticus was evidenced in all the four types of media namely CYA,
PDA, RBCA and SDA, with the highest reduction percentage of 80% in CYA medium. The percentage of aflatoxin
reduction in the medium was seen to reach a maximum of 97.3% on the seventh day of the co-culture (Table 1)
.pH measurements recorded during the simultaneous co-culture indicated a gradual decrease to 4.0 from an initial
pH value of 6.2 (Table 1). The morphology of the spores from co-cultured media appeared to be shrunken and dark
when compared to a 7 day old control A.parasiticus culture. The mycelial growth also showed a pronounced
reduction by 61.1% during the 7th day. The delayed co-culture showed reductions in aflatoxin levels by 9.6%
which were not as conspicuous as that in a simultaneous culture (Table 2). The percentage reductions in pH in
delayed co-culture were not marked when compared to those in simultaneous culture. Simultaneous co-culture in
corn kernels showed 95% reductions in aflatoxin levels.
DISCUSSSION
The hyphae of P.chrysogenum and A. parasiticus did not intermingle with each other, suggesting a competition
for resources. Both the fungi were found to be preventing each other from entering their zones.Thus, it can be
suggested that the type of hyphal interaction involved might be a direct interaction due to some secondary metabolite
produced by P. chrysogenum. The reduction in aflatoxin production might be due to lower metabolic rates, thus
providing meager opportunity for A. parasiticus to prepare for secondary metabolism. The decrease in pH was due
to the production of organic acids by P. chrysogenum and was found to be directly related to the decrease in
aflatoxin production levels. Further, sporulation has been disturbed in A. parasiticus MTCC2797 due to a decline in
aflatoxin production. The decrease in mycelial dry weights can also be associated directly with decreased levels of
aflatoxin in simultaneous co-culture. P. chrysogenum does not have the capacity to degrade the preformed toxin but
the reduced levels of toxin in simultaneous culture are indicative of the capacity to inhibit aflatoxin synthesis.
Table: 1. Variations in aflatoxin production and pH in simultaneous co-culture
Aflatoxin
Day of co-culture production levels (mg/ 100ml) pH variations
Control Co-culture % reduction Control Co-culture % reductions
1 10 10 0 7.0 6.2 11
2 80 60 25 6.9 5.8 15
3 220 150 31 6.8 5.4 20
4 380 200 47 6.7 5.2 22
5 520 170 67 6.5 5.0 23
6 640 80 87 6.3 4.6 26
7 760 20 97 6.0 4.0 33
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Table: 2. Variations in aflatoxin production and pH in delayed co-culture
Aflatoxin
Day of co-culture production levels (mg/ 100ml) pH variations
Control Co-culture % reduction Control Co-culture % reductions
0 10 10 0 7.0 6.2 11
24 80 60 25 6.9 5.9 14
48 220 150 31 6.8 5.7 16
72 380 250 34 6.7 5.6 16
96 520 470 9 6.5 5.6 13
REFERENCES
Hontanaya, C., Meca, G., Luciano, F. B. and Font, G. (2015), Inhibition of aflatoxin B1, B2 production by A.
parasiticus in nuts using yellow and oriental mustard flours. Food Control, 47: 154-160.
Kouchesfahani, M. M., Alimohamadi , M., Khaniki.J., Nodehi, N. R. and Rezai. (2015), Antifungal effects
of ozonated water on A. parasiticus. Journal of Food Safety, 35: 295- 302.
Wang, K., Yan, P and Cao, L. (2014), Chitinase from a novel strain of SerratiamarcescensJPP1 for Biocontrol
of Aflatoxin, Biomed Research International, 8 pg
Zanon, M. S.A., Chiotta, M. I. and Chulze,S. (2013), Evaluation of potential biocontrol agent for aflatoxin in
/Argentinian peanuts. Intl Jnl of Food Microbiology, 162: 220-225.
Grace, D., Mahuku, G., Hoffmann, V. and Atherstone. (2015), International Agricultural Research to reduce
food risks: case studies on aflatoxins, 7: 569-582.
87
Influence of geographical location of plant growth on its metabolite
accumulation: A comparative quantification of secondary metabolites in
invitro and invivo root samples of Withania
Sangeetha U, Kalaiselvi Senthil
Associate Professor, Department of Biochemistry, Biotechnology and Bioinformatics,
Email : aubio.ptc.kalai@gmail.com
Abstract
Withania is a principal medicinal plant that has been used in Ayurvedic and native medicine. In view of its varied
therapeutic prospective, it has also been the subject of considerable modern scientific attention. Its roots are an
integral of over 200 formulations in Ayurveda, Siddha and Unani medicine, which are used in the treatment of
various physiological disorders. The present study aims for quantification of withanolides and to analyze
phytochemical composition variance in Withania roots collected from different geographical areas. Field roots
of Withania coagulans and Withania somnifera were collected from different geographical locations and analyzed
for its withanolide accumulation in comparison to in vitro roots using HPTLC. Also other phytoconstituents
were quantified using phytochemical screening. It was found out that there was a great variation in metabolite
accumulation among the in vivo roots of Withania from different locations. HPTLC analysis revealed that the
banding patterns of in vitro adventitious roots of Withania coagulans were similar to the field grown roots. It
revealed that the in vitro conditions are favorable for the accumulation of withanolides as they are not diversely
affected by external factors.
Keywords : Withania somnifera, Withania coagulans, Secondary metabolites, HPTLC.
INTRODUCTION
Phytomedicines are dietary supplements in the form of powders, extract, fresh or dried plants and are
usually taken to improve health conditions and for well being. These herbal phytomedicines are considered to be
harmless and are increasingly consumed by people. The profiling of these traditionally used medicinal plants should
be accomplished in order to analyze the quality and quantity of phytocompounds embodied in them. It is the
efficacy and safety of these herbal medicines that has turned the major pharmaceutical population towards medicinal
plants research [1]. There are groups of bioactive chemicals called secondary metabolites in fruits, vegetables and
herbs, and each of these compounds work in different ways [2]. Changes in the environmental factors produce a
new and sometimes unexpected secondary metabolic profile resulting in their accumulation variance [3]. Hence the
production of these secondary metabolites is usually higher in in vitro tissue culture compared to that of wild
variety [4]. For this it is necessary to evaluate various qualitative and quantitative parameters, which may be helpful
in setting standards for particular medicinal plant/parts of the plant. With the help of these standards one can easily
identify and characterize single compounds, which may play a major role in maintaining quality and purity of that
particular drug [5].
The genera Withania plays an extensive role in the indigenous medicine of South East Asia. The twenty-
three known Withania species are widely distributed in the drier parts of tropical and subtropical zones [6]. Among
them, only two (Withania somnifera (L.) Dunal and Withania coagulans Dunal) are economically significant and
widely cultivated [7]. Although Withania have been extensively investigated yielding large number of steroidal
structures, withanolide A is one of the most important one among them that contributes to the therapeutic potential
88
of the plant [8]. In the present study, a sensitive, simple and accurate High Performance Thin Layer Chromatographic
(HPTLC) method has been established in order to analyze and quantify the withanolide accumulation variance
among the root samples of Withania coagulans from different locations and its comparison with the in vitro
cultivated adventitious roots.

Chemicals and Equipments
HIMEDIA chemicals and Elix-3 water were used for the entire study. HPTLC was performed on precoated
Silica gel aluminum 60F254 plates (E.MERCK, Germany) in a Semiautomatic CAMAG Linomat 5 device.
Spectrophotometer and colorimeter was used in the quantitative phytochemical analysis to measure the absorbance
of the samples.
Plant material
The invivo dried root samples of 21 isolates of Withania coagulans and 2 cultivars of Withania somnifera
from Iran, a Gujarat variety of Withania somnifera and in vitro root samples of both W.coagulans and W. somnifera
were used for assays.
Determination of Total Ash Value:

About 2 grams of air dried, powdered roots were accurately weighed and taken in a tarred silica crucible
and incinerated in a muffle furnace by gradually increasing the temperature to 4500 C to make it dull red hot and
free from carbon. Cooled in a desiccator, weighed and the percentage of total ash was calculated [9].
Preparation of extracts from roots

The dried root samples were ground to powders. One gram of each root sample was extracted using
200ml of Ethyl Acetate. Initially 1g of root was weighed and incubated at room temperature with 1ml of ammonia
for 20mins followed by sonication for 20 min with 50 ml of the solvent and placed in a shaker for 2 hours at 104
rpm at 220C. At the end of 2hrs the extract was filtered using Whatmann no: 1 filter paper and the residue were
again treated with 50 ml of the solvent. This step was repeated four times to obtain 200ml of the extract. using
Ethyl Acetate. These extracts were then concentrated by using a flash evaporator maintained at 450 C and 150rpm.
After complete solvent evaporation, the residue was dissolved using known volume of HPLC grade methanol [10].
Determination of extractive value: Extractive value was observed using ethyl acetate as a solvent in all the 23 root
samples. The extracts were obtained from 1g of root samples, and were completely evaporated to dryness in water
bath. The extract was then weighed by following the estimation procedure by Baccou et al.(1977) [11]. and the
percentage of compound extracted was calculated [9].
Quantitative estimation of selected phytochemicals

Estimation of total Carbohydrates: The procedure of Hedge and Hofreiter (1962) [12] was followed for the
estimation of total carbohydrates present in 1g of collected root samples.
Estimation of Proteins: The procedure of Lowry et al., (1951) [13] was followed for proteins present in 1g of
root extract samples. The optical density read at 660nm gave the protein content of the samples.
Estimation of Flavonoids: The procedure by Cameron et al. (1943) [14] was followed for estimation of flavonoids
in the root samples.
Estimation of Steroids: A modified procedure of Wall et al., (1952) [15] was followed for the estimation of
steroids in1g of root, where the green color developed was observed at 640nm and the total Steroids present in1 g
of the sample was calculated.
89
Estimation of Saponins: The amount of saponins present in 1g of roots was calculated by following the estimation
procedure by Baccou et al.(1977) [11].
Method development for HPTLC

The High Performance Thin Layer Chromatography analysis was carried out on 20x10cm precoated silica
gel aluminum plate 60F254 (E.MERCK, Germany). The sample extracts were applied to the plates as 6mm bands,
under a stream of nitrogen, by means of a CAMAG (Switzerland) Linomat V semiautomatic sample applicator
fitted with a 100l HPTLC Hamilton syringe. Linear ascending development to a distance of 8cm was carried out
in 20x10cm twin trough chamber saturated for 30mins at room temperature with 20ml mobile phase. The banding
patterns were visualized in 254nm, 366nm and white light and Densitometric scanning was performed with Camag
TLC scanner III in the reflectance absorbance mode at 540 nm after derivitization with 10% Sulphuric acid and
analysed by Win CATS software (1.3.0 Camag) [16].
The significance of the quantitative estimations were obtained by ANOVA using excel and cluster analysis
using NT sys software.
RESULTS
Quantitative analysis of phytochemicals
The Quantitative estimation of all field grown roots and in vitro root sample of Withania coagulans and
Withania somnifera were carried out. It is found that there is a major difference in accumulation of phytochemicals
among the samples(Table.1). From the analysis of AUF Wc 158 variety of Withania coagulans, it can be interpreted
that this plant might have grown in a geographical condition that has suppressed the production of proteins,
carbohydrates, saponins and flavonoids but has opposite effect on steroidal accumulation. Its also found that the
protein content of all the samples was higher compared to the carbohydrate and most of the samples except a few
had saponins content higher than that of protein. Among the five phytoconstituents studied, the carbohydrate
content was found to be the least in all the samples. The quantified values of the above phytoconstituents can be
used as a major tool for obtaining a quality control profile for a drug.
Plate 1: HPTLC fingerprint showing withanolide accumulation in field grown and in vitro roots of Withania
coagulans and Withania somnifera
90
Table 1: Quantitative analysis of phyto chemicals in field grown and in vitro roots of Withania coagulans and Withania somnifera
91
Table 2. Amount of Withanolide A field grown and in vitro roots of Withania coagulans and
S.No SAMPLE Rf WITHANOLIDE A(mg/g)
1. AUF Wc 001 0.68 0.27

2. AUF Wc 002 0.66 0.59
3. AUF Wc 003 0.66 0.83
4. AUF Wc 004 0.66 0.73
5. AUF Wc 005 0.66 0.46
6. AUF Wc 006 0.66 0.28
7. AUF Wc 007 0.68 0.73
8. AUF Wc 008 0.68 0.40
9. AUF Wc 010 0.67 0.65
10. AUF Wc 018 0.67 0.46
11. AUF Wc 019 0.67 0.92
12. AUF Wc 021 0.68 0.56
13. AUF Wc 022 0.69 0.91
14. AUF Wc 023 0.68 1.12
15. AUF Wc 024 0.68 1.17
16. AUF Wc 025 0.69 1.17
17. AUL Wc 0.69 0.40
18. AUF Wc 026 0.69 0.62
19. AUF Ws 025 0.66 1.30
20. AUF Ws 157 0.66 0.27
21. AUF Ws guj 0.65 0.37
22. AUL Ws 0.68 0.58
HPTLC fingerprinting of in vitro and field grown Withania root extracts

HPTLC fingerprint of the Ethyl acetate extracts of invitro and invivo roots of Withania coagulans and
Withania somnifera from different geographical locations of Iran was performed using mobile phase Toluene:
Ethyl acetate: Formic acid (5:5:1) to see the accumulation of various phytoconstituents. Standard Withanolide A
(0.1mg/ml) was spotted in varying volumes of 2l, 6l, 10l, 12l in order to quantitatively estimate the amount of
Withanolide A present in all the root samples. The HPTLC analysis was carried out with all the 26 samples. Among
all the samples analyzed the in vitro sample of Withania coagulans was found to have large number of spots
indicating a higher number of phytochemicals. All the field grown root samples gave nearly the same type of
banding pattern with the sample AUF Wc 010 showing more number of spots followed by AUF Wc 019(Plate 1).
The developed plates were then subjected to Densitometric scanning with Camag TLC scanner III using Savitsky-
Golay7 filter in the reflectance absorbance mode at 234 nm at the speed of 200mm/s, the D2 and W lamp was
chosen to scan the plates and the slit dimensions were set at 4.00 x 0.30 mm, Micro. All the tracks were scanned
92
and the peaks were displayed. The Withanolide A peak was viewed as a separate spot with Rf values around 0.65-
0.69. The peak analysis revealed increase in concentration of Withanolide along increase in volume. The amount of
Withanolide A in each of the samples was quantified comparing with the standards using peak area as an evaluation
mode at multilevel calibration. A linear regression graph was obtained using CAMAG software. The amount of
withanolide A varied considerably between the different root samples and is presented in Table 2. The Withanolide
A concentration in root samples ranged from 0.27 mg/g (AUF Wc 001 and AUF Ws 157) to 1.30 mg/g (AUF Ws
025). The variations in the Withanolide A concentration can be attributed to the growth condition which is influenced
by the geographical location in which it is grown. In addition to Withanolide A, four compounds with Rf 0.01, 0.40,
0.76 and 0.85 were found in all the samples including the in vitro samples. The in vitro samples of Withania
somnifera and Withania coagulans were found to have a same pattern of banding except that three compounds
with Rf 0.02, 1.07 and 1.18 present in Withania coagulans were absent in Withania somnifera indicating the
presence of additional compounds in Withania coagulans than Withania somnifera which is an established medicinal
plant. Among all the samples analyzed, the in vitro sample of Withania coagulans was found to have larger number
of spots indicating an increased number of phytocompound accumulations. To conclude, as observed, there is a
wide variation with the phytochemical contents of field grown roots collected from different locations, and hence
its quantification would be a useful tool for selecting the best source of the phytoconstituents for drug development
and also prevent contamination with other plant samples.
DISCUSSION
The secondary metabolites show a great variation among the samples. These variations among different
plant samples of the same species are due to the influence of the geographical locations on the metabolism.
Secondary metabolism is not a static process. Rather, it changes in response to numerous factors [17]. The effect
of modified external factors such as temperature and light intensity, and plant internal factors such as phenological
phase and their possible interactions was found to influence bioactive secondary metabolite accumulations in plants
[18]. Thus the concentrations of various plant secondary products are strongly dependent on the growing conditions
[19].
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anti-atherosclerotic agent. Drug Discov Ther 2010; 4: 334-340.
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grandiflorum leaves. Int J Pharm Frontier Res 2011; 1, 80-92.
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somnifera Root, and a Method for Quantitative Analysis of Withaferin A in Young and Old Roots. J Planar
Chromatogr 2009; 22: 283286.
11. Baccou JC, Lambert F, Sauvaire Y. Spectrophotometric method for the determination of total steroidal
sapogenin. Analyst 1977; 102: 458465.
12. Hedge JE, Hofreiter BT. In: Carbohydrate Chemistry (Eds. Whistler R.L. & Miller, J.N). New York: Academic
Press; 1962. p. 17.
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tissue. Anul Chem 1952; 24: 1337-1341.
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estimation of Betasitosterol D glucoside and Withaferin A. Int J Pharm Pharm Sci 2011; 3: 227-230.
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2007; 68: 29172936.
18. Raduien J, Karpaviien B, Stanius Z. Effect of External and Internal Factors on Secondary Metabolites
Accumulation in St. Johns Worth. Botanica Lithuanica 2013; 18: 101108.
19. Kannan ND, Kulandaivelu G. Drought induced changes in physiological, biochemical and phytochemical
properties of Withania somnifera Dun. J Med Plants Res 2011; 5: 3929-3935.
94
A comparative study of Enzymic Antioxidants in the selected
Beetroot Varieties
1
Raja Hepzi, F and 2Sivagami Srinivasan
1
Research scholar, 2Associate professor in Biochemistry, Department of Biochemistry, Biotechnology and Bioinformatics,
Email : aubio.ptc.kalai@gmail.com
Abstract
Beta vulgaris has long been regarded as a medicinal plant. Beetroot has been found to be beneficial to the blood,
heart and digestive system. Beetroot is rich in many important minerals and micronutrients; it is a nutritious
vegetable with many health-giving properties. Antioxidants are our first line of defences against free radical
damage and are critical for maintaining optimum health and well-being. Antioxidants are capable of stabilizing or
deactivating free radicals before they attack cells. The red pigment found in beets is loaded with antioxidants
that may help to protect the body against heart disease, certain cancers and even birth defects. Detroit dark red
and Ooty varieties of beetroot were analyzed for the enzymic antioxidant activities. The role of antioxidant
activities presents in the beetroot, mainly used for the oxidative stress. The enzymic antioxidants were found to
be high in the detroit dark red variety when compared to the Ooty variety.
Keywords : Beetroot, catalase, superoxide dismutase, peroxidase, GST, GPx, polyphenol oxidase.
INTRODUCTION
The colour of beetroot is an important characteristic quality. This vegetable has a beneficial health effect
against tumour cells and its mineral content is also high. It is also considered as a raw material for the production
of natural food colourant. Beetroot is a good purifier apart from its ability to remove unwanted impurities accumulated
in kidneys and gall-bladder. The iron found in beetroot helps our body to make new blood corpuscles (Kugler et al.,
2007). Beetroot is a good tonic for liver problems. If beetroot leaves are cooked like other greens and consumed,
diseases like ulcer and jaundice will be cured.
A high intake of vegetables and fruits can reduce the risk of developing cancer and other diseases. Beetroot
has unique chemicals (e.g. betalains) and high levels of important micronutrients, which make it a valuable vegetable
to include in the diet as a means of deterring the onset of cancer and other diseases. The betalains, for instance, act
as antioxidants. The antioxidants protect from the potentially damaging oxidative stress, which is a result of an
imbalance between the formation of ROS and the body antioxidant defence. Antioxidants have also been used in
food industry to prevent deterioration, nutritional losses and off-flavouring in various foods, especially those
containing polyunsaturated fatty acids. The antioxidant enzymes like glutathione peroxidase, catalase, and superoxide
dismutase (SOD) metabolize oxidative toxic intermediates and require micronutrient cofactors such as selenium,
iron, copper, zinc, and manganese for optimum catalytic activity.
Detroit dark red beets are considered one of the most outstanding beet varieties. It produced well formed,
almost round, blood-red 8cm diameter, nutritious roots. It grows well in almost all soil types and is heat and cold
tolerant. It contains a number of nutrients including iron, calcium, folic acid and vitamins A, B and C. The Ooty
beetroot is having a beautiful colour and plenty of nutrients. Hence a study was carried out to investigate the
enzymatic antioxidant activities in the above two varieties of the beetroot.

Detroit dark red and Ooty beet root variety were collected in and around Coimbatore, Tamilnadu. The fresh
95
beetroots are collected and air dried in the shade at room temperature for 20 days. In the laboratory, the beetroot
samples were peeled cut into small pieces and 1gm of each sample were homogenized. The enzymatic parameters
such as catalase, superoxide dismutase, peroxidase, glutathione-S-transferase, glutathione peroxidase, glutathione
reductase and polyphenol oxidase were analyzed by standared methods. The catalase activity was determined by
the method of Luck (1974). The superoxide dismutase activity was determination by the method of Mirsa and
Fridovich (1972). Peroxidase activity was determined by the method of Reddy et al. (1995). The glutathione-S-
transferase activity was determined by the method proposed by Habig et al. (1974). The method of Rotruck et al.
(1973) was followed to assess the activity of glutathione peroxidase. The activity of glutathione reductase was
determined by the method of David and Richard (1983). The polyphenol oxidase activity of the plant sample was
estimated by the method of Esterbauer et al. (1977). The statistical analysis like arithmetic mean and critical
difference were employed to predict the results of the experiment.

Reactive oxygen species (ROS) are generated from leakage of electrons onto oxygen from mitochondrial
electron transport chain, microsomal cytochrome P450 and their electron donating enzymes and other systems.
Inactivation and removal of ROS depend on reactions involving the antioxidative defence system. The endogenous
antioxidant defence includes enzymatic anti oxidants (eg. superoxide dismutase, catalase, peroxidase etc,). The
levels of catalase, peroxidase and superoxide dismutase of different beetroot varieties were represented in Figure 1.
Figure 1: Activities of Catalase, Peroxidase and Superoxide dismutase
Catalase is produced naturally in all living organisms. It helps the body to breakdown hydrogen peroxide
into oxygen and water. It prevents the accumulation of carbon dioxide in blood. Among the two varieties of
beetroot, the highest activity of catalase was observed in Detroit dark red variety (0.588U/g). It indicates that the
hydrogen peroxide formed by SOD is efficiently removed by catalase. Plant catalases are reported to be very
sensitive to environmental conditions. A highest activity of catalase was also reported by Kartikeyan and Rani
(2003) in Piper longum.
In plants, peroxidases are involved in many physiological processes, involving responses to biotic and
abiotic stress and biosynthesis of lignin. The peroxidase activity was found to be higher in Ooty variety (0.667U/
g) when compared to the Detroit dark red variety. Hydrogen peroxide was involved in peroxidase mediated oxidative
polymerization, which inturn results in cell wall strengthening, the activation of peroxidase may have a protective
role. Gomaa et al., (2008) has reported that the application of Nitrogen, phosphorus and potassium (NPK) fertilizer
had enhanced the peroxidase activity in wheat.
Superoxide dismutase play a major role in combating oxygen radical mediated toxicity. The highest superoxide
dismutase activity was found in Detroit dark red variety (0.524U/g) when compared to the Ooty variety (0.474 U/
g). Superoxide dismutases were reported widely in plant sources and have free radical scavenging effect. Gaballah
and Gomaa (2005) also reported that the SOD activity was found to be increased in fababean varieties by the
96
application of Rhizobium and sodium. The activity of Glutathione-S-transferase and Glutathione reductase in
beetroot varieties were predicted in Figure 2 and 3 respectively.
Figure 2: Activity of Glutathione-S-transferase Figure 3: Activity of Glutathione reductase
The role of glutathione-S-transferase is to catalyse the conjugation of electrophilic substrates to glutathione.

The Detroit dark red variety was found to be superior with the highest glutathione-S-transferase activity (2.57U/g)
compared to the Ooty variety (1.95U/g). Ali et al., (2005) also reported an increased level of enzymatic antioxidants
like glutathione peroxidase and glutathione-S-transferase in the roots and leaves of Phalaenopsis, which led to the
breakdown of oxidants such as H2O2, organic hydroperoxides and lipid hydroperoxide resulting in greater protection
against oxidative damage. The Ooty variety exhibited a very poor glutathione reductase activity. Again the Detroit
dark red variety was found to be superior registering highest glutathione reductase activity. It is the major endogenous
antioxidant produced by the cells, participating directly in the neutralization of free radicals and reactive oxygen
compounds as well as maintaining exogenous antioxidants such as vitamin C and E in their reduced forms (Amit et
al., 2009). Figure 4 and 5 represents the activities of glutathione peroxidase and polyphenol oxidase in the selected
beetroot varieties.
Figure 4: Activity of glutathione peroxidase Figure 5: Activity of polyphenol oxidase
Glutathione peroxidase protects the organism from oxidative damage. It reduces the lipid hydroperoxides
into corresponding alcohols and reduced free hydrogen peroxide to water. Glutathione peroxidase activity was
found to be significantly (P<0.05) higher in the Detroit dark red (0.88U/g) variety when compared to the Ooty
variety. Glutathione peroxidase is involved in the reduction of H2O2 and organic peroxide. A continuous flow of
reducing equivalents through glutathione system necessarily has to be balanced by continuous formation of NADPH
maintaining the steady state. A study by Karthikeyan and Rani (2003) had shown the highest activity of glutathione
peroxidase in Piper nigrum.
The Polyphenol oxidase activity in the beetroot varieties was found to be very low. Both the beetroot
varieties were shown comparable activities. Polyphenol oxidase or monophenol mono oxygenase is a tetramer that
contains four atoms of copper per molecule and binding sites for two aromatic compounds and oxygen. The
enzyme catalyzes the hydroxylation of monophenols to diphenols. They can also further catalyse the oxidation of
diphenols to produce quinones (Mayer, 2006). Solanum berthaultii has shown high polyphenol oxidase activity
(45% of soluble protein) in glandular trichomes (Kowalski et al., 1992).
97
CONCLUSION
The results revealed that the enzymic antioxidants of beetroot were found to be high in Detroit dark red
variety. The other Ooty variety of beetroot had shown the lowest level of the enzymatic antioxidants. The antioxidants
present in beetroot may wipe out the free radical bodies as well as lower the cholesterol level. Thus the regular
consumption of Detroit dark red variety of beet root may increase the blood supply, which in turn improve the
health conditions and prevent many disorders.
REFERENCES
1. Ali, M.B., Hahn, E.T and Paek, K.Y. (2005). Effect of temperature on oxidative stress defence systems, lipid
peroxidation and lipoxygenase activity in Phalaenopsis, Plant physiol and Biochem. 43, 213-223.
2. Amit, K., Arun, G., Shyam, S and Agrawal, C. (2009). Evaluation of Hepato protective Activity of Aerial
Parts of Tephrosia Purpurea L. and Stem Bark of Tecomella Undulate, Journal of Ethno pharmacology. 122,
1-5.
3. David, M. and Richard, J.S. (1983). Methods of enzymatic analysis 3, Bergmeyyer, J. and Mariare, G.B.
(ed), Verlag chemic Weinheina Dec, field Beach Floride based, 358.
4. Esterbauer, H., Schwarlz, E. and Hayan, M. (1977). A rapid assay for catachol oxidase and laccasee using
nitro-5 thio benzoic acid, Anal. Biochem. 77, 489-494.
5. Gaballah, M.S. and Gomaa, A.M. (2005). Interactive effect of Rhizobium Inoculation, Sodium Benzoate and
salinity on performance and oxidative stress in two Fababean varieties, International Journal of Agriculture
& Biology, 1560:8530, 495-498.
6. Gomaa, A.M., El-Mesiry, T. and Rady, M. (2008). The potential impact of Bio fertilization, Antioxidant and
Micronutrients Application in Reducing Salinity stress on two wheat cultivars, Australian Journal of Basic
and Applied Sciences, 2, 575-582.
7. Habig, W.H., Pabst, M.J. and Jacoby, W.B. (1974). Glutathione-S-transferase. The first enzymatic step in
mercapturic acid formation, J. Bio. Chem, 249, 7310-7339.
8. Karthikeyan, J. and Rani, P. (2003). Enzymatic and nonenzymatic antioxidant in selected Piper species. Ind
J. Exp. Biol., 41,135-140.
9. Kowalski, S.P., Eannetta, N.T., Hirzel, A.T and Steffens, J.C. (1992) Purification and characterization of
polyphenol oxidase from glandular trichomes of Solanum berthaultii. Plant Physiol 100, 677684.
10. Kugler, F., Stintzing, F. and Carle, R. (2007). Evaluation of the antioxidant capacity of betalainic fruits and
vegetables. J. Appl. Bot. Food Qual. 81, 69-76.
11. Luck, H. (1974). Methods of enzymatic analysis, Academic press, 885-894.
12. Mayer, A.M. (2006). Polyphenol oxidases in plants and fungi. Phytochemistry, 67:21, 23182331.
13. Misra, M.P. and Fridovich, I. (1972). The role of superoxide anion in the autoxidation of epinephrine and
simple assay for superoxide dismutase, J. Biol. Chem., 247, 31-70.
14. Reddy, K.P., Subhani, S.M., Khan, P.A. and Kumar, K.B. (1995). Effect of light and benzyladenine and desk
treated growing leaves, changes in the peroxidise activity, Cell physiol, 26, 984.
15. Rotruck, T.T., Ganther, H.E., Swanson, A.B., Hafeman, D.G., and Hoeckstra, W.G. (1973). Selenium:
Biochemical role as a component of glutathione peroxidise Science, 179, 588-590.
98
Identification of Protease - producing Bacteria from
Organic Waste Soil
Sujatha A.*, Anitha Subash, Shobana, A. & Sherin, A.
Department of Biochemistry & Biotechnology
Email : sujamphil@gmail.com
Abstract
Various applications of the enzyme protease in the detergent, tannery, food, pharmaceutical, metal recovery and
waste treatment industries enhance the interest for further studies on the enzyme. The aim of the study was to
identify microorganisms from selected organic waste soil for isolation of protease. The soil samples were
collected locally. Various methods including biochemical and morphological were used for the identification of
microorganisms which could produce protease. From the study, the main bacteria isolated were found to be
those of Pseudomonas sp. From this, five positive colonies were chosen and screened for the production of
cellulase, amylase, lipase and protease. The secreted enzymes were quantitatively assayed to identify the one
with maximum specific activity. Of all the enzymes, protease recorded the maximum specific activity. It was
purified by ammonium sulphate precipitation. Among the various concentrations used (0-20% - 80-100%), the
maximum activity was exhibited by the 80-100% precipitated fraction. Hence, this fraction was further purified
by dialysis. After partial purification by dialysis, the specific activity of the sample was found to be 2.49 U/mg,
the protein content 2.05 mg, the purification fold 3.4 and recovery percentage 2.3. The protocol developed in our
study for the isolation, characterization and media optimization for the production of protease from the isolate is
reproducible for further study and industrial applications.
Keywords : Enzymes, Pseudomonas sp., Ammonium Sulphate, Dialysis
INTRODUCTION
The new trend in biotechnology is the conversion of wastes into useful biomass by enhancing the
microorganism growth and thereby producing large amount of commercially important enzymes from them1.
Enzymes are considered as potential biocatalyst for a large number of reactions particularly, the microbial enzymes
have widespread uses in industries and medicine. The microbial enzymes are also more stable than plant and animal
enzymes2. Recent years have witnessed a phenomenal increase in the use of enzymes as industrial catalysts.
Proteases constitute a very large and complex group of enzymes, widely utilized in a host of industries. They differ
in properties such as substrate specificity, active site and catalytic mechanism, pH and temperature optima, and
stability profiles3. Microorganisms are potentially considered to be the most perfect source of protease for industrial
applications. There are various bacterial species found to be the source of protease, specifically Bacillus and
Psuedomonas4. Enzymes are proteinaceous in character. The manufacture and processing of enzymes is an important
fact of todays pharmaceutical, detergent, tannery, food, metal recovery and waste treatment industries. Hence the
present study was taken up to isolate, identify and purify protease from bacteria isolated from organic waste
(vegetable waste decayed) soil.

Isolation of bacterial strain: Organic waste soil sample was collected from Avinashilingam University, Coimbatore,
Tamil Nadu. The collected soil sample was kept in clean dry polythene bags and stored at 4C. The collected
sample was serially diluted and plated onto the nutrient agar with casein slants and incubated at 37 C for 24 hours
and stored at 4C5.
99
Identification of bacterial strains: The protease producing colonies were morphologically identified by simple
staining and gram staining techniques6 and the selected colonies were characterized by biochemical tests7 according
to the criteria of Bergeys Manual of Systemic Bacteriology8. The bacterial colonies were confirmed by MALDI-
TOF.
Screening for enzymes: The isolated colonies were qualitatively screened to identify the presence of enzymes
such as cellulase9, amylase10, lipase11 and protease12.
Preparation of crude homogenate: The crude homogenate was prepared by inoculating the screened colonies in
medium containing micro and macro nutrients. The enzyme producing bacterial broth was then centrifuge to
collect the supernatant and used as crude extract13.
Assay of Enzymes: Enzyme activity was assayed using the methods of Cellulase14, Lipase15, Amylase16, Protease17.
Estimation of Protein present in the Crude Homogenate: The protein content was estimated by the Method of
Lowry et al., 18 using bovine serum albumin as standard.
Purification of Protease
Ammonium Sulphate Precipitation: The crude enzyme was partially purified with 20% - 100% of ammonium
sulfate19.
Dialysis: To remove the excess salts present in the solutions obtained from ammonium sulphate precipitation the
sample were dialysed against phosphate buffer at 4C and the proteolytic activity was determined20.
Study of purification profile: The specific activity, recovery percentage and purification fold of the crude protease
and the partially purified protease was done by the Method of Lowry et al.,18.
RESULTS
Bacterial strains identified from organic waste soil: The bacteria isolated from organic waste soil showed the
presence of a single colony. The colony obtained was examined by simple staining and gram staining. The presence
of bacteria in organic waste soil sample as reported above is in accordance with the results of Alaria et al.,21.
Staining and Biochemical tests: Table 1 shows the results of the morphological identification and biochemical
characterization of bacteria from organic soil waste. From the table, it can be seen that the isolated bacterial strain
was gram negative and rod shaped. Thus it can be inferred that identified bacterial strain was Pseudomonas sp.,
Table 1 Morphological Identification and Biochemical Characterization of Organic Waste Soil Bacteria
Test Result Test Result
Simple Staining Rod shaped Oxidase Positive

Grams Staining Pink colour rods Starch Hydrolysis Positive
Indole Negative Voges Proskaur Negative
Nitrate Negative Triple Sugar Iron Agar Positive
Catalase Negative Citrate Utilization Positive
Methyl Red Negative Urease Positive
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MALDI-TOF Test
Table 2 shows the results obtained by MALDI-TOF test.
Table 2 Bacterial species present in the strain
Bacterial species Log (Score)

Pseudomonas mendocina (DSM50017THAM) 1.516
Pseudomonas aeruginosa(81472CHB) 1.400
Pseudomonas corrugate(DSM7228THAM) 1.308
Pseudomonas fragi(DSM3456THAM) 1.325
Pseudomonas marginalis(DSM13124THAM) 1.276
Figure 1: Pseudomomanas mendocina
From Table 2 it is clearly understood that the bacterial strain isolated from organic waste soil contains different
Pseudomomanas species. Among them Pseudomomanas mendocina was present in maximum concentration (1.516
log)-Fig 1.
Enzymes produced by bacterial strains: It clearly understood that the bacterial strains isolated from organic
waste soil can produce these four types of enzymes. The presence of proteases were identified by the formation of
a clear zone around the bacterial strain in the gelatin agar medium. The presence of cellulases were identified by the
formation of a clear zone around the bacterial strain in the starch agar medium. The amylases were identified by the
zone of inhibition in the starch agar medium with iodine and the presence of lipases were identified by olive oil
screening and orange fluoresence viewing under the UV light.
Table 3 Activity of Enzymes in Table 4 Ammonium Sulphate precipitated
Pseudomonas isolated from organic protease activity from Pseudomonas
waste soil
Enzymes Enzyme Specific Ammonium Protein Enzyme Specific
Activity Activity Sulphate Content Activity Activity
(U/ml) (U/mg) (%) (mg/ml) (U/ml) (U/mg)
Amylase 1.30 0.41 0-20 1.91 2.24 1.15
Cellulase 2.04 0.05 20-40 2.14 2.65 1.21
Lipase 1.84 0.61 40-60 1.37 2.86 2.05
Protease 5.53 1.5 60-80 1.50 2.49 1.61
80-100 1.40 2.37 2.07
Activity of enzymes in Pseudomonas crude homogenate: Table 3 depicts the enzyme activity and specific
activity of cellulase, amylase, lipase and protease. It can be inferred that protease isolated from Pseudomonas
recorded the highest enzyme activity as compared to the other enzymes and this was precipitated with ammonium
sulphate for further studies.
101
Activity of Protease in Ammonium Sulphate purified sample: The protease enzyme from isolated bacteria
was partially purified by ammonium sulphate precipitation. Table 4 depicts the protein content, enzyme activity and
specific activity of ammonium sulphate precipitation in various concentrations from 0-100%. It can be understood
that the protein content was maximum in 20-40% (2.14 mg/ml) followed by 0-20% (1.91 mg/ml) samples. The
maximum activity of protease in sample obtained by ammonium sulphate precipitation as shown in 40-60% of
saturation (2.86 U/ml), followed by 20-40% (2.65 U/ml). The high specific activity was found in 80-100% saturation
(2.07 U/mg) of protease by ammonium sulphate, followed by 40-60% saturation (2.05 U/mg). The present study
is in agreement with the study of Umayaparvathy et al.,22 who obtained maximum level of protease at 80-100% of
ammonium sulphate saturation. Thus it can be concluded that protease purification with 80-100% of ammonium
sulphate gave the highest specific activity. Hence this sample was taken for further purification by dialysis.
Activity of protease in dialysed samples:

Table 5 shows the enzyme activity and specific activity of protease after dialysis. It can be inferred that the
specific activity of the isolated protease indicated (2.49 U/mg) on dialysis as compared to the undialysed (80-100%
ammonium sulphate precipitated) sample (2.07 U/mg). Abirami et al.,23also purified extracellular alkaline protease
from Pencillium janthinellum & Neurospora crassa by dialysis after ammonium sulphate precipitation. Thus it can
be concluded that the protease can be purified by dialysis to increase its specific activity.
Table 5 Activity of Protease in Dialysed samples
Purification Step Total Protein Enzyme Activity Specific Activity

(mg/ml) (U/ml) (U/mg)
Dialysis 80-100% 2.05 3.17 2.49
Ammonium Sulphate 1.40 2.37 2.07
precipitated
Purification profile of crude and purified protease from Pseudomonas isolated from organic waste soil:
The purification profile of the protease after various steps of purification and also from crude, the protein content,
enzyme activity, specific activity, purification fold and recovery percentage are depicted.
Table 6 Purification profile of crude and purified protease from Pseudomonas
Purification Total Protein Enzyme Specific Purification Recovery

Steps (mg) Activity (U/ml) Activity Fold Percentage
(U/mg)
Crude 3.1 1.31 0.42 1.00 1.00
Ammonium
Sulphate 1.40 2.37 2.07 3.8 1.80
Precipitated
(80-100%)
Dialysed 2.05 3.17 2.49 3.4 2.3
From Table 6 it can be stated that the specific activity, purification profile and the recovery percentage of the
dialysed protease sample were higher (2.3) than that three of the ammonium sulphate precipitated & crude samples
(1.80 &1.00) respectively and dialysis showed a purification fold of 3.4 respectively.
102
DISCUSSION
The bacteria were isolated from the organic waste soil sample, and its morphology identified by staining
and biochemically characterized and identified as Pseudomonas sp. This was again confirmed by MALDI TOF
test. The isolated Pseudomonas was screened for the production of cellulase, amylase, lipase and protease. The
result showed that Pseudomonas can produce all the above mentioned enzymes. The secreted enzymes were
quantitatively assayed to identify the enzyme with maximum specific activity. Protease recorded the maximum
enzyme activity. Hence it was taken for further studies. The secreted protease by Pseudomonas was purified by
ammonium sulphate precipitation. Among the various concentrations used (0-20% - 80 100%), the maximum
activity was exhibited by 80 100% precipitated fraction. Hence this fraction was further purified by dialysis
which increased the specific activity of protease. The protein content was 2.05 mg in dialysed protease and the
purification fold 3.4 and recovery percentage was 2.3. Thus the result of the present study concludes that
Pseudomonas species were present in the organic waste soil and it has the ability to produce industrially important
enzymes such as cellulase, amylase, lipase and protease. Among various enzyme Pseudomonas produces protease
in maximum quantity than the other enzymes. Ammonium sulphate precipitation and dialysis purification is better
method to purify protease secreted by organic waste soil.
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J Taibah Univ Sci 9(1), 50-55.
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Res 1(3), 39-51.
4. Krishnaveni K, Mukesh Kumar D J, Balakumaran.M D, Ramesh S and Kalaichelvan P T (2012) Scholars Res
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5. Dam P, Das P M L, Rebecca J and Sharmila S (2013) Res J of Pharml, Biologl and Chem Sci 4(2), 1-8.
6. Norris, Berkeley and Logan ODonnell (1981) The genus Basillus and Sporolactobacillus 2, 1711-1742.
7. Arvinder K, Manjeet K, Manohar L S and Zabeer A J (2012) Microbiol Biotech 2(4), 573-579.
8. Vijayaram S, Kannan S and Muthukumar S (2016) J of Chem and Pharml Res 8(7), 883-889.
9. Basavaraj I, Patagundi C T, Shivasharan and Kaliwal B B (2014) Intl J of Current Microbio and App Sci
3(5), 59-69.
10. Rao K V B, Bose H, Richa K, Karthick L and Kumar G (2013) Res J of Pharml, Biologl and Chem Sci 4,
523-536.
11. Ranjitha P, Karthy E S and Mohankumar A (2009) Res J of Pharml, Biological and Chem Sci 1, 48-56.
12. Sayali D, Sahithya K, Karthik L, Gaurav Kumar and Bhaskara Rao K V (2013) Res J of Pharml, Biological
and Chem Sci 4(3), 1196-1204.
13. El-Hadj Ali N, Hmidet N, Ghorbel-Bellaaj O, Fakhfakh-Zouari N, Bougatef A and Nasri M (2011)Appl
Biochem Biotechnol 164, 10961110
14. Denison D A and Koehn R D (1977) Mycologia 69(3) 592-603.
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18. Lowry O H, Rosebrough N J, Farr A L and Randall R L (1951) J Biological Chem 193, 265-273.
19. Simpson R J (2004) Purifying Proteins for Protecmics: A Laboratory Manual Cold Spring Harbor Laboratory
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20. Reed R (2007) Practical Skills in Biomolecular Sciences, 3rd ed. Essex: Pearson Education Limited 379.
21. Alariya S S, Sethi S, Gupta S and Gupta B L (2013) Arch of Appl Sci Res 5(1), 15-24.
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104
Effect of Glycyrrhizaglabra root extract against letrozole induced
Poly cystic ovarian syndrome (PCOS) using rat model
Velvizhi S, Sowmiya V, Annapurani S, Suganthi B.
Email : sri.velvizhi@gmail.com
Abstract
Polycystic ovarian syndrome is a heterogeneous condition characterized by hyperandrogenic chronic anovulation
and is considered to be the most common endocrinopathy in women of reproductive age. Glycyrrhizaglabra is
an age old plant used in traditional medicine across the globe for its ethnopharmacological value to cure
varieties of ailments including AIDS, SARS and hepatitis. The aim of the present study was to assess the effect
of Glycyrrhizaglabra root extract in the treatment of letrozole induced PCOS in female Wistar rats. Testosterone
levels were used to evaluate the antiandrogenic effect of Glycyrrhizaglabra root extract in PCOS rats for 28
days. Serum FSH, LH, estradioland progesterone levels was also assessed. Administration of letrozole led to an
abnormalcy in serum sex steroid profile. Glycyrrhizaglabra root extract treatment significantly decreased
testosterone levels which were found to be elevated in PCOS rats induced by letrozole. Glycyrrhizaglabra
significantly restored the levels of other biochemical parameters such as estradiol, progesterone and FSH level.
The effect of Glycyrrhizaglabra was found to be comparable with clomiphene citrate, which is being used as a
major medicine in the treatment of PCOS. The findings of the present study suggested that the Glycyrrhizaglabra
root extract could be used as an adjuncttherapy forthe management of PCOS.
Keywords : Poly cystic ovarian syndrome, Glycyrrhizaglabra, letrozole,Testosetrone and FSH
Polycystic ovary syndrome (PCOS) is a complex endocrine disorder affecting 510 % of women of
reproductive age. It generally manifests with oligo/anovulatory cycles, hirsutism and polycystic ovaries together
with a considerable prevalence of insulin resistance1. The condition was first described in 1935 by the American
gynaecologists Irving F Stein and Michael L Leventhal, from whom its original name of SteinLeventhal syndrome
was originated.About 40% of teenagers are suffering from this type of condition. It is characterised by high levels
of androgens in the body. Many Women with this condition have high levels ofinsulin in their bodies. High levels of
insulin causethe ovaries to produce more male hormones2.
Women with PCOS have metabolic abnormalities that include insulin resistance, hyperinsulinemia, higher
levels of cholesterol, low-density lipoprotein (LDL), reduced high density lipoprotein (HDL) and hyper-
triglyceridemia3.Increased Luteinising hormone (LH) and increased insulin levels mainly amplify the intrinsic
abnormality of theca steroid genesis. Excess androgen activity hinders gonadotropin-induced estrogen and
progesterone synthesis in PCOS follicle. Normally, testosterone and androstenedione are converted to estradiol and
estrone respectively with help of P450 aromatase, which plays animportant role in the hormonal balance in the
ovary. However, decreased activity of this enzyme results in the increased ovarian androgen production; thus,
leadingto development of PCOS condition4.
Letrozole , a non-steroidal aromatase inhibitor,blocks conversion of testosterone and androstenedione to

estradiol and estrone respectively and simulates PCOS condition by causing hormonal imbalance , hyperandrogenism
and intra ovarian androgen excess leading to appearance of polycystic ovary. Follicular atresia and abnormal
follicular development is observed due to induced elevation of androgen levels inside the ovary. Letrozole induction
105
was reported to cause hyperglycemic condition which may contribute to insulin resistance, hyperlipidemia leading
to metabolic syndrome5, 6, 7.
The treatment for ovulation induction at present is by using allopathic medicine, such as clomiphene
citrate, metformin and tamoxifene. All these allopathic medicine usage leads to mild to severe side effects including
hot fluhes, arthritis, muscle pain and depression8.Traditional herbal medicines are naturally occurring substanceswith
minimal or no industrial processing that have been used to treat various illnesses. Traditional herbal medicines are
getting significant attention in global health debates. Traditional medicine has established preventive, curative and
rehabilitative role9.
Glycyrrhizaglabra, also known as liquorice and sweet wood, belonging to family Leguminaceae is native to
the Mediterranean and certain areas of Asia. Liquorice has been used in medicine for more than 4000 years. In
traditional siddha system of medicine, it is used as a demulcent, expectorant,laxativeand sweetener. Liquorice
extract has been used for more than 60years in Japan to treat chronic hepatitis, and also have therapeutic benefit
against other viruses,including humanimmunodeficiency virus (HIV), cytomegalovirus (CMV) and Herpes simplex10.
Glabridin, a prenylatedisoflavonoid ofGlycyrrhizaglabra root has been associated with many biological
properties such as antioxidants, anti-atherogenic and estrogenic compounds. Due to thishigh biological property
this root sample of Glycyrrhizaglabra is selected for the investigation11.

Collection of plant materials
The root sample of Glycyrrhizaglabra was collected from Ariyavaidhya pharmacy at Coimbatore.The
collected sample was grinded and used for the experiments. The root extracts were prepared with various solvents
(aqueous, methanol, petroleum ether, chloroform and ethyl acetate) using soxhlet apparatus. 10g of the root
sample were taken and homogenized with 150 ml of respective solvents. The crude extraction were prepared in
overnight condition and kept in the shaker at room temperature and then centrifuged at 4000rpm for 20 minutes.
The supernatant was transferred to the pre weighed beaker and then concentrated by evaporating the solvent at
70C.The aqueous extract was evaporated under reduced pressure and the yields were calculated. The residue
obtained was kept in dry clean bottles till used.
The study protocol was approved by Institutional Animal ethical committee (AUW.IAEC.2015.BC:01).
The experimental study was carried out using30 adult female Wistar rats body weight, 150-170g,were obtained
from theLaboratory Animal Department KMCH.Animals were housed in polypropylene cages lined with husk
beddings. The cages were maintained clean and hygienic.Animals were kept in the standard conditions of light and
temperature controlled roomwith a 12-12h dark-light cycle, temperature 252C and humidity 4550%and were
randomly divided into sixgroups. The rats were fed with commercial pellet rat feed and wateradlibitum and were
also weighed weekly throughout the duration of the experiment.
Grouping of animals
The rats were divided into six groups with 5 rats in each. Group I rats were kept as the control group
received 1% carboxy methyl cellulose. Group II rats were received oral dose of letrozole
(1mg /kg b.w) dissolved in 1% CMC once daily for 28 days.Group III rats given orally letrozole at a dose
of 1 mg/kgb.w in combination with aqueous extract of Glycyrrhiza.glabra (100 mg/ Kg.b.w) once daily for 28
days.Group IV rats received letrozole for 14 days and then supplemented withGlycyrrhiza .glabra root extract
(100mg/kg.b.w). Group V rats received standard drug ( clomiphene citrate 1 mg/kg.b.w) along with the plant
extract for 28 days. Group VI rats supplemented with G. glabraextract alone for 28 days.
106
Preparation of the sample for biochemical analysis:
After 24 hours of fasting period the rats were weighed and blood samples were collected by retino-orbital
punctured into different eppendorf tubes. The blood samples were centrifuged and serum was separated for
hormonal assay. The serum was stored in a freezer for further analysis.
Biochemical estimations:
Hormonal Assay:
Hormones were assayed by Competitive ChemiluminescentImmunoassay. Serum testosterone, Follicle

stimulating hormonesand Luteinizing hormone was estimated by the method of(Yilmazet al., 2001) 17.Estradiol and
progesterone was estimated by the method of (Taiebetal 2007)18.
The ovary comprises of two cellular components, which are stimulated independently by LH and FSH,
leading to the production of ovarian steroids. Androgen production from cholesterol and release during folliculogenesis
is dependent on the stimulation of cells by LH and FSH.
FSH is responsible of follicular growth and estrogen formation. LH is synergy with folliclestimulatiing
hormone stimulates follicular growth and ovulation. Thus normal follicular growth is the result of the complementary
action of FSH and LH12. In the present study, Letrozole-aromatase inhibitor, was usedto induce Polycystic Ovarian
Syndrome in female Wistar rats.Previous reports suggest that Letrozole induced PCOS conditiondepicts human
PCOS in many ways13.
Table-1: The levels of Testosteone and Estradiolin experimental rats
Groups Testosterone(ng/ml) Estradiol(pg/ml)
I 99.67+1.09 50.83+0.78
II 188.17+1.58a** 25.73+0.59a*
III 86.23+0.74+b** 46.37+0.74b**
IV 81.33+0.85b** 49.23+0.81b**
V 92.83+0.98b** 51.87+0.66b**
VI 76.27+0.87b** 53.6+o.72b**
Table 2: The levels LH, FSH and Progesterone in experimental rats
Groups LH ( mIU/ml) FSH( mIU/ml) Progesterone (ng/ml)
I 8.47+0.48 5.66+0.41 15.83+0.58
II 10.41+ 0.67a ** 6.69+0.56a 7.43+0.24a**
III 9.31+0.62b** 6.65+0.38b** 11.88+0.41b**
IV 9.34+0.51b ** 6.84 +0.47b** 10.83+0.28c*
V 8.71+0.35b** 5.88+0.43b** 13.32+0.36b*
VI 0.57+0.43b** 5.91+0.52b** 15.07+0.40b*
107
Values are mean + SD of six samples in each groups
Group comparison : a- GI vs. G2; b- G2 vs. G3,G4,G5 & G6
** Significant at 1% level (p< 0.01)
The rats treated with letrozole exhibited significant increase in thelevels of testosterone and LHsignificant
decrease inestradiol, and progesterone levels as compared to normal control.No significant change in the FSH
levels wasnoted in group II rats. Treatment with plant extract showed significant decreased in elevated testosterone
with significant increase in estradiol and progesterone as compared to group II. The group III and group IV rats
showed restoration of estradiol, FSH,and progesterone. The extract control was comparable to control group
indicating the protective effect of plant extract.The result were comparable that of standard drug treated group.These
points out the curative property of aqueous extract of Glycyrrhizaglabra.
De creas ed proge sterone le vels are also indicative of a novulation 5and aqueous e xtrac t of
Glycyrrhizaglabrasuccessfully restore its level to normal.Decreased estradiol concentration due to inhibition of
aromatase in PCOS induced group was significantly increased by repetitive administration of (100 mg/kg)
ofGlycyrrhizaglabra, confirming its earlier reported phytoestrogenic activity11,14.
PCOS is manifested primarily by anovulation and hyperandrogenism14. Abnormal follicular maturation or

acceleration of follicular atresia is reported with elevated intra ovarian androgens levels. Therefore, intraovarian
androgen excess resulting from either circulating hyperandrogenemia or abnormal steroid genesis may result in
abnormal follicular development and polycystic ovary15.Testosterone and LH levels in PCOS group were significantly
increased compared to normal rat indicating hyperandrogenism. Hyperandrogenism is the key feature of PCOS,
resulting primarily from excess androgen production in the ovaries to a lesser extent, in the adrenals. The primary
mechanism driving increased ovarian androgen production in PCOS included hyper secretion of LH and increased
LH bioactivity hyperinsulinemia due to insulin resistance and increased volume of theca cells in an expanded
ovarian stroma7.
Glycyrrhizaglabra has been demonstrated to reduce testosterone level 14. In the present study, we found a
reduction in estradiol level in PCOS group and this coincides with earlier studies16.
Isoflavones are a sub group of phytoestrogens,with structure similar to 17-B-estradiol and capable of
binding to es troge n rec eptors. Gly cy rr hizaglabra(licoric e) conta ins not only trite rpe ne sa ponins
(glycyrrhizin),flavonoids polysaccharides but also various isoflavonoids,glabrone,glyzaglabrinand glyzarin This
phytoestrogen may be responsible for the management of PCOS.
In the aqueous root extract of Glycyrrhizaglabra and standard drug treated animals testosterone level was
decreased when compared to the PCOS group thereby showing good anti-androgenic effect with reduction in the
abnormal androgens. Thus our findings are similar to the above cited literature.
CONCLUSION
It could be stated that the aqueous root extract of Glycyrrhizaglabra showed many beneficial effects
similar to Clomiphene citrate in treating PCOS condition and inducing ovulation. Gycyrrhizaglabra restored the
hormone profile in Letrozole induced PCOS animals. Therefore, the findings of the present study suggested that
the Glycyrrhizaglabra root extract could be used as an adjuncttherapy forthe management of PCOS.
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against letrozole induced polycystic ovary using rat model. Journal of coastal life medicine, 1(4), 309-
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17. Yilmaz, B., Kutlu, S., Canpolat, S., Sandal S., Ayar A., Mogulkoc, R. and Kelestimur, H. (2001):
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catecholamine content n the male rats. Bio.Pharma Bull,, 24(2):163-166.
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109
Simultaneous Determination of Proposed Anti-HIV Combination Com-
prising Of Elvitegravir and Quercetin in Rat Plasma Using the HPLC
ESI-MS/MS Method: Drug Interaction Study
Lubna Azmi [a b], Ila Shukla [a], Padam Kant [b] and Ch. V. Rao [a]
[a] Pharmacognosy and Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow-226 001, Uttar Pradesh,
India. [b] Department of Chemistry, Lucknow University, Lucknow, India.
Abstract
Elvitegravir is the mainstay of anti-HIV combination therapy in most endemic countries presently. However, it
cannot be used alone owing to its long onset time of action. 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-
4-one (Quercetin: QU) is a polyphenolic compound obtained from Argeria speciosa Linn. (Family:
Convolvulaceae) anti-HIV candidate In the present study, a sensitive, simple and rapid high-performance liquid
chromatography coupled with positive ion electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)
method was developed for the simultaneous determination elvitegravir and quercetin, in rat plasma. The method
was linear over a range of 0.2500 ng/ml. All validation parameters met the acceptance criteria according to
regulatory guidelines. LCMS/MS method for determination of elvitegravir and quercetin was developed and
validated. Results show the potential of drugdrug interaction upon co-administration this marketed drugs and
plant derived secondary metabolite.
Keywords : Anti-HIV Resistance, Extraction, HPLC-ESI-MS-MS
INTRODUCTION
Elvitegravir is a member of the integrase inhibitor class of antiretroviral compounds. Integrase inhibitors
block the ability of HIV to integrate into the genetic material of human cells1,2. The U.S. Food and Drug Administration
(FDA) have approved elvitegravir, an integrase strand transfer inhibitor for the combination treatment of human
immunodeficiency virus type 1 (HIV-1) infection in treatment-experienced adults. elvitegravir is an HIV integrase
strand transfer inhibitor that works by interfering with one of the enzymes that HIV needs to multiply3-5. It is
indicated in combination with an HIV protease inhibitor co administered with ritonavir and with other antiretroviral
drugs. It is one of the ingredients in the combination HIV drug Stribild, which was approved by the FDA in August,
2012. elvitegravir comes in 85 mg and 150 mg tablets. It was develope by the pharmaceutical company Gilead
Sciences, which licensed EVG from Japan Tobacco6-9. However, recently reports of resistance and side effect
have emerged in Indian market with big side effect , Owing to the emerging side effect to elvitegravir as well as
their low oral bioavailability, there is the need to find novel anti-HIV agents that are devoid of such drawbacks and
are more affordable to serve as an these synthetical drug alternative. To find a suitable fast-acting partner drug for
elvitegravir, Bioactive phenolic compound was carried out in study. One of the most promising compounds among
all plant isolated compound is quercetin (voucher specimen no. 13646), which has been identified for development
as a suitable combination of elvitegravir for use against drug resistant viral infection and side effect cases10-11. In the
present report, a simple, sensitive and specific liquid chromatography is described coupled with positive ion
electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method developed and validated for the
simultaneous quantification of elvitegravir and quercetin The validated method was then applied to study the
preclinical pharmacokinetic interaction of elvitegravir and quercetin combination to evaluate its prospects as a
potential Anti-HIV combination.
110
METHODS
Chemicals and reagents
Elvitegravir was a generous gift from Sunpharma Laboratories Ltd, Quercetin was isolated from 50 %
ethanolic extract of Argeria speciosa Linn. (Family: Convolvulaceae) in CSIR-NBRI. High performance liquid
chromatography (HPLC) grade acetonitrile was purchased from Sisco Research Laboratories (SRL) Pvt. Ltd,
Mumbai, India. HPLC grade methanol was purchased from Thomas Baker Pvt Ltd, Mumbai, India. Ammonium
formate and glacial acetic acid AR were purchased from E Merck Ltd., Mumbai, India. Sodium carboxy methyl
cellulose (CMC) was purchased from Sigma Aldrich Ltd., St Louis, USA. Ultra-pure water was obtained from a
Sartorius Arium 611 system. Heparin sodium injection IP (1,000 IU/mL, Biologicals E Ltd, Hyderabad, India) was
purchased from local pharmacy.
Animals and prerequisites

Blank, drug-free, plasma samples were collected from adult, healthy male SpragueDawley (SD) rats
purchased from CSIR- Central Drug Research Institute, Lucknow, India. Plasma was obtained by centrifuging the
heparinized blood (25 IU/mL) at 2,050 g for 10 min at 20C. Prior approval from the Institutional Animal Ethics
Committee (IAEC) was sought for maintenance, experimental studies, euthanasia, and disposal of carcass of
animals.
Instrumentation and chromatographic conditions

HPLC system consisting of Series 200 pumps and autosampler with temperature-controlled Peltier-tray
(Perkin- Elmer Corp, Norwalk, CT, USA) was used to inject 10-l aliquots of the processed samples on a Waters
Atlantis C18 column (4.6 50 mm, 5.0 m). The system was run in isocratic mode with mobile phase consisting
of acetonitrile: methanol (60:40, v/v) and 10 mM ammonium formate buffer (pH= 4.5) in the ratio of 95:5 (v/v) at
a flow rate of 0.65 mL/min. Mobile phase was duly filtered through 0.22 m Millipore filter (Billerica, MA, USA)
and degassed ultrasonically for 15 min prior to use. Separations were performed at room temperature. Auto-
sampler Carry-over was determined by injecting the highest calibration standard then a blank sample. Qurecetin
isolated form Argyeria speciosa Linn was quantified. Mass spectrometric detection was performed on an API 4000
mass spectrometer (Applied Biosystems / MDS Sciex, Toronto, Canada) equipped with an API electrospray ionization
(ESI) source. The ion spray voltage was set at 5,500 V. The instrument parameters: nebulizer gas, curtain gas,
auxillary gas, and collision gas, were set at 40, 13, 50, and 10, respectively. Compounds parameters: declustering
potential (DP), collision energy (CE), and entrance potential (EP), and collision exit potential (CXP) were 80, 33,
10, 10 V, 50, 30, 4, 10 V, and 90, 33, 6, 8 V for elvitegravir and quercetin, respectively. Zero air was used as source
gas while nitrogen was used as both curtain and collision gas. The mass spectrometer was operated at ESI positive
ion mode and detection of the ions was performed in the multiple reaction monitoring (MRM) mode, monitoring
the transition of m/z 529 precursor ion [M + H]+ to the m/z 511.3 product ion for elvitegravir, m/z 418.2 precursor
ion [M + H]+ to the m/z 119.1 product ion for Qurcetin Quadrupole 1 and quadrupole 3 were maintained at unit
resolution and well time was set at 200 ms. Data acquisition and quantitation were performed using analyst software
version 1.4.1 (Applied Biosystems/MDS Sciex Toronto, Canada). Preparation of standard and quality control
samples.Primary stock solutions of elvitegravir and quercetin and IS were prepared by dissolving the compounds
in acidified methanol (1% glacial acetic acid) to achieve desired concentration of 1 mg/mL. Working standard
solutions of elvitegravir and quercetin were prepared by combining the aliquots of each primary stock solution, and
diluting with methanol. A working stock solution of IS (50 ng/mL) was prepared by diluting an aliquot of primary
stock solution with acetonitrile. All the stock solutions were stored at 4C until analysis and were found to be stable
up to six months. Calibration standards of elvitegravir and quercetin (3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, and
500 ng/mL) were prepared by spiking 90 L of pooled, drug-free, rat plasma with the appropriate working standard
solution of the analytes (10 L). Quality control (QC) samples were prepared by individually spiking control rat
111
plasma at four concentration levels (4.0 ng/mL (lower limit of quantitation, LLOQ), 10 ng/mL (QC low), 100 ng/
mL (QC medium) and 400 ng/mL (QC high)) and stored at 75 10C until analysis.
Recovery
The extraction recovery of analytes through protein precipitation extraction procedure was determined by
comparing the peak areas of pre-spiked extracted plasma standard QC samples (n = 6) to those of the post-spiked
standards at equivalent concentrations. Recoveries of elvitegravir and quercetin were determined at three concentration
levels, LLOQ, QC low and QC high concentrations: 3.9, 10 and400 ng/mL, whereas the recovery of the IS was
determined at a single concentration of 50 ng/mL.
Recovery (%) = Concentration of prespiked sample 100 / Concentration of postspiked sample
Sample preparation
All frozen study samples and QC samples were thawed and allowed to equilibrate at room temperature
prior to analysis. A simple protein precipitation method was followed for extraction of analytes from rat plasma. To
100 L of plasma in a tube, 200 L of elvitegravir solution (50 ng/mL in acetonitrile), was added and vortexed for
10 min followed by centrifugation for 10 min at 15,000 g. The supernatant (200 L) was separated and was
injected onto analytical column.
Application to interaction study

Study was performed in male SpragueDawley rats (n = 5, weight range 200220 g). The rats were fasted
overnight (1416 hrs) prior to the experiment but given free access to water. Rats were divided into three groups
(n = 5 each): two control groups (Elvitegravir, 10 mg/kg, oral, suspension in 0.25% sodium CMC and quercetin,
70 mg/kg, oral, suspension in 0.25% sodium CMC), and one co-administration group (70 mg/kg of oral 9778 and
10 mg/kg of oral Elvitegravir). Blood samples (approximately 0.25 mL) were collected from the retro-orbital
plexus into heparinized microfuge tubes at 0.25, 0.50, 1, 3, 6, 9, 11, 13, 15, 24, 48, 72, and 120 hrs post-dosing
and plasma was harvested by centrifuging the blood at 15,000xg for 10 min and stored frozen at 70 10C until
bio-analysis. Pharmacokinetic and statistical analysis Plasma data were subjected to non-compartmental
pharmacokinetics analysis using WinNonlin (version 5.1, Pharsight Corp, Mountain View, CA, USA). The observed
maximum plasma concentration (Cmax) and the time to reach the maximum plasma concentration (Tmax)
wereobtained by visual inspection of the experimental data. Area under the plasma concentration-time curve from
time zero to the last quantifiable concentration (AUC0-t) was calculated using linear trapezoidal rule. The total area
under the plasma concentrationtime curve from time zero to time infinity (AUC0-) was calculated as the sum of
AUC0-t and Clast/kel, where, Clast represents the last quantifiable concentration and Kel represents the terminal
phase rate constant. The apparent elimination half-life (t1/2) was calculated as 0.693/kel and the kel was estimated
by linear regression of the plasma concentrations in the log-linear terminal phase. The clearance (Cl/F); where F
represents the oral bioavailability, was calculated as dose/AUC, and the volume of distribution (Vd/F) was calculated
as (Cl/F)/kel. The data is presented as a mean SD. The pharmacokinetic parameters were compared using
Students t test. A P-value of <0.05 was considered significant. The relative bioavailability (RB %) was Calculated
as follows:
Relative bioavailability (RB%) = AUC coadimin 100 / AUC control

LC-MS/MS optimization
It was important to optimize extraction technique, chromatographic conditions and mass spectrometry
parameters to develop and validate a selective and rapid assay method for simultaneously quantitation of Elvitegravir
and quercetin in rat plasma. Protein precipitation was chosen as the sample preparation method. Several organic
solvents, including acetic acid, trichloroacetic acid, acetonitrile and methanol, were investigated as the precipitation
112
Figures:
Figure 1 : Typical MRM chromatograms of Elvitegravir with Quercetin in rat plasma at LLOQ (3.9 ng/mL).
Figure 2 : Linear plots of plasma concentration elvitegravir after oral administration of elvitegravir alone or in
combination with quercetin (means SD)
Table 1 : The pharmacokinetic parameters of elvitegravir with quercetin in rats (n = 5 each)
Parameters Control Elvitegravir with Quercetin

Elvitegravir Quercetin Elvitegravir Quercetin
AUC0-t 16.42 2.94 9.10 1.46 10.64 0.71 8.04 1.26
AUC0-8 17.42 3.29 9.15 1.47 11.02 0.66 8.15 1.26
Cmax 1.82 0.75 1.34 0.14 1.92 0.62 1.79 0.69
Tmax 3.75 1.52 2.2 1.09 4 2.45 0.5
Vd/F 0.59 0.09 106.05 30.57 62.73 8.40 142.03 28.13
CL /F 46.94 6.52 9.27 1.33 0.91 0.06 8.76 1.38
extraction solvents. Acetonitrile was chosen because of higher extraction efficiency for Elvitegravir and quercetin,
and much cleaner samples than other solvents. Several column types and chromatographic conditions were tested
in order to develop a short, robust and sensitive analytical method. A short (4.6 50 mm, 5.0 m) Waters Atlantis
113
C18 column with mobile phase consisting of acetonitrile: methanol (50:50, v/v) and 10 mM ammonium formate
buffer (pH 4.5) in the ratio of 95:5 (v/v) at a flow rate of 0.65 mL/ min provided the best compromise between
selectivity and speed of analysis. The overall analysis time was 6.0 min. No carry-over was observed, as indicated
by the lack of Elvitegravir and quercetin peaks in the blank sample. Mass parameters were optimized by infusing
standard analyte solution (100 ng/mL) into the mass spectrometer.
In order to optimize ESI conditions for Elvitegravir and quercetin, quadrupole full scans were carried out
in positive ion detection mode. During a direct infusion experiment, the mass spectra for Elvitegravir and quercetin
revealed peaks at m/z 529, 418.2 and 502, respectively, as protonated molecular ions [M + H]+. Following detailed
optimization of mass spectrometry conditions (provided in instrumentation and chromatographic conditions section)
m/z 529 precursor ion [M + H]+ to the m/z 511.3 product ion for Elvitegravir, m/z 418.2 precursor ion [M + H]+
to the m/z 119.1 product ion for quercetin and m/z 502 precursor ion [M + H]+ to the m/z 142.2 product ion for
IS was used for the quantitation purpose.
Recovery
The extraction recovery of the Elvitegravir and quercetin ranged from 94.63 to 109.56%, and the extraction
recovery of the internal standard was 93.26%. Validation procedures were followed as per US FDA guidelines [12].
Specificity and matrix effect in the present study, the specificity and selectivity has been studied by using independent
plasma samples from six different rats. Figure 1 shows a typical chromatogram for the drug-free plasma (Figure
1) and drug-free plasma spiked with Elvitegravir and quercetin at LLOQ (Figure 1). As shown in Figure 1, there is
no significant interference from plasma found at retention time of either the analyte or the IS. The ion suppression
or enhancement by plasma was less than 13.25% for the analytes and 18.5% for IS which demonstrated that the
matrix effects do not cause quantitation bias. Therefore, matrix effect could be negligible under the experimental
conditions.
Application to interaction study

The rat plasma samples generated following administration of Elvitegravir and quercetin were analysed by
the newly developed and validated method along with QC samples. The mean plasma concentration-time profiles
of Elvitegravir administered (10 mg/kg) alone or in combination with quercetin (70 mg/kg) orally in rats, are shown
in Figure . 1Table 1summarizes the pharmacokinetic parameters of Elvitegravir and quercetin. The presence of
quercetin significantly (p <0.05) decreased the AUC0- (63.26%) of orally administered Elvitegravir. Consequently,
the RB% of Elvitegravir in the presence of Elvitegravir and querctin is remarkably decreased (64.41%) compared
to the quercetin alone. The Tmax and Cmax were not significantly altered by quercetin . Elvitegravir clearance (Cl/
F) was increased by 64.84% and Vd/F increased by 62.89% with quercetin co-administration. As both apparent Cl/
F and apparent Vd/F were increased almost proportionally, there was no significant effect on the t1/2 of Elvitegravir
in presence of quercetin (47.82 vs 46.94 hrs; p > 0.05). Mean plasma concentration-time profiles of querctin are
shown in Figure 2. The Tmax of 9778 was significantly decreased by querctin (2.2 vs 0.5 hrs; p < 0.05).
Elvitegravir had no significant effect on other PK parameters of querctin (Table 1). Further studies are required to
understand the mechanistic basis of alterations upon per-oral coadministration of anti-HIV as potential combinations.
These results should be taken into consideration while designing clinical proof of concept pharmacodynamic
studies and while designing dosage regimen.
CONCLUSION
In this study, a highly sensitive, specific, reproducible, and high-throughput LCESI-MS/MS assay has
been developed and validated to quantify Elvitegravir and quercetin following protein precipitation extraction
technique from rat plasma, Due to good sensitivity (LLOQ 3.9 ng/mL for both Elvitegravir and quercetin) of the
assay and its short run time of 6 min, it offers a suitable platform for the determination of Elvitegravir and quercetin
114
in preclinical studies. The results of the interaction study show that there are potential chances of pharmacokinetic
interactions between long-acting Elvitegravir and short acting quercetin.
ACKNOWLEDGEMENT
Authors are thankful to director of CSIR-NBRI, One of author Lubna Azmi special thanks to DST-INSPIRE,
and University of Lucknow.
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against gastric ulcer and antioxidant defence system in rats. Current Traditional medicine 1, 1-5.
12. US FDA. Guidance for industry: bioanalytical method validation. Rockville, MD: CDER; 2013.
115
Characterization Of Alkaloids from the Stem of
Toddalia Asiatica. L, A Wild Woody Liana
Arunambiga, S1, Praveena, A1* and Suriyavathana, M 2
1* Department of Biochemistry, K.S.Rangasamy College of Arts and Science, Tiruchengode, Namakkal DT
2 Department of Biochemistry, Periyar University, Salem-11.
Abstract
Natural products act as an important source of novel therapeutic agents for treating many diseases. Plants are
endowed with many phytoconstituents like alkaloids, phenols, flavanoids, tannins etc. These active compounds
with medicinal properties can be extracted and characterized to determine their therapeutic values. Alkaloids are
one such compound with active pharmacological properties, that can be extracted from natural sources and can
be purified using modern techniques. Due to their physiological effects they can preferably be used as medicines
for treating various ailments and serve as analgesics or anaesthetics, antimalarial, antihypertensive and local
anaesthetics in ophthalmology and have antihelminthic properties and acts as aphrodisiacs in the treatment of
erectile dysfunction. The present study is aimed at isolating and characterizing these alkaloids from the stem of
Toddalia asiatica. L The alkaloids were extracted in the crude form and it has been subjected to techniques like
HPTLC, GCMS and FTIR methods. The results from these techniques clearly confirms the presence of alkaloids
in the crude form. This investigation have clearly suggests that the alkaloids can be separated individually and
purified, so that it can commercially be used as therapeutic medicine for treating various illnesses.
INTRODUCTION
Plants have been used as medicine since time immemorial. There is an increased consciousness regionally
and globally in the production and use of plant with healing property (Devi and Meera, 2010). The active compounds
with high therapeutic values can be extracted and characterized from medicinal plants that have resulted in the
discovery of new drugs (Carmen, 2002). Alkaloids, widely existing in natural plants, are compounds containing
basic nitrogen atoms. Many alkaloids have important physiological effects on human and exhibit marked
pharmacological activity which is useful as medicine (Parker, 1997).
Today, various methods are needed for the separation, purification and identification of the many different
constituents present in plants. (Harborne and Rai, 1973). For such purposes of identifying the compounds, the
chromatographic techniques such as High Pressure Liquid Chromatography (HPLC), Gas chromatography Mass
spectrometry (GC-MS) and High Performance Thin Layer Chromatography (HPTLC) were used widely as reported
in numerous publications (Natalie and Peter, 1998). FT-IR is a quick and effective analysis method used for the
complicated mixture system had played an important role in pharmaceutical analysis in recent years (Druy, 2004;
Sohrabi et al., 2005). The extract of Toddalia asiatica plant leaf has been used as medicine for fever, wounds,
pains etc., (Veeramuthu Duraipanddian et al., 2006). Chen et.al. 1993 have reported the isolation and characterization
of an unusual alkaloid toddaquinoline, found in the root bark of Toddalia asiatica. Further David et al., 2001 have
synthesized toddaquinoline and benzo[h]quinoline alkaloid was synthesized by Chen et al., 1993.Since, this plant
possess many medicinal properties, the present study was designed to isolate and to characterize the alkaloids from
the stem of Toddalia asiatica.L.

In the present study, an attempt has been made to isolate the alkaloids in a crude form from the stem of
116
Toddalia asiatica . L and to characterize them by determining the molecular structure, functional groups, chemical
bonds, etc of the components of it with the help of IR, HPTLC and GC-MS spectrum
Plant Material
The Stem part of Toddalia asiatica were collected from Kolli Hills in Namakkal District, Tamilnadu, India.
The plant material was authenticated by Botanical Survey of India, Coimbatore. The samples were shade dried at
room temperature and then ground to a fine powder in a mechanic grinder and used for the extraction of the crude
alkaloids.
Extraction of crude alkaloids
The crude alkaloid from the dried stem was isolated by well established methods after preliminary detection
of alkaloids by the method of Harborne, 1984. The result obtained was presented in the previous article (Praveena
and Suriyavathana, 2014).
Characterization of the crude alkaloids by HPTLC ANALYSIS :
The given alkaloid extract of plant sample (25mg) was weighed accurately in an electronic balance (Afcoset),
dissolved in 400l of Methanol and centrifuged at 3000rpm for 5min (Test Sample - A). 2 l of test solution and 2
l of standard solution were loaded as 5mm band length in the 3 x 10 Silica gel 60F254 TLC plate using Hamilton
syringe and CAMAG LINOMAT 5 instrument. The samples loaded plate was kept in TLC twin trough developing
chamber (after saturated with Solvent vapor) with respective mobile phase (Alkaloid) and the plate was developed
in the respective mobile phase up to 90mm.The developed plate was dried by hot air to evaporate solvents from the
plate. The plate was kept in Photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images at
Visible light, UV 254nm and UV366nm.The developed plate was sprayed with respective spray reagent (Alkaloid)
and dried at 100C in Hot air oven. The plate was photo-documented in Visible light and UV 366nm mode using
Photo-documentation (CAMAG REPROSTAR 3) chamber. Before derivatization, the plate was fixed in scanner
stage (CAMAG TLC SCANNER 3) and scanning was done at UV 366nm. The Peak table, Peak display and Peak
densitogram were noted. The software used was winCATS 1.3.4 version.
Mobile phase: Toluene-Ethyl acetate-Diethyl amine (3.5:1:0.5)
Spray reagent: Dragendorffs reagent followed by 10% Ethanolicsulphuric acid reagent.
Gas Chromatography-Mass Spectrometry (GC- MS):

Presence of individual compounds in the given sample was analyzed using GC-MS/MS of Thermo Fisher
make, ITQ900 model. One micro liter of the sample was run in a DB-1 fused silica capillary column with helium
(1ml/min) as carrier gas, 250C injector temperature, 280C ion-source temperature and isothermal temperature
110C (2 min), with an increase of 10C/min to 200C then 5C/min to 280C and 9 min to 280C. The mass
spectrum interpretation was performed using the library of National Institute Standard and Technology (NIST) and
the compounds were identified.
Infrared spectroscopy:
Infra red spectroscopy was carried out with dried alkaloid fraction. This method exploits the property
that alkali halides become plastic when subjected to pressure and form a sheet that is transparent in the infrared
region. Potassium bromide (KBr) is the commonest alkali halide used in the pellets. Approximately 0.1 to 1.0 %
sample is well mixed into 200 to 250 mg fine alkali halide (KBr) powder and then finely pulverized and put into a
pellet-forming die. A force of approximately 8 tons is applied under a vacuum of several mm Hg for several minutes
form transparent pellets. Degassing is performed to eliminate air and moisture from the KBr powder. Inadequate
vacuum may result in easily broken pellets that scatter light. Before forming the KBr powder into pellets, pulverize
it to 200 mesh max. and then dry at approximately 110 C for two to three hours.
117
Extraction of alkaloids from T. A stem
Alkaloids were extracted by well established methods and the result obtained was presented in the previous
article as 3.3 0.22 g/100g (Praveena and Suriyavathana, 2014).
HPTLC analysis of the crude alkaloids
HPTLC requires very small amount of sample and can detect active principle concentration in nanogram
levels (Tripathi et al., 2006).
The alkaloid profile of the extracted sample was done with the reference standard nicotine and the developed
plate was sprayed with Dragendroff spray reagent (alkaloid-specific) and dried at 120C. The plate was photo
documented at daylight using Photo-documentation (CAMAG REPROSTAR 3) chamber. Blue coloured fluorescent
zone at UV 366nm mode were present in the track, it was observed from the chromatogram after derivatization,
which confirmed the presence of Alkaloid in the given standard and in the sample. From the chromatogram, the
presence of alkaloid in the extract and from the peak table the presence of six different alkaloids was confirmed.
Table 1: HPTLC Peak table for alkaloids
Track Peak Rf Height Area Assigned substance
Sample A 1 0.04 543.3 14384.1 Unknown
Sample A 2 0.15 16.3 207.6 Unknown
Sample A 3 0.20 55.9 1132.3 Alkaloid 1
Sample A 10 0.71 104.3 3449.8 Unknown
Sample A 13 0.93 74.3 1742.8 Unknown
STD 1 0.60 23.2 548.0 Alkaloid standard
118
Fig 1: Baseline display and peak densitogram of crude alkaloid sample (Scanned at 366nm)
Fig 2: Baseline display and peak densitogram of alkaloid standard
HPTLC analysis showed that Semecarpus anacardium seeds contained tetrahydroamentoflavone (Aravind
et al., 2008). Characterization of alkaloids from stem bark of Nyctanthes arbortristis was done by Bhavya Bandi, et
al., 2011. Likewise, our HPTLC fingerprinting also revealed the presence of alkaloids. The results obtained by
HPTLC showed excellent accuracy and precision when compared to those obtained by spectrophotometric methods.
3.3 GC/MS Analysis:
The crude alkaloid extract of Toddalia asiatica was subjected to GC/MS analysis, and it gave peaks as
shown in Figure 3 in the chromatogram. Capillary GC in combination with mass spectrometry was found to be the
method of choice for the analysis of complex mixtures (Wink et al., 1995).
Fig 3: Gas chromatogram of crude alkaloid extract of T. A stem
From the chromatogram, the compounds corresponding to the peak was identified and the results were
presented in Table 2. The GC-MS spectrum of the crude alkaloidal extract of Toddalia asiatica revealed the
presence of fifteen peaks. The interpretation of this spectrum confirmed the presence of nitrogen containing
119
alkaloid type of compounds, coumarins and steroidal fractions. The peak at RT 19.60 is alkaloid dictaminine and
other alkaloids like isoquinolinone were also present.
Table 2: Phyto-components identified in the crude alkaloid extract by GC-MS
S.No. Peak Compound

1. 19.60 Dictamnine
2. 21.46 Linoleoyl chloride
3. 22.16 Tetralin, 6-acetyl-8-isopropyl-2,5-dimethyl
4. 22.45 Brayelin (coumarin)
5. 23.35 5,5 methoxy3,3 dimethyl 2 pyrrolidinylidene methyl 2,3,3 trimethyl 3,4 dihydro
2H pyrrole 2 carbonitrile
6. 24.04 6,7-Dimethoxy-1 methyl-3(2H)-isoquinolinone(Alkaloid)
7. 24.42 2,3-Epoxypropyl p-tert-butylbenzoate
8. 25.09 1-Acetyl-2,3,4,5-tetrahydro-5-(2-hydroxyethyl)-1,5-benxodiazepine
9. 25.31 3-Buten-2-one, 4-(4,7-dimetoxy-1,3-benzodioxol-5-yl)-
10. 25.41 Androstan-17-one, 3,11-dimethoxy-,17-methoxime
11. 25.58 2-Thiazolamine, 4-(3,4-dimethoxyphenyl)-5-methyl-
12. 29.55 4-Amino-2-(3,4-dimethoxyphenyl)-5H-(1)benzopyrano(4,3-d)pyrimidin-5-one
13. 30.98 5,6dihydro-2-o nitrobenzylidene amino 4 H benzocyclohepta (2,1 - d) thiazole
14. 31.65 4-(4-Hydroxy-3,5-dimethoxy-phenyl)-3,4-dihydro-1H-benzo[h]quinolin-2-one
15. 34.09 Thaizolo[3,2-a]benzimidazol-3(2H)-one, 2-(4-formylbenzylideno)-6,7-dimethyl-
This technique allows much more precise alkaloidal identifications to be made than by comparative TLC
and GLC methods, and enables even trace constituents to be analysed (Kinghorn et al. 1980).
By interpreting these compounds by GC/MS, it was found that B.maderaspatensis leaves possess various
valuable phytoconstituents which will certainly finds application in drug discovery and will serve as pharmacological
tool to treat chronic diseases (Suriyavathana and Indupriya, 2014). Similarly our extract can also acts as a therapeutic
agent in future.
3.4 Infra Red Spectrum of Crude Alkaloid Extract

FT-IR is one of the most widely used methods to identify the chemical constituents and elucidate the
structures, and has been used as a requisite method to identify medicinal compounds. Owing to the fingerprint
characters and extensive applicability to the samples, FT-IR has played an important role in pharmaceutical analysis
in recent years
IR spectrum
In the IR spectra of alkaloids, an absorption band at ~3426 cm-1 shows the presence of N-H (amides and
amines) and at 1565 indicates C=C aromatic rings and 1401 shows the presence of C-H alkanes.In addition many
weak absorption band was also present indicating the other groups in the crude alkaloidal extract of Toddaliaasiatica.
120
Fig 4 : IR spectrum of crude alkaloids of T. A stem
Our results are in accordance with the IR spectroscopic analysis of hexane, ethyl acetate and ethanol
crude extracts of Plectranthus glandulous which suggested the presence of different functional groups ranging
from O-H stretching, hydroxyl, C-H stretching, alkyl, C=C stretching aromatic ring , C-O bending, and alcohols,
ethers, esters, carboxylic acid and anhydrides, C=O stretching, carboxylic, carbonyl and C-N bending, alkaloids
(Egwaikhide and Gimba, 2007).
4. CONCLUSION
This study was performed for the isolation of crude alkaloids from the stem of Toddalia asiatica and to
characterize the alkaloidal fraction. The experimental values of HPTLC, IR and GC-MS are used to predict the
molecular structure, atomic bond, possible molecular functional groups, etc., for the confirmation of alkaloid
present in the Toddalia asiatica plant and confirmed the content of alkaloids in the extract as crude form. Alkaloids
are also very important because of their high biological activites.
This investigation has helped to identify the compounds present in the alkaloid fraction of this plant which
could be further purified and used in the therapeutic field. The presence of various bioactive compounds justifies
the use of this plant for various commercial and therapeutic applications and for ailments by traditional practitioners.
ACKNOWLEDGEMENT
The authors are grateful to Department of Biochemistry, Periyar University, Salem-11 and K.S.Rangasamy
College of Arts and Science, Tiruchengode, Namakkal DT.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interests.
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1. Devi P. and Meera R. (2010), J. of pharma. Sci. and res. Vol. 2 (2), pp. 99-106.
2. Carmen W. H. (2002). Anal Bioanal Chem., Vol. 373, pp.2330
3. Parker, Sybil P. (1997). Chemistry; Dictionaries. McGraw-Hill (New York).
4. Harborne, J.B. and M. Rai .(1973). Phytochemical methods. A guide to modern techniques of plant analysis.
Chapman and Hall Ltd. London.
5. Natalie J. Lazerowych and Peter P. (1998). Drug information journal, 32; 497-512.
6. Druy, M.A. (2004) Spectroscopy, 19, 60.
7. Sohrabi M.R., Davallo M., Tadayyon F., Nabipoor F., Khamneifar A., (2005). Asian Journal of Chemistry;
17(1), 541-547.
8. Veeramuthu Duraipandian, Muniappan Ayyanar, Savarimuthu Ignacimuthu. (2006). BMC, Complementary
and Alternative Medicine. :35,1180/14,72-6882-6-35.
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9. Chen, I.S., Tsai,I.L,Wu, S.J.,Sheen,W.S.,Ishikawa, T.,Ishii, H.(1993). Phytochem, 34,1449.
10. David C. Harrowven, M.I.T.Nunn, N.J. Blumire, and D.R.Fenwick, (2001). Tedrahedron.
11. Tripathi, A.K., Verma, R.K., Gupta, A.K., Gupta, M.M. and Khanuja, S.P.S. (2006) .Phytochem. Anal., 17,
394397.
12. Aravind, S.G., Arimboor, R., Rangan, M., Madhavan, S.N. and Arumughan, C. (2008). J. Pharmaceut.
Biomed., 48, 808-813.
13. Bhavya Bandi, K. Venkatesan, Ujwala Mannarapu, M. Keerthi (2011). Int J Pharm Biomed Res., 2(3), 149-
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14. Wink M., C. Meibner and L. Witte, (1995). Phytochemistry 38, 139-153.
15. Kinghorn A. D., M. A. Selim and S. J. Smolenski, (1980). Phytochemistry 19, 1705-1710.
16. Suriyavathana M and Indhupriya. S. (2014). World Journal of Pharmaceutical Research, Volume 3, Issue 9,
405-414.
17. Egwaikhide, P.A. and Gimba, C.E. (2007) Middle-East Journal of Scientific Research, 2, 135-138.
18. Praveena. A. and Suriyavathana,M. (2014). World Journal of Pharmacy and Pharmaceutical Sciences,Vol 3,
Issue 3: 1781-1878.
19. Harborne, J.B.(1984). Phytochemical methods: A guide to modern techniques of plant analysis, 2nd ed:
Chapman and Hall ltd., London, New York. 1984.
122
Effect of Dietary Incorporation of Ksheerabala Residue on Growth Per-
formance in Wistar rats
Jasmine Rani K., Shyama K., Deepak Chandran, Minu Sara Jasmine, Ally K. and Gangadevi P.
Department of Animal Nutrition, College of Veterinary and Animal Sciences,
Mannuthy 680 651, Thrissur, Kerala. E mail ID jasminerani@kvasu.ac.in
Abstract
Ksheerabala residue is a byproduct obtained during the preparation of ksheerabala oil which is made by
incorporating bala (Sida cordifolia), cow milk and gingelly oil (Sesamum indicum). This residue is available in
considerable quantity and many of the farmers are using this byproduct for feeding livestock. But the level of
incorporation and the effect of Ksheerabala residue on growth in rats are not yet studied. Hence, the present
study is planned to evaluate the effect of dietary incorporation of Ksheerabala residue as a feed resource in the
diet of Wistar rats on their performance.
Twenty four male Wistar albino rats weighing 80 5g were used as experimental animals and were allotted
randomly to two treatments of six replicate each. Group 1 (T1) was fed with basal diet as per BIS specification
(control) and T2 was fed with diet containing 5% ksheerabala residue. The average body weight, body weight
gain, dry matter intake, haematological and biochemical parameters were found to be similar in both the groups
(P >0.05). The results of the present study indicated that Ksheerabala residue can be included in the rat ration up
to 5% level without any adverse effect on their growth performance.
Key words: Ksheerabala residue, Rat, Blood, Growth
INTRODUCTION
Inadequate production of farm crops to meet the needs of both humans and their domestic animals leads to
competition between man and livestock for these feed ingredients. Non conventional feedstuffs could be considered
as the best alternative to produce cheaper feed and ultimately lower the cost of meat and other animal products.
Kerala is famous for ayurveda and has various ayurvedic pharmaceuticals and byproducts from these pharmaceu-
ticals which are mainly composed of residues of medicinal herbs. Ksheerabala residue is a byproduct obtained
during the preparation of ksheerabala oil which is made by incorporating Sida cordifolia, cow milk and gingelly oil.
This residue is available in considerable quantity which can be used as an alternative feed resource for livestock.
Hence as a pilot study this research work was undertaken to evaluate the nutritive value of Ksheerabala residue and
to assess the effect of dietary incorporation of Ksheerabala residue as a feed resource in the diet of Wistar rats on
their performance.

Twenty four male Wistar albino rats weighing 80 5g selected from Small Animal Breeding Station,
College of Veterinary and Animal Sciences, Mannuthy, formed the experimental subjects for the study. They were
allotted randomly to two treatments of six replicates each for a period of one month. Rats belonging to group 1(T1)
were fed on basal diet (as per BIS specification) and animals of group 2 were fed on diet (T 2) containing 5%
ksheerabala residue. All the rations were made isonitrogenous and isocaloric. Proximate composition of Ksheerabala
residue and ingredient and chemical composition of experimental rations were analysed. Weighed quantity of feed
was given in the morning. Clean, fresh drinking water was provided ad libitum to all the animals. Animals were
maintained under normal ambient conditions. Data on quantities of feed offered daily were recorded. The left over
portion of the feed was weighed daily and their moisture content was analyzed to calculate daily dry matter intake.
Body weights of all the rats were recorded at weekly intervals. Based on the body weight, feed was reviewed
fortnightly.
123
Blood samples were collected from six animals from each group at the end of the experiment. Haematological
parameters were analysed with fresh blood samples using haematological analyzer (Mythic 18 vet), plasma total
protein, plasma glucose (GOP-PAP methodology using standard kits), serum total cholesterol and triglycerides
were determined by enzymatic colorimetric methods using standard kits from Agappe diagnostics Ltd, Ernakulam,
India. Alanine-aminotransferase (ALT) and aspartate-aminotransferase (AST) enzyme were measured according
to the method described by Reitman and Frankel (1957). [1]
Feed samples were analyzed for proximate principles (AOAC, 2005) [2]. Neutral detergent fibre (NDF)
and acid detergent fibre (ADF) were determined using the method described by Van Soest et al. (1991)[3]. Data
were analyzed statistically using Analysis of Variance (Snedecor and Cochran, 1994)[4].

Presented in Table 1 is the chemical composition of Ksheerabala residue. The dry matter obtained was
92.55%, Crude Protein-29.52 %,Total Ash-8.42%, Crude Fibre -6.39%, Ether extract -13.26%, Nitrogen Free
Extract -42.41%, NDF -33.34%, ADF-14.21%.
The data on body weight of calves have been presented in the Table 2. There was no significant difference
(P>0.05) in body weight between two dietary treatments. Total weight gain and average daily gain of the calves
were similar in both the dietary treatments. These results were in agreement with Ogungbemi et al. (2017) [5].
Total dry matter intake during the experimental period was 407.92 and 422.54 g respectively for T 1 and T2. (Table
2) and statistical analysis of the data did not reveal any significant difference (P> 0.05) among the groups. Manjula
et al. (2016) [6] observed similar total dry matter intake in rats.
Data on haematological and biochemical studies have been given in Table 3 and 4. The values of various
haematological and biochemical parameters were similar in both groups indicating that dietary incorporation of
Ksheerabala residue did not affect these parameters to any significant effect. Values recorded in the present study
falls in the normal range reported for the species. Akpanabiatu et al., (2003) [7] in a study of the biochemical effect
of Eleophorbia drupifera reported a decreased AST level in treated groups. Similar changes in ALT, AST activities
in animals treated with plant extracts were reported by Bumah et al, 2005[8]. The fact that total protein concentration
as well as levels of ALT and AST serum of both control and treated groups were similar implies that ksheerabala
residue may not pose any toxicological threat to the liver.
The results of the present study indicated that Ksheerabala residue an ayurvedic byproduct contains high
CP, EE and NDF value and can be included in the rat ration up to 5% level without any adverse effect on their
growth performance.
Table 1. Chemical composition of Ksheerabala residue and experimental feeds
Parameter Ksheerabala Residue T1 T2

Dry matter 92.55 91.90 91.98
Crude Protein 29.52 24.38 24.72
Ether extract 13.26 5.23 5.28
Crude fibre 6.39 5.26 5.78
Ash 8.42 6.77 7.85
Nitrogen free extract 42.41 58.36 56.37
Acid insoluble ash 0.06 0.82 0.78
Neutral detergent fibre 33.34 25.86 26.72
Acid detergent fibre 14.21 9.51 9.62
124
Table 2. Body weight (g) and dry matter intake (g) of experimental rats.
Parameters T1 T2
Initial body weight (g) 85.62 2.82 84.66 3.83
Final body weight (g) 132.56 8.90 137.33 9.07
Total weight gain (g) 46.94 3.52 52.67 4.52
Totaldrymatterintake(g/animal) 407.92 21.34 422.54 22.16
T1 and T2- mean of six values. (P > 0.05).
Table 3.Haematological parameters of experimental rats
Parameters T1 T2
Haemoglobin (mg/dl) 14.78 2.60 14.52 0.34
MCV (fL) 58.04 0.56 58.29 1.20
MCH (pg) 18.20 0.43 18.85 0.39
MCHC (g/dl) 34.15 0.39 33.28 0.57
RBC count ( x 106 /l) 8.84 0.51 8.08 0.32
WBC count ( x103/l) 10.32 0.14 10.44 0.37
T1 and T2- mean of six values. (P > 0. 05).
Table 4.Blood biochemical parameters of experimental rats
Parameter T1 T2
Albumin,(g/dl) 3.42 0.14 3.88 0.20
plasma protein, (g/dl) 6.30 0.05 6.33 0.06
Calcium, (mg/dl) 11.23 0.11 11.26 0.23
Phosphorus, (mg/dl) 7.04 0.10 7.05 0.11
Cholesterol, mg/dl 78.34 2.41 74.88 2.63
Triglyceride, (mg/dl) 44.88 5.89 38.33 4.88
Glucose, (mg/dl) 89.78 1.26 87.19 1.71
AST(u/l) 105 8.26 101 9.23
AST(u/l) 21.52 3.24 19.62 2.96
T1 and T2- mean of six values. (P > 0.05).
125
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Corchorus olitorius Int. J. Sci. Rep. 3:28-32.
6. Manjula, K. Alpha Raj and Remya Krishna. (2016). Feed efficiency and serobichemical profile of wistar rat
fed with spirulina as functional food. Curr. Res.Nutr. Food. Sci.4:2754.
7. Akpanabiatu, M. I., Igiri, A. O., Eyong, E. U. And Eteng, M. U. (2003). Biochemical and histological effects
of Eleophorbia drupifera leaf extract in Wistar albino rats. Pharmaceut Biol. 41: 96-99.
8. Bumah, V.V. Essien, E.U., Agbedahunsi, J.M. and Eka, O.U. (2005) Effect of Khaya grandifoliala (Melianceae)
on some biochemical parameters in rats. J. Ethnopharmacol. 102: 446-449.
126
Characterization and antibacterial activity of biosynthesized
Emblica officinalis iron oxide nanoparticles
Banupriya. SJS, Kavithaa. K, Padma. PR and Sumathi. S*
Avinashilingam Institute for Home Science and Higher education for Women University, Coimbatore-641 043
Email: sribanupriyanka@gmail.com
Abstract
Best method for biosynthesizing iron oxide nanoparticles is green technology, because it does not contain any
harmful chemicals. Nanoparticles have become a matter of great interest due to their numerous advantageous
properties and various applications in all fields. In the present study, the iron oxide nanoparticles were synthesized
by the reduction of Ferric chloride solution with Emblica officinalis fruit extract. The fruit plays a crucial role in
curing wide range of diseases like cancer, diabetes, liver disorders, anaemia and many other diseases. The plant
extract was prepared at 70C for 15minutes and the filtrate was used as an extract for the synthesis. The
synthesis was carried out in 1:1 proportion of Ferric chloride solution and plant extract. The particles were dried
in hot air oven and were characterized using UV- Vis and FTIR spectroscopy to confirm the presence of
nanoparticles. The synthesized particles were further analysed for their antibacterial activity against the
microorganisms such as E.coli and S.aureus. Antibacterial assay proved that these nanoparticles were effective
antimicrobial agent.
Keywords : Biosynthesis, Emblica officinalis, Iron oxide nanoparticles, Characterisation, Antibacterial activity
INTRODUCTION
Nanotechnology is one of the upcoming areas of research in the modern field of material science. It is
considered as a promising revolution of the world. Due to its unique biological and physicochemical properties it
deals with the matter at the large scale and it is also used to study the manipulating matter at the atomic and
molecular scale level1. Metal nanoparticles have different properties and these materials can be employed in various
catalytic, magnetic, photoelectronic, sensor and biomedical applications2.
A large number of physical, chemical, biological and hybrid methods are in use to synthesize different
types of nanoparticles. In biosynthesis method synthesis of metal nanoparticles from plants are under development.
This reaction has no harmful chemicals and this method is known as green technology3. Green nanotechnology is
used to restore our environment from toxic substances. This process has attracted many people. Magnetic
nanoparticles are non-toxic, environmental friendly and uses safe reagents2.
Synthesis of nanoparticles of different sizes, shapes, chemical composition and controlled dispersity are
considered for human benefits in nanotechnology. Biosynthesis of nanoparticles can be potentially useful in various
applications. For synthesizing magnetic nanoparticles reduction of Ferric chloride with different plant extracts
have been carried out through green technology4.
The current work describes the synthesis of magnetic nanoparticles from the fruit of Emblica officinalis
by the reduction of ferric chloride with the aqueous fruit extract. Nanoparticles are preferred to be prepared from
plant extracts as they cannot only act as a reducing agent, but also as a capping agent5.
In India for the treatment of various kinds of infective diseases mostly medicinal plants are used as traditional
medicines. Among the common medicinal plants Emblica officinalis plays a crucial role in curing wide range of
diseases like cancer, diabetes, liver disorders, heart diseases, ulcer, anaemia and various other types of diseases. In
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Indian tropical and subtropical regions the fruit is available6. Emblica officinalis is a deciduous free plant and it is
commonly known as Indian gooseberry or amla and nelli in Tamil. It belongs to the family Euphorbiaceae. It is also
known as Phyllanthus emblica. It is an edible fruit, pale yellowish and fleshy in nature. Emblica officinalis is a rich
source of naturally occurring vitamin C. Due to their advantageous properties and variety of applications in different
field synthesis of nanoparticles has become a great interest in the recent years7.
Green synthesis provides advancements over chemical and physical method as it is cost effective,
environmental friendly, easily scaled up for the large scale synthesis and in this method there is no need to use high
pressure, energy, temperature and toxic chemicals. In the present work we have reported for the first time the
synthesis of green iron oxide nanoparticles using the aqueous fruit extract of the Emblica officinalis plant.
The objective of the present study includes
(i) To synthesize the iron oxide nanoparticles by the reduction of ferric chloride with the fruit extract.
(ii) Characterization of synthesized iron oxide nanoparticles by UV-Visible spectroscopy and FTIR
spectroscopy.
(iii) To assess the antibacterial activity of the synthesized iron oxide nanoparticles and the fruit extract.

Preparation of Emblica officinalis fruit extract
Emblica officinalis fruits were taken and were washed thoroughly using sterile distilled water and blotted
dry using filter paper. The fruit was chopped into fine pieces and ground in a mixer grinder with triple distilled
water. The mixture was allowed to boil in the boiling water bath for 15 minutes at 70C. Then the contents were
filtered using Whatman No: 1 filter paper. The filtrate was collected in a sterile conical flask. The filtrate was
centrifuged and the supernatant was taken as the aqueous plant extract.
Preparation of Ferric chloride solution
0.001M Ferric chloride solution was prepared by dissolving 13.5mg of ferric chloride in 50ml of
triple distilled water and stirred for 15 minutes.
Synthesis of iron oxide nanoparticles
Iron oxide nanoparticles of Emblica officinalis was synthesized by taking the supernatant of aqueous
fruit extract of Emblica officinalis and Ferric chloride solution in 1:1 ratio and stirred well and kept in the shaker
at 60C to determine the color change.
Characterization
To understand the unique characteristics of the biosynthesised iron oxide nanoparticles and to determine
the tunable optical properties the UV-Visible spectrum analysis was carried out. To obtain the infra-red spectra of
absorption and emission FTIR spectroscopy analysis was done.
Antibacterial Assay
Nutrient agar slants were prepared and solidified and then the cultures Escherichia coli and Staphylococcus
aureus were streaked and kept overnight.
The cultures were taken from the slant and incubated in the nutrient broth at 37C for overnight. Muller
Hinton agar plates were prepared and allowed to solidify. The cultures grown in the nutrient broth were taken and
spread over the agar plate using L-rod. Then the well was punched in the plates and added chloramphenicol
(control), Ferric chloride solution, fruit extract and synthesized iron oxide nanoparticles in the wells. The plate was
incubated at 37C for 24 hours.
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A special group of materials with unique features and extensive applications in various fields are nanoparticles.
Because of their unique optical, electrical and photothermal properties iron oxide nanoparticles have acquired
importance in the fields such as chemistry, physics and biology. For improving the bioactivity of the materials
reducing the particle size is an efficient tool. As the size of the materials is reduced to the nanometre level the
resulting color changes noticeably8.
Change in color of the solution on iron oxide nanoparticles synthesis
Table: 1 Identification of iron oxide nanoparticle synthesis by colour change
After Colour
S.No Sample Before reduction Time
reduction intensity
1. Emblica officinalis Milky White Black +++ 24 hrs
fruit extract
2. 0.001M Ferric chloride Bright dark yellow - - -
+++ indicates dark colour

Figure: 1 Synthesised magnetic iron oxide nanoparticles from Emblica officinalis
From the Table: 1 and Figure 1 it is clear that the change in colour of the fruit extract after reacting with
Ferric chloride solution indicated that the iron oxide nanoparticles were synthesized.
Pattanayak5have reported that the reduction of nanoparticles solutions with the extracts such as Mango,
Clove, Rose, Black tea, Carom seeds, Neem and Curry leaves the reduced colour is black, from their initial colour.
Sometimes the colour change may be pale black, brownish black or dark green. The colour change may take place
immediately or within 24 hrs or 48 hrs based on the plant extract. This indicated that the magnetic iron oxide
nanoparticles were synthesized. Mahdavi 2also reported that due to the reduction of Fe3+ with the plant extract
(Brown seaweed, Sargassum muticum) the iron oxide nanoparticles were synthesised and the nanoparticles
were also stabilized because of the reducing capacity of Ferric chloride.
pH change on iron oxide nanoparticles synthesis
Table: 2 Change in pH of synthesised iron oxide nanoparticles
S.No Solution pH range Acidic/Basic
1. Plant extract 2.35 Acidic
2. Ferric chloride 2.37 Acidic
3. Ferric chloride and plant extract ratio (1:1) 2.46 Acidic
4. Iron oxide nanoparticle solution 2.33 Acidic
Table: 2 shows the reduction of pH from 2.4 (fresh mixture) to 2.33 (synthesized nanoparticles) showing
that the magnetic nanoparticles of Emblica officinalis is synthesized. This further indicates that the synthesized
magnetic nanoparticles will be always acidic in nature. The study of Mahdavi2showed that due to the involvement
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of the OH group in the reduction process iron oxide nanoparticles were produced and that was because of the
decrease in pH level.
UV-Visible Spectroscopy
Ultraviolet-Visible spectroscopy uses light in the visible and adjacent ranges (near UV and near infrared).
In this technique the electronic transitions have been undergone by electromagnetic spectrum. The chemicals
involved in this process are directly affected by absorption in the visible region. Stability and formation of iron
oxide nanoparticles are confirmed using UV-visible spectroscopy.
Figure: 2 UV-Visible spectrum of iron oxide nanoparticle
Figure: 2 shows that the UV-Visible spectrum of the synthesized magnetic iron oxide nanoparticles has
absorption spectra within the range of 330-380nm. The absorption peak at wavelength 340nm indicated the formation
of iron oxide nanoparticles. The peak was observed due to the surface plasmon vibration.
Arokiyaraj9have reported that the absorption peak of Argemone mexicana leaf extract was at 315nm.
Mahdavi2have reported that the peaks at a range of 402nm and 415nm for the iron oxide nanoparticles synthesised
from the extract of Sargassum muticum. From the study of Awwad10it was clear that the absorption peak was
absorbed at 233nm for the carob aqueous leaf extract. The normal range of iron oxide nanoparticles ranges from
200 to 400nm by Devi11. Our results are in tune with the above findings.
Fourier Transform Infra Red Spectroscopy

To identify the functional groups of the active components based on the peak value in the region of
infrared radiations the FTIR spectroscopy studies were done. This is mainly responsible for the stabilization and
capping of nanoparticles. The spectra were obtained in the range from 4000cm-1 to 400cm-1 using FTIR
spectrophotometer.
Figure: 3 FTIR spectroscopy of Emblica officinalis fruit extract
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Figure:3 shows the representative absorption peaks in FTIR spectra of nanoparticles located mainly at
3340cm , 2881cm-1, 1732-1, 1446-1, 1377-1, 1166-1, 840-1 in the region 400 to 4000cm-1. The peaks at 3340 cm-1
-1
indicates that hydroxyl group is present. 2881cm-1 could be assigned to the stretching vibrations of CH3 and CH2
functional groups.1732cm-1 is assigned for C=O stretching mode indicating the presence of COOH group in the
Emblica officinalis fruit extract.1446cm-1 are identified as the amide II which arises due to NH stretching vibrations
in the amide linkage of the protein. The peak at 1377cm-1indicates the presence of (COO-) carboxylate ions
responsible for the formation of Fe3O4 nanoparticles. The peak at 1166cm-1 has a characteristic stretching vibration
band denoting the asymmetric vibration of the sulphate groups.
From the study of Kumar6it is clear that the frequency ranges closely to 700cm-1 indicated the FeO vibration
of magnetite iron oxide nanoparticles. Arokiyaraj9have reported that the absorption peaks at 3340 cm-1 and 1732
cm-1 indicating the FeO stretching. Awwad10 observed a strong absorption peaks at 533cm-1 and 470cm-1 indication
the Fe-O stretching. Senthil12have reported that the peak ranges 3436cm-1, 1611cm-1 and 1113cm-1 are showing the
results similar to our studies. The results of our findings are similar to the other studies using different sources.
This confirmed that the magnetite iron oxide nanoparticles were synthesised from Emblica officinalis fruit extract.
ANTIBACTERIAL ACTIVITY
Table: 3 shows the antibacterial activities of the synthesised iron oxide nanoparticles. The zone of inhibition
was measured.
Table: 3 Antibacterial activities of iron
Zoneoxide nanoparticles
of inhibition (mm) of Emblica officinalis
Name of the
organism Chloramphenicol Emblica officinalis FeCl3 Synthesised Fe 2O3
(control) fruit extract solution nanoparticles
Escherichia coli 6 2 - 5
Staphylococcus 7.5 3 - 6
aureus
From the above table it is clear that the iron oxide nanoparticles synthesised from Emblica officinalis fruit
extract are having more antibacterial activity compared to the fresh fruit extract.
Being smaller in size the nanoparticles are having greater efficiency in controlling the growth of the
microorganism and that is mainly due to the reactive oxygen species and accumulation of nanoparticles13.Azam14have
reported that the iron oxide nanoparticles are having more antibacterial activity compared to the other type of
nanoparticles. Above studies clearly indicated that the iron oxide nanoparticles are having strong antibacterial
activity.
Biosynthesis of iron oxide nanoparticles is a non-toxic, reliable and eco-friendly method which is important
for their biomedical applications. The method which we have proposed was extremely reproducible. Compared to
chemical synthesis the biosynthesised particles are having high functional bioactivity. The spectral studies were
done using UV-visible and FTIR spectroscopy to confirm the structure and shape of the synthesised nanoparticles
and it also possessed good antibacterial activity.
REFERENCES
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cell uptake of titanium dioxide nanoparticles in the marine fish Trachinotus carolinus, Aquatic Toxicology,
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2. Mahdavi, M., Namvar, F., Ahmad, M.B. and Mohamad, R.(2013) Green biosynthesis and characterisation
of magnetic iron oxide (Fe3O4) nanoparticles using seaweed Sargassum muticum aqueous extract, Molecules,
18, 5954-5964.
3. Lodhia, J., Mandarano,G., Ferris, N. J., Eu, P. And Cowell, S.F. (2009) Development and use of iron oxide
nanoparticles, Journal of Biomedical Imaging and Intervention, 6(2) 352-363.
4. Wu, W., He, Q. and Jiang, C. (2008) Magnetic iron oxide nanoparticles: Synthesis and surface functionalization
strategies, Nanoscale Res Lett, 3, 397-415.
5. Pattanayak, M. and Nayak, P.L (2013) Green synthesis and characterization of zero valent iron nanoparticles
from the leaf extract of Azadirachta indica (Neem), World Journal of Nanoscience and Technology, 2(1),
06-09.
6. Kumar, B., Smita, K., Cumbal, L. And Debut, A. (2014) Biogenic synthesis of iron oxide nanoparticles for 2-
arylbenzimidazole fabrication, Journal of Saudi chemical society, 5, 432-438.
7. Khan, K.H (2009). Roles of Emblica officinalis in Medicine A Review, Botany Research International,
2(4), 218- 228.
8. Cheng, F., Su, C., Yang, Y., Yeh, C., Tsai, C., Wu, C., Wu, M. and Shieh, D. (2005) Characterization of
aqueous dispersions of Fe3O4 nanoparticles and their biomedical applications, Biomaterials, 26, 729-738.
9. Arokiyaraj, S., Saravanan, M., Prakash, U., Arasu, V., Vijayakumar, B. and Vincent, S. (2013) Enhanced
antibacterial activity of iron oxide magnetic nanoparticles treated with Argemone mexicana L. leaf extract:
An intro study, Materials Research Bulletin, 48, 3323-3327.
10. Awwad, A.M. and Salem, N.M. (2012) A green and facile approach for synthesis of magnetic nanoparticles,
Nanoscience and Nanotechnology, 2(6), 201-213.
11. Devi, H.S. and Singh, T.D. (2016) Iron oxide synthesis through a benign approach and its catalytic application,
Perspectives in Science, 8, 287-289.
12. Senthil, M. and Ramesh, C. (2012) Biogenic synthesis of Fe3O4 nanoparticles using Tridax procumbens leaf
extract and its antibacterial activity on Pseudomonas aeruginosa, Digest Journal of Nanomaterials and
Biostructures, 7(3), 1655-1660.
13. Kanthimathi, M. and Soranam, R. (2013) Antibacterial effects of Emblica officinalis and Phyllanthus niruri
crude extracts against bacterial pathogens, International Journal of Pharmaceutical and Clinical Science,
3(3), 20-23.
14. Azam, A., Ahmed, A.S., Khan, M.S., Habib, S.S. and Memic, A. (2012) Antimicrobial activity of metal oxide
nanoparticles against Gram-positive and Gram-negative bacteria: a comparative study, International Journal
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132
Anti-inflammation and Anti-oxidant activity of Lowsonia inermis Linn.
extract on Carrageenan induced paw edema in rats
Swarnakala, N. Sri Kumaran, R.Vijayara
Department of Marine Biotechnology, AMET University, Kanathur, Chennai 603112, Tamilnadu, India.
Email : vijayrradha@gmail.com
Abstract
In the present study, the anti-inflammatory and anti-oxidant activity was studied by using ethanolic extract of
Lawsonia inermis. The bioactive compounds from L. inermis were identified by GC-MS. The anti-inflammatory
& anti-oxidants assays were followed by the standard methods. The mice bioassay study was carried out by
dose of 50, 100, 150, and 200 mg/kg bw respectively. The ethanolic extracts showed the presence of Methyl
salicylate (8.846), Propanoic acid (9.608), ethyl (dimethyl) silyl ester 2, 1, 3-Benzothiadiazole (12.258), Diethyl
Phthalate (14.222), Ethanol, 2-bromo (16.985), Dibutyl phthalate (17.729), Phytol (19.671) and Disooctyl phthalate
(23.272) all the compounds are response for anti inflammatory activity. The extracts (100 and 200 mg/kg) showed
the maximum inhibition of 39.49% and 55.98% at the end of 3 hrs with Carrageenan induced in rat paw edema. A
significant increase in the activities of SOD, CAT, GPx, Vit-C and Vit-E were observed in tissue of tested groups
and compared with standard & control groups. Through this study as a conclusion, the L. inermis possesses
anti-inflammatory and antioxidant potential which may be used for therapeutic purposes mainly in the prevention
of oxidative damage that occur during inflammation.
Keywords: Lowsonia inermis, carrageenan, anti-inflammatory and antioxidants.
INTRODUCTION
Inflammation is a universal host defense process involving a complex network of cell-cell, cell-mediator
and tissue interactions. It occurs in response to a variety of stimuli viz. physical, chemical, traumatic, antigen
challenge and infectious agents. Synthetic drugs both steroidal and nonsteroidal are being used for acute and
chronic inflammation cause a number of side effects. Also they are not able to cure inflammation completely.
Hence, the traditional medicine practitioners and scientists are turning towards medicinal plants for curing these
ailments1.Herbal drugs can therefore be considered as a better alternative to synthetic anti-inflammatory drugs.
Medicinal plants are widely used in management of diseases all over the world. Historically, the use of
medicinal plants is as old as mankind and medicine. The relatively lower incidence of adverse reactions to plant
preparations compared to modern conventional Pharmaceutical, coupled with their reduced cost, is encouraging
both the consuming public and national health care institutions to consider plant medicines as alternative to synthetic
drugs.
Now-a-days herbal drugs are prescribed widely even when their biologically active compounds are unknown
because of their effectiveness and no side effect in clinical experience. Large numbers of plants belonging to
different families have been studied for their therapeutic properties. Previous studies have proved that the chemical
constituents such as flavonoids, alkaloids, tannins and terpenoids are promising agents in treatment of inflammation2.
However, Lythraceae belonging to family respectively which have many medicinal properties3, have not been
studied for their anti-inflammatory activity hence the present study focused on anti-inflammatory and antioxidants
properties were studied on carrageenan-induced paw edema in albino rats from ethanolic extracts of L. inermis.
133
Collection and Authentication of Plant
Fresh healthy L. inermis was collected from their natural habitat of Herbal Garden, AMET University
campus Chennai and authenticated by professionals in Department of Botany, St. Josephs College, and Tiruchirappalli.
According to Mukerjee4 the herbarium number of the plant is RVL001.
Preparation of Extraction
The coarse powder plant material was extracted with ethanol by using soxhlet apparatus. The solvent
were removed under reduced pressure to get crude extract. Standard methods were used for preliminary
phytochemical screening of the extract, which was performed to know the phytoconstituents in the extract5.
Gas Chromatography Mass Spectrometry analysis

GC-MS analysis of the sample was performed using a Shimadzu GCMS-QP2010 gas chromatograph
mass spectrometer interfaced with a Turbo Mass quadrupole mass spectrometer, fitted with an Rtx-5 fused silica
capillary column (30 X 0.25 mm, with 1 Cm film thickness). The oven temperature was programmed from
100RC to 320R C at 100RC/min and a hold for 10 min. Helium was used as carrier gas at flow 1.0 mL/min. The
injector temperature was 250RC, injection size 1 L neat, with split ratio 1:10. The interface and MS ion source
were maintained at 320RC and 200RC respectively and the mass spectra were taken at 70eV with a mass scan
range of 40-700 amu (atomic mass unit).
Identification of Compounds
Interpretation of mass spectrum of GC-MS was conducted using the mass spectral database of National
Institute of Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown
component was compared with the spectrum of the known components stored in the NIST library.
Animals
Wistar rats 7-8 week old, weighing 150-200 grams were used for the present study. To maintain the
animal house under the standard condition of temperature (242C) and relative humidity (30-70) with a 12:12
light: dark cycle. The animals were fed with stranded pellet diet and water. The animal handling was performed
according to good laboratory practice (GLP). Ethical clearance was obtained from institutional animal ethical
committee (CPCSEA/265 /2015) and conducted according to Indian national science academy guidelines for the
use and care of experiments.
Acute Toxicity Study

Animals were randomly allotted in 5 groups (Each group contain six mice). The ethanol extract was
administered orally at doses of 50,100, 200 and 300 mg/kg of body weight. The control group received only the
normal saline (10 mL/kg b.w). The animals were observed during the first two hours for toxic signs and then
mortality was recorded for each group at 24, 48 and 72 hrs after dose administration.
Evaluation of anti-inflammatory activity

Anti-Inflammatory activity was tested on extract of L. inermis against carrageenan induced paw edema in
rats. The reductions of paw edema of rats are compared with the standard drug i.e. Indomethacin.
134
Animal Grouping
Animals were divided into five groups (6 animals in each)
Group 1: Normal Control (Normal Saline)
Group 2: Negative Control (Carrageenan, 1%)
Group 3: Positive Control (Indomethacin, 5 mg/kg)
Group 4: Ethanolic Extract of L. inermis (100mg/kg)
Group 5: Ethanolic Extract of L. inermis (200mg/kg)
Preparation of Tissue Homogenate

The tissue samples were homogenized in a solution containing 5% trichloroacetic acid and 5mM EDTA at
4C and centrifuged for 10min at 15,000g in 4C6.
Estimation of Anti-Oxidants
Enzymatic Anti-Oxidants
The estimation of enzymatic anti-oxidants Superoxide Dismutase (SOD), Catalase (CAT), Glutathione
Peroxidase (GPx) and Glutathione (GSH) was measured by the following methods. The activity of superoxide
dismutase (SOD) was assayed by the method of Kakkar et al., 7Catalase (CAT) was estimated by the method of
Sinha et al., 8Glutathione peroxidase (GPx) measured by the method described by Rotruck et al.,9.
Non- Enzymatic Anti-Oxidants

The estimation of non-enzymatic antioxidants Vitamins E, Vitamin C and Glutathione was estimated. Vitamins
C by the method of Omaye et al.,10 Vitamin E was estimated by the method of Baker et al., 11and Glutathione (GSH)
was measured by the method of Ellman et al., 12 .

The plants are a rich source of secondary metabolites with interesting biological activities. In general,
these secondary metabolites are an important source with a variety of structural arrangements and properties. The
World Health Organization estimates that plant extracts or their active constituents are used as folk medicine in
traditional therapies of 80% of the worlds population13. There is growing awareness in correlating the phytochemical
constituents of a medicinal plant with its pharmacological activity. Phytochemical analysis conducted on the plant
extracts revealed the presence of constituents which are known to exhibit medicinal as well as physiological
activities14.
In the present study, the quantitative phytochemical investigations of ethanolic extract of L. inermis shows
the presence of tannins, carbohydrates, glycosides, phenols, alkaloids, terpenoids and flavonoids. The GC-MS
analysis, totally 8 compounds identified from the L. inermis extract (fig 1) such as Methyl salicylate (8.846),
Propanoic acid (9.608), ethyl (dimethyl) silyl ester2, 1, 3-Benzothiadiazole (12.258), Diethyl Phthalate (14.222),
Ethanol, 2-bromo (16.985), Dibutyl phthalate (17.729), Phytol (19.671) and Disooctyl phthalate (23.272). All these
compounds are of reported as pharmacological importance; they possess the properties such as response for anti
inflammatory activity.
135
Figure-1 : GC-MS spectra of L. inermis
The inflammation is a biological complex of vascular tissues in harmful stimulated by pathogens and
irritants and has been major health problems in the world16. The anti-inflammatory effects can be elicited by a
15
variety of chemical agents and that there is little correlation between their pharmacological activity and chemical
structure17. Carrageenan induced hind paw edema is the standard experimental model of inflammation. Carrageenan
is the phlogistic agent of choice for testing anti-inflammatory drugs as it is not known to be antigenic and is devoid
of apparent systemic effects. Moreover, the experimental model exhibits a high degree of reproducibility18. Carrageenan
induced edema is a biphasic response. The first Phase is mediated through the release of histamine, serotonin and
kinins whereas the second phase is related to the release of prostaglandin and slow reacting substances which peek
at 3 hrs19. It has been reported that the second phase of edema is sensitive to drugs like hydrocortisone, phenylbutazone
and indomethacin. The indomethacin is a cycloxygenase inhibitor, the ethanol extract has activity which is compareable
to indomethacin and can be said to inhibit the cycloxygenase enzyme but lipoxygenase inhibitors also possess
significant anti-inflammatory activity against carrageenan induced paw edema, so inhibition of carrageenan induced
paw edema by the crude extract. In the presence study th Acute toxicity showed that L. inermis was not a cause
of death at a single dose of even 200 mg/kgbw. A transient hypoactivity, loss of appetite, and piloerection were
observed at a dose of 2000 mg/kg bw and recovered within 12 h., while no sign or symptom of toxicity was
observed in a group of 50,100, 150, 200 mg/kg bw treated. So 1/5 (200 mg/kg bw) and 1/10 (100 mg/kgbw) were
considered as the appropriate dose range for further anti-inflammatory studies.
In the presence study of anti-inflammatory activity of ethanol extract of L. inermis against carrageenan
induced paw edema shows that the extracts have significant effect on inflammation and markedly reduced the
swelling. The percentage reduction in the paw volume in the group of animals treated with L. inermis extract 100
mg was 39.49 % and for the 200 mg/kg was 55.98 % at 3 hours. It shows that the plant extract have significant
(P <0.01; P< 0.001) anti-inflammatory effect and the results were compared with indomethacin 10 mg/kg and
show percentage paw volume reduction of 58.13% (Table 1).
Table 1: Antiinflammatory activity of L.inermis
Mean increase in paw volume % of paw volume

Treat ment (mg/ml) after 3 hrs
0 hrs 1 hrs 2 hrs 3 hrs
Control 39.632.16 85.114.15 1032.33 123.319.33 -
L. inermis extract 22.112.18* 39.734.05* 63.354. 18* 74.773.58* 39.49 %
(100 mg/kg)
L. inermis extract 31.371.98* 71.372.67* 71.162. 18* 54.411.69* 55.98 %
(200 mg/kg)
Indomethacin 25.711.69** 28.431.94* 49.111. 69* 51.752.15** 58.13 %
(10mg/kg)
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As evidenced by earlier studies Induction of inflammation in experimental animal models is a huge task.
Most studies revealed that so many factors play a role for the lack of uniformity in the induction of inflammation.
Moreover the acute model of inflammation is suited to evaluate the preventive effects of drugs while the delayed,
chronic model is better adapted for studies on healing or resolution of inflammation. The results of the study
supported the traditional use of this plant in some inflammation and painful conditions which confirm the presence
of active chemical compounds related to these activities. phytochemicals such as flavonoids, terpenoids, steroids
and phenolic compounds expressed their anti-inflammatory activity at least in part by modulation of proinflammatory
gene expression such as cyclooxygenase-2, inducible nitric oxide synthase and several pivotal cytokines. These are
considered to be reasonable candidates for new anti-inflammatory drugs20. The results of the present study showed
that, carrageenan injection induced the paw edema volume and observed edema volume was higher at 3 hrs21.
Sharma et al., (2010) observed that different doses of B. serrata and indomethacin pretreated rats showed an
inhibition of carrageenan induced paw edema in all observed time intervals. At high concentration of B. serrata
treated rats showed greater decreased in carrageenan induced edema as compared with standard drug (indomethacin),
low and mid doses of B. serrata.
Free radicals have long been implicated as mediators of tissue damage in inflammation patients, which are
released in large amounts into the surrounding tissue. To neutralize this charge, free radicals try to withdraw an
electron from, or donate an electron to, a neighboring molecule. Other antioxidants works against the molecules
that form free radicals, destroying them before they can begin the domino effect that leads to oxidative damage.
For example, certain enzymes in the body, such as Superoxide Dismutase (SOD), Catalase (CAT), Glutathione
Peroxidase (GPx) and Glutathione (GSH), work with other chemical to transfer free radical into harmless molecules22.
Oxidative stress is a condition of reduction in anti oxidative enzymes like SOD, CAT, GPx and GST23. The
antioxidant enzymes SOD and CAT play an important role in reducing cellular stress. SOD scavenges the superoxide
radical by converting it to hydrogen peroxide and molecular oxygen, while CAT brings about the reduction of
hydrogen peroxides and protects higher tissues from the highly reactive hydroxyl radicals24. Table .2, shows that
the activities of SOD, CAT and GPx were significantly decreased in tissue of inflammation control rats due to
inadequacy of the antioxidant defenses in combating ROS mediated damage. The decreased levels of enzymatic
antioxidants status were seen in erythrocyte lysate and inflammatory were observed in carrageenan alone treated
rats when compared with control group. Ethanolic extracts of L. inermis at a dose of 200 mg/kg b.w significantly
normalized the enzymatic antioxidant such SOD, CAT GPx in carrageenan treated animals
Table 2: Enzymatic antioxidants L. inermis
Groups Enzymatic antioxidants

GPX SOD CAT
(U/gmHb) (U/gmHb) (U/gmHb)
Control 15.79 1.10 8. 02 0.89 5.90 0.44
Carrageenan + L . inermis (100mg/kg) 10.54 1.37* 5.02 0. 50* 4.01 0.59*
Carrageenan + L . inermis (200mg/kg) 13.09 1.20** 6.45 0.28** 5.09 0.47**
Indomethacin (10mg/kg) 15.02 1.25** 6.77 0.81** 5.89 0.21**
Each Value is SEM 5 individual observations * P < 0.04; ** P< 0.01 Compared paw edema induced control vs
drug treated rats.
137
The decreased activities of CAT and SOD may be a response to increased production of H2O2 and O2 by the
autoxidation. These enzymes plays an important role in maintaining physiological levels of oxygen and hydrogen
peroxide by hastening the dismutation of oxygen radicals and eliminating organic peroxides and hydroperoxides
generated from inadvertent exposure to carrageen25. Treatment with extract of L. inermis increased the activity of
these enzymes and may help to control free radicals when compared to inflammation rats. The effect produced by
plant extract was comparable with that of standard drug indomethacin
Table 2: Non-Enzymatic antioxidants L. inermis
Groups Non-enzymatic antioxidants

GSH(mg/dl) Vitamin E(mg/dl) Vitamin C(mg/dl)
Control 27.361.81 1.410.15 1.640.11
Carrageenan + L. inermis (100 mg/kg bw) 21.761.66* 1.160.14* 1.310.10*
Carrageen+ L. inermis extract (200mg/kg) 23.791.80** 1.210.10** 1.540.10**
Indomethacin (5 mg/kg) 22.151.23** 1.260.18** 1.140.28**
Each Value is SEM 5 individual observations * P < 0.04; ** P< 0.01 Compared paw edema induced control vs
drug treated rats.
Vitamin C plays a central role in the antioxidant protective system, protecting all lipids undergoing oxidation
and diminishing the number of apoptotic cells 26and it also regenerates the oxidized vitamin E27. Vitamin E, on the
other hand, acts as a non-enzymatic antioxidant and reduces chain reactions of lipid peroxidation28. Table.3 showed
decreased levels of nonenzymatic antioxidant vitamin C and E was in inflammation rats, when compared to that of
control rats. The levels of these antioxidants were significantly increased in tissue of inflammatory rats by treating
with root extract of L. inermis. GSH has a multifaceted role in anti-oxidant defense. It is a direct scavenger of free
radicals as well as a cosubstrate for peroxide detoxification by glutathione peroxidases.
CONCLUSION
The results of present study justified and supported scientifically to the ethno pharmacological use of the
plant as an anti-inflammatory agent to treat pain and inflammation. Further attempts will be taken to isolate and
define the active analgesic and anti-inflammatory fraction and its components.
ACKNOWLEDGMENT
We sincerely acknowledged AMET University management for facilities and financial support to carry this
research.
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South India. In: Rakesh K Sharma, Rajesh Arora. Herbal Drugs: A Twenty First Century Prospective. I edn.
New Delhi: Jaypee Brothers Medical Publishers; 2006: 387-88.
2. Peraman M K, Ramalingam P, Bapatla J N N S. Anti-inflammatory and antimicrobial activities of the extracts
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13. Zaidan MRS, Rain NA, Badrul AR. (2005). In vitro screening of five local medicinal plants for antibacterial
activity using disc diffusion method. Trop Biomed, 22, 165-170.
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Prachayasittikul V. (2008). Antimicrobial and antioxidant activity of bioreactive constituents from
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15. Meena MK, Jain AK, Jain CP, Gaur K, Kori ML, Kakde A, Nema RK. Screening of Anti-Inflammatory and
Analgesic activity of Cassia grandis Linn. Academic Journal of Plant Sciences. 2009; 2(1): 51-55.
16. Li Z., Yang G., Khan M., Stone D., Woo S.L., Wang J.H. Inflammatory response of human tendon fibroblasts
to cyclic mechanical stretching. Am. J. Sports Med. 2004;32:435440.
17. Sertie JAA, Basile AC, Panizza S, Matida AK, Zelnik R (1990) Antiinflammatory activity and sub toxixity of
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18. Winter CA, Risley EA, Nuss GW (1963) Antiinflammatory, Antipyretic activities of indomethacin, 1-(p-
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20. Kim S, Takahashi H, Lin WW, Descargues P, Grivennikov S, Kim Y, Luo JL, Karin M Nature. 2009 Jan 1;
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22. Sachin Jain, Dinesh Kumar Jain , Neelam Balekar. In-Vivo Antioxidant activity of ethanolic extract of Mentha
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140
Anti Inflammatory Assay of Herbal Extracts of Leaves of
Pisonia grandis with Tamarindus indica, Ficus racemosa and
Pongamia pinnata
M.Nandhini, Shubashini K Sripathi and G. Poongothai
Department of Chemistry, Avinashilingam Institute for Home Science and Higher Education for Women University, Coimbatore.
Corresponding author email id : adusks@gmail.com
Abstract
Herbal extracts of leaves of the medicinal plant Pisonia grandis, Tamarindus indica, Ficus racemosa and
Pongamia pinnata were analysed for their in vitro anti inflammatory activity. It was found that maximum
inhibition of 96% was observed at 50g/ml for pet ether extract of leaves of Pisonia grandis (PGLPE), pet ether
extract of leaves of Tamarindus indica (TILPE) and pet ether extract of leaves of Pongamia pinnata (PPLPE).
A maximum inhibition of 96% was observed at 50g/ml for the herbal mixture F 1 (pet ether extract of leaves of
Pisonia grandis mixed with pet ether extract of leaves of Tamarindus indica) and F 5 (pet ether extract of leaves
of Pisonia grandis mixed with pet ether extract of leaves of Pongamia pinnata). The results were comparable
with that of standard aspirin at the same concentration of 50 g/ml.
Key words: Pisonia grandis R.Br, Tamarindus indica, Ficus racemosa and Pongamia pinnata.
INTRODUCTION
Inflammation is a normal, protective response to tissue injury caused by physical trauma, noxious chemicals
or microbiological agents. Inflammation is the bodys effort to inactivate or destroy invading organisms, remove
irritants and set the stage for tissue repair1. Inhibition of albumin denaturation is taken as a measure of in vitro anti
inflammatory activity. The extract samples were assessed for their anti-inflammatory potential by the in vitro
method of inhibition of protein denaturation. The ability of a plant extract to inhibit protein denaturation is taken as
a measure of its anti inflammatory activity.
Pisonia grandis R.Br. (Nyctaginaceae) commonly known as Lechai kottai in Tamil contains a number of
phytoconsituents such as sugar alcohols, terpenoids and tannins. Tamarindus indica L., commonly known as puli
in Tamil belongs to the Dicotyledonous family: Caesalpiniaceae3. Ficus racemosa Linn commonly known as atthi
belongs to the Moraceae4. Pongamia pinnata (Linn) Pierre commonly known as pungai belongs to Leguminosae
family (sub family-Papilionaceae)6.

Collection of Plant Materials
The leaves of Pisonia grandis, Tamaridus indica, Ficus racemosa and Pongamia pinnata plant were
collected from local areas of Coimbatore. The plant materials were air dried and pulverized.
Extraction
Method I
The dried plant material (250g) was extracted with pet-ether (60-800C) (1000 ml) by refluxing for 6
hours. The extract was filtered and concentrated to give a residue which was weighed. This procedure was done
for the leaves of Pisonia grandis.
141
Method II
The dried plant material (250g) was extracted with pet-ether (60-800C) (1000ml) by refluxing for 6 hours.
The extract was filtered and concentrated to give a residue which was weighed. The residual plant material was
extracted with hydro ethanol (80:20). This procedure was done for the leaves of Tamarindus indica, Ficus racemosa
and Pongamia pinnata.
The extract concentrates are designated as
PGLPE - Pet ether extract of leaves of Pisonia grandis
TILPE - Pet ether extract of leaves of Tamarindus indica
TILE 80% - Dewaxed hydroethanolic extract of leaves of Tamarindus indica
FRLPE - Pet ether extract of leaves of Ficus racemosa
FRLE 80% - Dewaxed hydroethanolic extract of leaves of Ficus racemosa
PPLPE - Pet ether extract of leaves of Pongamia pinnata
PPLE 80% - Dewaxed hydroethanolic extract of leaves of Pongamia pinnata
Preparation of plant extracts formulation for the anti-inflammatory activity

The non polar extract of leaves of Pisonia grandis was mixed with the equal amount of extracts of the
chosen plants Tamarindus indica, Ficus racemosa and Pongamia pinnata to investigate the in vitro anti-inflammatory
potential.
Table (1) Details of plant extract mixtures used for the formulation
Extract mixture Formulation code

PGLPE + TILPE F1
PGLPE + TILE 80% F2
PGLPE + FRLPE F3
PGLPE + FRLE 80% F4
PGLPE + PPLPE F5
PGLPE + PPLE 80% F6
The formulated plant extract (10 mg) was dissolved in 4ml ethanol and the solution was made upto 100 ml
with water. From aliquots of 50, 100, 200, 400, 800 l were taken for the study.
Assessment of in vitro anti-inflammatory activity by inhibition of albumin denaturation (Protein

denaturation)
Inhibition of albumin denaturation can be taken as a measure of in vitro anti inflammatory activity. The
extract samples were assessed for their anti-inflammatory potential by the in vitro method of inhibition of protein
denaturation which can be taken as a measure of in vitro anti inflammatory activity.
The test solution (5 ml) consisted of 0.2 ml of egg albumin (from fresh hens egg), 2.8 ml of phosphate
buffered saline (PBS, pH 6.4) and 2 ml of varying concentrations of extract so that final concentrations become
50, 100, 200, 400, 800 g/ml. Similar volume of double distilled water served as control. The mixtures were
incubated at 3720C in a BOD incubator for 15 minutes and then heated at 460C for 5 minutes. After cooling, the
absorbance of samples was measured at 660 nm with vehicle as blank. Aspirin at the final concentration of (50,
100, 200, 400, 800 g/ml) was used as reference drug and treated similarly for determination of absorbance. The
percentage inhibition of protein denaturation was calculated:
142
% of Inhibition = 100 X [Vt/Vc -1]
Vt = absorbance of test sample; Vc= absorbance of control
Table 2 gives the percentage inhibition of protein denaturation of individual plant extracts and the leaves of
Pisonia grandis with Tamarindus indica, Ficus racemosa and Pongamia pinnata of herbal mixture.

Table 2 Anti inflammatory activity of individual plant extracts
% inhibition & Absorbance at 660 nm

Concen
tration
(g/ml)
Aspirin TILE FRLE PPLE F1 F2 F3 F4 F5 F6
PGLPE TILPE FRLPE PPLPE
80% 80% 80%
50 95 96 96 90 85 82 96 85 96 91 91 90 96 87
0.003 0.002 0.002 0.007 0.010 0.012 0.002 0.010 0.002 0.006 0.006 0.007 0.002 0.009
100 94 95 90 82 79 76 91 79 94 88 88 87 93 85
0.004 0.004 0.007 0.012 0.014 0.016 0.006 0.014 0.004 0.008 0.008 0.009 0.005 0.010
200 92 82 85 75 75 69 85 75 88 78 87 84 91 82
0.005 0.012 0.010 0.017 0.017 0.021 0.010 0.017 0.008 0.015 0.009 0.011 0.006 0.012
400 91 60 81 69 68 63 81 72 87 76 85 82 90 78
0.006 0.041 0.013 0.021 0.022 0.025 0.013 0.019 0.009 0.016 0.010 0.012 0.007 0.015
88 40 75 59 54 59 73 66 85 63 81 79 88 76
800 0.008 0.120 0.017 0.028 0.031 0.028 0.018 0.023 0.010 0.025 0.013 0.014 0.008 0.016
From the results of the anti inflammatory assay, it was found that a maximum inhibition of 96% was
observed at 50g/ml for PGLPE, TILPE and PPLPE and was comparable with that of standard aspirin at the same
concentration.
Maximum inhibition of 96% was observed at 50g/ml for the formulation F1 and F5. The results were
comparable with that of standard aspirin at the same concentration.
CONCLUSION
The herbal mixtures of chosen plant extracts exhibited significant anti-inflammatory activity, these plant extracts
containing the non-polar extract portion of leaves of the medicinal plant Pisonia grandis can be taken up for
validating their in vivo anti inflammatory potential.
This study provides lead for the commercial marketing of anti inflammatory ointments containing Pisonia
grandis extracts.
REFERENCES
1. Barik C S, Kanungo S K, Tripathy N K, Panda J R and Padhi M (2015) International Journal of Pharmaceutical
Sciences and Drug Research 7(3), 211-228
2. Anitha Rani A, Mary Josephine Punitha. S, Rema. M (2014) International Research Journal of Pharmaceutical
and Applied Sciences 4(1), 57-60
3. Anupama A Suralkar, Kishor N Rodge, Rahul D Kamble, Kanchan S Maske (2012) International Journal of
Pharmaceutical Sciences and Drug Research 4(3), 213-217
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Sciences and Nanotechnology, Issue 3
5. Bandawane Deepti, Mayuri Hivarale, Ashish Mali, Nilam Mhetre (2013) International Journal of Pharmacy
and Pharmaceutical Sciences, Vol 5, Issue 4
6. Divya Singh, Rahul Nainwani, Amit Gupta (2014) International Journal of Pharmacy and Pharmaceutical
Sciences, Vol 6, Suppl 2
7. Santhosh kumar Gupta, Amit Gupta, Gupta A. K, Dhirendra pakash, Vedpal (2013) International Research
Journal of Pharmacy, 4(3)
143
Evaluation of anti-inflammatory activity of methanolic extract of
Foeniculum vulgare leaves in vitro and in vivo
Matheswaran Jagathambal1 and Palghat Raghunathan Padma
1
Technical Assistant, Food Corporation of India, Food Storage Depot, Panangadu, Salem
E-mail : padmapraghu@gmail.com
Abstract
Inflammation is the rapid and natural protective response elicited by our body to tissue injury. Uncontrolled
inflammatory response may result in chronic conditions. The anti-inflammatory drugs that are in use to treat the
inflammatory diseases can provoke adverse side-effects. Keeping this in mind, in the present study, a search has
been made to find out a novel anti-inflammatory drug candidate of plant origin. The methanolic extract of
F.vulgare leaves was chosen for the present investigation, based on the results of our previous studies.
Heat-induced hemolysis, HRBC membrane stabilization, albumin denaturation and proteinase inhibition assays
were carried out to evaluate the in vitro anti-inflammatory activity of the extract. The assays were carried out
using the optimal dose of 150g/ml and a higher dose of 300g/ml. The standard drug aspirin was used for
comparison. The results showed that the extract stabilized the HRBC membrane and inhibited hemolysis as well
as albumin denaturation and proteinase activity in a dose-dependent manner. The in vivo anti-inflammatory
activity the extract was carried out for both acute (carrageenan-induced paw edema model) and chronic (cotton
pellet-induced granuloma model) inflammations. Adult male Wistar rats were used for in vivo studies and two
doses (200 and 400 mg/kg bw) of the extract were compared. The results of carrageenan-induced paw edema
model showed that the extract treatment gradually decreased the paw volume dose-dependently. In cotton
pellet-induced granuloma model, a significant reduction in the wet and dry weights of the implanted cotton pellet
was noticed in the extract treated group, demonstrating the reduction in the formation of granuloma tissue.
Thus, the results of this study clearly showed that the methanolic extract of F.vulgare leaves could serve as a
potential candidate in the development of anti-inflammatory drugs.
Inflammation is the primary part of the innate immune response that is triggered when there is a threat to
tissue homeostasis1. Inflammation is beneficial, when it occurs as a response to microbial infection or mechanical
injuries. However, long-term inflammation or the dysregulation of inflammatory pathways, have the potential to
damage or harm the tissues and result in the development of several chronic diseases2. In addition, inflammation
may lead to the development of several autoinflammatory disorders (National Institute of Health, 2016)3.
Non-steroidal anti-inflammatory drugs (NSAIDs) are most commonly used worldwide for their anti-
inflammatory, analgesic and anti-pyretic properties. However, the long-term use of NSAIDs leads to several side-
effects such as nausea, heartburn, dyspepsia4, inhibition of platelets and prostaglandin formation, increased blood
pressure, drug-induced asthma, renal failure, hepatic injury, gastrointestinal and cardiovascular complications5,6.
Medicinal plants serve as natural sources of pharmacologically active compounds. They exert lesser or null side
effects when compared to allopathy medicines, due to which, most of the researches are diverted towards medicinal
plants7. The seeds of the plants belonging to Umbelliferae family have been reported to have anticancer, antidiabetic,
antioxidant, anti-spasmodic, anti-inflammatory, analgesic and diuretic properties8.
Foeniculum vulgare, commonly called as fennel, is a perennial aromatic plant belonging to umbelliferae
family. Fully ripened and dried seeds have culinary uses. Immunomodulatory, analgesic, antioxidant, antithrombotic,
hepatoprotective, antibacterial, antifungal, antidiabetic and anti-inflammatory activities of fennel have been reported9.
Scientific reports are not available on the leaves of F.vulgare so far. Based on the results of our pilot analysis10, the
144
leaves of F.vulgare were selected to screen their free radical scavenging/inhibiting potential and biomolecule protecting
property. The results of that study revealed that the methanolic extract of F.vulgare leaves exhibited strong antioxidant
property.
Hence, the methanolic extract of F.vulgare leaves was chosen for the present investigation. Since antioxidants
can serve as anti-inflammatory agents11, the present study was framed to evaluate the anti-inflammatory property
of the methanolic extract of F.vulgare leaves in vitro and in vivo. HRBC membrane stabilization, protein denaturation
and proteinase inhibitory activities were evaluated as a part of in vitro study, whereas carrageenan-induced paw
edema and cotton pellet-induced granuloma studies were done in rats for in vivo models.

Extract preparation
Seeds of Foeniculum vulgare were sown in the University garden in a pesticide-free location. Fresh leaves
(5.0g) of Foeniculum vulgare were collected, washed in running tap water, allowed to dry by blotting gently
between tissue paper and cut into thin pieces using a scissor. They were then taken in a conical flask containing
50ml of methanol, covered with aluminium foil and subjected to mild shaking at 4C for 72 hours. The solvent
containing the extract was filtered through Whatmann filter paper using a Buchner funnel connected with a vacuum
pump. The filtrate was then concentrated at low temperature (50-60C) to get a residue, which was used for the
study.
Dose optimization
The optimal dose of the methanolic extract of F.vulgare leaves was determined by conducting a dose-
response study by employing DPPH radical scavenging assay.
DPPH assay
DPPH radical scavenging ability of F.vulgare leaves was analyzed in different concentrations of the leaf
extract (25, 50, 75, 100, 150, 200, 250, 300, 500 and 1000g/ml) according to the method of Mensor et al.
(2001)12.
Anti-inflammatory activity of the methanolic extract of F.vulgare leaves
The anti-inflammatory activity of the methanolic extract of F.vulgare leaves was tested under in vitro and
in vivo conditions.
In vitro anti-inflammatory activity
The protocol for the collection and use of human blood cells from healthy human volunteers was reviewed
and approved by the Institutional Human Ethics Committee (Approval No. AUW/IHEC-14-15/XPD-08).
Heat induced hemolysis
The heat induced hemolysis assay was carried out using the method proposed by Shinde et al. (1999)13.
RBC suspension was prepared following the method of Sadique et al. (1989)14.
Membrane stabilization test
The method described by Gupta et al. (2013)15 was adopted with minor modifications.
Protein denaturation assay
The protein denaturation assay was carried out following the method of Elias and Rao (1998)16 with some
modifications.
Proteinase inhibitory activity
145
Proteinases are found to involve in inflammation and tissue injury17. The proteinase inhibitory potential of the
methanolic extract of F.vulgare leaves was tested following the method of Oyedepo and Femurewa (1995)18.
In vivo anti-inflammatory activity
To evaluate the in vivo anti-inflammatory activity of the methanolic extract of F.vulgare leaves, both acute
and chronic inflammations were studied. Carrageenan induced paw edema model was adopted to study acute
inflammation, whereas cotton-pellet induced granuloma model was used to study chronic inflammation. Healthy
male Wistar rats were used for the study. The experimental procedures used in this study were reviewed and
approved by the Institutional Animal Ethics Committee (Approval No. IAEC-KMCRET/Ph.D/06/2016-17) of KMCH
College of Pharmacy, Coimbatore and were in accordance with IAEC guidelines.
Carrageenan induced paw edema model (acute anti-inflammatory activity)

The study was carried out following the procedure of Walker et al. (2003)19. The animals were divided into
6 groups, each consisting of 6 rats. Group 1 served as a negative control receiving saline, Group 2 served as
positive control receiving only 0.1ml of carrageenan (1%), Group 3 served as standard receiving 1.0ml of the anti-
analgesic drug, diclofenac sodium (10mg/kg) and 0.1ml of carrageenan, Groups 4 and 5 received 1.0ml each of
200 and 400mg/kg b.w of the methanolic extract of F.vulgare leaves, along with 0.1ml of carrageenan. Group 6
received 400mg/kg b.w of the methanolic extract of F.vulgare leaves alone. Saline, standard drug and the extract
were administered orally.
Cotton-pellet induced granuloma model (chronic anti-inflammatory activity)

The cotton pellet induced granuloma model was studied according to the procedure of Mosquera et al.
(2011)20 with minor modifications. The animals were divided into 4 groups, each consisting of 6 rats. Group 1
served as a control receiving saline; Group 2 served as standard, receiving the drug dexamethasone (0.5mg/kg);
Groups 3 and 4 received 200 and 400mg/kg b.w of the methanolic extract of F.vulgare leaves. Saline, standard
drug and extract were administered orally.
Dose optimization
In order to select the optimal dose, DPPH radical scavenging assay was carried in the leaf extract for
concentrations ranged from 25g/ml to 1000g/ml. The leaf extract scavenged 13.29%, 36.04%, 56.43%, 80.36%,
87.97%, 88.31%, 88.18%, 88.56%, 88.48% and 88.10% of DPPH radicals at 25, 50, 75, 100, 150, 200, 250, 300,
500 and 1000g/ml respectively. Thus, the result clearly showed that the methanolic extract evinced a concentration-
dependent DPPH scavenging effect at150g/ml, beyond which dose, the increase in scavenging was not marked.
Fig.1-Optimization of dose by DPPH radical scavenging
146
Hence, 150g was selected as the optimal dose to carry out further experiments. The dose response curve obtained
is shown in Fig.1.
Anti-inflammatory activity of the methanolic extract of F.vulgare leaves
Following dose optimization, the in vitro and in vivo anti-inflammatory activities of the methanolic extract
of F.vulgare leaves were evaluated.
In vitro anti-inflammatory activity of the methanolic extract of F.vulgare leaves

The release of lysosomal constituents like proteases and bactericidal enzymes by activated neutrophils
during inflammatory response, triggers several reactions, which, in turn, induce biomolecule damage, protein
denaturation and membrane lipid peroxidation. Thus, the stabilization of lysosomal membrane is crucial in limiting
the inflammatory response 21. On the other hand, the release of proteases during inflammatory response denatures
the proteins, resulting in the production of auto-antigens and lead to the development of auto-immune disorders22.
Since, HRBC membrane is found to be analogous to the lysosomal membrane, the stabilization of HRBC membrane,
inhibition of protein denaturation and proteinase activity can be used as reliable measures to determine the anti-
inflammatory activity in vitro. For in vitro study, two different dose levels of the extract were used namely; the
optimal dose of 150g/ml and a higher dose of 300g/ml. Aspirin were used as the standard drug for comparison.
Heat-induced hemolysis
The inhibition of heat-induced hemolysis by the methanolic extract of F.vulgare leaves was checked and the result
is displayed in Fig.2. The result showed that the methanolic extract inhibited heat-induced hemolysis in a dose-
dependent manner. The extract inhibited 59.12% of hemolysis at 150g/ml concentration and 80.44% at 300g/
ml, which was comparable to that of the standard drug aspirin (89.28%). Thus, the inhibiting activity of the extract
increased with increasing concentration.
Fig. 2 - Effect of the methanolic extract of F.vulgare leaves on heat-induced hemolysis.
Tatiya et al. (2013)23 have reported a concentration-dependent inhibition of heat-induced hemolysis and
protein denaturation by crude saponin obtained from the methanolic extract of Sesbania sesban leaves. The
hydroalcoholic extract of fruits of Vernonia anthelmintica significantly reduced the hemolysis of human erythrocytes,
comparable to that of the standard drug indomethacin24. A comparison was made among the methanolic, ethanolic
and chloroform extracts of the bark of Erythrina variegate for their hemolysis inhibiting potential and the maximum
inhibition was observed in ethanol extract, followed by methanol and chloroform extracts25.
Membrane stabilization test
When erythrocytes are exposed to aggressive conditions, hemolysis occurs followed by oxidation of
hemoglobin26, release of inflammatory mediators and free radical-induced lipid peroxidation27. During inflammatory
response, the release of lysosomal contents by the neutrophils significantly contributes to tissue damage. NSAIDs
147
act by inhibiting the release of lysosomal contents, by stabilizing the lysosomal membrane. Hence, compounds
with membrane stabilizing property are implied to serve as effective anti-inflammatory agents28.
The result obtained for membrane stabilization test is shown in Fig..3. It is inferred from the result that the
extract rendered considerable protection to the membrane by inhibiting hypotonicity-induced hemolysis in a
concentration-dependent manner. The extract was found to exhibit 39.9% protection at a dose of 150g/ml, which
further increased to 67.55% when the concentration was doubled. Since, HRBC membrane is analogous to the
lysosomal membrane29, the membrane stabilizing ability of the methanolic extract of F.vulgare leaves can be
extrapolated to that of the lysosomal membrane. The methanolic extract of F.vulgare leaves might have stabilized
the RBC membrane by adjusting the calcium level inside the cell30, thereby increasing the surface area to volume
ratio of the cell. The membrane protecting property of the extract could also be attributed to the interaction of the
plant components with the cell membrane proteins31.
Fig. 3 - Effect of the methanolic extract of F.vulgare leaves on HRBC membrane stabilization.
The methanolic and butanol extracts of Amaranthus caudatus leaves stabilized the RBC membrane by
inhibiting its hypotonicity-induced lysis32. Similarly, Ansari et al. (2015)33 studied the erythrocyte membrane stabilizing
potential of the ethanolic extract of Ixora nigricans leaves and inferred the effect to be comparable with that of the
standard drug. The methanolic extract of Rumex vesicarius showed better anti-inflammatory activity by inhibiting
hypotonicity-induced hemolysis at a higher dose of 1000 g/ml34. The presence of alkaloids, flavonoids and tannins
in the ethanolic extract of the roots of Momordica charantia has been implied in its remarkable membrane stabilizing
and protein denaturation inhibiting potentials35. A significant decrease in hemolysis was observed in a methanol-
water (4:1) extract of Rosa damascene flowers, followed by Lawsonia inermis L leaves and Cuminum cyminum
seeds36.
Protein denaturation assay
Fig. 4 Effect of the methanolic extract of F.vulgare leaves on albumin denaturation.
148
Denaturation of tissue proteins has been evidenced in inflammatory and arthritic diseases and production
of auto-antigens37. Auto antigens can provoke delayed type hypersensitivity as effectively as native proteins 38.
Thus, compounds that prevent protein denaturation could serve as an effective anti-inflammatory drug. With this
view, in the present study, the methanolic extract of F.vulgare leaves was tested for its inhibiting potential of
protein denaturation.
The results (Fig.4) of anti-denaturation assay revealed that the extract inhibited albumin denaturation in a
concentration-dependent manner. At 150g/ml, about 60.10% inhibition was observed, which further enhanced to
75.89% on increasing the concentration to 300g/ml. The result was comparable with that of the standard drug
aspirin, which exhibited 84.23% inhibition. The conventional NSAIDs that are used to treat inflammation, act by
blocking COX enzymes (responsible for prostaglandins secretion) as well as by inhibiting protein denaturation39.
Chataut et al. (2015)40 compared the potential of the aqueous extract of Ocimum sanctum leaves from
tropical and alpine regions of Nepal and found that both the extracts inhibited albumin denaturation effectively. The
flavonoid rich fraction of the seeds of Monodora myristica showed a dose-dependent inhibition of albumin denaturation
(Akinwunmi and Oyedapo, 2015)41. A significant inibition of protein denaturation by the essential oil obtained from
the aerial parts of Helichrysum italicum has been observed by Djihane and Mihoub (2016)42. A concentration-
dependent inhibition of protein denaturation was reported in the methanolic extract of Piper betle leaf43.
Proteinase inhibitory activity

The serine proteinases that are abundantly present in the lysosomal granules promote the release of pro-
inflammatory cytokines during inflammatory response and cause tissue damage44. Thus, proteinase inhibitors are
needed to limit the inflammatory response from causing adverse effects. With this view, the proteinase inhibitory
activity of the methanolic extract of F.vulgare leaves was screened in vitro at two doses 150g/ml and 300g/ml
respectively. It can be inferred from the result (Fig.5) that the extract inhibited proteinases in a dose-dependent
manner. On increasing the concentration from 150g/ml to 300g/ml, the inhibition increased correspondingly
from 55.06% to 76.51%.
Fig.5-Effect of the methanolic extract of F. vulgare leaves on proteinase inhibition.
Our findings correlated with another study, in which Leelaprakash and Dass (2011)45 found the methanolic
extract of Enicostemma axillare to exhibit anti-inflammatory activity by inhibiting the proteinase trypsin in a significant
manner. Muthu et al. (2014)46 analyzed and reported a dose-dependent proteinase inhibiting activity of an ethanolic
extract of Boerhavia diffusa leaves. In a comparative study by Anyasor et al. (2015)47, the hexane, ethyl acetate,
n-butanol and aqueous fractions of 70% methanolic extracts of the leaf and stem of Costus afer were compared,
and the maximum proteinase inhibiton was rendered by the hexane fraction of the leaves, which was attributed to
the presence of terpenoids, coumaran and fatty acids. The methanolic extract of the leaves of Aster lanceolatus
was found to exhibit a significant anti-proteinase activity in vitro (Patel and Desai, 2016)48.
149
The findings of the present study showed that the methanolic extract of F.vulgare leaves stabilized the
HRBC membrane, and inhibited the heat- induced hemolysis of HRBC membrane, protein denaturation and proteinase
activity in vitro. These properties of the extract confirmed its strong anti-inflammatory activity.
In vivo anti-inflammatory activity of the methanolic extract of F.vulgare leaves

The anti-inflammatory activity of the methanolic extract of F.vulgare leaves was tested in vivo using
experimental rats, for both acute and chronic inflammations. For the in vivo study, a lower (200mg/kg b.w) and a
higher dose (400mg/kg b.w) were selected based on the available literatures 49-51.
Carrageenan-induced paw edema model (acute inflammation)

The carrageenan induced rat paw edema is considered to be a convenient experimental animal model for
determining the anti-edematous effects of plant extracts52. In this model, acute inflammation was induced in the
healthy male Wistar rats by injecting carrageenan in the intraplantar region of the left hind paw using a Hamilton
syringe, followed by measurement of paw volume of the rats at regular intervals (0th, 1st, 2nd, 3rd, 4th, 5th and 6th
hour). Six groups of animals were maintained, of which Group 1 served as the negative control and Group 6 was
given the higher dose (400mg/kg b.w), to check the effect of the extract alone. Carrageenan was not injected to
both these groups of animals.
The results obtained depicted in Figs.6 and 7. The results revealed that, in the animals to which only
carrageenan was administered (Group 2), the paw volume gradually increased from 0th hour (3.51 0.16 ml) till
3rdhour (8.19 0.16 ml) and then slowly decreased. In Group 3, where the animals were administered with the
standard drug diclofenac sodium and carrageenan, the paw volume increased till 1st hour (3.27 0.07ml) and
started to decrease at 2nd hour from 6.31 1.27 ml to 6.19 1.25 ml. A similar trend was observed in Groups 4 and
5, to which the plant extract of lower and higher doses was given, along with carrageenan. The methanolic extract
of F.vulgare leaves effectively inhibited the paw volume in the late phase, i.e., 2nd hour after injection.
Based on this observation, it may be suggested that the extract acts by inhibiting the release of prostaglandins,
the key vasodilator in acute inflammation. The reduction in paw volume was observed in a dose-dependent manner,
in which at a higher dose (400mg/kg b.w) the reduction in paw volume was higher, compared to the lower dose
Fig.6 - Effect of the methanolic extract of F.vulgare leaves on carrageenan-induced paw edema.
150
Fig.7 - Effect of the methanolic extract of F.vulgare leaves on carrageenan-induced paw edema.
(200mg/kg b.w). The results were comparable with that of the standard drug diclofenac sodium, which is a
cycloxgenase inhibitor53. The reduction in paw edema volume by the methanolic extract of F.vulgare leaves may
also be due to the inhibition of cycloxygenase enzyme.
Methanol extract of Vallaris solanacea leaves showed a marked decrease in paw volume after 4th hour of
carrageenan injection54. The aqueous bark extract of Quillaja saponaria exhibited a dose-dependent reduction in
edema volume in the late phase of acute inflammation induced by carrageenan55. Ghori et al. (2015)56 observed a
reduction in the paw volume induced by carrageenan in the 4th hour of injection by the ethanolic extract of Ficus
dalhousiae roots. A comparative study of the chloroform, ethyl acetate, methanol and butanol extracts of aerial and
root parts of Salvia fruticosa revealed promising protection against carrageenan-induced paw edema in the late
phase of inflammation57.
Cotton pellet-induced granuloma model (chronic inflammation)

Cotton pellet-induced granuloma model is based on the formation of granulation tissue in the pellet of
compressed cotton that is implanted in rats subcutaneously58. On implantation of the cotton pellet, the inflammatory
response is elicited by the proliferation of macrophages, neutrophils and fibroblasts, resulting in granuloma formation59.
The wet weight of the implanted cotton reflects the amount of fluid absorbed by the pellet, whereas the dry weight
indicates the amount of the granulomatous tissue that is formed60.
151
The results of the present study, as depicted in Fig.8, showed that the wet and dry weights of the implanted
cotton pellet increased on the 8th day in control. On the other hand, the administration of the methanolic extract at
a higher dose (400mg/kg b.w) and the standard drug dexamethasone (0.5mg/kg b.w) had significantly reduced the
wet cotton weight (0.5290.06 g and 0.4350.03 g) and dry weight of cotton pellets (0.1500.03 g and 0.1790.01
g) compared to the control (0.7510.02 g and 0.2150.02 g). A reduction in the weight of cotton pellet is a direct
measure of the granulomatous tissue. The results of the present study showed a reduction in the weight of the
cotton pellet, which in turn reflected the reduction in granuloma tissue formation and corresponded to the anti-
inflammatory property of the methanolic extract of F.vulgare leaves. This property of the extract may be due to the
suppression of proliferative phase of inflammation by inhibiting fibroblast proliferation and synthesis of collagen
and mucopolysaccharides, the major contributors of granulomatous tissue61.
Fig. 8 - Effect of the methanolic extract of F. vulgare leaves on cotton pellet-induced granuloma.
In several studies, the in vivo anti-inflammatory activity has been checked by inducing granuloma in animal
models. The ethanolic extract of Jasminum sambac was found to reduce the weight of granuloma in a dose-
dependent manner62. Panda et al. (2016)63 reported a considerable degree of inhibition of the proliferative phase of
inflammation by the methanolic extract of root of Curcumis callosus. Boukhira et al. (2016)64 prepared a formulation
cream from the ethanolic extract of Silene vulgaris and reported a significant reduction in the wet and dry weight
of implanted cotton than that of the standard drug.
Thus, the results of in vivo anti-inflammatory studies confirmed the potential of the methanolic extract of
F.vulgare leaves in inhibiting the acute and chronic inflammations, as evidenced from the reduction of rat paw
edema volume and inhibition of granuloma formation.
CONCLUSION
Several steroidal and non-steroidal anti-inflammatory drugs are in practice at global level to treat inflammatory
diseases. But, the use of those drugs results in adverse side-effects. Hence, the need arises for the determination of
safer and effective drugs of natural origin. The results of the present investigation have clearly showed that the
methanolic extract of F.vulgare leaves possessed strong anti-inflammatory property. Hence, the determination of
active principles in the extract could serve as a lead in the development of novel anti-inflammatory drugs of natural
origin.
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154
Hepatoprotective and antioxidant effects of Leucas indica leafextract
against Acetaminophen induced liver damage in rats
Suja Rani S.a, Devyani P.b, Prakruthi B.S.b, Rehna A.c
a
Assistant Professsor, College of Veterinary and Animal Sciences, Pookode, Wayanad, India.
b
M.V.Sc. scholar, College of Veterinary and Animal Sciences, Pookode, Wayanad, India.
a
Research Assistant, College of Veterinary and Animal Sciences, Pookode, Wayanad, India.
Abstract
Acetaminophen (paracetamol), one of the most widelyused analgesic-antipyretic drugs cause acute hepatotoxicity
upon overdose, though considered safe at therapeutic doses. Thus, there is a huge demand for development of
hepatoprotectiveagents,which are safer and easily available. Hence, the present study was designed to evaluate
hepatoprotective and antioxidant potentials of alcoholic leaf extract of Leucas indica against acetaminophen-
induced liver damage in rats. A preliminary phytochemical screening and acute oral toxicity test were conducted
to assess quality and safety of the extract. Administration of L. indicaextract to male albino Wistar rats at
200 mg/kg/day doses for 10 days p.o.produced hepatoprotective and anti-oxidant effectsagainst acetaminophen
(2g/kg) induced hepatotoxicity, as evident from significantly decreased liver marker enzymes (ALT, AST and
ALP)andimprovement in oxidative stress markers (LPO and GSH). Moreover, the findings of extract treated
group was comparable to the reference drug, sylimarintreated group.The phytochemical screening of extract
revealed active principles like alkaloids, tannins, flavonoids, terpenes, glycosides, phenolic compounds and
saponins, whiletoxicity test indicated that extract is safe up to limit dose level of 2000 mg/kg. Therefore, alcoholic
leaf extract of L. indicamay be used as a protective agent againstparacetamol induced hepatotoxicity.
Key words: Leucas indica, hepatoprotective, antioxidant, acetaminophen
INTRODUCTION
Liver diseases have become one of the major causes of morbidity and mortality to animals andmankind
worldwide, among whichdrug induced liver injury seems to be the most common factor that poses majorclinical
and regulatory challenges in the pharmaceutical industry[1]. Acetaminophen (APAP)/Paracetamol overdose is a
worldwide leading cause of acute liver failure and drug-induced hepatotoxicity, eventhough acetaminophen possesses
an excellent safety profile at therapeutic doses [2]. Acetaminophen induced hepatotoxicity is triggered by the
reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) that engender oxidative stress and glutathione depletion
[3]. The most effective therapy against paracetamol toxicity presently available is N-acetylcysteine (NAC), which
is a GSH precursor. However, the time constraints with regard to the NAC administration restrains its widespread
therapeutic application since NAC needsto be given within 12 to 24 hours of APAP ingestion.
Herbal medicines have a long history of use in the treatment of liver diseases, while most of themsans
scientific pharmacological validation [4]. Therefore, a huge demand prevails for exploringsafe and effective herbal
drugs for the management of various liver disorders against APAPtoxicity.Leucas indica, commonly known as
Thumba in India is a perennial herb reported for different pharmacological activities like antimicrobial, anti-
inflammatory, antioxidant, CNS depressant, wound healing activityand anti-diabetic effects[5]. Nevertheless, the
effect of L. indica leaf extract on hepatic ailments has not been elucidated so far. Hence the present study was
designed to evaluate the hepatoprotective and antioxidant potentials of alcoholic leaf extract of L. indicaagainst
acetaminophen-induced liver damage in rats.
155
Preparation of extract of L. indicaleaves: The leaves of L. indicawere procured from local area of Wayanad and
were identified and authenticated at Department of Botany, University of Calicut, Kerala. The fresh leaves were
cleaned, dried under shade and was powdered with pulverizer, which was further extracted with ethanol. The
liquid extract attained was filterd and dried under vacuum using rotary evaporator to prepare the alcoholic leaf
extract of Leucas.
Preliminary phytochemical screening: The alcoholic leaf extract of L. indica was tested for the presence of
various active chemical constituents as per the procedure quoted by Harborne [6].
Experimental animals: Albino Wistar rats (160180g body weight) of the present study were acclimatized for a
week before experimentation and maintained at optimal temperature and relative humidity with 12 h light/dark
cycle. The experimental protocol was approved by the Institutional Animal Ethics Committee of College of Veterinary
and Animal Sciences, Pookode, Kerala.
Acute oral toxicity test: The acute oral toxicity testing of alcoholic leaf extract of L.indica was carried out as per
organization for eonomic co-operation and development (OECD) test guideline 420 [7].
Experimental design: Twenty-four male albino Wistar rats were divided into four groups of six animals each.
Group I and II served as normal control and acetaminophen/paracetamol control respectively and were pretreated
with vehicle (water) for 7 days, whereas group III and IV were pretreated with silymarin (reference drug) at 100
mg/kg and L.indica extract at 200 mg/kg b.wt respectively for 7 days. On eighth day, paracetamol was administered
orally at 2 g/kg b.wt. to all the rats except from group I. Vehicle/drug/extract administration was continued for
three more days and on 10th day of experiment (after 48 hours of APAP administration), blood was collected for the
estimation of enzymes such as serum ALT, AST and ALP and liver samples were collected for anti-oxidant activities.
Statistical analysis: All values are expressed as mean SEM and statistical analysis was carried out using statistical
package for social science (SPSS). The data were analyzed using one-way ANOVA followed by Duncans post hoc
test for multiple group comparison. Statistical significance was set at 5C<0.05.
RESULTS
The phytochemical screening of the alcoholic extract of the L.indica leaf revealed the presence of flavonoids,
glycosides, saponins, tannins, phenolic compounds, diterpenes and triterpene (Table 1). In the acute oral toxicity
test, alcoholic leaf extract of L.indica was found to be safe upto the limit dose level of at 2000 mg/kg rat b. wt
upon oral administration and did not produce any signs of toxicity, mortality or any remarkable gross pathological
changes. The serum biochemical and hepatic antioxidant parameters in various experimental groups are depicted in
Table 2. Administration of paracetamol to rats in the present study has caused liver damage as indicated by a
significant increase in serum enzyme ALT, AST, ALP activity along with hepatic LPO level, while GSH was
decreased. However, administration of extract of L. indica leaves significantly ameliorated paracetamol induced
elevated serum levels of ALT, AST, ALP and hepatic LPO, whereas GSH level was increased, similar to reference
drug, silymarin.
DISCUSSION
Acetaminophen induced liver injury is a commonly used model for investigation into the efficacy of
hepatoprotective drugs. At normal therapeutic dose, acetaminophen is majorly glucuronidated or sulfated and then
excreted through the bile, while a minor quantity of the drug is transformed to toxic metabolite which is also readily
detoxified by glutathione (GSH) conjugation. On the contrary, when administered at overdose, majority of
acetaminophen is converted by the cytochrome P450 (CYP 450) enzymes to the reactive toxic metabolite, N-
acetyl-p-benzoquinoneimine (NAPQI) [2]. Reactive metabolites can exert initial cell stress through a wide range of
mechanisms including depletion of GSH or binding to enzymes, lipids, nucleic acids and other cell structures, and
156
thereby inducing hepatocyte death, which further trigger inflammatory signals and results in necrotic cell death/
acute liver failure [8]. Administration of paracetamol to rats in the present study has caused liver damage, which
was in confirmation with the facts cited above.
The presence of various phytochemical constituents in L. indica extract., as observed in the present study is in
close affirmation with the previous reports [5].However, administration of extract of L. indica leaves significantly
ameliorated paracetamol induced hepatic damages and oxidative stress, indicating hepatoprotectiveactivity of the
L.indicaextract, which might have ensued through the anti-oxidant mechanisms.
CONCLUSION
The present study shows the hepatoprotective and antioxidant potentials of Leucas indica leaf extract
against acetaminophen induced liver damage
ACKNOWLEDGEMENTS
This research work was supported by research grant from State Plan fund allotted to Kerala Veterinary and
Animal Sciences, Pookode.
Table 1. Phytochemical screening of L.indicaethanolic leaf extract
Name of phytochemical Ethanolic extract (L.indica leaf)
Flavonoids +
Glycosides +
Alkaloids +
Saponins +
Tannins +
Phenolic compounds +
Diterpenes +
Triterpenes +
Symbol (+) indicates presence of phytochemicals
Table 2. Effect of L.indica ethanolic leaf extract on serum biochemical and liver antioxidant
Hepatic GSH Hepatic LPO

Sl Serum AST Serum ALT Serum ALP
Groups (n moles of
No. (IU/L) (IU /L) (IU/L) (n mole/g)
MDA/g)
Group I
1 102.52 2.13 80.161.47 0.2230.19 36.020.54 53.010.68
(Normal control)
Group II
2 293.32 6.26a 280.876.37a 0.3500.008a 24.851.22a 183.672.27a
(Paracetamol control)
Group III
3 171.02 2.03b 136.285.17b 0.2160.004 b 34.340.95b 69.072.28b
(Silymarin treated group)
Group IV
4 188.74 2.70b 159.733.43b 0.2410.004 b 30.270.44b 65.252.64b
(L.indica treated group)
parameters against paracetamol induced acute liver damage in rats
157
Values are expressed as Mean SEM; n=6;
a
P <0.05 , Normal control Vs Paracetamol control; bP 0.05 treated groups Vs Paracetamol control.
REFERENCES
1. Adewusi EA, & Afolayan, AJ. (2010). A review of natural products with hepatoprotective activity. J Med.
Plants Res., 4(13), 1318-1334.
2. Karthivashan G, Arulselvan P, Tan SW, & Fakurazi, S. (2015). The molecular mechanism underlying the
hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen induced hepatotoxicity
in mice. J. Functional Foods, 17, 115-126.
3. Hurkadale PJ, Shelar PA., Palled S, Mandavkar YD, & Khedkar AS (2012). Hepatoprotective activity of
Amorphophallus paeoniifolius tubers against paracetamol-induced liver damage in rats. Asian Pacific J.
Tropic. Biomed., 2(1), S238-S242.
4. Chattopadhyay R. (2003) Possible mechanism of hepatoprotective activity of Azadirachta indica leaf extract:
Part II. J.Ethnopharmacol. 89(2), 217-219.
5. Ramani R, Sudini S, Boddupalli BM., & Anisetti RN. (2012). Antioxidant, free radical scavenging and invitro
cytotoxic studies of ethanolic extract of Leucas indica var lavandulifolia and Leucas indica var
nagalapuramiana. Asian Pacific J. Tropic. Biomed. 2(3), S1637-S1642.42.
6. Harborne AJ. (1998). Phytochemical methods a guide to modern techniques of plant analysis. springer
science & business media.
7. OECD [Organization for Economic Co-operation and Development]. 2001. OECD guideline for testing of
chemicals: Guideline 420, Acute oral toxicity- Fixed dose procedure. Organization for Economic Co-operation
and Development, Paris, France.
8. Pauli-Magnus C, Stieger B, Meier Y, Kullak-Ublick GA, & Meier PJ. (2005). Enterohepatic transport of bile
salts and genetics of cholestasis. J.Hepatol. 43(2), 342-357.
158
Identification of natural inhibitor from marine weeds to treat
Porphyromonas gingivalis induced Periodontitis :
An insilico and invitro approach
Chikoo Cherry Abraham Cherian* and J.Jannet Vennila
Department of Biosciences and Technology, Karunya University, Karunya Nagar, Coimbatore - 641114.
email : chikoozcherry@gmail.com, jannet_r@karunya.edu
Abstract
Periodontitis is a widespread disease which affects the gums and causes the destruction of the periodontium.
The keystone pathogen is a Gram negative bacteria Porphromonas gingivalis which produces many virulence
factors that are involved in initiation and progression of Periodontitis. Recent studies have reported that marine
algae (seaweeds) possess medicinally active metabolites. The present work aimed to study the antimicrobial
activity of sea weed metabolites against P.gingivalis, wherein, the structural characterization of the bacterial
virulence proteinPPAD was carried out. And, the Seaweed Metabolite Database was mined for potential
compounds that would serve as ligands and inhibit the the virulence proteins. Molecular docking (Schrodinger
GLIDE) and Molecular simulation (Desmond) was done to study the docking and stability of the metabolites.
The metabolites were further tested for in vitro antimicrobial activity against P.gingivalis. Out of 1052 sea weed
metabolites, bromophenolic metabolites (found in the marine red algae Rhodomela Confervoides) were observed
to have better docking score and reliably stable. The bromophenols also exhibited significant antimicrobial
activity against P.gingivalis in invitro condition. These findings suggest that the marine bromophenols can be
used to control the activity of P.gingivalis. However, in vivo studies can be done further to validate the results
and toxicity studies.
Keywords: P.gingivalis, bromophenols, docking, simulation, marine compounds
INTRODUCTION
Periodontitis is a gum disease that falls under the category of chronic oral inflammatory disorders. It
disrupts the harmony of the peridontium (1). If left untreated, can lead to the onset of many systemic and autoimmune
diseases. (2-7). The types of periodontitis includes Gingivitis, Chronic periodontitis, Aggressive periodontitis,
Gum diseases in systemic diseases, Abscesses of the periodontium and the Periodontium associated with endodontic
lesions (8).
The oral cavity is a natural habitat to over 700 species of both aerobic and anaerobic bacteria (9-10).
Dysbiosis of microbial flora is correlated to the prevalence of oral diseases. The keystone pathogen of Periodontitis
is Porphyromonas gingivalis- a Gram negative bacteria (11, 12). One of the virulence factors responsible for the
pathogenesis of P.gingivalis is Porphyromonas gingivalis peptidyl arginine deiminase (PPAD) (13). Citrullination
by human PADs is important in normal physiology and inflammation. P.gingivalis, is the only prokaryote described
to possess PAD. P. gingivalis infection may generate citrullinated peptides, which trigger anti-citrullinated peptide
antibodies. , host protein citrullination by human PADs in the joint probably perpetuates antibody formation, paving
the way for the development of Rheumatoid arthritis (13).
Marine macroalgae, or seaweeds, are plant-like organisms that generally live attached to rocks or other
hard substrata in coastal areas. Differentiated on the basis of color they can be classified as brown, red and green
algae (14). Studies show that seaweeds possess the property of anti-inflammation and antimicrobial activity (15).
159
Therefore, the present work aimed to study the inbibitory effect of sea weed metabolites against Porphyromonas
gingivalis peptidyl arginine deiminase (PPAD), thereby controlling the pathogenic activity of Porphyromonas gingivalis
in Periodontitis.

a) Insilico Molecular docking and Simulation
The structure and the properties of the seaweed metabolites were obtained by thorough literature search
and Seaweed Metabolite Database (www.swmd.co.in). The properties of each seaweed metabolites studied. A
total of 1052 metabolites were retrieved, tabulated and analyzed. The three dimensional structure of the virulent
factors Porphyromonas gingivalis peptidyl arginine deiminase (PPAD) was retrieved from Protein Data Bank
(www.pdb.org) (PDB ID: 4YTB). This protein was used as the target protein. The 1052 seaweed metabolites was
used as ligands and docked against Porphyromonas gingivalis peptidyl arginine deiminase (PPAD) using Schrodinger
GLIDE,which is a ligand docking program for predicting protein-ligand binding modes and ranking ligands. SP
(Standard Precision) and XP (Extra Precision) were the two different scoring functions used to rank the binding
compounds (25). The protein and the ligands were prepared for docking using the Protein Preparation Wizard and
Ligprep respectively. The seaweed metabolites (ligands) were ranked based on their docking score and Glide
energy. Metronidazole was used as the standard drug for comparison.
The seaweed metabolite which exhibited highest docking score in effective binding to PPAD was further
subjected to molecular dynamics (MD) simulation to capture the dynamic events of binding. The tool Desmond
was used for molecular simulation studies (26). The binding activity of the virulence protein PPAD with the
seaweed metabolite over a period of time can be viewed using this tool.
b) Invitro antimicrobial activity
The anaerobic culture of P.gingivalis was obtained from Father Muller Medical College, Mangalore. The
anaerobic bacteria was cultured in Brucella Blood Agar medium. The bacteria was well streaked to form a
lawn culture. It was then kept for incubation under anaerobic conditions at 37 C for 36 to 72 hrs. The
bromophenol was purchased from Sigma Aldrich and the stock solution were prepared by adding 0.50 gms of
the bromophenol to 10ml of DMSO (dimethyl sulfoxide) and mixed thoroughly. Different concentrations
(50l, 100 l) of bromophenol was loaded into the wells in the blood agar plates. Then, the plates were
incubated under anaerobic conditions for 3-4 days. After incubation, inhibitory zone formation was observed
around the wells loaded with the samples. The zone diameter was measured and further calculations were
done. Triplicates of plates was used to get concordant results.
RESULTS
A total of 1052 seaweed metabolites were retrieved by literature search and Seaweed Metabolite Database.
These were docked against Porphyromonas gingivalis peptidyl arginine deiminase (PPAD). The seaweed metabolites
(ligands) were ranked based on their docking score and Glide energy and the top 5 are listed in Table 1. Among all
the metabolites, (2S)-2-amino-3-(3-bromo-5-hydroxy-4-methoxyphenyl)propanoic acid (bromophenol) from
Rhodomela confervoides, illustrated highest docking score of 8.457996 with 3 hydrogen bonds, GLIDE
energy of 36.410622 which seems far potent than the commercial drug Metronidazole ( Figure1 and 2). The
dynamic properties of PPAD complex with (2S)-2-amino-3-(3-bromo-5-hydroxy-4-methoxyphenyl)propanoic
acid was analyzed from trajectory data obtained for 10 ns and found to be stable (Figure 3).
(2S)-2-amino-3-(3-bromo-5-hydroxy-4-methoxyphenyl)propanoic acid (bromophenol)was purchased
from Sigma Aldrich and tested for antimicrobial activity against Porphyromonas gingivalis. The bromophenol
at a concentration of 5 mg / 100 l exhibited good antibacterial activity with zone of inhibition of 1.33 0.18
mm (Figure 4).
160
Table 1: Seaweed metabolites docked against PPAD
S.No. Compound name Docking Glide Number of Amino acids

score energy hydrogen interacting
bonds
1. (2S)-2-amino-3- -8.457996 -33.450484 3 THR186:(O)

(3-bromo-5-hydroxy-4- GIU355:(O)
methoxyphenyl)propanoi PHE135:(H)
c acid
2. Metronidazole -5.870157 -35.120122 5 GLU 60:(H)

THR186:(H)
LYS242 :(H)
ARG 129: (H)
GLY181:(O)
161
Table 2 : Antibacterial activity of 4,5-dibromobenzene-1,2-diol (B) (50 l)
Plates 4,5-dibromobenzene-1,2-diol 4,5-dibromobenzene-1,2-diol

(B) (50 l) (B) (100 l)
Plate 1 0.4 mm 1.4 mm
Plate 2 0.3mm 1.1mm
Plate3 0.4 mm 1.5 mm
STANDARD DEVIATION 1.1 +-0.54 1.33+- 0.18
Figure 3: Dynamic properties of PPAD complex with Figure 4: Antibacterial activity of

(2S)-2-amino-3-(3-bromo-5-hydroxy-4-methoxy phenyl) seaweed metabolite against PPAD
propanoic acid
DISCUSSION
A structurally diverse product, marine algae is promising because of the presence of potent compounds
(16-18). One kind of these marine algae derived compounds is the bromophenols. Bromophenols share one or
several benzene rings, a varying degree of bromine and hydroxyl-substituents. (19-21). The same findings was
also observed in the present insilico and invitro study, wherein, one of the bromophenol found in Rhodomela
confervoides ie. (2S)-2-amino-3-(3-bromo-5-hydroxy-4-methoxyphenyl)propanoic acid (bromophenol) has shown
effective antimicrobial activity against periodontal pathogen Porphyromonas gingivalis. The potency of the
bromophenol might be due to the at it has the structure 3-bromo-4,5,dihydroxyl benzene unit as the bromination
promotes antimicrobial activity. This bromophenol potency was found to be well above the standard drug Metronidaole
and thus provides a promising drug because of its natural occurence and less chances of risk of side effects.
However, further invivo studies can be done to validate the results.
ACKNOWLEDGEMENT
I thank the Almighty for rendering me health and strength to complete the project successfully. I whole
heartedly thank the founders of Karunya University, for permitting me to do this work I also profoundly thank
Arathy Mohanan for helping me in this study.
162
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enolase connection (2010). Nature Rev. Rheumatol. 6, 727730.
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gingivalis. Microbiol. Mol. Biol. Rev. vol. 62, pp. 1244-1263, 1092-2172.
9. Aas J., Paster B.J., Stokes L.N., Olsen I., Dewhirst F.E (2005). Defining the normal bacterial flora of the
oral cavity.J. Clin. Microbiol.;43:57215732.
10. Paster B.J., Olsen I., Aas J., Dewhirst F.E (2000). The breadth of bacterial diversity in the human periodontal
pocket and other oral sites.Periodontol. 42:8087.
11. Abusleme, L. (2013). The subgingival microbiome in health and periodontitis and its relationship with
community biomass and inflammation. ISME J. 7, 10161025.
12. George Hajishengallis, Richard P. Darveau, and Michael A. Curtis 2012.
The Keystone Pathogen Hypothesis.
Nat Rev Microbiol. Oct; 10(10): 717725.
13. Nymphea Pandit, Radha Changela, Deepika Bali, Priyanka Tikoo, Shalini Gugnani Por phyr omonas
gingivalis : Its virulence and vaccine . 7 (1) 5:1-58.
14. Smith, G.M. 1944. Marine Algae of the Monterey Peninsula, California. Stanford Univ., 2nd Edition.
15. Choi JS, Bae HJ, Kim SJ, Choi IS, 2011. In vitro antibacterial and anti-inflammatory properties of seaweed
extracts against acne inducing bacteria, Propionibacterium acnes J Environ Biol. 32(3):313-8.
16. Wijesekara, I.; Pangestuti, R.; Kim, S.K. Biological activities and potential health benefits of sulfated
polysaccharides derived from marine algae. Carbohydr. Polym. 2011, 84, 1421.
17. Guven, K.C.; Percot, A.; Sezik, E. Alkaloids in marine algae. Mar. Drugs 2010, 8, 269284.
18. El Gamal, A.A. Biological importance of marine algae. Saudi Pharm. J. 2010, 18, 125.
19. Katsui, N.; Suzuki, Y.; Kitamura, S.; Irie, T. 5,6-dibromoprotocatechualdehyde and 2,3-dibromo-4,5-
dihydroxybenzyl methyl ether: new dibromophenols from Rhodomela larix. Tetrahedron 1967, 23, 1185
1188.
20. Katsui, N.; Suzuki, Y.; Kitamura, S.; Irie, T. 5,6-dibromoprotocatechualdehyde and 2,3-dibromo-4,5-
dihydroxybenzyl methyl ether: new dibromophenols from Rhodomela larix. Tetrahedron 1967, 23, 1185
1188.
21. Saenger, P.; Pedersn, M.; Rowan, K.S. Bromo-compounds of the red alga Lenormandia prolifera.
Phytochemistry 1976, 15, 19571958.
163
In silico docking studies of thymoquinone with cancer and
apoptotic targets proteins, inhibits proliferation and induces
apoptosis in breast cancer cells
Sonia Raj, K., Sumathi, S* and Padma, P.R
Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore
Abstract
Plants have been used for long periods as a medicinal source which plays a central role in healthcare system
around the world. In India, number of new breast cancer cases is about 1,15,000 per year. Thymoquinone is one
of the major active constituent present in Nigella sativa and it has numerous medicinal properties. In the present
study, the apoptosis inducing activity of thymoquinone (TQ) was tested using human breast cancer cell line
MCF-7 and MDA MB 231 in the presence/absence of standard chemotherapeutic drugs tamoxifen/etoposide in
vitro. The apoptosis inducing activity was assessed by cell viability, morphology and nuclear changes by
various staining techniques were carried out. In order to get a clear view about the interaction of the TQ with
cancer and apoptotic targets, in silico studies were performed. Our observation suggested that, on treatment
with TQ the percent viability was found to be decreased in cancer cells and increased in non-cancerous cells
HBL-100 and regulating the cancer and apoptotic target proteins thereby preventing the cancer interaction and
development.
Keywords: MCF-7, MDA MB 231, tamoxifen and etoposide.
INTRODUCTION
N.sativa L. belongs to family Ranunculaceae and the exceptional components of the plant are in use for
medicinal purposes to cure various maladies. TQ, is one of the active principles of N.sativa1. Carcinogenesis is a
multistep process which happens in three stages along with initiation, promotion, and progression that results in
malignant tumors2. Breast cancer has emerged as the leading site of cancer amongst women in India.
Among males and females, breast cancer alone is expected to go to the figure of 100,000 by the year of
20203,4. In silico studies are used for drug layout during the last few years. It helps in providing the fast indication
of capability risks to be used of a drug. Furthermore, it allows providing a mechanistic expertise of these predictions,
to give an explanation for why a compound is predicted to be active or inactive form5. Within the gift observe, we
embarked into study to assess the anticancer activity of TQ mediated inhibition in MCF-7 and MDA MB 231 breast
cancer cells. Our final results definitely determined out that TQ brought about apoptosis and shows excellent
interaction with cancer and apoptotic target proteins.

Drugs and Chemicals
TQ (Sigma-Aldrich), Human breast cancer cell lines MCF-7 and MDA MB 231 were obtained from the
National Centre for Cell Science (NCCS), Pune, India. ANNEXIN V/FITC staining become performed as according
to the protocol of BioVision.
Preparation of Compound
500mM stock solution was prepared in DMSO and stored in the refrigerator, wrapped in aluminum foil, to
avoid dimer formation and different concentrations of TQ were prepared from the stock solution using double
distilled water.
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Cell viability assay
The extent of survival of the cells induced by oxidative stress both in the presence and absence of TQ was
studied by way of sulphorhodamine B assay as proposed with the aid of 6.
Giemsa staining
The diluted Giemsa stain 10l was added to the slide and spread by placing another cover slip over it. The
cells were then observed for morphological changes using a phase contrast microscope (Nikon, Japan) at 400X
magnification. The number of apoptotic cells and normal cells were counted7.
DAPI staining
Apoptotic cells have been detected with DAPI (4-6-diamidino 2-phenyl indole) staining techniques as
explained by8.
PI staining
The nuclear changes in the apoptotic cells had been determined through PI staining as described by9.
Detection of cell death by Annexin V/ FITC staining

ANNEXIN V/FITC staining was done as per the protocol of BioVision.
In silico docking of apoptotic and cancer protein targets with TQ

To find the interaction between the protein and a ligand, molecular docking studies were performed. The 3-
dimensional structure of TQ changed into retrieved from PubChem while apoptotic and cancer proteins have been
retrieved from the protein data bank (PDB) (http://www.rcsb.org). The structures of the target proteins had been
loaded to the Maestro window of Schrodinger software (version 9.3). TQ was subjected to ADME profiling the
usage of QikProp 3.0 module of Schrodinger Drug Design Suite.
Preparation of the ligand and protein

LigPrep 2.3 Mastero 9.0 window of Schrodinger became used for a preparation of the ligand. LigPrep
additionally permits the addition of hydrogen atoms, removal of unwanted molecules and neutralizes charged
groups. The outcomes were stored and the prepared ligands have been then used for docking. The Maestro is the
graphical user interface (GUI) of the entire Schrodinger suite. The protein preparation wizard accepts a protein
from its raw state, (which can also include missing hydrogen atoms, incorrect bond order assignments, charge
states or orientations of various groups) to a state in which it is properly prepared for calculations.
All statistical evaluation had been carried out the use of one-way ANOVA with the level of significance at a
P value of not greater than 0.05 the use of Sigma Stat package version 3.1. The parameters of the experiments are
expressed as Mean S.D. All treatments and assays were carried out in triplicates.
RESULTS
SRB assay
The results of the SRB assay revealed that the viability of cancerous cells MCF-7 and MDA MB 231
decreased drastically on exposure to TQ but TQ improved the viability of non-cancerous HBL-100 cells. On the
other hand, when administrated with tamoxifen/etoposide the present viability was further decreased in cancer
cells and non-cancerous cells. Among the cancerous cells, the effect was more pronounced in MDA MB 231 than
MCF-7. The results are represented in Figure 1.
165
Morphology and Nuclear staining
The reduction of cell volume, cell shrinkage, chromatin condensation and cytoplasmic blebs were also
observed by visualizing the cells after Giemsa staining which stains the chromatin and nuclear membrane (Plate 1).
DAPI is a fluorescent stain that binds strongly to A-T rich regions in DNA that can pass through an intact cell
membrane and can be used to stain both live and fixed cells. the photographic pictures are shown in Plate 2.
Propidium iodide is a red-fluorescent nuclear and chromosome counter stain and used to detect dead cells in a
population (Plate 3).Membrane damage was determined by Annexin V/FITC-PI staining. the principle behind this
is, after initiating apoptosis, cells translocate phosphotidyl serine from inner plasma membrane to cell surface. By
this the phosphotidyl serine was easily detected by fluorescent stain (Plate 4). Here we found more number of
apoptotic cells in TQ treated group in cancer cells whereas, this effect was less pronounced in normal HBL-100
cell line. In all the staining techniques namely Giemsa, DAPI, PI and Annexin V/FITC/PI TQ showed similar
effects in MDA MB 231 which showed more cytotoxicity compared to MCF-7 breast cell line.
In silico docking studies

Our next attempt was to study the molecular interaction of TQ with major apoptotic and cancer target
proteins by using in silico approach. In silico modelling is a new approach to clinical chemistry for the optimization
of screening and testing by means of the observation of a particular compound. In this context, it was obvious that
TQ showed good human oral absorption, hydrogen bond donors and acceptors. TQ obeys perfectly the Lipinskis
rule of five and is found to have good absorption and permeability.
ADME studies of TQ
TQ was subjected to ADME profiling using QikProp 3.0 module of Schrodinger Drug Design Suite and the
results are presented in Table 3. The QikProp results for thymoquinone showed compliance to Lipinskis rule of
five, good hydrogen bond acceptor and excellent human oral absorption in GI tract and good MDCK permeability.
Since thymoquinone is hydrophobic in nature it showed no hydrogen bond donor. This study predicts that the
compound thymoquinone has good pharmacological activity, and was further subjected to docking using Glide 4.5
and the selected apoptotic and cancer proteins are given in Table 1 and 2.
The ligand showed efficient docking score -5.692 with Bax receptor, involved in the extrinsic pathway of
apoptosis. Among the various intrinsic proteins studied, TQ showed better interaction with the apoptotic proteins
as determined by the glide score. All the results were analyzed for their glide energy and good contacts, which also
showed the efficiency of docking. In cancer target proteins TQ showed good docking scores among which, the
166
highest docking was found in Tubulin -4.977. The results of docking showed that the thymoquinone showed good
interaction with the target proteins involved in apoptosis and cancer as seen in Table 4 and 5. The interactions are
given in Plate 5.
DISCUSSION
Our current research is focussed mainly on exploring TQ that are present in medicinal plants for the
treatment of various diseases and also provide scientific evidence for the use of the plants in the traditional folklore.
One such compound is TQ, which is one of the major active constituents of N.sativa, commonly known as black
cumin seed, belonging to the family Ranunculaceae. Nuclear changes such as nuclear fragmented bodies and
deformed nuclei can be visualized during apoptosis or necrosis by using DAPI staining10. The another study was
has reported that when five different solvent fractions from the leaves of S.grandiflora were tested on five cancer
cell lines such as MCF-7 cell line11. DAPI, Annexin V-FITC and PI triple fluorescence staining showed cell apoptosis
167
after HepG2 treatment with bufalin12. In silico molecular docking is one of the advanced technique to discover
novel ligands for receptors of known structure and thus play a key role in structure-based drug design13. Molecular
docking continues to hold great promise in the field of computer-based drug design which screens small molecules
by orienting and scoring them in the binding site of protein. As a result, novel ligands for receptors of known
structure are designed, and their interaction energies are calculated using the scoring functions14. In our results,
TQ showed good docking score in both apoptotic and cancer target proteins.
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168
Analysis of Citrus limetta and Citrus sinensispeel for their
phytoconstituents
G.T. Iswariya, P.R. Padma and R. Nirmaladevi*
Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore
E-mail id: nirmaladevi.saravanan32@gmail.com
Abstract
Emerging evidences suggest that secondary metabolites present in the medicinal plants play a critical role in
human health. Nearly13,000 secondary metabolites have been isolated from the medicinal plants which accounts
to 10% of the total metabolites. These secondary metabolites are chemically, taxonomically and extremely
diverse compounds with various biological functions. In the present study, Citrus fruit peel extracts were
screened for the presence of various phyconstituentsby preliminary phytochemical screening, spectral analysis
(UV absorption, FTIR) and chromatographic analysis (HPTLC, HPLC).Analysis of Citrus fruit peel revealed that
it contained various phytochemicals like flavonoids, phenols, alkaloids, tannins, terpenoids and steroids. The
distinct peaks observed in the UV absorption spectrum of methanol extracts of Citrus fruit peel indicates the
presence of various secondary metabolites which is further confirmed by HPTLC and HPLC analysis. The FTIR
spectrum revealed the presence of functional groups such as hydroxyl and NH which might be indicating the
presence of phytoconstituents such as phenol or flavonoid.To conclude, Citrus limetta and Citrus sinensispeel
extracts contains various phytochemicalsalong with the bioactive compound naringeninwhich attributes to the
medicinal property of the Citrus fruits.
Key words : Phytochemicals, Citrus limetta and Citrus sinensis
INTRODUCTION
Phytochemicals occur naturally in the leaves, roots, barks, flowers, fruits and seeds of the medicinal plants
which are known to combat and protect against various degenerative diseases including cancer, coronary heart
diseases, diabetes and other infectious diseases. Medicinal plants are the richest source of drugs in traditional
system of medicine1-3. Since ancient times, plants have been explored particularly in search of new drugs which
ledto the use of large number of herbs to treat various diseases. Approximately 80% of the world population relies
on traditional medicine for primary health care4. The plants of Citrus genes have been recognized for their medicinal
properties in many countries ever since the Middle Ages 5. Citrus fruits belong to the most important horticultural
crops, because of their nutritional value and special flavor.C. limetta is known to be an antihyperglycemic plant, its
fruit and leaves are used for common cold, decreasing cholesterol level, fever regulation, regulating inflammation,
digestive disorder and as well as blood pressure modulator6. C. sinensis is one of the most important sweet orange
varieties cultivated worldwide and grown primarily for juice production7. It is used in food and medicinal preparations
for its strong antioxidant potential8. Citrus fruit extracts have also been found to demonstrate anti-cancer, anti-
inflammatory, antitumour and blood clot inhibition activities9. The components responsible for these beneficial
effects are not fully known. Thus, the present study aimed to investigate the phytoconstituents present in the peel
of C. limettaand C. sinensis.
METHODOLOGY
Sample collection and preparation
Fresh fruits (Citrus limettaand Citrus sinensis)were procured from local markets in Coimbatore, India.
169
Then the fruits have been washed with the running tap water and dried. The peels were separated and sliced
into small pieces. The peel waskept in an incubator for 12 hours at 40C. About 20g of each sample is mixed
with 100ml of methanol and kept overnight in a shaker incubator. Then the extracts were filtered and used for
followinganalysis. Earlier studies performed revealed that among the various solvents extracts analyzed the
methanol extracts of C.limettaand C.sinensis mediated maximum activity under in vitro conditions and hence
methanol extract was chosen for present study.
Preliminary Phytochemical screening

The methanol extract of peel of C.limettaand C.sinensis, were screened for the presence of Phytochemicals
according to the method of (Khandelwal, 2002).
UV-Visible Spectral analysis

An absorption spectral analysis was done by a survey scan of methanol extract of C. limettaand
C.sinensispeelin a nanospectrophotometer (Optizen). The instrument was set to scan mode and the absorption
spectrum was obtained in the range of 200nm to 800nm.
HPTLC analysis
The methanol extractsof peel of Citrus limettaand Citrus sinensiswere loaded using a Hamilton syringe in
(CAMAG LINOMAT 5) instrument in the 10 x10 silica gel G60 F254 plate as 8mm band. The TLC plate loaded
with sample was kept in a TLC twin trough developing chamber which was saturated with the respective mobile
phase and developed up to 90mm.
HPLC analysis
The methanol extract of C.limettaand C.sinensispeel were dissolved in an appropriate volume of HPLC
grade methanol and 20l of the sample was injected using Hamilton syringe into the reverse phase C18 column of
the HPLC system (Sigma-Aldrich equipped with PDA detector). The sample analysis was performed at room
temperature in the wavelength range of 210-440nm at1000psi and the mobile phase used was 100% HPLC grade
methanol 60minutes at a flow rate of 1ml/minute.
FT-IR Spectral Analysis

IR spectroscopic study was carried out in the methanol extract of Citrus limettaandCitrus sinensispeel.
Infrared light from the source passes through a scanning Shimadzu interferometer and Fourier Transformation
gave a plot of intensity versus frequency.

Preliminary phytochemical screening
The results of the preliminary phytochemical analysis of the methanol extract of Citrus limettaand Citrus
sinensispeel are depicted in Table1.The results showed the presence of all the major phytochemicals such as
alkaloids, flavonoids, tannins, phenols, steroids and terpenoids. The spectral and chromatographic analyses were
performed to substantiate the data obtained.
170
Table 1: Preliminary Phytochemical analysis of peel extract of C.limettaandC.sinensis
S.No Components C.limetta C.sinensis

1. Alkaloids + +
2. Flavonoids + +
3. Phenols + +
4. Saponins - -
5. Steroids + +
6. Tannins + +
7. Terpenoids + +
(+ ) Present (-) Absence
UV-Visible Spectral analysis

The presence of secondary metabolites in the extract was identified using UV spectrophotometric analysis.
The peel extract of the selected Citrus fruits were studied usingUV-Visble spectroscopy at the wavelength range of
220 to 800nm. Distinc t pe aks we re obs erve d a t 220nm, 276nm a nd 334nm in the pee l e xtra ct of
C.limettawhileC.sinensisextractshowed spectra in the peaks at 221nm, 277nm and 328nm as observed in Figure 1
(a, b). A slight variation in the peaks obtained might reflect the presence of similar but derivatized compound.
a)
b)
FigureFigure 1- UV Spectrum
1- UV absorption absorption Spectrum
of methanol of(a)
extract of methano l extract of
and (b) C.sinensispeel
(a) and (b ) C .sinensispeel
HPTLC analysis
HPTLC profile of the standard alkaloids, flavonoids and phenols along with fruit extracts are given in the
Figures. 2, 3 and 4 respectively. The bands obtained in the peel extract were compared with the reference com-
pound indicates the compounds identified from both the samples were similar to the reference standard. HPTLC
profile shows the presence of alkaloids in both peel and pulp extracts in comparison with standard colchicine.
Similarly the presence of flavonoid was confirmed with standard naringenin while phenol was confirmed witheugenol.
HPLC analysis
The C. limettapeel extract showed the presence of one major peak and one minor peak while two major
peaks were observed in C.sinensispeel extract which is depicted in Figure 5. More spectral studies need to be
carried to understand the nature of the compounds corresponding to the peaks observed in HPLC profile.
FT-IR Spectral Analysis
In order to understand the nature of the bioactive compounds present in the Citrusfruit pulp extracts, FTIR
171
analysis was done. In FTIR spectrum, broad bands at 3324.46, 3317.71 cm-1 in peel extract showed the presence
of -OH group or -NH group whereas for C.sinensis the bands were observed at 3332.53, 3310.95, 3032.23 cm-
1.The presence of bands at 2943.50, 2832.59, 2609.80, 2526.66, 2504.67 cm-1; 2937.71, 2830.66, 2519.14 cm-
1; 2943.50, 2830.55, 2679.24, 2520.11 and at 2930.00 cm-1 indicates the presence of aromatic group.
Figure 2- HPTLC profile of alkaloids
a)Colchicine b)C.limettapeel C)C. sinensis peel
Figure 3- HPTLC profile of flavonoids

a)Naringenin b)C.limetta peel C)C.sinensis peel
Figure 4- HPTLC profile of Phenols

b)C.limetta peel C)C.sinensis peel
a)Eugenol
Figure 5- HPLC chromatogram

a)Naringenin b)C.limetta peel C)C.sinensis peel
The narrow bands which are seen at 1709.09, 1633.78, 1598.16, 1588.52 cm-1 in the peel extract of
C.limetta confirmed the presence of carboxyl group. The carboxyl group were also observed methanol extract
peel of C.sinensis at 1635.71, 1595.20, 1448.87 and
1409.06 cm-1. The spectrum showed 5 bands in the peel extract of C.limetta while 4 bands C.sinensispeel
confirmed the presence of hydroxyl (-OH) groups which are shown in Figure 6.
172
a)
b)
Fig 6- FT IR spe ctr um of P eel of a) C.l ime tta, an db) C. sinensi s
Citrus peels are the primary waste fraction of citrus fruits and have been used as a source for molasses,
pectin, cold-pressed oils, and limonene and also fed to livestock, used to scent perfumes and soap products10,11. In
traditional Chinese medicine, dried tangerine peel is used to treat a wide array of ailments, including bronchial
asthma, dysepsia, and cardiac circulation and have antioxidant, anticancer, anti-atherogenic, and anti-inflammatory
activities12. The results of the present study indicates that it contained various phytochemicals like flavonoids,
phenols, alkaloids, tannins, terpenoids and steroids in their methanol extract. However isolation and characterization
of these compounds and validating their therapeutic efficacy against different pathological condition is required for
clinical implementation.
CONCLUSION
Spectroscopic technique has become a powerful and analytical tool for the qualitative and quantitative
analysis of pharmaceutical and biological materials. Citrus limettaand Citrus sinensispeel are rich in bioactive
compounds. About 34% of Citrus fruits are made into juices, therefore; large amounts of residues or waste
generated resulting in an environmental pollution and loss of many valuable pharmaceutical components present in
Citrus fruits peels, can be redirected to use in pharmaceuticals and food industries. Further work is needed to
refine the techniques and to quantitatively analyze the compounds and structurally investigate the different active
constituents present in the peel of different Citrus fruits.
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2. Yadav RNS andAgarwala M, , J. Phytology, 3(12), 2011, 10-14
3. Wadood A, Ghufran M, Jamal SB, Naeem M, Khan A,Biochem Anal Biochem, 2, 2013, 144.
4. Pradeep A, Dinesh M, Govindaraj A, Vinothkumar D, Babu NGR, Int. J. Biol. Pharm Res,5(1), 2014, 48-50.
5. Perez YY, Ferrer EJ, Alonso D, Amaro CAB, Zamilpa A, J. Ethnopharm, 128 (2010) 611614
6. KunduSen S,Haldar PK, Gupta M,Mazumder UK,Saha P,Bala A, Bhattacharya S, Kar B, ISRN Endocrinology,
2011, 1-6.
7. Xu Q, Chen LL, Ruan X, Chen D, Zhu A, Chen C, Bertrand D, Jiao WB, Hao BH, Lyon MP, ChenJ,et
alNature Genetics, 45, 2013, 59- 68.
8. Chen ZT, Chu HL, ChyauCC, Chu CC, Duh PD, Food chem, 135, 2012, 21192127.
9. Goulas V andManganaris GA, Food Chemistry, 131, 2012, 3947.
10. Monajemi R, Oryan S, Roohani AH, Ghannadi A, Jafarian A, Iranian J. Pharma Res, 3, 2005, 183-187.
11. Jeong SM, Kim SY, Kim DR, Jo SC, Nam KC, Ahn DU, Lee SC, J. Agric. Food Chem, 52,2004,3389-3393.
12. Ho SC and Kuo CT, Food ChemiToxicol, 71, 2014, 176182.
173
Evaluation of thrombolytic potential of Basella alba L. and
Talinum portulacifolium
Pappa Ammal1 Pushpa2 A and Lourdu Silvi Sinduja2 R
1
Dept. of Science and Humanities, 2Dept. of Biochemistry, Biotechnology and Bioinformatics,
Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore- 641043 Tamil Nadu, India
Corressponding Author: pappins.in@gmail.com
Abstract
Green leafy vegetables have been used as medicine since ancient times. They contain diverse pharmacologically
active compounds which play a vital role in diet and nutrition. Leafy vegetables namely Basella alba L and
Talinum portulacifolium were selected for the present study. Aqueous and ethanolic extract of fresh leaves and
stem were analysed for the qualitative detection of phytochemicals. Thrombolytic activity of the selected plants
was determined using human blood and compared with that of standard streptokinase. Thrombolytic activity of
Basella alba L and Talinum portulacifolium revealed that the aqueous extract of both the plants exhibited
higher percentage of thrombolytic activity than the ethanolic extract. Of the two different selected plants
Basella alba L exhibited higher percentage of clot lysis. Total cholesterol was determined in the blood samples
collected for thrombolytic activity. A comparison was made between total cholesterol level and clot lysis by the
plant extract and a correlation analysis was also carried out. The results indicated that there is no correlation
between serum cholesterol and thrombolytic activity of the plant. The findings revealed that the selected plants
can be exploited for the development of thrombolytic drugs.
Key Words : Green Leafy Vegetables, Basella alba L , Talinum portulacifolium, Thrombolysis
INTRODUCTION
Plant products with medicinal properties have been extensively used in pharmaceutical and food industry
and have also been popular as health-promoting herbal products1. Thrombus is one of the major blood circulation
problems which can lodge in a blood vessel and block the flow of blood in that location and depriving tissues of
normal blood flow and oxygen2. Atherothrombotic diseases such as myocardial or cerebral infarction are serious
consequences of the thrombus formed in blood vessels3. Thrombolytic agents like tissue plasminogen activatore (t-
PA) Urokinase (UK) and Streptokinase (SK) are used all over the World for the treatment of these diseases. But these
drugs have certain limitations such as hyper risk of hemorrhage, severe anaphylactic reaction and lack of specificity4.
Plant based medicaments are used in the developing countries, but recent times there has been an increase in the use
of herbal medicines in the developed World too5. Hence the present study has been designed to evaluate the thrombolytic
properties of the plants namely Basella alba L and Talinum portulacifolium.
Identification of the selected plants
The selected plants namely Basella alba L and Talinum portulacifolium were identified and authenticated
by Botanical Survey of India, Coimbatore.
174
Phytochemical Analysis
Fresh leaves and stems of the selected plants were homogenized using water and ethanol and these extracts
were subjected to phytochemical analysis.
Determination of thrombolytic activity
Thrombolytic activity of the aqueous and alcoholic extract of the selected plants was analysed using
human blood and compared with the standard streptokinase 6.
Estimation of Total cholesterol
Total cholesterol was estimated in serum samples obtained from the above human blood samples 7.
RESULTS
Phytochemical Analysis
In the present study phytochemical screening was carried out and the results are given in Table1.
Many of the phytoconstituents were identified in ethanolic extract but not in the aqueous extract. But,
quinones were identified in the ethanolic stem extract of both the plants. Volatile oil was completely absent in all the
extracts.
Thrombolytic Activity
Clot lysis activity of aqueous and ethanolic leaf extracts was determined and the results are depicted in
Table 2
In in vitro clot lysis method the crude aqueous extract of Basella alba L and Talinum portulacifolium was
found to exhibit 13.61% and 12.56% respectively at dose of 0.1 mg/ml. The standard streptokinase showed
17.5% of thrombolysis. Ethanolic extract showed 9.46% and 9.19% respectively. There was a significant decrease
in the thrombolytic activity of both the plants as the concentration of the sample was increased. The thrombolytic
activity was more pronounced in the aqueous extract when compared with the ethanolic extract.
Comparison of serum cholesterol and thrombolytic activity of aqueous extract of Basella alba L and Talinum
portulacifolium
Serum cholesterol was determined and compared with thrombolytic activity of the plants. The values are
indicated in Table 3
Mild negative correlation was observed with aqueous extract of Basella alba L and Talinum portulacifolium.
This lack of correlation indicates that both the plants can render good protection against clot induced blocks in
blood vessels, irrespective of the cholesterol levels of the individual.
175
Table 1 Phytochemical Constituents of Basella alba L and Talinum portulacifolium
Basella alba L Talinum portulacifolium

Phytochemicals
Ethanolic extract Aqueous extract Ethanolic extract Aqueous extract
Leaf Stem Leaf Stem Leaf Stem Leaf Stem
Carbohydrates + + + + + + + +
Amino acids and + + + + + + + +
Proteins
Phenols + + - - + + - -
Glycosides + + - - + - - -
Saponins + + - - + + - -
Quinones - + - - - + - -
Flavo noids + + - - + + - -
Tannins - + - - + + - -
Volatile Oils - - - - - - - -
Terpenoids + + - - + + - -
Alkaloids + + - - + + - -
+ Present, - Absent
Table 2 Thrombolytic Activity of Basella alba and Talinum portulacifolium
Conc. Of the Thrombolytic Activity(%)

Sample
(mg/ml) Aqueous Extract EthanolicExtract
Basella alba L Talinum Basella alba L Talinum portulacifolium
portulacifolium
Streptokinase 17.51.04
Water 0.370..07
0.1 13.611.72 12.561.32 9.460.93 9.190.92
0.5 7.930.57 7.750.848 4.600.62 4.390.58
1.0 4.060.43 3.870.39 2.960.36 3.040.37
176
Table 3 : Comparison of Serum Cholesterol and Thrombolytic Activity of Aqueous Extract of
Basella alba L. and Talinum portulacifolium
Cholesterol(mg/dl) Clotlysis (%)

Basella alba L Talinum portulacifolium
5.79 5.42
270 5.9 5.78
210 6.1 5.49
240 6.2 5.67
210 6.24 6.15
275 6.4 5.95
230 6.87 6.12
150 9.5 9.34
185 9.5 8.82
195 9.6 8.74
200 9.65 9.34
200 9.75 8.73
175 10.01 9.61
130 10.35 9.46
140 10.55 9.75
180 11.29 10.56
160 11.35 10.56
190 11.97 11.05
165 13.35 12.25
150 14.36 13.43
DISCUSSION
Phytochemical analysis of selected plants contain a wide variety of primary and secondary metabolites.
The beneficial medicinal effects of plant materials typically result from the secondary products present in the plant
although, it is usually not attributed to a single compound but a combination of the metabolites8. Secondary metabolites
like flavonoids, carotenoids and phenolic compounds are present in Spinacia oleracea9. Thrombolytic activity of
the selected plants have moderate thrombolytic activity compared with the standard streptokinase. The aqueous
extract of Ocimum sanctum, Curcuma longa, Azadirachta indica and Anacardium occidentale showed moderate to
good clot lysis activity 10. Significant thrombolytic activity was demonstrated in the aqueous soluble fraction of
the stem bark of Bridelia tomentosa11. The findings revealed that aqueous soluble phytoconstituents have higher
thrombolytic activity compared to ethanol soluble fraction. Comparison of serum cholesterol and thrombolytic
activity suggests that both the plants can act as potential thrombolytic agents, irrespective of the cause of clot
formation. This property could preferably be explored in the treatment of thrombus in cardiovascular patients. The
present study might have important implications in cardiovascular health in developing countries where cheap and
effective medicines are the need of the hour. This investigation can also help in developing cost effective novel
thrombolytic agents from common plant sources, which are advantageous over synthetic drugs.
177
REFERENCES
1. Abourashed, E.A (2013) Antioxidants, 2: 309 325.

2. Thrombus (2011) Online http://adam.about.net/encyclopedia/ Thrombus.htm
3. Prasad, S., Kashyap, R.S., Deopiyari, J.Y., Purohit, H.J., Taori, G.M and Daginawala, H.F (2007) BMC
Complementary and Alternative Medicines, 7(36): 16.
4. Jennings, K (1996) BMJ, 312:393-394
5. Biswas, K.I., Chattopadhyay, A., Banerjee, Y.A (2002) Curr Sci., 82:1336-1345
6. Prasad, S., Kashyap, R.S., Deopujari, J.Y., Purohit, H.J., Taori, G.M and Daginawala, H.F (2006) Thrombosis
Journal, 4(14): 64 - 69.
7. Allain, C.C., Poon, L., Chan, S.G., Richmond, W and Fu, P (1974) Clinical chemistry, 20: 470.
8. Parekh, J., Jadeja, D and Chandra, S (2005) Turkish Journal of Biology, 29: 203 210
9. Subhash, G.P., Virbhadrappa, S.R and Vasant, O.K (2010) International Journal of Research in Ayurveda and
Pharmacy, 1(1): 78-84.
10. Khan, I.N., Habib, M.R., Rahman, M.M., Mannan, A., Sarkar, M.M.I and Hawlader, S (2011) Journal of
Basic and Clinical Pharmacy, 2(3): 125 127.
11. Anjum, A., Sikder, M.A., Haque, M.R., Hasan, C.M and Rashid, M.A (2013) Journal of Scientific Research,
5(2): 343 351.
178
Genetic diversity of Muntingiacalabura fruits collected from
various districts of Tamil Nadu
Dr.K.Preethia and K.Priyankab
a
Assistant Professor, Department of Microbial Biotechnology, Bharathiar University, Coimbatore.
b
Research Scholar, Department of Microbial Biotechnology, Bharathiar University, Coimbatore.
Abstract
Muntingiacalabura is the sole species in the genus Muntingia. This is a fast growing fruit tree. It is a pioneer
species that thrives in poor soil, able to tolerate acidic and alkaline conditions and drought. It is cultivated for its
edible fruit, and has become naturalized in some other parts of the tropics, including southeastern Asia. Monitoring
the antioxidant properties and the genetic diversity of M. calabura fruits collected from various districts of
Tamilnadu was the main aim of this research work. It was found that all the methanol extract of M. calabura fruits
collected from various districts were antioxidatively active and showed variations in the activity measured by
DPPH assay and super oxide radical scavenging assays. The present study constitutes a prerequisite for the
development of a molecular method suitable in the genetic polymorphisms. RAPD markers have found a wide
range of applications in gene mapping, population genetics, molecular evolutionary genetics and plant and
animal breeding. This is mainly due to the speed, cost and efficiency of the RAPD technique to generate large
numbers of markers in a short period compared with other methods. Therefore, RAPD technique can be performed
in a moderate laboratory for most of its applications
Key words: Antioxidants, RAPD, Genetic mapping.
INTRODUCTION
The use of traditional medicine is wide spread and plants still present a large source of novel active
biological compounds with different activities, including anti-inflammatory, anti-cancer, anti-viral, antibacterial and
cardioprotective activities. Antioxidants are gaining a lot of importance as a panacea for a large number of life-style
which scavenge free radicals and may there by break the reaction chains of pathogenesis of coronary heart disease
and cancer.
In general antioxidants may be obtained by two ways, natural and synthetic ones. Because of the high
carcinogenicity of synthetic antioxidants, developing effective natural antioxidants may have significant implications
for human health. Muntingiacalabura, the sole species in the genus Muntingia, is a flowering plant native to
southern Mexico. It is a pioneer species that thrives in poor soil, able to tolerate acidic and alkaline conditions and
drought. It is cultivated for its edible fruit, and has become naturalized in some other parts of the tropics, including
southeastern Asia. This research focuses on collecting the fruits from different places of Tamil Nadu and analysing
and differentiating their antioxidant activity through DPPH and super oxide radical scavenging activity and identifying
their genetic variation throughmolecular techniques using RAPD.It is an inexpensive and rapid method requires
only small amount of genomic DNA and can produce high levels of polymorphism and may facilitate more effective
diversity analysis in plants at molecular level (Ganesh et al., 2007).

Fruits
M.calaburafruits were collected from 10 districts of Tamilnadu namely Namakkal, Salem, Trichy, Tirupur,
Erode, Karur, Kanchipuram, Madurai, Dharmapuri, and Nilgiris for the analysis ofcomparison of antioxidant activity
among the individuals and their genetic diversity.
179
Extraction for antioxidant activity
Firstly, the fruits (100 g) were blended and extracted with methanol for 2 hours at room temperature in an
orbital shaker; then the extract was centrifuged and the supernatant was collected and dried using a rotary evaporator
at 40oC. The dried methanol extract was obtained, and various concentrations of the extracts were prepared from
the resultant extract to determine the in vitro antioxidant activity.
Evaluation of antioxidant activity of M. calabura fruit extract
DPPH radical scavenging assay
The effect of extracts on DPPH radical was determined using the method of Szabo et al. (2007.The ability
to scavenge DPPH radical was calculated by the following equation:
[(Abs control Abs sample)]
DPPH radical scavenging activity (%) = x 100
(Abs control)
Superoxide anion scavenging activity
Measurement of super oxide anion scavenging activity of M. calaburaextracts was based on the method
described by Liu et al.(1997). The percentage inhibition of super oxide anion generation was calculated using the
following formula:
(A control A sample)
Inhibition of super oxide generation (%) = A control x 100
Genetic diversity analysis:

DNA Extraction for genetic diversity analysis:Plant tissue (fruit) was sampled from 10
M. calabura individuals representing 10 districts of Tamilnadu, India. Total DNA was extracted according
to the procedure of Dellaportaet al.(1983). The quality andquantity of DNA were checked by analytic electrophoresis
in a 1%agarose gel containing ethidiumbromide (0.5 mg ml-1) in 1XTBE as described by Sambrooket al. (1989).
Primers and PCR Assays : Totally 10 primers were used for our current studies. The primer was obtained from
Sigma, India and used in the PCR. Primer sequences used in the study was given in Table 1 with their
sequences. Isolated DNA samples were amplified by PCR conditions described by Jones et al.(2002) with
minor modifications
Table 1. The primers used for the genetic diversity analysis
S.No Name of Primer Primer sequence (3 to 5)
1 OPAG-07 CACAGACCTGT
2 OPAG-O8 AAGAGCCCTC
3 OPAG-O9 CCGAGGGGTT
4 OPV-O6 ACGCCCAGGT
5 OPV-O9 TGTACCCGTC
6 OPV-2O CAGCATGGTC
7 OPX-O1 CTGGGCACGA
8 OPX-17 GACACGGACC
9 OPP-O1 GTAGCACTCC
10 OPP-O4 GTGTCTCAGG
180
AgaroseGel Electrophoresis:Agarose gel electrophoresis was carried out in a horizontal submarine electrophoresis
unit. Then the unit was switched off and was visualized under UV Transilluminator for the isolated DNA.
The DNA profiles were manually scored directly from gel photographs and only repetitive bands, i.e.,
those occurring in the two duplicates, were considered.

In our study, the fruits of M. calabura collected from various districts of Tamilnadu were evaluated for its
variation in their antioxidant activity. The IC50values were obtained for the tested assays and given in Table 2
Table 2.Showing the IC50 Value of DPPH assay and SOR scavenging assays
NAME OF IC50 VALUE G/ML

S. NO
DISTRICT DPPH SOR
1 Namakkal 78 70
2 Salem 91 80
3 Trichy 92 81
4 Tirupur 88 75
5 Erode 90 79
6 Karur 85 75
7 Kanchipuram 80 78
8 Madurai 76 74
9 Dharmapuri 72 68
10 Nilgiris 95 83
From the results obtained it was clearly notified that there was a moderate variation in the antioxidant
activity of the fruits collected from different place due to environmental factors which also play a major role in the
activity of the plant.
All the ten individual samples M. calabura were examined for RAPD genetic markers with ten decamer
primers. The primers were subjected to optimized conditions for PCR, were applied to all sample DNA.The
amplified fragments ranging from 100 bpto above 1000bp. These primers exhibited discriminatory band patterns
among the plant.
Among the 10 primers tested. primer 1, 3, 4 and 5 produce number of bands in all samples most of the
primers did not produced bands in 9th and 10th samples of the plant.
The lowest molecular weight bands observed in most of the samples (150 bp) and above 1000bp
molecular weight bands were observed from number of samples.In this RAPD analysisthe highest molecular
weight bands were observed in first primers pattern. In our investigation number of polymorphic bands was
observed.
CONCLUSION
Methanolic extract of M. calabura fruits collected from various districts of Tamilnadu were antioxidatively
active and showed variations in the activity measured by DPPH assay and super oxide radical scavenging assays
and RAPD technique was employed to analyse the genetic variations between each fruits.Therefore, RAPD technique
can be performed in a moderate laboratory for most of its applications (Bardakci, 2001).
181
Fig 1:RAPD analysis of M. calabura using different primers
Lane 1 Namakkal Lane 2 Salem Lane 3 Trichy
Lane 4 Tirupur Lane 5 Erode Lane 6 Karur
Lane 7 Kanchipuram Lane8 Madurai Lane 9 Dharmapuri
Lane 10 Nilgiris M - Marker
ACKNOWLEDGEMENTS
The authorsare thankful to the Department of Microbial Biotechnology, Bharathiar University for
providing the necessary facilities to carry out the research programme throughout the dissertation.
REFERENCES
1. Bardakci, F. (2001). Random Amplified Polymorphic DNA(RAPD) markers.Turkish Journal of Biology,25,
185-196.
2. Dellaporta, S. L., Wood, J. and Hicks, J.B.(1983). A plant DNA minipreparation: version II. Plant Molecular
Biology and Reproduction, 1, 19-21.
3. Ganesh, R.S., Parthyiban, K.T., Senthilkumar, R., Thiruvengadam, V. and Paramathma, M. (2007). Genetic
diversity among Jatropha species as revealed by RAPD arkers, Genetic Research and Crop Evolution.
4. Jones,R.C.,Mcnally,J. and Rossetto,M. (2002). Isolation of microsatellite loci from a rainforest tree,
Elaeocarpusgrandis (Elaeocarpaceae) and amplification across closely related Taxa. Molecular Ecology
Notes.2,179-181.
5. Liu, F., Ooi, V.E.C. and Chang, S.T. (1997). Free radical scavenging activity of mushroom polysaccharide
extracts. Life Science. 60: 763-771.
6. Sambrook, J., Fritsch, E. F., Maniatis, T.(1989). MoecularCloning:A LaboratoryManual, 2nd ed., Cold
Spring Harbor Laboratory Press: New York, NY, USA.
7. Szabo, M.R.C., Iditoiu, C., Chambre,D. and Lupea, A.X. (2007). Improved DPPH determination for
antioxidant activity, spectrophotometric assay. Chemical Papers.61: 214-216.
182
Metabolic engineering of phenyl propanoid pathway in Rice
(Oryza sativa)
Safia. Ashwini. M and Sathishkumar. R* 1
Plant Genetic Engineering laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India
E-mail : rsathish@buc.adu.in
Abstract
Nutraceuticals have established their potential roles in the protection of human health against diseases in
addition to nutrition. Isoflavones are phytochemicals similar in structure and function to the female hormone
estrogen. They act as estrogenic, anti-estrogenic, cancer enzyme inhibitors, antioxidants, immune enhancers
thereby protecting women from hormone related cancers - breast and uterine cancer, osteoporosis, menopausal
symptoms, hypertension and cardiovascular diseases. In the present study, we aim to generate transgenic rice
enriched with medicinally valuable natural flavonoids by manipulation of the phenylpropanoid pathway. To
achieve this, we use Glycine max - Isoflavone synthase (GmIFS) gene in rice, which will reroute the existing
pathway that is not normally present in plants. Isoflavone synthesis is restricted to leguminous plants due to the
absence of IFS gene in the non-leguminous members. In this study we have cloned GmIFS in binary vector
using Gateway cloning technology. This will result in nutritionally improved Rice, which should protect women
from many of the commonly occurring chronic diseases like breast cancer, osteoporosis, cardiovascular disease
etc.
Keywords: Isoflavonoids, Isoflavone Synthase (IFS), Naringenin, Gateway cloning, pEARLEY Gate 102 HA
INTRODUCTION
Rice is the staple food for more than half of the Worlds population providing 27% of caloric need and
20% of dietary proteins. Rice is the most studied cereal in animal, human clinical trials and for food fortification1.
The antioxidant content of rice can be linked to low incidence of certain chronic diseases in rice-consuming
regions of the world2. Highest antioxidant activity has been reported in black rice followed by purple, red, and
brown rice varieties. Phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols, tocotrienols, -
oryzanol, and phytic acid are the antioxidant molecules present in rice2.Phenylpropanoids, the plant organic
compounds synthesized from phenylalanine are ubiquitously present in all plants. This pathway is the starting point
for several biosynthetic branches namely, flavonoids, anthocyanins, lignins, stilbenes3, coumarins, plant pigments,
phytoalexins, antioxidants and UV light protectants4.
In leguminous plants, the key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids
to isoflavonoids is the cytochrome P450 mono-oxygenase, isoflavone synthase (IFS). The reported health benefits
of isoflavonoids include reduction of osteoporosis5, improvement in blood cholesterol level, lowering risk of
cardiovascular disease6, relief of menopausal symptoms5, 7, and lowering risk of certain hormone related cancers8,
9
. IFS catalyses the oxidation of 7, 4 dihydroxyflavonone (liquiritigenin) or 5,7,4 trihydroxyflavonone (naringenin)
to daidzein or genistein. Naringenin is ubiquitously present in all plants, liquiritigenin is produced by the legume
specific chalcone reductase10 (CHR). Isoflavone synthesis is restricted to leguminous plants due to the absence of
IFS gene in the non-leguminous members. Isoflavone synthase (IFS) is the entry point enzyme of isoflavonoid
biosynthesis and therefore a key step for engineering isoflavones production in non legumes.
183
Fig. 1-A Partial diagram of the phenylpropanoid pathway showing intermediates and enzymes involved in
isoflavone synthesis. The boxed part indicates the introduced conversion step catalyzed by IFS in the proposed
work. PAL - Phenylalanine ammonia lyase, C4H - Cinnamate 4-hydroxylase, 4CL - 4-coumarate: CoA ligase, CHS
- Chalcone synthase, CHI - Chalcone isomerase, F3H - Favanone 3-hydroxylase, F3H - Flavonoid 3-hydroxylase,
FLS - Favonol synthase, DFR - Dihydroavonol 4-reductase, LDOX - leucoanthocyanidin dioxygenase11.
Hence the present study aims to genetically engineer rice using Glycine max IFS (GmIFS), which is not
normally present by Gateway cloning technology. Flavonoids are barely detected in white rice grains, except for
small quantities of tricin12 (30,50 -dimethoxylated flavone). It has also been reported that black rice shows enhanced
catalytic efficiencies for naringenin than white rice12. Towards this, quantification of flavonoids in three locally
available white rice varieties was performed using HPLC, with an aim to check the existing levels of naringenin and
other intermediates in the phenylpropanoid pathway. There have been studies reporting the successful engineering
of the isoflavone pathway in non-legumes, including arabidopsis, rice, tobacco, maize, petunia and lettuce using
soybean-derived IFS genes IFS1 or IFS211,13,14,15.
Rice seeds of ASD 16, ADT 43 and IR64 were procured from Tamil Nadu Agricultural university,
Coimbatore, Tamil Nadu, India.
Flavonoid quantification in Rice
Standard preparation
Standards (HPLC grade) of naringenin, quercetin and kaempferol was weighed and dissolved in methanol
to get 1mg/ml (stock solution), the stock solution was further diluted to working concentrations of 0.001mg/ml.
This solution was used as working standard solution, for further chromatographic analysis.
Sample preparation
Three Indica rice varieties were used in the present study namely, ASD16, ADT43 and IR64. To 1 gm of
dehusked mature seeds, 1mL of HPLC grade methanol was added and homogenized to a fine paste. The paste was
184
extracted in a vacuum evaporator at 70-80C for 3-4 hours. The dried substance was again extracted with 1mL
HPLC grade methanol and filtered through 0.2m filter and stored at -20C in dark16.
HPLC conditions
Waters HPLC system equipped with a model 515 HPLC pump and model 2998 photo diode array detector
was used for analysis. Separation was performed on symmetry C18 column. The mobile phase was Acetonitrile:
Water (30:70) programmed at a flow rate of 1ml/min16. The injection volume was 20l and the column was
maintained at ambient temperature (24C to 26C). The concentration of flavonoids in sample was calculated by
using the following formula.
Where,
Cx = Concentration of the sample, Ax = Peak area of the sample, Cs = Concentration of the standard, As = Peak area
of the standard
Cloning of IFS
The Gateway cloning primers were designed specifically for the Glycine max IFS gene with four guanine
residues at 5 end followed by 25 bp sequence of recombination site, att B1 and then 15-20 bp of the gene of
interest in the forward primer. The same strategy was followed for the reverse primer with att B2 recombination
site. PCR was performed using the designed primers. The PCR reaction was performed using the following
conditions, initial denaturation at 98% C for 30 seconds, annealing at 56% C for 45 seconds, extension at 72% C
for 30 seconds and final extension at 72% C for 10 minutes using a MyCycler Gradient PCR machine (BioRad,
USA). The PCR product was purified from the agarose gel using the PCR purification kit (Macheray-nagal kit,
Germany) according to the manufacturer instructions.
Generation of entry and destination clones

Gateway compatible entry vector pDONR/Zeo (Life Technologies, USA) was used. The plasmid was
moved to one shot ccdB survivalTM 2 T1R strain (Life Technologies, USA) by the heat shock method. It was then
plated on low salt LB media supplemented with 50 mg L-1 Zeocin (Life Technologies, USA) and the plates were
incubated at 37% C overnight. Next day, single colony was inoculated in 10 ml of low salt LB broth with Zeocin 50
mg L-1 and incubated overnight at 37%C. the plasmid was isolated and used for BP reaction. BP recombination
reaction (Life Technologies, USA) was performed and the purified PCR products were used for generating entry
clones in pDONR/Zeo. E Coli DH5 competent cells were used to mobilize the entry clone by heat shock treatment
and the positive colonies were confirmed by PCR. The target genes in the entry vector pDONR/Zeo with the
recombination site att L1 and att L2 were used for the LR reaction with the plant expression vector pEARLEY Gate
102 HA which have att R1 and att R2 sites. The recombined pEARLEY Gate 102 HA with IFS was transformed to
E. coli competent cells following the same procedure and confirmed by PCR. The recombined plant expression
vector construct was transformed to Agrobacterium AGL1 by electroporation.
185
Fig. 2 - (A), (B) Vector map of destination vector pEARLEY Gate 102 HA (Source: Created with
Snapgene) (C) IFS in destination vector pEARLEY Gate 102 HA between att sites.
Preparation of explant and tissue culture
Mature dehusked rice seeds were rinsed in 70% ethanol for 3 minutes with constant shaking and then
washed with sterile water for four to five times. This was followed by 0.1% Mercuric chloride treatment with
constant shaking for 2 minutes followed by sterile water 4-5 times. Different concentrations of growth regulators
namely 2, 4-D and BAP were tried for callus initiation from explants using MCI (Modified Callus Induction). The
medium was supplemented with 2, 4-D and BAP and pH adjusted to 5.8 before sterilization. The culture medium
was sterilized at 121o C for 15 minutes. The surface sterilized seeds were inoculated with the embryo portion facing
the medium. The culture was maintained at 27 + 1 o C and 55 + 5% relative humidity in dark for 14 days. The
percentage of callus induction efficiency was calculated by considering the number of explants inducing callus
within 2 weeks after inoculation.
RESULTS
Flavonoid quantification in Rice
HPLC analysis was performed for quantitative comparison of naringenin, quercetin and kaempferol in the
3 rice varieties ASD16, ADT43 and IR64. The concentration of the three flavonoids in the samples is tabulated
below.
Table. 1 Quantification of flavonoids in three rice varieties
S.No Sample Concentration (ng/g)

Naringenin Quercetin Kaempferol
01 IR64 3.4 2.14 ND*
02 ADT43 ND* 29.7 ND*
03 ASD16 ND* ND* ND*
*Not detected
186
Fig. 3 HPLC chromatogram of naringenin Fig.4 HPLC chromatogram of IR64
Fig. 5 HPLC chromatogram of Quercetin Fig.6 HPLC chromatogram of IR64
Fig.7 HPLC chromatogram of ADT43 Fig. 8 HPLC chromatogram of Kaempferol
Cloning of IFS
Attachment sites att B1 and att B2 were added at the 5 and 3 ends of target generating a proof read error
free PCR product. The PCR amplified product was cloned in entry vector pDONR/Zeo by BP reaction followed by
transformation to E. coli. The plasmid was further cloned to the plant expression vector pEarly HA 102 by LR
reaction and subsequently to Agrobacterium strain AGL1
1 2 3 21 2 3 4 5 6 7 8 1 2 3 4 5
1 3 4
1.5 Kb ? 1.5 kb ? 1.5 kb?
500 bp?
250 bp ?
250 bp ?
Fig. 9 PCR amplification of GmIFS, Lane 1: Marker DNA, 2-3 GmIFS PCR product (B) PCR confirmation of destination
clones, Lane 1 Marker DNA, 2 positive control, 3-7 destination clones with IFS, 8 negative control (C) PCR confirmation
of entry clones, lane 1 Marker DNA, 5 positive control, 2-4 GmIFS clones Callus induction
187
Different hormonal combinations and concentrations of 2, 4-D and BAP were tried to optimize the frequency of
callus induction in the rice cultivar ASD16. Among the different hormone compositions tried maximum callus
induction was seen in 2.5 mg/L BAP and 3 mg/L 2, 4-D. The calli obtained were looking very healthy and yellow in
color.
Table. 2 Callus induction response of ASD16 rice variety in different hormone concentrations
Percentage of callus
Explants used Medium
induction (%)
MCI + 0.5 mg/L BAP
42% 1.53
and 1.0 mg/L 2,4-D
MCI + 1.5 mg/L BAP
Mature dehusked 69% 1.53
and 2.0 mg/L 2,4-D
seeds
MCI + 2.5 mg/L BAP
98% 0.58
and 3.0 mg/L 2,4-D
MCI + 3.5 mg/L BAP
73% 0.71
and 4.0 mg/L 2,4-D
Fig. 10 Callus culture in ASD16 using 2.5 mg/L BAP and 3.0 mg/L 2,4-D (A) Surface sterilized seeds inoculated in
MCI medium (B) Callus initiation from mature seeds (C) Callus from mature seeds
DISCUSSION
Our aim was to first check the presence of isoflavone precursor naringenin and two other flavonoids in
rice and quantitatively analyze them by HPLC. Naringenin in ubiquitously present in all plants, but in the present
study we could detect naringenin only in IR64 at a very low concentration of 3.4 ng/g. Quercetin was detected
only in IR64 and ADT43 at a concentration of 2.14 and 8.38 ng/g respectively, whereas kaempferol was not
detected in any of the three samples. As reported by Park et al., (2016), we could barely detect flavonoids in white
rice varieties. From these results it can be concluded that white rice is a promising system for metabolic engineering
of phenyl propanoid pathway for enhancement of flavonoids and isoflavonoids expression. Further optimization of
extraction methods and higher quantity of samples may help in better detection of flavonoids.
GmIFS was successfully cloned in plant expression vector pEARLEY Gate 102 HA for callus mediated
Agrobacterium transformation of rice. The Gateway system exploits the principle of site specific recombination
shuttling sequences between plasmids bearing flanking compatible recombination attachment (att) sites17. MCI
(Modified Callus Induction) medium was used in the present study to initiate callus formation from the rice seeds.
Different hormonal combinations and concentrations of 2, 4-D and BAP were tried to optimize the frequency of
callus induction in the rice cultivar ASD16. Among the different hormone compositions tried maximum callus
induction was seen in 2.5 mg/L BAP and 3 mg/L 2, 4-D. Different combinations of growth regulators directly
influence the frequency of callus induction and regeneration capacity18. The calli obtained were looking very
healthy and yellow in color.
188
In conclusion the present study provides a promising option for metabolic engineering of isoflavonoids in
non legumes (rice) by derouting the phenyl propanoid pathway towards the production of genistein and daidzein.
ACKNOWLEDGEMENT
The authors thank Dr. Oliver Yu and Prof. Brian Mc Gonigle from Donald Danforth Plant science center,
USA for providing us with the target gene for the current research. We are very thankful to Bharathiar University,
UGC-BSR, UGC-SAP and DST-FIST for funding support.
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1. Fardet A, Rock E & Rmsy C (2008) J of Cereal Sci 48, 258276
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13. Liu R, Hu Y, Li J & Lin Z (2007) Metab Eng 9, 1-7
14. Yu O, Jung W, Shi J, Croes R A, Fader G M, McGonigle B & Odell J T (2000) Plant Physiol 124, 781-94
15. Sreevidya V S, Rao C S, Sullia S B, Ladha J K & Reddy P M (2006) J Exp Bot 57, 1957-1969
16. Chien P J, Sheu F, Shyu Y T (2001) J Food and Drug Anal 9
17. Ashwini M, Murugan S B, Balamurugan S & Sathishkumar R (2016) Mol Biol 50, 1-6
18. Malla A, Srinivasan B, Shanmugaraj B M & Ramalingam S (2015) J Crop Sci Biotechnol 18, 37-43
189
Protein Profiling Of Honey Samples From Apis And Stingless Bee
AjithaNath K. G. Ra,*, AthulyaRaveendranb, A. Jayakumaran Naira, V. S. Sugunanc
a
Department of Biotechnology, University of Kerala, Karyavattom, Thiruvananthapuram, Kerala, India
b
Department of Biotechnology, Sree Narayana College, Kollam
c
Department of Zoology, University College, Thiruvananthapuram, Kerala, India
*
Corresponding author: AjithaNath K.G.R. Email: kgr.ajithanath@gmail.com
Abstract
The comparative analysis of honey produced by Apis and stingless bee species are described.The proteins from
honey were extracted using acetone, ammonium sulphate, ethanol and TCA. From the extraction procedure for
protein precipitation, acetone was found to be best for Apis and TCA for stingless bee honey. A total of eight
proteins were separated in SDS PAGE from both Apis and Stingless bee honey. Protein bands with a molecular
weight between 43 kDa and 66 kDa, 43 kDa were found to be common in both Apis and stingless bee honey
samples.
Keywords: Apis, Stingless bees, protein precipitation, SDS PAGE
Natural products from insects and their potential for development in to drugs to treat human disease have
been published. Honey is one of the natural products produced from honeybees, widely used throughout the world
several million years ago. The composition of honey varies depending on the plant species in which bees consume
nectar and the species that produce it. It has been reported that honey contain 200 substances 1. Each constituent
in the honey has its own medicinal and nutritional values and because of this honey has been utilize in variety of
applications. Honey from Apis species is a complex mixture of fructose and glucose (80-85%), water (15-17%),
ash (0.2%), proteins and amino acids (0.1-0.4%) and trace amounts of vitamins, enzymes and phenolic compounds2,3,4.
The demands for stingless bee honey are increasing nowadays because of their properties in antimicrobial, anti-
cancer, anti-inflammatory, wound healing properties and also promote cell functions in erythrocytes 5. Both types
of honey are different in terms of its colour, viscosity and taste 6, 7. Proteins are the minor components of honey
and limited studies available about the protein content of Apisand stingless bee honey. It is important to investigate
the amount of proteins in honey for understanding their functions and nutritive quality. Here we report the comparative
analysis of honey produced by Apis and stingless beespecies.
Studies were carried out using the honey samples collected from different location in Trivandrum.Apis
honey and stingless bee honey were harvested from the bee hive. The samples were stored at room temperature in
air-tight glass bottles until analysis. The protein extraction was performed using the honey samples in undiluted and
diluted form. Samples were diluted by mixing with water for uniform distribution of components. The four methods
of protein extraction were performed including, acetone, ethanol, TCA and ammonium sulphate precipitation. The
protein concentrations in all the four extracted samples were quantified according to the method of Lowry et al8
at 620nm using Bovine serum albumin (BSA) as standard for preparing the calibration curve. The four extracted
samples were analyzed using sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) according to the
method of Laemmeliet al9. Broad range of markers was used to assess the molecular weight of extracted proteins.
SDS PAGE electrophoresis was conducted using a constant current of 15mA.
The concentrations of protein in four extracted methods compared and are represented in Table 1. The
results indicates that for diluted and undiluted samples of Apishoney, acetone was found to be the best solvent for
protein extraction and TCA was found to be the best solvent for diluted and undiluted samples of stingless bee
honey. The results also indicate that protein content was higher in stingless bee honey compared to Apis. The
results confirm that acetone precipitates more acidic and hydrophilic proteins after removing salt and lipid soluble
190
contaminants10. Precipitation of proteins using TCA is best suited for both 1-D and 2-D electrophoresis, because it
can concentrate samples, removes salts and polysaccharides and denatures endogenous proteases 11.
Table 1: Protein estimation by Lowrys methodfrom honey
Precipitated by
Precipitated by Precipitated Precipitated
Honey Ammonium
Acetone by Ethanol by TCA
sulphate
0.5% (20%
Apishoney(undiluted) 1.2% 0.6% 0.2%
fraction)
0.1% (20%
Apis honey(diluted) 1% 0.5% 0.1%
fraction)
Stingless 0.4% (40%
0.9% 0.8% 1.9%
beehoney(undiluted) fraction)
Stingless bee honey 0.1% (60%
0.6% 0.6% 1%
(diluted) fraction)
The proteins extracted by different methods were analyzed by SDS PAGE. A total of eight proteins were
observed in diluted and undiluted samples of Apis and stingless bee honey. The protein pattern after acetone
precipitation in Apis honey of both undiluted, (Fig. 1(I)) and diluted form (Fig. 1(II)), showed similar protein band
with molecular weight of 43 and 66 kDa. The results indicate that proteins were well separated in acetone precipitation.
In contrast, for ethanol precipitation in undiluted Apis honey (Fig. 2(I)), no bands were observed and for diluted
Apis honey (Fig. 2(II)), proteins pattern were observed with a molecular weight of 29kDa. After ammonium
sulphate precipitation in undiluted (Fig. 3 (I)) and diluted (Fig. 3(II)), Apishoney showed a similar protein band of
43kDa. Additionally in undiluted Apis sample a 20.1kDa protein pattern was also present. After TCA precipitationboth
in undiluted honey (Fig. 4(I)) and diluted (Fig. 4(III)) Apis honey samples, three proteins were seen as distinctive
band with a molecular weight of slightly less than 29 kDa, greater than 20.1 kDa, 20.1 kDa. But in diluted (Fig.
4(III)) Apis sample another two protein were seen with a molecular weight of 43 kDa and 14.4 kDa.
In stingless bee honey samples, after acetone precipitation in both undiluted honey (Fig. 1(II) and diluted
honey (Fig. 1 (IV)) samples showed similar banding pattern with a molecular weight of slightly less than 66kDa.
But in diluted honey (Fig. 1 (IV)), other two proteins were present with a molecular weight between 43 and 66 kDa
and other between 66 and 116 kDa. Precipitation with ethanol showed (Fig. 2 (III)) a prominent band with a
molecular weight between 45 and 66kDa in stingless bee honey. Both undiluted honey (Fig. 2 (III)) and diluted
honey (Fig. 2 (IV)) also share bands with molecular weight between 29 and 45 kDa. After ammonium sulphate
precipitation, proteins were present in both undiluted (Fig. 3 (III)) and diluted (Fig. 3 (IV)) samples, but the band
was not clear. TCA precipitation with stingless bee honey showed proteins with a molecular weight of 43 kDa in
both undiluted (Fig. 4 (II)) and diluted (Fig. 3 (IV)) samples.Two protein bands were observed with a molecular
weight intermediate between 20.1 kDa and 29.0 kDa and one with slightly greater than 14.4 kDa were observed in
diluted samples of stingless bee honey. In common, two proteins with a molecular weight between 43 kDa and 66
kDa, 43 kDa are seen in both Apis and stingless bee honey samples after SDS-PAGE.
Out of the four methods used for precipitation, TCA produced the highest efficiency of protein precipitation
in stingless honey samples. In Apishoney samples, acetone shows greater yields of proteins than the other methods.
Fewer proteins were precipitated in other supernatants which were determined by Lowrys methods. There are
total eight proteins isolated from undiluted and diluted samples of both stingless and Apis honey samples. In
common, two proteins with a molecular weight between 43 kDa and 66 kDa, 43 kDa are seen in both Apis and
stingless bee.
191
Fig.1: SDS page separation of proteins extracted using Fig.2: SDS page separation of proteins extracted using
acetone ethanol
M I II III IV
M I II III IV
2 05.0 205.0
116.0
1 16.0 66.0
43.0
6 6.0
29.0
4 3.0
20.1
2 9.0
14.4
2 0.1
6.5
1 4.4
6 .5
M-marker; I- honey+ acetone (Apis) ;II - honey+ acetone M-marker; I- honey+ ethanol (Apis); II- honey+water+
(stingless bee); II-honey + water + acetone (Apis); IV- ethanol (Apis); III-honey + ethanol (stingless bee); IV-
honey + water + acetone (stingless bee). honey+ water+ ethanol(stingless bee)
Fig. 3: SDS page separation of proteins extracted using Fig.4: SDS page separation of proteins extracted using
Ammonium sulphate (AS) TCA
M I II III IV
M I II III IV
205.0
116.0
205.0
116.0
66.0 66.0
43.0
29.0
20.1 43.0
14.4 29.0
6.5
20.1
14.4
6.5
M- Marker; I-honey+ AS (Apis); II-honey+ water+ AS (Apis); M- Marker; I- honey + TCA (Apis); II - honey+ TCA
III- honey+ AS(stingless bee); IV-honey+ water+ (stingless bee); II- honey + water + TCA (Apis); IV-
AS(stingless bee) honey + water + TCA (stingless bee)
This research work was supported by the RGNF grant from University Grant Commission.
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2. National Honey Board (2003) Definition of honey and honey products. U.S. Secretary of Agriculture: US
Department of Agriculture.
192
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E O, De Souza A L & De Souza A G (2013) Food Chem 141, 35523558.
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193
Renoprotective capability of S-Allylcysteine against Streptozotocin-
Nicotinamide induced Diabetic Nephropathy in Rats
V. V. Sathibabu Uddandrao1, P. Brahmanaidu2 and Ganapathy Saravanan1*
1
Centre for Biological Sciences, Department of Biochemistry, K. S. Rangasamy College of Arts and Science (Autonomous),
Tiruchengode, Tamilnadu, India - 637215.
2
Department of Biotechnology, Vikrama Simhapuri University, Nellore, Andhra Pradesh, India- 524003.
Abstract
Diabetic nephropathy (DN) is the most familiar complication and leading cause of mortality connected with
diabetes. The present study was designed to evaluate the renoprotective capability of S- Allylcysteine (SAC)
against Streptozotocin (STZ)-Nicotinamide (NAD) induced diabetic nephropathy in experimental rats. SAC (150
mg/kg body weight) was administered orally for 45 days to STZ-NAD induced diabetic nephropathy rats and
Metformin (25 mg/kg body weight) was given as control. Effect of SAC on body weight, blood glucose, insulin,
hemoglobin, glycated hemoglobin, serum creatinine, serum uric acid, blood urea nitrogen and renal marker
enzymes such as creatinine kinase and lactate dehydrogenase were measured in both control and experimental
rats. Administration of SAC brought back blood glucose, glycated hemoglobin, creatinine, albumin and renal
marker enzymes to near normal and found high in diabetic control rats. In conclusion, according to these results
suggested that SAC hold effective potentiality to attenuate the diabetic nephropathy in STZ-NAD induced rats.
Key words: S-Allylcysteine, Diabetic nephropathy, Renal damage, Streptozotocin-Nicotinamide
DN is the most recognizable impediment and most important reason of death associated with diabetes. DN
is a foremost contributor to cases of kidney failure in urbanized countries, and both type I and type II forms of
diabetes can corollary in DN. DN is assessed clinically through a five-stage system of criteria, with each stage
featuring a distinct set of functional and structural changes and reflected modifications in standard renal function
markers1. The leading pathological conditions of DN encompass mesangial extension, extracellular matrix alterations,
tubulointerstitial fibrosis, and glomerular sclerosis2.
In view of the fact that the anti-diabetic drugs in develop for continuing therapy are instituted to be related
with miscellaneous side effects, the developmental process in the improvement of anti-diabetic drugs has reallocated
its centre of attentiveness towards native plant resources that have insignificant side effects3. For the burst through
of novel counteractive agents against diabetes, phyto-constituents with an influential antioxidant nature will be the
stupendous alternative and they have inward much deliberation as sources of biologically active substances, including
hypoglycaemic, hypolipidaemic and antioxidants agents4.
S-Allylcysteine (SAC), a derivative of garlic, is a sulfur containing amino acid5. SAC is well known for to
be anticancer, anti oxidative, and can also modest the incidence of stroke6. Until that time, it was made known that
SAC demonstrated insulin-like anti hyperglycemic effect in the STZ-NAD induced diabetic rats and also overturns
the changes in the levels of glucose metabolism in liver. For this reason, the current study was aimed to evaluate
the effect of SAC on renal damage amelioration in STZ-NAD induced renal damage in rats.

Animals
Male Wister/NIN rats of body weight 150-180g were acquired from National Centre for Laboratory Animal
Science (NCLAS), National Institute of Nutrition (NIN), Hyderabad. Animals were housed independently in standard
194
polycarbonate cages at 22 2C with top grill having facilities for holding pelleted feed and drinking water in
polycarbonate bottles and air changes per hour with a relative humidity 50-60% and a 12 hour light/dark cycle.
Before the initiation of the experiment, the rats were acclimatized for 7 days to the laboratory environment. The
protocol of this study was approved by Institutional animal ethical committee of National Centre for Laboratory
Animal Science, National Institute of Nutrition, Hyderabad (P7F/II-IAEC/NIN/2015/GS/WNIN Rats/42M).
Chemicals
SAC (99%) commercially obtainable and it was purchased from LGC Prochem, Bangalore, India.
Streptozotocin was purchased from Himedia, Bangalore, India. All the drugs and bio chemicals used in this research
were purchased from Sigma Chemical Company Inc., St Louis, MO, USA. All other chemicals used were of
analytical grade.
Induction of diabetes
The overnight fasted rats were made diabetic by a single intraperitoneal injection of freshly prepared STZ
(45 mg/kg body weight) in citrate buffer (0.1 M, pH 4.5), 15 min after the intraperitoneal administration of
Nicotinamide (110 mg/kg body weight) in 0.9% normal saline. Hyperglycemia was confirmed by the elevated
glucose levels (Above 250 mg/dl) in blood, determined at on day 7 after injection. After 15 days of diabetes
induction diabetic nephropathy was confirmed by renal markers.
Experimental design
After the successful induction of experimental diabetic nephropathy, the rats were divided into four
groups each comprising a minimum of six rats.
Group 1 : Normal control rats
Group 2 : Diabetic control rats
Group 3 : STZ-NAD treated Diabetic nephropathy rats given SAC (150 mg/kg body weight) in vehicle solution
orally for 45 days.
Group 4 : STZ-NAD treated Diabetic nephropathy rats given Metformin (25 mg/ kg body weight) in vehicle
solution orally for 45 days.
Effect on body
Body weights were measured weekly and at the end of the treatment blood was collected and sacrificed
the animals according to the ethical committee (IAEC) guidelines.
Estimation of glucose, insulin and glycated hemoglobin

Effect on the status of hyperglycemic markers such as plasma glucose, insulin, insulin sensitivity, heamoglobin
and glycated hemoglobin in experimental diabetes (anti hyperglycemic) were measured by the respective kits.
Biochemical Estimation of renal markers

The effect of S-Allylcysteine on nephrotic markers viz., serum creatinine, serum uric acid, blood urea
nitrogen and renal marker enzymes like creatinine kinase and lactate dehydrogenase were measured by the respective
kits as per manufacturer protocols in both control and experimental rats.
All the results were expressed as the Mean S.D. for six animals in each group. All the grouped data were
statistically evaluated with SPSS\10.0 software. Hypothesis testing methods included one way analysis of variance
(ANOVA) followed by least significant difference (LSD) test; significance level at p< 0.05 were considered to
195
indicate statistical significance.
RESULTS
Table 1 explained the body weight, blood glucose levels, insulin, and glycated hemoglobin in both control
and experimental animals. A noteworthy decrease in the level of insulin and bodyweight and a parallel increase in the
level of plasma glucose, glycated hemoglobin and insulin resistance were noticed in STZ-NAD rats and these levels
were standardized after treatment with SAC and Metformin.
Table 1: Effect of SAC on body weight, blood glucose, insulin and glycated hemoglobin in control and
experimental rats
Body weight (g) Blood glucose Insulin (g/dL) glycated

(mg/dl) hemoglobin
(mg/g
hemoglobin)
Control 261.36.25 110.14.17 3.210.98 0.280.10
Diabetic control 118.97.10 385.37.40 0.850.48 0.730.34
Diabetic + SAC
(150 mg/kg) 216.48.37 123.55.54 2.880.97 0.350.27
Diabetic + Met
(25 mg/kg) 225.56.78 117.64.86 3.140.56 0.310.38
Values are mean SD, n = 6, Values are statistically significant at p<0.05
Figure 1 & 2 explained the effect of SAC on serum creatinine, serum uric acid, blood urea and nitrogen in
both control and experimental rats. A remarkable raise in the levels of serum creatinine, serum uric acid, blood urea
and nitrogen were found in STZ-NAD induced diabetic rats and SAC and Metformin treated animals became
normalized after treatment of 45 days.
Fig 1: Effect of SAC on serum creatinine and serum uric acid in control and experimental rats, Values
are mean SD, n = 6, Values are statistically significant at p<0.05
196
Fig 2: Effect of SAC on blood urea and blood urea nitrogen in both control and experimental rats, Values are mean
SD, n = 6, Values are statistically significant at p<0.05
Table 2 was shown that the levels of renal marker enzymes like creatinine kinase and lactate dehydrogenase
in control and experimental rats. A significant elevate in the levels of creatinine kinase and lactate dehydrogenase in
diabetic nephropathy rats and these are reached normal after the treatment with SAC and Metformin.
Table 2: Effect of SAC on renal marker enzymes in control and experimental rats
Creatinine kinase (IU/ Lactate dehydrogenase

mg Protein) (U/L)
Control 4.070.97 203.832.73
Diabetic control 10.521.78 321.053.52
Diabetic + SAC (150 mg/kg) 5.211.22 221.854.39
Diabetic + Met
4.910.74 217.432.75
(25 mg/kg)
DISCUSSION
STZ-NAD induced diabetes in animals is well thought-out to be an excellent mould for the initial screening
of agents active against diabetes7. In the current study, the intraperitonially administration of STZ successfully
induced DN in rats. Consequently, in this study, we found an augmented level of blood glucose. The hypoglycaemic
efficiency of SAC has been authorized to the sulphur compound8. The mechanism of hypoglycaemic action
conceivably involves straight or tortuous stimulation of insulin secretion. This could be due to potentiating of the
insulin effect of plasma by increasing the pancreatic secretion of insulin from existing -cells or it does liberate
from bound insulin9.
DN refers to structural abnormalities include hypertrophy of the kidney, increase in glomerular basement
membrane thickness, nodular and diffuse glomerulosclerosis, tubular atrophy, and interstitial fibrosis. In our study
we observed the enhanced levels of uric acid and creatinine levels in untreated DN animals and noticed noteworthy
diminish in animals treated with SAC. Diabetes leads to the devastation of the glomerular filtration barrier, leading
197
to glomerular damage and ensuing in uric acid or creatinine leakage, exacerbating the progression of DN10. Consistent
with this interpretation, our study demonstrated that albuminuria and focal glomerular matrix expansion were
markedly increased in the vehicle-treated diabetic rats. The elevated blood glucose, altered insulin tolerance, increased
activities of renal creatinine clearance in the diabetic group of rats point out the progression of DN, glomerular
injury and renal dysfunction11. On the other hand, oral supplementation with SAC to DN group of rats customized
these distorted levels into near normal, suggestive of that SAC successfully ameliorates DN.
In conclusion, the current study disclosed the renoprotective potentiality of SAC in STZNAD induced
DN rats and make available confirmation that the defensive possessions are, perhaps, due to the turn down in
oxidant and pro inflammatory fabrication by the renal tissues. Moreover, SAC treatment to STZ-NAD induced
diabetic rats exhibited a significant ameliorative potential probably by attenuating the hyperglycemia. Further
comprehensive molecular level experiments are in progress to elucidate the accurate mechanism by which SAC
reduces its renoprotective potentials against DN.
ACKNOWLEDGEMENT
The authors thank Department of Science and Technology, India for providing financial assistance to this
work (SR/SO/HS/0227/2012). The authors also thank to the Management of K. S. Rangasamy College of Arts and
Science (Autonomous), Tiruchengode, Tamilnadu, India for their encouragement and for providing facilities to do
experiments.
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8. Augusti K T & Sheela C G (1996) Experientia 52, 115-120
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Mustapha Shabana Begum, Rajesh Pandiyan & Ganapathy Saravanan (2016) Mol and Cel End 419, 139-
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11. Palsamy P & Subramanian S (2011) Bioch et Biophy Act 1812, 719731
198
Ameliorative Potentiality of Asiatic acid against the High Fat Diet in-
duced Obesity in Sprague dawley Rats
P. Ramesh Reddy1, V. V. Sathibabu Uddandrao1, Parim Brahmanaidu2 and Ganapathy Saravanan1*
1
Department of Biochemistry, Centre for Biological Sciences, K. S. Rangasamy College of Arts and Science (Autonomous),
Tiruchengode, Namakkal Dt, Tamilnadu-637215.
2
Department of Biotechnology, Vikrama Simhapuri University, Nellore, Andhra Pradesh, India- 524003.
Abstract
Obesity, generally linked to hyperlipidemia, has been occurring of late with distressing alarm and has now
become a global phenomenon casting a huge economic burden on the health care system of countries around
the world. The present study was convened to evaluate the effects of Asiatic acid (AA), a pentacyclic triterpenoid
from Centella asiatica on lipid metabolic parameters in rat model, induced by high-fat diet for 45 days. The rats
were treated orally with AA(20 mg/kg b.w) once daily for 42 days with a Orlistat -treated group (10 mg kg1 b.wt)
included for comparison. Changes in body weight and lipid profiles of plasma such as namely LDL-C, HDL-C,
Triglycerides, Total cholesterol, adiponectin, leptin, amylase and lipase were observed in experimental rats. AA
(20 mg/kg b.w) exerts its helpful impacts like Orlistat in lessening body weight gain in, leptin, plasma lipids and
increasing adiponectin, amylase, lipase, in HFD-fed rats. In conclusion, from these findings it can be concluded
that AA possesses significant anti-obesity potential through the suppression of body weight gain and lipid
lowering action.
Key Words: Asiatic acid; High Fat Diet; Lipid; Metabolic disorders; Obesity
The obesity epidemic is widespread and its prevalence and incidence of obesity is escalating worldwide in
both developing and developed countries, which critically affect both males and females of different age groups1.
Obesity amplifies the risk of development of various sicknesses including Type 2 diabetes, hypertension,
hyperlipidemia and cardiovascular illnesses2. It is portrayed by an anomalous or unnecessary fat aggregation attributable
to irregularity between energy intake and energy expenditure3. The etiology of obesity is dissimilar one. On the
other hand, the root cause is energy discrepancy: more calories consumed than expended.
There are number of therapeutic interventions for the treatment of obesity, including low-calorie diets,
expanded physical action, behavioral treatment, pharmacological intervention, and bariatric surgery. However,
these are generally limited in efficacy and/or safety4. The pharmacotherapy of obesity has recently undergone
unique development. Along with lifestyle modifications, decrease of supplement assimilation and organization of
medications that influence lipid assembly and utilization will be the suitable remedial development for obesity. The
state of affairs as it currently stands, the market for anti-obesity drugs are mounting rapidly but the available drugs
are exceptionally meager in relation to require. Hence, there is necessitating searching for novel anti-obesity drug to
undertake the obesity problem5. Epidemiological studies have recommended the utilization of phytoconstituents
that could reduce the risk of diabetes, cardiovascular diseases and obesity6, suggesting that the consumption of
Asiatic acid may be of benefit in metabolic diseases.
Asiatic acid (AA), a triterpenes was found abundantly in Centella asiatica (L.) a perennial herbaceous
creeper of the Apiaceae family7. It possesses an extensive array of biological functions including antioxidant,
hepatoprotective and anti-inflammatory activities8. Besides this, AA shows antidiabetic property by escalating the
level of plasma insulin, decreases glucose level, switches the adjustments in the status of the lipid metabolizing
enzymes9. It also prevents lipid peroxidation and improves antioxidant status in rats with streptozotocin-induced
diabetes10. Furthermore, there is no information pertaining to the antiobesity effects of AA. Therefore, the purpose
of the present study is to appraise the antiobese action of asiatic acid in high fat diet fed rat model.
199
Chemicals
Asiatic acid (AA; 2, 3, 23-trihydroxyurs-12-en-28-oic acid) and Orlistat (04139 Sigma) were purchased
from Sigma Aldrich (Bangalore, India). All added reagents acclimated in the experiments were of analytic brand and
of the highest purity.
Animals
Male Sprague-Dawley rats were obtained from Department of Biochemistry, Muthyammal College of Arts
and Science, Rasipuram, Namakkal District, Tamilnadu, India. Experimental animals were kept up under standard
laboratory conditions (temperature; 222C: moistness; 40-60%), and permitted food and water ad libitum. Rats,
at first, weighing 180-200g were separated into four groups of six each (n= 6). All procedures involving laboratory
animals were in accordance with the institutional animal ethical committee of Muthyammal College of Arts and
Science, Rasipuram, Namakkal District, Tamilnadu, India.
High Fat Diet Formula:
Normal and high-fat diets were procured from National Center for Laboratory Animal Sciences, National
Institute of Nutrition, Hyderabad, India. Normal control rats were fed with standard rhodent chow pellet diet as per
AIN-93 guidelines, while the other groups of animals were fed with HFD throughout the experiment.
Experimental design
Group 1 : Normal Diet Control (Normal Diet)
Group 2 : High fat Diet group (Obesity control)
Group 3 : HFD induced obese rats orally treated with AA (20 mg/kg body weight) in a vehicle solution
for 45 days.
Group 4 : HFD induced obese rats orally treated with Orlistat (10 mg/kg body weight) in a vehicle
solution for 45 days.
Measurement of body weight

The body weight and the total amount of food consumption (Data are not shown) of each rat were
measured. Toward the end of the experiment, blood was collected from overnight fasted animals under inhalation
of anaesthesia by retro-orbital puncture strategy. Blood was collected in anticoagulant coated vials and permitted
for 15 minutes at room temperature. Plasma was separated by centrifugation at 2500 rpm for 15 minutes.
Estimation of biochemical profiles
Blood samples were centrifuged at 4000 rpm/min for 10 min to separate plasma for analyzing lipid profile.
The plasma levels of LDL-C, HDL-C, Triglycirides, Total cholesterol, adiponectin, leptin, amylase and lipase were
determined using commercially available kits.
Statistical analysis:
One-way ANOVA method was used and results were expressed as mean S. D followed by Least Significant
Difference (LSD) test. The level of statistically significance was set at P<0.05.
RESULTS
Figure 1 shows the changes in body weight in control and HFD group of animals during the experiment.
Intake of HFD for 42 days elicited a significant (p<0.05) increase in body weight, compared to the normal control
group. Oral administration with AA (20mg kg-1 b.w-1) or Orlistat significantly (P<0.05) reduced the increase in
body weight when compared to the HFD control group.
200
Fig 1: Effect of AA on the changes body weight in control and HFD induced obese rats,
Table 1 show the levels of lipid profile in control and HFD induced obese rats respectively. The level of
plasma lipids were significantly increased in experimental obese rats as compared to the normal rats. Oral treatment
with AA (20mg kg-1 b.w-1) or Orlistat significantly (p < 0.05) reduced the concentrations of plasma lipids in obese
rats to near normal level.
Table 1: Effect of AA on plasma lipid profile in control and experimental rats
Control HFD HFD + AA HFD + Orlistat

LDL (mg/dL) 20.121.09 31.991.78 23.641.36 21.191.05
HDL (mg/dL) 34.21 1.99 11.261.25 25.161.90 28.801.57
Triglycirides (mg/dL) 17.291.23 34.051.30 23.581.87 21.121.67
Total Cholesterol (mg/dL) 89.201.46 157.292.07 98.211.97 91.241.62
Figure 2 exemplifies the level of leptin and adiponectin in control and experimental obese rats. There was
a significant (p<0.05) elevation in leptin levels and decrease in adiponectin in HFD induced obese rats compared
with control rats. Oral administration with AA (20mg kg-1 b.w-1) or Orlistat brought back the above parameters
towards near normal levels.
Fig 2: Effect of AA on the level of leptin and adiponectin in control and experimental obese rats,
201
The activities of the amylase and lipase of normal and experimental obese rats were shown in Figure 3.
HFD supplemented rats showed significant elevation (p<0.05) in amylase and lipsae activity. Oral Administration of
AA (20 mg/kg bw) or Orlistat to obese rats significantly reduced the activity of amylase and pancreatic lipase.
Fig 3: Effect of AA on the activities of the amylase and lipase of normal and experimental obese rats,
Values are mean SD, n = 6, Values are statistically significant at p<0.05.
DISCUSSION
Diet induced obesity in animals has many features common with human obesity and is widely used to find
medicinal foods for reducing obesity. Signs of diet induced obesity are body weight composition and dyslipidemia2.
In the present study, enhanced body weight and fat was observed in HFD fed rats. Long-standing HFD fed to rats
resulted with increased bodyweight and plasma co morbidity factors11 and it might be due to the consumption of a
diet rich in energy in the form of saturated fats (lard) and its accretion in different body fat pads12 leads to
unwarranted development of adipose tissue and also the ratio of calorie ingestion to energy utilization determines
bodyweight. Comparable effects have been reported by earlier animal studies induced with high-fat rodent diets
having similar source of fat and experimental length2. Inclusion of AA showed a more significant lessening in body
weight development compared with that of HFD-fed control rats. In the present study, AA had no apparently
suppressive effect on feeding in HFD-fed rats (data not shown), exposed that the AA-induced weight loss involves
a mechanism that is independent of hypophagia. The control of body weight gain may be due to a number of
mechanisms including unusual food assimilation or decreased body energy storage and also the reduction in body
weight gain11.
Obesity is accountable for the escalating occurrence of metabolic disease. In the body, adipose tissue is a
key site of energy storage and is crucial for energy homeostasis12. It is well known that insulin can ultimately
instigate leptin secretion during the metabolism of nutrients, particularly on glucose exploitation in adipocytes.
Hyperlipidemia, hyperglycemia, insulin resistance, impaired glucose metabolism, distinctive visceral adiposity
hyperinsulinemia, and decreased bone mineral concentration and bone mineral density are the common characteristics
of diet-induced obesity in rodents13. Adipose tissue secretes a number of adipokines including leptin, adiponectin,
resistin, visfatin, tumour necrosis factor and interleukin-6 which participates a role in energy regulation14. Leptin
and adiponectin secreted by adipocyte are supposed to play imperative roles in the controlling of metabolic
homeostasis. A diminished plasma adiponectin levels were seen in obese animals and it additionally demonstrated a
positive connection with HDL-C1. Leptin resistance, the attribute of diet induced obesity, is a detectable authenticity
in which coursing leptin levels diminishes in answer to target tissues. Adipocytes secrete leptin in direct proportion
to adipose tissue mass as well as nutritional status15 where as adiponectin is inversely correlated with body mass16.
During the development of obesity, elevated level of leptin and concomitant decreased level of adiponectin were
observed17. Its secretion is positively interrelated with the extent of the triglyceride stores in adipocytes18. In the
202
present study, increased leptin and decreased adiponectin levels were observed in plasma of HFD control group
compared with the control group. Oral treatment with AA lowered leptin and enhanced adiponectin in plasma of
experimental rats. A few polyphenols have been reported to have an impact on lipid catabolism and diminishing
insulin resistance which assume fundamental parts in obesity19. These observations suggest that the decreased
leptin and increased adiponectin levels after AA supplementation may have ameliorated insulin resistance in obese
rats, resulting in decreased plasma lipid levels and weight loss20.
HFD induced obesity is associated with dyslipidaemia characterized by increasing triglycerides (TG),
VLDL, total cholesterol (TC), phospholipids (PL) and low density lipoprotein (LDL) and a decrease in plasma level
of high density lipoprotein HDL21. In the present study, dyslipidaemic changes like elevated TGs, VLDL, total
cholesterol (TC), and LDL, and declined plasma level of HDL has been observed. These findings were supported
by Park et al22. During fed state, elevation of chylomicrons is a typical element which prompts increment in TCs,
TGs level and endogenous VLDL generation. TGs rise was because of dietary cholesterol that had been appeared
to diminish unsaturated fat oxidation, which thus expanded the levels of hepatic and plasma TG and the excessive
accumulation of TGs in the lipid stores2. Noticeable TC levels intensify the risk of rising coronary heart disease by
uplifting LDL-C. On the other hand, high HDL-C is helpful in transporting surplus cholesterol to the liver for
excretion in the bile23. Oral supplementation of AA significantly lowered TC, TGs, and LDL-C levels in plasma of
rats with HFD-induced obesity. Yongqi et al24 reported that supplementation of phytoconstituents to fed animals
leads to hypolipidemic state by decreasing cholesterol absorption and secretion from the intestine which leads to
the lowered availability of FFAs to the liver. Our finding is in line with this report.
Amylase and lipase, the key catalysts in carbohydrate and lipid metabolism and inhibition of this digestive
enzyme is accordingly useful in the treatment of overweight1. -amylase, one of the key digestive compounds
discharged from the pancreas and salivary organs, is included in hydrolytic process of starch25. Activating lipase or
inhibiting pancreatic lipase would have an anti-obesity effect. Lowered pancreatic amylase discharge lessened the
assimilation of carbohydrates by suppressing their processing, prompting decreased energy intake26. Various
exploratory reports portrayed that -amylase inhibitors most of which are extricated from plants, can reduce the
post-prandial hyperglycemia, in this manner thus regulating the abnormal glucose metabolism and counteractive
action of metabolic issues27. In the present study, pancreatic lipase and amylase level was found to be increased in
HFD induced rats. Oral supplementation of AA inhibits the activity of pancreatic lipase and -amylase in treated
rats compared with HFD control rats. It is promising that the minimized levels of pancreatic amylase and lipase
may symbolize a component that would impede the assimilation and ingestion of starches and lipids which can
lesser the caloric admission in AA treated HFD-fed obese rats, in order to urge weight reduction.
CONCLUSION
In conclusion, these results suggested that dietary supplementation of Asiatic acid extracted from Centella
asiatica could inhibit the increment in body weight and fat percentage in high-fat diet induced obese rats. This
strongly suggests that Asiatic acid has therapeutic potential for the management of obesity and hyperlipidaemia.
ACKNOWLEDGEMENT
The authors thank the Management of K. S. Rangasamy College of Arts and Science (Autonomous),
Tiruchengode, India and Muthyammal College of Arts and Science, Rasipuram, Namakkal District, Tamilnadu,
India for their encouragement and providing facilities to do this research work.
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1. Despres J P (2012) Circulation 126, 13011313
2. Saravanan G, Ponmurugan P, Deepa M A & Senthilkumar B (2014) J Sci Food Agri 94, 29722977
3. Ferranti S & Mozaffarian D (2008) Clin Che 54, 6945955
203
4. Jindal A, Brietzke S & Sowers J R (2012) Cardio Renal Med 2, 314327
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6. Kishino E, Ito T, Fujita K & Kiuchi Y (2006) J of Nut 136, 433439
7. Yun Zhao, Ping Shu, Youzhi Zhang & Limin (2014) Oxid Med and Cel Long 154, 1-7
8. Huang S S, Chiu C S, Chen H J, Hou W C, Sheu M J, Lin Y C, Shie P H & Huang G J (2011) Evi-Ba Compl
and Alt Med 1, 110
9. Ramachandran V, Saravanan R & Senthilraja P (2014) Phytomed 21, 225232
10. Ramachandran V & Saravanan R (2013) J of fun foods 510, 77-87
11. Parim Brahma Naidu, Sathibabu Uddandrao, Ramavat Ravindar Naik, Pothani Suresh, Balaji Meriga, Mustapha
Shabana Begum, Rajesh Pandiyan & Ganapathy Saravanan (2016) Mol and Cel End 419, 139-147
12. Oben J, Kuate D, Agbor G, Momo C & Talla X (2006) Lip Heal and Dis 5, 24
13. Kim Y & Park T (2008) Nut Res 28, 414422
14. Garg A (2006) Clin Corner 8, 713
15. Cnop M, Landchild M J, Vidal J, Havel P J, Knowles N G & Carr D R(2002) Diab 51, 100515
16. Arita Y, Kihara S & Ouchi N (1999) Biochem Biophys Res Commun 257, 79-83
17. Kamohara S, Burcelin R, Halaas J L, Friedman J M & Charron M J (1997) Nat 389, 3747
18. Staiger H & Haring H U (2005) Exp Cli Endoc Diab 113, 6779
19. Crozier A, Jaganath I B & Clifford M N (2009) Nat Pro Rep 26, 10011043
20. Ono Y, Hattori E, Fukaya Y, Imai S & Ohizumi Y (2006) J Ethnopharmacol 106, 238-244
21. Duiyan Jin, Yi Xua & Xin Mei (2013) J of fun foods 5, 11421150
22. Park Y, Storkson J M, Liu W, Albright J, Cook M E & Pariza M W (2004) J of Nut Bioc 15, 561569
23. Garjani A, Fathiazad F, Zakheri A, Akbari N A & Azarmie Y (2009) J Ethnopharmacol 126, 52532
24. Yongqi G, Guanzhong W, Xin S, Hongxia Y & Juan Z (2009) Nut Res 29, 656663
25. Kei Nakajima, Toshitaka Muneyuki & Hiromi Munakata (2011) BMC Res Not 4, 419
26. Tundis R, Loizzo M R & Menichini F (2010) Mini Rev Med Chem 10, 315-331
27. Layer P, Rizza R A, Zinsmeister A R, Carlson G L & DiMagno E P (1986) Mayo Clin Proc 61, 442-447.
204
Investigation of the Polyethylene Degrading Potential of
the Isolated Bacterial Strain in Polyethylene Contaminated Soil by
Bioaugmentation Strategy
Kavithaa R and Bhuvaneswarib V
a
Department of Biochemistry, Bharathiar University,Tamil Nadu, India
b
Higher Education for women, Coimbatore 641043, India
Abstract
The bioremediation potential of the bacterial strain isolated from soil was investigated in low density polyethylene
(LDPE) polluted soil under laboratory conditions. Three soil model systems were developed wherein naturally
attenuated, biostimulated, and isolated bacterial strain inoculated (bioaugmented) soil samples were examined
for their ability to degrade LDPE. LDPE films were added to each of these microcosms and incubated for 60 days.
After the incubation period, soil samples were subjected to physicochemical analysis. The LDPE films were
removed from each treatment soil and subjected to FTIR analysis. During the incubation period the evolved CO2
was measured.
Significant changes in the physicochemical properties were noticed. The results of respirometric analysis revealed
the ability of isolated bacteria to degrade LDPE in polyethylene contaminated soil. It is evident from the FTIR
analysis that the values of KCBI, ECBI, VBI and DBI were increased in LDPE film samples taken from biostimulated
and bioaugmented treatments when compared to the LDPE film taken from naturally attenuated treatment. The
exogenous addition of isolated bacterial strain to LDPE contaminated soil causes an enhanced degradation.
Keywords : LDPE , Bacteria, Bioaugmentation, FTIR, Soil
Plastics are synthetic organic polymers that are derived from oil, coal and natural gas1. Though plastic and
polythene are durable and strong with wide applications, they appear to be non-degradable leading to severe
pollution issues by disturbing the whole ecosystem. Petroleum plastics are accumulating at the rate of 25 million
per year. Plastics pollution affects the soil and water bodies 2. It is well known that the impact of plastic pollution
through ingestion and entanglement of marine fauna, ranging from zooplankton to cetaceans, seabirds and marine
reptiles3. Several plastic-associated chemicals are linked to endocrine disrupting effects. Ingestion of plastic debris
may alter the endocrine system which functions in adult fish4. Therefore, considerable efforts have been undertaken
to degrade polyethylene.
Microorganisms are original recyclers, converting the toxic organic compounds into harmless products.
Studies were carried out to explore the microbial diversity, particularly of polluted areas in search of organisms that
can degrade a broad range of pollutants at high pollution load5. Bioaugmentation is a promising and low-cost
bioremediation strategy in which an effective bacterial isolate(s) or microbial consortium capable of degrading
xenobiotics is administered to contaminated sites. Biostimulation of indigenous microbes is a bioremediation strategy
which involves the addition of nutrients, either organic or inorganic, to improve the activities of indigenous microbes
that are capable of degrading pollutant from soil environment 6. The present study was designed to explore the
bioremediation potential of the isolated bacterial strain in LDPE polluted soil under laboratory conditions. Materials
and methods
Simulated microcosm experimentsSoil samples were collected 5 cm below the surface of the agriculture
field, sieved in order to remove stones, gravels, soil macrofauna, large roots and plant residues. The soil samples
205
collected at the agriculture field were subjected to three different treatments. The low density polyethylene (LDPE)
degrading bacterium previously isolated from the plastic waste contaminated soil was used in the study.
Treatment 1: Natural soil sample + LDPE
Treatment 2: Nutrients supplemented natural soil sample + LDPE
Treatment 3: Nutrients supplemented natural soil sample + LDPE + Isolated bacterial strain
In treatment 1, 450 g of non-sterile soil was taken. In treatment 2, 450 g of non-sterile soil was supplemented
with (NH4)2SO4 (250 mg kg1 of soil), K2HPO4 (100 mg kg1 of soil), and glucose (2 gkg1 of soil). In treatment
3, in addition to the nutrients added for biostimulation, the soil was inoculated with the isolated bacterial strain. (A
final concentration of approximately 105 colony forming units (CFU) g1 dry wt soil). For each treatment, three
replicates were prepared. Each microcosm was mixed with 450 mg of polyethylene film, covered with foil. Each
soil sample was incubated for 60 days at room temperature. During 60 days of experimental analysis, the systems
were protected from light. During this incubation period, each microcosm was kept moistened by adding sterile
water. The evolution of the carbon dioxide was measured during the incubation period. Experimental results were
subjected to statistical analysis of one-way analysis of variance (ANOVA) for comparison at 5 % level using the
software SPSS-16.
Physicochemical analysis of soil

After incubation, the soil samples were subjected to physicochemical analysis. The physicochemical
properties of the soil such as pH, electrical conductivity available nitrogen 7, available phosphorus8, available potassium9
and organic carbon10 were evaluated.
FTIR analysis
At the end of the incubation period, LDPE films were withdrawn from the soil model systems, carefully
washed and characterized by FTIR spectroscopy. From the FTIR spectra of LDPE film samples collected from
naturally attenuated, biostimulated and bioaugmented soil relative absorbance intensities of the ester carbonyl bond
at 1740 cm_1, keto carbonyl bond at 1715 cm_1, terminal double bond (vinyl) bond at 1650 cm_1 and internal double
bond at 908 cm_1 to that of the methylene bond at 1465cm_1 were evaluated using the following formulae11 : Keto
Carbonyl Bond Index (KCBI) = I1715/I1465; Ester Carbonyl Bond Index (ECBI) = I1740/ I1465; Vinyl Bond Index (VBI)
= I1650/I1465; and Internal Double Bond Index (IDBI) = I908/I1465.
CO2 evolution
To determine the metabolic activity in each microcosm, respirometric analyses were periodically performed
using CO2 evolution12. The experiments were carried out in triplicates and the results were expressed as mean SE
of triplicates.

Bioaugmentation is an approach that involves introduction of microorganisms that possessed biodegradation
potential into the contaminated environment to assist the indigenous microbes with biodegradative processes. In
the present study, for investigating the biodegradation and bioaugmentation effects of the isolated polyethylene
degrading bacterial strain three soil model systems were developed wherein naturally attenuated, biostimulated, and
isolated bacterial strain inoculated (bioaugmented) soil samples were examined for their ability to degrade LDPE.
The physiocochemical properties of the soil samples are presented in Table 1. The pH of the control, naturally
atteneuated, biostimulated and bioaugmented soil samples were found to be 7.5 0.005, 7.560.21, 7.30.40 and
7.580.371 respectively. The electrical conductivity of T1, T2 and T3 soil samples was found to be significantly
increased when compared to that of the control soil sample. High conductivity of soil indicated the high anion and
206
cation content. This might be due to the release of anions and cations during the degradation of polyethylene by the
microbes present in the soil.
Available nitrogen in T2 and T3 soil samples recorded a significant decrease when compared to T1 soil
sample. On comparison with control, T1 and T3 showed a significantly higher level of available nitrogen whereas
T2 showed a significantly lower level. It can be inferred from Table 1 that the soil samples from T1, T2 and T3 had
a significantly higher level of available phosphorus than the control soil sample. T2 soil samples showed a significantly
higher level of available phosphorus when compared to T1 and T3 soil samples. It is seen that there was a
significant increase in the available potassium levels of naturally attenuated, biostimulated and bioaugmented soil
samples when compared to that of the control soil. The available potassium level of T1 soil sample was found to be
significantly higher when compared to that of T2 and T3 soil samples.
The total organic carbon content was significantly increased in biostimulated (0.790.01) and bioaugmented
(0.670.00) soil when compared to naturally attenuated soil (0.620.01). On comparison with control (0.4760.005),
T1, T2 and T3 soil samples showed significantly higher levels of organic carbon. These results indicated that the
organic carbon content might have increased in the soil which is contaminated with the polythene waste due to the
action of indigenous microorganisms.
Despite the availability of C from the hydrocarbons for microbial growth,microorganisms need N and P to
metabolize carbon compounds. The availability of these two nutrients is a key parameter of biodegradation. N and
P must be available to ensure appropriate C/N and C/P ratios, sufficient for optimal microbial growth and metabolism,
which increases the rate of hydrocarbon degradation13. Studies suggested that microorganisms need an abundance
of key elements such as carbon, hydrogen, nitrogen, oxygen and phosphorusfor building macromolecules, addition
of fertilizer provides these microbes with essential elements to reproduce and thrive. Soil microorganisms may be
affected via variation in soil temperature, pH, nutrient status, heavy metals, oxygen level and which in turn can
affect the ecological processes linked with nutrients cycling14.
Table I Physicochemical properties of the soil samples
Treatment LSD (0.05)

Parameter
Control T1 T2 T3
PH 7.5 0.005 7.560.21 7.30.40 7.580.371 0.827

-1
EC dSm 0.290.90 0.990.147 1.3530.322 0.9360.37 0.006
Available N kg ha-1 1811.0 225.4146.94 177.687.023 207.3412.50 0.128
-1
Available P kg ha 14.86.057 30.338.083 37.108.76 37.0311.29 0.032
-1
Available k kg ha 6071.00 78037.54 765.3328.88 701.6750.52 0.001
Organic carbon % 0.470.005 0.620.02 0.790.02 0.670.015 0.001
Values are meanSD of the triplicates
FTIR analysis of LDPE
FTIR spectroscopic analysis is necessary to monitor the formation and disappearance of the carboxyl and
double bond bands to elucidate the mechanism of biodegradation process. In the biodegradation of polyethylene the
initial steps involve the oxidation of the polymer chain leading to the formation of carboxyl groups. These carboxyl
groups subsequently undergo beta oxidation and are completely degraded via the citric acid cycle resulting in the
formation of carbon dioxide and water. Since beta oxidation and citric acid cycle are catalysed by microbial
207
Figure 1. FTIR analysis. Bond indices of LDPE from T1, Figure 2. Cumulative CO2 emissions after 60 days of
T2 and T3 soil samples. Keto Carbonyl Bond Index (KCBI); incubation with LDPE. Values are the meanSE of
Ester Carbonyl Bond Index (ECBI); Vinyl Bond Index (VBI); triplicates
and Internal Double Bond Index (IDBI).The data repre-
sent the means of three replicates and the error bar indi-
cates the percent error.
enzymes, monitoring of these mechanisms by FTIR spectroscopy is essential15. The bond indices (KCBI, ECBI,
VBI and DBI) that were calculated by analyzing the FTIR Spectra of LDPE films taken from T1, T2 and T3 soil
samples are depicted in Figure 1. It is evident from the figure that the values of KCBI, ECBI, VBI and DBI were
increased in LDPE film samples taken from T2 and T3 treatments when compared the LDPE film from T1. These
results indicated the presence of oxidized products. Oxidation of LDPE leads to the accumulation carbonyl groups
and this resulted in increase of the carbonyl index values16.
CO2 evolution
In the present study to determine the metabolic activity in each microcosm, respirometric analysis was
periodically performed using carbon dioxide evolution. The cumulative CO2 emissions, expressed as average value
of three replicates, recorded after 60 days of incubation from the LDPE contaminated soil are shown in Figure 2.
The cumulative carbon dioxide evolution for the treatments T1, T2 and T3 were found to be 637.20 0.91 mg/kg,
644.840.45 mg/kg and 651.600.54 mg/kg respectively. The maximum CO2 evolution was noticed in the
bioaugmented soil. This increment in CO2 evolution of might be associated with large amount of easily available
soluble organic matter, nitrogen, phosphorous and other nutrient sources that would have triggered microbial
activity causing high rate of microbial-mineralization in the soil. The results indicated that the microbial metabolism
was enhanced with the addition of isolated bacterial strain. The highest level of LDPE degradation ability for the
microcosm bioaugmented with isolated bacterial strain might be due to the presence of a larger number of LDPE
-degrading microorganisms. It has been reported that the enhanced PES degradation in the bioaugmented microcosm
can be attributed to the increased microbial population and the result suggests that the bacterial strain Pseudomonas
sp. AKS2 has the potential to be a good bioaugmentation agent for in situ remediation of PES-contaminated soil 17.
In conclusion, the microcosm bioaugmented with isolated bacteria exhibited significantly more LDPE
degradation potential as compared with the degradation by the naturally attenuated and biostimulated microcosms.
Bioaugmentation of soil with the isolated bacteria might be a promising approach for LDPE degradation.
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1. Azeko, S. T., Etuk-Udo, G. A., Odusanya, O. S., Malatesta, K., Anuku, N., & Soboyejo, W. O. (2015)
Biodegradation of Linear Low Density Polyethylene by Serratia marcescens subsp. marcescens and its Cell
Free Extracts. Waste and Biomass Valorization, 6(6), 1047-1057.
208
2. Bhatia, M., Girdhar, A., Tiwari, A., & Nayarisseri, A. (2014) Implications of a novel Pseudomonas species
on low density polyethylene biodegradation: an in vitro to in silico approach. SpringerPlus, 3(1), 497.
1. Eriksen, M., Lebreton, L. C., Carson, H. S., Thiel, M., Moore, C. J., Borerro, J. C.,& Reisser, J. (2014)
Plastic pollution in the worlds oceans: more than 5 trillion plastic pieces weighing over 250,000 tons afloat
at sea. PloS one, 9(12), e111913.
2. Rochman, C. M., Lewison, R. L., Eriksen, M., Allen, H., Cook, A. M., & Teh, S. J. (2014) Polybrominated
diphenyl ethers (PBDEs) in fish tissue may be an indicator of plastic contamination in marine habitats. Science
of the Total Environment, 476, 622-633.
3. Abhay Raja, Sharad Kumara, Izharul Haqa, Sudheer Kumar Singhb (2014) Bioremediation and toxicity
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soils. Curr.Sci.. 25(8), 259-260.
6. Olsen, S. R. (1954) Estimation of available phosphorus in soils by extraction with sodium bicarbonate.
United States Department of Agriculture; Washington.
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journal, 41(9), 446-447.
8. Walkley, A. and I. A. Black. (1934) An examination of Degtjareff method for determining soil organic matter
and a proposed modification of the chromic acid titration method. Soil Sci. 37: 29-37.
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Polym. Degrad. Stab., 18, 7387.
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bioaugmentation strategy Environ Sci Pollut Res (2014) 21:25922602.
11. Semboung Lang, F. , Tarayre, C. , Destain, J., Delvigne, F. , Druart, P. , Ongena, M.1 and Thonart, P.
(2016) Int. J. Environ. Res., 10(4):583-592.
12. Tripathi, D. M., Tripathi, S., & Tripathi, B. D. (2011). Implications of secondary treated distillery effluent
irrigation on soil cellulase and urease activities. Journal of Environmental Protection, 2(05), 655.
13. Sudhakar M, Mukssh Doble, Sriyutha Murthy P, Venkatesan R (2008) Marine microbe-mediated biodegradation
of low and high-density polyethylenes. Int Biodeterior Biodegrad 61:203213
14. Abrusci C, Pablos JL, Corrales T, Lopez-Marn J, Marn I et al (2011)

Biodegradation of photo-degraded mulching films based on polyethylenes and
stearates of calcium and iron as pro-oxidant additives. Int Biodeterior Biodegrad
65:451459
15. Tribedi, P., & Sil, A. K. (2013). Founder effect uncovers a new axis in polyethylene succinate bioremediation
during biostimulation. FEMS microbiology letters, 346(2), 113-120.
209
In vitro antioxidant activity of Medicago sativa
R. Gomathi1 and K. Usha21
Ph.D., Scholar, 1Professor, Biochemistry, Department of Biochemistry, Biotechnology and Bioinformatics,
Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore -43.
e-mail ID: gomathi.biochemistry@gmail.com
Abstract
In the present study, the in vitro enzymic and non enzymic antioxidants were assessed in various parts of
Medicago sativa such as leaves, stem, root and germinated seeds. The leaves possessed the maximum levels of
all enzymic antioxidants analysed, except polyphenol oxidase, which was higher in roots. Stem and germinated
seeds were also found to contain considerable amounts of all the enzymic antioxidants. The non enzymic
antioxidants such as total carotenoid, vitamin C and vitamin E level was found to be higher in leaves when
compared to stem, roots and germinated seeds. The reduced gluthathione level was found to be higher in leaves.
The total phenol and flavonoid content was found to be less in stem, root and germinated seeds of Medicago
sativa when compared to leaves. The results indicated that the plant contain rich source of antioxidants.
Keywords: Medicago sativa, alfalfa, antioxidants
The interest in natural antioxidants, especially of plant origin has greatly increased in recent years (Bhuiya
et al., 2013).
The body has developed several endogenous antioxidant defense systems classified into two groups such
as enzymic and non-enzymic (Oluwaseun and Ganiyu, 2008). In this regard, the medicinal plant selected for the
study is Medicago sativa L. is commonly known as Alfalfa belongs to the family Fabaceae,found throughout the
temperate zone. Itis one of the forage species endowed with many socioeconomic and environmental advantages
(Farissi et al., 2013). So far no reports were published on in vitro antioxidant of this plant. Therefore, the present
study was carried out to assess the antioxidant activity of various parts of Medicago sativa.

Fresh plants of Medicago sativa were collected from Tamil Nadu Agricultural University, Coimbatore,
Tamil Nadu. The collected plant was authenticated by Botanical Survey of India, Coimbatore, (BSI/SRC/5/23/
2014-15/Tech/394). The collected plant samples were washed thoroughly in tap water. The different parts of the
plant were separated and shade dried. The shade dried plant materials such as Medicago sativa leaves, stem, roots
and seeds were ground into coarse powder separately and stored in clean containers at room temperature for
further studies.
Assessment of in vitro antioxidant activities

Estimation of enzymic antioxidants
i. Estimation of Catalase (CAT) activity
CAT activity was assessed by the method of Luck (1974).
ii. Estimation of Superoxide dismutase (SOD) activity
SOD is an important endogenous antioxidant enzyme which acts as the first line defense system against
ROS which scavenges superoxide radicals to H2O2. The activity of SOD was estimated by the method
of Misra and Fridovich (1972).
210
iii. Estimation of Glutathione S-Transferase (GST) activity
The activity of GST was estimated by the method of Habig et al. (1974).
iv. Estimation of Peroxidase (POD) activity

The activity of POD was assessed by the method of Reddy et al. (1995).
v. Estimation of Polyphenol oxidase (PPO) activity

The activity of PPO was assessed in different parts of M.sativa by the method described by Esterbauer
et al. (1977).
Estimation of non-enzymic antioxidants:

The non-enzymic antioxidants were analysed in Medicago sativa leaves, stem, roots and germinated seeds.
i. Estimation of total carotenoids

Total carotenoids were estimated by the method of Zakaria et al. (1979).
ii. Estimation of Vitamin C

The amount of Vitamin C present in different parts of M.sativa was estimated by the method of Roe and
Keuther (1953).
iii. Estimation of Vitamin E

The Vitamin E content in plant was determined by the method of Rosenberg (1992).
iv. Estimation of Reduced glutathione (GSH)

The activity of GSH was determined by the method of Moron et al. (1979).
v. Estimation of total phenol

The amount of total phenol present in various parts of Medicago sativa was estimated by the method
described by Mallick and Singh (1980).
vi. Estimation of flavonoids

The flavonoid content in different parts of the plant was assessed by the method of Cameron et al. (1943).
RESULTS
The activities of enzymic antioxidants were assessed in the different parts are presented in figure 1 (i, ii, iii,
iv, v).
The catalase activity was found to be maximum that is 278.45 3.89 U/g in leaves.The superoxide
dismutase (SOD) level was found to be 20.880.87U/g, 10.22 0.45U/g, 18.15 0.58U/g and 15.34 0.36U/g in
Medicago sativa leaves, stem, root and germinated seeds respectively. The SOD content was found to be highest
in the leaves.
Glutathione S-tranferase and peroxidase were also found to be higher in Medicago sativa leaves when
compared to other parts such as stem, roots and germinated seeds. The polyphenol oxidase level was found to be
high in root when compared to other parts. The leaves possessed the maximum levels of all enzymic antioxidants
analysed, except polyphenol oxidase, which was higher in roots. Stem and germinated seeds were also found to
211
contain considerable amounts of all the enzymic antioxidants.
The levels of non-enzymic antioxidants were estimated in different parts of Medicago sativa andare presented
in figure 2 (i, ii, iii, iv, v).
The total carotenoid content was found to be more in leaves and stem of Medicago sativa and less in roots
and germinated seeds. The vitamin C level was found to be higher i.e 89.34 2.89 mg/g in leaves. The vitamin E
level was found to be higher in leaves (3.560.09 mg/g) when compared to stem, roots and germinated seeds. The
reduced gluthathione level was found to be higher in leaves and stem compared to root.
The total phenol and flavonoid content was found to be 13.56 0.14 mg/g and 24.67 0.74 mg/g respectively
in Medicago sativa leaves. The total phenol and flavonoid content was found to be less in stem, root and germinated
seeds of Medicago sativa when compared to leaves. The results revealed that the leaves possess considerably
higher activities of all the non-enzymicantioxidants analysed compared to stem, root and germinated seeds.
DISCUSSION
Jaleel (2009) reported a higher amount of superoxide dismutase activity in Withania somnifera leaves.
Dhal et al. (2012) reported a higher amount of antioxidants such as superoxide dismutase, catalase and peroxidase
in Curcuma zedoaria leaves.
Bind et al. (2014) reported a higher level of non-enzymic antioxidants such astotal phenols, flavonoids and
carotenoid in the leaves of Argemone mexicana.
According to Sathishkumar et al. (2010) the enzymatic antioxidants such as polyphenol oxidase, peroxidase,
catalase, superoxide dismutase andnon-enzymic antioxidants such as tocopherol and phenols were found to be
significantly high in the leaves of Enicostemma littorale Blume when compared to roots and flowers.
The results indicated that Medicago sativa leaves possessed considerably higher antioxidant activities
when compared to other parts of the plant.
Figurei. 1: Enzymic dismutase

Superoxide antioxidant activity in different parts of Medicago sativa
ii. Catalase
30 400
300
) 20 )g
g/ /
U U 200
( 10 (
T 100
D A
O C
S 0 0
LEAVES STEM ROO T LEAVES STEM ROOT
V alues are expressed by mean SE

ii i.Glutathione S-transfera se iv. Peroxidase
0.8 8
0.6 6
) )g
/g 0.4 / 4
U U
T(S (
D 2
G 0.2 O
P
0
0
LEAVES STEM ROOT
LEAV ES STEM ROO T
V alues are expressed by mean SE

v. Polyphenol oxidase
8
6
3 )
-
0 4
1
x
(U 2
O
P
P 0
LEAVES STEM ROOT
Values are expressed by mean SE
212
Figure 2 : Non-enzymic antioxidant levels in different parts of Medicago sativa
Total Carotenoids Vitamin C
15 1 00
s )
d
io /gg 80
n 10) (m 60
te g/ C
o
r g n
aC 5m i 40
la ( m
at 20
t i
o V
T 0 0
LEAVES STE M ROOT GERMINATED SEE DS LEAVES STEM ROOT G ERMINATED SEE DS

Vitamin E Reduced glutathoine
4 150
) e
g/ n
g3 io 100
(m h
t g)
E2 at /s
N
I lu
g 50 le
o
M1 d m
A e
cu n (
TI
V0 d 0
e
R
LEAVE S STEM ROOT GERMINATED SEED S LE AVES STEM ROOT GERMIN ATED SEEDS

Total phenols Flavonoids
15
30
10 20
)g
5) /g10
/g
0g
m
m
0
L EAVES STEM ROOT GERMINATED L EAVES STEM R OO T GERMINATED SEEDS
SEEDS

ACKNOWLEDGEMENT
The Authors are grateful to Dr. Moorthy,Scientist, Botanical survey of India, Coimbatore. For their help
rendered for identification of Plants.
REFERENCES
1. Bhuiya M A M, Talukder B, Ajri M, Akter S & Sen R (2013) International Journal of Science and Technology,
14(2), 888 893.
2. Oluwaseun A A & Ganiyu O (2008) African Journal of Biotechnology, 7, 3138-3142.
3. Farissi M, Bouizgaren A, Faghire M, Bargaz A & Ghoulam C (2013) Turkish Journal of Botany, 37, 1166-
1175.
4. Luck H (1974) Estimation of catalase, Methods in enzymic analysis, 885, Academic Press, New York,.
5. Misra M P & FridovichI (1972) Journal Biological Chemistry, 247, 3170-3175.
6. Habig W H, PabstM J & Jakoby W B (1974) Journal of Biological Chemistry, 249, 7130-7139.
7. Reddy K P, Subhani S N, Khan, P A &Kumar, K.V (1995) Effect of light and benzyl adenine on dark-treated
growing rice (Oryza sativa) leaves, Change in peroxidase activity, Plant cell Physiology, 26, 987-994.
8. Esterbauer H, Schwarlz E & Hayn M (1977) Analytical Biochemistry, 77, 486-494.
9. Zakaria H, Simpson K, Brown R R & Krotwarie A (1979) Journal of Chromatography, 176, 109-117.
213
10. Roe J H & Keuther C A (1953) Journal of Biological Chemistry, 147, 399-407.
11. Rosenberg H R (1992) Chemistry and physiology of the vitamins, 452-453, Interscience Publishers, New
York.
12. Moron M S, Depierre J N & Mannerisk V C (1979) Biochimica Biophysica Acta, 582, 67-68.
13. Mallick C P & Singh M B (1980) Plant enzymology and plant histoenzymology, 286, Kalyani Publishers,
New Delhi.
14. Cameron G R, Milton R F & Allan J W (1943) Lancet, 179.
15. Jaleel, C A (2009) Plants Omics Journal, 2(4), 163-168.
16. DhalY, Deo B & Sahu, R K (2012) International Journal of Pharmacy and Pharmaceutical Sciences, 4, 3,
343-346.
17. Bind A, Kumar K, Prakash, V & Kumar, M (2014) Research Journal of Pharmaceutical, Biological and
Chemical Sciences, 5(2), 1175-1179.
18. Sathishkumar R, Lakshmi P T V & Annamalai A (2010)International Journal of Pharma and Bio Sciences,
6 (2),1-16.
214
Anticancer Activity Of Eugenol And Eugenol Rich Plant Extracts
(Piper Betle And Syzygium Aromaticum) On Oral Carcinoma Cell
Azhagumeena, C., Preethi, R., Archana, A.K. and Padma, P.R.
Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home science and
Higher Education for Women, Coimbatore 641 043. E-mail ID: jvcazhagubio@gmail.com
Abstract
Piper betle Linn. (Family: Piperaceae) is a common plant cultivated in Asian countries and it is used in our Indian
traditional medicine. Syzygium aromaticum is a tree of the family Myrtaceae, whose aromatic flower buds are
dried and used as a spice (clove). They also possess anti-malarial, anti-diabetic, anti-inflammatory, antinociceptive,
antioxidant, neuroprotective and hepatoprotective properties. Eugenol (4-allyl-2-methoxyphenol) is a natural
phenolic compound, which is the main active component in both Piper betle and Syzygium aromaticum. The
present study was aimed to study the cytotoxic effect of Piper betle and Syzygium aromaticum extracts and
eugenol in oral carcinomacells. HPLC analysis was carried out to confirm the presence of eugenol in the plant
extracts (Piper betle and Syzygium aromaticum). MTT and SRB assays were performed to assess the cytotoxic
effect in oral carcinoma (KB) cells. The results of the HPLC profiles proved that the selected plant sources
contained considerable amounts of eugenol in the methanolic extract. The results also revealed that both Piper
betle and Syzygium aromaticum, as well as their active phenol (eugenol), exhibited significant anticancer
activity towards oral carcinoma cells. To conclude, the presence of phenolics, especially eugenol, might contribute
to the anticancer effect of the extracts of the selected plants.
Key words: Piper betle; Syzygium aromaticum; eugenol, HPLC, MTT and SRB
INTRODUCTION
Eugenol (4-allyl-2-methoxyphenol) is a natural phenolic compound, which is the main active component in
both Piper betle and Syzygium aromaticum(Paranjpeet al., 2013). Piper plants are considered as ecologically and
economically important species and consistof about 1,000-2,000 species in this Piperaceae family (Socorro et al.,
2014). Piper betle L. belongs to the Piperacea family, commonly known as Betel and Paan. It is mostly found in
India, Taiwan, Sri Lanka, Thailand and Southeast Asian countries. It is a stout twisting climber with broadly ovate-
oblong leaves, tiny yellowish-green flowers and small sphere-shaped fruits (Islam et al., 2010; Periyanayagamet
al., 2012). Syzygium aromaticum is a tree belonging to family Myrtaceae, whose aromatic flower buds are dried
and used as a spice (clove). Cloves can be used to increase blood circulation and promote the flow of saliva and
gastric juices (Bhowmik, et al., 2012 and Anita et al., 2015). Piper betle and Syzygium aromaticumbeen reported
to possess anti-inflammatory, antibacterial, antiseptic, antimutagenic, antioxidant, antifungal, antiulcergenic,
antiplatelets, antidiabetic, antiaphrodisiac activity, antiamoebic, antinociceptive, antifertility, antihyperglycemic and
wound healing properties (Hewageeganaet al., 2011 and Wankhede, 2015).The aim of the present studyis to
confirm the presence of eugenol in Piper betle andSyzygium aromaticumto assess the cytotoxic effect of eugenol
along with plant extractsin oral carcinoma (KB) cells.

Preparation of betel leaf extract
Fresh leaves of Piper betle (Athur variety) and dry flower (clove) of Syzygium aromaticumwere purchased
from Coimbatore local market. The leaves were cleaned thoroughly with deinoized water to remove the dust
particles. The leaf sample (10g) was cut into small pieces and added to 100 ml of methanol. The cloves were
215
ground and the powder (10g) wasdissolved in 100 ml of methanol.Sample mixtures were stored at room temperature
in dark for 24 hours with occasional shaking and then filtered through Whatman No. 1 filter paper. The methanolic
extracts were stored at 4C until further experiments were carried out.
High Performance Liquid Chromatography Analysis

The methanolic extracts of the Piper betleand Syzygium aromaticum were dissolved in HPLC grade methanol
and 20l of the sample was injected into reverse phase C18 column of the HPLC apparatus (Shimadzu, Japan).
Sample analysis was performed at room temperature in the wavelength range of 210-600 nm at 1000 psi.Methanol
and water in the ratio of 40:60containing 1% methanol was used as the mobile phase, with a run time of 30 minutes
at a flow rate of 1ml/minute.
Culturing of KB Cell Line

The oral carcinoma (KB) cell line was procured from National Centre for Cell Science (NCCS), Pune,
India. The cells weremaintained in a CO2 incubator (Innova, UK) with 5% CO2 and 95% humidity, in DMEM
supplemented with 10% FBS and 1% of Penicillin and streptomycin (MP Biomedicals, USA) were also added to the
100X stock medium preparation. Once the cells had attained confluent growth, the cells were trypsinized using
TrypsinEDTA (MP Biomedicals, USA) and 105 cells were seeded into sterile 96 well plates. In each well of the 96-
well plate, 100l of the cells were seeded. Then the plates were incubated in a CO2 incubator (Innova, UK) with 5%
CO2 and 95% humidity.
The cells were treated with different concentrationssuch as 10 M, 25 M and 50 M of eugenol and
plant extracts. The cells were harvested after 24 hours of treatment and used for cell viability assays.
Cell viability assays

MTT dye reduction assay
The MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay wasemployed
to elucidate the cytotoxicity of the sample (Igarashi and Miyazawa, 2001).
Sulphorhodamine B (SRB) assay

The SRB assay was employed to determine the cell viability in oral (KB) carcinoma cells(Skehanet al.
1990).

HPLC analysis of the extracts
The HPLC analysis was carried out to confirm the presence of eugenol in both the plant extracts. HPLC
profile of stented eugenol was compared with both plant extracts. The HPLC profile of the eugenol standard
(Figure 1) peak observed at 9.2 min. This eugenol standard peak was compared with the methanolic extracts of
Piper betle leaves and Syzygium aromaticum flower. The Piper betle leaves extract (Figure 2) corresponded to
peak at 9.18 retention time.The Syzygium aromaticum extract showed a clear peak at 9.22 minutes. An additional
smaller peak was also observed in the extract, indicating the presence of other components. However, it is clear
from the HPLC profile that eugenol is the major component in the Syzygium aromaticum extract, which is depicted
in Figures 3.This observation proved the presence of eugenol in the methanolic extract of Piper betle leaves and
Syzygium aromaticum flowers. The extract also showed the presence of additional peaks, indicating the presence
of other compounds.Mahapatra and Roy (2014) reported the presence of eugenol in betel leaf using HPLC analysis.
Similarly the presence of eugenol was shown in the extract of Ocimumgratissimum(Banerjee and Shah, 2014).
Several studieshave proved that phenolic compounds present in spices that are used as food adjuncts possess
potent antioxidant,anti-inflammatory, antimutagenic and cancer preventive activities. The antioxidative effects of
216
eugenol in clove has been extensively studied (Srinivasan, 2014). Bruno et al., (2015) reported the presence of
eugenol in the Athur variety of Piper betle leaves and presence of eugenol, quercetin, rutin and catechin in the stem
of Piper guinneensis.
Peak retention time : 9.2 minutes
Figure 1 : HPLC profile of standard eugenol
Peak retention times

Peak 1 - 2.1 minutes
Peak 7 - 9.18minutes
Figure 2 : HPLC profile of methanolic extract of Piper betle leaves
Peak retention times

Peak 3 - 9.2minutes
Figure 3 : HPLC profile of methanolic extract of Syzygium aromaticumflower

MTT assay
Oral (KB) carcinoma cells were exposed to different concentrations(10 M, 25 M and 50 M) of eugenol,
Piper betle and Syzygium aromaticumextracts and the cell viability was asses MTT assay. The results are represented
in Figure 4. The results revealed that all the extracts induced cell death in a dose dependent manner. Among the
extracts treated Syzygium aromaticumshowed better cytotoxic effect at a concentration 50M towards the oral
217
(KB) carcinoma cells.Lee et al. (2013) depicted that the viability of colon carcinoma cells CT26.WT increased in
a dose-dependent manner when treated with aqueous garlic extract, compared with the control. The MTT assay
also reported that the cell viability on the -type Ti-13Zr-13Nb alloy was higher than the boron containing Ti-13Zr-
13Nb-0.5B composite in MG63 cell line (Majumdharet al., 2015).
Figure 4 : Effect of Piper betle,Syzygium aromaticum and eugenol on the viability of

oral (KB) carcinoma cells
Values are mean SD of triplicate, control group were fixed as 100% and the percent viabilities in the other
groups were calculated relative to this. Pb - Piper betle, Sa - Syzygium aromaticum, Eu Eugenol
SRB assay
SRB assay was performed to study the cytotoxic effect of the extracts. The results revealed that cell
viability was decreased with the increase in concentration. When compared, Syzygium aromaticum induced maximum
cell death at 50M than the other extracts. Form the results it can be confirmed that all the extracts were able
induce cell death in a dose dependent fashion (Figure 5). Olarteet al. (2013) depicted that the hexane extract of
Cassia alataleaves shown cytotoxicity to MCF-7, SK-BR-3 (breast cancer) cell line, bladder cancer cell line (T24)
and colorectal cancer cell line (Col-2) and no cytotoxicity to Chinesehamster ovarian cell line (CHO-AA8).
Figure 5 : Effect ofPiper betle,Syzygium aromaticum and eugenol on the viability of

oral (KB) carcinoma cells
Values are mean SD of triplicate, control group were fixed as 100% and the percent viabilities in the other
groups were calculated relative to this. Pb - Piper betle, Sa - Syzygium aromaticum, Eu - Eugenol
218
CONCLUSION
The presence of the phenolics in the plants was confirmed by HPLC. The HPLC profiles proved that the
selected plant sources contained considerable amounts of the phenolics under study. Based on the HPLC profiles,
the leaves of Piper betle and Syzygium aromaticum were taken for further study along with commercially available
eugenol. The MTT and SRB assays were used to determine the effect of the methanolic extracts of the Piper betle
andSyzygium aromaticum on the viability of KB cells. The results revealed that both Piper betle and Syzygium
aromaticum, as well as their active phenolic compound eugenol, exhibited good anticancer activity against oral
(KB) carcinoma cells. To conclude, the anticancer effect of the plant extracts might be due to the presence of
eugenol.
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3. Bokesch, H., Kenney, S. and Boyd, M.R. (1990) J. Natl. Cancer Inst., 82, 1107-1112.
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(2011)Int. J. Res. Ayur. Pharm., 2(5), 1601-1603.
6. Igarashi, M. and Miyazawa, T. (2001) Biochem. Biophys. Acta/Mol. Cell Biol. Lipids, 1530, 162-171.
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J. Int., 1(6), 384-387.
8. Mahapatra, S.K. and Roy, S. (2014) Asian Pac. J. Trop. Med., 7, 391-397.
9. Periyanayagam, K., Jagadeesan, M., Kavimani, S. and Vetriselvan, T. (2012)Asian Pacific J. Tropical
Biomedicine, 2(2), S506-S510. doi:10.1016/S2221-1691(12)60262-7.
10. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J.T., Socorro, M.M.L.D.,
Bendoy, C.P. and Dacayana, C.M.L. (2014)J. Multidisciplinary Studies, Vol. 3(1):100-111.
11. Wankhede, T.B. (2015)Int. Res. J. of Sci. Eng.,3(4): 166-172.
12. Lee, J., Gupta, S., Huang, J.S., Jayathilaka, L.P. and Lee, B.S. (2013) Anal. Biochem., 436, 187-189.
13. Majumdar, P., Singh, S.B., Dhara, S. and Chakraborty, M. (2015) Mater. Sci. Eng. C., 46, 62-68.
14. Paranjpe, R., Gundala, S.R., Lakshminarayana, N., Sagwal, A., Asif, G., Pandey, A. and Aneja, R. (2013)
Adv. Acc.Publ.,10, 1-9.
15. Olarte, E.I., Herrera, A.A., Villasenor, I.M. and Jacinto, S.D. (2013) Asian Pac. J. Cancer Prev., 14, 3191-
3196.
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Phytochem., 1(1):13-22.
219
Hypoglycemic and Hypolipidemic Potentials of Cinnamon
(Cinnamomum zeylanicum) on Metabolic Syndrome of Adults
*Balasasirekhar R and Lakshmi U K
*Assistant Professor, Department of Food Science and Nutrition,
Avianshilingam Institute for Home Science and Higher Education for Women, Coimbatore 43,
e-mail: balasasirekhar@gmail.com
Abstract
Medicinal plant of Cinnamomum zeylanicum has pharmacological effect in the treatment of hyperlipidemia and
diabetes mellitus. The objective of the study was to analyse the nutritive value, active principle, supplement and
evaluate the effect of cinnamon on hyperglycaemic and hyperlipidemic adults. Adults aged 40 to 60 years with
diabetes mellitus and hyperlipidemia was identified. Preliminary data was collected, screened and subjected to
biochemical tests relevant to their physiological status. Capsuled cinnamon powder was supplemented to
adults for 90 days. Respective control groups were also formed. Results revealed a mean reduction in total
cholesterol (68.87 mg/dl), triglyceride (47.73 mg/dl), LDL (62.77 mg/dl) and VLDL (9.55 mg/dl) cholesterol in
hyperlipidemic adults. A mean increase in HDL (3.83 mg/dl) cholesterol was observed. The adults with
hyperlipidemia and diabetics revealed that the fasting blood sugar showed a mean reduction of 47.07 mg/dl,
postprandial sugar (56 mg/dl) and glycosylated haemoglobin (1.95 %/100 ml). The mean reduction was 53.73,
36.20, 53.62 and 7.24 mg/dl in total cholesterol, triglyceride, LDL and VLDL respectively. The HDL showed a
mean increase of 7.13 mg/dl in the cinnamon supplemented group. The results of the experimental groups were
statistically significant at one per cent level. No significant change was observed in both the control groups.
Keywords: Cinnamon, spice, diabetes mellitus, hyperlipidemia
INTRODUCTION
Spices are derived from roots, barks, buds and fruits of plants. Herbs are usually taken from the leaves of
various plants. They are commonly divided into the categories of spices, spice seeds and herbs. Both herbs and
spices are referred to as herbal remedies and are excellent antioxidants, which work to neutralize the attacks made
by free radicals against the body. Spices also contain phytonutrients, which may prevent the mutation of healthy
cells into cancerous cells. Spices are the powerhouse of pleasure and health [1].
A growing body of research has demonstrated that the commonly used herbs and spices such as garlic,
black cumin, cloves, cinnamon, ginger, thyme, allspice, bay leaves, mustard and rosemary possess antimicrobial
properties that in some cases can be used therapeutically. Other spices such as saffron, a food colorant, turmeric,
a yellow coloured spice, tea, either green or black, ginger and flaxseed do contain potent plant substances including
carotenoids, curcumins, catechins and lignan respectively which provide significant protection against several
chronic health conditions including cardiovascular conditions and tumour prevention [2].
Cinnamon (Cinnamomum zeylanicum), a bushy evergreen tree of the Laurel family (Lauraceae) is light
brown in colour and has a delicately fragrant aroma and warm, sweet flavour. Ground cinnamon is the most
common baking spice. Cinnamon was once more valued than gold. In ancient Egypt, cinnamon was used in
embalming process. Pliny wrote that 350 g of cinnamon was equal in value to 5 kg of silver. In Hodoo, it is a
multipurpose ingredient used for purification, luck, love and money [3].
Cinnamon has been reported to have remarkable pharmacological effects in the treatment of type 2 diabetes
[4] and also used as an insect repellent. Half teaspoon of cinnamon per day can lower LDL cholesterol [5].
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Cinnamon has shown an amazing ability to stop medication resistant yeast infections [6]. In a study at Copenhagen
University [7], patients given half a teaspoon of cinnamon powder combined with one tablespoon of honey every
morning after breakfast had significant relief in arthritis pain after one week and continued to walk without pain
within one month. When added to food, it inhibits bacterial growth and food spoilage, making it a natural food
preservative [8]. Studies found that smelling cinnamon boosts cognitive function and memory. Researchers at
Kansas State University found that cinnamon fights the E.coli bacteria in unpasteurised juices [9]. It is a great
source of manganese, fiber, iron and calcium [10].
The benefits of spices may extend far beyond pumpkin pie recipes. There are evidences that spices can
boost insulin function and lower cholesterol. Diabetes Mellitus (DM) and Cardio Vascular Disease (CVD) share
several important characteristics. The occurrence of both conditions increases with age and both are associated
with an adverse lipid profile, obesity and a sedentary life style and the risk of both can be reduced by lifestyle
modifications of common risk factors [11].
Hence the objectives of the study were
To analyse the nutritive value and prepare the cinnamon spice capsules.
To supplement the diets of selected adults with hyperlipidemia and diabetes mellitus with the cinnamon
spice capsules for a period of three months and
To evaluate the effect of cinnamon spice capsules on hyperlipidemic diabetic adults using various parameters.

Five hundred adults in the age group of 40 to 60 years residing in Coimbatore with hyperlipidemia and diabetes
were identified for the survey. The controlled nature of the disease condition and their willingness to participate
and co-operate in the three months feeding trial formed the basis for selection of adults. One group of 30 men who
reported hyperlipidemia with symptoms of diabetes mellitus from Bharat Sanchar Nigam Limited, Bharathi Park
Road, Coimbatore were selected for the supplementation study.
Details regarding their socio-economic characteristics including age, sex, occupation, educational status, family
type, monthly income and family background were gathered through interview method using a pre-tested
questionnaire. Questions were also included to get information on their lifestyle pattern like yoga, exercise, alcohol
consumption, chewing habits, smoking, dietary pattern, food intake pattern, foods included and avoided, health
status like general health, history of the present condition, diabetic / hyperlipidemic trait in the family, duration and
treatment of the condition, physiological symptoms experienced and diseases if any. Consumption of spices and
awareness of the spices used for supplementation also formed part of the questionnaire. Five hundred adults
including the selected adults for supplementation and control were interviewed. Food consumption pattern of the
selected hyperlipidemic and diabetic adults was assessed using a three day recall method.
Anthropometric measurements such as height, weight, Body Mass Index, Waist Circumference, Hip
Circumference, Waist Hip Ratio, Blood Pressure were recorded for the 30 hyperlipidemic diabetic adults selected
for the supplementation study. All the selected adults were screened and examined by a physician for the presence
of clinical symptoms before and after the supplementation period. The selected adults were subjected to biochemical
tests. The biochemical tests include blood haemoglobin, lipid profile, total cholesterol, triglycerides, HDL, LDL
and VLDL cholesterol, fasting blood sugar, post prandial blood sugar and glycosylated haemoglobin. All these
biochemical parameters were done before and after the supplementation period of three months with the help of
trained laboratory technicians.
After an extensive appraisal of literature pertaining to spices, cinnamon was selected for the study. Cinnamon
was procured and cleaned to remove any impurities. It was washed and allowed to dry under shade to remove the
221
excess water. The cleaned spice was then spread in trays and dried in a cabinet drier at 40O C for one hour and
allowed to cool. The above process (i.e. heating at 40O C for one hour and cooling) is repeated until the moisture
content came to less than 10 per cent. The dried spice was then pulverized, sieved and then stored in air tight
container. Many of the adults of the present study preferred cinnamon in the form of capsules. Each capsule was
filled with approximately 490 to 500 mg of cinnamon powder. The nutrients present in the cinnamon was analysed
using the standard procedures [12].
The selected 30 adults with hyperlipidemia and diabetes mellitus were divided into two groups consisting of 15
adults in each group. One group constituted the experimental group who were given each two grams of cinnamon
in the form of four capsules daily for a period of three months. The remaining 15 adults constituted the control
group. To the control group two grams of roasted bengal gram flour in the form of capsules were given daily till the
end of the supplementation period. The effect of supplementation on the selected adults was evaluated using the
anthropometric measurements, clinical assessment and biochemical assessment before and after a period of three
months of supplementation.

Socioeconomic background
A majority of 30.4 per cent were in the age group of 40 to 45 years, whereas only 18 per cent of the males
were in the age group of 56 to 60 years. All the adults were literates. About 70 per cent of the adults have
completed either graduation or professional degree. About 70 per cent of the adults are either employed in private
or government sector or engaged in business. Only 23 per cent of adults were home makers and 8 per cent were
retired adults. Forty seven per cent of the families were joint and 53 per cent of them are nuclear type. Eighty three
per cent had 3 to 5 members in the families whereas remaining 17 per cent had above five members in the families.
Prevalence of the metabolic disorder i.e. hyperlipidemia and diabetes mellitus was high in high income group (66
per cent) compared to the low and middle income groups.
Life style pattern
Yoga was practised by 45 per cent of the adults either daily, weekly once or twice and 54 per cent of the
adults did not practice yoga. Thirty five per cent of the adults did exercises regularly from half an hour to one hour
and 65 per cent of adults did not do any exercise. All the adults who exercised either went for walking or jogging.
Almost 75 per cent of the adults had the habit of cigarette smoking. Among them 30 per cent discontinued
smoking after the onset of the disease. Forty three per cent of the adults developed the habit of smoking in the past
ten years. Also, about 47 per cent of the adults used less than 5 cigars per day. Only 25 per cent of the adults did
not have the habit of smoking. Out of 295 adult males, 110 adults did not use alcohol and others had the habit of
consuming it. With regard to the duration of alcohol consumption, 60 per cent consumed for the past 10 years.
With regard to the frequency of consumption, 31 per cent of the adults consumed regularly and 34 per cent
occasionally. After the onset of the disease condition, about 34 per cent of the adults stopped consuming alcohol.
Fifty six per cent of the adults did not have the habit of chewing neither tobacco, pan masala nor betel leaves. Forty
per cent of the adults had the habit of chewing for the past 11 to 20 years.
Food pattern
A majority of 61 per cent of the selected adults were non-vegetarians. Remaining 28 and 10 per cent of the
adults were vegetarians and ova-vegetarians respectively. All the male and female adults consumed three meals a
day i.e. breakfast, lunch and dinner with majority consuming either wheat or ragi based food items. Fifty three per
cent of adults included raw salad along with their diet. All the adults used steaming, roasting and boiling as a
method of cooking. About 53 and 61 per cent of adults adopted frying and stewing methods respectively. Bakery
items were used by 56 per cent of adults. Majority of the adults used gingelly oil (56 %) and groundnut oil (60 %)
222
for cooking. All the adults used refined oil for cooking various food preparations. Ten per cent of the adults used
olive oil for various food preparations. Regarding the consumption of beverages majority of adults consumed both
tea and coffee either with (17 %) or without sugar (19 %). Twelve per cent of the adults did not consume any
beverage. About 69, 49, 32, 29 and 16 males consumed fruit juices, tender coconut, health drinks, soups and green
or black tea respectively. Among females about 83 and 67 of them consumed fruit juices and soups. Majority of
adults consumed green leafy vegetables, vegetables and fruits. Fifty per cent of the adults included more pulses
and grams, green leafy vegetables and vegetables. Fifty seven per cent of adults added wheat products and 85 per
cent avoided coconut and salt in their diet. About 17 per cent of the adults did not follow any dietary modifications.
History of the Disease

Among the 500 adults, 255 adults were identified with hyperlipidemia and diabetes mellitus. Twenty six
per cent of adults had hyperlipidemia and diabetes mellitus for the past 5 to 10 years. Familial tendency of the
subjects revealed that heredity is playing a vital role in the precipitation of disease at a younger age. It is found that
179 adults had diagnosed the condition within ten years. Except 7.5 per cent all of them were taking treatment with
a majority of 28.6 per cent taking treatment from 1 to 5 years. With regard to the type of treatment a majority of
adults undertook allopathic treatment. Only 45 per cent adults were aware that spices could help to control or
fight against diseased conditions. About 53 males and 50 females were aware that fenugreek mixed with jeera
could control diabetes mellitus.
Nutrient analysis of cinnamon revealed that the energy content was 190 Kcal per 100 g. The trace
elements like lead, zinc, arsenic and chromium were found to be below detectable limits. The active principle
namely cinnamaldehyde was 36 mg %.
Food and Nutrient intake
Mean food intake was deficit in the consumption of greens, vegetables, fruits and milk products in adults
with hyperlipidemia and diabetes mellitus. The mean nutrient intake was deficit in beta carotene and vitamin C
when compared with RDA with respect to all nutrients.
Effect of Cinnamon supplementation on hyperlipidemic diabetic adults

Supplementation of cinnamon helped to reduce the clinical symptoms seen among the adults with
hyperlipidemia and diabetes mellitus for the experimental group. No change was observed in the control group
after three months. A significant weight reduction by 1.6 kg and 0.4 kg was observed in experimental and control
group respectively. It is notable that adults had normal BMI, whereas others fell under at risk categories. The waist
hip ratio of 49 adults was found to be above 0.95 before the supplementation and seven adults moved to normal
category after supplementation. Normal blood pressure was observed among 37 and 45 adults before and after
supplementation. The blood pressure of 38 adults were above normal before supplementation and eight adults
shifted to normal category after supplementation with cinnamon spice capsules. The mean blood haemoglobin
levels of the adults increased significantly with 0.17 mg/dl in the experimental group. No significant change was
observed in control group.
Lipid Profile
The total cholesterol levels of the adults in the cinnamon supplemented group was 249.67 mg/dl before
supplementation which were above the normal desirable level of < 200 mg/dl and placed in high risk group. A mean
reduction by 195.93 mg/dl was observed at the end of the supplementation period. The control showed a mean
reduction of 0.67 before and after supplementation. A mean difference of 36.2 mg/dl was observed in triglyceride
levels of the supplementation group with initial and final triglycerides values of 174.87 and 138.67 mg/dl. The mean
initial triglyceride levels was 177.07 mg/dl and mean final values was178.67 mg/dl. The mean increase of 1.6 mg/
223
Table I : Lipid Profile of the Hyperlipidemic Diabetic Adults
Mean SD t value
Biochemical
Groups Initial Final Between
Parameter (mg/dl) Difference I Vs F
(I) (F) Groups
Experimental 249.67 195.93 -53.73
20.44** HDA Vs
Group HDA 10.1 8.87 10.17
Total cholesterol HDE
Control Group 253.07 252.4 -0.67 17.94**
0.59 N S
HDE 7.59 8.86 4.37
Experimental 174.87 138.67 -36.2
13.83** HDA Vs
Group HDA 12.71 10.97 10.13
Triglyceride HDE
Control Group 177.07 178.67 +1.6 11.98**
1.02 NS
HDE 12.12 12.74 6.06
Experimental 36.95 44.08 +7.13
13.72** HDA Vs
Group HDA 2.22 2.28 2.01
HDL Cholesterol HDE
Control Group 38.93 38.91 -0.02
0.33 N S 12.10**
HDE 1.74 2.17 0.85
Experimental 177.74 124.12 -53.62
19.93** HDA Vs
Group HDA 9.66 8.45 10.41
LDL Cholesterol HDE
Control Group 178.73 177.76 -0.31 17.48**
0.26 NS
HDE 8.18 9.16 4.67
Experimental 34.97 27.73 -7.24
13.83** HDA Vs
Group HDA 2.54 2.19 2.03
VL DL Cholesterol HDE
Control Group 35.41 35.73 0.48 12.38**
1.61 NS
HDE 2.42 2.55 1.15
** P < 0.01; Significant at 1 per cent level; NS Not Significant
Table II : Blood Sugar Levels of the Hyperlipidemic Diabetic Adults
Mean SD t value
Biochemical Parameter Groups Initial Final Between
Difference I Vs F
(I) (F) Groups
Experimental 161.00 113.93 -47.07
19.03** HDA Vs
Fasting blood sugar Group HDA 9.51 6.3 9.57
HDE
(mg/dl) Control Group 165.87 165.33 +0.4
0.72NS 18.10**
HDE 9.24 9.48 2.16
22.65** HDA Vs
Post prandial blood Group HDA 11.69 9.7 9.57
HDE
sugar (mg/dl) Control Group 259.53 254.67 -4.87
3.62** 17.56**
HDE 13.31 14.97 5.21
Glycosylated 11.29** HDA Vs
Group HDA 0.52 0.55 0.67
haemoglobin (%/100 HDE
ml) Control Group 7.85 7.84 -0.01 10.78**
0
HDE 0.43 0.41 0.07
** P < 0.01; Significant at 1 per cent level; NS Not Significant
224
dl was observed after a supplementation period of three months. The mean HDL cholesterol levels increased from
36.95 mg/dl to 44.08 mg/dl before and after supplementation. A mean increase of 7.13 was observed. Whereas, a
mean reduction of 0.02 mg/dl was observed in the control group with mean initial and final values of 38.93 and
38.91 mg/dl respectively. The mean initial and final LDL cholesterol levels of the experimental group was 177.74
and 124.12 mg/dl respectively. A mean difference of 53.62 and 0.31 mg/dl was observed in the LDL cholesterol
levels of the experimental and control group respectively. The mean initial and final VLDL cholesterol levels of the
experimental group was 34.97 and 27.73 mg/dl and that of control group was 35.41 and 35.73 mg/dl respectively.
The mean difference of 7.24 mg/dl was found in the experimental group. But the control group showed an increase
of 0.48 mg/dl after a period of three months (Table I).
Blood Sugar Profile
The mean initial and final fasting blood sugar level among the supplementation group was 161 and 113.93
mg/dl; among the control group was 165.87 and 165.33 mg/dl respectively. A mean decrease of 47.07 mg/dl and
a mean increase of 0.4 mg/dl was observed in the experimental and control group respectively. The post prandial
blood sugar levels were 262.6 mg/dl in the experimental group, 259.53 mg/dl in the control group before the
supplementation. The mean values decreased to 206.6 mg/dl in the experimental group and 254.67 mg/dl in the
control group. Both the experimental and control groups showed a mean difference of 56 and 4.87 mg/dl respectively
after the supplementation period. The mean glycosylated haemoglobin decreased from 7.91 to 5.96 and from 7.85
to 7.84 %/100 ml in the experimental and control groups respectively. Majority of the cinnamon supplemented
adults moved from fair control to good control group after three months (Table II).
A significant difference at one per cent level was observed in the lipid profile of the initial and final values
experimental adults. No significant difference was observed in the control group. Also an one percent level of
significance was observed in the lipid profile within the groups.
A one per cent drop in serum cholesterol reduces the risk for Coronary Heart Disease (CHD) by two per
cent [13]. Lowering triglycerides and increasing HDL cholesterol is associated with a reduction in cardiovascular
events in patients with type 2 diabetes mellitus [14]. Elevated levels of HDL cholesterol actually lower the risk of
CHD (greater than or equal to 60 mg/dl) and is considered a negative risk factor for CHD [15].
CONCLUSION
Adults supplemented with cinnamon reported that they were very active and brisk throughout the study
period. They were able to work round the clock without any tiredness. All the adults of the experimental group
expressed that they did not come across any infections like cold, cough, asthma, wheezing and fever, and symptoms
like headache, giddiness, fatigue, stomach upsets like indigestion and diarrhoea during the three month period of
supplementation.
As far as the adults with hyperlipidemia and diabetes mellitus, results clearly indicated the positive role of
spices in maintaining the lipid profile and in the control of blood sugar and relieved them of painful symptoms in
perfect condition thus corroborating the results of some studies on cardiovascular condition from other countries
[16,17]. This positive impact on adults with hyperlipidemia and diabetes mellitus is encouraging and being a dietary
intervention it is devoid of other possible side effects, proving that cinnamon supplementation is a cost effective
and sustainable strategy in the management of metabolic syndrome.
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225
3. Scott, K., (2001), Medicinal seasonings, The healing power of spices, pp. 54
4. Agricultural Research Magazine (2000), Cinnamon Extracts Boost Insulin Sensitivity, July 2000
5. Khan, A., Safdar, M., Ali Khan, M.M., Khattak, K.N. and Anderson, R.A., (2003), Cinnamon improves
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7. Nessa, R., (2004), The benefits of cinnamon and honey, The Star magazine, Vol. 1 (150), pp. 245-254
8. Shelef, L.A., (2008), Antimicrobial effects of spices, Journal of Food Safety, Vol. 6 (1), pp. 29 44
9. Erlich, E.W., (2008), The effect of cinnamon on E.coli growth, Project Number J1409, California State
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10. Palmer, A.S., Stewart, J. and Fyfe, L., (1998), Antimicrobial properties of plant essential oils and and
essences against five important food borne pathogens, Letters in Applied Microbiology, Vol. 26, pp. 118
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11. Pyorala, K., Laakso, M. and Uusisupa, M., (1987), Diabetes and atherosclerosis: an epidemiological view,
Diabetes Metabolism Review, Vol. 3, pp. 463 - 524
12. Brahman, G.N.V., Laxmaiah, A., Mallikharjuna Rao, K. and Reddy, G., (2005), Methodology of assessment
of diet and nutritional status of community, Manual of National Institute of Nutrition, Hyderabad, pp. 7 9,
13, 16
13. Nathan, D.M., Cleary, P.A., Backlund, J.Y., et al., (2005), Intensive diabetes treatment and cardiovascular
disease in patients with type 1 diabetes, N Engl J Med, Vol. 353, p.2643.
14. Klausen, K., Borch-Johnsen, K., Feldt-Rasmussen, B., Jensen, G., Clausen, P., Scharling, H., Appleyard, M.
and Jensen, J.S., (2004), Very low levels of microalbuminuria are associated with increased risk of coronary
heart disease and death independently of renal function, hypertension, and diabetes, Circulation, Vol. 110,
pp. 3235
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on plasma lipid concentrations in adults with moderate hypercholesterolemia: a randomized clinical trial,
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226
Investigation of Hypoglycemic and Antioxidant Properties of Ethanolic
Extract of the Leaves of Boerhavia diffusa in
Streptozotocin-Induced Rats
C.C.S. Vasundhara1 and S. Gayathri Devi2
1
Ph.D Scholar, Department of Biochemistry, 2 Associate Professor, Department of Biochemistry, Biotechnology and Bioinformatics,
Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore 641 043, Tamil Nadu, India.
Email ID: gayathridevi.adu@gmail.com
Abstract
Diabetes mellitus, a chronic metabolic disorder characterized by hyperglycemia. It is characterized by abnormalities
in metabolic pathways of carbohydrate, lipid and protein. The aim of this study was to investigate the effect of
ethanolic extract of leaves of Boerhavia diffusa (ELBD) on glucose levels, haematological parameters, activities
of enzymic and non-enzymic antioxidants in the control and streptozotocin-induced rats (STZ). The rats were
made diabetic by single intraperitoneal injection of STZ (60 mg/kg body weight). After three days, the rats with
blood glucose level > 180mg/dl were separated and used for the study for a period of 45 days. The administration
of ELBD (250 mg and 500 mg/kg b.w.) as well as glibenclamide (10 mg/kg b.w) to STZ-induced rats was found to
reduce the glucose levels when compared to the control group. The haematological indices namely red blood
cell count (RBC), white blood cells (WBC) and its related indices were found to be decreased whereas the levels
of MCH, polymorphs and eosinophils were increased in STZ-induced rats. Treatment with ELBD and glibenclamide
has restored their levels to normalcy. The activities of the antioxidants were found to be decreased in STZ-
induced rats and on treatment with ELBD and glibenclamide a significant increase was observed. Hence, it can
be concluded that the ELBD possesses antihyperglycemic and antioxidant properties and hence can be used as
a curative medicine.
Keywords: Boerhavia diffusa, streptozotocin, glibenclamide, antihyperglycemic
INTRODUCTION
Diabetes mellitus is a metabolic disorder characterized by hyperglycemia with impaired metabolism of
carbohydrates, fat and proteins. Chronic hyperglycemia in diabetes mellitus induces multiple biochemical sequelae
including diabetes induced oxidative stress which plays a vital role in the symptoms and progression of the disease1.
Herbal medicine has become popular to cure diseases because of its safer and lesser side effects when
compared to the currently available drugs2. Boerhavia diffusa (one of the renowned medicinal plants) is used to
treat large number of diseases as mentioned in Ayurveda and other system of medicine3. Hence the present study
was undertaken to investigate the effect of ethanolic extract of the leaves of Boerhavia diffusa (ELBD) for its
hypoglycemic and antioxidant potentials in streptozotocin-induced rats.

Collection of plant material and extraction
The experimental plant Boerhavia diffusa was grown in the herbal garden of Avinashiligam Institute for
Home Science and Higher Education for Women, Coimbatore and authenticated by Botanical Survey of India,
TNAU, Coimbatore. The authentication number is BSI/SRC/5/23/2013-14/Tech/1041. 15 gm of fresh leaves of
Boerhavia diffusa was extracted in 95 per cent ethanol and the solvent was evaporated and used for the study.
227
Male Wistar rats weighing 150-200 g are used for the study. The experimental procedure was initiated
after getting approval from the Institutional Animal Ethics Committee (KU/IAEC/Ph.D/127/2013). The animals
were treated in accordance with Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) guidelines.
Diabetes mellitus was induced in fasting rats by intraperitoneal injection of streptozotocin (STZ) (60 mg/
kg b.w.) in 0.01 M sodium citrate buffer (pH 4.5).
Experimental design
The male Wistar rats with blood glucose level > 180 mg/dl were divided into four groups of six animals.
The grouping of the experimental animal is given in Table1.
Oral glucose tolerance test (OGTT)

The OGTT of ELBD was evaluated in the fasting normoglycemic and diabetic rats. The experimental
design for OGTT is presented in Table 2.
Table 1: Grouping of the experimental animals
Groups Designation Treatment

T1 UC Control
T2 STZ Streptozotocin-induced (60 mg/kg b.w/day)
T3 STZ + ELBD Streptozotocin + ethanolic extract of leaves of Boerhavia diffusa
(ELBD) (500 mg/kg b.w/day)
T4 STZ + glibenclamide Streptozotocin + glibenclamide (10 mg/kg b.w/day)
Table 2: Grouping of animals for OGTT
Groups Treatment (extract/drug dose)

1 Control
2 Streptozotocin-induced rats
3 Streptozotocin-induced rats + 250 mg/kg b.w. ethanolic extract of leaves of
Boerhavia diffusa (ELBD)
4 Streptozotocin-induced rats + 500 mg/kg b.w. ethanolic extract of leaves of
Boerhavia diffusa (ELBD)
5 Streptozotocin-induced rats + Glibenclamide (10 mg/kg b.w)
After 14 days of treatment the rats were fasted and fasting blood samples were collected from the tail vein.
Glucose load of (2 g/kg b.w) was given. The glucose levels at 30, 60, 90, 120 and 180 minutes was measured4.
The 1/5th (250 mg) and 1/10th (500 mg) of the dose were chosen to perform the OGTT.
After 45 days the rats were fasted overnight and anaesthetized. Blood was collected into tube which
contains EDTA for haematological parameters5 and liver was taken out, to determine the enzymic antioxidant
228
namely catalase6, superoxide dismutase7 and glutathione peroxidase8 and non-enzymic antioxidants vitamin C9,
vitamin E10 and reduced glutathione (GSH) 11.
Statistical Analysis
Statistical significance was determined using One-way Analysis of Variance (ANOVA) followed by Tukeys
test. p value of 0.05 or less was considered as significant.

Oral glucose tolerance test of experimental rats
The oral glucose tolerance test was evaluated in the fasting normoglycemic rats in order to evaluate the
efficacy of ELBD on glucose and the results are presented in Figure 1.
Figure 1: Oral glucose tolerance test of experimental rats
Values are mean S.E.M (n=6 rats in each group)
The administration of ELBD (250 mg and 500 mg/kg b.w.) as well as glibenclamide (10mg/kg b.w) to
STZ-induced rats was found to reduce (p<0.05) the glucose levels when compared to the control group. The
blood glucose lowering effect of the extracts was found to be dose dependent. The ELBD at 500 mg/kg b.w. was
found to be more effective when compared to 250 mg/kg b.w. and Glibenclamide. Hence in our study the glucose
tolerance observed in STZ-induced diabetic rats was altered to near normal by treatment with the ELBD indicating
the improved glucose homeostasis which may be due to insulin secretion from the pancreatic cells. Hence, 500
mg/kg b.w of the dose was chosen to study the antidiabetic activity in the experimental rats.
Haematological parameters
The haematological indices namely red blood cell count (RBC), hemoglobin (Hb), mean corpuscular volume
(MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet
count and packed cell volume (PCV) in the experimental rats are depicted in Table 3. The levels of white blood cell
(WBC), polymorphs, lymphocytes, monocytes and eosinophils in the experimental rats are shown in Table 4.
Table 3: Red blood cell indices in experimental rats
Treatment Gro ups To tal Hb RBC X M CV (fl) M CH (pg ) M CHC (% ) Platelets PCV %
(g/dl) 6 5
10 cmm ( 1 0 )
T1 Co n tro l 1 4.8 1 .0 1 5.6 0 .35 60 .9 0 .61 1 9.5 0 .5 8 36 .1 2.53 75 6. 0 24 .43 45 .0 2 .80
T2 ST Z 8. 5 1. 18 a 3.5 0 .78 54 .8 5. 94 a 1 9.6 2 .1 1 31 .8 1.32 a 7 14 .0 9. 84 32 .9 2 .02 a
T3 ST Z + E LBD 1 1 .6 0. 92 ab 4.8 0 .18 5 8 .5 2. 28 b 2 0.8 1 .4 0a b 3 4.0 1 .4 4a b 73 5. 3 13 .05 41 .6 1 .10
ST Z +
T4 G liben cla mide 1 1 .8 1.5 5a b 4.8 1 .01 56 .9 1. 70 a 2 1.3 1 .3 3a b 3 3. 3 0 .95 a b 73 3. 7 13 .57 40 .1 0 .98
229
Table 4: White blood corpuscles (WBC) and differential count in experimental rats
3
Treatment Groups WBC X 10 Polymorphs % Lymphocytes % Monocytes % Eosinophils %

cmm
T1 Control 10.8 0.50 5.3 8.91 5 2.0 8.91 2.3 1.15 2.6 0.58
T2 STZ 15.3 0 .96a 3.2 1.25 4 5.0 6.56 3.5 0.50b 4.3 1.15
T3 STZ + ELBD 11.2 0.75b 4.8 2.56 b 5 0.8 2.41 2.8 1.14 3.0 1.00
STZ +
T4 12.1 0.97b 4.2 1.60 4 9.9 2.67 3.6 1.02 3.5 1.85
Glibenclamide
Values are mean SEM (n=6 animals in each group)

a- (p<0.05) T1 vs T2-T10 b- (p<0.05) T2 vs T3-T4
c - (p<0.05) T5 vs T6-T7 d- (p<0.05) T8 vs T9-T10
(One Way ANOVA followed by Tukeys test)
The STZ-induced rats (T2) showed a notable decrease in the levels of haemoglobin, PCV, platelets and red
cell indices namely red blood cell (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin
concentration (MCHC), polymorphs and lymphocytes. On the other hand, a significant increase in the levels of
mean corpuscular hemoglobin (MCH), percentage of eosinophils and monocytes was seen along with increased
WBC levels was observed when compared to the control rats (T1). Following the treatment with ELBD and the
drug glibenclamide the level of RBCs and its related indices were significantly reverted. This gives an understanding
that both the extract and the drug stimulate the formation or secretion of erythropoietin, which induces stem cells
in the bone marrow to produce red blood cells.
A marked reduction in packed cell volume, haemoglobin content, red blood cells count, mean corpuscular
haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration and platelets with increased
white blood cells count. Treatment with leaf extract of Ficus sur has found to restore these anomalies to normal
levels12.
Antioxidants in the liver of experimental rats
The enzymic antioxidants namely catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx)
and non-enzymic antioxidants (vitamin C, vitamin E and reduced glutathione) were assessed and the results are
presented in Table 5 and 6.
Table 5: Enzymic antioxidants in experimental rats
Treatment Groups Catalase SOD Gpx

(U/mg protein) (U/mg protein) (U/mg protein)
T1 Control 57.6 12.67 8.00 1.64 8.99 1.68
T2 STZ 23.9 0.91a 2.52 0.69 a 3.90 0.78a
T3 STZ + ELBD 41.45 0.55b 6.95 0.85b 5.59 0.13a
T4 STZ + Glibenclamide 40.75 0.95a b 6.52 0.53b 4.42 0.59a
230
Table 6: Non-enzymic antioxidants in experimental rats
Treatment Groups Vitamin C Vitamin E Reduced glutathione

(mg/g tissue) (g/g ti ssue) (nmoles/g tissue)
T1 Control 2.67 0.69 2.00 0.93 25.21 4.86
T2 STZ 0.96 0.74 0.41 0.29 10.89 1.86 a
T3 STZ + ELBD 2.01 0.69 1.16 0.29 23.65 1.19b
T4 STZ + Glibencl amide 1.83 0.30 1.14 0.84 23.02 2.22b
Values are expressed in mean S.E.M (n=6)
a- (p<0.05) T1 vs T2-T10 b- (p<0.05) T2 vs T3-T4

c - (p<0.05) T5 vs T6-T7 d- (p<0.05) T8 vs T9-T10
(One Way ANOVA followed by Tukeys test)
A significant decrease (p<0.05) in the activities of enzymic antioxidants and non-enzymic antioxidants
was observed in comparison with that of the control rats (T1). The oral administration with the ELBD for 45 days
increased the antioxidant activities in the experimentally treated rats. The decreased activities of enzymes in the
STZ-induced rats (T2) may be due to the inactivation caused by reactive oxygen species. In the present study, the
ethanolic extract of leaves of Boerhavia diffusa rendered a significant change in the antioxidant potential which
may be due to the radical scavenging activity of the extract.
CONCLUSION
Thus, the results suggests that Boerhavia diffusa exhibits protection against the STZ-induced diabetes by
boosting endogenous antioxidant activity and restoring the levels of blood glucose and haematological parameters
thereby justifying the use of Boerhavia diffusa in polyherbal preparations.
REFERENCES
1. Kavitha S, Sivaraj R & Ravi D (2016) International Journal of Pharmacy and Pharmaceutical Sciences 8, 79-
84
2. Gavarnukulya Y, Abou-Elella F, Wamunyokoli F & Aei-Shemy H (2014) Asian Pacific Journal of Tropical
Medicine 7, s355-s363
3. Rao P P (2016) International Journal of Herbal Medicine 4, 05-09
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8. Rotruck J T, Pope A L, Ganther H E & Swanson A B (1984) Science, 179, 588-590.
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11.
10. Baker H, Frank O, Angelis, B. and Feingold, S. (1951), Nutrition Reports International 21, 531-536.
11 Moron M S, De Pierre J N and Mannervik V (1979) Biochimica Biophysica Acta 582, 67-68.
12. Chinonye A S, Ikechukwu O K & Nnah I S (2014) Comprehensive Journal of Agricultural and Biological
Science 2, 5 -11.
231
In vitro -amylase Inhibitory Kinetics and Antioxidant Potentials of
Trigonella foenum-graecum
Renuka R.*1 and Jeyanthi G. P 2
1. Department of Biochemistry, Sri Ramakrishna College of Arts and Science for Women, Coimbatore,Tamil Nadu, India.
2.Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home Science and
Higher Education for Women, Coimbatore, Tamil Nadu, India.
*Corresponding Author: E mail: senthilrenuka@gmail.com
Abstract
Diabetes mellitus is an endocrinological and metabolic disorder with an increasing global prevalence and incidence.
Today, inhibiting carbohydrate hydrolyzing enzymes is one of the therapeutic approaches for decreasing
postprandial hyperglycemia. Diabetes is also an oxidative stress related disorder and is emerging as a pandemic.
The immediate need is to identify novel food based bioactive agents or drugs for curing or preventing diabetes,
with comparatively fewer side effects. Trigonella foenum-graecum (Fenu greek) is one of the most promising
vegetable providing treasures of secondary metabolites known to have health benefits against various diseases.
In the present study alpha amylase inhibitory activity of Trigonella foenum-graecum leaf (TGL) and Trigonella
foenum-graecum seed (TGS) extracts were carried out. Maximum inhibition was found in ethyl acetate extracts of
seeds with 90.81.20 % and 80.32.5 % ethanol extracts of leaves. Mechanism of inhibition and its kinetics by the
method of Dixon Plot and Cornish- Bowden plot showed that competitive type for (TGL) and mixed type of
inhibition for (TGS). Total anti oxidant activity-FRAP, DPPH and Super oxide radical models for antioxidant
studies showed that TGS had higher free radical scavenging activities when compared to TGL extracts. The
combined effect of -amylase inhibitoryand antioxidant activity exhibited by Trigonella foenum-graecum shows
promising scope in treatment and prevention of complications in diabetes.
Keywords : Trigonella foenum-graecum, Diabetes mellitus, alpha amylase and antioxidants.
INTRODUCTION
Diabetes mellitus has become a growing problem in the contemporary world. India has today become the
diabetic capital of the world with over 20 million diabetes and this number is likely to increase to 57 million by
20251. The high blood sugar is a common effect of uncontrolled diabetes and it makes a serious damage to many
of the systems in the body, especially the kidney, nerves and blood vessels 2. More than 90% of diabetic patients
suffer from type 2 DM. Several epidemiological and clinical studies indicate a direct relationship between
hyperglycemia and long term microvascular and macrovascular complications, which develop as the disease
progresses, gradually decrease quality of life of diabetic patients. Therefore, it is essential to control blood glucose
levels during the early stages of the disease3.
In diabetes, oxidative stress has been found to be mainly due to an increased production of oxygen free
radicals and a sharp reduction of antioxidant defenses4. One therapeutic approach to treat diabetes is to decrease
post prandial hyperglycemia. This is done by retarding and reducing the digestion and absorption of glucose
through the inhibition of carbohydrate hydrolyzing amylase in the digestive tract. Inhibition of this enzyme
delays the digestion of the carbohydrates, causing a reduction in the rate of glucose absorption5. Searching for a
new class of compounds is essential to overcome diabetic problems. There is continuous search for alternative
drugs6. Compounds with both hypoglycemic and antioxidative properties would be useful antidiabetic agents 7.
Plants have long been used for the treatment of diabetes, particularly in developing countries where most
232
people have limited resources and do not have access to modern treatment. Phytoantioxidants from food and
medicinal sources are gaining importance because of their safety and availability 8.
Trigonella foenum-graecum (Fenugreek) is an annual leguminous bean, and belongs to Fabaceae family.
Its seeds and green leaves are used as food posses medicinal applications, and is an old practice of human history
9-11
. Recent advances in nutraceutical and phytochemical research stimulated a renewed interest in fenugreek to be
used as a functional food12. The present investigation was undertaken to assess the in vitro alpha- amylase Inhibitory
kinetics and anti oxidant potentials of Trigonella foenum-graecum leaves and seeds.

Plant Collection, Identification and Preparation of Extract
Trigonella foenum-graecum leaves and seeds were collected from local market identified and authenticated
by botanist from Botanical Survey of India, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India.
The plant parts were dried, finely powdered and stored in air tight containers at room temperature for further use.
Five grams of Trigonella foenum-graecum leaves (TGL) and Trigonella foenum-graecum seeds (TGS)
were macerated with 50 ml of different solvents (petroleum ether, chloroform, ethyl acetate, ethanol, acetone and
water) for 48 hours filtered and collected the solvent. The solvent was evaporated in water bath shaker to get dry
extract and used for further analysis.
Alpha-amylase Inhibitory Activity

The in vitro anti-diabetic activity of TGL and TGS in all the six extracts was determined by assaying the
inhibitory activity of the enzyme -amylase by modified method of Bernfield 195513.
Among the six solvent extracts ethanol extract of TGL and ethyl acetate extracts of TGS showed highest
-amylase inhibition, hence these extracts were used for further studies.
Mechanism of alpha amylase inhibition

Different concentrations of TGL and TGS (200-800 g) were used for studying of -amylase inhibition
with different substrate concentrations of starch (0.25, 0.5, 0.75, and 1.0%) as given by Dixon 195314 and method
of Bernfield.
Absorbance values were extrapolated in a standard graph for maltose and the amount of maltose produced
in presence of inhibitor was found. This was taken as the product concentration [V]. Calculated 1/ [V] values and
plotted graph with concentration of [I] taken along X-axis and (1/ [V]) along Y-axis.
From the nature of graph obtained, the mechanism of inhibition was studied.
Cornish- Bowden plot

The experiment was performed as mentioned in Dixon Plot. The difference here lies in plotting a graph
with the concentration of inhibitor [I] taken along X-axis and substrate concentration divided by product concentration
(S / [V]) along Y-axis. From the nature of graph obtained the mechanism of inhibition was studied as suggested by
Cornish-Bowden 197415.
Total Antioxidant Activity

FRAP Assay: The FRAP assayed according to the method of Benzie and Strain, 199616.
DPPH: The reduction capacity of TGL and TGS of DPPH was determined by method of Blois, 195817.
233
Superoxide Radical Scavenging Activity
Superoxide radical scavenging activity of TGL and TGS were studied by genearating Superoxide radicals
by a modified method of Beauchamp and Fridovich 197118.
RESULTS
Alpha-amylase Inhibitory Activity
Inhibition of alpha amylase by various solvent extracts of Trigonella foenum-graecum was studied and the
results are presented in the Table 1. Among the six solvent extracts maximum inhibition was found in Trigonella
foenum-graecum ethyl acetate extracts of seeds with 90.81.20 % and 80.32.5 % ethanol extracts of Trigonella
foenum-graecum leaves.
Table: 1 Alpha -amylase inhibitory activity
Solvent Extracts TGL % Inhibition TGS % Inhibition

Petroleum ether 10.41.25 82.5 2.65
Ethylacetate 50.90.55 90.81.20
Chloroform 14.20.40 160.58
Ethanol 80.32.5 50.41.73
Acetone 51.41.58 11.42.24
Water 75.41.75 67.991.55
Values are mean SD of triplicates
Mechanism of alpha amylase inhibition Trigonella foenum -graecum was studied by Dixon and Cornish-
Bowden plot as shown in the Fig 1a, 1b and 2a, 2b. Results of Dixon and Cornish- Bowden plot showed that the
type of inhibition in Trigonella foenum-graecum the mechanism was found to be competitive type in TGL and
mixed inhibition in TGS.
Fig 1a: TGL Dixon Plot Fig 1b: TGL Cornish- Bowden Plot
234
Type of Inhibition- Competitive
Fig 2 a: TGS Dixon Plot Fig 2 b: TGS Cornish- Bowden Plot
Type of Inhibition-Mixed
FRAP Assay: The ethyl acetate extracts of TGS showed (Table: 2) moderate ferric ion reducing activity of about
57.921.13 than TGL ethanol extracts 27.564.57 when compared to standard ascorbic acid with maximum of
195.318.37 (mmol (Fe(II)/g sample).
Table: 2 FRAP Assay
Standard / Plant Extracts FRAP (mmol (Fe(II) / g sample)

Ascorbic acid 195.318.37
TGL 27.564.57
TGS 57.921.13
Values are mean SD of triplicates
Fig 3: DPPH Radical Scavenging Activity Fig 4: Super oxide Radical Scavenging Activity
Values are mean SEM of triplicates
DPPH and Superoxide radical scavenging activity: The scavenging activity of DPPH (Fig 3 and Fig 4) by plants
were found to be higher TGS 56% ethyl acetate extracts and ethanol extract of TGL showed about 38% at 1000
g /ml Standard ascorbic acid exhibited scavenging of 62% at 1000 g /ml. The IC50 values of the plant extracts for
DPPH were found to be 1368.17(g) for TGL, 895.32(g) for TGS and 734.37(g) for standard Ascorbic acid.
The superoxide radical scavenging activity of TGL and TGS and standard ascorbic acid were found to be 55 %,
63% and 89%. The IC50 values of TGL, TGS and standard ascorbic acid were found to be 836.97 g, 703.28 g
and 319.02 g. Superoxide radical scavenging activity was found highest in TGS extracts when compared to
235
TGL.
DISCUSSION
Inhibitors of saccharide hydrolyzing enzymes (-amylase and -glucosidase) have been useful as oral
hypoglycemic drugs for the control of hyperglycemia especially in patients with Type 2 diabetes mellitus19. The
efficiency of alpha amylase inhibition may depend upon the solvents used for extraction. The polar solvents
extracts of Trigonella foenum-graecum leaf ethanol and seed ethyl acetate extracts showed maximum inhibition
when compared to other non polar solvent extracts. This may be due the active principles present in the solvent
extracts.
Competiive inhibition shown by TGL extracts were similar to that of Urtica dioica and Juglans regia
extracts which inhibited porcine pancreatic alpha amylase through competitive mechanism as shown by Dixon
plot20. Mixed type of inhibition shown by TGS may be due to binding of bioactive components to either in active
site or other sites to bring about the inhibition of alpha amylase enzyme. The kinetics of inhibition of green, oolong
and black tea extracts, through Dixon, Cornish-Bowden, and LineweaverBurk plots showed mixed-type inhibitors
with both competitive and uncompetitive inhibitory characteristics21.
Antioxidants are capable of stabilizing or deactivating, free radicals before they attack cells22. Ethyl acetate
extracts of Sechium edule leaves belonging to Cucurbitaceae species had the highest FRAP capacity of 4.54 %23.
Solvents play a vital role in the extraction of the plant constituents 24. DPPH is widely used to evaluate the free
radical scavenging effect of natural antioxidant. The ethyl acetate extracts of Arnebia benthamii exhibited considerably
higher DPPH radical scavenging activity. The free radical scavenging activities of different extracts decreased in
the order of ethyl acetate extract > ethanol extract > aqueous extract25. Superoxide is biologically important since
it can be decomposed to form stronger oxidative species such as singlet oxygen and hydroxyl radicals26. The
ethanol and ethyl acetate of whole fruit of Momordica charantia fruit extracts displayed a dose dependent activity
in inhibiting the superoxide radicals against reference agent curcumin27.
CONCLUSION
From the present work, it could be concluded that the solvents play a vital role in the extraction of the plant
constituents. The ethyl acetate extracts of seeds and ethanol extracts of leaves of Trigonella foenum-graecum
showed potent alpha amylase inhibition and the mechanism of inhibition was found to be competitive in Trigonella
leaves and mixed inhibition in seed extracts. The various antioxidant mechanisms of Trigonella foenum-graecum
may be attributed to their effectiveness as good scavengers of superoxide and free radicals. So Trigonella foenum-
graecum can be used in control of post prandial hyperglycaemia and due to its potent anti oxidant properties may
help in prevention of complications in diabetes.
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97-106.
2. WHO (2011) Diabetes fact sheet no: 312
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Pharmacotherapy 6, 24-45.
4. Oberley LW (1988) Free Radic Biol Med 5, 113.
5. Rhabaso Lhoret R & Chiasson JL (2004) International Textbook of Diabetes Mellitus 1, 2 ed. John Wiley &
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6. Syamsudin S (2010) Int. J. Phytomedicine 2, 430-435.
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7. Baynes JW, 1995. Mechanistic approach to diabetes. In: Inoannides C, Flatt FR, eds. Eths Horwood Limited,
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8. Rao Rak and Rehman (2012) F. J. Disper. Sci. Technol, 33:4, 472-481.
9. Thomas JE, Bandara M, Lee E , Driedger D & Acharya S N (2011) Biotechnol, 110117.
10. Parildar H, Serter R & Yesilada E (2011) J Pak Med Assoc 61, 1116-1120.
11. Vaidya HB, Ahmed AA, Goyal RK & Cheema SK (2013) J Pharm Pharm Sci 16 (4), 530 540.
12. Acharya SN, Basu SK &Thomas JE (2007) Advances in Medical Plant Research, 81-122.
13. Bernfeld P (1955) Methods in Enzymology, Academic Press, New York 1, 149-158.
14. Dixon M (1953) Biochemical Journal 55,170171.
15. Bowden AC (1974) Biochemical Journal 137,143144.
16. Benzie IFF & Strain JJ (1996) Anal Biochem 239, 70 76.
17. Blois MS (1958) Nature 26, 1199-1200.
18. Beauchamp C & Fridovich I (1971) Anal Biochem 44, 276-277.
19. Gin H & Rigalleau V (2000) Diab Metab, 26, 265-272.
20. MahsaRahimzadeh, SamanehJahanshahi, Soheila Moein & Mahmood Reza Moein (2014) Iran J Basic
Med Sci 17(6), 465469.
21. Lijun Sun, Fredrick J. Warren, Gabriele Netzel & Michael J. Gidley (2016) J Funct Foods 26, 144156.
22. Nazareth Arockiamary.S, Nadhiya. K & Vijayalakshmi. K (2014) International Journal of Biological &
Pharmaceutical Research 5(7), 566-570
23. Irda Fidrianny, Agung Darmawati & Sukrasno (2014) Int J Pharm Pharm Sci 6, 858-862
24. Syeda Birjees Bukhari, Muhammad Iqbal Bhanger & Shahabuddin Memon (2008) Pak. J. Anal. Environ.
Chem 9, 78- 83.
25. Showkat Ahmad Ganie, Tanveer Ali Dar, Rabia Hamid, Ovais Zargar, Shayaq Ul Abeer, Akbar Masood,
Shajrul Amin, & Mohammad Afzal Zargar (2014) Oxidative Medicine and Cellular Longevity, Hindawi
Publishing Corporation, Article ID 792574, 1-8 pages
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237
Anticancer activities of Methanolic leaf extract of Prosopis cineraria
in Breast Cancer cell lines
Dharani Bangaruswamy, Sivaprabha Jambunathan, Palghat Raghunathan Padma and
Sumathi Sundaravadivelu*
*Assistant Professor, Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home Science
and Higher Education for Women, Coimbatore 641 043, Tamil Nadu, India
Abstract
Free radicals interact with all the biomolecules in different ways, altering their natural properties and making
them more susceptible to damage. This leads to various diseases, including neurodegenerative diseases, chronic
inammatory diseases, cancer and cardiovascular disorders. Plants contain phytochemicals with strong
antioxidant activities which may prevent and control cancer and other diseases by protecting the cells from the
deleterious effects of the free radicals. Prosopis cineraria (Fabaceae), has a long history of use in herbal
medicine. Therefore the present was carried out to determine the phytochemical constituents present in the
leaves of Prosopis cineraria and to analyze the anticancer activity of the leaves using breast cancer cell lines.
The phytochemical analysis confirmed the presence of various phytochemicals in the methanolic extract of
Prosopis cineraria leaves, which forms the basis of their medicinal properties. Further, the cytotoxicity induced
by the methanolic extract of the leaves in the presence and the absence of a standard chemotherapeutic drug
(tamoxifen/etoposide) in the three cell lines were studied. The treatment with the extract decreased the viability
of cancerous cells MCF-7 and MDA MB 231 to a large extent but not in non-cancerous cells HBL-100. In order
to elucidate whether the leaf extract induced apoptotic cell death, various staining techniques were performed in
all the three cell lines. The results revealed that the methanolic extract of Prosopis cineraria leaves caused an
increase in the number of cells undergoing apoptosis in cancer cells compared to the non-cancerous ones. In
order to confirm that the cell death induced by the leaf extract was through apoptosis, immunocytochemical
analysis was performed to see the expression level of apoptosis-associated proteins namely p53, Bax and Bcl-2.
The results revealed that the leaf extract induced apoptosis in cancer cells. Cell cycle analysis showed accumulation
of the cells in the sub G0 phase and G0/G1 phase. Our studies demonstrate that the methanolic extract of P.
cineraria leaves can act as a new potential chemotherapeutic agent for the treatment of breast cancer, which is
not deleterious towards non-cancerous cells.
Keywords: phytochemicals, Prosopis cineraria, breast cancer, apoptosis, immunocytochemistry, cytotoxicity,
cell cycle
INTRODUCTION
Breast cancer is the second most common type of cancer, after lung cancer, worldwide1. Most of the
modern drugs that are available for treating cancers are mainly based on synthetic chemical compounds and they
are very expensive, toxic and found to have harmful side effects2. Therefore, there is a necessity to investigate the
agents derived from natural sources, described traditionally, for the prevention and treatment of cancer3. Medicinal
plants are a rich source of bioactive phytochemicals or bionutrients. Several anticancer agents from medicinal
plants such as vinblastine and vincristine from Catharanthus roseus, paclitaxel from the bark of Taxus brevifolia,
derivatives of camptothecin, topotecan and irinotecan, are used for the treatment of various cancers 4. With this
background, the candidate plant chosen for the present study is Prosopis cineraria, commonly called as Vanni
(Tamil) and Jhand tree (Hindi).
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The tree belongs to Fabaceae family and is used as a remedy for cough, cold, asthma and rheumatism. The
plant also possesses anti-inflammatory property. In spite of all these known uses in traditional medicine, there are
no reports of systematic study for scientific validation. Hence the study was formulated to identify the nature of
the active components present in leaf sample and the anticancer activity of leaf extract was tested using breast
cancer cell lines.

HPTLC analysis
The methanol residue (100mg) of Prosopis cineraria leaves was dissolved in 1ml of methanol and centrifuged
at 3000rpm for 5 minutes. The test sample (1l) and standard (2 l) was loaded as a 5mm band in the 3 10 Silica
gel G60 F254 plate using a Hamilton syringe and CAMAG INOMAT 5 instrument. The TLC twin trough developing
chamber was saturated with respective mobile phase. The samples loaded plate was then placed in a TLC twin
trough developing chamber and to run up to 90mm.The developed plates were then dried by keeping in hot air oven
for evaporation of solvents. The developed plates were kept in a photo-documentation chamber (CAMAG
REPROSTAR 3) and the images were captured in white light, UV 254nm and UV 366nm. The developed plates
were derivatized with the respective spray reagents and dried at 100C in hot air oven. The plates were photo-
documented at daylight, UV 254nm and UV 366nm.
Preparation of plant extract

1g of fresh leaf sample and 10ml of methanol were homogenized in a mortar and pestle. The extract was
then centrifuged at 2000 rpm for 5 minutes and the methanol was dried at 60C protected from light. The resulted
residue was weighed and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20mg/20l.
Culturing and maintenance of cell lines

The three cell lines namely MCF-7, MDA MB 231 and HBL-100 were purchased from National Centre for
Cell Science (NCCS), Pune, India. The cells were maintained in a CO2 incubator with 5% CO2 and 95% humidity
atmosphere supplemented with DMEM, 10% FBS, non-essential aminoacid and penicillin and streptomycin (PAA)
at 1X final concentration from a 100X stock. Once the cells attained confluent growth, the cells were trypsinized
using Trypsin-EDTA (PAA) and the required number of cells was seeded into sterile 6-well and 96-well plates for
carrying out various assays. All the cell viability and cytotoxicity assays were carried in 96-well plates and staining
was performed in 6-well plates. In each well of the 6-well plates, a clean, dry, sterile coverslip was placed before
the cells were seeded, followed by the incubation in a CO2 incubator with 5% CO2 and 95% humidity atmosphere
(Innova CO-170, United States).
Treatment groups
1. Cells alone
2. Cells + tamoxifen/etoposide
3. Cells + methanolic extract of Prosopis cineraria leaves
4. Cells + Prosopis cineraria leaves + tamoxifen/etoposide
Cell cytotoxicity assay

WST-1 cytotoxicity assay
CytoscanTM WST-1 cell cytotoxicity assay kit was used to determine the cytotoxicity of the extract in
cancer cell lines. Aliquoted 5x104 - 5x105 cells per well of a 96 well plate with a final volume of 100l/well of culture
medium and treatments were given after the cells becomes confluent. 10l WST 1/ CEC assay dye solution was
239
added at the end of the treatment to each well. The plates were then incubated for 4 hours in a cell culture
incubator. The plate was then shaken for a minute and the absorbance was measured at 420-480nm in a microplate
reader with the reference wavelength of more than 600nm. The per cent cytotoxicity was calculated using the
formula
Lactate Dehydrogenase Release (LDH) assay

The cells were treated with the leaf extract in the presence and absence of the drug. A spontaneous control
(PBS alone) and maximum LDH release control (cells in PBS lysed using lysis buffer) were also taken. 50l of the
supernatant was taken into a new ELISA reader plate; 50l of the reconstituted substrate mix (dissolve one vial of
the provided substrate mix in 11.4ml water and add 0.6ml of assay buffer provided with the kit) was added to each
well and incubated at room temperature for 30 minutes. The reaction was stopped by adding 50l of stop solution
and the absorbance was recorded at 490nm and the per cent cytotoxicity was calculated as follows:
Determination of apoptotic changes in the cells

Actively proliferating cells can be differentiated from quiescent cells by AO/EtBr staining technique. AO/
EtBr staining was performed as by described by Parks et al. (1979)5. The method proposed by Mercille and Massie
(1994)6 was followed for EtBr staining. The Hoechst 33342 staining assay was employed to observe morphology
alterations of the cells and performed as reported by Yamakawaet al. (2008)7.
Immunocytochemical analysis
Immunocytochemical analysis was done to study the expression level of pro-apoptotic proteins p53 and
bax and anti-apoptotic protein Bcl-2 in the presence and absence of methanolic extract of Prosopis cineraria
leaves. Sterile coverslips were placed inside 6-well plates and 0.1% gelatin was added and left for 30 minutes. The
gelatin was then discarded and cells were seeded onto the plates. The treatments were given according to the
optimized time for each cell line. 4% paraformaldehyde (in PBS) was added to the treated cells and incubated for
30 minutes. The fixed cells were washed three times with PBT for 10 minutes each. The cells were then treated
with 0.5% Triton X-100 (in PBS) for 5 - 60 minutes (often 15 minutes)and then washed three times with PBT for
10 minutes each. 200l of 10% normal goat serum (in PBT) was added and incubated for 30 minutes followed by
incubation with 200l of primary Ab (1:100-1:10,000, in PBS + 1% BSA) at 4C overnight. Then the cells were
washed six times with PBT for 10 minutes each followed by incubation with 200l of secondary Ab (1:100-1:500,
in PBS + 1% BSA) at 37C for 1 hour. After incubation, the cells were washed three times with PBT for 10minutes
each. Then the cells were incubated with 200l of DAPI for 10 - 15 minutes. Ater washing the cells six times with
PBT for 10 minutes each, 5l of antifading solution was placed onto clean glass-slide. The coverslip with cells was
mounted over it and the edges were sealed using manicure and were observed under 400X magnification in
fluorescent microscope.
Cell cycle analysis by flow cytometry

Cell cycle analysis was done to visualise the study at which maximum cell death occurred using flow
cytometer. Cell cycle analysis was performed using flow cytometry as per the protocol described by Krishan
(1975)8.
240
Values were expressed as mean SD. The parameters analyzed in the study were subjected to statistical
analysis using SigmaStat (Version 3.1) statistical software. Statistical significance was determined by one-way
analysis of variance for staining. The values with P<0.05 were considered to be significantly different.

HPTLC of methanolic extract of Prosopis cineraria leaves
HPTLC was carried out to identify the presence of different components that are responsible for the
anticancer property. The HPTLC indicated the presence of alkaloids, phenols, flavonoids, saponins,steroids, tannins
and terpenoids.
Figure 1. HPTLC
Abirami et al. (2014)9 recognized four compounds from methanolic extract of Grewiahirsuta leaves. The
HPTLC fingerprint of Gnidiaglauca revealedtwelve compounds in methanolic extract flower, ethyl acetate extract
of leaves, stemand petroleum ether extract of Dioscoreabulbifera10.
Cell cytotoxicity
In order to confirm the extent of cytotoxicity induced by the methanolic extract of Prosopis cineraria
leaves in the presence and the absence of a standard chemotherapeutic drug tamoxifen/etoposideWST-1 and LDH
release were performed in the three cell lines.The results of cytotoxicity assays indicated that the treatment with
the leaf extract caused a steep decrease in the viability of cancer cells. In MCF-7, the cytoxicity exerted by the leaf
extractby itself was similar to that of tamoxifen. The extract, in combination with the drug etoposide, further
reduced the viability of MDA MB 231.The Figure 2depicts the results of WST-1 and LDH release assay.
Figure 2. Effect of methanolic extract of Prosopis cineraria leaves on the viability of breast cells
Values are Mean SD of triplicates
The values of the negative (untreated) control group were fixed as 0% cytotoxicity and the percent cytotoxicity in
the other groups were calculated relative to this for WSt-1 assay
The main goal of many chemotherapy drugs is low cytotoxicity to healthy cells and high cytotoxicity to
cancerous cells. Khalil et al. (2014) [11] reported that curcumin was found to be more cytotoxic towards four
cancer cell lines [MCF-7, HepG2 (liver), HeLa, and HCT-116 (colorectal carcinoma)] compared to the metal-
complexedcurcumin. Treatment of HGC-27 (human gastric carcinoma) cells with oridonin (component of
Rabdosiarubescens) increased apoptosis by an increase in LDH release 12.
241
Apoptotic events in breast cell lines
Our laboratory has already reported the apoptotic activity in MCF-7 and HBL-100 13. In order to investigate
whether the inhibition of cell growth was related to apoptotic cell death, nuclear changes associated with apoptosis
were observed by various staining techniques such as AO/EtBr, ethidium bromide and Hoechst 33342. The results
exhibited a significant reduction in the number of viable cells and an increase in the number of apoptotic cells when
exposed to Prosopis cineraria leaf extract or drug alone in cancer cells. In HBL-100 cells, the number of apoptotic
cells wasless compared to that of cancer cells. This shows that extract is less toxic to normal cells.
The aqueous root extract of Vetiveriazizanioidesexhibited cytotoxicity towards the cancer cell line MCF-
7 by inducing nuclear fragmentation and chromatin condensation in acridine orange/ethidium bromide staining 14.
Esakkirajan et al. (2014) 15 reported that highly condensed chromatin in fragmented nucleus was observed in HCT
15 (colon) cells treated with compound isolated from Cassia auriculata. The present study correlates with these
supportive findings that the extract induced apoptotic cell death in cancer cells.
Immunocytochemical localization of apoptosis releated proteins
The expression level of apoptosis related proteins such as p53, Bax and Bcl-2 were analyzed by antigen-
antibody reaction. The leaf extract induced apoptosis in cancer cells by increasing the expression of pro-apoptotic
proteins p53 and bax (Figure 3Aand 3B). Bcl-2 protein expression was found to be decreased in the leaf extract
treated cancer cells (MCF-7 and MDA MB 231) than the untreated control group (Figure 3C).
Figure 3
In par with several reported studies treatment of HepG2 cells with Alantolactonetion reduced the expression
of anti-apoptotic protein Bcl-2 and increased the expression of proapoptotic protein Bax in a time dependent
manner 16. Exposure of T24 cells to costunolide increased Bax expression, down regulation of Bcl-2, survivin and
activation of caspase-317. This clearly shows that the methanolic extract of P. cineraria leaves acts as an anticancer
agent to inhibit cell growth and trigger apoptosis.
242
Flow cytometric analysis of breast cancer cell lines
Cell cycle analysis was performed to find whether the treatment with leaf extract caused apoptosis
induction by cell cycle arrest in the breast cancer cells. The results showed that the leaf extract arrested the growth
of both the ER positive and triple negative breast cancer cells at sub G0 and G0/G1 phases of the cell cycle, which
is an indicator of late and early apoptotic cell death respectively. The histogram of MCF-7 and MDA MB 231 cells
treated with leaf extract of Prosopis cineraria is shown in Figure 4.
Figure 4 Effect of Prosopis cineraria leaf extract on the per cent of apoptotic cells arrested in
sub G0 and G0/G1 phases of cell cycle
In tune with several other studies reported Rao and Prasad (2013)18 showed that Strychnosnux-vomica
root increased sub-G0/G1 phase population in U266B cells. Shenet al. (2014)19 reported that the treatment of PC3
cells with isobractatin from Garciniabracteata arrested the cell cycle in the G0/G1 phase.
CONCLUSION
Thus, our study demonstrates clearly that Prosopis cineraria leaves can be used as a source of drugs that
can be effective against all types of breast cancer. In case of non-cancerous breast cells, the toxicity exhibited by
the extract is less compared to that of cancerous cells. Thus, this differential effect validates the use of Prosopis
cineraria leaf extract as a promising candidate for cancer treatment, both individually and in combination with
standard chemotherapeutic drug. As it is a phytocomponent, the possibility of development of drug resistance may
also be very low; suggesting that long term administration without side effects for cancer treatment may be
possible. The phytochemical analysis of the methanolic extract of the leaves of P.cineraria showed that they are a
rich source of phytochemicals. Further analysis is needed for the elucidation of the exact structure of these
phytochemicals.
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D, Dinesh D, Babu G. and Dhanasekaran G ( 2014) SpectrochimicaActa Part A, Molecular and Biomolecular
Spectroscopy, 120, 462 - 466.
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420.
244
Role of Phytoproteins in In vitro antioxidant and In vivo antitumor ac-
tivity of Tabernamontana divaricata against Daltons Lymphoma Ascites
(DLA) cells bearing mice
Ms. M. Srilatha, Dr. R. Santhi* and Dr. S. Annapurani**
*Ph.D Scholar, Department of Biotechnology, *WOS (A), Project Staff, **Professor and Head,
Higher Education for Women, Coimbatore 641 043. Email ID: sripathy1331@gmail.com
Abstract
Medicinal plants have been a rich source of therapeutic agents and provided basis for several synthetic drugs.
Many of the currently available drugs are plant-based, and plant peptides and proteins have turned out to be a
critical source of biological compounds that exhibited bioactivities which can be exploited as drug. Based on
this, the present study was designed to investigate the in vitro antioxidant activity and in vivo antitumor
activity of protein fraction from Tabernaemontana divaricata leaves. In vitro scavenging activity was assessed
by DPPH radical scavenging activity and superoxide anion, the protein fraction showed its ability to scavenge
free radicals in a dose dependant manner. The IC50 values of T. divaricata in DPPH and SOD were found to be
43.861.85 and 73.762.43 respectively. In case of antitumor evaluation the hematological count, enzymic activity
and non-enzymic activity were restored towards the normal value in protein fraction treated group. Results
obtained in the present study indicate that the T. divaricata is a potential anticancer agent via its free radical
scavenging.
Keywords: Tabernaemontana divaricata, DPPH, Protein, Free radical
INTRODUCTION
Natural products are well recognized as sources for drugs in several human ailments including cancers.
Examples of natural pharmaceuticals from plants include vincristine, irinotecan, etoposide and paclitaxel1. Despite
the discovery of many drugs of natural origin, the search for new anticancer agents is still necessary, in order to
increase the range available and to find less toxic and more effective drugs. It has been recommended that samples
with pharmacological usage should be taken into account when selecting plants to treat cancer, as several ailments
reflect disease states bearing relevance to cancer or cancer-like symptoms2,3. Cancer is a leading cause of death
worldwide accounting for 8.2 million deaths in 20124. The American Cancer Society estimates that around one and
half million new cancer cases are expected to be diagnosed in the United States alone5. Lymphomas are a type of
cancer that affects the bone marrow, blood cells, lymph nodes, and other parts of the lymphatic system. DLA is a
cell lymphoma originating spontaneously in the thymus of DBA (H2D) strain of mice6. It has been noticed that
spontaneously arising animal tumors mimica situation more similiarty human neoplasia than experimentally induced
tumors7. However, there is no scientific validation of T. divaricata leaves being used against Daltons lymphoma
ascites tumour (DLA). Therefore, we aimed to evaluate the antioxidant and anticancer activities of T. divaricata
protein fraction against DLA tumor model.

Collection and identification
The fresh leaves of Tabernaemontana divaricata (Apocynacea) was collected from Coimbatore district,
India. Taxonomic authentication was done by Dr.G.V.S.Murthy Taxonomist, TNAU, Coimbatore and Tamilnadu.
India and the authentication number BSI/SRC/5/23/2015/Tech/2083.
245
Preparation of Protein extract of Tabernaemontana divaricata leaf (TdPf)
Protein was extracted by recrystallization of ammonium sulphate. Fresh leaves of Tabernaemontana
divaricata leaves 20% were taken and homogenized with PBS buffer pH 7.2 and were centrifuged for 5000 rpm
for 10minutes. Pellets were discarded and supernatant were saved. To the supernatant add known volume of
ammonium sulphate 10-100% and was centrifuged at 10,000 rpm, 4oC for 10minutes. The supernatant were
discarded and the pellet was suspended with Dialysis membrane for salting out. The crude extract was kept at -
20oC.
DPPH antioxidant activity

The antioxidant activity of the extracts was measured on the basis of the scavenging activity of the stable
1, 1- diphenyl 2-picrylhyorazyl (DPPH) free radical according to the procedure Brand williams with slight
modifications8. 1ml of 0.1mM DPPH solution in methanol was mixed with 1ml of plant extract solution of varying
concentrations (50, 100, 150,200 and 250 g/ml). Corresponding blank sample were prepared and L-Ascorbic
acid (1-100 g/ml) was used as reference standard. Mixer of 1ml methanol and 1ml DPPH solution was used as
control. The reaction was carried out in triplicate and the decrease in absorbance was measured at 517nm after 30
minutes in dark using UV-Vis spectrophotometer.
SOD antioxidant activity

Superoxide radical activities was determined by standard method9. The samples (20 l) were mixed using
the reaction mixture in the kit. Then, the mixtures were gently shaken and incubated at 37C for 20 min. Antioxidant
activity was measured at 450 nm using a Genesys 20 Thermo scientific (USA) spectrophotometer. The positive
control used was ascorbic acid (10 mg ml-1).The modifications included varying the amount of samples used and
the incubation period. The negative control to measure inhibition rates of SOD activity used all treatments without
sample.
Seventy days old Swiss albino mice weighing 202g were used for the present study. The mice were
procured from the Amala cancer Research Institute Kerala, India. The mice were divided into six groups (six mice
per group) and maintained in polyacrylic cages at a temperature of 252C, suitable humidity, dark/ light cycle,
with feed and water (ad-libitum). The mice were acclimatized to laboratory conditions for 7 days prior to the
commencement of the experiment. The animal care and handling was done according to the regulations of Council
Directive CPCSEA No: (IAEC.2015.BT:04) about good laboratory practice (GLP) on animal experimentation. All
animal experiments were performed in the laboratory according to the ethical guidelines suggested by the international
animal ethics committee (IAEC).
Induction of lymphoma
DLA cells were obtained from Amala Cancer Research Institute, Kerala, India. The cells were maintained
in vivo in Swiss albino mice by intraperitoneal transplantation of 1x106 cells/mouse. The DLA cells aspirated from
the peritoneal cavity of the mice were washed with saline and given intraperitoneally to the experimental animals to
develop ascitic tumors.
Hematological Profile
After 15th and 60th days of the repeated treatment, blood samples was collected from each mice via heart
puncture and collected in ethylenediaminetetraacetic acid (EDTA) tubes, following hematological parameters were
assessed using a Hemavet 1500S (Drew Scientific, Waterbury, CT, USA) and analysis of various parameters such
as total white blood cell (WBC), red blood cell (RBC), as well as Hemoglobin levels were then evaluated.
246
Antioxidant Assays:
The liver homogenate was used to analyze the enzymatic antioxidant activities by superoxide dismutase
(SOD), catalase (CAT), glutathione peroxidase (GPx) and nonenzymatic antioxidants like, vitamin A, vitamin E and
reduced glutathione (GSH) were evaluated in liver tissue homogenate by using Kit methods.
RESULT
2, 2-Diphenyl- 1-Picryl-Hydrazyl (DPPH) Radical Scavenging Activity of TdPf
The results showed better antioxidant activity of the protein fraction to scavenge free radicals when
compared to standard Ascorbic acid. The concentration of Ascorbic acid required for IC50 (57.11.03g/ml) was
found to be less than that of TdPf (79.02.01g/ml).It means that the protein extract of plant at higher concentration
captured more free radicals formed by DPPH resulting into decrease in absorbance and increase in IC50 value
(Figure 1).
Values are mean SD of three replicates

Figure 1:DPPH radical scavenging activity of T. divaricata
Superoxide Radical Scavenging Activity of T. divaricata

Protein fractions showed free radical scavenging effect on Superoxide in a concentration dependent manner.
The results clearly indicates that proteins isolated from T.divaricata leaves have higher superoxide scavenging
potential activity with IC50 value 46.02.0 g/ml highly effective antioxidant activity. It was also observed that Low
molecular weight proteins exhibited stronger superoxide activity as compared with the standard ascorbic acid as
shown in the Figure 2.
Figure 2: SOD radical scavenging activity of T. divaricata

Hematology Observation
Hematology response of TdPf treated DLA bearing mice is presented in table 1. There is an increase in
247
hemoglobin count, WBC and RBC TdPf treated mice when compared to the silymarin and paraffin treated group.
DLA tumor induced mice showed increased level in WBC, RBC and Hemoglobin count when compared with
control and TdPf treatment group. Coadministration of DLA+ TdPf showed increase level in WBC, RBC and HB
count when compared with Control, paraffin and silymarin on both days of treatment.
Table 1: Effect of TdPf on hematological values in different experimental groups
Values are mean SD. # CD=One way ANOVA; ## CD=Two way ANOVA
Estimation of Antioxidant activity:

Enzymic antioxidant
DLA cells induced mice showed significant decreased levels of SOD (0.830.03), CAT (1.930.03) and
GPx (0.990.01). The treatment of TdPf alone at 5.2 g/kg body weight showed a significant increased level of
SOD (1.740.46), CAT (1.940.03) and GPx (0.990.01) when compared to the normal mice group and also
highly significant in 60days of treated animals (Table 2).
Table 2: Activities of enzymic antioxidants in control and experimental groups
The values are mean SD of six animals; # CD=One way ANOVA; ## CD=Two way ANOVA a-amount of
enzyme that gives 50per cent inhibition of the extent of NBT reduction for SOD, a-amount of enzyme required to
decrease the absorbance by 0.05 units at 240nm for CAT and a- microgram of GSH utilized/minute.
Estimation of Nonenzymic Antioxidants.
Table 3 exhibits the levels of nonenzymatic antioxidants in liver of control and experimental animals. From
the table 3 it is found that the level of vitamin A, vitamin E and GSH was significantly reverted back to near normal
level when treated with the plant extracts when compared to group III treated (standard antioxidant).
248
Table 3 : Levels of nonenzymic antioxidants in control and experimental groups
Values are mean SD, # CD=One way ANOVA; ## CD=Two way ANOVA
DISCUSSION
Cancer is a major health problem in both developed and developing countries. Because of the high death rate
associated with cancer and because of serious the side effects of chemotherapy and radiation therapy, many
cancer patients seek alternative complementary methods of treatment. Natural products have played an important
role in treating and preventing human diseases such as cancer, and have been derived from various source materials,
especially terrestrial plants. The importance of plants with few side effects for use as antitumor agents in modern
medicine has been reported. DPPH is a stable, nitrogen-centered free radical which produces violet colour in
ethanol solution. It was reduced to a yellow coloured product, diphenylpicryl hydrazine, with the addition of the
fractions in a concentration-dependent manner. DPPH analysis the radical scavenging decreased in the order
sesame oil, mustard oil and groundnut oil10. The protein fraction isolated from Sundakai seeds showed better DPPH
radical scavenging activity when compared with standard antioxidant Vitamin C and the IC50 was found to be 76%.
The results of the present study are also in agreement with another study that showed a concentration dependent
radical scavenging activity in the extract of chick pea protein fraction also showed similar observation11. One of the
studies was proved with our results that the ethanolic extract of leaves of Annona muricata exhibited maximum
superoxide radical quenching of 84.6 per cent at 100 g/ml12. The aerial parts of Achyranthes aspera of both the
aqueous and ethanolic extract exhibited potent scavenging activity for superoxide radicals in a concentration dependent
manner13. The administration of C. indica increased the activity of antioxidant enzymes in order to counteract the
accumulation of carcinogens generated in serum and liver of DLA injected mice14. An enhancement in the activities
of CAT and SOD in EAC tumour bearing mice administered with the methanol extract of Indigofera linnaei and
ethanolic extract of Vitis vinifera respectively was reported15,16. Our results are comparable with the reports that
the administration of protein extract of Terminalia catappa leaves elevated the activities GPx, SOD and CAT in the
liver of ELA induced mice17. Vitamin E levels were decreased in mucosal tissue of reported that colorectal cancer18.
Significant increased level of vitamin A and E in the liver and kidney of rats induced with ammonium metavanedate
toxicity by the prior treatment with green tea was reported19. Methanol extract of Mucuna pruriens increased the
level of GSH in EAC treated animals20. The hematological observation was also agreement with our results that the
aqueous extract of Ziziphus jujube fruit showed increased level in WBC, RBC and HB content21.
CONCLUSION
The present study was carried out to evaluate the antitumor activity of TdPf offered protective effect
against DLA tumor by their in vitro and in vivo antioxidant and antitumorigenic potential. The extract treatment at
the dose of 48g/kg inhibited the tumor activity by Enzymic and Non enzymic antioxidant assay. Our results
suggest that whole plant extracts are promising anticancer reagents.
249
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13. Upadhyay R, Chaurasia J K, Tiwari K N & Singh K (2014) The Scientific World Journal 1-7
14. Jawahar G & Emilin R (2014) Aus J of Basic and Applied Sciences 8, 466-475
15. Kumar RS, Rajkapoor B & Perumal P (2011) Asian Pac J of Cancer Prevention 12, 613-618
16. Mahadik V J, Patil P B, Patil S B & Naikwade N S (2011) Int J of Pharma Res and develop 3, 65-72.
17. Santhi R & Annapoorani S (2009) Int J of Drug Dev and Res 1, 81-84
18. Skrzydlewska E, Sulkowski S, Koda M, Zalewski B, Koda LC & Sulkowska M (2005) World. J. Gastroenterol
11, 403-406
19. Soussi A, Croute F, Soleiharoup J P, Kammoun A & El-Feki A (2006) Comptes rendus biologies 329, 775-
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21. Reyhane H Zabihollah M Nihad T & Aliyeh A (2015) Asian Pacific J of Reproduction 4, 116-122
250
Toxicity Studies of Punica granatum From Cell Line to
Animal Model
Shalini H. Kumar1 and Pushpa A*
Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore, Tamil Nadu.
Email: pushpa.biochem@gmail.com
Abstract
Drugs based on herbs have become a common form of therapy because they are often perceived as being natural
and therefore harmless. Today, they are one of the hottest trends and most sought after in the field of nutrition
or herbal therapeutics. All toxicological assessment of a herbal medicine is aimed to identify its adverse effects
and to determine limits of exposure level at which such effects occur. The significance of the adverse effect and
its nature are the main factors that are considered while evaluating the safety of an herbal drug. In the present
study, the effect of fruit extracts on the extent of cell death was confirmed by the percent viability of the healthy
kidney epithelial cell line and by performing toxicity study in rats. The safety level of oral administration of
Punica granatum fruit extract was found to be greater than 5000 mg/kg. Hence, the present study provides
adequate preclinical proof of safety of the fruit that is recognised to own diverse biological activities.
Keywords: Punica granatum, herbal drug, toxicity study
INTRODUCTION
Drugs based on herbs have become a common form of therapy because they are often perceived as being
natural and therefore harmless. Today, they are one of the hottest trends and most sought after in the field of
nutrition or herbal therapeutics [1]. The important role of plant derived compounds is undeniable. About 79% of the
medicinal plants show some cytotoxicity, while 75% of the nonmedicinal plants exhibit bioactivity [2] Bioactive
compounds are almost always toxic in high doses. Pharmacology is simply toxicology at a lower dose or toxicology
is simply pharmacology at a higher dose [3].
All toxicological assessment of a herbal medicine is aimed to identify its adverse effects and to determine
limits of exposure level at which such effects occur. The significance of the adverse effect and its nature are the
main factors that are considered while evaluating the safety of an herbal drug. Toxicity testing can reveal some of
the risks that may be associated with use of herbs especially in sensitive populations [4]. Pomegranate is one of the
oldest known drugs as it is mentioned in the Ebers papyrus of Egypt written in about 1550 BC [5]. Pomegranate
has been considered as a healing food owing to its vast advantageous biological effects against several diseases
since ancient times [6]. To further emphasise on the cytotoxic nature of the fruit extracts, the present study was
carried out in cell lines and rat models.

Collection and preparation of extracts: Fresh fruits were collected, washed and homogenized using distilled
water and ethanol for the preparation of aqueous and ethanolic extract respectively. It was then filtered using
Whatmann No 1 filter paper and used for further study.
MTT assay: The extent of viability of normal kidney epithelial cell line (Vero) with different concentrations of aril
and rind of P. granatum was analysed by MTT assay using standard procedure [7].
Toxicology profiling: Animals - Male Swiss Albino rats were procured from Bangalore and were housed in
251
microloan boxes in a controlled hygienic environment at temperature 25 2C and 12 hr dark/light cycle. The
study was conducted after obtaining institutional animal ethical committees clearance (IAEC No: KMCRET / Ph.D
/ 12/ 2014 15). As per the standard practice, the rats were segregated and quarantined for 15 days before the
commencement of the experiment. They were fed on standard healthy diet and water ad libitium.
Acute toxicity study - The acute toxicity test was performed in rats, with a dose of 5000 mg/kg of aqueous
extract of pulp and peel of P. granatum by oral gavage. Utmost care was taken to avoid the entry of the gavage to
the breathing system of the rats during the gavage feeding. After the extract was administered, feed but not water
was withheld for 3 - 4 h and the animals were monitored for 14 days.
Subacute toxicity study - Subacute toxicity is a repeat dose study performed to expose any deleterious changes
in organ, hematological and biochemical indices that may arise in the course of repeated administration of a test
substance.
Body weight The body weight was recorded on day zero (before treatment) and day fourteen (after treatment).
Organ weight - The anesthetized animals were carefully dissected for brain, heart, kidney, liver and spleen. Each
organ was cleaned for connective tissue and fat, examined macroscopically and immediately weighed.
Hematological parameters - At the end of the experiment, the rats were sacrificed, blood was collected and
analysed for their hematological parameters namely RBC, haemoglobin, platelets and WBC.
Biochemical parameters Parameters such as alanine aminotransferase [8], alkaline phosphatase [9], aspartate
aminotransferase [8], creatinine [10], total protein [11] and urea [12] were also studied using standard protocols.
RESULTS
MTT assay:
In the present study, the effect of fruit extracts on the extent of cell death was confirmed by the percent
viability of the cells. The aqueous and ethanolic extracts of aril and rind of P. granatum were screened for the
cytotoxicity against Vero cell lines at different concentrations to determine the IC50 (50% growth inhibition) by
MTT assay. The results are presented in figure 1. The extracts did not confer any significant lethality to the healthy
Vero cell line with an LC50 value greater than 10 mg/ml confirming the safe nature of the extract.
Toxicology Profiling: Acute toxicity The extracts did not bestow any noticeable signs of acute toxicity and
there was no mortality and morbidity observed.
Body weight and organ weight - The increase in body weight of the animal administered with various extracts did
not differ significantly from that of control group. There was no alteration observed in the weight of the vital
organs also. Weight of the animal before and after treatment and the weight of brain, heart, kidney, liver and spleen
were also recorded (table 1 and table 2).
Fig 1: Percent lethality of Punica granatum rind extracts against Vero cell lines
252
Table 1: Body weight of control and experimental rats
Treatment groups Body weight (g)

Before treatment After treatment
Control 178 + 5.23 185 + 7.08
PgAA 170 + 6.71 190 + 4.22
PgAE 179 + 7.56 189 + 3.36
PgRA 170 + 8.84 180 + 7.12
PgRE 176 + 9.00 184 + 6.82
Table 2: Organ weight of control and experimental animals
Sample Organ weight (g)

Brain Heart Kidney Liver Spleen
Control 1.81 + 0.03 1.69 + 0.01 1.41 + 0.05 8.75 + 0.03 1.15 + 0.03
PgAA 1.81 + 0.02 1.70 + 0.02 1.38 + 0.04 8.77 + 0.04 1.17 + 0.02
PgAE 1.80 + 0.03 1.71 + 0.02 1.39 + 0.03 8.78 + 0.02 1.19 + 0.02
PgRA 1.82 + 0.01 1.68 + 0.03 1.42 + 0.04 8.75 + 0.02 1.18 + 0.03
PgRE 1.82 + 0.01 1.68 + 0.01 1.41 + 0.03 8.77 + 0.02 1.20 + 0.02
Hematological parameters - The effect of extracts on certain hematological parameters such as RBC, WBC,
lymphocyte, haemoglobin, platelet count and total packed volume was determined (table 3). There were no significant
changes in the blood indices that were scrutinized among the control and experimental groups which is a key
indication of the safety of the extracts.
Biochemical parameters - In the present study all the biochemical parameters were within the normal range and
there was no significant difference between the control groups and the extract treated groups (table 4). The
amount of ALT and AST remained unaltered in comparison with the control and treated group of animals. The
levels of urea, creatine and total protein were within the normal range as in control. Hence, the extracts do not pose
any negative ruinous or ill-effects in the normal functioning of liver and heart.
Table 3: Hematological profile of control and experimental rats

Parameters
Samples RBC WBC Platelets
Hb (g/dl) PCV (%)
(x 106 cmm) (x 103 cmm) (x 105 cmm)
Control 13.60 + 0.55 7.86 + 0.08 6.42 + 0.52 4.6 + 0.51 38.45 + 0.03
PgAA 14.56 + 0.41 8.18 + 0.15 6.90 + 0.35 3.9 + 0.32 37.45 + 0.05
PgAE 14.75 + 0.36 8.75 + 0.08 6.89 + 0.47 3.4 + 0.39 39.12 + 0.07
PgRA 14.01 + 0.34 8.01 + 0.09 6.73 + 0.31 4.4 + 0.74 38.14 + 0.04
PgRE 14.25 + 0.12 7.94 + 0.18 6.71 + 0.53 4.12 + 0.25 37.48 + 0.08
253
Table 4: Biochemical indices of control and experimental rats
Parameters
Samples Total
Creatinine Urea
ALT (IU/l) ALP (IU/l) AST (IU/l) protein
(mg/dl) (mg/dl)
(g/dl)
Control 54.8 + 3.67 107.61 + 15.13 151.95 + 17.24 0.75 + 0.02 6.9 + 0.8 39.51 + 8.20
PgAA 56.5 + 4.02 98.79 + 12.08 133.45 + 16.02 0.73 + 0.01 5.8 + 1.0 36.75 + 5.13
PgAE 51.7 + 6.34 126.47 + 16.21 130.5 + 13.12 0.74 + 0.02 7.2 + 0.75 40.2 + 6.32
PgRA 58.2 + 3.92 108.21 + 13.47 144.06 + 14.21 0.76 + 0.03 6.3 + 1.2 42.5 + 4.60
PgRE 53.4 + 5.89 120.95 + 10.51 128.94 + 16.35 0.76 + 0.02 6.5 + 2.3 38.74 + 6.43
DISCUSSION
Pharmacological research on the medicinal properties of phytochemicals has become mandatory, to establish
the claimed medicinal properties. As a result of proximity, reliability and age long practice, people still rely on
traditional medicines which lack scientific explanations [13]. Metabolic changes occurred by any toxicants affect
the primary organs namely heart, liver, kidney and spleen. The weight of major organs of the fruit extract treated
animals did not significantly differ from those of the control group implying that the fruit extract is nontoxic to
these organs, even after administering a dose of 5000 mg/kg. Similar results were observed by Sireeratawong [14]
who carried out acute toxicity study of water extract from dried fruits of Terminalia bellerica in rats and found the
oral LD50 to be greater than 5000 mg/kg body weight.
A nonsignificant increase was observed in WBC levels which could be attributed to the beneficial effect of
the extracts in improving the general immunity of the animals. A lack of significant change in the haemoglobin level
was observed among the groups, justifying the fact that the extracts do not induce anaemia in animals in the
aforesaid concentration.
The present findings are in agreement with the study of Hosseinzadeh [15] who analysed the biochemical
parameters during the acute and subacute toxicity study of saffron and found that parameters like ALT, AST and
creatinine were normal even at a concentration of 0.5ml/kg.
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14. Sireeratawong S Jaijoy K Panunto W Nanna U Lertprasertsuke N & Soonthornchareonnon N (2013) Afr J
of Trad Comp and Alt Med 10(2), 223-231.
15. Hosseinzadeh H Shakib S S Sameni A K & Taghiabadi E (2013) Iranian J Pharm Res 12(1), 93-99.
255
Protective effect of A.malabarica leaves against hyperlipidemia and
oxidative stress in Streptozotocin induced diabetic rats
Peddanna Kotha1, Kameswara Rao Badri2* and Appa Rao Chippada1*
1
Department of Biochemistry, Sri Venkateswara University, Tirupati, 517 502 A.P, India
2
Minority Mens Health Initiative, Transdisciplinary Collaborative Center, Hampton University, Hampton, VA 23668, USA
Email: chippadar@yahoo.com
Abstract
The aim of the present study was to evaluate the antihyperlipidemic activity of active fraction of ethyl acetate
extract of A.malabarica leaves in STZ induced diabetic rats. The active fraction was isolated from ethyl acetate
extract of A.malabarica leaves by column chromatography and its antihyperlipidemic activity was evaluated
with a dose 50 mg/kg.bwt in STZ induced diabetic rats. Lipid profiles in Serum and tissue, Hepatic HMG Co-A
reductase activity, non-enzymatic antioxidants (Vit.C, Vit.E and GSH) in plasma, liver and kidney were estimated.
Elevated levels of serum and tissues lipids, HMG Co-A reductase enzyme activities in liver and decreased levels
of non-enzymatic antioxidants were brought back to normal levels after the treatment with the fraction. The
results show that the active fraction of ethyl acetate extract of the leaves of the A.malabarica has potent
antihyperlipidmic and antioxidant activity.
Key words: Anisomeles malabarica, Hyperlipidemia, HMG Co-A reductase, non-enzymatic antioxidants
INTRODUCTION
Hyperlipidemia is a condition of elevated levels of free fatty acids (FFAs) in circulating system of obese and
diabetic subjects by insulin resistance or/and inability of insulin action on suppression of hepatic glucose production.
The oxidation of circulating free fatty acid activates hepatic gluconeogenis and inhibits the glucose utilisation. The
American Heart Association (AHA) found that elevated levels of total cholesterol (TC) and triglycerides (TG) in
serum are responsible for the progression of atherosclerotic lesions1. Among the diabetic complications hyperlipidemea
is the major risk factor for the premature development of atherosclerosis and its cardiovascular complications2,3.
Atherosclerosis related diseases are leading cause of death, accounting for more than 70% of mortality in all forms
of diabetes1. Accelerated circulating FFAs and oxidation would be reduced by improving the hyperglycemia, lipid
lowering diet, reducing insulin resistance and drugs4. Lipid-lowering drugs, Statins and hypoglycemic agents
(insulin and antidiabetic drugs) are used for the treatment of hyperlipidmea. Indeed, there are unusual side effects
caused by the presently used treatments for hyperlipidmia5.
Hence, there is continuous search for the discovery of lipid-lowering drugs and natural compounds from
medicinal plants are preferred to be useful in the treatment of diabetic hyperlipidemia6. The Hypolipidemic effects
of number of Phytochemicals have been evaluated to achieve significant improvement lipids and lipoprotein profiles
of animal models and diabetic patients7.
Herbs from Lamiaceae family are also commonly used for food preservation, culinary flavors and treatment
of common illnesses as traditional medicine due to the presence of phenolics Phytochemicals and antioxidants8.
Phenolics Phytochemicals and antioxidants are playing a potential role in managing chronic oxidation linked
complications, such as cardiovascular diseases and type 2 diabetes 9. Anisomeles malbarica plant belonging to
Lamiaceae family is a great source of phenolics Phytochemicals, natural antioxidants and is an aromatic herb. So
the aim of the present study was to assess the antihyperlipidemic activity of the active fraction of ethyl acetate
extract of Anisomeles malabarica leaves in STZ induced diabetic rats.
256
Collection of plant material and isolation of active fraction/s
Anisomeles malabarica was collected from Tirumala, Tirupati regions and identified by the taxonomist in
Depot.of Botany. The leaves were shade dried, mechanically ground and used for extraction. Bioassay guided
fractionation was carried out according to Fathima et al. (2010)10 To prepare ethyl acetate extract the leaf powder
(1 kg) was soaked in 5 liters of ethyl acetate for 48 hours at room temperature and the solvent was filtered with
whatman no.1 filter paper. This was repeated 3-4 times until the filtrate gave no coloration. The filtrates obtained
were distilled and concentrated under reduced pressure at low temperature (400C to 450C) in Buchi rotavapor R-
200 and final concentrate was freeze dried. The ethyl acetate extract (10g) was subjected to column chromatography
(60-120 mesh size). Fractionation was performed using gradient system solvents in the following order; n-
Hexane, different proportions of hexane and ethyl acetate (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1:9) and ethyl
acetate. Various fractions were collected and monitored by thin layer chromatography (TLC) and were pooled into
six fractions viz. F1, F2, F3, F4, F5, and F6 from n-hexane:ethyl acetate solvent system. Each of these fractions
was evaluated for antidiabetic activity in STZ induced diabetic rats and the fraction which has shown the maximum
antidiabetic activity (data not shown) was considered as a active fraction (AMAF).
Induction of diabetes mellitus and Experimental design

Type 2 diabetes was induced in overnight-fasted rats by a single intraperitoneal injection of freshly prepared
STZ (50 mg/kg, dissolved in 0.1 M cold citrate buffer, pH 4.5)11. The animals were divided in to 5 groups of 6 rats
each. Group I: Normal untreated rats (NC), Group II: Normal treated rats (NT) with 50 mg active fraction/kg b.w.
(AMAF), Group III: Diabetic untreated rats (DC), Group IV: Diabetic rats treated (DT) with 50mg active fraction/
kg b.w. (AMAF), Group V: Diabetic rats treated (DG) with 20mg glibenclamide/kg b.w. Active fraction and
glibenclamide were administered to the respective group of rats once every day morning for 30 days by gastric
intubation using oral gavage. At the end of study after overnight fasting animals were sacrificed by asphyxiation
followed by cervical dislocation. Then blood, liver and kidney were collected and immediately stored at -20C until
further analysis. This study was approved by Institutes Animal Ethics Committee vide Resolution no: 27a/2012-
2013/(i)/a/CPCSEA/IAEC/SVU /CAR-KP.
Effect of the AMAF on lipid profiles and non enzymatic antioxidants
Estimation of cholesterol by Zlatkis method12, triglycerides levels by Foster and Dunn method13 were
carried out in serum, liver and Kidney. Serum HDL-cholesterol was determined by Burstein method14 and LDL
cholesterol was calculated by Friedwald formula15. The VLDL cholesterol was calculated using the formula, TG/5
mg/dl11.The ratio between HMG-CoA and mevalonate in hepatic tissue was taken as an index of the activity of
HMG-CoA reductase as described by Philip and Shapiro16. The levels of Vitamin C17, Vitamin E 18 and GSH19 were
estimated in plasma, liver and kidney.
The data was analyzed statistically using Student t-test and one way ANOVA test followed by Duncans
multiple range test (DMRT).
RESULTS
The diabetic control rats showed increased levels of serum TC (145.336.15mg/dl), TG (162.6614.01mg/
dl), LDL (76.162.51mg/dl) and VLDL-cholesterol (66.678.02mg/dl), and decreased levels of HDL-cholesterol
(15.538.45) when compared to those in normal rats (fig.1). After treatment with AMAF, there was a significant
decrease in the levels of TC (81.6618.03mg/dl), TG (86.3311.27mg/dl), LDL-cholesterol (41.1711.13mg/dl)
and VLDL-cholesterol (51.2521.51mg/dl) and also significant increased in HDL-cholesterol (23.710.57mg/dl)
257
(fig.1) in the treated diabetic rats. On other hand tissue lipid profiles, TCs and TGs also increased in diabetic
untreated rats (fig.2A&B), but the administration of AMAF to diabetic rats significantly decreased TC and TG in
hepatic (TCs- 6.0850.043mg/g and TGs-6.4320.031mg/g) and kidney tissues (TCs-7.490.082mg/g and TGs-
7.1050.055mg/g) (fig.2A&B respectively). Lower the ratio of HMG-CoA/Mevalonate, higher is the HMG-CoA
reductase activity and vice-versa, In the diabetic rats, the HMG-CoA/Mevalonate ratio was very low (0.9320.035)
indicating an increased HMG-CoA reductase activity(fig.3). The ratio of HMG-CoA/Mevalonate(1.2480.005)
was significantly increased in the AMAF treated diabetic rats after the treatment (fig.3).
Figure: 3 Figure: 5
Figures: 1) Effect of AMAF on Serum levels of lipid profiles, 2) Effect of AMAF on Total triglycerides and
cholesterol in liver(A) and kidney(B) 3) Effect of AMAF on HMG Co-A/Mevalonate ratio in Liver 4) Effect of
AMAF on Vit.C and Vit.E in plasma(A), Liver(B) and Kidney(C) 5) Effect of AMAF on GSH in Plasma(A),
liver(B) and Kidney(C). Values represent meanS.D. Values not sharing same superscript letter differ significantly.
at p<0.05(DMRT).
258
To study the effect of AMAF on non enzymatic antioxidants, we assayed Vit.C, Vit.E and GSH in plasma
and tissues. The levels of vitamin C, vitamin E and GSH were significantly decreased in plasma, liver and kidney
tissues of Diabetic untreated rats compared normal rats (fig.4&5). The administration of AMAF to the Diabetic rats
significantly restored there near to normal levels (fig.4&5).
DISCUSSION
Cellular free fatty acids homeostasis play very crucial role in the prevention of atherosclerosis diseases.
The circulatory and tissue free fatty acids are directly proportional to severity of diabetes mellitus. The increased
levels of TC and TG of serum and tissues, serum LDL-C and decreased levels of HDL-C in serum are exhibited in
hyperglycemic state20. In insulin resistance, since the blood glucose is not utilized by the adipose, hepatic and
muscle tissues, which in turn resulting in the free fatty acids mobilization from the adipose tissue to liver for
conversion to glucose and energy purposes. Therefore, the excess free fatty acids are accumulated in serum and
hepatic tissue and converted to TG. In the present study, the AMAF has significantly reduced the TCs and TGs in
serum, liver and kidney of diabetic rats.
Several previous reports show that STZ induced diabetic rats also have increased levels of low density
lipoprotein cholesterol (LDL-cholesterol) and VLDL-cholesterol21. These effects are associated with the STZ which
strongly disturb insulin secretion cells and loss of insulinotropic effect could alter the lipid profiles. Treatment with
AMAF has significantly decreased the levels of serum LDL-C and VLDL-C in diabetic rats. This effect could be
due to improvement in secretion of insulin and it promote the positive regulation of lipoprotein lipase enzyme and
thus blocking fatty acid and triglyceride synthesis ultimately leading to a fall in the levels of LDL-C, VLDL-C and
triglycerides.
The hepatic HMG CoA reductase is a key rate limiting enzyme responsible for conversion of HMG Co-A to
Co-A and mevalonic acid in the de novo synthesis of cholesterol. The higher activity of HMG Co-A reductase
enzyme and reduced level of HMG-Co-A/mevalonate ratio are signs for cholesterol synthesis in hepatic tissue,
which is a characteristic of type 2 diabetes22. Insulin and some of statins stimulate the feedback inhibition of HMG
Co-A reductase there by inhibited cholesterol synthesis. In the present study, increased HMG CoA/mevalonate
ratio was observed in hepatic tissue of diabetic rats treated with the AMAF after the treatment. Our previous
reports (data not shown) showed that the AMAF has increased the secretion of insulin from b-cells of pancreas,
which in turn decreased the levels of HMG CoA/mevalonate ratio and TCs in hepatic tissue. The AMAF could
strongly inhibit the HMGCo-A reductase and opposes the accumulation of cholesterol in hepatic tissues.
The increased level of free fatty acids in blood and tissues leads to increased -oxidation and production of
free radicals, which raise the oxidative stress causing damage to the cellular membranes and contents. Vitamin C is
playing central role in the detoxification of free radicals via singlet oxygen radical to hydroxyl radical and rejuvenates
the reduced vitamin E and GSH back to active state in non enzymatic antioxidant system. In diabetic rats, the
decreased levels of Vit.C in plasma and tissues, could be due to utilization of Vit.C against free radicals or rejuvenating
the reduced vitamin E and GSH back to active state23. The treatment with AMAF could significantly revert to
normalcy of the altered levels of Vitamin C in hepatic and kidney tissues. Vit.E a lipophilic antioxidant, plays
protective role against lipid peroxidation. In the present study decreased levels of Vit E were observed in plasma
and tissues of diabetic rats, which could be due to decreased levels of Vit C and increased lipid peroxidation by free
radicals24. The administration of AMAF to the diabetic rats have significantly restored the normal levels of Vit E in
plasma and tissues by decreasing lipid peroxidation. These effects may be due to the phenolic compounds of
AMAF, which transfer their hydrogen to a peroxyl free radical of peroxidized poly unsaturated fatty acids, preventing
the free radical chain reaction and restore the antioxidant mechanism. GSH is non enzymatic antioxidant, substrate
for GST antioxidant enzyme and can scavenge the free radicals in body. In the present study, GSH was significantly
decreased in plasma, liver and kidney of diabetic control animals. In our study, we observed significantly increased
GSH content in plasma, liver and kidney of diabetic animals after treatment with the AMAF. The increased GSH
259
content may promote the activity of GST enzyme and prevent the oxidative damage of cell membranes from free
radicals.
CONCLUSION
The present findings indicated that AMAF has potent antihyperlipidemic activity. Further studies are in
progress for the isolation of active principle responsible for antihyperglycemic and antihyperlipidemic activities
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260
Protective Effect of Ethanolic Extract of Fruits of Terminalia bellirica
on Experimentally Induced Obesity in Wistar Rats
Mary Shoba Das, C. and Gayathri Devi, S
Higher Education for Women, Coimbatore -641043. E-mail : gayathridevi.adu@gmail.com
Abstract
Obesity is one of the most common nutritional problems resulting mainly from an energy imbalance caused by
an increased ratio of caloric intake to energy expenditure. From the ancient days world has been facing a
challenge that is none other than obesity. The prevalence of obesity increasing day by day. Therefore, the
present study was designed to investigate the protective effect of ethanolic extract of fruits of Terminalia
bellirica (EFTB) on High Fat Diet (HFD) induced obesity in experimental rats. Obesity was induced by oral
feeding of HFD for 45days. Experimental rats were divided into 4 groups (n=6): Group I served as control rats fed
with normal pellet chow, Group II served as obese control rat fed with HFD, Group III rats received 500mg/kg
body weight of EFTB along with HFD and Group IV received 10mg/kg body weight of sibutramine along with
HFD. Body weight gain, liver marker enzymes (AST, ALT and ALP) and selected kidney function parameters
(creatinine, urea and uric acid) were assessed in the experimental rats. Oral administration of EFTB for 45 days
was found to significantly reduce the activities of liver marker enzymes and the levels of selected kidney
function parameters. These results clearly indicate that the EFTB can evoke a potent antiobesity activity and
hence it could be a useful intervention in the treatment of obesity.
Keywords: Obesity, High Fat Diet, Sibutramine, Terminalia bellirica
INTRODUCTION
Obesity is an increasingly serious socioeconomic and clinical problem, results from a positive energy
balance i.e., when calorie intake chronically exceeds energy expenditure. The World Health Organization has
described obesity as an escalating epidemic and one of the greatest neglected public health problems1. Obesity is
also known to be risk factor for the development of metabolic disorders, dyslipidemia, atherosclerosis and type 2
diabetes. Since many factors influence energy balance and they interact at many levels, determining the
pathophysiology of obesity is difficult. The main determinant is a disturbance of the homeostatic mechanisms that
control energy balance. Other factors such as food intake and lack of physical activity also contribute. In recent
years, there has been a great increase in the use of herbal medicines for the treatment of obesity2.
Terminalia bellirica commonly known as dhandrika in Tamil and it belongs to the family Combrataceae. It
has lot of medicinal values. It is reported that the fruits promote digestive power, wound healing, curative of
ulcers, local swelling, anemia and chronic recurrent fever3. With this back ground, the present study was designed
to investigate the protective effect of ethanolic extract of fruits of Terminalia bellirica (EFTB) on HFD induced
obesity in experimental rats.
Plant material and extract preparation
The fruits of Terminalia bellirica were collected from Velliangiri hills, Coimbatore and duly authenticated
by Botanical Survey of India, TNAU, Coimbatore. The authentication number is BSI/SRC/5/23/2014/Tech 510.
The collected fruits were washed, dried and powdered. The powdered fruits were then subjected to ethanolic
extraction by using Soxhlet apparatus.
261
Male Wistar rats of 8 10 weeks old, weighing 100 200 grams were used for this study were procured
from KMCH college of Pharmacy, Coimbatore, India. They were acclimatized to animal house conditions and it
was housed in polypropylene cages at room temperature (25 - 30 C) and at 45 - 55% relative humidity for 12h,
each of dark and light cycle and had free access to drinking water and fed with standard pellet diet. All animal
experiments were carried out in accordance with the guidelines of CPCSEA. The animal ethics approval no: KU/
IAEC/Ph.D/128.
Induction of obesity to experimental rats

The rats were made obese by the induction of High Fat Diet (HFD). The feed was prepared, dried and
administered every day morning with water. The male Wistar rat was divided into four groups with six rats each
for each treatment period. The groupings are as follows:
Group I : Control
Group II : HFD- induced obese control
Group III : HFD + EFTB (500mg/kg)
Group IV : HFD + Sibutramine (10mg/kg)
The experiments were carried out for 45 days. At the end of the study the animals were sacrificed after an
overnight fasting. The blood samples were collected from the cardiac puncture and used for the biochemical
analysis like liver marker enzymes namely AST4, ALT5 and ALP4 and selected kidney function parameters (creatinine6,
urea7 and uric acid8).
The values were expressed as mean S.D for six rats in each group. All the data were analyzed with SPSS
student software. Hypothesis testing method included one way analysis of variance (ANOVA) followed by Tukeys
test. A value of P<0.05 was considered as significant.

The body weight of the experimental rats was measured before and after the treatment periods. The
changes in body weight of the experimental rats are given in the Table 1.
Table 1: Body weight of experimental rats
Treatment Groups Body Weight

Before After
Control 17212.86 1909.34
HFD-induced 1789.62 2235.81
HFD+EFTB 1714.76 1884.73
HFD+Sibutramine 1766.28 1903.62
Values are expressed as mean S.E.M (n=6)
The control rats showed a linear proportion of growth in terms of body weight whereas there was a
significant (p<0.05) increase in the body weight of rats fed with HFD from the first week till the end of the
treatment period. The rats which received the ethanolic extract of fruits of T.bellirica and the standard drug
262
sibutramine significantly reduce the body weight near to the initial weight on the 45th day.
Liver Marker Enzymes in Experimental Rat The activities of liver marker enzymes namely AST,
ALT and ALP were assessed in the serum of the experimental rats and the results are recorded in Table 2.
Table 2: Activities of liver marker enzymes in experimental rats
Treatment Groups AST ALT ALP

Control 63.3 1.53 31.0 1.15 85.33 1.53
HFD-induced 94.67 2.52a 58.67 2.08 105 2.00a
HFD+EFTB 66.33 3.51 37.67 2.08b 68.67 2.08ab
HFD+Sibutramine 67.00 2.00a 40.67 3.06b 65.33 1.53ab

a- (p<0.05) G1 vs G2-G4
b- (p<0.05) G2 vs G3 and G4
A significant elevation in the activities of AST, ALT and ALP (P<0.05) were observed in the serum of obese
control rats when compared to the untreated control rats. The continuous oral administration of ethanolic extract
of fruits of T.bellirica and standard drug to the obese rats for 45 days was able to restore all the functions of liver
marker enzymes back to normalcy.
The liver uses transaminase enzymes, AST and ALT to metabolize amino acids to make proteins. When a
liver cell gets damaged, AST and ALT leak into the blood stream. Fatty liver is a reason for the hepatic damage and
frequent causes of fatty liver are diabetes, obesity and alcohol abuse. Obesity is also associated with the increase
in adipose tissue accumulation in the abdominal region9. The obese rats administered with ethanolic extract of fruits
of Terminalia bellirica were found to show activities of AST, ALT and ALP back to normal. This might suggest the
possibility of the fruits of Terminalia bellirica to normalize the hepatocytes, so as to cause accelerated regeneration
of parenchymal cells, thus providing fortification against membrane decreased fragility and leakage of these marker
enzymes.
Selected Biochemical Parameters of Kidney Function in Experimental Rats

The biochemical parameters of kidney function namely urea, uric acid, creatinine and protein were evaluated
and the results are presented in Table 3.
Table 3: Biochemical parameters of kidney function in serum of experimental rats
Treatment Groups Creatinine(mg/dl) Urea(mg/dl) Uric acid(mg/dl)

Control 0.47 0.04 27.3 3.06 7.38 0.04
HFD-induced 0.72 0.03 28.0 4.00a 12.43 0.14a
HFD+EFTB 0.42 0.03b 26.3 1.53ab 8.43 0.04ab
HFD+Sibutramine 0.44 0.04b 26.3 2.52ab 8.70 0.02ab

a- (p<0.05) G1 vs G2-G4
b- (p<0.05) G2 vs G3 and G4
263
The obese rats revealed a significant increase (p<0.05) in the levels of urea, uric acid and creatinine. These
biochemical markers were reverted back to near normalcy upon continuous oral administration of the ethanolic
extracts of fruits of T.bellirica and standard drugs to obese rats.
The capacity of the hepatocytes in obese rats to synthesize urea from precursors is decreased and the
uptake of amino acids by liver and the hepatic activity of the enzymes of the urea cycle are also decreased10. The
observed alteration in the levels of blood urea and serum creatinine in group of obese rats reverted to near normalcy
by treatment with fruit extract of T.bellirica, indicating renal protective nature of the extract.
CONCLUSION
The present study indicated that the administration of ethanolic extract of fruits of Terminalia bellirica at
a dose of 500mg/kg b.w produced significant antiobesity activity in HFD induced obesity rats. Besides this, the
drug administered to treat HFD induced obesity rats significantly reduce the activities of liver marker enzymes and
the levels of selected kidney function parameters. These observations concluded that the EFTB possess antiobesity
effects. Further studies are needed to explore the underlying mechanisms.
REFERENCES
1. Supriya K, Kotagiri S, Swamy B M V, Swamy, P A & Vishwanath K M (2012) Research Journal of
Pharmaceutical, Biological and Chemical Sciences 3, 542-554
2. Ramgopal M, Attitalla I H, Avinash P & Balaji M (2010) Academic Journal of Plant Sciences 3, 104-107
3. Manohar V R, Chandrashekar, R & Rao S N (2012) World Journal of Pharmacy and Pharmaceutical
Sciences, 1, 1376-1383
4. Reitman S & Frankel A S (1957) American Journal of Clinical Pathology, 1, 56-63
5. King J (1965) The hydrolases-acid and alkaline phosphatases, In practical clinical enzymology (Van D.
Nostrand Co. Ltd.), London, 191-196.
6. Owen J A, Iggo B, Scandrett F J & Stewart C P (1954) Biochemistry Journal 58, 426-437
7. Netelson S (1957) Microtechniques of clinical chemistry for the routine laboratory, C.C. Thomas, Springfield,
Illinios, 381
8. Caraway W T (1963) Uric acid in: Standard methods of clinical chemistry (Ed. Selingson, D), Academic
press, New York 4 239-247
9. Paschos P & Paletas K (2009) Hippokratia 13, 9-19
10. Kanthlal S K, Suresh V, Arunachalam G, Frank R P & Kameshwaran S (2012) International Research
Journal of Pharmacy, 3, 157-161
264
Pharmaceutical and Cost Benefit A nalysis of
Commercially Important Pomegranate Varieties
Sree Preethy. K and Sathishkumar. R1
Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University,
Coimbatore, Tamil Nadu, India. E-mail: rsathish@buc.edu.in
Abstract
Pomegranate, the fruit of Paradise has gained increased popularity among the producers and consumers worldwide
due to its low cultivation cost, high tolerance capacity, high yielding potential, better keeping quality and higher
nutraceutical value. Pomegranate being pharmaceutically important fruit, the commercially available six
pomegranate varieties were studied for its antioxidant capacity, pigmentation level and Vitamin C content in both
peel and pulp. The economic importance of these varieties were also analyzed by cost benefit analysis. This
study clearly proved that Mridula variety showed the maximum antioxidant activity and Kesar variety found to
be highly resourceful in anthocyanin pigment level and Vitamin C content. Thus, from this study, it can be
concluded that both the peel and pulp of different varieties of this super fruit proved to be highly resourceful in
nutritional levels. Lastly, coming to the cost benefit analysis, we conclude that Mridula variety is a commercially
viable, due to its low cost and high yield.
Keywords: Pomegranate; Pharmaceutical; Nutritional; Antioxidant; Anthocyanin; Vitamin C; Cost Benefit;

Yield.
INTRODUCTION
The genus Punica was described by Linnaeus for the first time in 1753. It is traditionally treated under
the Punicaceae, a monogeneric family of two species i.e., Punica granatum and Punica protopunica. Punica
granatum, commonly known as Pomegranate has been used since the dawn of human civilization. Besides
consumption as a raw fruit and for juice, pomegranate has tremendous therapeutic applications and hence, used in
the treatment of many diseases like, cancer1, diarrhea, diabetes, blood pressure2, leprosy, dysentery3, tapeworm
infection, hemorrhage, bronchitis4, gums bleeding, dyspepsia and throat inflammation. Due to its unique properties
and its efficiency as a traditional medicine to cure many diseases compared to other fruits, it is also called as Fruit
of Paradise.
In India, the pomegranate research was first started at the College of Agriculture, Pune in 19055. The
important commercial pomegranate varieties cultivated in India are Bhagwa, Alandi or Vadki, Dholka, Kandhari,
Kabul, Kesar, Muskati Red, Paper Shelled, Spanish Ruby, Ganesh (GB I), G 137, P 23, P 26, Mridula, Aarakta,
Jyoti, Ruby, IIHR Selection, Yercaud 1 and Co 1. Varietal improvement in pomegranate has been developed both by
selection of promising types from the indigenous ones and through controlled hybridization. Breeding objectives are to
develop cultivars with high yield potential, better fruit quality with respect to fruit colour, soft seeds, red coloured arils
and resistance to pests and diseases through different breeding methods viz., selection, hybridization. Thus, based on
the popularity, Ruby, Kabul, Kesar, Bhagwa, Ganesh and Mridula varieties were chosen for this study to analyze
their molecular level sequence variations and to check their varying nutrient levels.
The pomegranate has been considered as a food medicine of great importance for therapeutic purposes,
due to the presence of betulic and urosolic acids and different alkaloids, such as pseudo pelletierine, pelletierine and
some other basic compounds6, 7. It has been also widely used in the traditional Asian medicines both in Ayurvedic
and Unani systems. A remarkable increase in the commercial farming of the pomegranates is an global phenomena,
265
due to its potential health benefits, as it possess high antioxidant, anti-mutagenic8, anti-hypertension9 activities and
the ability to reduce liver injury10. Hence, since time immemorial, pomegranate had been a universal therapeutic
agent owing to the presence of biologically active ingredients in its different parts.
The polyphenolic compounds of pomegranate help to elevate the antioxidant capacity of the human body.
Anthocyanin pigment present in the Pomegranate fruit possess scavenging activities and its well known for its anti-
inflammatory and anti-atherosclerotic effect activity against osteoarthritis, prostate cancer, heart disease and HIV11,
12
. It was reported that the commercial pomegranate juices had three times higher antioxidant activity than those of
red wine and green tea13. It also increases the bodys resistance against infections, acts as cooling beverage and
tones up the function of kidney, liver and heart. These have recently been found to boost activity of an enzyme,
which protects the cardiovascular risks. Thus, pomegranate can be valued as an important medicinal fruit crop.
Pomegranate cultivation has got commercial importance in many countries, including India. Interestingly,
every part of pomegranate, namely the root and trunk bark, wood, sprouts, leaves, flowers, fruit, rind and seeds
have economic value14. In global market, there is a high demand for pasteurized fresh juice. The juice is mainly
used to prepare grenadine, extracts, liquors, wines, jelly and also for culinary purpose15, 16. The arils are used to
prepare jam or its dried form as condiments. The young leaves may serve as tea surrogate. The wild fruit is used
for citric acid production and its byproducts can be used to produce alcohol, tannin, waxes and frothers. The
flowers and rind are good sources of natural dye. Its seed oil can be used for producing enamels, and oilcake for
feed meal and some sorts of medical compounds. Hard wood of pomegranate is used for making small articles,
canes and handles. It is also a valuable honey-producing plant, which can produce 11.5 kg of honey/ha. Besides, it
also has a certain reclamation value due to its capability to thrive on saline soils, loose sands and mountains or
hills17. Due to its excellent flavor, nutritive value and medicinal properties, the pomegranate fruit has got a high demand
and this indicates its good potentiality in the development of its economic value too.
Thus, this study emphasis on the analysis of pigment content and antioxidant levels of commercially
available six pomegranate varieties in comparision with its economic importance.
MATERIALS AND METHODS:

Sample collection
Fruit samples of six Pomegranate varieties were collected from Tamil Nadu Agricultural University,
Coimbatore and other local markets in Coimbatore for the analysis of Antioxidant activity in the available varieties
of Pomegranate species, namely Ruby, Kabul, Kesar, Bhagwa, Ganesh and Mridula (Figure. 1).
Antioxidant Activity
Antioxidant profiling was carried out for commercially available six pomegranate varieties in our studies.
Sample Preparation
One gram of sample (Peel and Pulp) was weighed separately and homogenized with 1 ml of acidified
methanol (85ml Methanol: 15ml 0.1M HCl), until the tissue gets into a fine paste in a mortar and pestle. The
samples were then centrifuged at 10,000 rpm at 4C for 10 minutes. The supernatant was separated and stored at
-20C for the further biochemical studies.
266
Fig. 1- Six Commercially Important Pomegranate varieties used in this study
DPPH Radical Scavenging Assay

The scavenging activity on DPPH radical of different sample extracts was determined based on the method
reported by Okonogi et al. (2007).
The percentage of DPPH scavenging activity was determined using the below formula,
AO A1
Scavenging Activity (%) = 100
AO
Where, AO Absorbance of Blank and A1 Absorbance of Sample Extract
Determination of Total Anthocyanins using pH Differential Spectroscopic Method

Total anthocyanin was determined according to the pH differential spectroscopic method reported by
Cheng and Breen (1991). For calculation of total anthocyanins, the molar absorptivity coefficient ([) value 26900
M-1cm-1 was used. The results were calculated similarly to Giusti and Wrolstad (2001) as given below:
Asp = (A510 A700) pH1.0 (A510 A700) Ph4.5
The content of total anthocyanin (TA) was calculated as follows:
TA = (Asp M DF 1000) / ([ m)
Where, DF Dilution Factor, M Molecular weight of Anthocyanin (449), Cuvette optical path length (1 cm),
m Weight of the sample (g).
The total anthocyanin content was expressed as mg of anthocyanin per g fresh weight.
Vitamin C (Ascorbic acid) Estimation

Total Ascorbic acid of the commercially important pomegranate varieties was quantified according to the
method described by Roe and Keuther (1943). A standard graph was constructed and the concentration of ascorbic
acid in the samples were calculated and expressed in terms of mg/g of fresh weight of the sample.
267
Statistical analysis of data
All samples were analyzed in triplicates. The values are expressed as means of triplicate analysis of the
samples (n = 3) SD. These Biochemical data were further analyzed using one-way analysis of variance (ANOVA)
followed by Duncans multiple range test (P < 0.05) with the aid of SPSS (17 version) statistical package program.
P < 0.05 was considered as indicative of significance, as compared to either control or between the varieties.

Antioxidant activity, Anthocyanin pigment and Vitamin-C content analysis
In recent years, more attention has been paid to the antioxidants contained in fruits because epidemiological
studies revealed that high fruit intake was associated with reduced mortality and morbidity of cardiovascular
disease and some types of cancer and one of possible mechanisms was attributed to the antioxidant activity
presented by the fruits18, 19. Besides these antioxidant molecules, the biologically active compounds had also been
identified as important antioxidants contained in fruits20. Interestingly, the peel and seed fractions of some fruits
possess higher antioxidant activity than the pulp fractions. For example, grape seeds had been demonstrated to be
higher than grape pulps in antioxidant capacity and were rich sources of proanthocyanidins, which were very effective
in scavenging various ROS21. Another example is the pomegranate peel22. Therefore, the peel and seed fractions of fruits
may potentially contain more antioxidants quantitatively or qualitatively than the pulp fractions.
Thus, in the present study, the DPPH Radical Scavenging assay was employed and the DPPH value of
peel, pulp and seed fractions of different pomegranate varieties were determined in an attempt to make a systematic
comparison among their antioxidant activities.
Fig. 2 - Antioxidant levels (%) of the Pomegranate Varieties derived using

DPPH Radical Scavenging Assay
Each value in the table is represented as mean SD (n = 3). Superscript letters with same letters in the
same column indicate not significant and others are significantly different (P < 0.05) analyzed by Duncans multiple
range test.
Pomegranate has the highest antioxidant activity as compared to other fruits. With evident to such proposed
studies, the antioxidant activities of the different pomegranate varieties were analyzed and it was observed that the
highest antioxidant value was found in the Pulp of the Mridula variety (65.4%) and also in the peels of the Kesar
variety (67.1%) as compared to other varieties (Figure 2).
Several factors influence the stability of the anthocyanin, such as their chemical structure (number of
hydroxyls, methylation degree and glycosylation); the pH, temperature, light, presence of oxygen; enzymatic degradation
and interactions among other food components, such as ascorbic acid, metal ions, sugars, and co-pigments. Thus,
anthocyanin also has a significant role as a natural antioxidant or inhibitor of other toxic substances.
268
In this study, the anthocyanin pigment levels of the commercially available six pomegranate varieties were
determined by pH differential method. It was observed that the highest anthocyanin content (mg/ml) was found in the
pulp of the Bhagwa variety and also in the peel of the Kesar variety. The total anthocyanin content was found in the Kesar
variety as compared to the other varieties taken for this study. Therefore, we can conclude that the highest anthocyanin
content was observed in the peel of the Kesar variety and pulp of the Bhagwa variety (Figure 3).
Fig. 3 - Total Anthocyanin Content (mg/ml) of the Pomegranate Varieties

Each value in the table is represented as mean SD (n = 3). Superscript letters with same letters or * symbol in
the same column indicate not significant and others are significantly different (P < 0.05) analyzed by Duncans
multiple range test.
Besides antioxidant molecules, there are other biologically important and efficient compounds like Vitamin
C, E and other such polyphenols or flavonoids which are found essentially in fruits. These compounds also play an
important role in enhancing the metabolic activity of an individual and also help in supporting the normal metabolism.
Hence, in this study, vitamin C content was determined in the pomegranate varieties. Interestingly, high vitamin C
content was observed in the peel of the pomegranate varieties as compared to the pulp. The total vitamin C content was
found in the Bhagwa variety (218.9 mg/g) as shown in Figure 4.
Fig. 4 - Vitamin C content of the Pomegranate varieties
269
Each value in the table is represented as mean SD (n = 3). Superscript letters with same letters or * symbol in
the same column indicate not significant and others are significantly different (P < 0.05) analyzed by Duncans
multiple range test.
Cost Benefit Analysis

The pomegranate fruit is considered as the suitable fruit for the processing and utilization due to its excellent
flavour, colour, physico-chemical constitution and therapeutic properties. It is referred as the Super fruit due to
its high nutritional value, high antioxidant capacity and consumer appeal. The pomegranate can be processed into
products like minimally processed fresh arils, juice, squash, beverage, molasses, juice concentrates, frozen seeds,
jam, jelly, marmalades, grenadine, wine, seeds in syrup, pomegranate spirits, pomegranate powder, pomegranate
rind powder, anardana, confectionery and pomegranate seed oil. Thus, the keen and immediate attention is required
in meeting the research and developmental gaps for the commercialization and popularization of pomegranate
processing technology and pomegranate products.
Pomegranate Varieties Cost per kg (Rs) Annual Yield per plant

(Kg)*
Ruby 100 65
Kabul 128 60
Kesar 130 50
Bhagwa 105 55
Ganesh 118 45
Mridula 77 65
*The data collected from ICAR-National Research Center on Pomegranate, Solapur, Maharashtra, India
Table 1 - Comparison between the Commercial Value and the Annual yield per plant of the Pomegranate
VarietiesThis study focused on the importance of the pomegranate varieties and their antioxidant profile with
respective to the cost and annual yield per plant (Kg) as given in Table - 1. Our data suggests that the Mridula
variety shows the highest antioxidant activity as compared to other commercially available varieties. As an added
advantage, its market price is the lowest and also one of the high yielding variety per plant. Hence we can conclude,
Mridula variety is the best one from the consumer point of view.
ACKNOWLEDGEMENT
The authors Special gratitude towards Dr. K. Dhinesh Babu, Principal Scientist (Horticulture- Fruit Science),
ICAR - National Research Center on Pomegranate, Solapur, Maharashtra, India for their support in providing
essential details about the pomegranate varieties. We are also thankful to Bharathiar University, UGC-SAP and DST-
FIST.
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2. Aviram M & Dornfeld L (2001) Atherosclerosis 158, 195-198
3. Lansky E P & Newman R A (2007) J Ethnopharm 109, 177-206
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12. Sumner M D, Elliott-Eller M, Weidner G, Daubenmier J J, Chew M H & Marlin R (2005) The American J
Cardio 96: 810-814.
13. Gil M I, Tomas-Barberan F A, Hess-Pierce B, Holcroft D M & Kader A A (2000) J Agri & Food Chem
48, 4581-4589
14. Still D W (2006) Pomegranates: a botanical prospective. In: Pomegranates: Ancient Roots to Modern Medicine
(Seeram P, Schull- man RN, Heber D, Eds), CRC Press Taylor and Francis Group, Boca Raton, Florida, pp
199-209
15. Elyatem S M & Kader A A (1984) Scientia Horticulturae 24, 287-298
16. Holland D & Bar-Yaakov I (2008) Chronica Horticulturae 48, 12-15
17. Levin G M (2006) In Pomegranate (Ist Edn), Third Millennium Publishing, East Libra Drive Tempe, AZ, pp
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18. Guo C J & Yang J J (2001) China Public Health 17, 8788.
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271
Phytochemical Screening and Antioxidant Potential of the
Selected Coconut Products
Lalitha Ramaswamy*, Madhan Shankar**, T.Benoite***
*Associate professor and HoD, Department of Nutrition and Dietetics, **Associate professor and HoD, Department of Biotechnology
*** PG Student, Department of Nutrition and Dietetics, PSG College of Arts and Science,
Affiliated to Bharathiar University, Coimbatore, Tamil Nadu, India. benoite.thomas@gmail.com
Abstract
Coconut is a product of having important components, which are identified to have medicinal properties and
have been used in Ayurvedic medicine. The aim of the study is to assess the phytochemical components and
antioxidant potential of the selected coconut products namely coconut oil, virgin coconut oil, coconut water and
coconut milk. The ethanol extracts of the selected coconut products were screened for the presence of
phytochemicals and their effect on 2, 2-diphenyl-1-picryl-hydrazyl-hydrate radical (DPPH) was used to determine
their free radical scavenging. Phytochemical screening of the extracts showed the presence of flavonoids, alkaloids,
phenols, saponins, tannins, terpenoids and steroids. However, alkaloids were absent in virgin coconut oil, saponins
in coconut water, flavonoids in coconut milk, tannins in coconut oil and steroids in virgin coconut oil and
coconut milk..DPPH assay showed a maximum inhibition of 83.33% in the extracts of virgin coconut oil at a
concentration of 500g/ml, followed by coconut water, coconut milk, and coconut oil with an inhibition of
79.16%, 75% and 70.83% respectively. The selected coconut products were found to have phytochemicals and
showed potent inhibition of DPPH radical scavenging activity, with virgin coconut oil being the most potent.
Keywords: Coconut Products, Phytochemical Screening, Antioxidant Potential.
INTRODUCTION
Phytochemicals with significant health potentials include flavonoids, carotenoids, phenolic acids, saponnins,
glycosides, tannins, alkaloids [1]. Nuts are complex plant foods that are not only rich sources of unsaturated fat but
also contain several non-fat constituents such as plant protein, fiber, micronutrients (eg, copper and magnesium),
plant sterols, and phytochemicals [2] . Coconut has been found to be a typical example of plant possessing such
quality [3]. Thus for the present study coconut products were chosen to evaluate their pharmacological activity
with the following objective: To screen the phytochemical components present in coconut products and evaluate
the antioxidant properties of coconut products.

Sample Collection and Extraction
Coconut products namely coconut oil, virgin coconut oil, coconut water and coconut milk were selected for
the study and were procured from local markets of Coimbatore (India). Five ml of the sample was taken. Sample
extracts were prepared in 20 ml of 70% Ethanol containing 0.1% HCl by shaking for four hours at room temperature.
The sample suspension was centrifuged at 10,000 rpm for 15minutes at 10C, the supernatant was collected and
filtered through Whatman no 1 filter paper and the resultant filtrate was stored at 20C. Analysis was completed
within a month of extraction [4].
Phytochemical Screening
Phytochemicals namely flavonoids, phenol, tannins, saponins, alkaloids, terpenoids, steroids were tested
for the above mentioned coconut products using the standard procedure [5] to indentify the components.
274
DPPH Radical Scavenging Activity
The percentage of antioxidant activity (AA %) of each substance was assessed by DPPH free radical assay.
The measurement of the DPPH radical scavenging activity was performed according to methodology described by
[6]. The samples of various concentrations were reacted with the stable DPPH radical in an ethanol solution. The
reaction mixture consisted of adding 0.5 mL of sample, 1 mL of absolute ethanol and 0.5 mL of DPPH radical
solution 0.4 mM in ethanol. When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is
reduced. The changes in colour (from deep violet to light yellow) were read at 517 nm after 30 minutes.

Results of Qualitative Analysis
Results (Table-I) of qualitative analysis carried out on the extracts of coconut oil, virgin coconut oil,
coconut water and coconut milk showed the presence of phytonutrients namely flavonoids, alkaloids, phenols,
saponins, tannins, terpenoids and steroids.
Flavanoids were present in coconut water in high concentration, coconut oil and virgin coconut oil in
moderate concentration. Coconut milk showed the absence of flavonoids. Flavonoids constitute a wide range of
substances that play important role in protecting biological systems against the harmful effects of oxidative processes
on macromolecules, such as carbohydrates, proteins, lipids and DNA [7]. Therefore the presence of flavonoids in
coconut oil, virgin coconut oil and coconut water makes them good antioxidant component in the diet.
Moderate concentration of phenols was present in coconut water, while low concentration was observed
in coconut oil, virgin coconut oil and coconut milk. Phenolics possess diverse biological activities, for instance
anti-inflammatory [8] and antioxidant [9].Therefore all the selected coconut products have the above stated
pharmacological activities.
Table I : Qualitative Analysis

S.No Phytoconstiuents Coconut Oil Virgin Coconut Oil Coconut Water Coconut Milk
1. Flavonoids ++ ++ +++ -
2. Alkaloids ++ - +++ +
3. Phenols + + ++ +
4. Saponins + + - ++
5. Tannins - + +++ ++
6. Terpenoids ++ + ++ +
7. Steroids + - ++ -
Key: + = Present; ++ = Moderate Concentration; +++ = High Concentration and - =Absent Co conut wat er
showed high concentration of alkaloids, moderate concentration in coconut oil, low concentration in coconut milk, while
it was absent in virgin coconut oil. Purely synthesized alkaloid can be used as medicinal agents because of their analgesic
and antibacterial properties [10]. Therefore coconut oil and coconut milk are also analgesic and antibacterial in nature.
Tannins were present in high concentration in coconut water, moderate amount in coconut milk, low in
virgin coconut oil and was absent in coconut oil. Tannins may be employed medicinally in antidiarrheal, haemostatic,
and antihemorrhoidal compounds [11]. Therefore coconut water, virgin coconut oil and coconut milk can also be
used for antidiarrheal, haemostatic, and antihemorrhoidal treatment.
Coconut milk had moderate concentration of saponins, while it was low coconut oil and virgin coconut oil.
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Coconut water showed absence of saponins. Saponins serve as antioxidants as they prevent degeneration of DNA
and also help to reduce colon damage and risk of cancer [12]. Therefore the presence of saponins in the coconut
products makes them a source of antioxidants and antimicrobial components.
All the products showed the presence of terpenoids with coconut oil and coconut water containing moderate
concentration. Terpenoids used as antimicrobial, anti-diarrheal agent [13]. Therefore coconut products can be used
in antimicrobial and antidiarrheal treatment.
Moderate concentration of steroids was present in coconut water. Steroids were absent in virgin coconut
oil and coconut milk. Plant steroids possess many medicinal, pharmaceutical and agrochemical activities like anti-
tumor, immunosuppressive, hepatoprotective, antibacterial, plant growth hormone regulator, cytotoxic and cardiotonic
activity [14]. Therefore coconut oil and coconut water have medicinal, pharmaceutical and agrochemical activities.
Results of DPPH Radical Scavenging Activity Assay
The results of DPPH radical scavenging activity (Figure I) of sample extracts in comparison to ascorbic
acid showed extract of virgin coconut oil appeared to be as potent as Vitamin C with a maximum inhibition of
83.33% at 500g/ml which is same to 83.33% for vitamin C at the same concentration. Coconut water was less
potent than the standard with a maximum inhibition of 79.16% at 500g/ml, followed by coconut milk with a
maximum inhibition of 75% at 500g/ml. Coconut oil was the least potent showing a maximum inhibition of
70.83% at 500g/ml. The extract was capable of neutralizing the DPPH free radicals via hydrogen donating activity
[15]. This study suggests that these samples possess antioxidant activities which can counteract the oxidative damage.
Figure I : Inhibition of DPPH by the Different Samples and Vitamin C
Key: s= standard ascorbic acid: c= coconut oil: v= virgin coconut oil: w= coconut water: m= coconut milk
CONCLUSION
The present study showed that the presence of phytochemicals in coconut products and also possess
antioxidant activity thus indicating that these coconut products can be utilized in treatment of several disease
conditions.
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of triphala determined by using different in vitro models. Journal of Medicinal Plant Research 7(39), 2898-
2905
277
Phytomedicines for the Treatment of Postmenopausal Osteoporosis
A Review
Anitha, S1 and Gayathri Devi, S2
1
DST WOS-A Project Staff and Ph.D. Research Scholar, Department of Biochemistry, 2Associate Professor, Department of Biochemistry,
Biotechnology and Bioinformatics, Avinshilingam Institute for Home Science and Higher Education for Women, Coimbatore 641 043,
Tamil Nadu, India, Email ID: gayathridevi.adu@gmail.com
Abstract
Osteoporosis is the widespread bone disorder in postmenopausal women, characterized by diminished bone
mass, micro-structural deterioration of bone tissue and greater the bone turnover that leads to increased fracture
risk with morbidity and mortality. Along with the advancing age, lack of estrogen, vitamin D and calcium
deficiency, low body weight or low body mass index (BMI), endocrine disease, the use of obvious medications
have been represented as risk factors of osteoporosis. The synthetic drugs which are used to treat osteoporosis,
acts by reducing the frequency of bone resorption, thereby lowering the rate of bone loss, but they are associated
with the side effects such as hypercalcemia, hypercalciuria, increased risk of endometrial and breast cancer,
breast tenderness, menstruation, thrombo-embolic events, dyspepsia and gastro intestinal ulcers. To overcome
the broad range of side effects produced by synthetic drugs, there is a rise in demand for Phytomedicines
which are believed to be healthier and safer for the therapy of postmenopausal osteoporosis. From the ancient
days many herbal plants have been used for treating this disease. A few of them namely, Rubia cordifolia,
Puerariae radix, Wedelia calendulacea Less., Cicer arietinum L and Nigella sativa were found to be useful for
bone disorders.
Keywords : Bone disorders, postmenopausal osteoporosis, pharmacological agents, phytomedicines
INTRODUCTION
Bone tissue has got numerous functions as it gives shape to the body, helps in movement, involved in the
production of blood cells and weight bearing. This is the reason for the area of concern in drug development for the
bone related problems namely osteoporosis, rheumatoid arthritis and osteomalacia. Osteoporosis is the disease of
interest among the other bone diseases, since it affects the elders and particularly the postmenopausal women
because of estrogen deficiency after cessation of menopause [14]. Menopause is the cessation of menstruation for
twelve consecutive months from the last menstrual period, a definition that has been consistent for decades.
Anticipation, agitation and even anxiety are the frequently raised problems followed the arrest of menstrual cycle
in women. Physiologically, the changeover takes years, during which time there is a lowering of ovarian function,
its production of estrogen and progesterone and, finally, the end of ovulation [9]. Postmenopausal osteoporosis is a
slowly emerging global health problem by depending on several factors. The lifetime risk of dying from hip fracture
is likewise as that from breast cancer. In India one among three women between the age group of 50-60 years
suffers from osteoporosis [16].
Pathogenesis of Postmenopausal Osteoporosis
In healthy normal women, the reduction of estrogen level is the causative for accelerated bone turnover
and the subsequent decline in bone density and the deterioration of bone structure, leading to an increased fragility
fracture risk [3]. The main determinant of the peak bone mass is genetic factors. Low-density lipoprotein receptor
related protein 5 (LRP5), osteoprotegerin (OPG), sclerostin (SOST), estrogen receptor 1, and the receptor activator
of NF-B (RANK) pathway genes were the several genetic variants related to bone mass. The accumulation of
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bone mass found to be regulated by hormone, especially estrogen. The other factors may also involved in the
process of building the peak bone mass includes nutrition, smoking and exercise. Through the following mechanism
the hormone estrogen affects the bone by lowering the sensitivity of bone mass to PTH (parathyroid hormone),
proliferating the production of calcitonin, hastening calcium resorption by the intestine, reducing the calcium excretion
from the kidney direct effect of estrogen on bone [7].
Hormone Replacement Therapy
Hormone replacement therapy (HRT) has been used to treat menopausal estrogen and increase durability
in postmenopausal women for more than 60 years, in which the estrogen is analogous to natural ovarian production
[4]. It is crucial for incorporation of a progestogen or micromised progesterone, to prohibit endometrial hyperplasia
and cancer. Estradiol can be delivered orally (micronised estradiol, estradiol valerate, estrone, estriol or conjugated
equine estrogens), or transdermally (17b-estradiol). In postmenopausal women, topical vaginal administration of
estrogen is used for localised symptoms. Discrete progestogens are used in combination with estradiol, either in a
sequential cyclical regimen or as continuous combined therapy [2]. Since 1960s, hormone replacement therapy for
postmenopausal women has been subjected to much disputation and consideration. Before 2002, in order to a
reduction in risk of cardiovascular disease, osteoporosis and colon cancer, the effects of hormone replacement
therapy were assumed to be beneficial. Principally on the basis of results from observational studies of HRT, the
negative side effects such as an increased risk of breast cancer and thrombo-embolic disease were thought to be
outweighed by the advantages [12].
Role of Phytomedicines in Osteoporosis
The use of hormone replacement therapy for long term regrettably caused numerous unwanted side effects
which were interconnected with these powerful steroids and extended risk for breast and endometrial cancers.
Therefore, to prevent bone loss in osteoporotic condition, further investigation of alternatives and supportive
approaches that can produce clinically significant data of the interest. For the treatment and prevention of osteoporosis
the non-hormonal therapy or natural product therapy may be more acceptable [10]. The various types of medicinal
plants approaches osteoporosis in different manner by supplementing dietary calcium, modulating the bodys use
of calcium and increasing the level of certain hormones in the body [11]. The largely available natural products
which share structural and functional similarities with naturally occurring or synthetic estrogens for the management
of osteoporosis are largely phytoestrogens which include isoflavones, lignins, flavonoids and coumestan. At different
reproductive and non-reproductive tissues the phytoestrogens exhibit its effect similarly like estrogen [8]. List of
plants that have been used as herbal medicine for the treatment of osteoporosis includes Rubia cordifolia, Puerariae
radix, Wedelia calendulacea Less., Cicer arietinum L, Nigella sativa, Podophyllum emodi, Cissus quadrangularis,
Glycine max, Cimicifuga racemosa L, Glycyrrhiza glabra, Trifolium pretense, Petroselinum crispum, Equisetum
arvense, Persea americana, Dioscorea villosa, Coriandrum sativum. A few of them were discussed below:
Rubia cordifolia
Manjishta, the common name of Rubia cordifolia is a climbing or scrambling herb, with red rhizomatous
base and roots. It is an eminent medicinal plant often seen in India, Africa and Southeast Asia. In ayurveda, the root
of Rubia cordifolia have been described as bone mender, since it is used to heal fractured bones. In ovariectomised
rats, the administration of ethanolic extract of shade dried powdered root of Rubia cordifolia significantly increased
biomechanical strength, higher osteoblastic activity and minimal osteoclastic activity for bone formation were
observed. Furthermore improved serum calcium absorption, bone ash composition and ash calcium in Rubia cordifolia
extract treated groups were observed [14].
Puerariae Radix
Gegen (Puerariae radix) is one of the preliminary and most essential edible crude herbs used in Chinese
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medicine. Initially, in China, Japan and Korea, this herb has been used to treat the common cold, influenza and
wrist and shoulder stiffness or as an anti-alcohol abuse agent [17]. Puerarin, 3-hydroxy puerarin, puerarin-wood
glycoside, daidzin and fermlononetin are the isoflavone compounds found in Gegen. Among these Puerarin is the
peculiar composition of this genus which is present highest in content. The ovariectomized mice are acknowledged
animal models for studying postmenopausal bone loss. An assured dose of Gegen, which was identical to estradiolin
therapeutic effect, was effective in improvising calcium content and bone density of ovariectomized mice, apparently
it also improves the pathological change of bone microstructure without increasing uterus mass and estrogen-like
side effects. Osteoclast number of mice found to be significantly reduced after the treatment with Gegen water
extract, which indicated that Gegen could directly (or indirectly) change osteoclast number, modulate osteogenesis
osteoclasts coupled response, promote bone formation, inhibit bone absorption and reduce bone turnover rate,
consequently reducing bone loss and achieving the effect of anti-osteoporosis [18].
Wedelia calendulacea Less.
Wedelia calendulacea Less., a perennial herb found in Uttar Pradesh, Assam, Arunachal Pradesh and Tamil
Nadu. It contains isoflavanoids which are used in the treatment of disease such as liver disorders, uterine hemorrhage
and menorrhagia. The treatment of ethanolic extract of Wedelia calendulacea Less. in rats at the end of 3 months
following ovariectomy, the biomechanical parameters, viz., load testing of the femoral head of the femur (Femur),
three-point bending (Tibia) and compression test of vertebra (Vertebra) showed a significant increase in biomechanical
strength. It also showed an enhancement of osteoblastic activity and a reduction of osteoclastic activity by showing
a significant increase in alkaline phosphatase and tremendous decrease in tartarate resistant acid phosphatase (TRAP).
The protective effect of the drug were revealed in this histological examination of extract treated groups showed
ossification, mineralization, calcified cartilaginous deposits and a marginal osteoclastic activity due to an increase
in bone formation and a reduction in bone resorption [1].
Cicer arietinum L
Cicer arietinum L is one of the oldest and most widely planted legumes in the world and commonly called
as chickpea. The chickpea has been a classical Uighur herb for over 2500 years in Xinjiang, China and it is a crucial
food because it is rich in high-quality protein, carbohydrates and essential mineral elements. It also has been widely
used in traditional Uighur medicine for the treatment of various diseases such as hypertension, hyperlipidemia,
diabetes, itchy skin, flatulence, low libido, tumor formation and osteoporosis. It contains 8 phytoestrogens which
includes, biochanin A, formononetin, genistein, biochanin A-7-O--D-glucoside, calycosin, trifolirhizin, ononin
and sissotrin. Isoflavones are the important component present in chickpea seed and sprout, the level of it was
found to be higher in sprout than in its seed. The significant estrogenic effects on the uterus has observed in
ovariectomized (OVX) rats treated with chickpea sprouts (ICS) and also exhibits increase in uterine weight, epithelial
height and gland number, as well as in the expression of the cell proliferation marker PCNA. The serum E2 level
was significantly increased, while serum LH and FSH levels were decreased. Chickpea sprouts exhibits moderate
estrogenic activities as compared to E2 in ovariectomized rats, recommending the potential use of chickpea sprouts
for the treatment of menopausal symptoms and osteoporosis caused by estrogen deficiency [5].
Nigella sativa
Nigella sativa is a herbal plant which belongs to Ranunculaceae family. It is commonly known as black
cumin or Habbatus sauda. It is vastly distributed in the southern region of Asia. The active ingredients of the
Nigella sativa (NS) present in its seed, found to be a most valuable part of the plant has been used in Islamic
medicine for its healing powers. The various studies have revealed its therapeutic value in the treatment of disease
such as anticancer, antioxidant, antibacterial, antifungal, antiparasitic and antiasthmatic. Nigella sativa consists of
3638% fixed oils, proteins, alkaloids, saponin and 0.42.5% essential oil [15]. Ovariectomised (OVX) rats showed
significant decrease in plasma Ca2+, accompanied by a significant increase in plasma Alkaline phosphatase, amino
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terminal collagen type 1 telopeptide, nitrates, TNF- and IL-6. These changes were overcome by Nigella sativa
supplementation in OVX-NS. Histological examination of the tibias revealed discontinuous eroded bone trabeculae
with widened bone marrow spaces in Ovariectomised rats accompanied by a significant decrease in both cortical
and trabecular bone thickness. These parameters were markedly reversed in OVX-NS rats which showed its positive
effects towards osteoporosis in postmenopausal women [13].
CONCLUSION
The estrogen deficiency following the menopause is linked with the osteoporosis, a serious concern should
be taken on postmenopausal women which reduces the quality of life during their postmenopausal period. In
clinical practice, the impact of osteoporosis associated with higher morbidity and mortality. Several studies have
been conducted to investigate the action of different medicinal plants to reverse the osteoporosis in postmenopausal
women. To avoid side effects of hormone replacement therapy, the phytomedicines could be considered as natural
heritage from mother nature as a source of medicine for the treatment of postmenopausal osteoporosis.
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1. Annie S, Prabhu R G & Malini S (2006) Phytomedicine 13, 43-48.
2. Bakour S H & Williamson J (2014) The Obstetrician and Gynaecologist 17, 20-8.
3. Gambacciani M & Levancini M (2014) Przeglad Menopauzalny 13, 4, 213-220.
4. Gerval M G & Stevenson J C (2017) The Pharmaceutical Journal 1-12.
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Akber A. (2013) Acta Pharmacologica Sinica 34, 380386.
6. Ji M X & Yu Q (2015) Chronic Diseases and Translational Medicine 1, 1, 9-13.
7. Kumar M, Rawat P, Dixit P, Mishra D, Gautam A K, Pandey R, Singh D, Chattopadhyay N & Maurya R
(2010) Anti- Phytomedicine 17, 993999.
8. Nardone A G (2014) Contemproary Ob/Gyn 1-4.
9. Nurrochmad A, Leviana F, Wulancarsari C G and Lukitaningsih E (2010) International Journal of

Phytomedicine 2, 363-372.
10. Rajagopal P L, Amrutha C, Premaletha K & Premkumar N (2013) International Journal of Advances in
Pharmacy, Biology and Chemistry 2, 4, 605-608.
11. Schierbeck L L, Rejnmark L, Tofteng C L, Stilgren L, Eiken P, Mosekilde L, Kober L and Jensen J E B
(2012) British Medical Journal 1-11.
12. Seif A A (2014) BMC Complementary and Alternative Medicine 14, 22.
13. Shivakumar K, Mukund H & Rabin P (2012) International Journal of Drug Development and Research 4,
3, 163-172.
14. Shuid A N, Mohamed N, Mohamed I N, Othman F, Suhaimi F, Ramli E S M, Muhammad N and Soelaiman
I N (2012)Evidence-Based Complementary and Alternative Medicine 1-6.
15. Thulkar J & Singh S (2017) Journal of Mid-life Health 6, 3, 104-107.
16. Wang X, Wu J, Chiba H, Yamada K & Ishimi Y (2005) Metabolism 54, 1536 1541.
17. Yan-qiong S, Cong M A & Lei L I (2016) Journal of Translational Medicine 4, 2, 132-136.
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Influence of Elicitors on Major Withanolide Accumulation in in vitro
Shoots of Withania somnifera Dunal: Winter cherry
Parameswari. Da Estri Laras Ab, Joni Kc and Kalaiselvi, Ka*
a
b
Professor Department of Biology, University of Brawijaya, Malang, c Professor Department of Food Science and Technology ,
University of Brawijaya, Malang. E-mail : Susiparam526@gmail.com
Abstract
Withania somnifera, a multipurpose medicinal herb is a rich repository of pharmaceutically active steroidal
lactones known as withanolides. In the present study, adventitious shoot multiplication protocol of W. somnifera
was optimized in media supplemented with different concentration and combinations of Biotic (A. niger) and
Abiotic (Salicylic acid, SA) elicitors. The maximum number of Shoots (31.000.57) was observed in MS
basal medium supplemented with Salicylic acid (150M) and A. niger (1.0 v/v), 3% sucrose and 4.44 M BAP
in suspension. An increase in shoot mass of 38.6g/L and presence of bioactive compound withanolide A
(186.340.33) and withaferin A (1240.84 0.45) was observed in in vitro shoot. In vitro adventitious shoots in
bioreactors could be used as a quick and efficient process of generating multiples that would offer unique
opportunities for producing shoot based pharmaceutical products without having to depend on field cultivation.
Keywords: BAP-Benzyl adenine purine; SA-Salicylic acid; Withania somnifera
INTRODUCTION
Withania somnifera also known as Ashwagandha belonging to the family Solanaceae is a plant of immense
medicinal value largely due the presence of array of bioactive withanolides 1. The bioactive constituents of this
plant have been isolated and reported to possess adaptogenic, anticancer, anticonvulsant, immunomodulatory,
antioxidative and neurological effects 2. Among the bioactive constituents, Withanolide A and Withaferin A were
reported to be principal metabolites 3, 4, distributed among various tissues 5.
The annual production of this plant is not sufficient to meet the global requirement 6. Therefore, Tissue
culture techniques paves an alternate way for mass production of plants in a short time span to meet the increasing
demand and offer a viable tool for mass propagation and multiplication in a variety of medicinal plant species7. In
vitro shoot suspension culture has become an alternative method for the production of valuable secondary metabolites
on a commercial scale. Adventitious shoots are natural, grown vigorously in phytohormones supplemented medium
and have shown tremendous potentialities of accumulation of valuable secondary metabolites. exogenous supplement
of cytokinine may be influence for to promote adventitious shooting 8.
Bioproduction of secondary plant metabolites using elicitation of suspension cultures is well documented
for plants producing minute quantities of useful phytochemicals 9. The accumulation of secondary metabolites in
plants is part of the defense response against pathogenic attack, which is triggered and activated by elicitors, the
signal compounds of plant defense responses10. Elicitors are one of the important factors that can act as a switch for
increasing the yield of secondary metabolites of useful bioactive compounds in plant tissue cultures 11.
Since it is impossible to consider the great variety of elicitors assayed in plant cell cultures in their entirety,
in this work we have focused mainly on the action of the most commonly used and effective biotic elicitors and
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abiotic elicitors for the biosynthesis and accumulation of secondary compounds. With this background the present
study was formulated to increase the production of secondary metabolites in in vitro maintained shoot cultures of
Withania somnifera by feeding elicitors.

Plant materials
Seeds of a released variety of Withania somnifera, Jawahar Asgandh-20 were purchased from Tamil Nadu
Agricultural University (TNAU), Coimbatore. Surface sterilized seeds were then germinated in vitro in Murashigae-
Skoog (MS) media supplemented with 2% sucrose and seedlings produced were maintained on MS basal medium
with regular subculturing. The 15 days old in vitro solid medium with 4.44 M BAP containing growing shoot
cluster were used as explants for the study.
Elicitors Treatments
To establish a shoot elicitor culture, the 15 days old in vitro shoot cluster were transferred to 20mL of MS
suspension medium supplemented with (4.44 M) BAP and allowed to grow in orbital shaker with 45 RPM,
2000lux photo intensity under 252C. After 20 days, the shoots mass were subjected to the elicitated with various
concentration and combination as given in Table 1.
Table 1. List of Treatments
S.No. Salicylic acid (M) Aspergillus niger (mg/L)

T0 Control (4.44 M IBA)
T1 150 0
T2 0 0.5
T3 0 1.0
T4 0 1.5
T5 150 0.5
T6 150 1.0
T7 150 1.5
Elicitation period varies for biotic and abiotic elicitor. The biotic elicitor was added on the 20th day to the
shoot cultures grown on medium amended with 4.44 M and left in the medium till the date of harvesting whereas
the abiotic elicitor was added on the 20th day to the shoot culture and allowed for 2hrs in the medium amended with
4.44 M. Then the media was decanted and the shoots were again allowed to grown in liquid MS suspension media
fortified with 4.44 M and allowed to grow after 30 days they were harvested. The media without the addition of
elicitors is considered as control. After exposure period, control and treated tissues were harvested and preceded
for secondary metabolite extraction followed by HPTLC analysis. The experiment was done with three replications
and repeated thrice.
Extraction of Secondary Metabolites
One gram of dried in vitro shoot powder of Withania somnifera was taken and the evaluation of secondary
metabolites was carried out. Dried shoots were ground thoroughly using mortar and pestle and shoot powder was
obtained. Initially one gram of shoot powder was weighed and treated with 1 mL ammonia for 20 mins at room
temperature followed by sonication for 20 min with 50 mL of methanol and placed in a shaker for 2 hours at 150
283
rpm at 22 C. At the end of 2 hours the extract was filtered using Whatmann no.1 filter paper and the residue were
again treated with 50 mL methanol. This step was repeated four times. Finally, the extracts were pooled together
and concentrated by evaporation using flash evaporator maintained at 40C and 150 rpm. After complete evaporation,
the residue was dissolved using HPLC grade methanol.
Quantification of Major Withanolides
HPTLC was performed on precoated silica gel aluminum plate (20 cm X 20 cm) 60F254 (E.MERCK,
Germany). The methanolic extract of W. coagulans were loaded to the plates as 6 mm bands, under a stream of
nitrogen using the CAMAG (Switzerland) Linomat V semiautomatic sample applicator fitted with a 100 l of
Hamilton HPTLC syringe. The HPTLC plates were developed up to 80mm using the mobile phase Toluene: Ethyl
Acetate: Formic acid in the ratio of 5:5:1 respectively in a Camag Twin trough glass tank. It was pre saturated with
the mobile phase solvents for 30 minutes at room temperature (25 2 C). The developed plate was air dried and the
image was captured at 245 nm and 366 nm. Densitometric scanning was performed at 235 nm for withanolide A
and 530 nm for withaferin A using Camag TLC scanner III controlled by CAMAG CATS 4 integration software.
The Rf values of the resolved spots were noted. The peak areas were evaluated using linear regression12.
The amount of withaferin A and withanolide A in the samples were quantified using peak area. The plates
were derivatized using anisaldehyde sulphuric acid (85mL methanol: 10mL glacial acetic acid: 5mL sulphuric acid:
0.5mL anisaldehyde) reagent and placed in hot-air oven for 2 min at 110C for detection of spots.
Influence of elicitors on micro shoots, biomass and withanolide production in suspension culture of W. somnifera
Elicitors are compounds that stimulate plant cell secondary metabolism. Feeding of elicitors has been
proven to be an effective way to enhanced biomass and secondary metabolites production in plant cell cultures.
Different concentration and combination of biotic and abiotic elicitors were used to optimize the best elicitation for
shoot proliferation in suspension culture of W. somnifera. In present study, exogenous supplement of biotic and
abiotic elicitors is an important factor for micro shoots formation, biomass accumulation and withanolides production
in shoot suspension culture. The maximum number of micro shoots (31.000.57) and shoot biomass of 38.6g/L
was observed in MS basal medium supplemented with Salicylic acid (150M) and A. niger (1.0 v/v), 3% sucrose
and 4.44 M BAP in suspension (Fig 1and Plate 1).
Figure.1. Influence of elicitors on microshoots, biomass and withanolide production in

shoot suspension of W. somnifera
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Plate1.Effect of elicitors on shoot multiplication in liquid culture of W. somnifera
The liquid culture system were successfully using BA and TDZ in Phalaenopsis and Alocasia amazonica
13
and the maximum number of multiple shoots were achieved in half strength MS medium with 5.0 M BAP (35
3.25) in W. somnifera 14. SA (salicylic acid) showed an effect of biomass growth insignificantly related to the
control15.
The quantity of withanolide A and Withaferin A content of proliferated shoots in liquid medium varied
with culture treatments. Fig 1 represented the influence of different concentration of elicitors on withanolide
production in proliferated shoots harvested after 4th week of culture. 150M salicylic acid and 1.0 v/v with 2 hours
exposure time produced higher amount of withanolide A 186.340.33 g/DW (1.12 fold) and Withaferin A 1240.84
0.45 g/DW (2.26 fold) when compared to control.
According to the above report it was concluded, that shoots are a good source of withaferin A compared to
withanolide A, this finding was consistent with previous reports of withanolide production by shoot suspension
cultures of W. somnifera 16. The higher amount of Withaferin A and maximum number of micro shoots were reached
in MS liquid medium supplement with BAP and coconut water17. Influence of 50M Salicylic acid enhanced the
production of hypericin and pseudohypericin in H. hirsutum15 and increased furanocoumarin accumulation in shoot
cultures of R. graveleons after SA elicitation at 200 M18.
CONCLUSION
Enhanced production of withanolides in shoot culture of W. somnifera was directly influenced by different
concentration of biotic and abiotic elicitors and exposure time. SA (150 M) and 1.0v/v A.niger was the most
effective elicitor at enhancing the level of withanolides production (1.12 to 2.24-fold higher than control) on 4th
week of culture with 2 h exposure time in MS liquid medium supplemented with a combination of 4.44 M BAP.
This in vitro protocol could be provide unique opportunities for large scale propagation than field grown plants.
285
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286
Antimycobacterial Activity of Tylophora Indica Against Isolates
from Female Infertility Cases
Anchana Devi .C1
1
PG & Research Department of Biotechnology, Womens Christian College, Chennai.
Email : dr.anchanababu@gmail.com
Abstract
Challenges to fertilityarise from genetic abnormalities, infectious or environmental agents, delayed child bearing,
behavior, and certain diseases. Many microorganisms seem to be involved in female reproductive failure in
various ways. However, it seems to have a greater impact on female fertility. Genital tract tuberculosis is an
extremely indolent infection, it tends to manifest as menstrual irregularities, infertility, and chronic pelvic or
lower abdominal pain, and is always acquired by hematogenous spread from a pulmonary source to extra
pulmonary source. Nearly 350 Indian medicinal plant species have been reported to be used in different Ayurvedic
formulations for tuberculosis. Tylophora indica has been used traditionallyfor treatment of jaundice, bronchitis,
asthma and tuberculosis. Apart from the antituberculosis activities, Tylophora indica plant extracts have also
demonstrated immunomodulatory effects on different cell cultures and in experimental animals. Petroleum
ether, Diethyl ether and ethyl acetate extracts of Tylophora indica was used for the study, the extracts were
purified using column chromatography and standardized using preparative thin layer chromatography and different
purified fractions were obtained and subjected to Antimycobacterial studies using Rapid Versa trek culture
system- 528 model. The bioactive compound from fraction expressing antimycobacterial activity was analysed
using GC MS and it was identified as 1, 2-Benzenedicarboxylic acid, diisooctyl ester.
Keywords: Antituberculosis activity, Tylophora indica, 1, 2-Benzenedicarboxylic acid, diisooctyl ester
INTRODUCTION
The problem of infertility in India has to be interpreted in a context of poverty, class and gender inequality
and unequal access to health-care resources (Widge, 2002). The causes of infertility arise from genetic abnormalities,
infectious or environmental agents. Of all the couples whom classified as infertile, female infertility accounts for
about 4050%. Some conditions, however, seem to have a greater impact on female fertility. Vaginal infections are
of doubtful impact and cases of endometritis, leading to uterine synechiae, are less common than tubal occlusions
resulting from salpingitis. Tuberculosis is a persistent and stubborn disease that might have been present on this
planet since life began. WHO statistics indicates it kills two million people each year (World Health Organization,
2002) and around eight million new cases of tuberculosis are detected each year. Tuberculosis is also known to be
a major health hazard and genital tuberculosis forms an under diagnosed part of it and is still responsible for a
significant proportion of female infertility. Genital tuberculosis has been steadily declining in developed countries
(Jahromi et al., 2001) While 510% of infertile females all over the world have genital tuberculosis, the incidence
varies from less than 1% in USA to nearly 13% in India (Jones et al., 1988).
A considerable number of plant species have been mentioned in Ayurveda for the treatment of tuberculosis
and related disorders. In Ayurvedic Formulary of India (Anon, 2003), Over 350 plant species used in traditional
medicines have been assessed for their antituberculosis activities worldwide (Eldeen and Van Staden, 2007). Apart
from the antituberculosis activities, plant extracts have also demonstrated immunomodulatory effects on different
cell cultures and in experimental animals (Eloff, 2001).
287
The perennial plant Tylophora indica (Burm.F) Merill, belonf to the family Asclepiadaceae.. There are nearly 35
species widely distributed among the Asian continent.Commercially important species include T. indica, T.fasciculata,
T. rotundifolia, T.apiculata etc., The plant has been reported to have a alkaloids, sterols , flavonoids , wax, tannins
and resins. The leaves and the root of T.indica has been used for traditional preparation of medicines to treat
Asthma, Bronchitis, Whooping cough and other common respiratory problems. Apart form this, there are reports
of T.indica possessing Antiinflammatory activity, Antioxidant activity, Antimicrobial activity , cytotoxic effect
and diuretic activity.
MATERIALS & METHODS
Collection of Sample
The leaf samples were collected from places in and around Chennai and authenticated and ensured that the
plant was healthy and uninfected. The leaves were washed with running tap water and distilled water to clean the
leaves thoroughly to free from dust particles and dried under shade until complete moisture is withdrawn.
Solvent Extraction
The dried leaves were finely powdered and mixed with 1 : 10 ratio with petroleum ether , diethyl ether and
ethyl acetate separately. The extractions were obtained by filtering using Whattman No.1 filter paper . Then the
filtrates were dried and concentrated and stored at 4 C for further studies.
Thin Layer Chromatography
Each solvent extract was subjected to thin layer chromatography (TLC) using silica gel 60F254, 7x6 cm
(MERCK) readymade plates. The plates were cut with in to different dimensions depending on the number of spots
to be analyzed. Glass capillaries were used to spot the samples in TLC. The mobile solvent phase were Petroleum
ether : chloroform (9:1 ) , Benzene : ethylacetate ( 6.5:3.5) , Diethyl ether : ethylacetate (7:3). After presaturation
with the mobile phase for 15min, the plates were dried and viewed under UV (254-366), and sprayed with 1%
vanilin in concentrated sulphuric acid.
Purification and separation of bioactive compounds using column Chromatography

Different fractions of purified bioactive compounds were obtained by column chromatography. Column of
silica gel (60-120) mesh was used to pack the column. The dried extracts obtained were used as samples. The same
mobile phase used in TLC was also used for column chromatography . The purity of the separated bioactive
compounds were tested using TLC.
Preparation of sample for Antimycobacterial studies
The endometrium was taken in a sterile test tube to which 4% NaOH was added just to cover the whole
tissue material. The test tube was vortexed for 5 minutes and incubated for 30minutes at 37C. Intermittently the
tube was vortexed every 10 minutes. Then the sample was centrifuged at 3000rpm for 20minutes and supernatant
solution was discarded safely. To the cell pellet, sterile distilled water was added for pH neutralization.
Mycobacterial inoculum preparation
The colonies of Mycobacterium tuberculosis from Lowenstein Jensen Mediumwere suspended in 10ml of
sterile normal saline. Vortex the suspension with sterile glass beads and allowed 30minutes for larger particles
settled down. The upper solution was removed without disturbing the settled particles. The solution was adjusted
with sterile normal saline, to a turbidity matching that of a 1.0Mc Farland Standard. The standard was further
diluted in sterile normal saline of 1:10 ratio.
288
Mycobacterial susceptibility assay
The following concentrations of Tylophora indica purified extracts (100mg, 200mg, 300mg, 400mg, 500mg)
were used for the study.1 ml of growth supplement and 500ml of standard inoculums were aseptically injected into
Versa Trek mycobottle. Like vise all the variable concentrations were inoculated and labeled. The controlled
mycobottle was also prepared with the addition of 500ml sterile normal saline in place of antimicrobial substance.
Then the mycobottles were fitted with the connector and loaded into the Versa-Trek rapid culture system with
appropriate labeling identification in the touch screen inbuilt computer monitor.
Analysis of bioactive compounds in plants by GC MS
The GC MS analysis was carried out only for the fractions which showed antimycobacterial activity using
a Clarus 500 Perkin Elmer (Auto system XL ) Gas Chromatograph equipped and coupled to a mass detector .
Turbo mas gold Perkin Elmer Turbomass 5.1 spectrometer with and Elite -1 (100% Dimethyl poly siloxane), 30m
X 0.25mm ID x 1mic.m df capillary column.
RESULTS & DISCUSSION

Collection of sample
Fig 1 :Tylophora indica

The plant sample was collected from authenticated sources , leaves were shade dried , powered and stored
in separate air tight containers.
Solvent Extraction
Fig 2a. Filteration of extract Fig 2b. Collection of crude extract
The plant sample was cold extracted using Diethyl ether , Ethylacetate and Petroleum ether. The solute
solvent were mixed in the ratio of 1: 10 and left over night and filtered using Whattman No.1 filter paper after
24hrs. The extracts were collected separately and the dry weight of the crude extracts were obtained.
Purification of the crude extracts
The crude extracts were subjected to Column chromatography for separation of fraction containing active
compounds. Column was prepared using silica gel 60-120 mesh. Nearly 34 fractions were obtained from different
solvent extracts and number of compounds in the fractions were identified using Thin layer chromatography. The
column was repeated until single compound was obtained in a fraction and affirmation was done using TLC.
289
Fig 3a.: Identification & Isolation of bioactive compounds from T.indica
Antimycobacterial Susceptibility Assay using Versa Trek Culture System
The organisms Mycobacterium tuberculosis and MOTT (Mycobacterium other than tuberculosis were isolated
from endometrial samples using LJ medium. MOTT appeared as glossy colonies where as Mycobacterium
tuberculosis appeared as powdery colonies. (Fig 4a, 4b). Ethical committee permission no. DM/2011/101/6 for
collection of endometrial samples.
Fig 4a. Mycobacterium tuberculosisFig 4b : MOTT in LJ medium
Fig 5 : Versa trek Culture system with Mycobottles
The Versa Trek system mycobottles contain modified Middle brook 7H9 broth with cellulose sponges.
Then 0.5ml of antibiotic supplement of PVNA (polymyxin B, vancomycin, Nalidixic acid) was injectedthrough the
septum using syringe. Finally 0.5ml of decontaminated specimen was aseptically injected using a syringe. The
culture bottle and stopper was immediately disinfected with sterile solution. The antibiotic supplement eliminates
the growth of other bacteria, the modified medium enriches the growth of Mycobacterium sp
290
Mycobacterial susceptibility test by Versa Trek Culture System
DATE: 22/2/2016
Purified extract Day of identification growth Status
Petroleum ether Early growth (10.2 hrs)

Susceptible
fractions Compound harbors with bacteria
Diethyl ether Early growth (10.2 hrs)

Susceptible
fractions Compound harbors with bacteria
Ethyl acetate
No growth upto 6 days Resistant
fractions
Table 1: Antimycobacterial susceptibility assay using Versa Trek culture system
3.5 Analysis of bioactive compounds in plant leaf sample by Gas Chromatography Mass Spectroscopy
(GC-MS)
The bioactive compounds in the selected plant extract were analysed by GC-MS spectra. Tylophora indica
showed the presence of five peaks viz, 14.38 corresponds to 3,7,11,15 Tetramethyl 2 hexadecen -1 -01, 16.17
corresponds to n-hexadecanoic acid , 18.37 corresponds to phytol, 24.56 corresponds to 1,2 benzene dicarboxylic
acid diisoocytyl ester and 25.01 corresponds to 5 (7a Isopropenyl -4,5 dimethyl octahydroinden -4 yl -3 -
methyl pent -2-3n-1-01) respectively.
Fig. 6 Analysis of Bioactive compounds in Tylophora indica plant leaf extract by GC- MS
DISCUSSION
The urgent need for the development of new drugs to help reduce the global burden of tuberculosis (TB) is
well documented in the current biomedical literature (Andries et al., 2005; Barry et al. (2000), Tripathi et al.
(2005). The leaves of the plant Tylophora indica or Tylophora asthmatica was used in Ayurveda, the traditional
Indian system of medicine, to treat various allergic and inflammatory disorders like bronchial asthma, rhinitis,
whooping cough etc., (Chopra et al., 1958). Various extracts of the leaves of Tylophora indica were found to have
antiasthmatic and antiallergic properties (Shivpuri et al., 1968; 1969 and 1972). In the present study, ethyl acetate
extract of the selected plant leaf of Tylophora indica was found to have inhibitory activity against Mycobacterium
sp. The solvent extracts of the plant was prepared using petroleum ether, diethyl ether and are found to be susceptible
to Mycobacterium tuberculosis by Versa Trek Rapid culture system. Sparg. (2004) studied on the synergistic effect
291
of the plant extract against several potential pathogens. The plant materials possessed antimicrobial activity with
greater efficiency when used synergistically on the test organisms especially in MDR strain. Tylophora indica
showed the presence of five peaks among which the retention time was 24.56 with peak area of approximately
88.01% corresponds to 1,2 benzene dicarboxylic acid diisoocytyl ester . Penecilla, G.L et al.,2011 reported the
antimicrobial activity of 1,2 benzene dicarboxylic acid diisoocytyl ester, which was also reflected in the present
study.
CONCLUSION
The rationale behind studying the extraction and isolation of the bioactive constituents present plants,
possess medicinal properties and are of therapeutic value. These compounds aids in synthesizing and isolating
compounds that are potent analogs, thus fueling the discovery and use of new and cheap drugs in therapy. The
bioactive compound 1,2 benzene dicarboxylic acid diisoocytyl ester from Tylophora indica, a potent candidate
to be explored further to develop as a antimycobacterial agent
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1. Widge A (2002). Sociocultural attitudes towards infertility and assisted reproduction in India. In: Vayena
E, Rowe PJ, Griffin PD (eds). . Current Practices and Controversies in Assisted Reproduction. Geneva,
Switzerland World Health Organization: 6074.
2. WHO (August 2002 ). Fact sheet No. 104.
3. Jahromi BN, Parsanezhad S, Shilazi RG (2001). Female Genital tuberculosis and infertility.Int J Gynaecol
Obstet 75(3): 269272.
4. Jones HW, Went AC, Burnett LS (1988). Novaks textbook of gynecology. Williams & Wilkins, Baltimore:
557569.
5. Anon. (2003). Ayurvedic Formulary of India ,Part I. . Department of ISM & H, Ministry of Health and
Family Welfare, Govt. of India. .
6. Eldeen I.M.S. and J. van Staden (2007). Antimycobacterial activity of some trees used in South African
traditional medicine. South African Journal of Botany 73: 248251.
7. Eloff, J. N. (2001). Antibacterial activity of Marula (Sclerocaryabirrea (A. rich) Hochst. Subsp. Caffra
(Sond Kokwaro) (Anacardiaceae) bark and leaves. Journal of Ethnopharmacology 76: 305308.
8. Gilchrist, M. (1991). Biosafety Precautionjs for AirbonePathogens.In: Flemng, D.O., J.H. Richardson,
J.J.Rylis, D. Vesely eds., . Laboratory Safety: Principles and Practices 2nd edition. Washinton, D.C. American
Society for Microbiology Press, 4: 207 - 241.
9. Penecilla, G.L., Magno, C.P.,(2011). Antibacterial activity of extracts of twelve common medicinal plants
from phillipines .J.med.plants research, 5(16),:3975-3981
10. Andries K, Verhasselt P, Guillemont J (2005). A diarylquinoline drug active on the ATP-synthase of
Mycobacterium tuberculosis. Science 307: 223 - 227.
11. Barry C.E., Slayden R.A., Sampson A.E., Lee R.E. (2000). Use of genomics and combinatorial chemistry
in the development of new antimycobacterial drugs.BiochemPharmacol 59: 221 - 231.
12. Tripathi, RP., N. Tewari, Dwivedi, N., Tiwari, VK. (2005). Fig hting tuberculosis: an old disease with new
challenges.Medicinal Research Reviews 25: 93 - 131.
13. Chopra, RN., Chopra, IC., Handa, KL., Kapur, LD: (1958). Tylophora asthmatica. In: Indigenous drugs
of India, 2nd Ed. Published by Dhur, Calcutta 431.
292
14. Shivpuri DN., Menon MPS.,Prakash D (1969). Crossover double-blind study in leaves of Tylophora indica
in the treatment of asthma and allergic rhinitis. J. Allergy 43(3): 145 - 150.
15. Shivpuri DN., Singhal SC., Prakash, D.: (1968). Preliminary studies in Tylophora indica in the treatment of
asthma and allergic rhinitis. J. Ass. Phys. India 15(1): 9 - 15.
16. Shivpuri DN., Singhal SC., Prakash, D.: (1972). Treatment of asthma with an alcoholic extract of Tylophora
indica a cross over double-blind study. . Ann. Allergy 30(7): 407 - 409
17. Gkaravela*, A. Foka, M. Sevdali, F. Kolonitsiou, A. Spiliopoulou, E.D. Anastassiou, I. Spiliopoulou. (2011).
Mycobacterial infections (including diagnosis)Diagnosis of tuberculosis and susceptibility testing by
conventional and molecular methods in Southwestern Greece L. 21st European Congress of Clinical
Microbiology and Infectious Diseases (ECCMID) 27th International Congress of Chemotherapy (ICC).
18. S. G. Sparg and M. E. Light and J. van Staden (2004). Biological activities and distribution of plant saponins.
Journal of Ethnopharmacology 94(2-3).
293
Antimicrobial activity of isolated compounds from
Hybanthus enneaspermus .L
P. Muneeswari, S. Deepika and K. Poornima
Department of Biochemistry & Bioinformatics, Karpagam University, Coimbatore, Tamil Nadu, India 641 021
Abstract
Medicinal plants are used by 80% of the world population for their basic health needs. The relationship between
human, plants and drugs derived from plants describe the history of mankind. Plants are the important source of
natural drugs. Secondary metabolites have been used for centuries in traditional medicine. Nowadays, they
correspond to valuable compounds such as pharmaceutics, cosmetics, fine chemicals and nutraceutics. The
present study is aimed to evaluate the antimicrobial activity of isolated compounds from Hybanthus enneaspermus
L. Two compounds were isolated by using column and thin layer chromatography and their structural elucidation
was done by using NMR spectroscopy. The anti-microbial activity of the isolated compounds were analyzed
against five different bacteria and three different fungus. From the results of the present study, Compound I and
II possesses good antibacterial activity compared to standard drug ampicillin at the concentration of 50mg/ml.
The maximum zone of inhibition of the compound I and II were found to be 13mm and 15mm respectively and
also it possesses very good antifungal activity against Aspergillus terrus. In future Compound I and II can be
effectively used for the treatment of bacterial and fungal infection. Future trials may be carried out for further
clarification.
Key words: Hybanthus enneaspermus .L, Hybanthus enneaspermus L, Secondary metabolites, Compound
isolation, Antimicrobial activity.
INTRODUCTION
Medicinal plants are considered as rich resources of ingredients which can be used in drug development
and synthesis. Besides that these plants play a critical role in the development of human cultures 1. Medicinal
plants are great importance to the health of Individuals and communities. An antimicrobial is an agent that kills
microorganisms or inhibits their growth. Antimicrobial medicines can be grouped according to the microorganisms
they act primarily against. For example, antibiotics are used against bacteria and antifungal are used against 2. They
can also be classified according to their function. Agents that kill microbes are called microbicidal, while those that
merely inhibit their growth are called biostatic 3. The use of antimicrobial medicines to treat infection is known as
antimicrobial chemotherapy, while the use of antimicrobial medicines to prevent infection is known as antimicrobial
prophylaxis 5 . Infectious diseases are one of the leading causes of morbidity and mortality worldwide, especially
in developing countries 6.
Hybanthus is from the Greek hypos (humpbacked) and anthos (flower), referring to the spurred anterior
petal; enneasperus is from the Greek ennea (nine) and spermus (seed), referring to the capsule which contains
about nine seeds 7. Hybanthus enneaspermus (L). F. Mull, belonging to family Violaceae, is a rare, perennial herb
found in some of the warmer parts of the Deccan peninsula in India. Popularly called Ratan Purus and in Tamil it is
Orithaztamarai. By the local villagers and herbalists, this ethanobotanical herb is known to have unique medicinal
properties. It grows up to 15-30cm in height with many diffuse or ascending branches and is pubescent in nature.
294
Plant collection
The whole plant powder of Hybanthus enneaspermus was purchased from Ayurveda pharmascy at pollachi.
The powdered material is used for the study. The plant specimen is authenticated by Botanical Survey of India,
Coimbatore (No: BSI/SRC/5/23/10-11/Tech.

1kg of the powdered whole plant material of Hybanthus enneaspermus was mixed with 5Litre of ethanol
and kept at room temperature for 72 hrs with occasional shaking.After 72hrs filtered and the filterate was air dried
and stored in refergerator for further use.
Column chromatography
The individual fractions collected were subjected to thin layer chromatography to separate identify the
individual components following the method 8. Thin layer chromatograms was then subject to identify the components
Ultraviolet- visble spectroscopy

To visible and ajacent (near-UV and near- infrared ranges to identify the components. In this region of the
electromagnetic spectrum, molecules undergo electronic transitions.
Analysis of the Functional Groups by Fourier Transform Infrared (FTIR) Spectroscopy

The infrared spectrum originates from the vibrational motion of the molecule. The vibrational frequencies
are a kind of fingerprint of the compounds. This property is used for characterization of organic, inorganic and
biological compounds. The band intensities are proportional to the compound and hence quantitative estimations
are possible.
Identifications of the structure of the active constituents using nuclear magnetic resonance (NMR) spectroscopy
NMR spectroscopy was used to determine the molecular structure based on the chemical environment of
the magnetic nuclei like 1H,13C,NMR,etc.,even at low concentrations. This is one of the most powerful non
destructive techniques in elucidating the molecular structure of biological and chemical compounds. A fraction the
colleted fractions was also subject to NMR spectroscopy to characterize the individual components.
P REPARATION OF PLANT EXTRACTS OF VARIOUS CONCENTRATIONS FOR ANTIMICROBIAL

SUSCEPTIBILITYTESTS
Different concentration (5 mg, 10 mg, 15 mg, and 20 mg) of the plant extracts were made by diluting in
Dimethyl sulpoxide (DMSO).
Microorganisms
Different strains of bacteria and fungi were obtained from Microbiology lab of Karpagam University,
Coimbatore. Strains used are Staphylococcus aureas Streptococcus aureas, E.Coli, Pseudomonas aureas, Klebsilla,
A.terrus, A.niger and A.flavus.
Culture Media
The bacterial and fungal strains were sub cultured in nutrient broth and Potato dextrose agar medium
respectively. The bacterial sub cultured should be 12 hours where as the fungal subculture should be 72 hours.
295
Composition
Nutrient Agar Medium (100 ml) for Bacterial Assay
Peptone : 0.5 g
Beef extract : 0.3 g
Yeast extract : 0.2 g
Sodium chloride : 0.5 g
Agar : 2-3 g
Distilled Water : 100 ml
Potato Dextrose Agar Medium (100 ml) for fungal Assay
Potato : 2g
Dextrose : 2g
Agar : 2g
Antimicrobial assay
Antimicrobial activity test was carried out following the modification of the method originally described
by (Groove and Randall, 1955).
Preparation of culture medium and Inoculation
The petriplate and the media (Nutrient agar medium and PDA medium) were sterilized for 20 minutes at
0
120 C. The rest of the procedure was carried out in Laminar Air flow. Approximately 20 ml of the media was
poured in to the sterile petriplate and allowed to get solidify for 15-20 minutes. After the media gels solidified,
swabbed using cotton.
COMPOUND I
The result of the present study Antioxidants activity of isolated compounds from the plant extract of
Hybanthus Enneaspermus L. is discussed under the following headings. Isolation of the active compounds using
Column chromatography and TLC. Characterization of active compounds using UV-Vis Spectroscopy and FTIR
Identification and structural elucidation of isolated bioactive compounds using13C NMR,1H, 13C NMR and 2D
NMR. The ethanolic plant extract of Hybanthus Enneaspermus L. was subjected to column chromatography by
using the solvents Petroleum ether, ethyl acetate Chloroform and Methanol in the increasing order of polarity and
fractions were collected. A total of 305 fractions was collected in test tube. The isolated fractions were spotted in
TLC plate coated with silica gel. The solvent system used for running is Chloroform: Methanol in the ratio 4:6 and
3:7. The spots were identified by placing in iodine chamber.The fractions (fraction 231-235 to 245-252) collected
from column chromatography showed single spotmas mentioned earlier solvent system in iodine chamber. So these
two were used for further studies such as UV-Visible spectroscopy , FTIR analysis and NMR to determine the
absorption spectra ,the functional groups of the compounds that is present in the defatted ethanolic extract of
Hybanthus Enneaspermus L.
296
Fig.1 compound I Fig.1 compound II
Antibacterial activity of isolated compound from ethanolic extract of Hybanthus enneaspermus L.

different species of bacteria (Compound 1)
Table 1
Tested Amphicilin(Posi DMSO(Neg 10mg/ml 20mg/ml 30mg/ 40mg/ml 50mg/1

organisms tive control) ative ml ml
(10mg/ml) control)
Staphylococcus 14 mm Resistance Resistance 7mm 9mm 10mm 11mm
aureas
Streptococcus 12mm Resistance Resistance Resistance 6mm 7mm 9mm
aureas
E. coli 14mm Resistance Resistance Resistance 6mm 7mm 9mm
Pseudomonas 14mm Resistance Resistance Resistance 6mm 8mm 9mm
aureas
Klebsilla 15mm Resistance Resistance 6mm 9mm 10mm 13mm
(+) Positive control Ampicilin (-) Negative control DMSO
The isolated compound 1 showed be very good antibacterial activity against (Staphylococcus aureas,
Streptococcus aureas, E. coli, Pseudomonas aureas, Klebsilla) at 50mg/ml concentration. At 10mg/ml concentration
these bacterial species does not show any zone of inhibition. It shows if activity from 20mg/ml concentration from
20mg/ml concentration. The maximum zone of inhibition is showed at 50mg/ml concentration against all the
(Staphylococcus aureas, Streptococcus aureas, E. coli, Pseudomonas aureas, Klebsilla) pathogens. The zone of
inhibition at 50mg/ml concentration against (Staphylococcus aureas, Streptococcus aureas, E. coli, Pseudomonas
aureas, Klebsilla) was found to be 11mm, 9mm, 9mm, 9mm & 13mm respectively. The compound showed a very
good antibacterial activity against Staphylococcus aureas and Klebsilla species. Ampicilin was used as the positive
control and DMSO was used as the negative control. The isolated compounds have shown better antibacterial
activity as that of standard drug. So the compound may be used as antibiotic against.
297
Fig 3 Antibacterial activity of isolated compound from ethanolic extract of Hybanthus enneaspermus L.
Table 2
Tested Amphicilin DMSO 10mg/ml 20mg/ml 30mg/ml 40mg/ml 50mg/
organisms (Positive (Negative ml
control) control)
(10mg/ml)
Staphylococcus 10mm Resistance Resistance Resistance 6mm 9mm 11mm
Streptococcus 10mm Resistance Resistance Resistance Resistance Resistance 7mm

aureas
E. coli 13mm Resistance Resistance Resistance 9mm 13mm 15mm
Pseudomonas 10mm Resistance 5mm 7mm 9mm 10mm 11mm

aureas
Klebsilla 15mm Resistance Resistance 5mm 8mm 13mm 15mm
Fig 4 Antibacterial activity of isolated compound from ethanolic extract of Hybanthus enneaspermus L.
298
ANTIFUNGAL ACTIVITY OF ISOLATED COMPOUNDS FROM ETHANOLIC EXTRACT OF Hybanthus
enneaspermus L.
Antifungal activity of isolated compounds 1 and 2 from ethanolic extracts of Hybanthus enneaspermus L.
was carried out with the concentration range of 10- 50mg/ml was used for this study. The percentage of growth
inhibition was determined after 48 hrs and 72 hrs. Chloramphenicol was used as positive control. Unfortunately the
compound 1 and 2 did not show antifungal activity against (Aspergillus flovus and Aspergillus niger). But the
isolated compounds 1 and 2 from ethanolic extract of Hybanthus enneaspermus L. possess very good antifungal
activity against aspergillus terrus.
Antifungal activity of isolated compounds from ethanolic extract of Hybanthus enneaspermus L. fungi
(Compound 1)
Antibiotic 40 50
Tested Chloramphenicol DMSO 10 20 30 mg/ml mg/ml
organisms mg/ml mg/ml mg/ml mg/ml
(10mg/ml)
A.terrus 15mm Resistance 10mm 12mm 12mm 13mm 14mm
A.niger N/A N/A N/A N/A N/A N/A N/A
A.flavus N/A N/A N/A N/A N/A N/A N/A
A.terrus
Fig 5 : Antifungal activity of isolated compounds from ethanolic extract of

Hybanthus enneaspermus L. fungi (Compound.1)
Table 4
40 50
Antibiotic mg/ml mg/ml
Tested Chloramphenic DMSO 10 20 30
organisms ol(10mg/ml) mg/ml mg/ml mg/ml mg/ml
A.terrus 14 mm Resistance 10mm 10mm 12mm 13mm 13mm

A.niger N/A N/A N/A N/A N/A N/A N/A
A.flavus N/A N/A N/A N/A N/A N/A N/A
(+)Positive control Chloramphenicol (-) Negative control DMSO

From the results, it is found that both the compounds are having very good antifungal activity against
A.terrus. So these compounds can be used against opportunistic infection caused by A.terrus.
A.terrus
Fig 6 : Antifungal activity of isolated compounds from ethanolic extracts of Hybanthus enneaspermus L.
against pathogenic fungus (COMPOUND.2)
299
Mean measurement zone of inhibition (mm), N/A- No activity or no zone of inhibition isolated compounds
from ethanolic extracts of Hybanthus enneaspermus L., Disc size 6 mm in diameter
CONCLUSION
From the present study, it can be concluded that the isolated compounds from ethanolic extract of Hybanthus
enneaspermus L. is tested against five different bacterial organisms (Staphylococcus aureas, Streptococcus aureas,
E. coli, Pseudo aureas and Klebsilla). They should good antibacterial activity compared to standard drug ampicillin
at the concentration of 50mg/ml. The maximum zone of inhibition of the compound 1 and 2 were found to be 13mm
and 15mm respectively. Isolated compounds from ethanolic extract of Hybanthus enneaspermus L. was tested
against three different fungus (A.terrus, A.niger, A. flavus). But the isolated compound 1 and 2 from ethanolic
extract of Hybanthus enneaspermus L. possesses very good antifungal activity against aspergillus terrus. So these
two compounds can be effectively used for the infections and inflammations caused by antibacterial and fungus.
Future trials may be carried out for further clarification.
REFERENCE
1. Aiyelaagbe OO, Adeniyi BA, Fatunsin OF, Arimah BD (2007). In vitro Antimicrobial activity and
photochemical analysis of Jatropha curcas roots Intern. J. Pharmacol. 3(1): 106-110.
2. Aiyelaagbe OO, Adesogan EK, Ekunday O, Adeniyi BA (2000). The antimicrobial activity of roots of Jateopha
podagrica Hook. Phytother. 14:60-62.
3. Akinpelu D.A. and Onakoya Z.T.M. (2006). Antimicrobial activities of medicinal plants used in folklore
remedies in South-Western, Afr. J. Biotechnology. 5:1078-1081.
4. Araiza, J., Canseco, P. & Bonifaz, A.Otomycosis (2006). Clinical and mycological study of 97 cases. Rev
Laryngol Otol Rhinol (Bord). 127:251254.
5. Barbara et al., Entophytic fungi: (2002). A source of novel biologically active secondary metabolites. 106
(9):996-1004
6. Castello MC, Phatak A, Chandra N, Sharon M. (2002). Antimicrobial activity of crude extracts from plant
parts and corresponding calli of Bixa Orellana L. Indian J. Exp. Biol. 1378-1381.
7. Coates A, Hu Y, Bax R, Page C. (2002). The future challenges facing the development of new antimicrobial
drugs. . Nat. Rev. Drug Discov. 1:895-910.
8. Sadasivam, S. and A. Manickam.( 1996) . Biochemical method. New Age International (P)
300
Alteration in biochemical profiles by Macrotyloma uniflorum (Lam.)
Verdc. leaves in streptozotocin-nicotinamide induced diabetic rats
Suriyamoorthy Priyanga1, Subrahmanian Hemmalakshmi1 and Kanakasabapathi Devaki1,2*
D1Department of Biochemistry, Karpagam University, Coimbatore 641 021
2
Department of Bioinformatics, Karpagam University, Coimbatore 641 021
Abstract
Diabetes is one of the most familiar metabolic disorders and is interconnected to oxidative stress. It is characterized
by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects
in insulin secretion, insulin action or both. In view of the role of M. uniflorum in control and prevention of
diabetes, this study was carried out. The animals were divided into five groups of six animals each. Group I
served as control; Group II served as streptozotocin-induced diabetic rats (Streptozotocin 45mg/kg b.w-
administered as a split dose of 20 mg/kg b.w and 25 mg/kg b.w with a two day break between the doses); Group
III served as streptozotocin-induced diabetic rats treated with M. uniflorum (200 mg/kg b.wt.); Group IV served
as streptozotocin-induced diabetic rats treated with standard drug glibenclamide (2 mg/kg b.wt) and Group V
were normal rats treated with ethanolic extract of M. uniflorum (200 mg/kg b.wt.). After the experimental
period of 45 days, the animals were sacrificed by cervical dislocation under mild chloroform anesthesia. Blood
was collected by decapitation and serum was separated by centrifugation and used for the estimation of
biochemical parameters (Glucose, protein, Hb, HbA1c, urea, uric acid, creatinine, SGOT and SGPT).
Streptozotocin induced diabetic rats restores the altered levels of various biochemical profiles upon treatment
with ethanolic extract of Macrotyloma uniflorum (200 mg/kg b.wt.) which proves the anti diabetic potential of
the plant.
Keywords: M. uniflorum, Antidiabetic, Streptozotocin, Glibenclamide
INTRODUCTION
Diabetes is a metabolic disorder characterized by increased fasting and postprandial blood sugar levels.
The prevalence of diabetes is likely to be increased by 35%1. Development of an adverse event is one of the
complications in the treatment of any systemic disorder; hence, countless of the research institutes and pharmaceutical
companies are implicated in drug development to find innovative medicines for a broad range of diseases with
good therapeutic potential and less adverse events2.
Macrotyloma uniflorum, commonly known as horse gram (Fabaceae) is herbaceous plant with annual
branches, sub erect or twining, leaflets 2.5-5 cm and widely distributed throughout Asia, Africa and Australia. It is
famous for its medicinal uses because different parts of the plants are used for the treatment of heart problems,
asthma, bronchitis, leucoderma and for treatment of kidney stones. Literature survey showed that Dolichin A and
B, pyroglutaminyl glutamine along with some flavonoids were isolated from this plant3. The present study was
carried out to assess the protective effects of the ethanoic extract of Macrotyloma uniflorum L. on impaired glucose
level and other biochemical profiles induced by streptozotocin.

Collection of plant material
The plant specimens for the proposed study were collected from Kothavadi village, Coimbatore district,
Tamil Nadu, India. The plant was taxonomically authenticated by Dr. G.V.S Moorthy, Botanical Survey of India,
TNAU campus Coimbatore, with the voucher number BSI/SRC/5/23/2013-14/Tech/1309. The leaves were washed
301
with running tap water and shade dried at room temperature for 10 days in the absence of sunlight and coarsely
powdered using a mixer grinder. Then they were weighed and kept in an airtight container.
Sample extraction
50g of dried plant powder was weighed and extracted in 250 ml of ethanol in an orbitory shaker for 72hrs.
Repeated extraction was done with the same solvent till clear colorless solvent is obtained. Obtained extract was
evaporated to dryness by using a rotary vacuum evaporator at 40-50C and stored at 0-4C in an air tight container
for further use.
Preparation of animals
Adult female albino rats weighing about 150-180 g were obtained from the animal house of Karpagam
University, Coimbatore and were used for the study. Rats were housed in polycarbonate cages in a room with a 12
hrs day-night cycle, at constant temperature of 22C and humidity of 45-64%. During the experimental study rats
were fed on pellets (Gulmohur Rat Feed, Lipton India, Bengaluru) with free access to tap water. The rats received
humane care according to the criteria outlined in Principles of Laboratory Animal Care, 1985. The study was
approved by Institutional Animal Ethics Committee (IAEC) and the experiments were conducted according to the
ethical norms and IAEC guidelines. Experimental design
Nicotinamide was administered intraperitoneally in the concentration of 110mg/kg body weight of rats 15
minutes prior to streptozotocin induction.
Table 1: Experimental design of animal groups
Groups Experimental setup
Group I Control, normal healthy rats
Group II Diabetic control (Streptozotocin 45mg/kg b.w- administered as a split dose of 20 mg/kg
b.w and 25 mg/kg b.w with a two day break between the doses)
Group III Diabetic rats treated with M. uniflorum (200 mg/kg b.w)
Group IV Diabetic rats treated with Glibenclamide (2 mg/kg b.w)
Group V Normal healthy rats treated with M. uniflorum (200 mg/kg b.w)
After the experimental period of 45 days, the animals were sacrificed by cervical dislocation under mild
chloroform anesthesia. Blood was collected for serum separation. The blood was then centrifuged at 3000 rpm for
20 mins to separate the serum which were used for biochemical analysis.
In diabetic condition, elevated blood glucose, reduction in body weight, polyuria, polydipsia and polyphagia
are commonly observed. In the present study, induction of diabetes by alloxan produced increase in blood glucose
level, decrease in body weight and polyuria. In diabetic rats, observed reduction in body weight was possible due to
catabolism of fats and protein4.
Figure 1 shows a significant (p< 0.05) increase in glucose observed in diabetic rats after induction with
streptozotocin, when compared with control rats. On oral administration of the ethanolic extract of M. uniflorum
for 45 days, there was a significant decrease in blood glucose after the treatment with plant extract and standard
drug with highest percentage.
302
Figure 1-Effect of ethanolic extract of Macrotyloma uniflorum on glucose. Values are expressed as Mean SD for
six animals. Values not sharing common superscript letters (a-c) differ significantly at p< 0.05 (DMRT)
Figure 2 represents the effect of M. uniflorum on serum that the diabetic rats had decreased levels of serum
protein when compared with normal control rats. After treatment with M. uniflorum extract and glibenclamide the
protein contents were brought back to near normal levels and there is no significant difference between the control
and plant alone group.
Figure 2- Effect of ethanolic extract of M. uniflorum on protein in serum of control and experimental rats. Values
are expressed as Mean SD for six animals. Values not sharing common superscript letters (a-b) differ significantly
at p< 0.05 (DMRT)
STZ induced diabetic rats account for the observed decrease in the total protein content. Increased urea
production in diabetes might be due to enhanced catabolism of protein in liver and other tissues 5.
Figure 3- Effect of ethanolic extract of M. uniflorum on the hemoglobin level of control and experimental rats.
Values are expressed as Mean SD for six animals. Values not sharing common superscript letters (a-d) differ
significantly at p< 0.05 (DMRT)
303
In DM, the hemoglobin level is decreased, because the surplus glucose produced in the body reacts with
the hemoglobin to form glycosylated hemoglobin6. Hematological complications consist mostly of abnormalities in
the function, morphology and metabolism of erythrocytes, leukocytes and platelets. Literature has shown that
intake of medicinal compounds or drugs can modify the normal range of hematological parameters7,8. Furthermore,
it has been exposed by the present study that hemoglobin levels in diabetic control (untreated group) showed
abnormalities. This might due to the destruction of matured Red Blood Cells (RBC) leading to low haemoglobin
count (Hb), because of reaction of excess glucose with the haemoglobin to gives rise to glycosylated haemoglobin
with diminish in RBC9.
Figure 3 implies that group 2 showed decreased haemoglobin levels upon treatment with the M. uniflorum
and glibenclamide the stage was brought back to near normal. This might also be responsible in improving the
immune system being weak due to the generation of reactive oxygen species as a result of streptozotocin induction
and shows that the aqueous extract may not have negative effect on the bone marrow, kidney and haemoglobin
metabolism.
HbA1c is considered as an investigative marker and helps to know about degree of protein glycation,
longterm blood sugar level and correlation of diabetes connected complications10,11. HbA1c has been found to be
increased over a long period of time in diabetes because the excess glucose present in blood reacts with haemoglobin
to form glycosylated haemoglobin12. The rate of glycation is proportional to the concentration of blood glucose. In
the present study, streptozotocin induced diabetic rats showed significant increase in HbA1c level compared with
normal rats (Figure 4). The treatments with the ethanolic extract of M. uniflorum and glibenclamide showed a
significant decrease in the content of glycosylated haemoglobin that could be due to an improvement in glycemic
status.
Figure 4- Effect of ethanolic extract of M. uniflorum on the glycosylated hemoglobin level of control and experimental
rats. Values are expressed as Mean SD for six animals. Values not sharing common superscript letters (a-b) differ
Evidence showed that glycation itself may persuade the formation of oxygen-derived free radicals in diabetic
condition, and the level of HbA1c is considered as one of the markers of degree of oxidative stress in diabetes
mellitus13. Administration of M. uniflorum to diabetic rats reduced the glycosylation of haemoglobin by virtue of its
normoglycaemic activity and thus decreases the levels of glycosylated haemoglobin in diabetic rats. This normalisation
of glycosylated haemoglobin indicates decreased glycation of proteins.
Several studies reported that STZ administration elevated serum renal markers in rats14 the indicator of
diabetic nephropathy with altered glomerular filtration rate. The current study also revealed that serum renal markers
such as urea, uric acid and creatinine levels were increased. The daily administration of M. uniflorum extract for 45
days caused a significant decrease in serum urea, uric acid and creatinine levels (Figure 5). This data indicates that
the plant extract improved the renal functions and reversed the damage in the kidney tissues of diabetic rats.
304
Figure 5- Effect of ethanolic extract of M. uniflorum on the biochemical parameters in the serum of control and
experimental rats. Values are expressed as Mean SD for six animals. Values not sharing common superscript
letters (a-b) differ significantly at p< 0.05 (DMRT)
The amount of urea in the blood is increased with affiliated decrease in plasma protein levels in experimental
diabetes as a result of increased breakdown of plasma and tissue proteins due to negative nitrogen balance. Further,
the supra physiological concentration of glucose in diabetic state causes severe derangement in protein metabolism
that result in the development of negative nitrogen balance. This in turn elevates urea and creatinine levels which
act as biochemical diagnostic markers for assessing renal impairment and drug induced toxicity15. The level of
purines is elevated due to accelerated muscle wasting. These accumulated purines are the main source for the
production of uric acid by the activity of xanthine oxidase16.
In the present study, streptozotocin induced diabetic rats showed a significant increase in the liver marker
enzymes such as SGOT and SGPT, while the treatment with ethanolic extract of M. uniflorum showed a significant
decrease in the marker enzymes which shows that the extract possess protective action against tissue damage.
There was no significant difference between the control and plant alone treated groups (Figure 6).
Figure 6- Effect of ethanolic extract of M. uniflorum on liver marker enzymes in serum of control and experimental
rats. Values are expressed as Mean SD for six animals. Values not sharing common superscript letters (a-b) differ
The transaminases (AST and ALT) are healthy known enzymes used as biomarkers to predict possible
toxicity to the liver17. Damage to structural integrity of the liver is reflected by an increase in the activity of these
two enzymes in the serum, probably as a result of leakage from altered cell membrane structure. Therefore, the
increase in serum AST and ALT activities in the untreated diabetic rats confirms damage to the plasma membrane,
leading to a negotiation of membranal integrity18.
305
CONCLUSION
From the above discussion it conclude that ethanolic extract of M. uniflorum (200 mg/kg) leaf exhibited
significant antihyperglycemic activity in streptozotocin-nicotinamide induced diabetic rats. These investigations
lead a path for the isolation of active principles responsible for the antidiabetic activity.
ACKNOWLEDGMENT
We, the authors are thankful to our Chancellor, Chief Executive Officer, Vice Chancellor and Registrar of
Karpagam University for providing facilities and encouragement.
We declare that we have no conflict of interest.
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1. Okigbo R N, Anuagasi C L & Amadi J E (2009) J Med Plants Res 3, 86-95
2. Parasuraman S, Kumar E, Kumar A & Emerson S (2010) J Pharmacol Pharmacother 1, 38-41
3. Kawsar S M A, Mostafa G, Huq E, Nahar N & Ozeki Y (2009) Int J Biol Chem 3, 42-48.
4. Veeramani C, Pushpavalli G & Pugalendi K V (2008) J Appl Biomed 6, 19- 26.
5. Hassan H A, Agmy S M, Gaur R L, Fernando A, Raj M H G & Ouhtit A (2009) Int J Biol Sci 5, 249-255
6. Alamgeer M, Mushtaq N, Bashir S, Rashid M, Malik M N H, Ghumman S A, Irfan H M, Akram M, Khan A

Q & Rashid H U (2012) Afr J Pharm Pharmacol 6, 2845-2850
7. Comazzi S, Spagnolo V & Bonfanti U (2004) Comp Clin Path 12, 199-205
8. Sakuljaitrong S, Chomko S, Talubmook C & Buddhakala N (2012) ARPN J Sci Tech 2, 1049-1054
9. Mansi K M S (2006) Am J Pharmacol Toxicol 1, 5-10

10. Deguchi Y & Miyazaki K (2010) Nutr Metab 7, 9
11. Lanjhiyana S, Garabadu D, Ahirwar D, Bigoniya P, Chandrana A, Chandrapatra K, Lanjhiana S K & Karuppaih
M (2011) Adv Appl Sci Res 2, 47-62
12. Alagammal M, Nishanthini A & Mohan V R (2012) JApplPharmSci 2, 143-148

13. Bravi M R, Armiento A, Laurenti O, Cassano F M, De Luca O & Morettia A (2006) Metabolism 55, 591-596
14. Balasubramanian T, Senthilkumar G P, Karthikeyan M & Chatterjee T K (2014) Exp Pharmacol 4, 1-7
15. Chang C L, Chen Y C, Chen H M, Yang N S & Yang W C (2013) Med Chem 20, 899-907
16. American Diabetes Association (2010) Diabetes Care 33, 11-61
17. Rahman M F(2001) Hum Exp Toxicol 20, 243-249.
18. Yakubu M T, Bilbis L S, Lawa M & Akanji M A (2003) J Pure Appl Sci 18, 1395-1400.
306
In vitro antioxidant activity and phytochemical analysis of Citrus
limettaand Citrus sinensis
Kavitha, M, Iswariya, G.T, Padma, P.R and Nirmaladevi, R*
Avinashilingam Institute for Home Science and Higher education for Women, University, Coimbatore-641 043
E-mail : nirmaladevi.saravanan32@gmail.com
Abstract
Free radicals are electrically charged molecules, react with other substances in order to neutralize themselves by
a process called oxidation. Antioxidants are vital substances which possess the ability to protect the body from
damage caused by free radical induced oxidative stress. There is an increasing interest in natural antioxidants,
which is present in medicinal and dietary plants, which might help in preventing oxidative damage. With this
background the present study was carried out to screen the antioxidant potential of Citrus limettaand Citrus
sinensis. The antioxidant activity of the methanol extract of Citrus limetta and Citrus sinensis were analysed by
DPPH, ABTS, H2O2 scavenging assays, inhibition of nitric oxide and superoxide generation, chelating and
reducing ability assay. The results of the various free radical scavenging systems revealed that the methanol
extract of Citrus limetta and Citrus sinensis extracts were individually a strong and rich source of antioxidants,
with varying scavenging abilities for different ROS at different magnitudes of potency. To conclude, Citrus
limetta and Citrus sinensis extracts exhibited good radical quenching ability due to the presence of various
phytochemicals.
Keywords: Free radicals, Antioxidants, Citrus fruits and Phytochemicals
INTRODUCTION
Free radicals are unstable molecules, which contains an unpaired electron spinning on the outermost layer
around the nucleus.Due to excessive production of reactive species, induced by exposure to external oxidants or a
failure in the defense mechanisms, damage to cell structures, DNA, lipids and proteins occur which increases the
risk of different diseases1.These radical reactions are implicated in the pathology of many human diseases including
atherosclerosis, ischemic heart disease, ageing, hepatotoxicity, inflammation, diabetes, immunosuppression, and
neurodegenerative conditions 2. Antioxidants stabilize these free radicals as quickly as they are generated in the
human body due to metabolic activities by preventing orquenching free radical formation or chelate redox metals3.
Currently, there is a global interest in finding new and safe antioxidants from natural sources, to minimize oxidative
injury of living cells through free radicals. Citrus speciesis one of the most important fruit crops. Citrus limettabelongs
to the Rutaceae family and are rich in flavonoids and phenolic compounds, which might be responsible for the anti-
microbial and anti-inflammatory effects4. Citrussinensis or sweet orange originated from south East Asia, but is
consumed all over the world as an excellent source of vitamin C, a powerful natural antioxidant that builds the
bodys immune system5. The aim is to investigate the free radical scavenging activity of methanol extract of
Citruslimettaand Citrus sinensispulp.
METHODOLOGY
Sample preparation
Fresh fruits (Citrus limettaand Citrus sinensis)were procured from local markets in Coimbatore, India.
The peels were separated and sliced into small pieces. The pulp was kept in an incubator for 12 hours at 40C.
307
About 20g of pulp is weighed and mixed with 100ml of methanol and kept overnight in a shaker incubator. Then the
extracts were filtered and used for following analysis.
DPPH radical scavenging activity

The ability of the extracts to scavenge the stable free radical DPPH and convert it into Diphenylpicryl
hydrazine was determined by the method described by Mensoret al.6.
ABTS scavenging activity

The percent inhibition of ABTS radical by Citrus fruit extracts was determined as per Shirwaikeret al.,7.
Hydrogen peroxide scavenging activity

The ability of the methanol extract of C. limetta and C.sinensis pulp to scavenge hydrogen peroxide radical
was determined by measuring the decrease in absorbance at 230nm spectrophotometrically, explained by Ruchet
al.,8.
Inhibition of nitric oxide generation

The extent of nitric oxide generation was studied using Griess reagent, a method explained by Green et
9
al., .
Inhibition of superoxide generation

The extent of inhibition of superoxide generation was studied by the method described by Winterbournet
10
al., .
Chelating activity
The chelating activity of methanolic extract was measured by the method of Hegazy and Ibrahium. The
absorbance was measured at 522 nm11.
Reducing power ability

This was carried out as described by Yildrimet al., and the absorbance was measured at 700 nm12.

The DPPH assay is a simple and popular method to establish the free radical scavenging potential of a
sample. In order to determine the antioxidant potential of the methanol extract of C.limettaand C.sinensis, different
concentrations of the extracts (10, 50, 100, 150and 200g) were tested against the battery of free radicals. In DPPH
assay, both the Citrus fruit extracts were able to scavenge DPPH radical. At a lower concentration of 10g, the
methanol extract of C.limettaand C.sinensisshowed moderate activity while at higher concentration (200g) both
the pulp extracts exhibited maximum scavenging activity. It was also noted from the graph (fig 1A) that C.limetta
showed enhanced activity than the C. sinensis.
ABTS radical scavenging activity

The ABTS free radical scavenging activity is based on the ability of these stable free radicals, to be decolourized
in the presence of antioxidants13. The results of ABTS scavenging assay revealed that Citrus fruit pulp extracts
exhibited good ABTS radical quenching activity. It is also observed that the radical quenching ability was found to
be increased with increase in the concentration of the pulp extracts. In addition, methanol extract of C.limetta
showed slight increase in antiradical activity when compared toC.sinensis as presented in fig 1B.
308
Hydrogen peroxide scavenging activity
Hydrogen peroxide is a biologically relevant, non-radical reactive oxygen species and is inevitably generated
as a by-product of normal aerobic metabolism. In the present study, the Citrus fruit pulp extracts were subjected to
oxidative stress by adding H2O2. The scavenging activity of the methanol extract of C.limettaand C.sinensis at 10g
concentration was found to be 37% and 34%respectively. As the concentration of the pulp extracts increases, the
H2O2 was neutralized to a greater extent. Also, C.limetta showed better activity than the C.sinensis. The result is
depicted in fig 1C.The results strongly suggest that these extracts contain the necessary compounds for radical
elimination. Many reports have already proven that nutritive phenols play a significant role in protecting mammalian
and bacterial cells from cytotoxicity induced by H2O2, indicating that the observed activity of plants extracts could
be due to the presence of phenols13. Thereby proving that the phytoconstituentssuch as phenols present in the pulp
extracts of C.limetta and C.sinensis might be responsible for H2O2 scavenging activity thereby rendering protection
to ROS mediated diseases.
Determination of inhibition of nitric oxide generation

The scavenging activity of the extract against nitric oxide was detected by its ability to inhibit the formation
of nitrite through direct competition with O2 and oxides of nitrogen in the reaction mixture. During pathological
conditions, nitric oxide reacts with superoxide anion and forms potentially cytotoxic molecules such as
peroxynitrite15.The methanol extract of C. limettaand C. sinensis pulp were tested for its inhibitory potential
against nitric oxide in vitro. When the results are compared,C. limettapulp extract showed better inhibition against
NO generation than C. sinensis pulp extract. The methanol extract of C. limettapulp showed considerable inhibition
at 50, 100, 200g/ml concentration while C. sinensis pulp extract showed slightly lesser inhibition at indicated
concentration as given in fig 1D.
Determination of inhibition of Superoxide generation

Superoxide is one of the most harmful radicals and its scavenging is necessary because it is a precursor for
other major ROS like H2O2, hydroxyl and singlet oxygen that are liable to induce oxidative damage in proteins,
lipids and DNA in the body16. Figure 1E shows the inhibition of superoxide radical by the methanol extract of C.
limettaand C. sinensis pulp. The results showed that the methanol extract of C. limetta inhibited superoxide in a
slightly better manner than the C. sinensis pulp extract. At a lower concentration the SO inhibition by the pulp
extracts was found to be 29% and 16% which was increased to considerable amount of about 49 and 43% respectively.
Even though both the Citrus fruit pulp extracts showed better scavenging activity against DPPH, ABTS and H2O2
and a remarkable inhibition was observed only against NO radicals rather than SO generation which was inhibited
only to a moderate extent.
100 100 100
A) DPPH y B) ABTS
it yt C) H2 O2
yt ivt iv
i c
tiv a it
c
c g50 50 a
a50 in g
g g n
in n
ev
i
g
g n
n
e ac e
va S0 v
ac0
cS 0 % S
% 10g 50g 100g 150g 200g % 10g 50g 100g 150g 200g
10g 50g 100g 150g 200g
Concentration Concentration Concentration
C.limetta C.sinensis C.limetta C.sinensis C.limetta C.sinensis
100 100
n D) NO generation E) SO generation
io
it n
ib io
h it
In50 ib
50
t h
en In
c t
r n
e e
P c
r
0 e0
P
10g 50g 100g 150g 200g 10g 50gCo ncentration
100g 150g 200g
Concentration
C.lim etta C.sinensis C.limetta C.sinensis
Fig 1- Radical scavenging activity of Citrus limetta and Citrus sinensis
309
3
100 A) Chelating ac tivity
yt B) Reducing power Ability
vii yt
is
tc n 2
a e
g50 D
la
in ci
gn t 1
ev p
O
ac
S0
% 0
10g 50g 10 0g 150g 2 00g
Concentration 10g 50 gConcentra
10 0g tion
15 0g 2 00g
C.limetta C.sinensis C.limetta C.sinensi s
Fig 2 - Chelating activity and Reducing power ability of methanol extract of C. limettaand C. sinensis
Chelating activity and reducing power ability

Reducing power assay is based on reduction of ferric ions into ferrous ions by antioxidantis monitored by
recording absorbance at 700nm. Iron, in nature, can be found as either ferrous (Fe2+) or ferric ion (Fe3+), with ferric
ion predominating in foods. Ferrous ions (Fe2+) chelation may render important antioxidative effects by retarding
metal-catalyzed oxidation. Ferrous ions (Fe2+) are the most powerful pro-oxidants among the various species of
metal ions. Minimizing ferrous (Fe2+) ions may afford protection against oxidative damage by inhibiting production
of ROS and lipid peroxidation. The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity17. The metal chelating activity of the fruit pulp extract of C. limettaand C. sinensis are
shown in fig 2A. In this graph, the methanol extract of both the fruit showed significant chelating activity. On
comparison with the fruit pulp extracts, C. sinensis showed better activity than the C. limettaextract. It is clearly
evident from the graph that both the extracts showed good chelating activity in a dose dependent manner.The
results are given in fig 2B. The results revealed that reducing activity was significantly increased as the concentration
of C. limettaand C. sinensis extracts were increased. The presence of antioxidant reductants in the methanol extracts
of fruit pulp causes the reduction of Fe3+ to the ferrous form. The methanol extract of C. sinensis pulp has significant
reducing power when compared with the C. limetta extract.
Since both the fruit varieties were able to scavenge the radicals such as DPPH, ABTS and H2O2 and inhibit
the generation of superoxide and nitric oxide radicals. It is clearly evident that some of these phytochemicals
constituents might have been responsible for their antioxidant potency and radical quenching abilities. Therefore, it
becomes imperative to assess the phytochemicals/ secondary metabolites that are present in the methanol extract of
Citrus fruit pulp.
CONCLUSION
The results from various free radical scavenging systems revealed that the methanol extract of C. limetta
and C.sinensis extracts were found to have rich antioxidant status, with varying scavenging capabilities for different
ROS at different magnitudes of potency. Thus the antioxidant activity of the extracts may be due to the presence of
polyphenolic compounds such as flavonoids and tannins and this, in turn, may be attributable to the hydrogen or
electron donating ability of the group present in the structure. Therefore, the Citrus fruit pulp extracts could be
potentially used for the prevention of free radical mediated diseases and these extracts can be exploited in future for
pharmaceutical use, involving the preparation of drugs against dreadful diseases and disorders.
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311
Attenuation of biochemical changes by ethanolic extract of Erythrina
variegata L. flowers on streptozotocin induced diabetic rats
Subrahmanian Hemmalakshmi1, Suriyamoorthy Priyanga1 and Kanakasabapathi Devaki*1,2
1
Department of Biochemistry, Karpagam University, Coimbatore, 641 021, India
1,2
Department of Biochemistry and Bioinformatics, Karpagam University, Coimbatore, 641 021, India
Abstract
Diabetes mellitus is probably the single most important metabolic disease and is widely recognized as one of
the leading causes of death and disability. The aim of the present study was carried out to evaluate the antidiabetic
activity of ethanolic extract of Erythrina variegata L. flowers in streptozotocin induced diabetic rats. Diabetes
was induced by intraperitoneal injection two consecutive doses of streptozotocin (20 and 25 mg/kg body wt)
with one week interval in Wistar albino rats and divided into six groups. Diabetic animals were treated with
Erythrina variegata L. flowers (200 mg/kg body weight) for 45 days. Several indices such as blood glucose,
glycosylated hemoglobin (HbA1c), lipid profiles, hepatic marker enzymes and renal marker enzymes were
examined after 45 days of experimental period using standard protocols. Oral administration of ethanolic extract
of Erythrina variegata L. flowers to diabetic rats for 45 days significantly reduced the altered levels of blood
glucose, HbA1c, lipid profiles, hepatic marker enzymes and renal marker enzymes. The current study clearly
evidenced the antidiabetic potential of Erythrina variegata L. flowers in Wistar albino rats and this study
established pharmacological evidence to support its use in ethnomedicine which can also be extrapolated for
the management of diabetes.
Keywords : Erythrina variegata L., streptozotocin, glucose, hepatic and renal markers
INTRODUCTION
Diabetes mellitus is a chronic metabolic disorder associated with long term damages, dysfunctions, failure
of organs especially the eyes, liver, kidneys, nerves and cardiovascular system1. Different phytoconstituents of
herbal products are safer than synthetic medicine and beneficial in the treatment of diseases caused by free radicals
and it also protect the body from tissue injury2. Herbal medicines involve the integration of several therapeutic
experiences and practices of indigenous systems of medicine that may span many previous generations, which
often provide valuable guidelines to the selection, preparation and application of herbal formulation with a view to
providing therapeutic benefits3. Erythrina variegata L. belongs to the family- fabaceae, it is a medium sized quick
growing tree found in deciduous forests throughout India4. The whole plant (Erythrina variegata L.) is used in
alternative system of medicine for wind damp obstruction syndrome, rheumatic joint problems, lower back and
knee pain, asthma, nerve depression, epilepsy and insect bites 5. In the some experiments, it has potential effects for
treatment of several diseases like convulsion, fever, inflammation6, bacterial infection, insomnia, helminthiasis,
cough, ulcer, cuts, liver ailment and injury 7.The aim of the present study was carried out to evaluate the alteration
of biochemical parameters in the antihyperglycemic activity of ethanolic extract of Erythrina variegata L. flowers
in Streptozotocin induced diabetic rats.

Plant Collection and Authentication
The fresh plant samples were collected in the month of August from Kodaikannal and its surroundings,
Dindigul District, Tamil Nadu, India. They were botanically authenticated by Dr. G.V.S Moorthy, Botanical Survey
of India, TNAU campus, Coimbatore. The specimen was deposited in the Herbarium for future reference (voucher
312
number: BSI/SRC/5/23/2013-14/Tech/1500). The plant samples were thoroughly washed in the running tap water
to remove adhering dust particles and dried under the shades for about two weeks. It was grounded into a coarse
powder and stored in an airtight container, which can be use for the further investigations.
Preparation of Ethanolic Extracts of Flowers

The powdered plant sample of flowers (100g) were weighed and mixed with 500 ml of ethanol. Then it is
kept in an orbital shaker at 190-220 rpm for 48 hours. The supernatant was collected, filtered through Whatman
No.1 filter paper and then concentrated by evaporating to dryness which gave a solid amorphous residue and it was
dried thoroughly to remove the solvent used. The obtained dried extract was then accurately weighed, stored in a
small vials at -20C and used for the subsequent studies.
Animals
Wistar albino rats of either sex weighing about 150180 g were procured from the animal house of Karpagam
University, Coimbatore, India. The animals were under standard conditions and were housed in polypropylene
cages with a wire mesh top and a hygienic bed of husk in a specific pathogen free animal room under controlled
conditions of 12 hr light and 12 hr dark cycle, with temperature of 24 2C, relative humidity of 50 10% and fed
with rodent diet and water ad libitum. The study was accepted by Institutional Animal Ethical Committee constituted
for the purpose of CPCSEA (Approved No: KU/IAEC/Ph.D.,/145). Government of India.
Diabetes was induced by intraperitoneal injection two consecutive doses of freshly prepared streptozotocin
(20 and 25 mg/kg body wt) ) in 0.1 M citrate buffer (pH 4.5) in the volume of 1 ml/kg body weight using sterile 25G
needle with one week interval in Wistar albino rats and divided into six groups. Diabetes was identified in rats by
moderate polydipsia and marked polyuria. After 48 hrs of STZ administration, blood glucose levels were estimated
in rats following overnight fasting. Rats with a blood glucose level ranging between 200 and 300 mg/dl were
considered diabetic and used for the experiments.
Experimental design
The experiment was conducted with a total of 36 rats divided into six groups of six animals each. Based on
the GTT results 200 mg/kg of Erythrina variegata was selected for this study.
Group 1: Untreated rats (Control), Group 2: Untreated rats (Vehicle Control),Group 3: Diabetic control (two
consecutive doses of Streptozotocin - 20 and 25 mg/kg, b.wt)),Group 4: Diabetic rats treated with Glibenclamide
(2mg/kg b.wt), Group 5: Diabetic rats treated with Erythrina variegata L. (200mg/kg b.wt), Group 6 Normal
healthy rats treated with Erythrina variegata L. (200mg/kg b.wt)
The test drug and reference standard drug was fed orally for a period of 45 days. After the experimental
period, rats were sacrificed by cervical dislocation after giving chloroform in mild dose.
Collection of blood
After the experimental period of 45 days, the animals were sacrificed by cervical dislocation under mild
chloroform anesthesia. Blood was collected by decapitation and serum was separated by centrifugation for 10 min
at 1500 rpm and used for the estimation of various biochemical estimations.
Biochemical estimations
Whole blood was used for estimate the glucose and HbA1c level in the experimental rats by using standard
kit method and serum was used for Urea, uric acid, creatinine, serum glutamate oxaloacetate transaminase (SGOT),
313
serum glutamate pyruvate transaminase (SGPT) and lipid profiles of cholesterol, triglycerides and HDL were
estimated by using diagnostic kits manufactured by Agappe Diagnostics Ltd.
All samples were tested and analyzed in triplicates. Results were calculated as the Mean SD (standard
deviation) for each sample. Statistical analysis was done with one way analysis of variance (ANOVA) using SPSS
version 16.0 and the individual comparisons were obtained by the Duncan Multiple Range Test (DMRT). The
significant difference was judged to exist at a level of p < 0.05.

Many plant extracts and plant products have been identified as good protectors against the free radicals by
triggering antioxidant gene expression. For that account, natural antioxidants from plant sources have been viewed
as promising therapeutic drugs against diabetes mellitus and other diseases 8.
Fig 1-Effect of ethanolic extract of Erythrina variegata L. flowers on glucose (A) and HbA1c (B) level in control
and experimental rats. Values are expressed as Mean SD for six animals, Values not sharing common superscript
letters (a-c) differ significantly at p< 0.05 (DMRT).
In the present study, streptozotocin induced diabetic rats showed significant increase in blood glucose and
HbA1c level compared with normal rats (Figure 1). The treatments with the ethanolic extract of E. variegata and
glibenclamide showed a significant decrease in the content of blood glucose and glycosylated haemoglobin that
could be due to an improvement in glycemic status. Administration of E. variegata to diabetic rats reduced the
glucose and glycosylation of haemoglobin level by virtue of its normoglycaemic activity and thus decreases the
levels of glycosylated haemoglobin in diabetic rats. This normalisation of glycosylated haemoglobin indicates
decreased glycation of proteins. There are countless reports available to support the multiple mechanisms of
antidiabetic plants to exert their blood glucose lowering effect, such as inhibition of carbohydrate metabolizing
enzymes, enhancement of insulin sensitivity, regeneration of damaged pancreatic islet -cells and enhancement of
insulin secretion and release9. HbA1c has been found to be increased over a long period of time in diabetes because
the excess of glucose present in blood reacts with haemoglobin to form glycosylated haemoglobin10.
314
Fig 2-Effect of ethanolic extract of Erythrina variegata L. flowers on cholesterol(A), Triglycerides (B) and HDL (C)
levels in control and experimental rats. Values are expressed as Mean SD for six animals, Values not sharing
common superscript letters (a-d) differ significantly at p< 0.05 (DMRT).
Figure 2 reveals the total cholesterol and triglycerides level in serum was significantly elevated in diabetic
control group as compared to control group. On treatment with the Erythrina variegata L. produced significant
reduction in total cholesterol and triglycerides levels in serum when compared to diabetic control. Whereas HDL
cholesterol was decreased in diabetic animals as compared to control animals. The level of HDL cholesterol were
returned to near normal range in diabetic rats after treatment with Erythrina variegata L.
Fig 3-Effect of ethanolic extract of Erythrina variegata L. flowers on Urea(A), Uric acid (B) and Creatinine (C)
levels in control and experimental rats. Values are expressed as Mean SD for six animals, Values not sharing
common superscript letters (a-d) differ significantly at p< 0.05 (DMRT).
Generally an increase in serum urea, uric acid and creatinine levels is a sign of damaged renal function that
evidenced in diabetic rats. In our data clearly indicated that serum urea, uric acid and creatinine points were decreased
when treated with Erythrina variegata L. flowers and recover to near normal levels, shows enhanced renal function
by normalizing the DNA and protein metabolism by E. variegata L in Figure 3.
315
Fig 4-Effect of ethanolic extract of Erythrina variegata L. flowers on SGPT(A) and SGOT(B) levels in control and
experimental rats. Values are expressed as Mean SD for six animals, Values not sharing common superscript
letters (a-d) differ significantly at p< 0.05 (DMRT).
From the figure 4 shows the effects of Erythrina variegata L. on the serum levels and activities of markers
of liver damage (SGOT and SGPT) in all groups of rats. No significant enzyme alterations were seen in the control,
vehicle control rats and treated with the aqueous extract of Erythrina variegata L. flower alone (200 mg/ kg). On
the other hand, the activities of SGOT and SGPT were significantly elevated (p<0.05) in streptozotocin diabetic
rats when compared with the normal controls rats. Alternatively, Rats administrated ethanolic extract of Erythrina
variegata L. flower showed significant reduction (p<0.05) in these hepatic marker enzyme activities. Although
glibenclamide resulted in significant decreases in the activities of these enzymes, their levels remained significantly
higher as compared to control group and diabetic rats treated with the ethanolic extract of Erythrina variegata L.
flowers. Therefore an increase in the activities of SGOT and SGPT in serum might be mainly due to the leakage of
these enzymes from the liver cytosol into the blood stream which provide an symptom of the hepatotoxic effect of
STZ11.
CONCLUSION
The present research clearly indicated that the ethanolic extract of Erythrina variegata L. flowers have
promising hypoglycemic activity against Streptozotocin induced diabetic rats. It may be difficult to find the exact
mechanism responsible for the hypoglycemic effect. Studies are needed to identify the active compound responsible
for the hypoglycemic effect for further management of diabetes mellitus and prevention of diabetic complications.
ACKNOWLEDGEMENT
The authors are thankful to the Chancellor, the Chief Executive Officer, the Vice Chancellor and the
Registrar of Karpagam University for providing facilities and encouragement.
We declare that we have no conflict of interest.
REFERENCES
1. Gomathi D, Kalaiselvi M, Ravikumar G, Devaki K & Uma C (2013) Indo Ameri J. Pharm Res 3, 33679
2. Sen S, Chakraborty R, Sridhar C, Reddy YSR & De B (2010) International Journal of Pharmaceutical
Sciences Review and Research 3, 91-100
3. Chikezie PC, Ojiako OA & Nwufo KC (2015) J Diabetes Metab 6, 546 doi:10.4172/2155-6156.1000546)
4. Hemmalakshmi S, Priyanga S & Devaki K (2016) Int. J. Pharm and Pharm sci 8, 217
316
5. Devaki K, Hemmalakshmi S & Priyanga S (2015) Israel Journal of Plant Sciences 1-4 (doi:10.1080/
07929978.2015.1096608)
6. Kumar A, Lingadurai S, Shrivastava TP, Bhattacharya S & Haldar PK (2011) Pharma Biology 49, 577582
7. Karunambigai M & Venkateswarlu B (2013) Int. J. Pharmaco Res 3, 66
8. Zengin G, Cakmak YS, Guler GO & Aktumsek A (2011) Records of Natural Products 5,123132
9. Nadkarni KM (2004) Medcinal plants of India, 2004 edition, Reprint Publication, Dehradun, India,10-11
10. Alagammal M, Nishanthini A & Mohan VR (2012) Journal of Applied Pharmaceutical Science 2,143 148
11. Shanmugasundaram R, Devi KV, Soris TP, Maruthupandian A & Mohan VR (2011) Int J Pharm Tech
Res,3756
317
Phytoconstituents From Essential Oil Of Cyanthillium Cinereum (L.)
H. Rob And Their Molecular Docking Studies
J. Dharani And S. Ravi*
Department of Chemistry, Karpagam University, Coimbatore-641021, Tamilnadu, India.
Abstract
Cyanthillium cinereum belongs to the genus Cyanthillium, in the family Asteraceae. The fresh aerial parts of C.
cinereum is subjected to hydro-distillation by using Cleavenger type apparatus. The extracted essential oil was
dried over anhydrous sodium sulphate and the yield of the essential oil is 0.521%. The constituents were
identified by co-TLC using standard compounds. Docking studies has been performed for the constituents
present in the C. cinereum species (Lupeol, 12-oleanen-3-ol-3-acetate, Stigmasterol, -sitosterol) using bacterial
protein 2X5O.
Key words: Cyanthillium cinereum, essential oil, Docking, protein
INTRODUCTION
Cyanthillium cinereum also known as ironweed. In Malayalam, it is known as Poovankurunthila. It is a
species of perennial plants in the sunflower family. It is an annual herb up to 120 cm tall with flat topped arrays of
numerous flower heads, each with pinkish or violet ray florets. It can be found in roadside, open waste places, dry
grassy sites. It is located especially in different Asian countries such as India, Bangladesh and Nepal. The whole
plant has great medicinal value in diverse traditional usage in different nations and also gets recognition in the
Ayurvedas. Various extracts of the plant are antiviral, anticancer and diuretic. The plant possess antimicrobial[1],
antibacterial [2], antioxidant [3], antihelmentic [4], anti-inflammatory, analgesic, antipyretic [5,6], antiflautulent,
antispasmodic and antidiuretic properties[7]. Some of the phytochemical compounds present are sterols, flavonoids,
sesquiterpene lactones[8] and a terpenoid, leupeol acetate which shows antihyperglycaemic and antiulcer properties.
In Tamil Nadu, India, root extract is taken 3-4 times daily to treat diarrhea. Leaf juice is taken twice daily to
treat cough. Leaves were used for skin diseases: Handful of leaves pound and boiled in coconut oil and oil extract
applied three times daily to treat leprosy and scabies. In the Philippines infusion of plant taken internally for cough.
Plant also used for wounds infections and fever. It is considered a more potent abortifacient and better treatment for
menstrual pains when C. cinereum is decocted with Justicia secunda. Leaf: Boiled with Stachytarpheta jamaicensis
for treating albuminuria; sprains.
It is well-known that the same taxon growing in different areas and in different seasons may have widely
differing chemical components and hence differing biological properties. Ingredients of the essential oil from C.
cinereum are determined by collecting the plant from the same locality but in different seasons. Essential oils
obtained from the aerial part of C. cinereum collected from Dindigul, are cut down into small parts and exposed to
hydro distillation using a Clevenger type apparatus. The chemical constituents were identified by Co-TLC. The
following four compounds were identified.
Docking studies (Fig-1) have been carried out for the four identified bioactive compounds from the essential
oil of Cyanthillium cinereum against bacterial protein. The results were given in the below table-1.
318
Table-1: Molecular docking studies of the compounds from the essential oil of C. cinereum
Docking Details
Ligands
Binding Scores Alkyl Bond Pi-Alkyl Bonds
12-Oleanen-3-Ol-3b Acetate -8.8 - His:238
Lupeol -8.4 Met:234 His:238, His:238
Stigamasterol -7.0 Met:234 His:238

-Sitosterol -6.6 His:238 His:234, His:238
Fig-1: Molecular docking of the compounds from the essential oil of C. cinereum with 2X5O protein
319
CONCLUSION
Essential oil was obtained from C. cinereum and the compounds were identified by Co-TLC. Docking
studies has been performed for the constituents present in the C. cinereum species (Lupeol, 12-oleanen-3-ol-3-
acetate, Stigmasterol, -sitosterol) using bacterial protein 2X5O. In these Lupeol and 12-oleanen-3-ol-3-acetate
shows good scoring and bonding.
REFERENCES
[1] K.M.Nadkarni,IndianMateriaMedica withAyurvedicUnaniTibbi,Siddha,Allopathic, Home opathic,
Naturopathic & Home Remedies, Popular Prakashan, Mumbai, India, 1954.
[2] M. Daniel, Medicinal Plants: Chemistry and Properties, NH: Science Publishers, 2005.
[3] Smithsonian Institution, some rights reserved.
[4] M. Yusuf, Chowdhury JU, Wahab MA, Begum J., Medicinal plants of Bangladesh. BCSIR, Dhaka (1994)
17266.
[5] Y.T.Hsu,Study on the Chinese drugs used as cancer remedy, Journal of South Asian Researches, vol. 3, p.
63, 1967.
[6] T.Johnson, CRCEthnobotany DeskReference, CRCPress,Boca Raton, Fla, USA, 1998.
[7] K. W. Lin, Ethnobotanical study of medicinal plants used by the Jah Hut peoples in Malaysia, Indian
Journal of Medical Sciences, vol. 59, no. 4, pp. 156161, 2005.
[8] Kirtikar KR, Basu BD Indian Medicinal Plants, Sri Satguru Publication, New Delhi, 2000.
[9] Medicinal Plants Of The Guianas ( Guyana, Surinam, French Guiana) By Robert A. Defilipps, Shirley L.
Maina And Juliette Crepin; Online At The Biological Biodiversity Of The Guiana Shield. Smithsonian Natural
Museum Of Natural History 2004.
[10] Maruthupandian A, Mohan VR Observations of ethnomedicinal plants from Sirumalai hills in Western Ghats
of Tamilnadu, India. J Herbal Med Toxicol, 2010; 4
320
Evaluation of Sensory and Nutritional values of Carrot juice Incorpo-
rated Cow Milk Paneer
P. Nivedha Raveendran, V. V. Sathibabu Uddandrao, Ganapathy Saravanan,
and Vadivukkarasi Sasikumar*
Centre for Biological Sciences, Department of Biochemistry, K. S. Rangasamy College of Arts and Science (Autonomous),
Tiruchengode, Tamilnadu, India - 637215.
Department of Biochemistry, Centre for Biological Science, K. S. Rangasamy College of Arts and Science (Autonomous),
Thokkavadi, Tiruchengode, Tamil Nadu, India - 637215. Email: vadivusai16@gmail.com
Abstract
Milk and dairy foods are considered to be one of the main food groups important in a healthy balanced diet. In
the present study we did an attempt to enhance the nutritional values by incorporating carrot juice in to cow milk
paneer. 20% carrot juice was added to cow milk to prepare carrot incorporated paneer and pure cow milk
panner was taken as control. The both paneers were subjected to evaluate the basics of proximate composition
and sensory evaluation by the sensory panelists according to 9-point Hedonic scale evaluation. Sensory evaluation
studies revealed that 20% carrot juice incorporated cow milk paneer has highest acceptability in terms of colour,
flavour, appearance and taste. Incorporation of carrot in paneer illustrated that an enhancement in calcium,
phosphorus, -carotene, protein and lipid when compared to normal paneer. In conclusion, our study revealed
that incorporation of 20% carrot shown highest acceptability by the panellists and proximate analysis demonstrated
that rich in nutritional values when compared to pure cow milk panner, this study may suggest that to incorporation
of natural products such as carrot in paneer can be used as better source to enhance nutrition.
Key words: Paneer, Cow milk, Carrot, Sensory evaluation, proximate analysis
INTRODUCTION
Paneer is native gelatinous product from milk made by addition of organic acids to milk at lofty temperature.
Paneer is consumed as pedestal essence for the manufacturing of an enormous number of cookery dishes and it is
a well-liked product at the household level as well as still its make the most of is increasing in prearranged food
chains. It is an exceptional counterpart of non vegetarian food1. It is a wealthy source of vitamins, fat, high class
animal protein and minerals. Due to convenience of various kinds of milk and variations in milk composition, a
variety of techniques have been instituted for the production of paneer as per the requirements of the consumers
with noteworthy improvement in the capitulation and other quality characteristics2. Good quality paneer can be
obtained from milk via some modifications in the developing procedure or through apply of additives or natural
products. Impregnation of vegetables not only declines the cost of paneer but also provides resourceful worldly
goods to it3.
Carrot (Daucus carota L.) is the for the most part crucial yield of Apiaceae family. It is a root vegetable that
has universal circulation. Carrot is a superior source of nutritional fiber and of the trace mineral molybdenum,
occasionally institute in diverse vegetables4. There are outstanding reasons to take in carrots into human food,
because they are supplemented with phenolic compounds, polyacetylenes, carotenoids, and vitamins and by this
cause they may assist reduce the threat of some disorders. Research data has acknowledged that these carrot
compounds exert anticarcinogenic, immunoenhancer, and antioxidative effects5. Hence, the present study was selected
to develop carrot incorporated paneer from cow milk and to appraise the sensory and nutritional properties of the
product.
321
Materials
The cow milk was procured from local milk producers and standardized according to Pearsons square
method to 6% fat using cow skim milk. Carrot and lemon were purchased from the local market at Erode, Tamilnadu,
India.
Preparation of cow milk paneer

1500 ml of cow milk was heated to 85C then temperature reduced to 80C and then removed all the froth
and skim. Later, added two spoons of lemon juice to the milk. Then milk was allowed to curdle at 75C and strained
it with muslin cloth. The curdled milk was then washed in muslin cloth with fresh cold water for four times. Then
the curdled milk was squeezed in a muslin cloth and a heavy weight was placed over it and after half an hour all the
particles of paneer was came close together to form a firm block of paneer and stored the paneer at 4C in a
refrigerator for further analysis6.
Preparation of carrot juice incorporated cow milk paneer

Fresh carrots were washed in distilled water then they were grinded to form a fine juice using a mixer.
Carrot juice was incorporated in fixed ratio of 20% to 1200 mL of cow milk and mixed well. The milk was heated
to 85C then temperature reduced to 80C. All the froth and skim was removed and added two spoons of lemon
juice. The milk was begun to curdle at 72C and strained it with muslin cloth. The curdled milk was then washed in
muslin cloth and a heavy weight was placed over it. After half an hour all the particles of paneer came close
together to form a firm block of paneer and stored at 4C in a refrigerator for further analysis7.
Organoleptic evaluation of product

Organoleptic evaluation of carrot incorporated paneer was carried out by sensory testing along with cow
milk panner as control. Members were selected for evaluation by polite solicitation. Panelists were selected based
on their interest. The order in which participants tasted the samples was not controlled. Sensory evaluation was
carried out under controlled temperatures. The appearance, color, texture, flavor, mouth feel, taste, and overall
acceptability of the products were evaluated by the sensory panelists according to 9-point Hedonic scale evaluation
as described by Karl Ruther3.
Proximate analysis
The carrot incorporated paneer and cow milk paneer were subjected to nutrient composition, namely
moisture, fat, protein, total ash, carbohydrate, crude fiber, -carotene, calcium, iron, and phosphorus by the methods
of AOAC (2003).
All results for the triplicates in each sample are expressed as the Mean S.D. All the samples data were statistically
evaluated using SPSS\10.0 software (Genericom, Germany). Hypothesis-testing methods used were the one-way
analysis of variance and the least-significant difference test. The significance levels were set at p< 0.05.
RESULTS
Cow milk paneer and carrot incorporated cow milk paneer were prepared by coagulated it with acidulate
like lemon 0.3%. Organoleptic evaluation was done by using hedonic scale, carrot incorporated paneer was
significantly higher acceptability than cow milk paneer as shown in table 1. Panel results were revealed that freshly
made paneer from cow milk incorporated with 20% carrot juice was liked more due to its superior flavour, colour,
appearance, taste and overall acceptability as compared to non carrot incorporated cow milk paneer.
322
Table 1: Organoleptic evolution of 20% carrot incorporated paneer and cow milk paneer
Attributes Cow Milk Paneer (Control) Cow Milk With 20% Carrot Paneer
Color 6.6 0.08 7.5 0.07
Appearance 7.6 0.10 8.4 0.08
Flavor 8.0 0.13 8.8 0.10
Taste 7.2 0.05 8.6 0.08
Overall Acceptability 7.4 0.18 8.7 0.25

Proximate analysis of carrot incorporated paneer revealed that rich in protein, iron, calcium, phosphorus
and -carotene when compared to cow milk paneer, and also revealed that reduced carbohydrates in carrot
incorporated paneer than to the control (Table 2).
Table 2: Proximate composition of 20% carrot incorporated paneer and cow milk paneer
Nutrients Cow Milk Paneer Cow Milk With 20% Carrot Paneer
Carbohydrate (gm/100 gm) 18.09 1.29 10.68 0.75

Lipid (gm/100 gm) 25.32 1.89 26.52 1.39
Protein (gm/100 gm) 10.73 0.75 20.36 1.29
Moisture (%/100 gm) 40.31 2.61 45.20 2.10
Total ash (%/100 gm) 1.10 0.25 1.54 0.59
Iron (mg/100 gm) 0.33 0.11 0.73 0.23
Calcium (mg/100 gm) 240.99 5.45 261.23 4.12
Phosphorus (mg/100 gm) 373.83 4.30 484.12 5.94
-carotene (mg/100 gm) 0.00 0.00 0.27 0.31

DISCUSSION
Paneer is a south Asian variety of soft cheese accomplished by acid and heat coagulation of milk. It is non-
fermentative, unripened, non-melting and non-renneted type of cheese8. For the vegetarian population as live in
India and across the globe, vigorous vegetable foods are scrupulous outcome. Such vegetable protein products
incorporated into milk products can make possible plummet the worth of milk manufactured goods9. A selection of
suitable foods can be produced from native products to all together in the peoples diet. Carrot is rated tremendously
high among vegetable foods when correctly utilized. It is a commendable source of considerable quantity of fat and
vegetable protein. Subsequently, in the current study an attempt was done to create carrot juice incorporated cow
milk paneer to make use of as enhanced nutritional resource.
Sensory evaluation is significant feature that reciprocally with taste, aroma, and visual appearance includes
the sensory distinction of foods. The only way to evaluate sensory characteristic by the grades of intuition experienced
by human being when consuming food which attributed by questioning the decision of consumer10. In the present
study we analyzed the sensory assessment which showing that carrot incorporated cow milk paneer publicized
323
superior satisfactoriness when compared to control. The assorted features for which the sample was painstaking
were flavor, taste, texture, colour and overall acceptability as given away in Table 1. It can be observe that the
appearance scores of carrot incorporated cow milk was comparable (P>0.05). The score for receipt is in agreement
with that represented by Singh and Kanawjia11 for recombined milk paneer.
In this study, we were used lemon juice as coagulant for the production of paneer. Sachdeva and Singh12
and Kumar et al13 observed that lessen in the flavour scores with the hoist in the attentiveness and level of acid. The
flavour scores more remarkable in carrot incorporated paneer (P>0.05) than control paneer. At lower coagulant
concentrations, coagulation was slower and more reliable due to extra systematic exploit of coagulant throughout
the milk assortment, while at higher concentration coagulation was immediate and non-uniform with higher level
of denaturation at a diversity of points in milk14. Sensory assessment revealed that carrot incorporated paneer has
the utmost acceptability than cow milk paneer. The improved acceptability and organoleptic virtues of carrot
incorporated paneer may be legitimate to carrot and its goodness.
In the present study, we observed that protein content was affluent in carrot incorporated paneer than cow
milk paneer, we also noticed that important demarcation in protein content of carrot incorporate paneer and cow
milk paneer which may be due to reposition of protein resource from carrot. Our study also revealed that carbohydrate
content was condensed in carrot incorporated paneer when compared to cow milk paneer, more exploit in budding
food products and formulate assured their reputation may be the comfortable of precise carbohydrates or groups of
carbohydrates15.
The proximate composition of foods take account of ash, protein, lipid, moisture, and fibre, carbohydrate,
iron, phosphorus, calcium contents. These food products may be of consideration in the food developing for product
enhancement, quality control, or dictatorial reasons16. The moisture content of foods is momentous for reasons
such as food expediency of transport, price, superiority, and reliability for expressing other analytical reasons17. In
our study found that carrot incorporated paneer has the supplementary moisture content than normal paneer. This
may be due to prejudiced segregation of moisture from paneer samples. Ash in a food product communicates to the
on the whole mineral content, and point out to the inorganic residue endures after either explosion or complete
deterioration of organic matter18. Our study revealed that carrot incorporated cow milk paneer contains rich in
minerals such as calcium, iron, and phosphorus than normal paneer. A statistically significant difference was observed
in ash content of carrot incorporated paneer and normal paneer samples, our results were in agreement as statemented
by Krishna19.
In conclusion, was made an attempt develop a product such as incorporation of carrot juice into traditional
milk paneer and evaluated nutritional and organoleptic aspects on the basis of chemical composition and sensory
evaluation. The 20% carrot juice incorporated paneer was shown greater in quality with respect to its physical and
chemical parameters and found that paneer with 20% carrot incorporated shown additional nutritional values. This
study may helps to paneer producers in India and other countries for mounting dietary standards by including
natural products such as carrot.
ACKNOWLEDGEMENT
The authors thank the Management of K. S. Rangasamy College of Arts and Science (Autonomous),
Tiruchengode, India for their encouragement and providing facilities to do this research work.
REFERENCES
1. Agnihotri M K & Pal UK (1996) Small Rut Res 20, 75-81
2. Shahnawaz Umer Khan & Mohammad Ashraf Pal (2011) J Food Sci Technol 48, 645660
3. Karl Ruther (2007) Int J of Dev Res 5, 2230-9926
4. Simon P W (2000) Pla Bre Rev 19, 157-190
324
5. Joao Carlos da Silva Dias (2014) Fd and Nut Sci 5, 2147-2156
6. Agrawal S P (2007) Dairy India Yearbook, 6th edn, pp. 413-414, Priyadarshini Vihar, New Delhi
7. Butool & Buttol (2015) Int J of Dev Res 5, 4124-4130
8. Supekar A L, Narwade S G, Kadam R P & Syed Imran Hashmi (2014) The Asi J of Ani Sci 9, 189192
9. Biradar G S, Gujar S K, Dande K G & Gaikwad SM (2012) J Anim Prod Adv 2, 142-145
10. Desai H K, Gupta S K, Patel A A & Patel G R (1991) Jap J Dairy Sci 40, 15-21
11. Singh S & Kanawjia SK (1991) Indian J Dairy Sci 44, 7679
12. Sachdeva S & Singh S (1988) J Food Sci Tech 25, 142145
13. Kumar S, Rai D C & Verma D N (2008) Indian J Anim Res 42,145149
14. Khan S U, Mohammad Ashraf Pal, Sarfaraz Ahmad Wani & Mir Salahuddin (2014) J Food Sci Technol 51,
565-70
15. Pomeranz Y & Meloan C E (1994a) Carbohydrates, in Food Analysis Theory and Practice, 3rd edn, pp.
625677, Chapman & Hall, New York
16. Pomeranz Y, Meloan CE (1994) General Remarks, in Food Analysis Theory and Practice, 3rd edn, pp.
567574, Chapman & Hall, New York
17. Bradley R (1998) Moisture and Total Solids Analysis, in Food Analysis, 2nd edn, pp. 119139, Aspen
Publishers, Gaithersburg, MD
18. Harbers LH (1998) Ash Analysis, in Food Analysis, 2nd edn, pp. 141149, Aspen Publishers, Gaithersburg,
MD
19. Krishna A (2003) J Food Sci Tech 40, 490-492
325
In vitro propagation and phytochemical studies of Anoectochilus elatus
- A medicinally important terrestrial orchid
Natarajan Parimala Devi*, Narmatha Bai*, Thangaraj Parimelazhagan1,
Sivaraj Dhivya1 and Lakkakula Satish2
*Plant Tissue Culture Lab, Department of Botany, School of Life Sciences, Bharathiar University,
Coimbatore 641 046, India. *Email: parimalaraj87@gmail.com
1
Bioprospecting Lab, Department of Botany, School of Life Sciences, Bharathiar University, Coimbatore 641 046, India.
2
Department of Biotechnology, Alagappa University, Karaikudi, India
Abstract
Anoectochilus elatus Lindl. is an endangered monopodial terrestrial South Indian Jewel Orchid grown for
beautiful foliage and its medicinal properties. The nodal explants were cultured on MS (half strength medium)
supplemented with various types and concentrations of cytokinins such as, BAP, KIN and TDZ (0.25, 0.5 1.0
and 2.0 mg/L) individually. Among the different combinations tested half strength MS medium supplemented
with BAP (0.5 mg/L) in combination with NAA (0.25 mg/L), IBA (1.0 mg/L), AC (0.5 g/L) and GA3 (1.0 mg/L)
induced maximum number of multiple shoots and roots after 135 days. A survival rate of 95 % was observed.
Screening of antioxidant activities using acetone and methanol extracts of micro propagated plants were carried
out and compared with that of the wild plant extracts showed higher antioxidant activities.
Keywords: Jewel orchid, BAP, NAA, DPPH, ABTS and FRAP
INTRODUCTION
Orchidaceae is the largest and most diverse family of the flowering plants, consisting of 30-35 thousand
species under 850 genera (Hossain et al., 2013). The highly fascinating flowers of orchids with brilliant coloration,
varied forms and sizes make them one of the most expensive ornamentals today. Orchid extracts and purified
compounds are shown to exhibit several bioactivities such as antimicrobial, antioxidant, anthelmintic and anti-
inflammatory activity (Kiran et al., 2013). The genus Anoectochilus consists of approximately 40 species distributed
from the Himalayas to New Caledonia and Hawaii. They are commonly called as Jewel orchids and King of
medicines. Anoectochilus species produce a wide range of biological compounds, including an alkaloid called
kinsenoside that is used to treat diabetes, hyperliposis, and breast cancer (Zhang et al., 2013).
Anoectochilus elatus Lindl. is an endangered monopodial terrestrial South Indian Jewel Orchid distributed
in Southern Western Ghats. In Kolli hills of Tamil Nadu it is commonly called as Mayilraegai saedi or Kairaegai
saedi. This plant has been used to cure chest and abdominal pains and to treat snake bites by tribal of Kolli hills.
Several studies related to this genus have revealed the presence of antidiabetic, antibacterial and antihyperliposis
activities (Ratnaweera et al., 2014). However, this plant has not been scientifically validated for its phytochemical
studies.
Healthy plants of Anoectochilus elatus were collected from the Valparai hills in the Southern Western
Ghats of Tamil Nadu, India and a voucher specimen (Accession no:176967) has been deposited in the herbarium of
BSI.
Effect of different growth regulators on multiple shoot and root induction

Surface sterilized nodal explants were inoculated on half strength MS medium supplemented with various
326
cytokinins such as BAP, KIN and TDZ (0.25, 0.5, 1.0 and 2.0 mg/L) individually or in combination with NAA
(0.25, 0.5 and 1.0 mg/L) and IBA (1.0mg/L) for multiple shoot and root induction. Addition of GA3 (1.0 mg/L) was
added to the same medium for elongation of shoots. Activated charcoal (0.5 mg/L) was added to the same medium
to remove the phenolic exudates.
Phytochemical studies
The in vitro propagated and wild Anoectochilus elatus plants (whole plants) were collected and the powdered
sample (10 g) was extracted by maceration using different solvents for further antioxidant studies. DPPH, ABTS,
FRAP and Phosphomolybdenum was observed for both in vitro propagated and wild plant extract according to the
standard methods. The results were expressed as Mean Standard Deviation and the data were statistically analyzed
using one way ANOVA followed by Duncans test for tissue culture and antioxidant studies. Mean values were
considered statistically significant at p 0.05.
RESULTS
Of the various cytokinins tested, half strength MS medium supplemented with TDZ (1.0 mg/L) was found
to be superior in inducing maximum number of multiple shoots 6 in 72 days (Table - I). Among the different
combinations of growth regulators tested, half MS + BAP (0.5 mg/L) + NAA (0.25 mg/L) + IBA (1.0 mg/L) + AC
(0.5 g/L) and GA3 (1.0 mg/L) (Fig IA) induced maximum number of multiple shoots and roots (Fig IB) (Table
II). A survival rate of 95% was observed when maintained under culture room condition (252 C).
Phytochemical studies
The in vitro propagated and wild plants of A. elatus percentage yield for the extracts were calculated and
presented in the Figure II.
DPPH scavenging activity
Acetone extract (253.20 g/mL) of in vitro propagated plant and methanol extract (146.83 g/mL) of wild
plant, showed the lowest IC50 value (Figure: III and IV).
ABTS + scavenging activity
The highest ABTS radical cation scavenging activity was found in acetone extract (253.09 10.07 M TE/g
extract) of in vitro propagated plant methanol extract (95.70 1.23 M TE/g extract) of wild plant (Table - III).
FRAP (Ferric Reducing Antioxidant Power) assay
Acetone extract (904.20 4.35 mM Fe(II) E/g extract) of in vitro propagated and wild plant (930.28 4.30
mM Fe(II) E/g extract) showed higher ferric reducing ability (Table - III).
Phosphomolybdenum assay
Acetone extract of in vitro propagated plant (18.97 2.17 mg AAE/g extract) and wild plant (12.31 1.59
mg AAE/g extract) depict the better antioxidant activity when compared to all the other extracts (Table - III).
Discussion
Habitat destruction and over exploitation are the two important factors threatening the survival of orchids
in India. Plant tissue culture technology has been successfully used for the commercial production of pathogen-free
plants and to conserve the germplasm of rare and endangered species. Anoectochilus elatus Lindl. is commonly
known as Jewel orchids grown for its beautiful foliage and various medicinal properties. In the present study, an
attempt has been made to develop a standardized protocol for mass propagation using the nodal explants.
327
Half strength MS medium supplemented with different cytokinins promoted shoot proliferation. Among
the different cytokinins tested, TDZ at 1.0 mg/L was favorable for inducing maximum number of multiple shoots.
Similar promotive effect of TDZ has also been reported in A. elatus (Sherif et al., 2012). In A. elatus, of the
different combinations of growth regulators tried MS medium + BAP (1.0 mg/L) + NAA (0.25 mg/L) along with
GA3 (1.0 mg/L) and AC (0.5 mg/L) was found to be best for the formation of multiple shoots. Similar results were
also obtained in Malaxis acuminata (Arenmongla and Deb, 2012) from the nodal segments. Root initiation was
achieved with IBA in conjunction with AC resulted more vigorous growth, rooting and production of healthy
leaves. Similar observations were made in A. formosanus and A. elatus (Ket et al., 2004 and Sherif et al., 2012).
The extract yield percentage of Anoectochilus elatus showed maximum yield in water and ethyl acetate
extract of in vitro propagated and wild plant extracts. DPPH assay is simple, sensitive and rapid assay for measuring
the capacity of several types of samples including herbal extracts to scavenge free radicals. In A. elatus, the
phytoconstituents of acetone extract (253.20 g/mL) of in vitro propagated plant and methanol extract (146.83 g/
mL) of wild plant might be capable enough to donate hydrogen atom thereby reducing hydrazyl to hydrazine thus
giving more yellow complex. The result obtained from the present study relates to the previous accounts as in and
Aphyllorchis Montana (Mahendran and Narmatha Bai, 2016). Synthetic antioxidant BHT was used as reference
compound.
ABTS assay of acetone extract (253.09 10.07 M TE/g extract) of in vitro propagated plant and methanol
extract (95.70 1.23 M TE/g extract) of wild plant in A. elatus showed higher radical scavenging activity when
compared to that of other solvent extracts (Table IV). The present study is in agreement with Pholidota pallida
(Nagananda et al., 2014).
The FRAP assay is a simple and reproducible method to study the antioxidant efficiency of pure dietary
antioxidants. In A. elatus acetone extract (904.20 4.35 Fe(II) E/g extract) of in vitro propagated plant and wild
plant (930.28 4.30 Fe(II) E/g extract) showed the highest ferric ion reducing ability (Table IV) was found to be
similar when compared to the other quantification and antioxidant assays (ABTS+ and DPPH+ radical scavenging).
Similar results were also observed in Dendrobium thyrsiflorum (Bhattacharya et al., 2015).
Phosphomolybdenum assay is successfully used to determine the ability of extracts to reduce Mo (VI) to
Mo (V) and subsequent formation of green phosphate/Mo (V) complex at an acid pH. Acetone extract (18.97
2.17 mg AAE/g extract) of in vitro propagated and wild plant (12.31 1.59 mg AAE/g extract) showed superior
results in A. elatus (Table IV). Similar results were also obtained in Flickingeria nodosa (Nagananda et al., 2013).
From these the inference can be made sure that all the extracts can scavenge different kinds of free radicals in a way
or the other. The phytochemical profiles of in vitro propagated and wild plant samples varied, which may be due to
the influence of cultural environment and also in vitro conditions the exogenous factors such as organic and inorganic
components in the medium, growth hormones, light and temperature have strong influence on growth and secondary
metabolism.
ACKNOWLEDGEMENT
I am extremely thankful to Dr. K. Gopinath, Alagappa University, Karaikudi for his kind support and co-
operation. I am also thankful to my family who provided me these opportunities, supported and strengthen me at
every face of this study.
REFERENCES
1. Bhattacharya, P., Kumaria, S., Job, N., & Tandon, P. (2015). Phyto molecular profiling and assessment of
antioxidant activity within micropropagated plants of Dendrobium thyrsiflorum: a threatened, medicinal
orchid. Plant Cell Tissue and Organ Culture, 122 535-550
328
2. Ket, NV., Hahn, EJ., Park, SY., Chakrabarty, D., & Paek, KY. (2004). Micropropagation of an endangered orchid
Anoectochilus formosanus. Biologia Plantarum, 48 339344
3. Kiran, R., Kekuda, PTR., Kumar, PHG., Hosetti, BB., & Krishnaswamy, K. (2013). Biological activities of
Sarcanthus pauciflorus. Journal of Applied Pharmaceutical Science, 3 105-110
4. Mahendran, G., & Narmatha Bai, V. (2016). An efficient in vitro propagation, antioxidant and antimicrobial
activities of Aphyllorchis Montana (Reichenb.f). Plant Biosystems, 150 10871095
5. Nagananda, GS, Ashwini Patil., Jayakumar, V Kambli., & Rajath, S. (2014). Phytochemical evaluation and
in vitro free radical scavenging activityof cold and hot successive peudo bulb extracts of medicinally important
orchid Pholidota pallida Lindl. Advances in Bioresearch, 5 100-105
6. Nagananda, GS., Rajath, S., Anagolakar, SP., & Lohar, RM. (2013). Phytochemical evaluation and in vitro
free radical scavenging activity of successive whole plant extract of orchid Cottonia peduncularis. International
Journal of Pharmacy and Biological Sciences, 3 9197
7. Sherif, NA., Benjamin, JHF., Muthukrishnan, S., Senthil Kumar, T., & Rao, MV. (2012). Regeneration of
plantlets from nodal and shoot tip explants of Anoectochilus elatus Lindley, an endangered terrestrial orchid.
African Journal of Biotechnology, 11 7549-7553
8. Zhang, F., LVY., Zhao, Y., & Shun- xing, GUO. (2013). Promoting role of an endophyte on the growth and
contents of kinsenosides and flavonoids of Anoectochilus formosanus Hayata, a rare and threatened medicinal
orchidaceae plant. Journal of Zhejiang University- Science B (Biomedicine and Biotechnology), 14 785
792
329
Mechanism of In vitro Inhibition of Alpha-amylase and Alpha-glucosi-
dase Activities By Ethanolic Extracts of Polyalthia longifolia Bark
P. Gayathri and G.P. Jeyanthi
DDepartment of Biochemistry, Biotechnology and Bioinformatics,
Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore-641043.
Abstract
Diabetes mellitus is a metabolic disorder characterized by hyperglycemia and is considered to be one of the free
radical mediated ailments. One of the current trends in the management of diabetes mellitus is reducing intestinal
glucose absorption by inhibition of carbohydrate metabolizing enzymes alpha-amylase and alpha-glucosidase.
In the present study, various solvent extracts of Polyalthia longifolia bark has been screened for the inhibition
of alpha-amylase and alpha-glucosidase activities. Ethanolic extract of the bark was found to exhibit highest
enzyme inhibitory activities. Hence the mechanism of alpha-amylase and alpha-glucosidase inhibitory activity
of the ethanolic extract of the bark was evaluated by Dixon plot and Cornish-Bowden plot. Dose dependent
increase in alpha-amylase and alpha-glucosidase inhibitory activities was observed. Results revealed that the
ethanolic extract of the bark of Polyalthia longifolia exhibited competitive mode of inhibition of alpha-amylase
and alpha-glucosidase activities.
Key words: Polyalthia longifolia, Bark, Alpha-amylase, Alpha-glucosidase, Dixon plot, Cornish-Bowden
plot, Ethanolic extract
INTRODUCTION
Type 2 diabetes mellitus is a clinical syndrome due to relative or absolute deficiency of insulin or resistance
to the action of insulin at the cellular level resulting in hyperglycemia and glycosuria. The World Health Organization
(WHO) has estimated that by 2025, India, China and the United States will be the top three countries to have large
number of diabetic patients1. Modern medicines which offer variety of effective treatment options can have several
side effects like hepatotoxicity, flatulence, abdominal pain and hypoglycemia2.
Herbal drugs are popular for their safety and efficacy. Scientific investigations are needed to prove the
efficacy of these wonder drugs to make them reach a wider population. One of the emerging trends in the management
of diabetes mellitus is exploring the medicinal plants / natural products for potent inhibitors of carbohydrate
metabolizing enzymes alpha-amylase and alpha-glucosidase which would help in the regulation of blood glucose
level 3,4.
Polyalthia longifoliaSonn.Thwaitesisanevergreentreewithconicalcrown,belongingtothefamilyof
Annonaceae. P.longifoliacv.pendulareferredasKasthadharuinAyurveda,isotherwisecalledfalseAshoka.Itis
referred as Asokamu in Telugu; Nettulingam, Asogu in Tamil, Aranamaram, Assoti in Malayalam. In Ayurveda, the
bark of P. longifoliaisusedforthetreatmentofskindisease,diabetes,hypertensionandhelminthiasisItisalso
being used as a substitute for Ashoka (Saraca indica) bark due to its easy availability in nature. Almost all parts of
the plant are used in the Indian traditional system of medicine for the treatment of various human ailments5. Reports
on the antidiabetic property of Polyalthia longifolia leaves are available, yet no scientific investigation proving the
antidiabetic potential of the bark is available.
Hence, the present study was designed to study the antidiabetic property of Polyalthia longifolia bark in
terms of inhibition of alpha-amylase and alpha-glucosidase activities and the mechanism of inhibition using Dixon
plot and Carnish-Bowden plot.
330
Collection and processing of plant material
The bark of Polyalthia longifolia was collected from Central Institute for Medicinal Plants Heritage (CIMH),
Kanjikode, Kerala and authenticated by the botanist in Institute of Forest Genetics and Tree Breeding (IFGTB),
Coimbatore and Voucher specimen is deposited at IFGTB, Coimbatore. The bark sample collected was shade dried,
pulverized by mechanical grinder, made into powder and stored in air tight container.
Preparation of extracts and screening studies

The solvent extracts of Polyalthia longifolia bark namely aqueous (PLA), ethyl acetate (PLEA), ethanol
(PLE), chloroform (PLC) and hexane (PLH) were prepared by placing the bark in five parts of respective solvent in
mechanical shaker for 48 hours at 40C with 100 revolutions per minute, filtered, concentrated by lyophilization/
flash evaporation and stored at 4C. The extracts were screened for inhibition of alpha-amylase activity using
starch as substrate by the method of Bernfeld6, inhibition of alpha-glucosidase using para-nitro phenyl -D
glucopyranoside7. The ethanolic extract was found to possess highest -amylase and -glucosidase inhibitory
activities. Hence, further experiments were performed with the ethanolic extract of the bark.
Inhibition of alpha-amylase activity

The ethanolic extract of Polyalthia longifolia (PLE) was tested for its dose dependent effect on inhibition
of alpha-amylase activity by the method of Bernfeld6 with slight modifications. The reducing sugars produced by
the action of -amylase react with dinitrosalicylic acid and reduce it to a coloured product, which is measured at
540nm.Theamylaseinhibitorinhibitstheactionofamylasethathydrolysesstarchtomaltose.Inbrief,25Lto
200LofPLE(1mg/mL)wereallowedtoreactwith200Lofporcinepancreatic-amylaseenzyme(dissolved1
mg-amylase/mL of0.1Msodiumacetate buffer,pH4.7)and100 Lof0.1Msodiumacetatebuffer,pH4.7.
After20minofincubation,0.5mLof1%starchwasadded.Thesamewasperformedforthecontrolwhere200L
of enzyme was replaced by the buffer. Incubated for 15 minutes and added 1 mL of DNS (dissolved by stirring 1 g
dinitrosalicylic acid, 200 mg crystalline phenol in 100 mL of 1% NaOH solution, 50 mg sodium sulphite added at
the time of use) to both the control and test. The tubes were kept in a boiling water bath for 10 minutes. At hot
conditions added 0.5 mL of 40 % potassium sodium tartarate and made up the volume to 5 mL with distilled water.
The absorbance was recorded at 540nmusingaspectrophotometerandthepercentageof-amylaseinhibitionwas
calculated using the formula,
Inhibition (%) = 100 (Absorbance Control - Absorbance Test / Absorbance Control)
Suitable reagent blank and inhibitor controls were also carried out and subtracted the absorbance values
from the test value. Acarbose was used as standard inhibitor and tested in parallel at varying concentrations (10-100
g).
Inhibition of alpha-glucosidase activity

PLE was tested for its dose dependent effect on inhibitionof-glucosidaseactivity.Thiswasdeterminedby
measuring the release of 4-nitrophenol from p-nitrophenyl -D glucopyranoside7. The assay mixtures for these
experiments contained 0.3 mL of 10 mM paranitrophenyl alpha-D-glucopyranoside, 1.0 mL of 0.1 M potassium
phosphatebuffer,pH-6.8,0.2mLofenzymesolutionand25Lto200LofPLEallinafinalvolumeof1.7mL.
Following an incubation time of 30 min at 37C, the reaction was terminated by the addition of 2.0 mL of 100 mM
sodium carbonate. The liberated p-nitrophenol was determined at 400 nm using spectrophotometer. The percentage
inhibition rates were calculated using the formula
Suitable reagent blank and inhibitor controls were also carried out and the absorbance values were subtracted.
Acarbose was used as standard inhibitor and tested in parallel at varying concentrations (10-100 g).
331
Mechanism of inhibition of alpha-amylase
Dixon plot
Differentconcentrationsof PLEat40-200g/mLwereusedforstudyingthemechanismof-amylase
inhibition as given by Dixon8. 100 L of enzyme (1 mg / mL of 0.1 M sodium acetate buffer, pH 4.7) was kept for
15 min incubation along with various concentrations of inhibitor (40-200 g) and three different concentrations of
starch say 0.9, 1.0, 1.1 % was added and incubated for 20 min. This serves as the test in the presence of inhibitor.
Inhibitor control was carried out in a similar way as the test, but buffer was taken in the place of enzyme. Incubated
for 15 minutes and added 1 mL of DNS to both the control and test. The tubes were kept in a boiling water bath for
10 minutes. At hot conditions added 0.5 mL of 40 % potassium sodium tartarate and made up the volume to 5 mL
with distilled water. The absorbance was recorded at 540 nm using a spectrophotometer and the percentage of
-amylaseinhibitionwascalculatedusingtheformula
Test and control for enzyme assay at two different starch concentrations were also prepared. Read the
absorbance at 560 nm. Absorbance values were extrapolated in a standard graph for maltose and found the amount
of maltose produced in the presence of inhibitor. This was taken as the product concentration [V]. Calculated 1/[V]
values and plotted a graph with the concentration of inhibitor [I] taken along X-axis and (1 / [V]) along Y- axis.
From the nature of graph obtained, the mechanism of inhibition was studied as suggested by Eisenthel and Cornish-
Bowden9.
Cornish - Bowden plot

The experiment was performed as mentioned in Dixon plot. The difference here lies in plotting a graph
with the concentration of inhibitor [I] taken along X-axis and substrate concentration divided by product concentration
(S / [V]) along Y- axis. From the nature of graph obtained the mechanism of inhibition was studied as suggested by
Eisenthel and Cornish-Bowden9.
Mechanism of inhibition of alpha-glucosidase

Dixon plot
DifferentconcentrationsofPLEat20-100g/mLwereusedforstudyingthemechanismof-glucosidase
inhibition as given by Dixon8. The assay mixtures for these experiments contained varying volume of 10 mM
paranitrophenyl-D-glucopyranoside(275, 300 and 325 L), 1.0 mL of 0.1 M potassium phosphate buffer of pH-
6.8, 0.2 mL of enzyme solution and PLE taken at different concentrations all in a final volume of 1.7 mL. Following
an incubation time of 30 min at 37C, the reaction was terminated by the addition of 2.0 mL of 0.1 M sodium
carbonate. The liberated p-nitrophenol was determined at 400 nm using spectrophotometer. Suitable reagent blank
and inhibitor controls were also carried out and subtracted. This was taken as the product concentration [V].
Plotted a graph with the concentration of inhibitor [I] along X-axis and 1 / [V] along Y-axis. From the nature of
graph obtained, the mechanism of inhibition was studied as suggested by Eisenthel and Cornish-Bowden9.
Cornish-Bowden plot
The experiment was performed as mentioned in Dixon plot. The difference here lies in plotting a graph
with the concentration of inhibitor (I) taken along X-axis and substrate concentration divided by product concentration
([S] / [V]) along Y-axis. From the nature of graph obtained, the mechanism of inhibition was studied as suggested
by Eisenthel and Cornish-Bowden9.
Alpha-amylase and alpha-glucosidase inhibitors have potential value in the control of kinetics of carbohydrate
digestion and absorption which could be exploited in the prevention and control of conditions such as diabetes,
obesity and hyperlipidaemia10,11. Inhibitors of these enzymes have been recently developed from natural sources.
332
Study on the plants and plant parts for alpha-amylase, alpha-glucosidase inhibition, antioxidant potential along
with testing biosafety of plant extracts in selected cell-lines becomes important in choosing good candidature
plants for extensively exploring on the antidiabetic property using animal models12.

Inhibition of alpha-amylase and alpha-glucosidase actvities by various extracts of P.longifolia bark
Inhibitionof-amylaseand-glucosidaseactivitiesbyaqueous,ethylacetate, ethanol, hexane and chloroform

extracts prepared from the bark of P. longifolia is presented in Table 1.
Table 1 - Alpha-amylase and Alpha-glucosidase inhibition by various extracts of the bark of

Polyalthia longifolia
Extract Alpha-amylase Alpha-glucosidase

% Inhibition
PLA 83 0.50 81 0.12
PLEA 85 0.82 86 0.20
PLE 91 0.51 88 0.22
PLH 58 0.70 55 0.18
PLC 36 0.50 42 0.14
Values are mean SD of triplicates; PLA- P. longifolia aqueous extract; PLC - P. longifolia chloroform extract ;
PLE- P. longifolia ethanol extract; PLH- P. longifolia hexane extract
Among all the extracts tested, the ethanolic extract of P. longifolia (PLE) bark showed 91 % and 88 %
inhibitionof-amylaseand-glucosidase activities respectively. This was followed by ethylacetate, aqueous, hexane
and chloroform extracts respectively. Depending upon the efficiency of the solvents in extracting the active principles,
varioussolvent extractsexhibit differencein theirinhibitionof -amylaseand -glucosidase. The polarsolvent
extracts namely aqueous, ethylacetate and ethanol extracts of P. longifoliaweremorepotentinhibitorsof-amylase
and-glucosidaseactivities,whereasthenon-polarsolventextractsnamelyhexaneandchloroformextractsshowed
moderate inhibition of these enzyme activities. This may be attributed due to the difference in the class of
phytochemicals getting extracted in various solvent systems exhibiting alpha-amylase and alpha-glucosidase
inhibition.
The-amylaseinhibitorypropertiesoftheethanolicextractsofMurrayakoeniigiiandTamarindusindica
have been reported13.Pancreatic-amylaseinhibitorsofferaneffectivestrategytolowerthelevelsofpostprandial
hyperglycemiavia controlofstarch breakdown.Several-amylase inhibitorsincludingacarbose, vogliboseand
miglitol are clinically used for treatment of Type 2 diabetes mellitus (Type 2 DM), but their cost is high and clinical
side effects are likely to occur. Natural-amylaseand-glucosidaseinhibitorsfromtraditionallyvaluedfoodand
medicinal plants can provide benefits by controlling postprandial hyperglycemia without side effects for the
management of diabetes 14.
Inhibition of Alpha-amylase and alpha-glucosidase inhibitory activities by the ethanolic extracts of Polyalthia
longifolia bark and its mechanism of Inhibition
The ethanol extract of the bark was further investigated for the dose dependent inhibitory activities and the
mechanism of inhibition.
333
Alpha-amylase inhibitory potential
Alpha-amylase inhibition by PLE and acarbose is presented in Figures 1a and 1b respectively. PLE exhibited
highest -amylase inhibition of 96% at 175 g and 92% at 200 g respectively. The inhibition attained saturation
withfurtherincreaseintheconcentrationofextracts.Theinhibitionof-amylasebystandardacarbosewasfound
to be 94% at a concentration of 70 g. The IC50 values of PLE and acarbose were found to be 41 g, 82.3 g and 7
g. A positive correlation between percentage inhibition of alpha-amylase and the concentrations of the extracts/
acarbose was observed which was significant at 5% level. This proved the dose-dependent inhibition by the extract
and standard acarbose.
A dose-dependent increase in alpha-amylase inhibition by the ethanolic extracts of Evolvulus alsinoides
have been reported15.
Alpha-glucosidase inhibitory potential

The alpha-glucosidase inhibition by PLE and standard acarbose are presented in Figures 2a and 2b
respectively. PLE exhibited highest -glucosidase inhibition of 91% at 175 g and 88% at 200 g respectively. Alpha-
glucosidase inhibition by the standard acarbose was found to be 92% at a concentration of 50 g. The IC50 values
of PLE and acarbose were found to be 77 g and 5 g. A positive correlation between percentage inhibition of
alpha-glucosidase and the concentrations of the extract/acarbose was observed which was significant at 5% level.
This proved the dose-dependent inhibition of alpha-glucosidase shown by the extract and standard acarbose.
Fig. 1. Alpha-amylase Inhibition by ethanolic extract of the bark of Polyalthia longifolia and acarbose
a. Ethanolic extract of Polyalthia longifolia bark b. Acarbose
Similar results have been reported in the study using the methanolic extracts of Psidium guajava leaves16.
Many herbal extracts have been reported for their anti-diabetic activities and are being used in Ayurveda for the
treatment of diabetes. The results of in vitro inhibition of alpha-amylase and alpha-glucosidase indicate that the
bark of the chosen medicinal plant Polyalthia longifolia can act as good source of natural inhibitors for these
enzymes.
334
Mechanism of alpha-amylase inhibition
Mechanismofinhibitionof-amylaseby PLEisdepictedinFigures2aand2b,respectively.Resultsof
DixonplotandCornish-BowdenplotrevealedthatPLEexhibitedcompetitiveinhibitionof-amylase.
The competitive mode of inhibition by PLE obtained from the Dixon and Cornish-Bowden plot emphasizes
the fact that the active principles in the extract compete with the substrate for binding to the active site of the
enzyme, thereby preventing or slowing down the breakdown of oligosaccharides to disaccharides.
Competitivemodeofinhibitionof-amylaseactivitybyUrtica dioica and Juglans regia extracts17,18 supports

our results. The variation in the mechanism of inhibition among different medicinal plants may be attributed due to
the active constituents present.
S
PLE
a. Dixon plot b. Cornish-Bowden plot plot

Fig 2. Mechanism of alpha-amylase inhibition by ethanolic extract of Polyalthia longifolia bark
Mechanism of alpha-glucosidase inhibition
The mechanism of -glucosidaseinhibitionbyPLEisdepictedinFigures3aand3brespectively.Results

ofDixonplotand Cornish-BowdenplotrevealedthatPLEexhibitedcompetitiveinhibitionof-glucosidase.
This implies that the active components of the extract compete with the substrate for binding to the active
site of the enzyme, thereby preventing or slowing down the breakdown of disaccharides to monosaccharides19.
Similar type of inhibition of alpha-glucosidase was observed in Picralima nitida leaves20.
PLE S PLE
a. Dixon plot b. Cornish-Bowden plot
Fig 3. Mechanism of alpha-glucosidase inhibition by ethanolic extract of Polyalthia longifolia bark
335
In conclusion, the present study demonstrated that the ethanolic extract of the bark of Polyalthia longifolia
exhibited efficient inhibition of alpha-amylase and alpha-glucosidase activities and total antioxidant potential. The
mechanism of inhibition of these two enzymes was found to be by competitive mode. This is the first report
showing the alpha-amylase and alpha-glucosidase inhibitory properties of the bark of Polyalthia longifolia and its
mechanism of action.
ACKNOWLEDGMENT
The authors wish to express their sincere thanks to Dr. B. Thayumanavan, Rtd Professor of Biochemistry,
Tamil Nadu Agricultural University, Coimbatore for the guidance rendered during the study.
REFERENCES
1. Poongothai K, Ponmurugan P, Ahmed K S Z, Kumar B S and Sheriff S A, Asian Pacific Journal of Tropical
Medicine, (2011) 4, 10, 778-785.
2. Kathirvel A, Prabhakar T, Vinoth Kumar, Sadasivam, S and Thayumanavan, B (2012) IJPSR 3, 316-322.
3. Bhat M, Zinjarde S S, Bhargava S Y, Kumar A R and Joshi B N (2008), Evidence - Based Complementary
and Alternative Medicine 1, 324-328.
4. Doshi G M, Zine, S P, Chaskar, P K and Une, H G (2014), Pharmacognosy Res 6, 234-239.
5. Sampath M and Vasanthi, M (2013) Int J Pharm Pharm Sci, 5, 1.
6. Bernfeld, P (1955) Methods in Enzymology, Vol 1, Academic press, New York, 149.
7. Sun Z, Duke, S H and Henson, C A (1995) Plant Physiol, 108, 211-217.
8. Dixon, M. (1953) JBC 55,170-171.
9. Eisenthal, R and Carnish-Bowden, A (1974) JBC 139, 721-730.
10. Kim, S D and Nho H J (2004) Journal of Microbiology 42, 223-227.
11. Alagesan K, Thennarasu P, Kumar V, Sankarnarayanan S and Balsamy T (2012) IJPSR 3, 316-322.
12. Gayathri P and Jeyanthi G P (2013), IJCPS 4, 87-96.
13. Narkhede M B (2012) Asian J of Pharm Clin Res 5, 75-76.
14. Sudha P, Smita S, Zinjarde A, Bhargava Y S and Kumar A R (2012) BMC Complement Altern Med 11, 1-10.
15. Gomathi D, Muthulakshmi C, Guru Kumar D, Ravikumar G, Kalaiselvi M and Uma C (2012) Asian Pacific
Journal of Tropical Biomedicine, s67-s73.
16. Manikandan R, Vijaya anand A and Duraimuthumani G (2013) Int J Curr Microbiol Appl2,15-19.
17. Karthick K, Kirthiram K S, Sadasivam S and Thayumanavan B (2008) IJEB 6, 677-680.
18. Rahimzade M, Jahanshahi, S, Moein, S and Moein, M R (2014) IJBMS 17, 465-469.
19. Ogunwande A, Matsui T, Fujise T and Matsumoto K (2007) Food Sci Technol Res, 13, 169-172.
20. Kazeem M I, Ogunbiyi J V and Ashafa A O T (2013) TropJPharmRes, 12, 719-725.
336
Bioactive Optimization For Antioxidant Activity Of Whey Protein
Hydrolysate A Natural Antioxidant
K. Devi1, M. Suriya, Srilekha, Sravanthiand Sundaramoorthy Haripriya 2
1
Assistant Professor, Dept. Food Science and Nutrition, Avinashilingam Institute for Home Science and Higher Education for Women,
Coimbatore 641 043. devi.karuthapandian@gmail.com
2
Assistant Professor, Pondicherry University, Puducherry 605 014.
Abstract
Whey proteins are not only nutritionally enriched protein, also bioactive protein encrypted with bioactive peptides
in primary sequence. Hence the present study was based on the enzymatic hydrolysis of whey protein isolate
(WPI) to release antioxidative peptides in the production of antioxidative whey protein hydrolysate (WPH)
using trypsin. One factor at a time method and response surface methodology were sequentially employed to
optimize hydrolysis conditions such as substrate concentration (2-14 % w/v), Enzyme substrate ratio (E/s, 0.1
3.1 % w/w), pH (6 -12), temperature (30-50C) and time (2 24 hrs). Antioxidant activity of hydrolysate was
measured in term of reducing power. Results showed that the substrate concentration (10 % w/v), E/s ratio
(1.14%w/w), pH (8.9), temperature (39.79C) and time (6 hrs) were optimum for antioxidative whey protein
hydrolysate with maximum reducing power (0.595 at 700 nm) than WPI (P<0.05). SDS PAGE analysis showed
the presence of low molecular weight peptides in WPH as compared to WPI. Hence the present study proved
that RSM optimization in enzymatic hydrolysis that could enhance the antioxidant activity of WPI. Antioxidative
WPH couldbe used as a natural antioxidant in the explorative field of natural antioxidants and also an antioxidative
protein supplement in the context of functional foods.
Key words: Antioxidant activity, whey protein hydrolysate, reducing power, optimization, response surface
methodology.
INTRODUCTION
Enzymatic hydrolysis of dietary proteins has been explored in the production ofantioxidative peptides in
the explorative field of natural antioxidants.Antioxidative peptides from the enzymatic hydrolysis of proteins such
as whey protein (Penget al., 2009), soy protein (Kong andXiong, 2006)), fish protein (Thiansilakul, Benjakul,
&Shahidi, 2007), egg yolk protein (Senji&Yumi, 2006), canola protein (Cumby, Zhong, Naczk, &Shahidi, 2008)
have been reported. Whey proteins have been widely exploited among dietary proteins for the production of
antioxidative peptides based on its underutilization as the byproduct of cheese industry. Whey protein are actually
20 percent of milk proteins with a mixture of bioactive proteins such as -lactalbumin, -lactogloublin,
immunoglobulins, lactoferrin, lactoperoxidase etc.
Antioxidant activity of whey protein hydrolysatehas been demonstrated in an iron-catalyzed liposome

oxidation system (Xiong, et al., 2001) or a copper-catalyzed liposome emulsion (Colbert et al, 1991), in cooked
meat pork patties (Pena, et al., 2003). These studies reveal that the antioxidant activity of protein hydrolysate
depends on the proteolytic activity of enzyme on the substrate resulting in the production of peptide composition
which depends on the hydrolytic conditions including pH, temperature, enzyme and substrate ratio and hydrolysis
time. Hence the production of antioxidant peptides could be maximized by tailoring theses hydrolytic conditions in
the cost effective manner.
Response surface methodology (RSM) is a multivariate statistical technique to optimize the bioprocess
with many independent variables over the traditional one factor at a time method (OFAT). The application of RSM
has been demonstrated to optimize the hydrolysis conditions to result in maximum antioxidant activity of peptides
337
generated from food proteins such as fish protein hydrolysates (Guerardet al.,2007&Renet al., 2008). However,
there are lacks of studies in preparation of antioxidative whey protein hydrolysatesusing RSM. Hence the present
study was aimed at the optimization of the production of antioxidative whey protein hydrolysate using trypsin
enzyme by the application of RSM in the context of cost-effective bioprocess technology.
Whey protein isolate (WPI) was purchased from Marin Agro private limited, New Delhi Trypsin enzyme
from Himedia, DPPH (1,1-diphenyl-2-picrylhydrazyl) from Sigma-Aldrich and methanol, ferric chloride, potassium
ferricyanide, trichloro acetic acid, sodium dodecyl sulphate, mercaptoethanol, Tris-HCl, Coomassie Blue dye,
Bromophenol blue dye from Himedia, India were purchased.
Preparation of whey protein hydrolysate

Whey protein isolate was dissolved in distilled water at the substrate concentration (protein content basis)
and the hydrolysis of suspension was carried out by the addition of enzyme at the hydrolytic conditions as per the
experimental design of optimization. The hydrolytic pH was maintained using 0.1N NaOH or HCL. The termination
of hydrolysis was carried out at 85C for 20 min after the required time of hydrolysis and the supernatant of the
hydrolysate was collected by centrifugation at 10000 rpm for 30 min and stored at 4C for the analysis of antioxidant
activity.
Optimization
Optimization was carried out through two steps. (i) One factor at a time method (OFAT) and Response
surface methodology (RSM)
(i). OFAT method: This method was first used to identify the possible optimum range of hydrolytic conditions for
maximum antioxidant activity of whey protein hydrolysate (WPH). In this method, the level of one hydrolytic
variable was varied keeping the other hydrolytic variables at the constant level.
(ii). Response surface methodology (RSM) was appliedto further optimize the possible optimum range of hydrolytic
conditions out of OFAT method. Central composite rotatable (CCRD) design was chosen to obtain design matrix
and actual experiments were carried out accordingly. Second order polynominal model was obtained out of actual
experimental value and adequacy of the model was analyzed for predictability to predict hydrolytic conditions for
maximum antioxidant activity. The individual, interactive and quadratic effect of hydrolytic variables on reducing
powder of WPH was expressed through surface plots.
Reducing power assay

Reducing power was analyzed based on the reduction of potassium ferricyanide by WPH (Oyaizu, 1986)
and protein content of WPH was estimated by the method of Bradford (1976).
RSM methodology was carried out using Design Expert software (Version 6.0.8, Stat-Ease Inc., Minneapolis,
USA). The significance of difference in reducing power of WPH was analyzed using one way ANOVA and Duncun
multiple range test (DMRT) using SPSS version.

Optimization by One factor at a time method
In this first phase, the possible optimum range of hydrolytic conditions were around the substrate ratio of
the substrate concentration of 10% (w/v on the basis of protein content), E/s ratio of 1.1 ( % w/w), pH of 9,
338
Table I : CCRD matrix and experimental value of reducing power
S.No E/s ratio pH Temperature Hydrolysis time Reducing power

(%) (C) (hr) at 700 nm
1 0.60 7.50 35.00 14.00 0.285
2 1.60 7.50 35.00 14.00 0.244
3 0.60 9.50 35.00 14.00 0.281
4 1.60 9.50 35.00 14.00 0.329
5 0.60 7.50 45.00 14.00 0.263
6 1.60 7.50 45.00 14.00 0.329
7 0.60 9.50 45.00 14.00 0.271
8 1.60 9.50 45.00 14.00 0.293
9 0.60 7.50 35.00 20.00 0.259
10 1.60 7.50 35.00 20.00 0.299
11 0.60 9.50 35.00 20.00 0.337
12 1.60 9.50 35.00 20.00 0.388
13 0.60 7.50 45.00 20.00 0.226
14 1.60 7.50 45.00 20.00 0.292
15 0.60 9.50 45.00 20.00 0.390
16 1.60 9.50 45.00 20.00 0.255
17 0.10 8.50 40.00 17.00 0.206
18 2.10 8.50 40.00 17.00 0.354
19 1.10 6.50 40.00 17.00 0.249
20 1.10 10.50 40.00 17.00 0.552
21 1.10 8.50 30.00 17.00 0.259
22 1.10 8.50 50.00 17.00 0.288
23 1.10 8.50 40.00 11.00 0.318
24 1.10 8.50 40.00 23.00 0.350
25 1.10 8.50 40.00 17.00 0.543
26 1.10 8.50 40.00 17.00 0.586
27 1.10 8.50 40.00 17.00 0.610
28 1.10 8.50 40.00 17.00 0.568
29 1.10 8.50 40.00 17.00 0.595
30 1.10 8.50 40.00 17.00 0.625
*Substrate concentration 10%(w/v) at the fixed level
339
Table II : ANOVA for response surface quadratic model
Source Sum of Degree of Mean square F value P Prob>F

squares freedom value
Model 0.46 14 0.033 11.12 <0.0001 Significant

A-E/S ratio 7.107E-003 1 7.107E-003 2.40 0.1419
B-pH 0.038 1 0.038 12.80 0.0028
C-Temperature 8.437E-005 1 8.437E-005 0.029 0.8681
D-Hydrolysis 1.926E-003 1 1.926E-003 0.65 0.4323
time
AB 1.314E-003 1 1.314E-003 0.44 0.5151
AC 3.901E-004 1 3.901E-004 0.13 0.7215
AD 3.331E-004 1 3.331E-004 0.11 0.7418
BC 1.388E-003 1 1.388E-003 0.47 0.5038
BD 3. 630E-003 1 3.630E-003 1.23 0.2853
CD 1. .173E-003 1 1.173E-003 0.40 0.5383
A2 0.18 1 0.18 61.22 < 0.0001
B2 0.072 1 0.072 24.23 0.0002
C2 0.19 1 0.19 63.69 < 0.0001
D2 0.13 1 0.13 42.56 < 0.0001

Residual 0.044 15 2.957E-003
Lack of Fit 0.040 10 4.003E-003 4.62 0.0525 not
significant
Pure Error 4.331E-003 5 8.662E-004
Cor Total 0.50 29
Std. Dev.0.054 R-Squared 0.9121
Mean0.360 Adj R-Squared 0.8301
C.V. % 3.30% Pred R-Squared 0.5308
PRESS0.240 Adeq Precision9.5790
340
Table III : Model based Optimization and validation
Process parameters Optimum level Validation of the model

by experiment
E/s ratio 1.14 1.14

pH 8.9 8.9
Temperature C 39.79 39.79
Hydrolysis time (hrs) 17.33 17.33
Predicted value of reducing power 0.597a 0.595a
(OD at 700nm)
Desirability 0.933
*Desirability close to 1 indicates the reliability of the prediction of the model
A B
C D
Figure I : Interactive effect of hydrolytic factors on reducing power of WPH

(A)Temperature and E/s ratio (B) Hdrolysis time and E/s ratio (C) Temperature and pH
(D) Temperature and pH
341
Figure II : SDS PAGE of whey protein isolate (1) and whey protein hydrolysate by trypsin (2)
temperature of 45 C, hydrolysis time of 6 hrs (Data not shown). Each of hydrolytic variable was varied at the
constant level of substrate ratio of 10% (w/w), E/s ratio of 1.1% (w/w), pH of 9, temperature of 45C, and hydrolysis
time of 6 hrs.Ovissipouret al., (2012) has observed the similar rate of hydrolytic reaction in the present study.
Optimization by Response Surface Methodology

TableI explains the CCRD design matrix of 30 experimental combinations and corresponding experimental
value of reducing power in the range of 0.206 to 0.625 OD at 700nm. The second order polynominal model was
obtained out of CCRD design experiment as shown in equation 1. As shown in ANOVA Table II, the model was
significant and lack of fitness was not significant indicating the adequacy of the model for predictability.The validity
of the model was further confirmed by R- squared value (0.91, P<0.05), R2Adj (0.83), coeffient of variation (C.V) )
(3.36 %).
0.00000195 Equ - I
Equ - I
The individual, interactive and quadratic effect of the model are expressed through surface plots (Figure I
A- D), wherein, the maximum reducing power was observed around the centre level of the experimental domain of
hydrolytic factors in study.
Optimization of response and validation of the model
The optimum hydrolytic conditions including E/s ratio of 1.14 (%w/w), pH of 8.9, temperature of 39.79 C
and hydrolysis time of 17.3hrs were predicted for the maximum reducing power of 0.597 OD at 700 nm from the
model. Actual experiments were conducted in triplicates at the same hydrolytic conditions and reducing power was
0.595 OD at 700 nm comparable to the predicted value validating the predictability of the model (P > 0.05). There
was a comparable effect between hydrolysis time of 6 hrs and 17.3 hrs and hence hydrolysis time of 6 hrs was taken
as optimum time instead of 17.3 hrs of hydrolysis time.
342
SDS PAGE Analysis
Figure II depicts the changes in molecular mass of whey protein isolate with the effect of hydrolysis by
trypsin. Whey protein isolate consists of a mixture of proteins mainly bovine serum albumin, - lactalbumin,
-lactoglobulin. Trypsin has been reported to be effective in proteolysis against -lactoglobulinand resistant to -
lactalbumin (Bayramet al., 2008) and thus resulting hydrolysate enriched with - lactalbumin and peptides inherited
from -lactoglobulin wouldexhibit enhanced antioxidant activity. As shown in Figure I, WPI was observed with
higher molecular weight proteins, whereas in WPH, the appearance ofsmaller peptides was observed relatively. As
reported by Bayramet al.,(2008), it could be assumed the breakage of higher molecular weight protein could be of
-lactoglobulin by trypsin and it could be further attributed to the increased antioxidant activity observed in WPH
as compared to that of WPI.
CONCLUSION
Hence the present study concluded that the hydrolysis conditions including E/s ratio (1.14 %w/w), pH (8.9),
temperature (39.79C), and hydrolysis time (6hrs) at fixed substrate concentration (10% (w/v on the basis of protein
content in WPI)) could be optimum level for the preparation of whey protein hydrolysate with the maximum
antioxidant activity. The present study demonstrates the application of response surface methodology in the
optimization of enzymatic hydrolysis of whey protein and paves the way for optimization of complex biological
processes using response surface methodology.
REFERENCES
1. XinyanPeng X, Xiong, Y L &Kong B (2009) Food Chem 113, 196201.
2. Herminia A D & Juan C P (2006) Process Biochem 4, 447456.
3. Kong B H &Xiong Y L (2006) J Agri Food Chem 54, 60596068.
4. Senji S &Yumi T (2006) Food Chem 95, 243249.
5. Thiansilakul Y, Benjakul S &Shahidi F (2007) Food Chem 103, 3851394.
6. Cumby N, Zhong Y, Naczk M &Shahidi F (2008) FoodChem 109, 144148.
7. Pena-Ramos E A, Xiong Y L (2001) J Dairy Sci 84, 25772583.
8. Colbert L B & Decker E A (1991) J Food Sci 56,12491250.
9. Pena-Ramos E A &Xiong Y L (2003) Meat Sci 64, 259263.
10. Ren X, Ma L, Ju Cha, Want Y H, Zhuang Y P, Zhang S (2012) J Aqua Food Product Tech 21, 99 -110.
11. Oyaizu, M. (1986). Japanese Journal of Nutrition, 44, 307315.
12. Bradford M M (1976) Anal Biochem 72, 248 254.
13. Ovissipour M, Abedian A, Motamedzadegan A (2010) Int Jap Aqua Res 2, 87 95.
14. Bayram T, Pekmez M, Arda N, suhaYalcin A (2008) Talanta 75, 705 -709
343
Emodin (1, 3, 8-trihydroxy-6-methylanthraquinone), a natural
anthraquinone derivative induces oxidative stress and apoptosis in
hepatocellular carcinoma cells
Aruljothi Subramaniam, Kiruthika Sundarraj1, Azhwar Raghunath and Ekambaram Perumal*
Molecular Toxicology Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore 641 046, Tamilnadu, INDIA.
Email: ekas2009@buc.edu.in
Abstract
Hepatocellular carcinoma (HCC) is one of the most common cancers and the third most common cause of
cancer mortality worldwide. Though HCC management has improved in recent years through the advances
in prevention and treatment, it still requires the effective mechanism based approaches. It is well known that
cancer cell evades oxidative stress and apoptosis. Thus, novel chemotherapeutic drug that exerts cytotoxic
effects through oxidative stress mediated apoptosis is warranted. Emodin is a natural anthraquinone derivative
has been shown to possess anti-tumour and immunosuppressive properties. This study has been aimed to
explore the impact of emodin on oxidative stress and apoptosis using HepG2 cells. In order to evaluate
the mechanism of action of emodin on oxidative stress and apoptosis, we used techniques such as MTT
assay, oxidants and antioxidants assay, live/dead assay, Comet assay, and Western blotting. We observed that
treatment of emodin with HepG2 cells effectively induced oxidative stress and apoptosis by reactive oxygen
species. Further, emodin treatment results in mitochondrial membrane damage to release cytochrome c via
altering Bax/Bcl-2 ratio. These results reveal that emodin exposure causes oxidative stress mediated cell death
in HCC cells.
Keywords: Apoptosis, emodin, hepatocellular carcinoma, mitochondrial damage, oxidative stress, reactive
oxygen species
Introduction
HCC is the fifth most common human cancer, with approximately 7,50,000 new cases occurring worldwide
each year and it ranks third in annual global cancer mortality rates and has the shortest survival time of any cancer
in both males and females1. The HCC is distinguished as a highly chemoresistant cancer with several aetiological
factors being classified as high-risk factors, including exposure to aflatoxin B1, alcohol abuse, infection with
hepatitis B virus and hepatitis C virus 2-4. Surgical treatment is considered to be the best choice for HCC, however
tumor size, hepatic functional reserve and/or portal hypertension may all restrict surgical ablation 5-6. Chemotherapy
is the most common treatment for advanced cancers7. Compared with local treatments, such as radiation and surgery,
chemotherapy is a systemic treatment that may reach cancer cells wherever they have spread8. Chemotherapy using
conventional cytotoxic drugs, such as doxorubicin, cisplatin and fluorouracil is one of the commonly treatment
options, especially for patients with unresectable tumors. Though, because of poor response rates, severe toxicities
and high recurrence rates, the mean survival time is approximately 6 months9. The exploration for new chemo-
preventive and anti-tumor agents that are more effective and less toxic has magnetized enormous interest in
phytochemicals.
Emodin (1, 3, 8-trihydroxy-6-methylanthraquinone), a natural anthraquinone derivative isolated from root

and rhizome of Rheum palmatum L is one such compound10. Emodin has been demonstrated to display a number of
biological activities such as antiviral, antimicrobial, immunosuppressive hepatoprotective, anti-inflammatory and
antiulcerogenic11. Emodin has been exhibited to regulate many gene expression associated with cell proliferation,
cell apoptosis, oncogenesis, DNA repair and cancer cell invasion and metastasis12-17. Recent studies confirm that
emodin can induce apoptosis in several human cancer cells such as the human lung cancer, leukemia, ovarian
cancer, colon cancer cervical cancer, hepatoma and prostate cancer17-22.
Results in HeLa cells demonstrated that the emodin-mediated generation of ROS inhibits the prosurvival
344
transcription factors NF-kB, thus accounting for the cytotoxicity23. The molecular mechanism by which emodin
induces cell apoptosis is through inactivation of ERK and AKT and the decrease of anti-apoptotic protein Bcl-
2 levels in human A549 cells24. Emodin has been proposed as a potential agent in the management of tumors.
Also emodin is considered as a strong ROS-producing agent and induction of DNA damage. Emodin-mediated
oxidative injury triggers mitochondrial dysfunction, cytochrome c release and caspase activation, which leads to
apoptosis in lung cancer A549 cells24. The generation of ROS may contribute to mitochondrial damage, reduction
of mitochondrial trans-membrane potential, release of cytochrome c and Smac, and subsequent caspase activation
including apoptosis25.
In the case of rats or mice given emodin during pregnancy period, neither prenatal mortality nor morphologic
development was affected and genotoxicity of emodin would be distinct26-27. It has been discovered that cytotoxicity
of emodin on human fetal hepatic cell line L-02 and found that emodin lacks significant effect on non-cancer liver
cells. These data suggest that normal cells are more resistant to emodin-induced cytotoxicity than cancer cells25.
Above observations made clear that emodin can serve as a best chemotherapeutic drug.
It is well recognized that both growth inhibition and apoptosis are interconnected in establishing the
response of cancer cells to chemotherapeutic agent. A moderate increase in ROS can promote cell proliferation
and differentiation, whereas excessive amounts of ROS can cause oxidative damages to lipids, proteins and DNA
and lead to cell death28-31. Several chemotherapeutic drugs exert their cytotoxic effects through the generation of
ROS32. It is now clear that the generation of ROS can be exploited therapeutically in the treatment of cancer. Emodin
mediated chemotherapeutic response to HCC cells remains obscure. Here we explored the possible mechanism that
emodin could induce oxidative stress and apoptosis in HepG2 is demonstrated.
Materials and Methods
Cytotoxicity by MTT assay
Cell viability was assayed by the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT)
assay, because the reduction of tetrazolium salts is widely accepted as a reliable method to examine cell viability/
proliferation. Briefly, the cells (5x103/ml) were incubated in triplicate in a 96-well plate in the presence or absence
of different concentrations of emodin (0, 5, 10, 25, 50 and 60 M) in a final volume of 0.2 ml for 24, 48 and 72h
intervals at 37C. After the exposure times, the wells were treated with a final concentration of 0.5 mg/ml MTT and
incubated at 37C for 4h. The purple formazan crystals formed were dissolved in 0.2 ml DMSO and read at 570 nm
in a microquant plate reader (Bio-Tek Instruments).
Biochemical evaluation for oxidative stress and antioxidant enzymes
Cells were cultured in a 75 cm2 culture flask and exposed to emodin at the concentration of (0, 5, 10, 25 and 50
M) for 24 h. After the treatment, cell extracts were prepared by sonication in 50 mM Tris, 5 mM ethylene diamine
tetraacetic acid (EDTA), 10 g/ml phenyl methyl sulfonyl fluoride (PMSF), pH 7.6. The cell debris was removed
by centrifugation at 4000 rpm for 5 min at 4C and the protein content of the supernatant was determined by the
Lowry method and used for antioxidant enzyme assays. Both reagent blanks and enzyme blanks were measured
for all assays. All assays were carried out in the linear range and expressed as specific activities. Lipid peroxidation
(LPO) was determined by Thiobarbituric acid (TBA) reaction with malondialdehyde (MDA)33. The activities of
antioxidant enzymes SOD, CAT, GPx and levels of total protein was measured with previously described standard
methods34-37.
Live/Dead Assay
Apoptosis of cells was also determined by Live/Dead assay (Molecular Probes, Eugene, OR, USA) that
measures intracellular esterase activity and plasma membrane integrity. It is a two-color fluorescence assay that
simultaneously determines: Live cell numberLive cells have intracellular esterases that convert nonfluorescent,
cell-permeable calcein acetoxymethyl (calcein AM) to the intensely fluorescent calcein. Cleaved calcein is retained
within cells. Dead cell numberDead cells have damaged membranes; the ethidium homodimer-1 (EthD-1)
enters damaged cells and is fluorescent when bound to nucleic acids. EthD-1 produces a bright red fluorescence in
345
damaged or dead cells. Briefly, 1 X106 HepG2 cells were incubated with emodin in various concentrations (0, 5, 10,
25 and 50 M for 24 h) at 37C. Cells were stained with the Live/Dead reagent (5 M ethidium homodimer, 5 M
calcein-AM) and then incubated at 37C for 30 min. Cells were analyzed under a fluorescence microscope (DP 70,
Olympus, Tokyo, Japan) by counting live (green) and dead (red) cells in 20 random fields.
Alkaline single cell gel electrophoresis (Comet) assay
Harvested cells were resuspended in Hanks balanced salt solution (Sigma) were mixed with 0.7% low melting
point agarose, applied on Comet slides and subjected to lysis (2.5 M NaCl, 0.1 M pH 8 EDTA, 10 mM Tris base, 1%
Triton X) at 42C for 1h. The slides were loaded into a gel electrophoresis tank in 0.3 M NaOH, pH 13 with EDTA,
allowed to denature for 40 min and run at constant 25 V/300 mA for 20 min. Samples were neutralized with 0.5 M
TrisHCl pH7.5 for 15 min, dehydrated in 70% ethanol and dried at 37C. DNA was stained with SYBR Green. One
hundred randomly selected cells were examined per sample using Komet Imager Software. Extent of DNA damage
was expressed as a measure of comet tail moments, which corresponds to the fraction of DNA in the comet tail.
Measurement of intracellular ROS
Intracellular production of ROS was measured using 2,7-dichlorofluorescein diacetate (DCFH-DA).
The DCFHDA passively enters the cell, where it reacts with ROS to form the highly fluorescent compound
dichlorofluorescein (DCF). In brief, 10 mM DCFH-DA stock solution (in methanol) was diluted in culture medium
without serum or another additive to yield a 100 M working solution. HepG2 cells were treated with emodin with
increasing concentration (0, 5, 10, 25 and 50 M of emodin and 50 M of H2O2) for 24 h. At the end of exposure,
cells were washed twice with HBSS and then incubated in 1 ml of working solution of DCFH-DA at 37C for
30 min. Cells were lysed in alkaline solution and centrifuged at 2300 g for 10 min. A 200 l supernatant was
transferred to a 96-well plate and fluorescence was measured at 485 nm excitation and 520 nm emission using a
microplate reader (FLUO star Omega). The values were expressed as a percent of fluorescence intensity relative to
control wells.
Evaluation of morphology by phase contrast microscopy
To further confirm the oxidative stress mediated cell damage in HCC cells, HepG2 cells with a density 1
105 cells/ml were plated in 6-well plates and the next day it was treated with emodin (25 M) or without emodin
and with pre-incubation of 10 mM NAC for 4 h along with or without emodin (25 M) for 12 h. The cells were
observed for the morphological characteristics under phase contrast inverted microscope. Morphological changes
like rounding up of cells, apoptotic body formation and cell detachment were observed.
Western Blot Analysis
1105 cells exposed with various concentration emodin were lysed and protein concentrations were determined
by BCA method. For protein determination of each sample, the cell lysates (40 g of each) were separated by SDS
PAGE on a polyacrylamide gel followed by electrotransfer onto a PVDF membrane (Immobilon-P; Millipore,
Bedford, MA, USA). The blots were then incubated with primary antibodies (1:1000 dilutions in blocking buffer)
overnight at 4C. After being washed, secondary antibodies conjugated with horseradish peroxidase (HRP) were
applied at a dilution of 1:20,000 in blocking buffer for 1 h at room temperature. HRP-conjugated goat anti-rabbit or
antimouse IgG was used as a secondary antibody for enhanced chemiluminescence (ECL Kit, Millipore, Billerica,
MA, USA). The protein levels of cytosolic and mitochondrial cytochrome c were carried out according to the
manufacturers protocol (Mitochondria/Cytosol Fractionation Kit, BioVision, Inc., Mountain View, CA, USA).
Western blotting for examining the effects of emodin on the levels of Bax, Bcl-2, -actin, cytochrome c and COX
IV were performed for emodin-treated HepG2 cells.
All data are expressed as mean S.E.M of 3 independent experiments. Statistical significance was determined
using unpaired Students t-test and the probability P value less than 0.001 were considered statistically significant.
346
Results
Exposure of emodin resulted in cell morphological changes and loss of viability in HepG2 cells
Growth inhibition of HepG2 cells was assessed following 24, 48 and 72 h treatment with emodin by the
MTT assay. Emodin inhibited the proliferation of HepG2 cells in a dose-dependent manner with various time
courses. Treatment of emodin at 60 M concentration inhibited the cell proliferation to 50% at 24 h time point.
50 M of emodin significantly decreased almost 50% the viable cells at 48 h and the 50% of the cell death were
detected in the treatment of 25 M emodin with 72 h incubation (Figure 1).
Emodin altered the oxidant/antioxidant balance of human liver cancer cells in concentration dependent
manner
It has been suggested that oxidant generation and antioxidant depletion are the common pathways through
which anticancer drugs trigger apoptosis in cancer cells. In order to investigate whether the cytotoxic effect of
emodin are related to the induction of ROS as well as significant fall in antioxidant enzymes, HepG2 cells were
treated with emodin with increasing concentration (0, 5, 10, 25 and 50 M) and the activities of LPO and antioxidant
enzymes including SOD, GPx and CAT were measured. MDA levels, the end product of LPO were used to reflect
the level of oxidative damage in cells, were significantly increased in dose dependent manner. As the evidence
of dose dependent increase in lipid peroxidation, MDA content showed a significant elevation of 61.55% in 10
M, 94.96% in 25 M and 155% in 50 M of emodin treated groups when compared to untreated group in a dose
dependent manner. However, 13.34% in 5 M emodin treated group was not shown significant difference in MDA
content when compared to that of control group.
Increased SOD, GPx and CAT enzymes are regarded as the first line of the antioxidant defense system against
ROS generated during oxidative stress. Significant dose dependent decrease in SOD, catalase and GPx activities were
observed in the cells challenged with emodin at various concentration. A dose-dependent decrease was observed in
the activity of GPx. 7% in 5 M, 42.5% in 10 M, 70.7% in 25 M and 84.4% in 50 M of emodin treated groups
were observed. SOD activity was found to be decreased in dose dependant manner in emodin treated groups with
various concentration of emodin when compared to the control cells (10.45% in 5 M, 55.74% in 10 M, 68.16%
in 25 M and 81.11% in 50 M). Emodin induced depletion in catalase activity 9.56%, 32.23%, 57.77%, 71.43% in
5 M, 10 M, 25 M and 50 M respectively. The results were found to be statistically significant when compared
to the control group (Figure 2).
Emodin Induces ROS generation determined by DCF-DA fluorescent dye fluorimetry
ROS play critical roles in the regulation of diverse functional pathways involved in apoptosis, proliferation
and survival in cancer cells. Therefore, we investigated whether generation of intracellular ROS is part of the
mechanism by which emodin induces apoptosis through oxidative stress in HepG2 cells. The generation of ROS
by emodin was assessed by using the fluorescent probe H2O2DCFDA. H2DCFDA is cleaved intracellularly by
non-specific esterase to non-fluorescent 2,7-dichlorofluorescin (DCFH), which leads to the fluorescence compound
2,7-dichlorofluorescein (DCF) upon oxidation by ROS. Treatment with 0, 5, 10, 25 and 50 M of emodin significantly
increased ROS in a dose dependent manner. The value of ROS was expressed as relative fluorescence compared to
control value which was taken as 100%. The cells were also treated with 50 M of H2O2 as a positive control, which
showed a significant increase in relative fluorescence (Figure 3).
Emodin enhances % of apoptotic population with increasing concentration
We also determined whether emodin can enhance apoptosis using esterase staining Live and Dead assay. We
found that emodin induces apoptosis in HepG2 cells in concentration dependent manner. Control cells have very
few numbers of dead cells about 2%. The percentage of dead cell population increased with increasing dose of
emodin. 0-2%, 5-4%, 10-11%, 25-25%, 50-34% and 60-48% respectively (Figure 4).
Emodin induced apoptosis is mediated through oxidative stress mechanism in liver cancer cells
To confirm that the effect of emodin induced DNA damage in HepG2 cells as noted in COMET assay was due
to oxidative stress, HepG2 cells were treated with 0, 5, 10, 25 and 50 M for 12 h (Figure 5). Cells were harvested
347
from each treatment and then were lysed and total proteins from each sample were prepared for Western blotting
analysis for apoptosis associated proteins expression. The western blot results indicated that the levels of Bax and
cytosolic cytochrome c were increased and the level of Bcl-2 and mitochondrial cytochrome c was decreased, which
led to cell apoptosis in emodin-treated HepG2 cells (Figure 6).
Oxidative stress is involved in emodin-induced apoptosis
Emodin-mediated oxidative stress was studied by pre-treating the cells with water soluble thiol antioxidant 10
mM NAC to quench ROS. Morphological changes by treatment with emodin (25 M) were also examined using
phase-contrast microscopy, emodin treatment led to increase the cell morphological changes, including early effects
like rounding up of cells, cell shrinkage, cells floated on the well and cell number were reduced compared to the
control after 24 h exposure while the addition of NAC almost completely eliminated the floating cells. These results
suggest that emodin significantly inhibits the growth of HepG2 cells through ROS generation. Emodin induced
cell death via oxidative stress was confirmed with cell proliferation assay (MTT). N-acetylcysteine (NAC, a ROS
Quencher) decreased emodin-induced cell death when compared with emodin-treated cells alone and this result
is in agreement with LS1034 human colon cancer cells treated with emodin and NAC. The thiol NAC is a source
of cysteine for the synthesis of the endogenous antioxidant glutathione (GSH), thus, NAC is postulated to protect
cells by increasing intracellular GSH levels and scavenging ROS. NAC was able to completely abrogate emodin-
induced apoptosis. In contrast, emodin induced cell death significantly increased in HepG2cells with the addition of
H2O2. These data suggest that emodin induced cell death might be triggered by oxidative stress. Thus, the protection
afforded by antioxidants against the induction of cell death by emodin suggests that free radicals may be involved in
this phenomenon. To examine this possibility, we measured the levels of intracellular free radicals before and after
exposure to emodin using the cell permeate dye DCFH-DA. Measurements of cellular fluorescence revealed that
emodin generated 5 fold increases of intracellular ROS in HepG2 cells. A non-cytotoxic dose of NAC completely
suppressed emodin-induced ROS generation. Emodin substantially enhanced oxidative stress mediated apoptosis
in HepG2 cells and pre-treatment of cells with NAC markedly reduced this emodin-induced enhancement (Figure
7). These results suggest that ROS is needed for the oxidative stress mediated apoptosis by emodin in HepG2 cells.
Discussion
Current developments in cancer therapeutic research suggest that a number of apoptotic stimuli share
common mechanistic pathways characterized by the generation of ROS through oxidative stress38-39. ROS played
a very important role in apoptosis induction under both physiological and pathological conditions. Interestingly,
mitochondria were both the source and target of ROS. ROS typically include the superoxide radical (O2-), hydrogen
peroxide (H2O2) and hydroxyl radical (.OH), which cause damage to cellular components including DNA, proteins
and lipids40. Exploiting the cancer cell killing potential of ROS could be performed by two means, namely, (1)
inducing the generation of ROS directly in tumor cells and (2) inhibiting the antioxidative enzyme (defense) system
of tumor cells. Many studies have shown that diverse chemotherapeutic agents can induce apoptosis in cancer cell
lines by elevating oxidative stress41. Several natural compounds such as gallic acid, -phenylethyl isothiocyanate
and sulforaphane were also reported to induce apoptosis in cancer cell lines through ROS generation41-42.
Previous treatments of diseases with herbs were experimental more than theoretical. Therefore, clarification
of the mechanisms of action of components in herbs may be important in developing their applications. Emodin
(1,3,8-trihydroxy-6-methyl-anthraquinone), a natural anthraquinone derivative isolated from Rheum palmatum L,
has been reported to exhibit anticancer effect on several human cancers such as liver cancers and lung cancers.
However, the molecular mechanisms of emodin-mediated tumor regression have not been fully defined. Emodin,
a natural anthraquinone derivative, was selected because of its derived semiquinone structure which is likely
to increase the generation of intracellular ROS. It is a type of natural anthraquinone with a molecular structure
similar to that of 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ). DMNQ is considered as a ROS generator because
its property of quinone and derived semiquinone, like mitochondrial ubiquinone, allows it to transfer electrons43.
Emodin has a structure similar to those of DMNQ and ubiquinone. Quinones are well known redox active molecules
capable of forming a redox cycle with their semiquinone radicals leading to the formation of ROS. Thus, it has been
348
suggested that the quinoid structure of emodin could be activated to the semiquinone radical intermediate, which in
turn could react with oxygen to produce ROS and ROS-induced apoptosis44.
In the present report, we found that emodin decreased the percentage of viable HepG2 cells and these effects
are a concentration-dependent manner, which is in agreement with other reports showed that emodin induced
cytotoxic effects in human prostate cancer LNCaP cells and LS1034 colon cancer cells45-46.
A previous study demonstrated that treatment with emodin rapidly increases ROS generation in vascular
smooth muscle cells47. The inhibition of RhoA activation and induction of apoptosis is associated with an increase
in oxidative stress in emodin-treated gastric carcinoma cells48. Emodin has been characterized as a strong ROS-
producing agent that can generate superoxide radical anions, hydrogen peroxide and the hydroxyl radical, which
eventually cause DNA strand break and apoptosis49. In the present study, emodin significantly altered the oxidant/
antioxidant status of human liver cancer cells. ROS generation and membrane LPO were significantly higher,
whereas antioxidant enzymes SOD, CAT and GPx were significantly lower in emodin-treated cells. The activity of
GPx can provide important clues about the consumption rate of GSH during detoxification of organic hydroperoxides
by it. SOD is specialized to convert highly reactive O2- to less toxic H2O2. CAT enzyme reduces H2O2 to H2O50-51.
Higher production of intracellular ROS and membrane LPO in emodin-treated cancer cells, along with depletion of
antioxidant components, suggests that ROS generation and oxidative stress might be the primary mechanisms for
toxicity of emodin to human liver cancer HepG2 cells. The results from live and dead assay revealing that increasing
dead cell population along with increasing concentration of emodin confirmed the apoptotic efficacy of emodin in
dose dependent manner.
It is reported that apoptosis can be divided into mitochondrial dependent and -independent pathways.
Furthermore, it was reported that if the loss of the outer mitochondrial membrane integrity and the release of
cytochrome c from the mitochondria to the cytosol of cells after exposed stimulator then the cells are committed to
apoptosis52. In the present study, our results already showed that emodin altered the levels of cyctocrome c release.
In order to investigate the possible molecular signal pathway of apoptosis in HepG2 cells after exposure to emodin,
western blotting analysis was used for examining the apoptotic protein levels, which indicated that anti-apoptotic
Bcl-2 significantly decreased in dose dependent manner in HepG2 cells and pro-apoptotic Bax was increased in
comparison to control, which led to the decrease the ratio of Bax/Bcl2 then caused mitochondrial dysfunction then
induced apoptosis. It was reported that Bax/Bcl-2 ratio indicates whether and how a cell will respond to an apoptotic
signal53. Our results showed that the Bcl-2/Bax ratio therefore decreased with increasing emodin concentration. This
decrease may contribute to the oxidative stress mediated apoptosis via the mitochondrial apoptosis pathway. This
result is agreement with previous results in various cancer cells treated with emodin54-58.
There is evidence which suggests that ROS may act as signalling molecules for the initiation and execution of
the apoptotic cell death program59. In this investigation, we studied the role of ROS formation in emodin-induced
apoptosis of HepG2 cells. NAC (10 mM) was shown to inhibit emodin-induced cytotoxicity and morphological
changes in HepG2 cells treated with 25 M of emodin. Oxidative stress is implicated in a number of cellular
processes including apoptosis and many chemotherapeutic agents are known to induce their cytotoxic effects to
tumor cells by an ROS mediated mechanism. Quinones are known to modulate oxidative stress as part of their
mechanism of anticancer action and thus we examined ROS production by emodin, an anthraquinone. We report
in that emodin (25 M, 12h) induces apoptosis and pre-treatment of HepG2 cells with 20 mM NAC inhibited this
effect. Measurements of cellular fluorescence revealed that emodin generated a 5-fold increase of intracellular ROS
in HepG2 cells. A non-cytotoxic dose of NAC completely suppressed emodin-induced ROS generation.
In conclusion, we found that emodin induced oxidative stress and apoptosis through generating ROS and
apoptosis in HepG2 cells. Thus, we proposed that emodin treatment results predominantly in ROS generation
through mitochondrial cytochrome c release and apoptosis induction via altering Bcl-2/Bax ratio, a mechanism
drawing much current interest with respect to its therapeutic potential in HepG2 cells in the future. These findings
may aid in the understanding of the mode of actions of the emodin and provide a theoretical basis for the therapeutic
use of this compound in further investigations.
349
Acknowledgements
Dr. Aruljothi Subramaniam is highly indebted to Dr. Gautam Sethi for his scientific insight to carry out this work.
Ms. Kiruthika Sundarraj greatly acknowledges the financial assistance from Department of Science and Technology
- Science and Engineering Research Board (SB/EMEQ-246/2014). Mr. Azhwar Raghunath acknowledges the UGC-
BSR fellowship (UGC-BSR No. F7-25/2007) funded by UGC-BSR, New Delhi, India. This work was supported by
the University Grants Commission Special Assistance Programme (UGC-SAP-II: F-3-20/2013) and Department
of Science and Technology, Fund for Improvement of Science and Technology infrastructure in universities and
higher education institutions (DST-FIST:SR/FST/LSI-618/2014), New Delhi, India.
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Figures
140
Emodin M
120 0 5 10
25 50 60
100
* *
*
% Cell Viability
80
*
60 * * * *
* *
* *
40
20
0
24 h 48 h 72 h
Time Course (h)
Figure 1. Emodin induced loss of total cell viability in HepG2 human liver cancer cells. HepG2 cells were
plated in triplicate, treated with indicated concentrations of emodin and then subjected to MTT assay after 24, 48
351
and 72 h to analyzed proliferation of cells. Each bar represents mean SEM of 3 experiments. *P < 0.001compared
to control (One way ANOVA followed by Tukeys multiple comparison tests).
30 90
A B
80
25 *
70
M of MFD/mg protein
20 * 60
*
* 50
U/mg protein
15
40
10 30 *
20 *
5
10
0 0
0 5 10 25 50 0 5 10 25 50
Emodin M Emodin M
2.5 600
C D
2 500
400 *
1.5
U/mg protein
U/mg protein
300 *
*
1
*
* 200
0.5 * 100
0 0
0 5 10 25 50 0 5 10 25 50
Emodin M Emodin M
Figure 2. Emodin induced oxidant generation and diminishes anti-oxidant defence system in human liver
cancer (Hepg2) cells in a dose dependent manner. Lipid peroxidation (a) Glutathione peroxidase (b) SOD (c) and
Catalase (d) in the cells treated with various concentration of emodin for 24h (0, 5, 10, 25 and 50 M of emodin).
Each bar represents mean SEM of 3 experiments *P < 0.001compared to control (One way ANOVA followed by
Tukeys multiple comparison test). 1000
*
900
DCF flurescence (%) of control
800
700
600
*
500
400
*
300 *
200
100
0
0 5 10 25 50 H2O2
Emodin M
Figure 3. Induction of ROS generation in HepG2 cell lines. Cells were treated with emodin in various
concentration for 24h and H2O2 (50 M) was used as positive control. These cell lines were then labelled with
H2DCFH-DA and ROS production was quantified by fluorimetry. Each bar represents mean SEM of 3 experiments.
* P < 0.001compared to control (One way ANOVA followed by Tukeys multiple comparison test).
352
Figure 4. Emodin induced apoptotic cell death in HepG2 cells treated with increasing concentration: HepG2
cells were treated emodin (a-0, b-5, c-10, d-25, e-50 and f-60 M for 24h) and the cytotoxicity was determined by
the live/dead assay and 20 random elds were counted.
Figure 5. Emodin induced DNA damage is oxidative stress mediated. Sybr Green-stained comets of a typical
comet from nuclei of control and cells treated with increasing concentration of emodin (a: 0 or control; b: 5 M of
emodin; c: 10 M of emodin; d: 25 M of emodin; e: 50 M of emodin).
Figure 6. Emodin affected the apoptosis-associated protein levels on HepG2 cells. Cells were incubated with
various concentrations of emodin (0, 5, 10, 25 and 50 M) for 12h and the cells were collected for Western blotting
as described in Materials and methods. (A). The protein levels of Bcl-2 and Bax were shown and (B). The fractionate
cytosolic (top) and mitochondrial (bottom) cellular cytochrome c protein were performed. -actin and COX IV are
353
used as an internal control, respectively.
1000
900
DCF Fluorescence (fold change)

800
700
*
600
500
400
300
200
#
100
0
Control NAC Emodin Emodin+NAC H2O2
Figure 7. The intracellular generation of ROS was measured using the oxidationsensitive
fluorescein DCFH-DA. Exponentially growing cells (2x105/ml) were treated with or without 25 M emodin,
pre-treated with NAC (10 mM), treated with NAC along with 25 M emodin and with 50 M H2O2 for 1h. The
cells were collected and loaded with DCFH-DA for 30 min at 37C and then incubated for 4h with antioxidant
before loading with DCFH-DA. The fluorescence intensity of the cell suspension was measured using a fluorescence
spectrophotometer with excitation at 488 nm and emission at 525 nm. Each value represents the mean S.E. of
duplicate assays in three independent experiments (*, p< 0.001 when emodin compared with control and Emodin +
NAC compared with emodin treated cells).
354
Aloe vera extract mitigates arsenic induced neurotoxicity in rats
Sasi Priya Gopalakrishnan, Vinothini Subramaniam1, Lakshmikanthan Panneerselvam and
Ekambaram Perumal*
Molecular Toxicology Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, Tamilnadu, India.
Email: ekas2009@buc.edu.in
Abstract
Arsenic induced neurotoxicity has been extensively studied in both humans and animals. However, the
mechanism underlying in the neurotoxicity of experimental arsenicosis remains obscure. The severity of arsenic
toxicity was reduced by oral administration of certain plant derived extracts. The present study was undertaken
to evaluate the protective effect of aqueous extract of Aloe vera leaves against sodium arsenite (NaAsO2)
induced neurotoxicity in female Wistar rats. Animals were treated with sodium arsenite (20 ppm NaAsO2) in
drinking water ad libitum, alone or in combination with A. vera extract (100 mg/kg bw by oral intubation)
daily for 30 days. The serum arsenic concentration increased markedly in the NaAsO2 treated animals and was
accompanied by impairment in spontaneous motor activity and motor coordination, decreased total protein
and acetylcholinesterase activity in brain. Arsenic intoxication also decreased the neuronal antioxidants-SOD,
CAT, GPx, GSH, ascorbic acid and increased the levels of lipid peroxidation, protein carbonyl content and
nitric oxide. Further, arsenic treatment altered histo-architecture in brain was also found in these animals.
A. vera extract supplementation prevents arsenic induced behavioural impairment and oxidative neuronal
damages in rats. So, these findings reveal that A. vera can be used as nutraceutical against arsenicosis.
Keywords: Aloe vera, Arsenic toxicity, Oxidative stress, Neuronal degeneration, Rats
Introduction
Arsenic (As) is a naturally occurring metalloid found in water, soil and air. As is released into the environment
from both natural and anthropogenic sources1. The environmental As exists in both organic and inorganic states.
The inorganic forms of As pronounce more toxic than the organic forms and the trivalent (arsenite) forms are more
toxic then the pentavalent forms (arsenate)2. Exposure of As mainly occurs through accidental, occupational and
major cause via environmental contaminated drinking water in many parts of the world including India3. As is a
potent neurotoxin known to cause severe neurological disorders such as hyperpyrexia, convulsion tremor, axonal
degeneration and mental retardations4,5. The mechanism by which As induced neuronal degeneration is directly
associated with reactive oxygen species (ROS), oxidative stress, DNA damage and alteration in its methylation
pattern, clevage of p35 to p25 by calpin, alterations in brain biogenic amines and nitric oxide levels, apoptotic
and necrotic signaling molecules5-7. Thus, ancient folk treatement shows that consumption of antioxidants rich
medicinal plants extracts known to chealte the metal toxicties and prevents As induced behavioural impairment and
oxidative neuronal damages in rat brain.
Aloe vera (Aloe vera var. barbendensis) is a perennial plant belonging to the family of liliaceae, which includes
360 species8. A. vera is the most widely used both commercially and for its therapeutic properties. A. vera has
traditionally been used around the world as a folk remedy for various diseases and exhibits number of activities such
as analgesic, anti-microbial, anti-inflammatory, anti gastric ulcer, powerful immune modulator, antitumor agent
and antioxidant9. Earlier report showed that protective value of A. vera against some toxic effects of As in rats10.
However, the effects of A. vera on neurotoxicity and its behavioural impairment induced by As have not been
investigated to date. Therefore, this study was designed to investigate the therapeutic potential of A. vera aqueous
extract on As-induced neurotoxicity and examine the underlying mechanism whether involved its anti-oxidant
activity in rats brain.
355
Material and methods
Chemicals
Sodium arsenite was purchased from Sigma Chemical Co., St. Louis, Mo., USA. All other chemicals used in
the study are of high standard.
Animals
Adult female Wistar rats with the body weight range of 130 150 g were used throughout the experiment.
The rats were caged in groups (3 per cage) and were maintained at room temperature (22-26C) with a normal 12-
hr light/dark cycle. The animals were fed with balanced commercial available pelleted rat chow (Sai Durga Feeds
Pvt Ltd, Bangalore India) containing energy: 3620 Kcal/kg, crude protein: 22.11%, crude oil: 4.10%, crude fibre:
3.16%, ash: 5.13% and water ad libitum. The experiments were designed and conducted in accordance with the
ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Institutional
Animal Ethics Committee Guidelines (722/02/A/CPCSEA).
Experimental Design
The animals were divided into four groups with a minimum of six rats in each group. Administration of sodium
arsenite at 20 ppm with drinking water for 9 weeks did not produce lethality in mice in previous study11. A. vera (100
mg/kg) were orally administered to rats and indicated that A. vera did not have any toxic effects on central nervous
system12. Group I: Rats received tap water alone, served as control. Group II: Rats received sodium arsenite ad
libitum in tap water at concentration of 20 ppm daily for 30 days. Group III: Rats received A. vera extract orally
along with water at a concentration of 100 mg/kg bw daily for 30 days. Group IV: Rats received A. vera extract
at a concentration of 100 mg/kg bw/day along with sodium arsenite (20 ppm) in drinking water ad libitum for 30
days. Experiments were carried out 24 hr after the 30th day of treatment in test and control animals. The animals
were sacrificed by cervical decapitation. Brain tissue was immediately processed and used for various biochemical
estimations.
Behavioral Parameters
Spontaneous motor activity (SMA)
SMA was measured using an activity monitoring apparatus13. The capacitance sensors implanted in the floor of
the apparatus was sensitive to the vibrations caused by the locomotor as well as scratching and grooming activities
of the animal. Each animal was placed in the chamber for one minute to get accustomed and then the activity was
measured for duration of 10 min.
Rota-Rod Motor Coordination
A Rota rod apparatus consisted of a horizontal rod (5 cm diameter; 30 cm long with partitions for testing
3 animals at a time) with a roughened surface. The rod moved on its axis at 14 rpm. The rational of this test was
that the animal was forced to stand on the moving rod and that animal having defective motor function would drop
off from the moving rod to a tray placed 20cm below the rod. The test was carried out as described previously13 in
another group of test animals. A test period of 90 seconds was allowed to each animal and the endurance time was
determined by measuring the time between the placing of the rat on the moving rod and the moment the animal fell
down.
Serum As content
Serum (500 L) was mineralized by acid (nitric acid, sulphuric acid and per chloric acid respectively) digestion.
The samples were incubated in a water bath for 4 h at 80C and diluted to make up 5 mL with deionized water.
Serum As concentration was estimated using ICP-OES and expressed as g/L.
Biochemical Assays
The brain tissue was homogenized with Tris-HCl buffer (pH 7.6, w/v) and then was used for further analysis.
Brain oxidants- lipid peroxidation (LPO)14, protein carbonyl content (PCC)15, nitric oxide (NO)16 and antioxidants
- superoxide dismutase (SOD)17, catalase (CAT)18, glutathione peroxidase (GPx)19, reduced glutathione (GSH)20,
356
ascrobic acid21, protein content22 and acetylcholinesterase (AChE)23 were measured using standard methods.
Histopathological studies
The brain tissues were xed in 10%, v/v neutral buffered formalin and dehydrated with differently graded
ethanol series, embedded in parafn wax, sectioned with 5 micometer thickness and stained with hematoxylin and
eosin stains.
All the values were expressed as meanSD of six rats in each group. The data were analyzed with one-way
analysis of variance (ANOVA) followed by Turkeys multiple comparison test. P < 0.05 was considered to indicate
statistical significance.
Results
Effect of A. vera on SMA and motor coordination
The endurance time on Rota-rod was shortened and the SMA was significantly decreased (Fig. 1) in As treated
animals than that of control animals. Supplementation of A. vera extract to the As intoxicated rats showed significant
increase in Rota-rod endurance time and the SMA when compared to As treated groups. Administration of A. vera
extract alone did not show any significant changes in these parameters.
Effect of A. vera on arsenic content, total proteins and AChE activity
Figure 2 shows the levels of As content in serum of control and test animals. The As content in serum was
found to be increased and declined the brain total protein and AChE activity in As intoxicated rats when compared
to control. Supplementation of A. vera extract to the As intoxicated rats showed significantly decreased level of As
and restored the protein and AChE activity when compared to As treated groups.
Effect of A. vera on neuronal oxidants levels
The status of neuronal oxidant markers are presented in Fig 3. As treatment significantly increased the levels
of neuronal LPO, PCC, and NO when compared to control. Co-treatment of A. vera with As showed significant
decrease in the levels of LPO, PCC and NO when compared to As treated rats. Administration of A. vera alone did
not show any significant changes in these parameters when compared to control.
Effect of A. vera on neuronal antioxidants
As intoxication significantly declined the levels of neuronal antioxidants such as SOD, CAT, GPx, GSH,
and ascorbic acid when compared to control rats. A significant increase in the antioxidant levels was found in rats
treated with A. vera and As when compared with As alone. Administration of A. vera extract alone did not show
any changes in the levels of antioxidants (Fig. 4).
Effect of A. vera on brain histopathological changes
The histoarchitecture in the brain of control rats was showing the three distinct layers: a. Molecular layer, b.
granular layer and c. purkinje layer. These demarcations were not observed in the brain of rats treated with As and
the peripheral layer was found to be collapsed and merged with the second layer. Also the purkinje neurons of the As
treated rat brains showed chromatolysis, which was not observed in the control brains (Plate-1). Supplementation
of A. vera extract to As intoxicated rats has reduced the above histological aberrations when compared to As treated
rats (Fig. 5).
Discussion
In the present study there was a decrease in locomotor activity in As exposed animals when compared to
control. It has been showed that short-term exposure to 20 mg/kg As resulted in decreased locomotor activity24.
The changes observed in locomotor behavior could be a result of the general malaise evident in these animals, or
alternatively, it could reflect the deleterious effects of As on metabolism, particularly in the central nervous system25.
Other studies using high doses of arsenic trioxide have also found decrements in locomotor parameters in rodents26.
357
A significant decrease in spontaneous motor activity was observed in As exposed animals. The spontaneous activity
measured by summing up different types of movement of rat on a digital top pan balance showed diminished
activity at all dose levels of As with significant reduction at 0.3 and 3.0 ppm27. The results indicate that the As
exposure affects neurobehavioral parameters of the developed as well as the developing brain. The neurotoxicity
of As could be due to the adverse effects of the parent compound or to the effects of the methylated metabolites on
antioxidant enzymes and neurotransmitter metabolism, which could account for the behavioral alterations reported
in animal models24.
In the present study, AChE activity was greatly reduced in the brain of As treated animals. The result agree
with previous studies that demonstrated a decrease activity of AChE in Sprague-Dawley rats28, in neuroblastoma
cells of mice29, in rat whole brain30 and in two models of fish31. A disturbance in the activity of cholinesterase is
likely to impair cholinergic transmission at neuromuscular junction resulting in a suppression of motor system.
Accumulation of As in the brain leads to excessive generation of ROS. As reported previously4, As is a potent
neurotoxin induces ROS and causes LPO, PCC, and peroxynitrite formation. In this study, the above parameters
were found to be increased in As intoxicated rats. This result confirms that As causes deletrious effect in the
membrane degeneration, cleavage of functional groups in the amino acids and formation of peroxynitrite through
ROS resulting in neuronal damages. A significant decrease in the levels of the antioxidant enzymes like SOD,
Catalase and glutathione peroxidase was observed in brain of As treated animals. Decreased levels of SOD, catalase
and glutathione peroxidase may be due to oxidative stress produced by free radicals. Antioxidant enzymes such as
SOD, catalase and glutathione peroxidase are very important in protecting organisms from free radicals generated
from As exposure. Similar results of decreased SOD, catalase and glutathione peroxidase were reported in As
exposed male albino mice brain32 and rat neonatal brain27. A marked reduction was seen in glutathione (GSH)
content in As exposed animals. Similar GSH reduction was observed in As exposed rat brain30 and male albino
mice32. In its trivalent form, As binds with high affinity to GSH, restricts its antioxidant activity and inhibits various
enzymes, which require GSH as cofactor. The result of the present work shows decrease of GSH in brain of As
exposed animals indicating oxidative stress.
The ascorbic acid content was decreased in brain of As exposed rats. These results were concomitant with
the earlier reports32-33 reveals that reduced ascorbic acid levels in As intoxicated mice and rats. Under As induced
stress, ascorbic acid gets rapidly oxidized and gets converted to the dehydro form (DHA); resulting in its increased
form, subsequently, DHA did not get converted to reduced form (RHA) due to the lack of GSH. Histopathological
studies revealed the marked brain cell damage in As treated rats. Spheroid bodies were observed in the neuroplasm
of neurons and chromatolysis was also observed in the purkinje neurons. The normal brain showed the three
distinguishable layers namely the molecular layer, granular layer and purkinje layer. The As treated brain lacked the
demarcations and also showed clumping of purkinje neurons and chromatolysis34.
In the present study, A. vera extract prevented the toxic effects of As when it was administered along with
sodium arsenite. The motor activity and motor coordination was increased compared to those As exposed animals.
The AChE activity was also increased in As+A. vera extract treated animals. This may be due to choline content
present in A. vera extract, which is a memory booster and directly effects nerve impulses from the brain through
central nervous system. The aminoacids in A. vera are the building blocks of protein and influence the brain function.
So the activity of AChE was increased in As+A. vera extract treated animals in the present study.
The protein concentration of As+A. vera extract treated rats showed a preventive effect over As induced
impairment of the protein synthetic machinery. The decrease in MDA level was found in As+A. vera extract treated
animals than As treated groups and it showed that the ability of the extract to prevent lipid peroxidation. GSH
content was increased in As+A. vera extract treated rats. This may be due to the extract can either increase the
biosynthesis of GSH or reduce the oxidative stress leading to less degradation of GSH, or have both effects. Nitric
oxide, glutathione peroxidase, SOD, Catalase was significantly increased due to the presence of antioxidants like
vitamin E, C and Vitamin B12 present in A. vera which prevents it from free radical attack. The increase in LPO,
358
GSH, SOD, catalase, glutathione peroxidase activity was observed in A. vera treated diabetic rats35. The similar
increase in antioxidants were found in the liver of A. vera and As exposed rats10. The As content of As+A. vera
extract was slightly decreased compared to As treated animals. This is due to A. vera have little chelating activity on
As. The brain of As+A. vera extract group showed normal purkinje neurons similar to that of control. Hence A. vera
has a property to prevent chromatolysis in As treated animals. It has been reported that antioxidant compounds found
in A. vera36. They examined lipid peroxidation using rat liver microsomal and mitochondrial enzymes. Among the
aloesin derivatives examined, isorabaichromone showed a potent antioxidant activity. In this study, the toxic effects
of As were prevented by A. vera extract may account for its vitamin E, C, B12. A. vera also contains Aloe-emodin
which is an antioxidant and it has scavenging activity on free radical and ROS37. Tocopherol is a fat soluble vitamin
found in A. vera gel that has proven antioxidant activity. The antioxidant phytoconstituens in A. vera extract may
be responsible for the protective effect against As induced neurotoxicity in rats.
Conclusion
The above results suggest that A. vera has some protective value against As induced oxidative stress, but these
effects are independent of As depletion from serum of animals. Thus A. vera in combination with chelating agents
greatly prevents the oxidative stress and eliminates As from the body. Hence, A. vera in combination with chelating
agents should be considered in future studies.
Acknowledgments
This work was supported by the UGC-SAP DRS II: F-3-30/2013 and DST-FIST: SR/FST/LSI-618/2014, New
Delhi, India for the partial nancial assistance. The author LP also extends his thanks to the Indian Council of
Medical Research, New Delhi, for nancial assistance in the form of a senior research fellowship (No. 45/25/2013/
BMS/TRM).
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Figures 300
250 Control As
A. vera As + A. vera +
200
*
150
Units
+
100
50 *
0
Roto-rod SMA
Fig. 1. Effect of A. vera on motor coordination and SMA activity in control and test animals. Units: Motor
coordination -Sec; SMA Counts/min. Values are mean SD of six rats in each group. *p < 0.05 signicantly
different from control. +p < 0.05, signicantly different from As treated rats (One way ANOVA followed by Tukeys
multiple comparison test).
6
Control As
5
A. vera As + A. vera
4 + *
+
3
Units
* +
2 *
0
Protein Serum As AChE
Fig. 2. Effect of A. vera on arsenic content, total proteins and AChE activity in control and test animals.
Units: Protein -mg/g; As g/L; AChE - M/min/mg/g tissue. Values are mean SD of six rats in each group. *p
< 0.05 signicantly different from control. +p < 0.05, signicantly different from As treated rats (One way ANOVA
followed by Tukeys multiple comparison test).
360
45
40
*
test).
35
Control ML GL Diffused cortical As A. Vera + As
+
30 layer
Control As PL
25
Units
20
40X
15
10 *
+
5 *
+
0
LPO PCC NO
400X
Fig. 3. Effect of A. vera on neuronal oxidants in
control and test animals. Units: LPO-nM of MDA/mg
protein; PCCnM carbonyl/mg protein; NO- M of
nitrite/100 mg tissue. Values are mean SD of six rats
in each group. *p < 0.05 signicantly different from
Fig. 5. Effect of A. vera on brain histological
control. +p < 0.05, signicantly different from As treated
abnormalities in control and test animals. Structure of
rats (One way ANOVA followed by Tukeys multiple
control brain shows Molecular layer (ML), Granular
comparison test).
layer (GL) and Purkinje layer (PL) of neurons. As
18 induced rat brain shows chromatolysis in the purkinje
16 neurons whereas A. vera supplementation reduced the
Control As
14 +
As induced histological aberrations.
12
10
*
Units
8 +
+ +
6
*
+
4 *
* *
2
0
SOD CAT GPx GSH Ascorbic acid
Fig. 4. Effect of A. vera on neuronal antioxidants

in control and test animals. Units: SOD- Units/mg
protein; CATM of H2O2 utilised/min/mg protein;
GPx-g of GSH utilised/min/mg protein; GSH- g/
mg protein. Values are mean SD of six rats in each
group. *p < 0.05 signicantly different from control. +p
< 0.05, signicantly different from As treated rats (One
way ANOVA followed by Tukeys multiple comparison
361
Comparison on antioxidant potential of pulp and seed extracts of
Elaeocarpustectorius fruits
Poorani Gurumallesh and Baskar Ramakrishnan*
Department of Biotechnology, Kumaraguru College of Technology, Coimbatore 641049
*E-mail: baskar.r.bt@kct.ac.in
Abstract
Free radicals are highly reactive species having unpaired electrons in their outermost shell. The balance
between the production and neutralization of reactive oxygen species by antioxidants is very delicate, and if
this balance tends towards the overproduction of ROS, the cells start to suffer the consequences of oxidative
stress. Free radicals react rapidly with the membranes eventually causing cellular degeneration and finally
death. To cope with these radicals, the living system produces many antioxidants or takes the supplement
through diet. Elaeocarpus tectorius is an evergreen tree found abundantly in Nilgiris district. The present
study aims to evaluate the antioxidant potential of Elaeocarpus tectorius. Extracts of pulp and seed of the fruit
were prepared using methanol and ethyl acetate using Soxhlet extraction unit and subjected to various in vitro
antioxidant assays. The results proved that the extracts exhibited potent antioxidant properties even in their
low concentration. Therefore, it is suggested that further work could be carried out on the identification and
isolation of the antioxidative components from Elaeocarpus tectorius. Thus, the study suggests that the extract
could be developed as a potent drug molecule or as a food additive.
Keywords: Free radicals, Antioxidant, Ethanobotany, Western Ghats, Elaeocarpus tectorius,
Introduction
A proper diet and healthy nutrition are very important parameters to maintain good health throughout the
lifetime. A free radical could be noted as a molecular species containing an unpaired electron in atomic orbital [1].
The characteristic feature of the free radical is their highly unstable nature which is capable of causing irreversible
damage to the cells. Free radicals are produced in our body through various metabolic processes. Rarely, our bodys
own immune system produces free radicals to neutralize pathogens. Other factors that contribute to the free radical
production are certain environmental factors such as pollution, radiation and herbicides. The body system can
handle only a few numbers of free radicals. More numbers of free radicals can lead to cancer, chronic inflammation,
age related diseases and vascular diseases. These are non communicable diseases and can be cured by antioxidants
[2,3].
The common term used for free radicals are Reactive Oxygen Species (ROS). The half life of the ROS is
relatively high so that it can travel longer distance in the cellular environment and cause oxidative stress to the cells
[4]. It is to be noted that the ROS includes radicals of oxygen, hydrogen peroxide, hydroxyl and super oxide. A few
antioxidants such as superoxide dismutase, catalase and glutathione are naturally produced in the body to counteract
the free radicals. Additionally, plants can also contain certain phytochemicals that can neutralize the free radicals.
Elaeocarpus tectorius, belonging to the Elaeocarpaceae family in one such plant, an evergreen tree found
predominantly in Western Ghats region. The ethanobotanical knowledge of the fruit was obtained from badaga
community people. Elaeocarpus tectorius was traditionally used for the treatment of skin allergy purposes. But no
scientific evidence is available which depicts the therapeutic potential of the Elaeocarpus tectorius fruits [5].
The present study aims to evaluate and compare the antioxidant potential of the pulp and seed extracts of the
Elaeocarpus tectorius fruits using antioxidant assays like total antioxidant capacity assay, DPPH (2,2 diphenyl
dipicrylhydrazyl) radical scavenging assay, ABTS (2,2 Azino-bis 3-ethylbenzthiazoline-6-sulfonic acid)radical
scavenging assay, nitric oxide radical scavenging assay, metal chelating assay (Ferrous ion chelation), FRAP (Ferric
reducing antioxidant power) assay.
362
Materials and methods
Plant material
The Elaeocarpus tectorius fruits were obtained from Oranally hamlet of The Nilgiris District. The pulp and
seed were separated, shade dried and ground into coarse powder. The antioxidant components were extracted using
Soxhlet Extraction Unit. Two solvents, methanol and ethyl acetate were used for extracting 10 g of sample powder.
Following extraction, the extracts were dried allowing the solvents to evaporate. The residues were then used for
various antioxidant assays.
All the solvents, reagents and standard chemicals used in the assays were procured from HiMedia and all are
of analytical grade. Ascorbic acid was used as the standard antioxidant.
Total antioxidant capacity assay
The total antioxidant capacity assay gives an estimate of overall antioxidant potential of methanol and ethyl
acetate solvent extract of Elaeocarpus tectorius pulp and seed. The activity was expressed in terms of ascorbic acid
equivalence. The reagent consisted of 28 mM disodium hydrogen phosphate, 4mM ammonium molybdate and 0.6
M sulphuric acid. 1.0 ml of the reagent was added to the 0.1 ml sample aliquots at a concentration of 20 - 100g.
The reaction mixture was incubated at 95C for 90 minutes. Absorbance was measured at 675 nm. Ascorbic acid
was used as the standard [6].
Different concentrations (20, 40, 60, 80 and 100 g/ml) of the extracts were prepared dissolving extract in
methanol. Varying concentrations of sample triplicates (20 to 100g/ml) were prepared and made up to 2ml with
methanol. 0.5ml of freshly prepared DPPH reagent (0.02g in 75ml of methanol) was added to all test tubes and
mixed well. The reaction mixture was incubated for 30 minutes in dark and absorbance measured at 517 nm [7].
Standards were prepared using ascorbic acid and percentage inhibition of the DPPH radical by the extract was
calculated from the formula:
% inhibition = Optical density of control sample Optical density of test sample

______________________________________________________
Optical Density of control sample
2.4 ABTS radical scavenging assay
In this assay, 2, 2 Azino-bis 3-ethylbenzthiazoline-6-sulfonic acid diammonium salt is converted to its radical
cation by the addition of ammonium persulfate which results in the reduction of blue colour. Equal volumes of ABTS
solution (7mM) and ammonium persulfate (2.45mM) was mixed well and was kept in the dark for 16 hours at room
temperature to form a dark coloured solution. The initial absorbance of the solution was measured at 745 nm and
was found to be exceeding the visible range. Final absorbance of 0.700(0.02) was obtained by diluting the solution
with ethanol at room temperature. Varying concentration of the sample triplicates (20 - 100g) was prepared.0. 3
ml of the ABTS standard solution was added to 0.5ml of extract sample and made upto 1.0 ml with ethanol. After a
period of 6 minutes, decrease in absorbance of the mixture was read at 745 nm. 0.5 ml of the methanol and 0.3 ml
of the ABTS was taken as a control and methanol was set as a blank. The percentage inhibition of the ABTS radical
by the antioxidants in the sample was calculated as above [8].
Nitric oxide radical scavenging assay
NO- is generated in biological tissues by specific nitric oxide synthases, which metabolizes arginine to
citrulline with the formation of NO- via a five electron oxidative reaction. The compound sodium nitroprusside is
known to decompose in aqueous solution at physiological pH (7.2) producing NO-. Under aerobic conditions, NO-
reacts with oxygen to produce stable products (nitrate and nitrite), the quantities of which can be determined using
Griess reagent. 1.0 ml of different concentrations of extracts (20 100g) is mixed with 1.0 ml of 5mM sodium
nitroprusside (in PBS, pH 7.4)and incubated in room temperature for 30 minutes. After the incubation period,1.0
ml of Griess reagent (1% sulphanilamide, 2% orthophosphoric acid, 1% naphthalene ethylene diamide) was added
363
and the coloured complex formed was read at 546 nm The percentage inhibition of the nitric oxide radical by the
antioxidants in the sample was calculated [10].
Reducing power assay is based on the reduction of Fe3+ to Fe2+ by direct electron transfer. Prussian blue colour
complex is formed by the addition of free Fe2+ ions to the reduced product, which could be read at 700 nm. Samples
of varying concentration (20 100 g/ml) were taken and made up to 2.5 ml with methanol. To all the reaction
mixtures 2.5 ml of 1% potassium ferricyanide and 2.5ml of 0.2 M phosphate buffer were added and incubated at
50C for 20 minutes. Excluding the sample, rest of the reaction mixture was taken as control. After the incubation
time, the reaction was terminated by adding 2.5ml of 10% trichloroacetic acid followed by centrifugation at 3000
rpm for 10 minutes. 2.5ml of supernatant was taken and 2.5ml of deionized water and 0.4ml of 0.1% ferric chloride
solution was added. The coloured complex formed was read at 700 nm [9].
Ferrous Ion chelation assay
Under acidic conditions, iron is liberated from transferring. Ascorbate reduces the released Fe3+ ions to Fe2+
ions, which then react with ferrozine to form a coloured complex. The colour intensity is directly proportional to
the iron concentration and can be measured spectrophotometrically. Varying concentration of the sample triplicates
(20 - 100g/ml) was prepared. 1 ml of each extract was added with 0.1 ml of 2mM FeCl2 and 3.7 ml of methanol.
Then, it was added with 0.2 ml of 5mM ferrozine and incubated at room temperature for 10 minutes. The decrease
in absorbance was measured at 562 nm [11].
Results and Discussion
Total antioxidant activity
The total antioxidant capacity of pulp and seeds of Elaeocarpus tectorius was depicted in Figure 1(a) and
1(b). There is a formation of phosphomolybdenum complex, the intensity of which indicates the potential of solvent
extracts as a scavenger of free radicals. The results are expressed in ascorbic acid equivalence / gram of tissue. From
the figures, it was found that the antioxidant activity was high in extracts, that were prepared using solvent with high
polarity index. Thus, methanol extracts showed maximal antioxidant activity, when compared to extracts of ethyl
acetate. The result obtained was in accordance with that obtained from total antioxidant capacity of Sapota peel[12].
Fig. 1(a) Total antioxidant capacity of methanol Fig. 1(b) Total antioxidant capacity of ethyl acetate
extract of Elaeocarpus tectorius pulp and seed extract of Elaeocarpus tectorius pulp and seed
DPPH is a nitrogen centered radical that shows absorbance at 517 nm. Change of colour from purple to yellow
was observed, because of reduction of 2,2 diphenyl dipicryl hydrazyl. Decrease in the absorbance with increase
in the concentration of the extract was obtained for pulp and seed extracted using both methanol and ethyl acetate.
Figure 1(a) and 1(b) shows the % inhibition of the extracts prepared from methanol and ethyl acetate. From the
figure, it was inferred that at 20 g/ml concentration of the methanol extract exhibited maximum radical scavenging
activity. The results are in concordance with antioxidant potential evaluation from different solvent extracts from
leaves, flowers and bark of Gold Mohar [13].
364
Fig. 2(a) Scavenging effect (%) on DPPH radical by Fig. 2(b) Scavenging effect (%) on DPPH radical by
methanol extract of Elaeocarpus tectorius pulp and ethyl acetate extract of Elaeocarpus tectorius pulp
seed and seed
ABTS radical scavenging activity
ABTS assay is based on the inhibition of the absorbance of the radical cation ABTS+, which has a characteristic
long wavelength absorption spectrum. This radical scavenging assay is used to determine the efficiency of seed and
pulp extract of Elaeocarpus tectorius. The absorbance of the sample decreases with increase in the concentration
of the sample and thus increasing the percentage inhibition [14]. Free radical scavenging potential of methanol and
ethyl acetate extracts of seed and pulp of Elaeocarpus tectorius under hot extraction is represented in Figure 3(a)
and 3(b) respectively.
Fig. 3(a) Scavenging effect (%) on ABTS radical by Fig. 3(b) Scavenging effect (%) on ABTS radical by
methanol extract of Elaeocarpus tectorius pulp and ethyl acetate extract of Elaeocarpus tectorius pulp
seed and seed
Nitric oxide radical scavenging activity
Nitric oxide scavenging effect is a reduction of nitric oxide. The nitric oxide generated from sodium
nitroprusside reacts with oxygen to form nitrite. The nitrite ions diazotize with sulphanilamide acid
and couple with naphthyl ethylenediamine, forming pink colour, which was measured at 546nm. As
antioxidants donate protons to the nitrite radical, the absorbance decreases. The decrease in absorbance
was used to measure the extent of nitrite radical scavenging [15]. Nitric oxide scavenging effect on methanol
and ethyl acetate solvent extracts of Elaeocarpus tectorius are depicted in figure 4(a) and 4(b). From the figure
it was inferred that methanol and ethyl acetate extracts of seed material exhibited high scavenging activity when
compared to the pulp.
Fig. 4(a) Scavenging effect (%) on Nitric oxide Fig. 4(b) Scavenging effect (%) on Nitric oxide
radical by methanol extract of Elaeocarpus tectorius radical by ethyl acetate extract of Elaeocarpus
pulp and seed tectorius pulp and seed
365
In this method, antioxidant compounds form a coloured complex with potassium ferricyanide, trichloroacetic
acid and ferric chloride that was measured at 700 nm. Increase in absorbance of the reaction mixture indicates the
increase in reducing power of the sample [16]. Reducing power of different solvent extract of Elaeocarpus tectorius
under hot extraction condition are indicated in Figure 5(a) and 5(b). From the figures, it was inferred that ethyl
acetate pulp extract exhibited maximal reducing power capacity.
Fig. 5(a) Reducing power capacity of methanol Fig. 5(b) Reducing power capacity of ethyl acetate
extract of Elaeocarpus tectorius pulp and seed extract of Elaeocarpus tectorius pulp and seed
3.6 Ferrous ion chelation assay

The ferrous ion chelating activity can be used to evaluate the antioxidant potential of the sample extract. It is
measured by decrease in the absorbance with increase in the concentration of the sample. The ferrous ion chelating
activity of pulp and seed extract using solvents methanol and ethyl acetate are depicted in figure 6(a) and 6(b). It
was observed that methanol and ethyl acetate seed extracts showed higher chelation ability when compared to pulp
extracts.
Fig. 6(a) Ferrous ion chelation effect (%) of Fig. 6(b) Ferrous ion chelation effect (%) of ethyl
methanol extract of Elaeocarpus tectorius pulp and acetate extract of Elaeocarpus tectorius pulp and
seed seed
Conclusion
The present work has proved that the methanol and ethyl acetate extracts of pulp and seed from Elaeocarpus
tectorius, an endemic fruit used by Badaga tribe in Western Ghats possessed strong antioxidant properties. High
potential activity was obtained at very low concentration of the sample extract (20 100 g/ml). The results serve
as a scientific basis to further development of the pulp and seed extract into potent drug molecule or a food additive
that can be consumed as a dietary supplement.
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367
Protein interactions may influence the effectiveness of phytochemicals
as anticancer agents
Padma, P.R.* and Sivapriyadharshini, S.#
* Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home Science and
Higher Education for Women University, Coimbatore 641 043
# Department of Biotechnology, Sri Shakthi Institute of Engineering and Technology, Coimbatore 641 062
Abstract
The global search for effective anticancer drugs has led to the recognition of several phytochemicals and
herbal extracts to possess good anticancer activity. In the effort to understand the mechanism of action of such
phytochemicals, various cellular and molecular events, including cell death, gene expression profiles, protein
expression profiles, signal transduction profiles and the pathways involved in cancer (like angiogenesis, invasion
and metastasis) are being studied in depth. Several studies using the expression profiles of cancer-related genes
and the cancer genomes have revealed genetic signatures associated with different types of cancer. However,
the understanding of the influence of the anticancer phytochemicals on the gene expression pattern in cancer
cells is not complete, eventhough some studies have been reported. Quercetin, a flavonoid found in abundance
in several fruits and vegetables, has been shown to have good anticancer effect against several types of cancer.
However, the mechanism of action of this compound is yet to be completely understood. We have studied the
effect of quercetin on the cancer genome of oral cancer cells (KB) and found that it profoundly influences the
expression of several genes, both by upregulation and downregulation. In the present study, we have made an
attempt in silico, to study the effect of quercetin on the interaction profile of the protein products of the genes
influenced by quercetin. Protein interaction maps of the upregulated proteins (20), downregulated proteins
(11) and all the influenced proteins (31) were constructed using STRING (ver.10.0). The maps showed very
strong interactions between the proteins coded by the genes responding to quercetin exposure. The analysis
of these interactions revealed the possible mechanism of the anticancer action of quercetin. Our study shows
that the analysis of protein interactions may provide insights into identifying novel protein targets for cancer
therapy.
Keywords: Anticancer phytochemical, flavonoid, quercetin, protein interactions
Introduction
Cancer continues to be a killer disease worldwide, in spite of the enormous advances made by researchers to
develop better diagnostic tools, identify potential anticancer agents, understand their mechanism of action and to
develop them into effective drugs1. The treatments of cancer include surgery, radiation, chemotherapy, hormone
therapy, immune therapy and targeted therapy, but they are not without limitations, such as, toxicity, serious side
effects and development of resistance to therapeutic agents by cancer cells2. These limitations have paved way to
the rise in interest in the use of plant-derived natural products as sources of cancer chemotherapeutics. An important
area of cancer research in recent times is the search for natural compounds that effectively kill cancer cells, while
exerting low toxicity towards normal cells3. In this pursuit, researchers all over the world have identified many
phytocomponents that hold promise as cancer chemotherapeutics. One of the important group of such phytochemicals
are the flavonoids.
Quercetin is a major plant-derived flavonoid found in several foods and bevarages, apart from many fruits
and vegetables4. The significant anti-tumor effects of quercetin, both in vivo and in vitro, have been reported widely
and these actions have been linked to its effect on the molecules involved in carcinogenesis5. Yet, the underlying
mechanism of action of quercetin is generally unknown. Understanding a compounds target mechanisms is vital in
predicting its phenotypic effects. Revealing the mechanism of action of any potential chemopreventive compound
will open up newer avenues of therapy, resulting in improved efficacy and therapeutic effects of the compound. In this
attempt, recently the awareness has dawned on the scientists that, quite often, it is not an individual target molecule,
but the interplay of several target molecules, that influences the final outcome of the effect of an anticancer drug.
368
The present study is one such attempt, wherein the possible interactions between the various cancer-related proteins
that are influenced by the treatment of quercetin in oral cancer cells has been studied using an in silico platform.

All reagents used in the study were of analytical grade. Oral carcinoma cells (KB) were procured from
National Centre for Cell Sciences, Pune and maintained in our laboratory under standard conditions. The cells were
exposed to quercetin at a final dose of 50M, for 18 hours. The dose and duration of exposure were determined
by an elaborate pilot study (results not shown). Appropriate control cultures without quercetin exposure were
also maintained alongside. After the completion of the incubation period, the cells were harvested and the total
RNA was isolated using RNEasy Kit (Qiagen). The first strand synthesis (cDNA) was done using realtime PCR.
Gene expression profiling was done using RT-PCR Cancer Pathway Finder Array (Qiagen), by following the
manufacturers instructions. A total of 84 cancer-related genes that are involved in the pathways of Adhesion,
Angiogenesis, Apoptosis and cell senescence, Cell cycle control and DNA damage, Invasion and metastasis and
Signal transduction molecules and transcription factors were studied using the array. The results of this part of the
study have been communicated for publication in a different source.
In the present study, the gene expression data were used to analyze the interactions between the differentially
expressed gene products. For this, the online protein interaction networking database, STRING (version 10), was
used (www.string-db.org). The upregulated genes and the downregulated genes by quercetin in the KB cells were
listed out separately, using the fold-change in the intensities of gene expression in the gene expression array. These
two lists were submitted separately to the STRING server and the interaction maps were constructed. No clustering
algorithms were applied and the default clustering values were accepted to construct the maps. After the maps were
constructed, the pathway-specific gene products were manually enclosed in circle- and oval-shapes as appropriate
and labelled to identify the pathways. After the analysis of upregulated and downregulated components separately,
the total interaction maps of all the modulated gene products were constructed. This was done to analyze the
interplay of differentially modulated components. The maps were constructed in the interactive flash mode of the
software. The nature of interactions between the key proteins was analyzed by mouse-over the lines of interactions
and inferences were made regarding the possible mechanisms.
Results
The gene expression profiling of the cancer-related genes showed that 31 genes were significantly influenced by
the exposure of oral cancer cells to quercetin. Of these, 20 genes were upregulated and 11 genes were downregulated
(results communicated for publication in journal). The lists of upragulated genes (ANGPT1, IGF1, FGFR2,
COL18A1, IFNA1, IFNB1, INF, TEK, TERT, BCL2, CASP8, TNFRSF25, SYK, ITGB3, MTSS1, CDC25A,
TIMP3, TWIST1, MMP2 and ERBB2) and downregulated genes (CXCL8, BAX, HTATIP2, CDKN1A, CDKN2A,
MDM2, TIMP1, NME1, PLAUR, S100A4, NFKBIA) were prepard. The two lists were submitted separately to
STRING (ver.10.0). The interaction maps obtained for the upregulated gene products and the downregulated gene
products are presented in Figure 1 (a and b) respectively.
Figure 1
Protein Interaction Map from Genes Upregulated and Downregulated by Quercetin
(a) Upregulated Proteins (b) Downregulated proteins

Twenty proteins were upregulated upon quercetin treatment, which, when plotted on the interaction map,
showed 54 actual interactions, against 10 predicted (expected) interactions. Of the 20 proteins plotted in the map, 19
369
showed interactions, while one protein (MTSS1 metastasis suppressor 1), which was a component of the adhesion-
related molecules, did not show any interaction with other proteins in the quercetin-upregulated network. However,
it was positioned in close proximity to the other adhesion-associated molecules in the network, suggesting that they
function together, albeit without direct interaction. Very high Combined interaction score was observed between
four pairs of proteins (TIMP3 and MMP2, ANGPT1 and FGFR2, COL18A1 and MMP2, CASP8 and BCL2). Of
these, three pairs of proteins fell into the same cancer-related pathways (angiogenesis, invasion and metastasis,
apoptosis), while COL18A1 and MMP2 were components of different pathways (angiogenesis and invasion-and-
metastasis respectively). This observation suggests that the process of invasion and metastasis is closely influenced
by angiogenesis.
Angiogenic components showed good interactions with the signal transduction protein, adhesion molecules,
apoptotic molecules and the components of the invasion and metastatic pathway. The only protein upregulated in the
cell cycle control and DNA repair process, namely CDC25A (a tyrosine protein phosphatase that influences mitosis)
showed interaction only with BCL2, an anti-apoptotic protein. This pattern implies that the anti-apoptotic factor,
Bcl2, may exert its action, not only by suppressing apoptosis, but also by exerting an influence on the cell cycle. The
two adhesion-related proteins, SYK (a tyrosine kinase) and ITGB3 (integrin beta 3), showed very high combined
interaction score in their interaction with the other proteins. This observation also suggests that quercetin improves
adhesion, thus acting against metastasis.
Out the eleven proteins that were negatively regulated by quercetin, two proteins (HTATIP2, which is a Tat-
interactive protein 2, and PLAUR, which is plasminogen activator-urokinase receptor) did not show any interaction
with the downregulated proteins in the network. The role of HTATIP2 in cancer is also quite unclear, but is implied
to play a role in apoptosis and cell senescence, as per the legend of the PCR array. The maximum combined
interactive scores were observed between the proteins involved in cell cycle control and DNA damage repair. This
observation denotes a strong tripartite association between these three proteins, all of which are associated with
the control of cell cycle and DNA repair. The co-suppression of all the three proteins by quercetin implies that the
anticancer effect of quercetin is not mediated through the p53-mediated CDK inhibitory control of the cell cycle.
Figure 2
Combined Protein Interaction Map from Genes Modulated by Quercetin in KB Cells
Following this, all the 31 modulated gene products were submitted together and the interaction map was
constructed (Figure 2). The recorded 124 observed interactions against 67 total interactions when upregulated (54
interactions) and downregulated (13 interactions) components were considered separately, clearly proved that there
were interactions among the upregulated and downregulated proteins. An interesting observation was that, MTSS1
(adhesion-related protein) and PLAUR (invasion-and-metastasis pathway component) showed interactions with
other proteins when all the modulated gene products were plotted together. Both these components did not show
any interaction with other proteins when the upregulated and downregulated components were plotted separately.
HTATIP2 did not exhibit any interaction, even when all the altered components were plotted together.
Discussion
Proteins and their multitude interactions play an undeniable central role in the cellular processes. The protein
components of the complex networks in a cell are functionally interdependent. The resultant multiple downstream
370
functions are important in both disease and normal state6. In the present study, the complexity of the interactions
between the proteins coded by the genes that were profoundly influenced by the exposure of quercetin in oral
carcinoma cells showed that the pathways involved in the process of carcinogenesis are inter-related and their
components influence each other.
Several studies reported in the literature also provide evidence to support our findings. The genes modulated
by artemisinin, sesquiterpene lactone extracted from the Artemisia Sp. in cancer cells were investigated by STRING
analysis and the protein interaction maps revealed targeting of membrane receptors and key signaling pathways7.
Recently, protein interaction network analysis used to investigate the effect of dihydroartemisinin in prostate cancer
cells showed that the main pathways altered were aminoacyl-tRNA biosynthesis and metabolic pathways8. In a
study9 on the effect of curcumin in gastric cancer (BGC-82) cells, using STRING analysis, identified that the major
networks affected by curcumin were associated with cellular function and maintenance, cellular assembly and
organization and metabolism. In another study10, Lasonolide A, an alkaloid from marine sponge, showed significant
protein-protein interactions of the candidate genes in pathways related to protein phosphorylation, cell cycle and
survival, in HCT-116 colon cancer cells.
Wang et al.11 used STRING analysis to study the interactions between 55 differentially expressed proteins
in leukemia cells (CCRF-CEM) exposed to 8a, a synthetic acridone derivative, wherein it was reported that the
protein-protein interactions revealed that proteins associated with nucleosomal assembly and energy metabolism
formed the largest cluster. RH1, a novel anticancer drug that has entered Phase I trials has been studied for its ability
to modulate cancer related pathways in hepatoma cancer cells and STRING analysis revealed a multifactorial effect
on cell cycle, DNA repair and other cellular pathways12. These reports add validity to the use of protein-protein
interaction studies in analyzing the major proteins and their participating pathways and the use of STRING database
in constructing the networks.
Conclusion
The results of the present study revealed that there is profound influence of the protein products that are
modulated by quercetin exposure in the KB oral cancer cells, at the gene level. This suggests that in-depth analysis
of the proteomic profile of the exposed cells may throw more light into possible sure-fire target proteins that may
respond positively to anticancer phytochemicals.
Acknowledgement
The financial support to SPD from DST, under the WOS-A scheme, to carry out this study is gratefully
acknowledged.
Conflict of Interest
The authors declare no conflict of interest.
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371
A Study on Basic Knowledge of Urban Community on Spices and
Condiments
Salma. S 1 and Lalitha Ramakrishnan
Department of Nutrition and Dietetics, PSG College of Arts and Science, Affiliated to Bharathiar University,
Coimbatore, Tamil Nadu. India. smartsalma@outlook.com. Mobile No: 9025529929/9843162880
Abstract
Spices play an important role as a flavouring agent in the diet. This study has attempted to identify the knowledge
of urban community (Coimbatore) on some key areas of Spices and condiments. The survey method using
questionnaire was carried out to elicit the required information from 100 Household women respondents
of different areas in Coimbatore district. The collected data was analysed using descriptive statistics, Cross
tabulation and Chi-square test. More than 80 % of respondents preferred to buy spices on basis of its quality
and believed to possess medicinal properties such as peppercorns for cold, cumin for digestion. Similarly,
more than 40 % of respondents believed that the excessive usage of spices may be the causative factors for
an ulcer and gastritis. The findings indicated that the illiterate old women respondents were more aware of
the medicinal properties and risk of using excess spices than educated respondents. It is mandatory to create
awareness among the young aged respondents about medicinal values of spices and condiments.
Keywords: Spices and condiments, urban community, perception, basic knowledge.
Introduction:
Spices are defined as dry plant material to be used as flavor/additive in foods [1]. Consumption of spices is
generally higher in Asian countries such as India, China, and Thailand, there has been an increasing trend in their
intake in developed countries such as Europe and the USA, because of changing food habits and preference for
ethnic and spicy food [2, 3]. Ancient Indian philosophy promoted the use of natural resources to prevent disease
and battle all ills related to human body. Today, the world looks towards India as it makes use of spices and herbs
to cure many illnesses, lifesaving drugs and various health foods. India is endowed with enormous varieties of
spices grown in the major land area of the country and one of the largest exporters of spices in the world. Many
household spices are being used regularly in the Indian foods [4]. Indiais beatified with a variegated climate each
of its states produces some spice or the other.India is an ideal setting to evaluate consumption of these food items,
as they are the key components of the traditional diets. Moreover, in developing countries with limited access to
diverse foods, certain spices may also be important dietary sources of micronutrients, such as iron and calcium [6].
Apart from adding colour, flavour and taste, consumption of spices provide countlesshealthbenefits.It is important
to document the uses and perform studies to ensure their efficacy and safety. Hence the present study was aimed to
identify basic knowledge of the urban community (Coimbatore city), focussing specifically on medicinal uses of
spices and condiments.
Materials and Methods:
Study Area:
The study is based on a primary survey of 100 respondents belonging to Coimbatore City. Coimbatore lies
at1116N765821Einsouth Indiaat 411metres (1349ft) above sea level on the banks of theNoyyal River,
in south-western Tamil Nadu. It covers an area of 642.12km2 (247.92sqmi). It is the second largest city in the
state afterChennaiand 16th largesturban agglomeration in India. The rich black soil of the region has contributed
to Coimbatores flourishing agriculture industry and hence remains major occupation. In spite of its industrial and
technological growth, traditions and age-old customs are still held in high esteem and so the city has been selected
as study area.
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Data Collection and Analysis:
Direct Interviews with the people living in Coimbatore city was conducted
using a semi-structured questionnaire. The respondents to the interview were
selected using random sampling technique. Recruiter visited respondents in their
homes and asked them the questions in the questionnaire. Examples of some
commonly used spices and condiments were given to make the idea of the study
easier for the respondents. The interview was done in Tamil, language spoken by
the people living in the state. The information was collected from 100 Household
respondents whose age ranged from 21-70 years. The socio-demographic profiles
of the respondents were also recorded on the parameters such as Age, Gender,
Educational Qualification, Occupation and Monthly Household Income. For easy
recognition of various herbs, spices and condiments by the respondent, a chart
showing samples of herbs, spices and condiments were shown to the respondent
while administering the questionnaire. The collected data was consolidated and
interpreted using data analysis techniques such as descriptive statistics; cross-
tabulation and chi-square test using SPSS 19.0.
Results:
Socio -Demographic profile of respondents:
If an individual does not cook most of their own food, they may not know all the food items contained in
mixed dishes, therefore the food preparer may be best suited to provide information on items added during cooking.
Therefore, All 100 respondents selected were females who performed cooking activities in the households. The
majority of the respondents were married and Homemakers. Monthly Household Income is evenly distributed across
from 20000 to 50000 above. The respondents also cover both low and high-income groups as illustrated in Table 1.
Table 1: Socio-Demographic information of the respondents
Socio Demo graphs Criteria Percent
Age 21-30 Years 19
31-40 Years 36
41-50 Years 26
51-60 Years 15
Above 60 Years 4
Education Illiterate 03
Up to 10th Standard 37
High School 11
Under Graduation 24
Post-Graduation 24
Higher Education 01
Occupation Home Maker 72
Business 06
Salaried 22
Monthly Income Up to 20000 41
20000-50000 40
Above 50000 19
Marital Status Married 97
Single 03
373
Knowledge on medicinal properties of spices, herbs and condiments:
Of all the respondents, more than 80 percent believed that spices, herbs and condiments possess medicinal
properties. The knowledge of respondents on medicinal properties is represented in Table 2 in which spices, herbs and
condiments are arranged in the alphabetical order. For each spice, herbs and condiments the following information
is provided: Name of the spice/condiments, its botanical name, local name and ailments treated.
Table 2: Respondents perception towards medicinal values of spices, herbs and condiments
Spice and condiments Name Botanical Name Use/Ailments Treated
Peppercorns Piper nigrum L. Cold, Cough
Cumin seeds Cuminum cyminum L. Digestion, Gastritis
Fenugreek seeds Trigonella foenum-graecum L Body Coolant
Turmeric Curcuma longa L. Antibiotic
Ginger Zingiber officinale Digestion
Garlic Allium sativum Digestion, Gastritis
Asafoetida Ferula asafoetida Whooping cough
Coriander seeds Coriandrum sativum L Sore throat, Digestion
Perceptions about excessive usage of spices, herbs and condiments:
About 44 percent of the respondents believed that excessive consumption of spices, herbs and condiments
like red chillies, cinnamon, cardamom and pepper may cause serious illness such as ulcer, stomach pain, gastritis,
heartburn and acidity. The majority of the respondents conceived that high usage of spices and condiments may
cause ulcer as they cause stomach burning sensation soon after consuming spicy foods.
Figure 1 shows the perception of respondents on excessive usage of spices
Interpretation of consumer perception upon sociodemographics:

Data analysis revealed that peoples demographic background had a significant impact on their perceptions
towards spices and condiments. The statistical findings indicated that the illiterate old women respondents (p = 0.01)
were more aware of the medicinal properties and risk of using excess spices than educated respondents (p=0.159)
at 5 % level of significance.
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Table 3: Different attributes upon sociodemographics
Socio-demographics Medicinal Properties Excessive Usage
Age 0.01 0.02
Occupation 0.159 0.09
Education 0.04 0.05
Monthly Income 0.320 0.349
5% level of significance
Discussion:
Spices have been of particular interest with regard to diseases in both cell and animals studies, but there
are little-known facts about the impact of these foods at the human or epidemiologic level. Yet, researches do
not indicate that all spices have solely beneficial properties in relation to disease outcomes. Though these spices
provide innumerable benefits they should be used sparingly. The excessive use of spices in food can cause harm to
the health. Making specific use of these spices will help consumers to make optimal use of the resources provided
by nature. These type of studies compels immediate action on the loss of traditional knowledge and consumer
education is highly recommended as a contribution to protecting the health, safety, economic and legal interests of
society. It is mandatory to create awareness among the young aged respondents about medicinal values of spices
and condiments.
Acknowledgements:
Authors are grateful to all 100 interviewed respondents for their patience during the busy time of their day.
References:
Sultana S, Ripa FA, Hamid K. Pak J Biol Sci 201013(7):340 3.
Williams PG. Publ Health Med J Aust. 2006; 185:S178. http://ro.uow.edu.au/cgi/viewcontent.cgi?
article=1034&context=hbspapers.
CBI Ministry of foreign Affairs. Spices and Herbs 1999. http://www.faoda.org/download/Spices_and_Herbs_Survey.pdf
Rahman K. Clin Interv Aging 2007;2(2):219-36
Leah M Ferrucci et al Asian Pacific J Cancer Prev, 11, 1621-1629
Ramasastri BV (1983) Plant Foods Hum Nutr, 33,11-5.
Slavica Grujic et al Acta Sci. Pol., Technol. Aliment. 12(2) 2013, 215-222.
375
Differential Cytotoxic Effect of Majorana Hortensis Leaf Extract on
Chick Embryo Cells and Hep2 Cell Lines
Radha Palaniswamy1 and Padma, P.R.2
1
Department of Biotechnology, Dr. NGP Arts and Science College, Coimbatore -641048
2
Department of Biochemistry, Biotechnology & Bioinformatics, Avinashilingam University for Women,
Coimbatore 641043.
Abstract
Nature has been a source of medicinal agents for thousands of years and an impressive number of modern
drugs have been isolated from natural sources. Herbal medicines have been found to cater to cure various
disorders including devastating diseases like cancer. It is a class of diseases in which a group of cells display
uncontrolled growth, invasion and metastasis. Programmed cell death, known as apoptosis, is an essential
cellular homeostatic mechanism that ensures correct development and function of multicellular organisms.
A wide variety of natural products have been recognized to have the ability to induce apoptosis in various
tumour cells of human origin and many of these substances are from herbal source. The present study throws
light on cellular events of apoptosis in the presence of Majorana hortensis leaf extract on chick embryo
fibroblasts and Hep2 (laryngeal carcinoma) cells as model systems. Here, oxidative stress was induced by
etoposide. The results revealed that, in both the cell types studied, oxidative stress imposed by etoposide
caused a steep increase in the number of cells that commit to apoptosis. Majorana hortensis leaf extract
administration showed no cytotoxicity in the normal cells (chick embryo cells), but significant cytotoxicity
towards Hep2 cells. Additionally, it was also significant that Majorana hortensis leaf extract protected normal
cells from the death induced by etoposide, while no such response was exerted against the Hep2 (cancer cells)
treated with etoposide.
Key Words: Majorana hortensis, apoptosis, cytotoxicity, Hep2 cells, oxidative stress.
Introduction: Use of plant sources has gained lot of importance, however due to lack of credible experimental
evidences, it has not been popularized yet. Nevertheless, it is being used for various diseases like cancer and many
more1. The plant used in this study is Majorana hortensis (M.hortensis), a perennial herb belonging to lamiaceae
family, grown extensively in Mediterranean region. It is used as a seasoning agent and as sacred leaf in India,
commonly called as sweet majoram2. Several antibacterial, antitussive, antifungal activities, have been reported
apart from its use as perfume3. The current study aims to find out various other properties of the extract like
anticancer activity.
Cancer is a multifactorial heterogeneous disease characterized by the multistage nature of pathogenesis4.

Damage to DNA by reactive oxygen species (ROS) has been widely accepted as a major cause of cancer. It is now
assumed that ROS are involved both in the initiation and the progression of cancer5. In this direction, alternative
methods such as in vitro cell-based assays, genomics and computational analysis have gained more attention6.
Replacement, Reduction and Refinement of animal procedures (the Three Rs) are implemented wherever possible.
Over the last decade, there has been substantial progress with applying in vitro and in silico methods to both drug
efficacy and safety testing 7.
Preparation of Plant Extract: Leaves from pot culture grown in the campus was shade dried. One gram of
leaf extract was homogenized with 1 ml of methanol. The extract was centrifuged in a microfuge at 5000 rpm for 10
minutes. The supernatant was collected and dried at 60 degrees Centigrade well protected from light. The residue
obtained after drying the solvent was weighed and redissolved in a known amount of dimethyl sulfoxide (DMSO)
to yield a concentration of 20 mg/5 l. DMSO was maintained at a minimum level to avoid DMSO induced events
if any.
376
Treatment Groups:
The following treatment groups were setup for each parameter
1. Primary cells alone (negative) control.
2. Primary cells + etoposide (positive) control.
3. Primary cells + methanolic extract of M.hortensis leave extract.
4. Primary cells + etoposide + methanolic extract of M.hortensis leaf extract. The etoposide and /or leaf extract exposure was
given for 24 hours.
The etoposide concentration was 200 M and leaf extract concentration was 20 mg/mL.
Parameters Analyzed: The parameters analyzed to follow the apoptotic death in the various treatment groups
were the extent of cell death (as determined by SRB and MTT), morphological changes (by Giemsa staining),
nuclear changes (by EtBr, PI, DAPI staining) and DNA fragmentation (by agarose gel electrophoresis).
Cell Viability Study: SRB and MTT was used to quantify the rate of cell survival. For SRB the protocol used
as Skehan et al, 19908. The MTT assay was used to quantify the cell viability which depends on both the cell number
and mitochondrial activity of the cell as proposed by Igarashi and Miyazawa, 20019.
Morphological Changes during apoptosis: The morphological changes that occur during the stages of
apoptosis in etoposide and M. hortensis leaf treated groups were observed by performing Giemsa staining according
to the method of Chih et al., 200110.
Nuclear Changes during apoptosis: the changes in nuclei characterizing apoptosis was visualized with the
help of Ethidium Bromide (EtBr) as described by Mercille and Massie 199011 and Propidium iodide by Sarker et
al., 200012. A comparative study was done between the etoposide treated and leaf extract treated groups of fibroblast
and cells.
DNA Fragmentation: This was studied by agarose gel electrophoresis using the method proposed by Yin et
al., 199413 which showed the typical laddering pattern in electrophoresis of the treated groups with and without leaf
extract both in the cancer cell lines and the normal chick embryo fibroblasts.
Statistical analysis: The parameters analyzed in all the phases of the study were subjected to statistical analysis
using SigmaStat (Version 3.1) statistical software. Statistical significance was determined by one-way ANOVA,
followed by post-hoc Fischer analysis was used and the values with P<0.05 were considered to be significantly
different.
Results: It was observed that M.hortensis leaf extract when administered alone, evoked a differential response
depending on the cell type. In chick embryo fibroblasts, the extract did not induce apoptosis. However, there was
a marked increase in the number of apoptotic cells in the cancer cells exposed (Hep2) to the methanolic extract
of M.hortensis leaves. All staining results showed a similar trend suggesting that the leaf extract does possess
anticancer activity and this action is mediated by selective toxicity towards the cancerous cells namely, Hep2. The
results of Giemsa, EtBr, PI, and DAPI are shown in Figure 1, 2, 3, and 4 respectively. The arrows in the figures
indicate the apoptotic cells. Also, the oxidative stress induced cellular events or death was counteracted by the
presence of the leaf extract in the normal cells, namely chick embryo fibroblasts. But in the cancer cells, the extract
caused no change in the extent of cytotoxicity of etoposide. The cell viability study conducted by MTT and SRB
results are shown in Figure 5.
In the chick embryo cells, the viability is more and along with the leaf extract it increases further. The same
trend was observed for both MTT and SRB assay. In the case of cancer cells, the leaf extract alone showed less
viability and when co-administered along with etoposide, showed further decrease in the viability of cells, indicating
anti-cancer activity of the leaf extract.
The DNA fragmentation study on an agarose gel electrophoresis was calculated by the integrated density
377
values (IDV) of the DNA bands that were visualized as shown in the table 1.
Etoposide administered to the Hep2 cells exerts a decline in the survival of the cells. The methanolic extract
of the leaf caused a remarkable decrease in the viability of Hep2 cells which could decrease further in the presence
of etoposide. The M.hortensis leaf extract by itself, without etoposide, also could cause a decrease in the Hep2 cell
survival. Cells treated with etoposide shared an increased DNA fragment which was depicted by the integrated
density values and the administration of the leaf extract augmented the extent of DNA damage caused by etoposide
as reflected by occurrence of DNA fragmentation in Hep2 cells indicating the anti cancer effect and exerting a
protective effect in the normal fibroblasts.
Discussion: Similar studies have shown same trend when carried out with various other plant extracts like
Rhinacanthus nasutus14. A study conducted in Artemesia vulgaris has shown similar results supporting our trend15.
Morphological changes were observed in human neuroblastoma cell line treated with thimerosal16. Celecoxib
treatment induced apoptosis associated morphological changes in K562 cells17. The above results are in accordance
with a study reported by Amirghofran et al. 200718 who demonstrated the antitumour activity of an extract of Dionysia
terneana against K562 leukemia cell line and Jurkat cells using DNA fragmentation as an index. The combination
of STI-571 with radiation or cisplatin had an additive antiproliferative effect in SKNMC cells. A similar effect was
observed in human MCF-7 breast cancer cells, which was determined by sulphorhodamine B cytotoxicity assay as
described by Yerushalmi et al., 200719. The M.hortensis leaf extract showed signification protection against the cell
death in the oxidatively stressed chick embryo fibroblasts which not affecting the cytotoxic effects of etoposide in
cancer cells (Hep2). Thus, M.hortensis leaf extract administration showed no cytotoxicity in the normal fibroblasts,
but significant cytotoxicity towards the cancer cell line, namely, Hep2.
Acknowledgement: The authors are grateful for the financial assistance provided by Women Scientist Scheme
(WoS- A), Department of Science and Technology, New Delhi.
378
References:
Suriyavathana M, Usha V & Shanthanayaki M (2010) J Pharm Res 3, 260-262.
Ietswarrt JH (1980) A taxonomic revision of the genus origanum (Labiatae), 4th edn., pp.57 Leiden University press, Leiden.
Verma RS (2010) Chem Bull 55, 9-11.
Surch YJ & KyungNa HN (2007) Genes Nutr 6, 62-65.
Waris G & Ahsan H (2006) J Carcinog 5, 3163-3164
Suggitt M & Bibby MC (2005) Clin Cancer Res 11, 971-981.
Combes RD, Berridge T, Connelly J, Eve MD, Garnes RC, Toon S & Wilcon P (2003) European J Pharma Sci 19, 1-11.
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S & Boyd MR (1990) J
Natl Cancer Inst 82,1107-1112.
Igarashi M & Miyazawa T (2001) Biochem. Biophys. Acta/Molecular and Cell Biology of Lipids, 1530, 162-171.
Chih HW, Chill HF, Tang KS, Chang FR & Wu YC (2001) Life Sci 69, 1321-1331.
Mercille S & Massie B (1994) Biotech. Bioengin., 44, 1140-1154.
Sarker, K.P., Obara, S., Nakata, M., Kitajima,I. & Maruyama, I. (2000) FEBS Lett 472, 39-44.
Yin D, Kondo, S and Takeuchi, J (1994) FEBS Lett, 339, 73-75.
Nirmaladevi R (2008) Antioxidant responses evoked by Rhinacanthus nasutus leaf extracts in oxidatively stressed in vitro
systems, Ph.D. thesis, Avinashilingam University for Women, Coimbatore.
Haniya AK (2010), Antioxidant and apoptosis-modulating effects of Artemisia Vulgaris leaf extracts on in vitro systems
subjected to oxidative stress, Ph.D. thesis, Avinashilingam University for Women, Coimbatore.
Amirghofran Z, Bahmani M, Azadmehr A, Ashouri E & Javidnia K (2007) Cancer Invest 25, 550-4.
Humphery ML, Cole MP, Pendergrass JC & Kiningham KK (2005) Neurotoxicol 26, 407-416.
Subhashini J, Mahipal SVK & Reddanna P (2005) Cancer Lett 224, 31-43.
Yerushalmi R J, Nordenberg E, Beery O, Uzie M, Lahav D, Luria E & Fenig J (2007) Exp Oncol 29, 126131.
379
Identification of ER agonist from Agele marmelos
Ganesh K. Prasanth, Divya Lakshmanan M., and C. Sadasivan
Department of Biotechnology and Microbiology, Kannur University,
Thalassery Campus, Kerala, India e-mail: gpkmahe@hotmail.com
Abstract
The Aegle marmelos commonly known as Bael tree is one of the most useful medicinal plant. All the parts
of this tree including stem, bark, root, leaves and fruit at all stages of maturity has medicinal virtues and
has been used as traditional medicine in India since Vedic period. Phytoconstituents present in this plant has
shown diverse medicinal properties like inhibitory effect in vitro proliferation of human tumor, anti-cancer
activity against thyroid cancer, anxiolytic and antidepressant, anti spermatogenic activity etc. Considering the
diverse medicinal properties of Aegle marmelos, the study was done to isolate and characterize an ER agonistic
compound. Phytoconstituents of leaf of this tree were Soxhlet extracted using methanol and fractioned using
HPLC followed by fractionation, identification and verification by LC-ESI-MS and Yeast estrogen assay. The
activity of the purified compound was compared to 17-beta estradiol standard in terms of their relative
ability for binding to ER. It gave agonist responses at a higher concentration and gave only sub-maximal
response compared with 17-beta estradiol, thus the compounds may be acting as partial agonist of human
ER alpha. MS data was compared with the Wiley 275.L mass spectral library and was identified as
Marmesin (dihydrofuranocoumarin). The study has identified Marmesins phytoestgroenic potential using
Yeast Estrogen Assay.
Keywords: Estrogen, Aegle marmelos, phytoestrogen, marmesin
The Aegle marmelos commonly known as Bael tree is one of the most useful medicinal plant. All parts of
Bael tree has been used as traditional medicine in India for a long time. Use of its fruit has been mentioned in
Yajurveda and its medicinal properties have been described in the ancient Charaka Samhita. In traditional
medicine it is also commonly used as a reproductive normalizer, to stimulate delayed menstruation, for
reproductive or intestinal cramps, to normalize digestion (Kirtikar and Basu, 1993). The leaf of Aegle marmelos
has been shown to posses contraceptive properties (Bhattacharyya, 1982). The leaf extract has been reported to
regenerate damaged pancreatic beta cells in diabetic and reduces the blood sugar level in alloxan induced
diabetic rats (Sabu and Ramadasan, 2004). The leaf extract was found to be a potential antioxidant drug,
increased the activities of peroxidase in the liver tissues of Isoproterenol treated rats (Rajadurai et al., 2005).
The extract of Aegle marmelos showed inhibitory effect in the in vitro proliferation of human tumor cell
lines such as leukemic K562, T-lymphoid Jurkat, B-lymphoid and Breast cancer MCF7 (Lambertini et al.,
2004). The compound lupeol, was found to stimulate and increase the expression of Era gene in MDA-MB-231
ERa-negative breast cancer cells and also inhibited cell proliferation. The methanol extract of leaves poses
anxiolytic and antidepressant activities in mice (Kothari et al.., 2010). The ethanolic extract of leaf possesses
extracts had a considerable effect on the motility of sperm (Remya,et al., 2009). Considering the diverse medicinal
properties of Aegle marmelos, the study was done to isolate and characterize an ER agonistic compound
Methods
Recombinant yeast culture was obtained though the courtesy of John Sumpter (Brunel University, UK).
In vitro cell cultures were performed adhering to Good Cell Culture Practice (GCCP) by Coecke (Coecke,
2005). Soxhlet extracts of Agele marmelos leafs using methanol which showed estrogenic activity was further
purified using a Shimadzu 210 reverse phase HPLC system combined with a Shimadzu UV-visible detector.
Phytoestrogens were separated using a Luna II C18 reverse phase column (4.6 x 250 mm Phenomenex, Torrance,
CA). A guard column containing the same packing was used to protect the analytical column. All sample preparation
and procedures for HPLC analyses were done using HPLC grade solvents.. Injection volume of sample was 20
l with a flow rate of 1.0 ml/min with the following solvent system: 70% Acetonitrile, 30% water. The spectra
were recorded at detector wavelength of 280 nm. Semi preparative HPLC (C18 10 mm x 500 mm column) at a
380
flow rate of 3.0 ml/min with the above solvent system, Peaks which were above 100mV were collected
using a FRC-10A (Shimadzu) fraction collector. HPLC purified fractions were dried and assayed for the estrogen
agonist assay, the fraction which showed estrogenic properties were further analyzed. A concentration response
curve was plotted. The assay was done in triplicate and the estrogenic activity of the compound was expressed as
a percentage of the -galactosidase activity induced by 2.70 ng/ml of estradiol, the OD of which i.e. 2.76 equated
with 100%. To confirm the identity of the peaks obtained by HPLC-UV, the samples were analyzed by LC-ESI-MS.
Analyses were performed on a Finnigan Quadrupole (MAT LCQ) ion trap mass spectrometer (Thermo Finnigan,
San Jose, CA, USA) coupled with an electrospray ionization (ESI) source and equipped with a TSP 4000
HPLC system, which includes UV600LP PDA detector, P4000 quaternary pump and AS3000 auto-sampler. The
ionization technique used for the structural elucidation of the compound was done by electrospray ionization
in the positive mode. LC-MS analysis was carried out using the same conditions used for the HPLC analysis. Single
peak obtained was further extracted using the +ve acquisition mode over an m/z range from 50-1000 with a scan
speed of 1 sec per scan. The spray voltage was set at 4.5 kV and the capillary temperature was set at 250C. The m/z
ratio was recorded and the compound was identified by comparison with that of the retention time and mass
spectral data of the standards from NIST Library (version 8).
Results
Soxhlet extracts of Agele marmelos leafs using methanol which showed estrogenic activity was further
purified using a reverse phase HPLC system, chromatogram recorded at a wavelength of 280nm. The mobile
phase was a mixture of Acetonitrile: Methanol (70:30) delivered at a flow rate of 1.0 ml /min. Fraction with retention
time 2.675, 4.017, 4.958 and 6.417 were collected using FRC20A fraction collector and were further assayed for
estrogenic activity using the yeast assay system.
Figure 1a: Yeast estrogen assay of 17-beta Estradiol standard. 1b:. Assay of purified compound plotted
against 17-beta estradiol standard (Values are the mean SD)
The assay results showed that only fraction with RT 6.417 possesses distinct estrogenic activity,
Concentration response curve of natural agonist estradiol in YES assay is shown in Figure 2a (Values are
the mean SD). Figure 1b shows the partial agonistic activity of purified compound plotted against 17- beta
estradiol, Estrogenic activity of the compound is expressed as a percentage of the -galactosidase activity
induced by 2.70 ng/ml of Estradiol, OD of which i.e. 2.76 equated with 100% (Values are the mean SD).
To confirm the identity of the peaks obtained by HPLC-UV, the samples were analyzed by LC-ESI-MS. LC-MS
analysis was carried out using the same conditions used for the HPLC analysis. Fraction with RT 6.417 which
showed positive result was analyzed with LC-ESI-MS analyzer obtained spectra is shown in figure 2.
Figure 2a. LC chromatogram of Agele marmelos fraction with RT 6.417; Figure 2b: ESI-MS Spectrum of
HPLC fraction (RT: 6.417) confirming the presence of a compound with m/z 247.12
381
The ionization technique used for the structural elucidation of the compound was done by electrospray
ionization in the positive mode Single peak obtained was further extracted using the +ve acquisition mode over an
m/z range from 50-1000 with a scan speed of 1 sec per scan. The m/z (Mass/Charge) ratio was recorded as shown
in figure 3b.
Figure 4a: Tandem Mass Spectrum of the analyte with m/z 247.12. Figure 4b: Mass Spectrum of compound
Marmesin matching the analyte m/z 246 (Wiley275.L) 7H-Furo[3,2-g][1] benzopyran-7-one, 2,3-dihydro-
2-(1-hydroxy-1-methylethyl)-, (S)- Marmesin $$ CAS- 013849-08-6; Mol Formula C14H14O4; Mol Weight
246.089; Entry Number 131085 from C:\Database\WILEY275.L(version 8).
and the Tandem Mass Spectrum of the analyte is shown in figure 4a, the spectra was identified by comparison
with the mass spectral data as shown in figure 4b of the standards from NIST Library
Discussion
The present investigation was undertaken to analyze if any phytoestrogens were present in Aegle marmelos.
The estrogen agonist property of purified extract was compared with 17-beta estradiol in terms of their relative
ability for binding to ER. It gave agonist responses at a higher concentration in all assays and gave only sub-
maximal response compared with 17-beta estradiol, thus the compounds may be acting as partial agonist of human
ER alpha. . The compound induced 78% 2.01 betagalactosidase activity when compared with that of 2.70
ng /ml of 17-beta estradiol. A maximum of 450 ng/ml of purified compound was required to elicit beta-
galactosidase activity similar to that of 2.02 ng/ml of 17-beta-estradiol suggesting it to be a partial agonist. The
results of the ESI-MS analysis produced characteristic fragmentation pattern with major peaks (m/z) at 60,
164, 187 and 246, this data was compared with the Wiley 275.L mass spectral library and the compound
was identified as Marmesin. Marmesin is one of the most prevalent linear dihydrofuranocoumarin, originally
isolated from Aegle marmelos (Chatterjee and Mitra, 1949). The study has identified Marmesins phytoestgroenic
potential using Yeast Estrogen Assay
References
Bhattacharya, S. (1982). Medical importance of Aegle marmelos.Chironjibi Banoushadi. Kolkata: Annada Publisher, 341-346.
Chatterjee, A., & Mitra, S. S. (1949). On the Constitution of the Active Principles Isolated from the Matured Bark of Aegle
marmelos, Corre.Journal of the American Chemical Society,71(2), 606-609.
Kirtikar, K. R., & Basu, B. D. (1993). Phylanthus emblica.Indian medicinal plants, 2220.
Kothari, S., Minda, M., & Tonpay, S. D. (2010). Anxiolytic and antidepressant activities of methanol extract of Aegle marmelos
leaves in mice.
Lambertini, E., Piva, R., Khan, M. T. H., Lampronti, I., Bianchi, N., Borgatti, M., & Gambari, R. (2004). Effects of extracts from
Bangladeshi medicinal plants on in vitro proliferation of human breast cancer cell lines and expression of estrogen receptor
gene.International Journal of oncology,24(2), 419-423.
Rajadurai, M., & Prince, P. S. M. (2005). Comparative effects of Aegle marmelos extract and alpha-tocopherol on serum lipids,
lipid peroxides and cardiac enzyme levels in rats with isoproterenol-induced myocardial infarction.Singapore medical
journal,46(2), 78.
Remya, M., Sharma, R. C., Shoaib, H., Asad, R. J. U., & Swati, S. (2009). In vitro effect of Aegle marmelos on human sperm
motility.Journal of Medicinal Plants Research,3(12), 1137-1139.
Sabu, M. C., & Kuttan, R. (2004). Antidiabetic activity of Aegle marmelos and its relationship with its antioxidant
properties.Indian journal of physiology and pharmacology,48(1), 81-88.
382
Isolation and characterization of phytosterols having 5 reductase
inhibitory activity
P Rani and Anisha A
Department of Biotechnology, PSG College of Technology, Coimbatore 641004, Tamilnadu, India
Abstract
Androgenetic alopecia is a typical pattern of receding hairline and hair thinning caused by several factors
including androgens, genetic factors and age. Excess conversion of testosterone to dihydrotestosterone (DHT)
by 5 reductase is the main reason for androgen dependent alopecia. DHT also causes prostate cancer by
binding to androgen receptors with higher affinity. 4-azasteroid compounds are widely used as 5-reductase
inhibitors Due to the adverse reactions caused by these chemical drugs, phytosterols could be looked as an
alternate drug candidates. The current study is mainly focused on screening and characterizing the potent
phytosterols establishing 5 reductase inhibitory activity from various plant sources. Organic extracts of plant
sources namely seeds of Fenugreek (Trigonella foenum-graecum), leaves of Moringa (Moringa oleifera) and
Sage (Salvia officinalis) were screened for 5 reductase inhibitory activity using NADPH utilization assay.
Isolation and characterization of phytosterols were carried out using HPLC and FTIR analysis. Among three
plant extracts, Moringa oleifera leaf extract was identified as a potent inhibitor of 5 reductase. HPLC analysis
of Moringa leaf extract revealed that the active compound has a sterol moiety and structurally similar to
commercial drugs namely finasteride and dutasteride. FTIR analysis concluded that it consists of -sitosterol
as its bioactive compound and is responsible for inhibitory activity of 5 reductase. Inhibition kinetics proved
that it is a competitive inhibitor with an IC50 value of 1.5 mg/mL. The study pinpoints that -sitosterol from
Moringa oleifera can be a prospective drug candidate for alopecia and prostate cancer treatment.
1. INTRODUCTION
Androgenetic alopecia is the most common type of hair loss in men as early as late adolescence. It usually
follows a typical pattern of receding hairline and hair thinning leading to baldness. It is caused by several factors
including androgens, genetic factors and age. In the hair follicle cells, testosterone gets converted into the biologically
more active metabolite, dihydrotestosterone (DHT) catalyzed by the enzyme 5-alpha reductase. This hormone binds
to androgenic receptors in the hair follicle and triggers cellular processes which reduce the anagen phase (growth
phase) of the hair cycle and miniaturization of hair follicles leading to excessive hair loss (Bienova, Kuerova,
Fiurakova, Hajduch and Kolar, 2005).
Prostate cancer is one of the most complex oncologic problems in medicine. It is highly prevalent, particularly
in elderly males. Testosterone is taken up from the systemic circulation by the prostatic glandular and stromal cells.
Once within the prostate, testosterone is rapidly and irreversibly converted to DHT by the enzyme 5 reductase.
This leads to a five-fold higher concentration of DHT versus testosterone within the prostate. DHT then binds to the
androgen receptor within the cytosol and actively transported into the nucleus. It serves as a transcription factor for
prostatic gene expression and cellular proliferation. The higher concentration of intracellular DHT and its higher
affinity for the androgen receptors, support the importance of 5-alpha reductase in normal and pathologic prostate
physiology (Hudak, Hernandez and Thompson, 2006)
Topical and systemic drugs, individually or combined may be used for the treatment of alopecia. Finasteride,
a selective type 2 5 reductase competitive inhibitor with nanomolar affinity reduces plasma dihydrotestosterone
levels. It is a widely used oral therapeutic agent in the treatment of benign prostatic hyperplasia and it is used in
androgen deprivation therapy to treat prostate cancer and androgenetic alopecia (Drury, Costanzo, Penning and
Christianson, 2009). Oral administration of Dutasteride inhibits type 1 and type 2 5-reductase, leading to a 95%
decrease in serum DHT concentrations (Amory, Anawalt, Matsumoto, Page, Bremner, Wang, Swerdloff and Clark,
2008). Minoxidil 2% or 5% solution is the most frequently used drug for topical application. It has a specific direct
383
effect on the proliferation and differentiation of follicular keratinocytes which leads to prolongation of the anagen
phase (Bienova et al., 2005).
5 reductase type 1 play important roles in the hepatic clearance of steroid hormones, suggesting that high
dose finasteride may have an adverse effect on hepatic steroid metabolism (Drury et al., 2009). Adverse reactions
such as erectile dysfunction, loss of libido, a small volume of ejaculate or gynecomastia are prevalent. The effect
of the therapy is temporary and hair loss progresses. In addition, irritative dermatitis or contact allergic dermatitis
is also observed (Bienova et al., 2005). The most common adverse events occurring in 10% or more of patients
were ear, nose and throat infections, malaise and fatigue, headaches, musculoskeletal pain and dizziness (Clark,
Hermann, Cunningham, Wilson, Morrill and Hobbs, 2004)
Phytosterols have been commercially used to treat various ailments particularly for their capability to lower
serum cholesterol levels in humans and thereby reduce the risk of cardiovascular diseases (Hicks and Moreau,
2001; Jones, MacDougall, Ntanios & Vanstone, 1997). They are also considered to have anti-inflammatory, anti-
bacterial, anti-ulcerative and antitumor properties (Beveridge, Li and Drover, 2002). Phytosterols compete with
testosterone and its metabolites for the active sites of 5 reductase and thus alter androgen metabolism (Awad,
Hartati and Fink, 1998). Phytosterols act through multiple mechanisms of action, including inhibition of carcinogen
production, cancer-cell growth, angiogenesis, invasion and metastasis, and promotion of apoptosis of cancerous cells
(Woyengo, Ramprasath and Jones, 2009). Importantly, these botanicals have not been linked with the spectrum of
adverse reactions, or teratogenicity, associated with the pharmaceutically derived alternatives (Klepser and Klepser,
1999). Thus the current study was focused on screening various plant sources for 5 reductase inhibitory activity,
characterizing the potent phytosterol and establishing the inhibitory activity for prostate cancer and androgenetic
alopecia treatment. Plant sources for the invitro screening were chosen on the basis of traditional treatments that
were used in ancient times for treating baldness.
2. MATERIALS AND METHODS
2.1 Sample Preparation: All chemicals have been purchased from S. D. Fine Chemical Limited from Mumbai,
Maharashtra, India. Leaves of Moringa (Moringa oleifera) and Sage (Salvia officinalis) and seeds of Fenugreek
(Trigonella foenum-graecum) were collected, shade dried and weighed before extraction. Extraction of phytosterols
was done using Soxhlet method (Wrolstad et.al, 2005). Hexane solvent was used for sterols extraction from the
above mentioned sources. The extraction was carried out at 69oC for 10 h. The sample was cooled and concentration
of the product was subsequently done in a rotary evaporator at 40oC under reduced pressure. The extract was then
stored at 4oC for further analysis.
2.2 Analysis of Phytosterols: A small quantity of the dried plant extract was dissolved in Dimethyl Sulfoxide
(DMSO) and filtered through a 0.2 m syringe filter before being used for qualitative analysis by Thin Layer
Chromatography (TLC) and Spectrophotometric analysis.
Thin Layer Chromatography was done on 20 x 20 cm glass plates using Silica Gel F254. The extracts
dissolved in DMSO were spotted on the surface using glass capillary tubes. The standards used include a mixture of
Testosterone, Estradiol and Cholesterol. The mobile phase used was Acetone: Chloroform (1:9). The plates were run
for 30-45 minutes, in a closed chamber. Staining was accomplished by spraying 1% Iodine solution in chloroform
using an atomiser.
Spectrophotometric analysis was done by doing a wavelength scan from 200-800 nm using uv-visible
spectrophotometer. The standards used include Testosterone (HiMedia), Estradiol (HiMedia), Cholesterol and
Finasteride. The absorbance peaks were studied.
2.3 Characterization of bioactive compound: The sterolic extract of the Moringa leaf extract was
characterized using HPLC and FTIR analysis.
For this purpose, the standards of finasteride and dutasteride were extracted from tablets (Finast and Dutalfa
384
from DR.REDDYS) using silica gel column chromatography with ethyl acetate as solvent. The elute was
concentrated by evaporation at 80C and analysed by biochemical analysis to confirm the elution of Finasteride
and Dutasteride. The elute stored at 4C was used for further analysis.
2.3.1 HPLC Analysis: The extracted samples from finasteride and dutasteride tablets, Moringa leaf extract,
cholesterol (100 gmL-1), testosterone (100 gmL-1) and estrogen (100 mM) samples were run in HPLC (Agilent
Technologies) using C18 reverse phase column with Acetonitrile: Water (95:5) as mobile phase at a flow rate of 1
mLmin-1. Injection volume was 20 L. Analysis was done using a UV detector at 210 nm. All samples were filtered
using 0.22 m filter before analysis.
2.3.2 FTIR Analysis: The leaf extract was further purified by repeating the extraction step. The dried
leaf extract was mixed thoroughly with water and extracted with hexane. The organic layer was collected
and evaporated in water bath at 70 C. The dried extract was then analysed using Fourier Transform
Infrared Spectroscopy (FTIR 8400S, Shimadzu) with KBr pellet.
2.4 Isolation of 5 reductase: The prostatic tissue samples were isolated from 2-4 month old Wistar rats and
from a man undergoing transurethral resection for benign prostatic hyperplasia stored in 0.9% sodium chloride
solution at 4oC. It was then homogenized in eight volumes of ice-cold homogenization buffer containing 100 mM
potassium phosphate buffer (pH 7), 150 mM potassium chloride and 1 mM EDTA. Subsequently centrifugation
was done at 23000xg for 40 minutes at 4oC. The crude enzyme was collected from the supernatant. Aliquots of the
crude enzyme extracted were made and stored at -70oC for further analysis. Total and specific activity of the enzyme
was determined by studying the utilization of NADPH spectrophotometrically at 340 nm (Drury et al., 2009) using
UV-Visible spectrophotometer (UV 1601, Shimadzu) for a time period of 10 minutes at an interval of 30 seconds.
2.5 Inhibition Kinetics
2.5.1 Determination of Inhibition Mechanism
5 reductase- NADPH assay was performed spectrophotometrically at 340 nm to check for the mechanism
of inhibition with varying substrate concentrations ranging from 5-100 M and constant enzyme concentration
(100L) - both with (100 L at 1mgmL-1) and without inhibitor (Drury et al., 2009). The mechanism of inhibition
of the extract was analysed using Lineweaver Burke plot. All experiments were conducted in triplicates and mean
absorbance values were used for analysis.
2.5.2 Determination of IC 50
To measure IC50 of the inhibitor, 5 reductase- NADPH assay was done with varying inhibitor concentrations
ranging from 0-1.5 mgmL-1 with constant enzyme (100L) and substrate (100 M) concentration. Absorbance at
340 nm was recorded for 10 minutes at an interval of 30 seconds to determine the enzyme activity.
3. RESULTS AND DISCUSSION
3.1 Qualitative Analysis: From thin layer chromatography, it can be concluded that the retention factors
of the plant sterols are similar to that of estradiol and testosterone indicating that the enzyme inhibitors maybe of
competitive type.
From spectrometric analysis, it could be seen that the phytosterolic extracts had similar spectra as that of
finasteride and thus they could be structural analogues of Finasteride.
3.2 Activity Assay: Due to the commercial non-availability of 5 reductase, the enzyme was isolated from
rat and human prostatic tissues. Total Activity of 5 reductase is defined as the amount of enzyme that catalyzes
the oxidation of moles of NADPH per minute per gram of prostatic tissue. The total and specific activity of the
extracted 5 reductase from rat ventral prostatic tissue measured by NADPH utilization assay was found to be
2.709x10-2 U/g of tissue and 0.263 U/mg of protein respectively. Similarly for human prostatic tissues total and
specific activity was found to be 2518.71 U/g of tissue and 204.94 U/mg of protein.
385
3.3 Screening of Phytosterols: The sensitivity and effectiveness of an inhibitor can be expressed in terms of
its IC50 values. Initial screening of the phytosterols was done using enzyme isolated from human prostatic tissue. IC
50 for each of the phytosterolic extracts is depicted in Table 1 and it was observed that the Moringa leaf extract had
the least IC50 value i.e., it is a potent inhibitor of 5 reductase.
Table 1: Comparison of inhibitory effects of the three phytosterols
Source of the phytosterols IC50 values for the phytosterols (ng/mL)
Fenugreek seeds 33.00
Sage leaves 12.60
Moringa leaves 6.38
3.4 Characterization Studies
3.4.1 HPLC Analysis: Biochemical analysis of eluted samples from tablets usingferric chloride and conc
H2SO4 confirmed the presence of steroids by the appearance of characteristic green colour. The extracted samples
from finasteride and dutasteride tablets, cholesterol, testosterone, estrogen and Moringa leaf extract were run on
HPLC and analysed using uv detector at 210 nm. The retention times of various samples are depicted in Table 2.
From HPLC analysis, a peak at about 10.5 similar to cholesterol, testosterone and estrogen suggests that the plant
extract has a sterol moiety. Another peak around 3.9 observed in cholesterol, testosterone, finasteride and dutasteride
is found in plant extract also. This further confirms its structural similarity with other steroids and existing chemical
drugs used for alopecia and prostate cancer treatment.
Table 2: HPLC Analysis of Sterolic extracts
SAMPLE RETENTION TIME(min) SAMPLE RETENTION TIME(min)
3.907 3.877
Cholesterol Finasteride
10.557 10.293
3.987
3.917 5.223
Testosterone Dutasteride
10.573 6.443
10.263
3.913
5.087 5.417
Estrogen Moringa leaf extract
10.770 6.747
10.513
3.4.2 FTIR Analysis: The Moringa leaf extract was analysed using FTIR spectroscopy and the IR spectra
obtained is shown in Fig 1. The spectra reveal the following data: The C=C stretching was observed as peak at
1462.09 and 1637.62 cm-1. The bending vibrations of C=C bond was observed as a peak at 669.32 cm-1. The out of
plane C-H deformation peaks were found at 719.49 and 802.41 cm-1. Their corresponding overtone and combination
bands were seen at 1716.70, 1732.13 and 1992.53 cm-1. The aromatic C-H stretching was seen as peak at 3173.01
cm-1. These results confirm that the compound has aromatic carbon skeleton structure.
The O-H bond vibrations were seen at 1261.49 and 669.32 cm-1. The methyl substituent group stretching and
bending was observed at 2848.96 and 1462.09 cm-1. The methylene group vibrations were found at 2848.96 and
1377.22 cm-1. From literature review, it is found that this spectral data confirmed with IR spectra of -sitosterol
(Badgujar and Jain, 2009 & Kemp, 1991) which is a commercially used phytosterol currently employed for alopecia
and prostate cancer treatment.
3.5 Inhibition Kinetics: As the initial screening of various phytosterols revealed Moringa leaf extract with
- sitosterol to be the potent inhibitor, further analysis to appreciate its mechanism of inhibition was carried out.
386
Fig 2. FTIR Spectra of Moringa leaf extract
3.5.1 Determination of Inhibition Mechanism: The mechanism of inhibition by an inhibitor was studied to
understand the pattern of binding of inhibitor and to determine the inhibitory constant Ki. The assay was done using
the enzyme isolated from rat prostatic tissue. The substrate concentrations used and the corresponding velocities
both with and without inhibitor are given in Table 3.
Table 3: Inhibitory Activity of Moringa Leaf Extract on 5 reductase
Velocity x 10-2 (moles of NADPH oxidised/min/g of prostatic tissue)
[S] (M)
Without Inhibitor With Inhibitor
5 0.3870.007 0.2910.029
10 0.4840.017 0.2930.029
15 0.5810.010 0.3870.018
20 0.7740.011 0.4840.020
25 0.8710.009 0.6770.016
50 1.6450.016 0.9680.028
75 2.0320.027 1.3540.025
100 2.7090.023 1.8380.032
The experiment was done in triplicates and values are represented as MeanSD.
The Lineweaver- Burke plot (double reciprocal plot) demonstrating the data graphically for interpretation
of inhibition mechanism is given in Fig 4-2. From the graph it is seen that Vmax remains unaltered for both with
and without inhibitor. But Km increased on addition of inhibitor indicating the decrease in activity proving that
-sitosterol from Moringa leaf is a potent competitive inhibitor for the substrate at the enzyme active site. The
values of enzyme kinetics constants are given in Table 4-3. The Ki value was found to be 6.799 mgmL-1
Fig 2. Lineweaver- Burke Plot for Inhibition of 5 reductase Activity by Phytosterol.
387
Table 4. Enzyme Kinetic Constants of 5 reductase isolated from rat prostatic tissues.
Sample Km (M) Vmax (moles of NADPH oxidised/min/g of prostatic tissue)
Without Inhibitor 116.7 10
With Inhibitor 171.4 10
3.5.2 Determination of IC50: The enzyme activity recorded at 340 nm for various inhibitor concentrations is
given in Table 5 and the IC50 value of the inhibitor- Moringa leaf extract is found to be 1.5 mgmL-1.
Table 5. 5 reductase activity at various inhibitor concentrations.
[I] (mgmL-1) Enzyme Activity x10-2 (U/g)
0 2.709
0.5 2.225
1.0 1.838
1.5 1.258
Earlier studies regarding inhibition of 5 reductase by ann-hexane lipid/sterol extract ofSerenoa repenswas
non-competitive and, when expressed in terms of recommended therapeutic doses, was 3-fold greater than for
finasteride. These studies suggest that the phytosterolic extract from Serenoa repensmight exert a regulatory
inhibitory activity due to its specific lipid/sterol composition. (Delos, Lehle, Martin and Raynaud, 1994)
Extracts from fruits of Sabal serrulata were studied for their influence on androgen metabolism in the human
prostate. It was found that the extract from S.serrulata consisting mainly of phytosterols inhibited 5-reductase
activity dose dependently and noncompetitively in the epithelium and stroma of human at a testosterone
concentration of 580 nM as substrate for 5-reductase. It showed a mean inhibition of 15% and 10% respectively
against 5-reductase in the epithelium and stroma. (Weisser, Tunn,Behnke andKrieg, 1996)
Some derivatives of 6-methylene-4-pregnen-3-one were studied as inhibitors of4-3-ketosteroid 5-reductase.

Maximum inhibitory activity was shown by 17-acetoxy-6-methylene-4-pregnene-3, 20-dione (AMPD). Irreversible
inactivation was observed following preincubation of the enzyme with NADPH and AMPD. It seemed likely that
the observed inactivation was derived from an irreversible combination of the enzyme with AMPD. This case
was established by kinetic studies which revealed a competitive inhibition mechanism. (Petrow, Wang, Lack and
Sandberg, 1981)
Thus the current study proves the invitro potential of Moringa leaf organic extract with - sitosterol, as
competitive inhibitor of steroid 5 reductase. But its Invivo efficacy is yet to be studied in detail. It is also essential
to study the drug-drug interactions when coadministered with chemical drugs. Experiments can be carried out with
androgen sensitive prostate cancer cell line for invitro bioassays and LD 50 of the drug candidate is to be determined.
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389
Antimicrobial efficiency of Chromolaena odorata extracts treated spun
lace viscose nonwoven fabric
Resmi.G* and Dr.S.Amsamani**
Research Scholar*, Professor and HOD**, Department of Textiles and Clothing, Avinashilingam Institute for Home
Science and Higher Education for Women, Coimbatore, Tamil Nadu, India. E-mail: gangadharan.resmi@gmail.com
dr.amsamani@gmail.com
Abstract
The antimicrobial efficacy of Methanolic extracts of C.odorata (5%) was applied on spun lace viscose
fabric with the aid of an ultrasonic atomiser. The phytochemical screening revealed the presence of tannins,
alkaloids, flavonoids and phenolic compounds. The antimicrobial assay done as per AATCC Test method 90-
2011 revealed that 5% C.odorata extracts the treated fabric is having 3mm zone of inhibition towards E.coli
and S.aureus. The presence of carbonyl acid and organic aromatic group in treated fabric was identified by
FTIR analysis. SEM analysis shows good adsorption of C. odorata micron size particles on fabric surface.
Since C. odorata extracts treated fabric are having antimicrobial quality and also the phytochemicals
are able to adher on the fibre structure, the fabrication of eco-friendly, safe and economical wound dressing
material medicated with C. odorata extracts will be of high potential.
Key words: Medical textiles, Chromolaena odorata extracts, wound dressings, antimicrobial textiles.
Introduction
Conventional textile based wound dressing materials are cost effective and highly absorbent, but when used
alone fails to provide optimal wound healing conditions like homeostasis, non-adherence, maintenance of a moist
wound bed. An ideal wound dressing material should have certain important properties such as moist environment,
diffusion of gases and removal of excess exudates, mechanical protection, comfortable, non-allergenic and
biocompatible [1]. The concept of an active involvement of a wound dressing in establishing and maintaining
an optimal environment for wound repair and this cognizance resulted in the development of functional active
dressings [2]. The incorporated drugs play an active role in the wound healing process either directly or indirectly as
cleansing or debriding agents for removing necrotic tissue, antimicrobials which prevent or treat infection or growth
agents to aid tissue regeneration [3].
Plant extracts and other biologically active compounds isolated from plants have gained widespread interest
as they have been known to cure diseases and illnesses since ancient times and are widely accepted due to the
perception that they are safe [4]. Chromolaena odorata (L.) King and Robinson (Siam weed) is a perennial scandent
or semiwoody shrub in the Asteraceae family. The extracts of the plant have been used to stop bleeding and in
wound healing in many tropical countries [5]. The medicinal values of plants have been claimed to lie in their
phytochemical component including alkaloids, tannins, flavonoids and other phenolic compounds, which produce
a definite physiological action on the human body [6]. Hence the objective of this research is to evaluate the wound
healing potential of Chromolaena odorata extracts treated spun lace viscose wound dressings.
2. Materials and methods
Collection of raw materials
Fabric selection
Nonwovens are extensively used in the medical field. Spun laced nonwovens are an attractive alternative
to woven gauze for medical/surgical applications. The spun lace wound dressing can comply with body outline
and curves without any obstacle to muscle action [7]. The nonappearance of dust and easiness in production of
non-woven fabric makes it an excellent substrate for wound dressing application [8, 9, and 10]. High absorbency,
390
hydrophilicity, excellent moisture management ability and skin-friendliness are the features of viscose fibers which
makes it an excellent wound dressing material [11]. Hence spun lace viscose non-woven fabric was chosen to
fabricate wound dressing material.
Collection of plants
Fresh leaves of Chromolaena odorata plants were collected from Kerala. The collected plants were washed
in tap water, and rinsed in distilled water. The leaves were separated and shade dried for one week, powdered and
kept in air tight polyethylene bags.
Plant extraction
The extractions of bioactive agents were done in a Soxhlet apparatus with methanol as solvent. After complete
extraction process the methanolic extract was filtered with what man No.1 filter paper and excess methanol was
evaporated at room temperature to obtain extracts and stored at 4 0 C.
Phytochemical analysis
Phytochemicals like tannins, flavonoids present in the plant accelerates wound healing activity
by producing certain biological reaction inside the body [12]. The presence of alkaloids, flavonoids,
tannins and phenolic compounds were analyzed.
Flavonoids: One ml of 1% ammonia solution was added to 2 ml of each extract. Appearance of
yellow colour indicates the presence of flavonoids [13].
Tannins: One or two drops of ferric chloride solution were added to 1 ml of each extract. Brownish
green or blue black colour solution indicates the presence of tannins [13].
Alkaloids: To one mL of the extract, a few drops of Wagners reagent were added and the
formation of a reddish-brown precipitate indicates the presence of alkaloids [14].
Phenolic compounds: A small quantity of extract was dissolved in 0.5ml of 20% sulphuric acid
solution. Followed by addition of few drops of aqueous sodium hydroxide solution, it turns blue
presence of phenol [14].
2.4 FTIR analysis
Identification of various components on finished fabric surface was done qualitatively using
FTIR spectrometer as per AATCC Test Method 94- 2007 standards (AATCC Technical manual, 2012).
C.odorata extracts treated fabric, one gram weight was taken and dried at10510C and desiccated to
constant weight prior and subjected to various solvent extraction the compounds were identified by
spectrophotometer in the spectral range of 4000 to 500 cm-1 at a resolution of four wave numbers with
signal averaged over 30 scans [15].
2.5 Application of plant extracts on spun lace viscose fabric
Methanolic extracts of C odorata, five per cent, dissolved in ethanol and applied on spun lace viscose fabric
with the aid of an ultrasonic atomiser with the solution dispensing speed fixed at 10 ml/minute. The finished fabric
was dried at room temperature.
2.6 SEM Analysis of C.odorata extracts treated fabric
A Scanning Electron Microscopy to study the particle size and its adhesion to fabric surface was done.Bio
extracts treated samples were subjected to SEM analysis with a magnification of X100 to X5500, 20 kv.
2.7 Evaluating the Antimicrobial efficiency of the finished fabric
AATCC Test method 90 2011, Agar plate method [16] was followed to assess the antimicrobial efficiency of
the treated fabric against various microorganisms like Escherichia coli (NCIM No. 2065), Staphylococcus aurous
(NCIM No. 2079), Aspergillus niger (NCIM No.596) and Candida albicans (NCIM NO. 3471).
391
3. Results and discussion
Presence of phyto constituents
The screening of phyto constituents confirms the presence of alkaloids, flavonoids, tannins and phenolic
compounds in the methanolic extracts of C odorata. The flavonoids and phenolic compounds in plant have been
reported to exert multiple biological effects including antioxidant, free radical scavenging abilities, anti-inflammatory
properties and wound healing efficiency [18].
3.2 FTIR analysis of the C.odorata extract treated fabric
The Fourier-transform infrared spectrum of the C.odorata extracts treated fabric is shown in Figure. I.The distinctive
functional groups of C. odorata extracts bound on to the surface of spun lace nonwoven fabric are identified.The presence of
organic functional compounds were indicated by the highest peak 3650.79cm-1 revealing the presence of alcohol with O-H,
stretch free vibration type. It is followed by postions 2853.53cm-1 and 1514cm-1 indicating the occurance of carbonyl
acid and organic aromatic group with O-H and C=C stretch respectively. The band at 1157.79cm-1 represents the
occurance of the C-H stretch of the organic amines. These infrared bands confirm the presence of phenolic group
in the treated fabric.
Figure I- FTIR spectrum of the C.odorata treated spun lace fabric
3.3 SEM Analysis

SEM analysis shows good adsorption of C. odorata micron size particles on fabric surface as evidenced at
different magnifications by the images as below.
Figure II SEM image of C. odorata extracts treated fabric
392
3.4 Antimicrobial efficiency of bio extracts and Chitosan
Table I -Antibacterial efficacy of 5% C.odorata extracts treated fabric to microorganisms
Antimicrobial Activity
(zone of Inhibition mm)
Source Concentration
S aureus
albicans
A niger
E coli
C
Chromolaena Odorata 5% 3 3 3 0
Control fabric (untreated) - 0 0 0 0
From Table I, Figure III it is clear that the plant extract of C.odorata had formed a zone of inhibition 3 mm for
E. coli, S. aureus and C. albicans however no activity was observed against Aspergillus niger. No zone of inhibition
was exhibited by control untreated fabric.
Figure III- Zone of Inhibition formed by C odorata (5%) extracts to microorganisms
Conclusion
The presence of phytochemicals like alkaloids, flavonoids, tannins and phenolic compounds ensure the wound
healing efficiency of the C.odorata extracts. The spun lace viscose fabric treated with C.odorata extracts can be
used as a wound dressing material. Since C. odorata extracts are eco-friendly, safe and of economical in nature, the
fabrication of wound dressing material treated C.odorata extracts can be a promising and feasible medical textile
product.
Acknowledgement
The support by University Grants Commission Senior Research Fellowship is gratefully acknowledged and
Spun laced viscose fabric was sponsored by Ginni filaments Ltd, Gujarat for this study are thankfully acknowledged.
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394
In vitro Inhibitory Potential of -amylase and Proximate Analysis of
Selected Medicinal Plants
Geethanjali A, Lalitha P*, Smina CS**
Department of Chemistry, Avinashilingam Institute for Home Science and Higher Education for Women,
Coimbatore 641 043
Abstract
Numerous plants are known for its anti-diabetic activity. Traditional knowledge needs to be substantiated by
scientific validation of its activity. Hence researchers are involved in exploiting the ethnopharmacological
knowledge and validating the activity of medicinal plants. Wet lab studies may be tedious and hence this
necessitates a screening tool to narrow down the plants that can be taken up for study. Contemplating on
the aforesaid need, the present work is aimed at providing a screening tool for identifying plant extracts with
anti-diabetic activity. Seventeen commonly used plants were taken up for study and its extracts were gauged
for i)anti-diabetic activity by a simple spectrophotometric method and ii) proximate nutrient composition.
Proximate analysis in plants gives beneficial information and helps to access the quality of the plant sample.
The results disclose Coccina indica to have highest percentage ash content value (22.06%) portraying the
presence of more number of organic compounds whereas the low value of ash content of Trigonella foenum
graceum (3.36%) reveals less number of mineral or inorganic constituents. The high percentage value of
acid insoluble ash for Trigonellafoenum graecum, Curcuma longa and Annona squamosa show the easy
digestibility of the plants when consumed. The -amylase inhibitory potential of all 17 plants showed
appreciable alpha amylase inhibition efficiency (70-98%) compared to acarbose the standard anti diabetic
drug used for reference. The IC50 value of Aegle marmelos and Hibiscus rosasinensis were found to be 50%
at 20 ml concentration .
Keywords: -amylase, Inhibition efficiency, Anti-diabetic plants
Introduction
Diabetes, the worlds largest endocrine disease involves metabolic disorders of carbohydrate, fat and protein
(Abdel- Hassan et al., 2000). It is a chronic metabolic disorder characterized by raised blood sugar level resulting
from the defective insulin production, secretion, or utilization and is characterized by raised glucose concentration
in the blood and alterations in carbohydrate, protein and fat metabolism. A balance between insulin and glucagon
activity maintains blood glucose in a normal range. The micro vascular complications of diabetes include
nephropathy, retinopathy, and neuropathy (Lakshmi et al, 2012; Dixen et al, 2008)). Diabetes increases the risk
of myocardial infarction, acceleration of atherosclerosis, increases the probability of renal failure , foot ulceration,
subsequent amputation etc. (Thakur et al., 2010).
In order to overcome the above mentioned problems it is necessary to control the Blood Glucose Level (BGL)
in diabetic patients within specified limits, with a controlled and restricted diet and lifestyle changes. The use of
pharmacologically-active agents has gained prominence in Diabetes mellitus management and the patient can be
treated with either insulin or with insulin secretagogue agents (Thakur et al., 2010).
The aforesaid complication of diabetes, the killer disease prevalent in India, is the impetus behind the choice
of this research work.
India is a country with a vast reserve of natural resources and a rich history of traditional medicine. In India
indigenous remedies have been used in the treatment of Diabetes mellitus. Approximately 80% of the world
population is almost entirely dependent on traditional medicines (Srinivasan., 2005). Traditional medicines use
herbs as its primary constituents. Knowledge of the nutrient composition of plants is vital for the preparation of
traditional medicines, knowing the metabolite responsible for action and the mechanism of action in varying disease
conditions or disorders. The plants chosen for study were hence subject to proximate analysis to evaluate the nutrient
composition.
395
Methods and Materials
Collection of plants: The plant samples were collected from local places in Coimbatore district and Palladam.
Chemicals
Starch, 3,5dinitrosalicylic acid and -amylase were purchased from Hi-Media Pvt. Ltd., Mumbai, Potassium
Sodium Tartrate, DMSO etc were purchased from Merck Specialities Pvt. Ltd., Mumbai, Fischer inorganics and
Aromatics, Madras. Doubly distilled water was used throughout the study.
Plant extract(10g) was boiled with 60ml water and filtered. The solvent was distilled off to get residue. This
was dried and used for further study.
Proximate analysis of plants
The total ash value, acid soluble ash value, acid insoluble ash value, extractive values, the moisture content
were determined by standard procedures (AOAC, 1990).
- Amylase inhibition assay
Plant extract (100mg) was shaken well with DMSO. From this solution 10l was taken and added again 1ml
of DMSO. 20l was taken from the later solution, added 1ml of DMSO again and shaken well and to this added 1ml
starch solution and cooled for 3 min. Then added 1ml enzyme solution and 1ml colouring reagent. This mixture was
heated in a water bath for 15min. After the solution was cooled, the absorbance was noted at 540nm using photo
colorimeter (Giancarlo et al., 2006).
Calculation
The inhibition efficiency of the plant extracts against the enzyme alpha amylase was calculated using the
formula:
Inhibition efficiency% = [Ab-(As- -Ab)/Ab] 100
where Ab Absorbance of blank,As - Absorbance of sample

IC50 value of ethanol extract of plants
100 mg of plant extract was dissolved in 1 ml DMSO .10l of prepared solution was taken and again made
upto 10ml using DMSO. Various concentrations like 10 l, 20 l, 30l, 50l, 60l were taken to check the IC50
value.
Proximate analysis of plants
Proximate analysis of plants helps us in getting information about the nutrient composition of plants. It gives
us information about the qualitative and quantitative metabolite richness of a plant.
Ash content
The ash content of a plant signifies the organic and inorganic content of a plant material. The results (Table 2)
show the percentage of ash content to be highest for Coccina indica(22.06%)
Acid insoluble ash
In medicinal plants, there may be meager amounts of silica which can be estimated by knowing the value
of acid-insoluble ash. Some of the ashes get dissolved in dilute acid which shows that they are physiological ash
and those that do not dissolve are the non-physiological ash. So from this value the digestibility of plant when
consumed can be understood. The results of the study (Table 3) reveal Trigonellafoenum graecum, Curcuma longa
and Annona squamosa to have high percentage of acid insoluble ash value when compared to other plants. So these
plants with high value can be digested easily when consumed (Patel et al., 2011).
396
Water soluble ash
The water soluble ash content values gives an idea about the extent of contamination of plant with metal ions.
From table 4 it can be known that the value of water soluble ash is less than the acid insoluble ash. So these plants
may be more contaminated with metal ions. Trigonellafoenum graceum has the highest value of water soluble ash
(24.93% ) implies more contamination of metal ions when compared to other plants having water solube ash ranging
from 5 to 20% (Shellard.,1958).
Moisture content
Moisture content of plant samples are essential criteria in determining the stability of plant materials or drugs
that are plant-based. Greater the moisture content lesser is the stability as moisture tends to deteriorate the quality
and nature of the metabolites in the plants. Moisture content is also related to the increased microbial contamination
as moisture favours the growth of organisms (Rajesh, 2012).
Methanol soluble extract
The plant materials when extracted with 60% methanol and tested for its phytochemicals by preliminary colour
tests revealed the presence of constituents like sterols, tannins, flavonoids, saponins, glycosides, carbohydrates and
alkaloids.
The methanol soluble extract percentage was found to be more in Eugenia jambolana, Hibiscus rosasinesis
and Aloe vera having value above 30% compared to other plants.
- Amylase inhibition assay
Inhibition of a-amylase based on starch iodine colour complex formation was performed with 17 plant
sample extracts in order to evaluate the anti-diabetic potential of the extracts and also to establish alpha amylase
assay as a facile screening tool. The results of the study revealed alcohol extract in general to show more inhibition
efficiency compared to water extract.
Hibiscus rosasinesis alcoholic extract showed high percentage of inhibition efficiency (Table 7). Likewise
among aqueous extracts Gymnema sylvestre showed higher percentage of inhibition efficiency (Table 8) when
compared to the alpha amylase inhibition efficiency of alcoholic extract.
All the samples studied showed appreciable alpha amylase inhibition efficiency (70-98%). This result not only
justifies the traditional use of these plants in the control of diabetes but also validates the ethnobotanical knowledge.
Also this study ascertains this assay as a simple and facile screening tool to establish the anti-diabetic potential of
plants.
The higher inhibition efficiency of alcohol extracts suggests the use of these plants as alcoholic extract. The
extract can be powdered, dried and taken in instead of cooking in water or boiling the plant material in water. The
extractives in alcohol are more contribute to higher antidiabetic activity of alcohol extract than aqueous extract.
IC50 value of Aegle marmelos and Hibiscus rosasinensis extracts
The inhibition efficiency of two plants extracts namely Aegle marmelos and Hibiscus rosasinensis was found
to be above 95%. Hence these two plant extracts were taken for determining the IC50 value at different concentrations
(10l, 20l, 30l, 50l, 60l). The IC50 value calculated for 20 l concentration of extracts is tabulated (Table 9
and Table 10).
Conclusion
Traditionally used 17 anti-diabetic plants were taken and analyzed for nutrient composition.. Ash content value
for Coccina indica has highest percentage 22.06% which shows the presence of more number of organic compounds
whereas the low value of ash content of Trigonellafoenum graceum (3.36%) reveals less number of mineral or
inorganic constituents. It was noted that the value of acid insoluble ash for the plants Trigonellafoenum graecum,
Curcuma longa and Annona squamosa has the highest percentage which shows the easy digestibility of plant when
397
consumed. The highest value of water soluble ash of Trigonella foenum graceum (24.93%) gives the maximum
contamination of metal ions when compared with other plants.The stability of natural product in the plants promote
microbial contamination and chemical degradation can be given by knowing the moisture content of plants. The
inhibition efficiency of a-amylase using alcohol extract was high compared to aqueous extract.Also it was noted
that, the plants with high inhibition efficiency in alcoholic extract have low inhibition efficiency in aqueous extract.
So the alcoholic extract can be used to know the inhibition of a-amylase. The IC50 value of two plants, namely, Aegle
marmelos and Hibiscus rosasinensis were found to have 50% inhibition efficiency at 20 l concentrations.
Acknowledgement
The authors thank the Avinashilingam Institute for Home Science and Higher Education for Women,
Coimbatore for providing laboratory facilities during the work.
References
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colocunthis fruit aqueous extract in normal and alloxan diabetic rabbits, J.Ethnopharmol 71:325-300.
Acosta-Patin.O.J.L, Jume nez Balderas.E, Juarez-Oropeza.M.A and Dyaz.zagoya.J.C, 2001, Hypoglycemic action of Cucurbita
ficifolia on Type-2 diabetic plants with moderately high blood glucose levels, J.Ethnopharmol, 77: 99-101.
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therapy for type 2 diabetes: a randomized controlled trial, J. Am. Med. Assoc., 299 (3): 316323.
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4(7):685-688.
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used for its Treatment, Asian Journal Of Pharmaceutical And Clinical Research, 5.
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Mann J. Secondary Metabolism, Oxford University press, London 154.
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19:155-156.
Shukla. R, Sharma. S. B, Puri. D, Prabhu. K. M and Murthy. P. S, 2000, Medicinal Plants for Treatment of Diabetes Mellitus,
Indian Journal of Clinical Biochemistry,169-177.
Table 1. Sample code assigned to plant extracts
Plant name Sample code Ethanol extract Aqueous extract
Aegle marmelos AM ETAM AOAM
Azadiachta indica AI ETAI AOAI
Annona squamosa ASq ETASq AOASq
Aloe vera AV ETAV AOAV
Citrus aurantifolia CA ETCA AOCA
398
Coccinia indica CI ETCI AOCI
Curcuma longa CL ETCL AOCL
Catharanthus roseus CRO ETCRO AOCRO
Coriandrum sativum CS ETCS AOCS
Eugenia jambolana EJ ETEJ AOEJ
Ficus bengalensis FB ETFB AOFB
Gymnema sylvestre GS ETGS AOGS
Hibiscus rosasinesis HR ETHR AOHR
Mimordica chirantia MC ETMC AOMC
Mangifera indica MI ETMI AOMI
Ocimum sanctum OS ETOS AOOS
Trigonella foenumgraecum TF ETTF AOTF
Table 2. Percentage of ash content of plant samples obtained in proximate analysis
S.No Sample code Percentage ash value S.No Sample code Percentage ash value
1. AM 15.36 10. EJ 10.06
2. AI 11.16 11. FB 12.28
3. ASq 6.14 12. GS 10.11
4. AV 9.24 13. HR 5.86
5. CA 10.52 14. MC 10.00
6. CI 22.06 15. MI 8.12
7. CRO 10.64 16. OS 13.48
8. CS 19.52 17. TF 3.38
9. CL 6.38
Table 3. Percentage of acid insoluble ash content of plant samples obtained in proximate analysis
Percentage acid insoluble Percentage acid insoluble
S.No Sample code S.No Sample code
ash ash
1. AM 68.47 10. EJ 68.12
2. AI 70.93 11. FB 55.52
3. ASq 81.66 12. GS 79.52
4. AV 75.16 13. HR 59.46
5. CA 61.24 14. MC 77.56
6. CI 79.04 15. MI 58.55
7. CRO 76.96 16. OS 77.92
8. CS 79.68 17. TF 98.00
9. CL 82.40
399
Table 4. Percentage of water solube ash content of plant samples
S.No Sample code Percentage water soluble ash S.No Sample code Percentage water soluble ash
1. AM 5.93 10. EJ 15.12
2. AI 11.00 11. FB 12.36
3. ASq 20.73 12. GS 13.36
4. AV 17.40 13. HR 16.26
5. CA 18.04 14. MC 16.00
6. CI 17.28 15. MI 5.20
7. CRO 18.88 16. OS 6.76
8. CS 10.64 17. TF 24.93
9 CL 19.80
Table 5. Percentage moisture content of plants
S.No Plant code % Moisture content S.No Plant code % Moisture content
1. AM 8.14 10. EJ 10.74
2. AI 4.24 11. FB 7.62
3. ASq 2.44 12. GS 6.6
4. AV 0.36 13. HR 1.2
5. CA 3.88 14. MC 3.14
6. CI 2.92 15. MI 2.26
7. CRO 1.64 16. OS 8.42
8. CS 1.64 17. TF 1.76
9. CL 8.76
Table 6. Percentage of methanol soluble extract of plants
S.No Plant Code % of methanol soluble extract S.No Plant Code % of methanol soluble extract
1. AM 25.96 10. EJ 35.84
2. AI 29.22 11. FB 14.52
3. ASq 19.96 12. GS 18.72
4. AV 32.18 13. HR 33.96
5. CA 22.78 14. MC 27.32
6. CI 17.44 15. MI 25.60
7. CRO 27.18 16. OS 14.82
8. CS 27.32 17. TF 17.68
9. CL 21.04
Table 7. Alpha amylase inhibition efficiency of ethanol extract of plants
Plant Optical density Plant Optical density
S.No IE % S.No IE %
code (540nm) code (540nm)
1. ETAM 0.35 97.05 10. ETEJ 0.52 86.96
2. ETAI 0.37 91.17 11. ETFB 0.60 69.57
3. ETASq 0.57 81.25 12. ETGS 0.50 91.30
4. ETAV 0.40 82.35 13. ETHR 0.47 97.82
5. ETCA 0.50 95.83 14. ETMC 0.52 86.96
6. ETCI 0.59 71.73 15. ETMI 0.54 87.50
7. ETCRO 0.41 79.41 16. ETOS 0.41 89.13
8. ETCS 0.51 93.75 17. ETTF 0.55 80.43
9. ETCL 0.64 60.86
400
Table 8 . Alpha amylase inhibition efficiency of water extract of plants
Plant Optical density Plant Optical density
S.No IE % S.No IE %
code (540nm) code (540nm)
1. AOAM 0.57 76.08 10. AOEJ 0.47 69.44
2. AOAI 0.44 77.77 11. AOFB 0.44 77.77
3. AOASq 0.42 83.32 12. AOGS 0.49 97.91
4. AOAV 0.48 95.65 13. AOHR 0.40 88.88
5. AOCA 0.48 66.66 14. AOMC 0.54 87.50
6. AOCI 0.43 80.55 15. AOMI 0.45 75.00
7. AOCRO 0.52 86.95 16. AOOS 0.41 86.11
8. AOCS 0.46 72.22 17. AOTF 0.40 88.88
9. AOCL 0.44 77.77
Table 9. IC50 value for Aegle marmelos plant extract

S.No Concentration of extract (l) Optical density (540nm) Inhibition efficiency %
1. 10 0.55 47
2. 20 0.50 52
3. 30 0.48 66
4. 50 0.38 88
5. 60 0.39 91
Table 10. IC50 value for Hibiscus rosasinensis plant extract
S.No Concentration of extract (l) Optical density (540nm) Inhibition efficiency %
1. 10 0.56 44
2. 20 0.51 50
3. 30 0.47 69
4. 50 0.31 91
5. 60 0.38 94
401
Reverse pharmacological validation of a clinically successful
Ethnoveterinary herbal preparation against mastitis.
N.Punniamurthy, Ramakrishnan N, Elamurugan. A, Vijay Anand. J
Veterinary University Training and Research Centre, Thanjavur, TANUVAS, Pillayarpatti, Thanjavur- 613 403, Tamil
Nadu. Corresponding author:murthyvcri@hotmail.com, Ph:+91-9842455833
Abstract
Mastitis, the inflammation of udder parenchyma, is multi-etiological and complex in nature. Mastitis causes
heavy economical loss to the farmer directly by decrease in milk yield and indirectly by treatment cost and
negative implications for milk hygiene and quality. Recent works suggest prevalence of mastitis to be the
highest in high yielder cross bred dairy cows. More than 100 recent studies spread over 32 states of India
indicate that the overall prevalence of mastitis ranges from 25 to 97 % with an average prevalence of 45 %
(NDRI News, 2012). In India, marginal producers and small scale farmers own over 60% of all milch animals
and constitute the core milk production sector. One of the earlier studies pegs the overall burden of mastitis in
excess of USD 1 million (INR 60,532.10 million annually. These losses are primarily on account of clinical
mastitis and with a prevalence rate of 46.85 for sub-clinical mastitis. Under Indian Council for Agricultural
Research (ICAR) the EVM research and training centre, TANUVAS validated fresh herbal combination of
Aloe vera, Curcuma longa, Calcium hydroxide. The parameters such as somatic cell count, pH, electrical
conductivity along with the physical observation of nature and consistency of udder tissue. Milk yield before
and after EVM intervention was recorded. In early interventions 80-100% recovery possible in 3-5 days time
without using any other form of drugs or antimicrobials. Based on clinical success over thousands of cattle
in field under controlled conditions, the recipe was tested for its phytochemical profile quantitatively and
qualitatively. Field isolated pathogens of mastitis and standard cultures were tested against the herbal extract
involving four different solvents for the anti-inflammatory, antioxidant activities. These were confirmed
through in vitro studies and Scanning Electron Microscope analysis.
Keywords: Mastitis, Ethnoveterinary, Aloe vera, Turmeric, CaOH
More than 100 studies spread over 32 states of India indicate an average prevalence of mastitis at 45 %. The
loss per animal was estimated to be INR 1,390 (USD 23/animal). A 49% of this loss per animal was due to loss of
value from milk production and 37% on account of veterinary medicines expenses. (NDRI News, 2012)1
Under Indian Council for Agricultural Research (ICAR) EVM network programme the VUTRC, TANUVAS
validated fresh herbal combination of Aloe vera, Curcuma longa, Calcium hydroxide. In early interventions 80-
100% recovery is found in 3-5 days time without using any other form of drugs or antimicrobials. Based on clinical
success over thousands of cattle in field conditions, the recipe was tested for its phytochemical profile. Field isolated
pathogens of mastitis and standard cultures were tested against the herbal extract involving four different solvents for
the anti-inflammatory, antioxidant activities. These were confirmed through in vitro studies and Scanning Electron
Microscope analysis.
Inhibition of heat induced albumin denaturation activity was assayed as described by Williams et al., 20022
with slight modifications. The experiment was performed in triplicate. The superoxide radical scavenging activity
of solvent extracts of fresh and dried anti-mastitis herbal preparation was assessed by the method described by
Govindarajan et al., 20033 with slight modifications. The absorbance was measured at 560nm and Vitamin C was
used as a positive control. The experiment was performed in triplicate
Agar well diffusion method was followed to determine the antimicrobial activity of the fresh and dried
anti-mastitis herbal preparations against E.coli, S.aureus and P. aerugenosa. using standard procedures. Control
experiments comprised DMSO 5% (Negative control) and Ciprofloxacin/Tetracycline (Positive control).
402
The bacteria that were susceptible to the plant extracts were prepared for SEM. Small agar pieces were cut
out from the inhibition zone and mounted to the conductive carbon stubs and allowed to dry for 5 min. The samples
on the conductive stubs were coated with gold nano particles and examined using scanning electron microscope.
Results
Inhibition of heat induced albumin denaturation was observed in the solvent extracts of the anti-mastitis herbal
preparation in a dose dependent manner . The results revealed 80.50% inhibition of protein denaturation by aqueous
extracts form fresh herbal preparation. Comparative study on the reactive oxygen species scavenging activity of
aqueous, ethanol and ethyl acetate extracts of anti-mastitis fresh herbal preparations revealed scavenging activity in
a dose dependent manner. The ethanolic extracts revealed maximum scavenging activity of 88.81% at 300 g/ml.
Solvent extracts of fresh anti-mastitis herbal preparation showed significant antimicrobial activity against
E. coli, S. aureus and P. aerugenosa. Among different solvent extracts ethyl-acetate extract showed greater
antimicrobial activity .
Morphostructural damage in bacteria studies using Scanning Electron Microscopy
Bacterial cells treated with extracts of fresh anti-mastitis herbal preparations revealed various morphostructural
changes such as cell disruption, partial disruption of cytoplasmic membrane and plasmolysis. These findings indicate
antimicrobial activity of the anti-mastitis herbal preparation ( Fig 1)
Bioinformatic analysis-Molecular docking
Using bioinformatics this herbal combination against mastitis was subjected to molecular docking studies
using Staphylococcus aureus as a model pathogen. In-silico analysis revealed that the herbal combination has affinity
towards number of Staphylococcal proteins, such as biotin protein ligase, DNA gyrase, opuCB, sirA, Penicillin
binding protein 3, sortase A. and shows that the herbal preparation has antimicrobial potential.
The Reverse pharmacology approach using in vitro studies indicates that the herbal combination which is
being used successfully in the field for treating clinical and subclinical mastitis has anti-inflammatory, anti-oxidant
and anti-microbial properties. The recipe against mastitis has been successfully used widely in the southern states
of India i.e. Tamil Nadu, Kerala, Karnataka and certain pockets of northern India such as Gujarat and Uttar Pradesh
(Nair et al 20154; Punniamurthy and Nair, 20165).
References
NDRI news (2012). 17(1)
LAD Williams, OConnar A, Latore L, Dennis O, Ringer S, Whittaker J A, Conrad J, Vogler B, Rosner H &Kraus W (2008)
West Indian Med 57 (4),327-331
Govindarajan R, Rastogi S, awat A K S, Vijayakumar M,Shirwaikar A, Mehrotra S & Palpu P(2003) Pharmaceut.Bulletin 26,
1424-1427
Nair BM, N Punniamurthy, Seethakempanahalli Kempanna. (2015). Role of Ethnoveterinary Practices (EVP) in reducing
antimicrobial resistance in livestock production systems: a field experience Planta Medica ; 81 -SL3C_06
Punniamurthy N, B M Nair.(2016).A decade of clinical research replace synthetics in animal health and production. Planta
Medica ; 82(S 01): S1S381
403
Fig.1- Scanning electron microscopic images of morpho-structural changes induced by extracts of fresh
anti-mastitis herbal formulation in E.coli (A1 to A4), S. aureus (B1 to B3) and P.aeruginosa (C1 to C4). A1,
B1 and C1-Untreated; A2, B2 and C2-Treated with Aqueous extract; A3, B3 and C3-Treated with Ethanolic
extract; A4, B4 and C4-Treated with Ethyl acetate extract.
404
A novel method for protein quantification from mice and zebra fish
L.Surendran1, D.S.Ranjith Santhosh kumar1, Nasir hazza1, Dr. P. Senthilkumar1*
1
PG and Research Department of Biotechnology, Kongunadu Arts and Science College, Coimbatore-641 029,
Tamilnadu, India.
Abstract
Thecolorimetricdye binding assays are important to resolve the qualification and quantification of proteins and
its specificity, which helps researchers to find the nature of the protein. The present study deals with comparison
of protein assays, from mice and zebra fish of protein samples and its major impurities of sugars and lipids can
also be identified. In connection with this present research, we used three types of dyesCommassiebrilliant
blue,Biuretreagent and Lowrys assay reagent. WhenCommassiebrilliant blue dyeturn into pale blue to
dark blue, due to the positive amino acids, which measured by the peak at595nm. In case of Biuretreagent
turn into blue, denotestheamide bond of proteins with copper ions, measured by peak at550nm. Then at
last, when Lowrys reagent turns into blue color, measured by peak at750nm. To find impurities, we have
performed all the three type of dye binding assays for sugars and lipids. From these assays, we measured a
single peak on each desiredwave length. But when impurities mixed with protein sample manually in volume
dependantway, we obtained abnormal peaks with desired peak wavelength. It is reasonable to concludethat,
dye binding assays are more sensitive and reliable method for protein quantification and qualification from
mice and zebra fish.
Key words: Total Proteins, Dye Binding assay, Commassie brilliant blue, Biuretreagent Lowrys assay, UV-
Visible spectroscopy.
Introduction
The quantification of biomolecules is an essential technique to understand the regulation of the cell and its
byproducts (Horisawa et al., 2017). Since quantification history there were varieties of techniques employed to
quantify the desired products such as DNA by diphenyl amine, RNA by orcinol, sugars by benedicts and protein
by Bradford etc. While performing protein extraction we have observed so many impurities (Sun et al., 2002),
which can be avoided by washing the sample many times prior to quantify. The present study focused on extraction
and quantification of protein by colorimetric method and to find major impurities such as sugars and lipids. For the
extraction of protein, we have selected model animals, mice and zebra fish. The research has employed dye binding
techniques to quantify the samples containing protein and impurities.
Model animals:
Swiss Albino Mice (20 - 30g) were purchased from PSG institute of Medical Research, Animal facility
laboratory, Coimbatore and hosted into a cage with suitable diet. Zebra fishes were purchased from Aquatic fisheries
farm house town Pollachi With aeration facilities and diet at room temperature. The animal applications were
carried out according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on
Animals (CPCSEA). The Institutional Animal Ethical Committee approved added experimental design performed
in this study for the use of Swiss Albino Mice as an animal model for protein extraction.
Extraction of Total protein:
The current research obtained tissue sample from mice and zebra fish for extraction of protein and quantification.
The research has employed two types of chemical methods and the extracted protein was quantified by dye binding
assays and qualified by gel electrophoresis.
405
Isolation of protein by Radio Immuno Precipitation Assay buffer (Franquesa, Marcella et al., 2014):
The tissue samples and minced into very small pieces with treating of Liquid nitrogen using a dean razor blade
.Frozen tissue thawed in Radio Immuno Precipitation Assay buffer containing (Protease Inhibitors) and (Phosphatase
Inhibitors). We used 3ml of precold RIPA buffer per gram of tissue and further disruption was done with a sonicator.
While sonication and protein extraction 4C was maintained throughout the procedures. Incubate on ice for 30 min.
Transfer to micro centrifuge tubes, centrifuge at 10,000 x 9 for 10 min at 4C. Remove supernatant and centrifuge
again. The supernatant fluid is the total celllysate. A longer centrifugation may be necessary to obtain a dear Iysate.
Isolation of protein by TCA-Acetone precipitation:
1 ml of cell lysate , 8 ml of 100% ice-cold acetone and 1 ml 100% trichloroacetic acid added to tissues and
(100%TCA, w/v) Precipitate at -20 C for 1 hr. 3. Centrifuge at 11,500 rpm for 15 min at 4 C. Discard supernatant.
Wash with 1 ml ice-cold acetone and Centrifuge at 11,500 rpm for 15 min at 4 C. 5. Repeat step 4 twice to remove
all of the TCA. Store the pellet at -20 C for late use or dissolve the protein pellet in the appropriate volume of
2-D rehydration buffer by repeatedly pipetting up and down to break up the pellet. Allow sample to settle at room
temperature for 1 hr, vortexing approximately every 10 min. 10. Transfer to eppendorf tube and centrifuge at 14,000
rpm for 10 min at room temperature.
Dye-Binding assays:
The protein of interest was extracted and purified then quantified by standard dye binding methods Brad ford,
Lowrys and Biuret. After that the sample containing protein was loaded through SDS-PAGE gel electrophoresis.
Brad ford method (Bradford, M. M 1976):
The protein sample was diluted into three aliquots and then an undiluted sample was added. Bovine serum
albumin was added as standard. The Bradford reagent containing Commassie brilliant blue was added and then the
color was developed into blue. Last two samples were diluted and the contaminants sugars, lipids added manually
to protein sample. After 5-60 minutes the O.D values were recorded 595nm.
Lowrys method (Lowry et al., 1951):
The total protein sample was diluted into three aliquots and then an undiluted sample was added. Bovine serum
albumin (2mg/ml) was added into 10 dilutions (10 to 90) as standard. The Lowrys solution A and B containing folin-
ciocalteu reagent was added and then the color was developed. Last two samples were diluted and the contaminants
sugars, lipids added manually to protein sample. After incubation, the O.D values were recorded at 750nm.
Biuret method (Franquesa, Marcella et al., 2014):
The total protein sample was diluted into three aliquots and then an undiluted sample was added. Allow all of
the tubes to stand at room temperature for 15 minutes. While you are waiting, turn on the spectrophotometer and
allow it to warm up; this may take 1-2 minutes. Do not push any buttons until the machine is ready (when the ABS
reading at 542 nm appears on the screen). Adjust the wavelength to 550 nm.
SDS-Poly acrylamide gel electrophoresis (Fortis F et al., 2006):
The total protein was extracted and resolved through the 12% polyacrylamide gel. At first the separating gel
was prepared and then stacking gel was made. After polymerization the comb was removed and the protein samples
were loaded by dissolving it in loading dye. Then the gel was stained with commassie brilliant blue and detained by
method. The gel visualized under white light .The BSA (2mg/ml) 25 added as standard.
406
Result and discussion
SDS-PAGE gel electrophoresis:
Figure I: 12%SDS-PAGE gel for total protein extract.Lane1:

Dye binding methods:
A. Bradford method B. Lowrys method C. Biuret method
Figure II: Dye binding methods for Total protein

Protein extraction and quantification:
The protein sample was extracted from the mice and zebra fish by two different types of methods. Those are
RIPA buffer method and TCA-Acetone method, and then the extracted protein sample was examined for quality
and quantity.
Quantitation by colorimetric method:
A. RIPA Buffer extraction B. TCA-Acetone Method
Figure III: Extraction methods and its protein quantification
407
From the above results, we discussed that the protein extraction and quantitation was fully depending on the
method we employed and from Figure I the gel electrophoresis showed us, the quality of the protein was slightly
reduced then in the RIPA method (Franquesa, Marcella et al., 2014), Because RIPA containing protease and
phosphate inhibitor cocktail instead of TCA-Acetone, which have prevented the maximum degradation of samples
from mice and zebra fish, Confirmed through the gel (Fortis F et al., 2006) and quantitation assays. Also the
quantitative results clearly stating that pure protein sample gave a positive colour change (Dark blue, and purple)
and peak at desired wavelength on brad ford 595nm, Lowrys 720nm and Biuret 550nm) (Lowry et al., 1951)
Figure II. But the sample containing sugar or lipid (Snyder,F et al., 1959), gave a value gives false absorbance
results and an abnormal peak wave length instead of normal wave length on brad ford (true 595nm and false
620nm), Lowrys (true 720nm and 680nm) and Biuret (true 550nm and false 620nm )Figure III.
Conclusion
So the current study identified the protein impurities and its interference in the protein sample through simple
dye binding methods and helps to improve the protein extraction and quantification of biological sample and how
the impurities will change the protein quality measurements.
Reference
Horisawa, Kenichi. Specific and Quantitative Labeling of Biomolecules Using Click Chemistry.Frontiers in Physiology5
(2014): 457.PMC. Web. 15 Feb. 2017.
Sun, Liping, and Thomas Kodadek. Removal of Impurities from Transcription Factor Preparations That Alter Their DNA-
Binding Properties.Nucleic Acids Research30.16 (2002): e88. Print.
Fortis F,Guerrier L,Righetti PG,Antonioli P,Boschetti E. Electrophoresis.2006 Aug: 27(15):3018-27.
Franquesa, Marcella et al. Update on Controls for Isolation and Quantification Methodology of Extracellular Vesicles Derived
from Adipose Tissue Mesenchymal Stem Cells.Frontiers in Immunology5 (2014): 525.PMC. Web. 15 Feb. 2017.
Snyder,F., and N. Stephens.1959. A simplified spectrophotometric determination of ester groups in lipids.Biochim. Biophys.
Acta.34:244245.
Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 72, 248-54 (1976).
Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (November 1951).Protein measurement with the Folin phenol reagent.J.
Biol. Chem.193(1): 26575.
408
Modulation of GLUT2 expression by aqueous extract of Erythrina
variegata L. bark in streptozotocin induced diabetic rats
Kanakasabapathi Devaki*1,2
Subrahmanian Hemmalakshmi1, Suriyamoorthy Priyanga1 and Muthusamy Sridhar1
1
Department of Biochemistry, Karpagam University, Coimbatore, 641 021, India
1,2
Department of Biochemistry and Bioinformatics, Karpagam University, Coimbatore, 641 021, India
Email: devakirajasekar@gmail.com
Abstract
GLUT2 is a bidirectional transporter, allowing glucose to flow in two directions. The aim of the present
study was to investigate and to evaluate the expression of Glucose Transporter 2 (GLUT2) in the intestine of
streptozotocin induced diabetic rats treated with or without aqueous extract of Erythrina variegata L. bark.
Diabetes was induced by intraperitoneal injection two consecutive split doses of streptozotocin (20 and 25 mg/
kg body wt) in Wistar albino rats. The animals were divided into five groups of five animals each. Diabetic
animals were treated with Erythrina variegata L. bark extracts (200 mg/kg body weight) for 30 days. The gene
expression data showed significant differences in Glut2 gene expression between the experimental groups
(p< 0.05). The elevated Glut2 gene expression in the intestine of the diabetic treated rats were significantly
reduced. Western blot analyses of glucose transporter GLUT2 from control, STZ-induced diabetic rats,
standard treated, plant treated and treated non-diabetic rats were assessed to explore the inhibitory mechanism
of E. variegata and glibenclamide on glucose absorption. mRNA and Protein expression of Glut 2 were
elevated in diabetic rats and Erythrina variegata L. is able to inhibit the expression of Glut 2. The present
findings suggest the regulatory effect of E. variegata on GLUT 2 expression, which could be exploited for the
treatment of diabetes.
Key words: Erythrina variegata L., streptozotocin, GLUT2
INTRODUCTION
Diabetes mellitus is a chronic endocrine disorder caused by inherited or acquired deficiency in the production
of insulin by pancreas or in effectiveness of insulin on target organs1. It is associated with reduced life expectancy
because diabetes in chronic condition induce microvascular complications (retinopathy, nephropathy and
neuropathy) and increase the risk of macrovascular complications (ischaemic heart disease, stroke and peripheral
vascular disease)2.Worldwide the numbers of cases of diabetes is rising gradually. There are 366 million people
have diabetes in 2011; by 2030 this will have risen to 552 million.
The number of people with type 2 diabetes is increasing in every country 80% of people with diabetes live in
low- and middle-income countries3.
The worsening metabolic control often seen after the initiation of an oral hypoglycemic agent4 and associated
side effects like hypoglycemia, digestive discomfort, permanent neurological deficit, idio syncratic liver cell injury,
headache5 have led to the use of phytochemical from medicinal plants in the treatment of diabetes. Earlier results
provide pharmacological credence to the suggested use of the phytochemicals in the management and treatment of
diabetes mellitus associated with hyperlipidaemia and related cardiovascular complications. Some phytochemicals
have either antioxidant property or enhancing the insulin activity or both (Tiwari and Rao, 2002)6.
Various parts of E. variegata is reported to have antibacterial / dental caries prevention, antioxidant, anti-
inflammatory, cardioprotective, neuroprotective, antiosteoporetic, cytotoxic properties. Hypoglycemic activity
of Erythrina variegata leaf in streptozotocin-induced diabetic rats is also reported7.Studies to validate the use of
E.variegata as anticarcinogenic8,antilipidemi9 and also for the removal of heavy metals from contaminated water10
were reported. Study on enzymatic alteration in the vital organs of streptozotocin diabetic rats treated with aqueous
extract of Erythrina variegata bark reduced the hyperglycemia and maintain the antioxidant status 11.
409
Agents that delaying or inhibiting glucose absorption have a substantial impact in managing diabetes and
obesity. Emerging results indicates that apical or luminal facing facilitated glucose transporter 2 (GLUT2) is
a major pathway of sugar absorption, and therefore an attractive target of potential hypoglyemic agents12. This
leads to design a study to establish whether the E.variegata treatment to diabetic rats would results in changes in
expression of glucose transporters especially the GLUT2 transporter.
Plant material
The plant materials for the proposed study were collected from Kodaikannal, Dindigul district, Tamil Nadu,
India. The plant was authenticated by Dr. G.V.S Moorthy, Botanical Survey of India, TNAU campus Coimbatore,
with the voucher number BSI/SRC/5/23/2013-14/Tech/1500.
Sample extraction
The bark was air dried at 25C for 15 days in the absence of sunlight and coarsely powered well using a mixer
and stored in an air tight container. Aqueous extract was prepared by taking 1;5 ratio plant material and water. The
extraction was carried out for 16 hours with occasional shaking and filtered extract was concentrated by rotary flask
evaporator. The extracted sample was used for the study.
Experimental design
Based on our earlier studies 11, 400 mg/kg body weight dose was chosen for antidiabetic research. The
animals were divided into five groups comprising five animals each. Group 1 rats were untreated rats and serve as
control. Group 2 were made diabetic using streptozotocin (45mg/kg) served as diabetic control. This group was
divided in to three groups (Group 2-4), which were left treated or treated with 2 mg/kg of Glibenclamide, 400 mg/
kg of Erythrina variegate. Group 5 rats treated with 400 mg/kg of Erythrina variegata alone. The animals were
weighed and dosed through oral intragastric tube every day. The test drug and reference standard drugs were fed
orally for 30days.
Expression of GLUT2 gene in intestine
(i) Tissue collection
The intestine was weighed and immediately used or frozen in liquid nitrogen until analysis.
(ii) Isolation of total RNA
Total RNA was isolated from the tissue using Trizol (Invitrogen) kit method. The concentration and quality of
isolated RNA were assessed by measuring absorbance at 260 and 280nm using spectrophotometer.
(iii) Synthesis of cDNA from RNA and RT-PCR
The isolated RNA was subjected to reverse transcription and polymerase chain reaction using gene specific
primers following the manufacturers protocol (Invitrgen)
GLUT2: Forward 5-GTC CAG AAA GCC CCA GAT ACC-3
GLUT2: Reverse 5-GTG ACA TCC TCA GTT CCT CTT AG-3.
-actin: Forward 5-GAA GAG CTA CGA GCT GCC -3 and the
-actin: Reverse primer 5-TGA TCC ACA TCT GCT GGA -3.
Expression of GLUT 2 by Western blotting
Homogenate for Western blot analysis was made by grinding tissue samples (0.5-1g) with ice-cold buffer
(30ml) (250mM sucrose containing 20mM HEPES and 1mM EDTA, pH7.4) with grinding tubes resting in ice water
bath and subjected to Western blot analysis using rabbit anti-rat GLUT2 antibody (Santa Cruz Biotechnology, CA-
sc 31833).
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E. Variegata regulate the mRNA expression of Glut 2 in diabetic rats
GLUT2 is a bidirectional transporter which facilitates bidirectional glucose transport. It is identified as primary
defect in diabetic individuals. Therefore, drugs that target Glut2 can have potential impact in controlling the blood
glucose level. It is expressed by renal tubular cells, small intestinal epithelial cells, liver cells and pancreatic beta cells.
It is also present in the basolateral membrane of the small intestine epithelium. All three monosaccharides (glucose,
galactose and fructose) are transported from the intestinal mucosal cell into the portal circulation by GLUT213. It
has been shown that the GLUT2 is the major glucose transporter in the plasma membrane of hepatocytes for glucose
uptake. This protein is also involved in the second step of trans-epithelial glucose transport in the small intestine and
the proximal convoluted tubule of kidneys14.
The gene expression data showed significant differences in Glut2 gene expression between the experimental
groups (P< 0.05). The mRNA analysis showed increased Glut2 gene expression in the intestine of the diabetic rats.
The increase in the mRNA expression were not evident in diabetic rats treated with E. variegate and standard drug
(Figure 1). These results suggest that the aqueous extract of decreased the high levels of Glut2 gene expression in
rat intestine.
Figure 1: Fold changes of GLUT2 gene expression in

rat intestine. 1: Untreated rats (Control), 2: Diabetes induced
rats (45mg/kg of streptozotocin), 3: Rats treated with 400 mg/
kg of Erythrina variegata, 4: Rats treated with 2 mg/kg of
Glibenclamide, 5: Rats untreated with 400 mg/kg of Erythrina
variegata alone. Values are expressed as Mean SD for six
animals. Values not sharing common superscript letters (a-c)
differ significantly at p< 0.05 (DMRT).
This assumes larger significance as the facilitative

transporter GLUT2 transports sugars accumulated in the enterocytes across the basolateral membrane to the blood
and the capacity of the small intestine to absorb D-glucose in type 2 diabetic individuals and experimentally
induced diabetic rats15,16 (Drozdowski and Thompson, 2006; Dyer et al.,2002).
Expression of GLUT 2 by Western blotting
To corroborate the findings of mRNA expression, Western blot analysis of glucose transporter GLUT2 from
control, STZ-induced diabetic rats, standard treated, plant treated and treated non-diabetic rats were performed.
The small intestine of STZ induced diabetes rats showed increased expression of GLUT2 protein compared to
non-diabetic control rats (Figure 2). The increase of GLUT2 expression in diabetic rats was significantly abrogated
by aqueous extract of E. variegata indicating that the extract reduced small intestine glucose absorption in part via
inhibition of glucose transporters.
Figure 2: Representative immunoblots

showing the GLUT2 protein expression in the rat
intestine. Lane 1: Untreated rats (Control), Lane 2:
Diabetes induced rats (45mg/kg of streptozotocin),
Lane 3: Rats treated with 400 mg/kg of Erythrina variegata, Lane 4: Rats treated with 2 mg/kg of Glibenclamide,
Lane 5: Rats untreated with 400 mg/kg of Erythrina variegata alone.
It has been shown in rats with experimentally induced diabetes that the capacity of the small intestine to
absorb glucose increases at least in part, due to enhanced activity and abundance of brush border and basolateral
GLUT217. Therefore, it is possible that down regulation of these transporters by E. variegata reduces the total
glucose concentration in the circulation. Collectively, it can be suggested that the extract of E. variegata modulates
the activity of intestinal GLUT2 at mRNA and protein level.
411
CONCLUSION
Decreased expression of GLUT2 RNA and protein in intestine of diabetic treated rats demonstrate that the
Erythrina variegata L. is able to reduced postprandial hyperglycemia. By all these findings, it is concluded that the
aqueous extract of Erythrina variegata L. bark have promising activity against streptozotocin induced diabetic rats
and regulate Glut 2 in diabetic rats.
REFERENCE
1. Thirumalai T, Therasa SV, Elumalai EK & David E (2011) Asian Pacific J Trop Biomed 1, 197-199
2. Sreekumar R, Halvasiotis P, Schimke JC & Nair. KS (2002) Diabetes, 51, 1913-1920
3. American Diabetes Association (2009) Diabetes Care. 2009;32(suppl 1): S13-S61
4. Turner RC, Cull CA, Frighi V& Holman RR (1999) JAMA, 281,2005-2012
5. Joseph B & Jini D (2010) Res J Med Plant, 5, 352-376
6. Tiwari AK & Rao M (2002) Current Science, 83,30
7. Kumar A, Lingadurai S, Shrivastava TP, Bhattacharya S & Haldar PK, (2011) Pharma Biology, 49, 577582
8. Baskar N, Parimala Devi B & Jayakar B. (2012) Int J Res Pharm Pharm 1,30-33
9. Balamurugan G & Shantha A (2010) J Pharm Bioallied Sci. 2:350-355
10. Anupama V, Narmadha R, Gopalakrishnan VK & Devaki K (2012) Int J Pharm Pharm Sci 4,134-147
11. Venkateswarlu1 P, Durga GV, Babu NC & Rao MV (2008) Int J Phys Sci 3,197-204
12. Kwon O, Eck P, Chen S, Corpe CP, Lee JH, Kruhlak M & Levine M (2007) FASEB J , 21, 366-377
13. Anuradha G, Malini S & Jyoti S (2015) International journal of current microbiology and applied sciences 4, 58-77
14. Thorens B (2015) Diabetologia 58, 22132
15. Drozdowski LA & Thompson ABR (2006) World Journal of Gastroenterology 12, 16571670
16. Dyer J & Wood IS, Palejwala A, Ellis A, Shirazi-Beechey SP (2002) American Journal of Physiology Gastrointestinal and
Liver Physiology 282, 241248
17. Khathi A, Serumula MR, Myburg RB, Van Heerden FR & Musabayane CT (2013) PLOS ONE 8,1-8
412
Antioxidant activity of isolated compounds from Hybanthus
enneaspermus .L
*K.Poornima, P. Muneeswari and S. Deepika
Department of Biochemistry & Bioinformatics, Karpagam University, Coimbatore, Tamil Nadu, India 641 021
Abstract
Medicinal plants are considered as rich resources of ingredients which can be used for drug development
and synthesis. Focus on the plant secondary metabolites for treating various ailments have been increasing
over the past few years. These molecules are known to play a major role in the adaptation of plants to
their environment, and represent as an important source of active pharmaceuticals. The present study is
aimed to evaluate the antioxidant activity of the compounds isolated from Hybanthus enneaspermus L. Two
compounds were isolated by using column and thin layer chromatography and their structural elucidation
was done by using NMR spectroscopy. DPPH (2,2 Diphenyl-1-picryl-hydrazyl) radical scavenging activity
,FRAP radical scavenging activity, nitric oxide radical scavenging activity and hydroxyl radical scavenging
activity were done to evaluate and compare the antioxidant capacity of the compounds using ascorbic as a
reference standard. Compound II exhibit good radical scavenging activity compared to compound I. In future
Compound I and II may be used as a good antioxidant agent for the treatment of oxidative stress induced
diseases with further detailed study.
Key words: Hybanthus enneaspermus L, Secondary metabolites, Compound isolation, Antioxidant activity.
Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation
reactions can produce free radicals, which start chain reactions that damage cells. As oxidative stress might be an
important part of many human diseases, the use of antioxidants in pharmacology is intensively studied, particularly
as treatments for stroke and neurodegenerative diseases. However, it is unknown whether oxidative stress is the
cause or the consequence of disease. 1. Low levels of antioxidants, or inhibition of the antioxidant enzymes, causes
oxidative stress and may damage or kill cells. Antioxidants are widely used as ingredients in dietary supplements
in the hope of maintaining health and preventing diseases such as cancer,diabetes, inflammation and coronary heart
disease 2.
Now a days, most food & pharmaceutical products contain synthetic antioxidants. These compounds are added
to food in order to prolong product shelf life, mainly by preventing the oxidation of unsaturated double bonds
of fatty acids. In pharmaceutical products antioxidants are added to enhance the stability of therapeutic agents
that are susceptible to chemical degradation by oxidation. Dietary antioxidants include ascorbate, tocopherols,
carotenoids and bioactive plant phenols. The health benefits of fruits and vegetables are largely due to the
antioxidant vitamins supported by the large number of phytochemicals, some with greater antioxidant properties.
There is great importance in maintaining the fragile balance between these antioxidants and the ROS molecules.
For instance, in humans, disturbing this balance can cause serious health problems, such as cancer, cardiovascular
and neurodegenerative diseases, and premature aging. metabolites are small molecules which are not essential for
the growth and development of the producing organism have importance because of their biological activities on
other organisms and which are chemicals produced by plants such as alkaloids, flavonoids, tannins, saponins and
terpenoids. These compounds are very efficient against free radicals and also against various free radical induced
diseases like cancer.So these phytochemicals called as antioxidants it has the ability to scavange the free radicals.
Secondary metabolites are a wide range of compounds from different metabolite families that can be highly inducible
in response to stresse. It is mentioned that the complete phytochemical investigations of medicinal plants of India
should be carried out, because these secondary metabolites are responsible for medicinal activity of the plant 3.
Hybanthus enneaspermus L. Muell., (Syn. Ionidium en-neaspermus DC) is a perennial ethanobotanical herb and
it belongs to the family Violaceae. It grows 15 - 30 cm in height with many diffuse or ascending branches and is
413
pubescent in nature. It has common names like spade flower and pink ladys slipper. The plant is a perennial herb
or small herb. In Ayurveda, it is known as Sthalakamala. The whole plant is used in Ayurveda, Siddha and other
traditional systems of medicine for curing various ailments.
Plant collection
The whole plant powder of Hybanthus enneaspermus was purchased from Ayurveda pharmascy at pollachi.
The powdered material is used for the study. The plant specimen is authenticated by Botanical Survey of India,
Coimbatore (No: BSI/SRC/5/23/10-11/Tech
1kg of the powdered whole plant material of Hybanthus enneaspermus was mixed with 5Litre of ethanol and
kept at room temperature for 72 hrs with occasional shaking.After 72hrs filtered and the filterate was air dried and
stored in refergerator for further use.
Column chromatography
The individual fractions collected were subjected to thin layer chromatography to separate identify the
individual components following the method of sadasivam and manickam, 1991 4 . Thin layer chromatograms was
then subject to identify the components
Ultraviolet- visble spectroscopy
To visible and ajacent (near-UV and near- infrared ranges to identify the components. In this region of the
electromagnetic spectrum, molecules undergo electronic transitions.
Analysis of the Functional Groups by Fourier Transform Infrared (FTIR) Spectroscopy
The infrared spectrum originates from the vibrational motion of the molecule. The vibrational frequencies are a
kind of fingerprint of the compounds. This property is used for characterization of organic, inorganic and biological
compounds. The band intensities are proportional to the compound and hence quantitative estimations are possible.
Identifications of the structure of the active constituents using nuclear magnetic resonance (NMR)
spectroscopy
NMR spectroscopy was used to determine the molecular structure based on the chemical environment of the
magnetic nuclei like 1H,13C,NMR,etc.,even at low concentrations. This is one of the most powerful non destructive
techniques in elucidating the molecular structure of biological and chemical compounds. A fraction the colleted
fractions was also subject to NMR spectroscopy to characterize the individual components.
Free radical scavenging activities
The free radical scavenging activities of the methanolic extract of Hybanthus enneaspermus.L was determined
by using various in vitro assays like DPPH radical scavenging assay, Nitric oxide radical scavenging assay, Hydroxyl
radical scavenging assay, Reducing power assay.
The DPPH radical scavenging activity of the methanolic extract of Hybanthus enneaspermus.L was analyzed
by the standard method 5. Briefly, the reaction mixture contained 100 M DPPH in methanol, various concentrations
(100-500 g/ml) of the extracts and incubated for 30 minutes at room temperature. The decrease in absorbance was
measured at 517 nm. The scavenging activity was calculated as a percentage of the radical reduction. All tests were
performed in triplicates. Ascorbic acid was used as a reference compound.
Nitric oxide radical scavenging assay
The Nitric oxide was generation by sodium nitroprusside and measured at 514nm 6 . The reaction mixture
contained 10 mM sodium nitroprusside (SNP), phosphate buffered saline (pH 7.4) and various doses (100500 g/
ml) of the test solution in a final volume of 3 ml..
414
Hydroxyl radical scavenging assay
The hydroxyl radical scavenging activity of the methanolic extract of Hybanthus enneaspermus.L was measure
at 532nm 7 . All the solutions were freshly prepared. To 1ml of the reaction mixture contained, 2-deoxy-2-ribose
(2.8 mM); KH2PO4-KOH buffer (20 mM, pH 7.4); FeCl3 (100 M); EDTA (100 M); H2O2 (1.0 mM); ascorbic
acid (100 M) and various concentrations (100500 g/ml) of the test sample. After incubation for 1 hour at 37C,
0.5 ml of the reaction mixture was added to 1 ml of 2.8% TCA, then 1 ml 1% aqueous TBA was added and the
mixture was incubated at 90C for 15 minutes to develop the color.
The reducing power capacity of the plant was assessed 8. Various concentrations (100-500 ug/ml) of the
extract were mixed with 0.5 ml phosphate buffer (0.2 M, pH 6.6) and 0.5 ml potassium hexacyanoferrate (0.1%)
and incubated at 50C for 20 minutes. After incubation, 0.5 ml of TCA (10%) was added to the complete reaction.
The upper portion of the solution (1 ml) was mixed with 1 ml distilled water, and 0.1 ml FeCl3 solution (0.01%)
was added. The reaction mixture was left for 10 minutes at room temperature and the absorbance was measured at
700 nm against a suitable blank solution. All tests were performed in triplicates. A higher absorbance of the reaction
mixture indicated greater reducing power. Ascorbic acid was used as a positive control.
COMPOUND I
The result obtained of the present study Antioxidants activity of isolated compounds from the plant
extract of Hybanthus Enneaspermus L.. Isolation of the active compounds using Column chromatography and
TLC. Characterization of active compounds using UV-Vis Spectroscopy and FTIR Identification and structural
elucidation of isolated bioactive compounds using13C NMR,1H, 13C NMR and 2D NMR. The ethanolic plant
extract of Hybanthus Enneaspermus L. was subjected to column chromatography by using the solvents Petroleum
ether, ethyl acetate Chloroform and Methanol in the increasing order of polarity and fractions were collected. A total
of 305 fractions was collected in test tube. The isolated fractions were spotted in TLC plate coated with silica gel.
The solvent system used for running is Chloroform: Methanol in the ratio 4:6 and 3:7. The spots were identified
by placing in iodine chamber.The fractions (fraction 231-235 to 245-252) collected from column chromatography
showed single spotmas mentioned earlier solvent system in iodine chamber. So these two were used for further
studies such as UV-Visible spectroscopy, FTIR analysis and NMR to determine the absorption spectra ,the functional
groups of the compounds that is present in the defatted ethanolic extract of Hybanthus Enneaspermus L.
Fig. 1 Compound I
Fig. 1 Compound II
COMPOUND II
The percentage inhibition of isolated compound and standard drug are more are less similar at 500g/ml
(56%,56%and 75% respectively). In this assay, these results confirmed that, when the bio active compounds of
ethanolic extract has high inhibition at low concentration compared with standard. . This suggests that the plant
extract contain compounds that are capable of donating hydrogen to a free radical in order to remove odd electron
which is responsible for radicals reactivity 7.
415
Fig.3 DPPH radical scavenging activity of ethanolic extract of Hybanthus enneaspermus .L
Hydroxyl radical scavenging activity
The hydroxyl radical can easily cross cell membranes at specific sites and , react with most biomolecules and
furthermore cause tissue damage and cell death. Thus, removing hydroxyl radical is very important to the protect
of living systems 10
Fig.4 Hydroxyl radical scavenging activity

activity of ethanolic extract of Hybanthus enneaspermus .L
The percentage inhibition of ROS by compound I & II at 500g/ml was found to be extract similar effect when
compared with ascorbic acid 11 . In this assay, these results confirmed that, the of bio active compounds ethanolic
extract has high inhibition at low concentration compared with standard. The IC50 value of compound I,II &
standard drug is almost same where as compound I showed increased inhibition at 100 g/ml conc & gradually
reduced.
The hydroxyl radical in the cells can easily cross cell membranes at specific sites, react with most biomolecules
and furthermore cause tissue damage and cell death. Thus, removing hydroxyl radical is very important for the
protection of living systems (Yang et al., 2008).
Nitric oxide radical scavenging activity
Nitric Oxide is an important bio regulatory molecule, which has a number of physiological effects including
control of blood pressure, neural signal transduction, platelet function, antimicrobial and antitumor activity 12
Fig.5 Nitric oxide radical scavenging activity of ethanolic extract of Hybanthus enneaspermus .L
416
The percentage inhibition of plant extract and standard drug are more are less similar at 500g/ml (54%,85%and
51% respectively, Figure 16). In this assay, these results confirmed that, the bio active compounds of ethanolic
extract has high inhibition at low concentration compared with standard.
The reducing power of the extract was investigated by the Fe3+ Fe2+ transformation in the presence of the
fractions as described by 13 The Fe2+ can be monitored by measuring the formation of Perls Prussian blue at 700
nm 14.
Fig.6 Reducing power assay of ethanolic extract of Hybanthus enneaspermus .L

These results were illustrated in Figure 19. At 500 g/ml, the absorbance of plant of the isolated bioactive
compounds and ascorbic acid were 0.54 , 0.60 and 0.51 respectively. These results indicated that the plant extract
has high activity than ascorbic acid.
Conclusion
From the results of free radical scavenging assays, isolated bioactive compounds of the ethanolic extract
of Hybanthus Enneaspermus L. showed very good antioxidant activity compared to the standard drug ascorbic
acid. In DPPH, reducing power, FRAP assay, nitric oxide scavanging activity and hydroxyl scavanging activity
and has good activity obtained in compound II while compare to the compound I. Tetrahydro-2,3,5-trihydroxy-
6-(hydroxymethyl)-2H-pyran-4-yl stearate can act as a good antioxidant agent in future that can be tested for its
efficacy in ROS related diseases.
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418
Anticancer Effect of Partially Purified Phenolic fraction in MCF-7
Breast Cancer Cell Lines
K. Pavithra1*, S.Vadivukkarasi1
1
Department of Biochemistry, Centre for Biological Science,
K.S. Rangasamy College of Arts and Science, Thokkavadi, Tiruchengode, Tamil Nadu, India.
Abstract
Kedrostis foetidissima (Jacq.) Cogn., a traditional herb, belongs to Cucurbitaceae family. The indigenous tribal
peoples in Tamilnadu used the boiled form of leaves of K.foetidissima as edible food source. Various crude
extracts of K.foetidissima were found to have anti microbial, antioxidant and cytotoxic properties. To validate
the ethnopharmacological claims against cancer, the antiproliferative effects of partially purified phenolic
fraction of K.foetidissima, was investigated in Breast cancer Cell lines (MCF). The partially purified phenolic
fraction (ppPFKF) was highly cytotoxic to MCF cell lines and the cytotoxicity values were significantly
similar to that of standard Doxorubicin. The study also revealed that ppPFKF at 10 and 25g/ml concentrations
induced G2/M phase arrest accompanied by a reduction in the G0/G1 phase of the cell cycle while at 50g/ml
concentration ppPFKF induced G0/G1 arrest with a concomitant decrease in the G2/M phase of the cell cycle,
hence indicating that ppPFKF targeted different stages of the cell cycle suggesting different targets of action
in increasing concentrations. These results support the traditional use of K.foetidissima as a potent source of
anticancer compounds that could be utilized pharmaceutically.
Keywords: Anticancer, K.foetidissima, phenols, HPTLC, cytotoxicity
Introduction
Plants are rich in secondary metabolites, which are produced as a byproduct of primary metabolism1. Secondary
metabolites include alkaloids, flavonoids, terpenoids, phenols, saponins, tannins, glycosides, oils and waxes etc2.,
These bioactive compounds play an important role in the prevention of many diseases like cancer, inflammation
and brain dysfunction3. Phenolic compounds are known as powerful chain breaking antioxidants, which may
contribute directly to antioxidative action. These compounds are very important constituents of plants and their
radical scavenging ability is due to their hydroxyl groups4. In recent years, many plant derived substances which are
classified as polyphenols are increasingly known for their various biological effects5.
Cancer, as a chronic disease, represents the largest cause of mortality in the world and over 6 million lives
are claimed each year6. Many factors are involved in these abnormal cellular changes and may influence progress
of the cell cycle and cell development7. Breast cancer is becoming the second leading cause of cancer deaths in
India, almost 10% of women between 35-54 years, have the risk of developing breast cancer8. Although great
improvements have been made in curing breast cancer, the overall five-year survival rate remains less than 50% and
many patients relapse after surgical resection because of the dispersion of undetectable cancer cells9. Therefore, it is
necessary to establish sensitive and specific techniques and therapies for the early detection and treatment of tumor
cells10.
Kedrostis foetidissima (Jacq.) Cogn., is generally called as Appakovai in Tamil is a medicinal climber
which belongs to the family of cucurbitaceae. Traditionally K. foetidissima was found to be very effective in the
treatment of asthma, chest pain, urinary tract infection11. diarrhoea, small pox, skin diseases12, snake bite13, measles
and livestock problems 14. The aim of the present study is to isolate phenolic compounds and to determine its
cytotoxicity against breast cancer cell lines.
419
Isolation of phenolic c.ompounds from K. foetidissima leaves
30g of K. foetidissima leaf powder was extracted with 200 ml of methanol at room temperature for 24 hrs and
filtered using Whatman No.1 filter paper to remove extractable substances, at every 3 hrs interval. The combined
extracts were then evaporated at 40C to dryness. The dried extracts were defatted with petroleum ether thrice.
Then the defatted extract was dissolved in water and extracted with ethyl acetate thrice. The combined ethyl acetate
fraction was concentrated and evaporated to dryness on water bath. The crude ethyl acetate residue was then
subjected to partial purification of phenolic compounds using column chromatography.
Partial purification of phenolic compounds from crude ethyl acetate residue of K.foetidissima leaf extract
using column chromatography
10g of silica gel for column chromatography [laboratory grade] was mixed with methanol and activated in hot
air oven at 110C for 1-2 hrs. The activated silica gel was packed into the column with the small quantity of solvent
at the top of the column (about 4 cm) to avoid drying. 0.2g of crude ethyl acetate residue was dissolved in 2 ml of
methanol and chromatographed on silica gel column and eluted with ethyl acetate: methanol (1:1) solvent system.
Fractions were collected until the eluted fractions showed negative for phenolic compounds. All the fractions eluted
were evaluated for the presence of phenolic compounds with 2% ferric chloride and lead acetate solutions. Positive
fractions were pooled into major fractions and evaporated to dryness. This partially purified phenolic fraction
(ppPFKF) was then subjected to HPTLC analysis for the confirmation of the compound.
HPTLC analysis of ppPFKF leaf extract for phenolic profile
1mg of dried ppPFKF of leaf extract was dissolved in 1 ml of methanol and centrifuged at 3000rpm for 5
min. This solution was used as test solution for HPTLC analysis. 2 l of test solution and 2 l of standard solution
were loaded as 5mm band length in the 3 x 10 Silica gel 60F254 TLC plate using Hamilton syringe and CAMAG
LINOMAT 5 instrument.
The plates loaded with sample were kept in TLC twin trough developing chamber. The developed plate was
dried by hot air to evaporate solvents from the plate. The plate was kept in photo-documentation chamber (CAMAG
REPROSTAR 3) and captured the images at white light, UV 254nm and UV366nm.
The developed plate was sprayed with Folin Ciocalteaus spray reagent and dried at 100C in Hot air oven. The
plate was photo-documented in day light and UV 366nm mode using photo-documentation (CAMAG REPROSTAR
3) chamber. Before derivatization, the plate was fixed in scanner stage (CAMAG TLC SCANNER 3) and scanning
was done at UV 254nm. The peak table, peak display and peak densitogram were noted using win CATS 1.3.4
version software.
Blue, brown and brownish blue colored zone at daylight and UV mode were observed from the chromatogram
in both before and after derivatization process, for the confirmation of the presence of phenolic compounds in the
standard and sample.
Cytotoxic screening of ppPFKF of leaf extract using MCF-7 cancer cell lines
Cell Culture
MCF-7 (Michigan Cancer Foundation - 7) cells lines were obtained from the King Institute of Preventive
Medicine, Chennai. The cells were cultured in Minimal Essential Medium (MEM) (pH: 7.2-7.4) supplemented with
10% heat inactivated foetal calf serum (FCS), 3% L- glutamine, 100U/ml penicillin G and 100 g/ml streptomycin.
The cells were grown as monolayer in tissue culture flasks (BD Falcon) in humidified atmosphere under the
conditions of 37C/5% of CO2 in incubator.
In vitro cytotoxic activity by MTT Assay 15
MTT assay was widely used method for testing the cytotoxicity of the compound and plant extracts. MTT
is cleaved by mitochondria dehydrogenase in viable cells, yielding a measurable purple product formazan. This
420
formazan production is proportionate to the viable cells and inversely proportional to the degree of cytotoxicity. 24
well tissue culture plates were used to culture cells for MTT assay. 1 ml of cell culture was seeded into 24 well plates
and kept in desiccators in 5% CO2 atmosphere. Then the 48 hrs monolayer cultures of MCF-7 cells at a concentration
of one lakh /ml /well were seeded in 24 well titer plates. The plates were microscopically examined for confluent
monolayer, turbidity and toxicity. The growth medium was removed and the monolayer of cells was washed twice
with MEM without FCS to remove the dead cells and excess FCS. To the washed cell sheet, added 1 ml of medium
(without FCS) with varying concentration of doxorubicin and sample (ppPFKF of leaf extract) in respective wells.
Each dilution of the standard and sample ranges from 1:1 to 1:512 with the concentration of 1000, 500, 400, 300,
200, 100, 50, 25, 10, 5g/ml respectively. The cell control wells contained 1 ml of MEM without FCS. The plates
were incubated at 37C in 5% CO2 incubator for 48 hrs and observed for cytotoxicity using inverted microscope.
After incubation, remove the medium from the wells for MTT assay. The wells were washed with MEM without
FCS and then added 200 l of MTT (5mg/ml) and incubated for 6-7 hrs in 5% CO2 incubator. After incubation 1 ml
of DMSO was added in each well and mixed gently and left it for 45 seconds to dissolve the violet formazan crystals
formed within metabolically viable cells. This formazan production is directly proportional to the viable cells and
inversely proportional to the degree of cytotoxicity. Then the suspension was read spectrophotometrically at 595nm,
percentage of cell viability was calculated using the formula.
% cell viability = (OD of sample/OD of cell control)*100
Non-treated cells were used as negative control and IC value was calculated as the concentration of fractions
50
and compounds causing 50% inhibition of cell viability. Graph was plotted using the % of cell viability at Y-axis
and concentration of the sample in X-axis. Cell control and sample control was included in each assay to compare
the full cell viability in cytotoxicity and antitumor activity assessments.
Flow cytometric analysis
Cellular DNA content was determined by flow cytometric analysis of propidium iodide (PI) labeled cells16.
MCF-7 cells were grown to exponential phase, seeded at a density of 1X106 cells/6 well dish and treated with
the indicated concentrations (IC50) of ppPFKF (10, 25 and 50 g/ml) for 48 h. After treatment, the cells were
collected by trypsinization and fixed in 70% ice-cold ethanol at -20C overnight. The cells were re-suspended in
PBS containing 1% Triton X-100, 0.5 mg/ml of RNase and 4 mg/ml of propidium iodide at 37C for 30 mins in dark
room. A FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA) was used for flow cytometric analysis.
The DNA content of 10,000 cells per analysis was monitored using the FACS Calibur system. The cell cycle was
determined and analyzed using cell quest software.
Results
Isolation and partial purification of phenolic compounds
Fractionation of crude ethyl acetate residue of K.foetidissima leaf extract on silica gel column chromatography
using ethyl acetate and methanol as solvents yielded 0.05g of partially purified phenolic fraction of K.foetidissima
(ppPFKF) leaves.
HPTLC analysis of ppPFKF leaf extract
The Peak table of HPTLC analysis of ppPFKF (Sample A) leaf extract was illustrated in Table 1. The
ppPFKF was run along with the standard phenolic compound (Quercetin) and viewed under UV and Daylight
conditions before and after derivatization. The ppPFKF of the plant extract showed different spots in before and
after derivatization chromatograph both in Daylight and UV, which confirmed the presence of phenolic compounds
in the sample fraction (Figure 1 and 2).
The Rf value of different phenolic compounds in the extract was found to be 0.01, 0.07, 0.10, 0.21, 0.28,
0.39, 0.45, 0.66, 0.74, 0.80, 0.90, 0.97 with corresponding peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 respectively. The
baseline display, peak densitogram and 3D display of ppPFKF and Quercetin was represented in Figure 3- 6.
421
Cytotoxic screening of ppPFKF in MCF-7 breast cancer cell lines
The cytotoxic screening of ppPFKF on the viability of MCF-7 breast cancer cell lines was presented in Figure
7. Cells were treated with various concentrations (5 to 1000g/ml) of ppPFKF and standard (Doxorubicin) for 48
hrs and percentage cell viability was determined by MTT assay.
A dose dependent growth inhibition of MCF-7 cancer cells were observed at concentrations ranging from
5-1000 g/ml. The IC50 value was found to be 22g/ml for ppPFKF and 15g/ml for Doxorubicin. Signicant
differences (P<0.05) were found between cells treated with ppPFKF and untreated control. As the concentration of
the ppPFKF increases the viability of cancer cells decreases which was greater than Doxorubicin with respect to
percentage inhibition at 1000 g/ml (ppPFKF-2.52 % and Doxorubicin - 12.35%).
Flow cytometric analysis of ppPFKF
Different concentration treatment of ppPFKF exhibited strong inhibitory effect on MCF-7 cell growth, so
further experiments were carried out to ascertain whether ppPFKF could interfere with the cell cycle arrest. The
progression of cell cycle was followed for 48 hrs and the percentages of the cells in different phases were analyzed
by flow cytometry after staining with propidium iodide (Figure 8).
Analysis of cell cycle after treatment of MCF-7 cells with differing concentration of ppPFKF revealed that
the proportion of cells in G2/M phase was markedly increased and G0/G1 phase of the cells decreased as the
concentration of ppPFKF decreases from 50g/ml to 10g/ml. ppPFKF had a concentration dependent effect on
the percentage of G0/G1 and G2/M phase of cells, indicating that the ppPFKF exhibited the cellular proliferation
of MCF-7 cells via G2/M phase arrest at 25 and 10g/ml concentrations and G0/G1 phase arrest at 50 g/ml
concentration.
Discussion
Phytoconstituents play a potential role in interfering with the oxidation process by reacting with the free
radicals generated in biological systems. Free radicals play an important role in carcinogenesis which comes from in
vitro studies describing their role in DNA damage, protein structural and functional modifications. In present study
column chromatography is used to isolate the phenolic compounds from the leaves of K.foetidissima. HPTLC
analysis of the partially purified phenolic fraction of K.foetidissima leaves confirmed the presence of 12 phenolic
compounds.
Flavonoids and phenolic acids make up one of the most pervasive groups of plant phenolics 17. Phenolics
and flavonoids possess diverse biological activities like antiulcer, anti-inflammatory, antioxidant, cytotoxic and
antitumor, antispasmodic and antidepressant activities. It was also reported that plant phenolic compounds increases
bile secretion, reduces blood cholesterol and lipid levels and exhibit antimicrobial activity18.
K.foetidissima leaves found to contain high number of phenolic compounds when compared to aerial parts of
M.heterophylla (3 phenolic compounds with the Rf value of 0.48, 0.68, 0.77)19 and Mukia maderaspatana leaves
(3 phenolic compounds with Rf values 0.02, 0.20 and 0.87)20 Thus the presence of phenolic compounds in the leaf
extract of K.foetidissima attributes to the traditional use of plant leaves in the treatment of common cold, measles,
asthma, urinary tract infection and skin diseases.
Cytotoxic activity is estimated as the capacity of the test compound to selectively inhibit the growth of certain
cell lines. Cytotoxicity screening models provide important preliminary data to help selecting plant extracts with
potential antineoplastic properties for future work21. MTT assay is a well-established in vitro method for cytotoxicity
screening against cancer cell lines to determine the selective activity of the extracts22.
Several plant species rich in phenolic compounds are reported to have disease preventive and therapeutic
properties23. This observation is of particular importance since phenols and flavonoids are ingredients of many
vegetables and fruits and the association of vegetable and fruit consumption with reduced cancer risk has been
reported24.
422
The results of the present study indicate that ppPFKF at 1000g/ml shows significant cytotoxic activity than
the standard drug and IC50 value of ppPFKF was very closer to doxorubicin. Phenolic fraction of K.foetidissima leaf
extract found to have greater cytotoxic activity than crude leaf extract of Solanum anguivi (IC50 value 1.25 mg/ml)
25
and root extract of Bryonia aspera (IC50 value 4.65)26.
Cell proliferation and death are involved in the maintenance of homeostasis in normal cells, however,
homeostasis is often disrupted in tumor cells with uncontrolled proliferation. Anti-tumor effects could be attributed
to altered biochemical mechanisms, including inhibitions of proliferation, induction of cell cycle arrest at various
cell cycle checkpoints, enhanced apoptosis and regulation of signal transduction pathways, which are related to
altered expressions of key enzymes27.
Cell growth is controlled by several genetically defined checkpoints which ensure the coordinated progression
of cells through the different stages of the cell cycle and monitoring the integrity of the DNA28. Cell cycle is guarded
at three main checkpoints at G1/S, G2/M and metaphase/anaphase. Progress through the cell cycle can be halted at
any of these checkpoints if the condition for successful cell division was not met29. Cell cycle checkpoint control
mechanisms can be disrupted in cancers through accumulation of mutations 30, which can be measured by flow
cytometric analysis.
Many anticancer agents arrest the cell cycle at G0/G1 and G2/M phase and then induce apoptotic cell death.
The G0/G1 phase cell cycle arrest inves regulation of proteins such as p53 or the cyclin-dependant kinases31. The
G2/M phase cell cycle arrest inves interactions targeting tubulin or disrupting the tubulin-microtubule equilibrium32.
Dietary polyphenols have been correlated with a reduced risk of developing cancer. Quercetin, a natural polyphenolic
compound induced apoptosis in breast cancer MCF-7 cells by regulating cell cycle. It was reported that quercetin
caused a remarkable increase in the number of S phase and sub-G1 phase cells in a dose dependent manner33.
Bouallagui et al., in 201134 reported that hydroxytyrosol (polyphenolic compound) rich olive leaves extract
exhibited potential anti-tumor activity in a dose dependent growth inhibition of MCF-7 cells and the inhibition of
cells was due to the cell cycle arrest in the G0/G1 phase. Ray et al., in 2010 35 revelaed that Momordica charantia
extract inhibits breast cancer cell (MCF-7 cells) proliferation by modulating cell cycle regulatory genes and
promotes apoptosis. Fluorescence-activated cell sorting analysis suggested that MCF-7 cells treated with Momordica
charantia extracts accumulated cells during the G2/M phase of the cell cycle.
Partially purified phenolic fraction of K.foetidissima leaves demonstrated anticancer activity by reducing cell
viability and cell cycle regulation. These findings suggest that K.foetidissima leaves possess anticancer properties
and may be suitable for the application in the pharmaceutical industry. The results of the study clearly indicates that
K.foetidissima provides a source of biologically active phytochemicals and it was found to have potentially active
antioxidant and cytotoxic properties, which can able to protect the cells from free radicals induced oxidative damage
in the biological system. The anticancer activity of the K.foetidissima leaves might be due to the synergistic action
of the bioactive compounds present in them. Further studies have to be carried out to assess the in vivo biological
activities and to isolate the specific compounds responsible for the antioxidant and anticancer activity.
Acknowledgement
Authors are grateful to K. S. Rangasamy College of Arts And Science (Autonomous), Tiruchengode, for
providing facilities for the research work.
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Table 1 : Peak table of HPTLC analysis of ppPFKF leaf extract
Track Peak Rf Height Area Assigned substance
Sample A 1 0.01 82.0 772.3 Phenolic 1
QUE 1 0.71 486.1 10826.7 Quercetin standard
Figure 1. HPTLC profile of ppPFKF
Chromatogram - Before derivatization
Daylight UV 366nm UV 254nm
425
Figure 2. HPTLC profile of ppPFKF Chromatogram - After derivatization
Figure 3. Baseline display (Scanned at 254nm) of Figure 4. Peak densitogram display (Scanned at
ppPFKF 254nm) of ppPFKF
Figure 5. Baseline display (Scanned at 254nm) of Figure 6. Peak densitogram display (Scanned at
(Quercetin) standard 254nm) of (Quercetin) standard
Figure 7: Cytotoxic screening of ppPFKF using MCF-7 cancer cell lines

Values expressed in mean SD of n = 3 samples, P<0.05 by one way ANOVA and student t test using Graph
Pad prism 5 software
Figure 8. Cell phase distribution of MCF-7 cancer cells Control (untreated with ppPFKF) and cells treated
with 50, 25 and 10 g/ml of ppPFKF
426
427
Attenuation of Hyperglycaemia through Asiatic acid in High Fat Diet
induced Obese Rats
K. Swapna1, V. V. Sathibabu Uddandrao1, P. Brahmanaidu2 and Ganapathy Saravanan1*
Centre for Biological Sciences, Department of Biochemistry, K. S. Rangasamy College of Arts and Science
1
(Autonomous), Tiruchengode, Tamilnadu, India - 637215.
Abstract
High fat diet and eventual obesity predispose an individual to the development of a number of morbidities
such as heart disease, hepatic disease, hypertension, diabetes mellitus, and dyslipidemia. The current study
was aimed to find out the anti hyperglycaemic potentiality of Asiatic acid in high fat diet induced obese rats.
Obesity was induced by using high fat diet and the body weight of each rats were measured. Hyperglycaemic
markers namely blood glucose, oral glucose tolerance test, insulin, haemoglobin, glycated haemoglobin,
hexokinase, glucose-6-phosphatase, fructose-1, 6-bisphosphatase, glycogen synthase, phosphorylase, and
liver glycogen were assayed by the commercially available kits. Oral administration of Asiatic acid (20 mg/
kg body weight) significantly reduced the levels of glucose and glycated hemoglobin and tissue glucose-
6-phosphatase, fructose1, 6- bis phosphatase activity and glycogen phosphorylase in high fat diet induced
hyperglycaemic rats. The decreased hemoglobin, hepatic glycogen and the activity of hexokinase and
glycogen synthase in the liver of hyperglycaemic state was found to be significantly increased by Asiatic acid
treatments. In conclusion the decreased level of blood glucose accompanied with the changes in the activities
of the glucose metabolizing enzymes showed the anti hyperglycaemic effect of Asiatic acid.
Key Words: Hyperglyceamia; Asiatic acid, High fat diet; Natural medicine
Introduction
Obesity amplifies the risk of development of various sicknesses including Type 2 diabetes, hypertension,
hyperlipidemia and cardiovascular illnesses1. It is portrayed by an anomalous or unnecessary fat aggregation
attributable to irregularity between energy intake and energy expenditure2. The etiology of obesity is dissimilar one.
On the other hand, the root cause is energy discrepancy: more calories consumed than expended. Modal of HFD-
induced obesity in rats has many features common with human obesity. Hyperlipidemia, hyperglycemia, insulin
resistance, impaired glucose metabolism, distinctive visceral adiposity hyperinsulinemia, and decreased bone
mineral concentration and bone mineral density are the common characteristics of diet-induced obesity in rodents3.
Diabetes mellitus is a multiple etiology metabolic disorder with abnormalities in the metabolism of
carbohydrate, fat and protein, being characterized by chronic hyperglycemia and defects in insulin secretion, insulin
action, or both4. Epidemiological studies have recommended the utilization of phytoconstituents that could reduce
the risk of diabetes, cardiovascular diseases and obesity5, suggesting that the consumption of Asiatic acid may be
of benefit in metabolic diseases. Asiatic acid (AA), a triterpenes, was found abundantly in Centella asiatica (L.)
a perennial herbaceous creeper of the Apiaceae family6. It possesses an extensive array of biological functions
including antioxidant, hepatoprotective and anti-inflammatory activities7. Furthermore, there is no information
pertaining to the anti hyperglycaemic capability of AA in HFD-fed obese rats. Therefore, the purpose of the present
study is to appraise the antidiabetic action of asiatic acid in high fat diet fed rat model.
Chemicals
Asiatic acid (AA) and Orlistat (04139 Sigma) were purchased from Sigma-Aldrich (Bangalore, India). Fasting
blood glucose kits and insulin kits were procured from Stanbio Laboratory USA, Bio-Merieux, RCS, Lyon, France.
All added reagents acclimated in the experiments were of analytic brand and of the highest purity.
428
Animals
Male Sprague-Dawley rats were obtained from Department of Biochemistry, Muthyammal College of Arts
and Science, Rasipuram, Namakkal District, Tamilnadu, India. Experimental animals were kept up under standard
laboratory conditions (temperature; 222C: moistness; 40-60%), and permitted food and water ad libitum. Rats,
at first, weighing 180-200g were separated into four groups of six each (n= 6). All procedures involving laboratory
animals were in accordance with the institutional animal ethical committee of Muthyammal College of Arts and
Science, Rasipuram, Namakkal District, Tamilnadu, India.
High Fat Diet formula
High fat diet was obtained from National centre for laboratory animal sciences, National institute of nutrition
(NIN-ICMR), Hyderabad. High fat diet was composed by Corn starch-15%, Sugar-27.5%, Lard oil-17.6%, Vitamin
mixture-1%, Mineral mixture-3.5%, Casein-20%, Cellulose powder-5%, Corn oil-9.9%, Choline bitartrate- 0.2%.
Experimental design
Group 1: Normal Diet Control (Normal Diet)
Group 2: High fat Diet group (HFD)
Group 3: HFD + AA (20 mg/kg body weight)
Group 4: HFD + Orlistat (10 mg/kg body weight)

Measurement of body weight
The body weight and the total amount of food consumption (Data are not shown) of each rat were measured.
Toward the end of the experiment, blood was collected from overnight fasted animals under inhalation of anaesthesia
by retro-orbital puncture strategy. Blood was collected in anticoagulant coated vials and permitted for 15 minutes at
room temperature. Plasma was separated by centrifugation at 2500 rpm for 15 minutes.
Blood glucose and insulin:
Plasma glucose was estimated using kits (Cat No 1060-500, Stanbio laboratory, USA). A plasma level of
insulin was determined using kits from Bio-Merieux, RCS, Lyon, France.
Oral glucose tolerance test (OGTT):
OGTT was performed toward the end of the experiment; after overnight fasting, glucose was administered
orogastrically at a dose of 2.0 g/kg body weight and blood samples were collected at 0, 30, 60 and 120 minutes and
glucose level was evaluated.
Hemoglobin and Glycosylated hemoglobin were estimated by using commercially available kits. Liver was
immediately dissected out, washed in ice-cold saline to remove the blood and homogenized in 0.1 M Tris - HCL
buffer, pH 7.4. The supernatant was used for the assay of enzymes activity. Hexokinase, glucose-6-phosphatase and
fructose-1, 6-bisphosphatase, Glycogen synthase, phosphorylase activities, and Liver glycogen were assayed by the
commercially available kits.
All the grouped data were statistically evaluated with SPSS/10.0 software. Hypothesis testing methods
included one way analysis of variance (ANOVA) followed by least significant difference (LSD) test; p value of less
than 0.05 were considered to indicate statistical significance. All the results were expressed as the mean S.D. for
six animals in each group.
Results
Table 1 shows the changes in body weight in control and HFD group of animals during the experiment. Intake
of HFD for 42 days elicited a significant (p < 0.05) increase in body weight, compared to the normal control group.
429
Oral administration with AA (20mg kg-1 b.w-1) or Orlistat significantly (P<0.05) reduced the increase in body weight
when compared to the HFD control group. Table 1 also depicts the level of plasma glucose, plasma insulin and
insulin resistance in control and experimental obese rats. There was a significant (p<0.05) rise in plasma glucose,
plasma insulin and insulin resistance in rats administered with HFD. Oral supplementation of AA (20mg kg-1 b.w-1)
or Orlistat tended to bring blood glucose and plasma insulin towards near normal levels.
Table 1: Effect of AA on body weight, blood glucose and insulin in control and experimental rats
Body weight (g) 19010.89 321.3415.21 23112.32 212.2213.67
Glucose (mg/dL) 1102.89 2986.89 1433.89 1254.89
Insulin (g/dL) 4.971.09 0.840.06 3.210.67 3.990.95
Figure 1 shows the level of oral glucose tolerance in the control and experimental obese animals. In the control
rats, there was a significant raising of blood glucose level to a maximum value at 30 min after glucose load and
declined to near basal levels at 120 min, whereas, in HFD-induced obese rats, the peak increase in blood glucose
level was observed even after 60 min and remained high over the next 90 min. Oral supplementation with AA (20mg
kg-1 b.w-1) or Orlistat to obese rats resulted a significant reduction in blood glucose level at 90 min when compared
with HFD control rats.
Fig 1: Effect of AA on the level of oral glucose tolerance in the control and experimental obese animals,
Figure 2 summarized the level of hemoglobin and glycated hemoglobin level in control and experimental
animals. A significant reduction (p<0.05) in hemoglobin level and concomitant increase in glycated hemoglobin
level was observed in HFD-fed rats and it was normalized after treatment of AA and Orlistat.
Fig 2: Effect of AA on level of hemoglobin and glycated hemoglobin level in control and experimental
animals, Values are mean SD, n = 6, Values are statistically significant at p<0.05
430
Table 2 explained the level of Hexokinase, glucose-6-phosphatase and fructose-6- phosphatase level in
control and experimental animals. A noteworthy reduction in hepatic hexokinase level and concomitant increase
in glucose-6-phosphatase and fructose-6- phosphatase level was observed in HFD-fed rats and it was normalized
after treatment of AA and Orlistat. Table 2 also showed the hepatic glycogen content, and in the activity of glycogen
synthase and glycogen phosphorylase in control and experimental group of rats. There was a significant reduction
in hepatic glycogen content and activity of glycogen synthase and concomitant increase in the activity of glycogen
phosphorylase in HFD-fed rats when compared with control rats. Oral treatment of AA and Orlistat tended to bring
glycogen content, glycogen synthase and glycogen phosphorylase towards near normal levels.
Table 2: Effect of AA on the Glucose metabolism markers in control and experimental rats
Hexokinase (U/L) 30012.09 179.909.67 25610.12 2789.00
Glucose-6-phosphatase (U/L) 90013.89 18008.97 120612.90 10006.90
Fructose-6- phosphatase (U/L) 4007.98 70010.90 5637.90 501.897.99
Glycogen content (mg/g wet tissue) 1014.26 521.99 824.98 914.21
Glycogen synthase (U/L) 7009.90 1702.89 6318.90 6799.90
Glycogen phosphorylase (U/L) 5607.42 90010.21 7009.89 62110.09

Discussion
such as heart disease, hepatic disease, hypertension, diabetes mellitus, and dyslipidemia8. Diet-induced obesity in
animals has many features common with human obesity and diabetes is widely used to find medicinal foods for
reducing obesity1. In the present study, high-fat diet treatment to experimental animals confirmed the nature of
obesity as evidenced by increasing body weight, hyperglycemic condition and increased insulin resistance. This is
in line with previous reports9. It might be due to the consumption of a diet rich in energy in the form of saturated
fats and its deposition in various body fat pads10 leads to excessive growth of adipose tissue resulting in obesity
which includes two growth mechanisms: hyperplastic and hypertrophic of adipose tissue. In the present study,
enhanced body weight and fat was observed in HFD fed rats. Long-standing HFD fed to rats resulted with increased
bodyweight and plasma comorbidity factors11 and it might be due to the consumption of a diet rich in energy in
the form of saturated fats (lard) and its accretion in different body fat pads10 leads to unwarranted development of
adipose tissue and also the ratio of calorie ingestion to energy utilization determines bodyweight.
Obesity is accountable for the escalating occurrence of metabolic disease. In the body, adipose tissue is a key
site of energy storage and is crucial for energy homeostasis. It is well known that insulin can ultimately instigate leptin
secretion during the metabolism of nutrients, particularly on glucose exploitation in adipocytes. Hyperlipidemia,
hyperglycemia, insulin resistance, impaired glucose metabolism, distinctive visceral adiposity hyperinsulinemia,
and decreased bone mineral concentration and bone mineral density are the common characteristics of diet-induced
obesity in rodents. In the present study, high-fat diet treatment to experimental animals confirmed the nature of
obesity as evidenced by increasing body weight, hyperglycemic condition and increased insulin resistance12. Under
physiological conditions, insulin enhances lipogenesis and inhibits lipolysis leading to increased circulating levels
of glucose and lipids resulting in impaired glucose-stimulated insulin secretion13. In the present study, we observed
that AA treatment lowered plasma insulin level, suggesting AA could improve insulin resistance in the experimental
animals. AA promotes insulin sensitivity, thus lowering insulin resistance, decreasing glucose level in obese rats,
perhaps by controling the cell energy metabolism or diminishing free fatty acids. Further, it was reported that
administration of AA significantly improved glucose homeostasis in metabolic syndrome like diabetes14.
431
Hyperglycemia is the clinical attribute of improperly controlled diabetes, which is known to cause protein
glycation, also well-known as non - enzymatic glycosylation15. During diabetes, the overindulgence of glucose
presence in the blood reacts with hemoglobin to form glycosylated hemoglobin. The rate of glycation is relative
to the concentration of blood glucose. HbA1C consists of 3.4% to 5.8% of total hemoglobin in normal human red
blood cells but it is increased in patients with unconcealed hyperglycaemia. It was found to increase in diabetic
patients up to 16%, and the level of HbA1C is monitored as a reliable index of glycemic control in diabetes. In our
study, the obese rats have showed higher levels of glycosylated hemoglobin compared to control rats indicating
their poor glycaemic control16. HFD-fed rats treated with AA and Orlistat showed a noteworthy diminish in the
glycosylated Hb levels that might be due to the anti hyperglycemic effect of AA.
Hexokinase, one of the key enzymes of glucose metabolism is distinctly altered to produce hyperglycemia,
which lead to the pathogenesis of diabetic complications. Hexokinase, the first regulatory enzyme of glycolytic
pathway, is an insulin-dependent and insulin-sensitive enzyme and are almost entirely introverted or inactivated
in hyperglycaemic rat liver in the deficiency of insulin17. In our study, we also have observed a decrease in hepatic
hexokinase activity in HFD-fed hyperglycaemic rats. Administration of AA and Orlistat to HFD-fed rats resulted in
an increased activity of hexokinase in liver.
The hepatic gluconeogenic enzymes, glucose-6-phosphatase and fructose-1, 6-bisphosphatase were increased
in HFD-fed hyperglycaemic rats. Increased glucose-6-phosphatase activity in diabetic rats provides hydrogen,
which binds with NADP+ in the form of NADPH and enhances the synthesis of fats from carbohydrates (i.e.
lipogenesis) and at last, contributes to improved level of glucose in blood. Increased hepatic glucose production
in diabetes mellitus is associated with impaired restraint of fructose 1-6 bis phosphatase1. In the present study, the
activities of the glucose-6-phosphatase and fructose-1, 6-bisphosphatase were found to be increased significantly in
HFD-fed hyperglycaemic rats. The diminishment in the activities of these gluconeogenic enzymes can bring about
diminished concentration of blood glucose by the supplementation of AA and Orlistat. Hyperglycaemia is linked
with evident reduction in the level of liver glycogen18. The reduced glycogen has been embraced to the reduction in
the action of glycogen synthase and increased activity of glycogen phosphorylase. In HFD-fed hyperglycaemic rats,
the condensed activity of glycogen synthase and increased activity of glycogen phosphorylase were found. This may
be the outcome of low level of circulating insulin, which is an inducer of glycogen synthase19. Administration of AA
and Orlistat to HFD-fed hyperglycaemic rats restored back the activity of enzymes to normal on 45 days treatment.
In conclusion, these results suggested that dietary supplementation of Asiatic acid extracted from Centella
asiatica could inhibit the increment in body weight and blood glucose levels in high-fat diet induced hyperglycaemic
rats through switching glucose metabolism enzymes. This strongly suggests that Asiatic acid has therapeutic
potential for the management of hyperglycaemia.
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433
Anticancer Activity of the isolated compound from Alpinia purpurata
using PC-3 cell lines
K. Swapna1, V. V. Sathibabu Uddandrao1, P. Brahmanaidu2 and Ganapathy Saravanan1*
Centre for Biological Sciences, Department of Biochemistry, K. S. Rangasamy College of Arts and Science
1
(Autonomous), Tiruchengode, Tamilnadu, India - 637215.
Abstract
such as heart disease, hepatic disease, hypertension, diabetes mellitus, and dyslipidemia. The current study
was aimed to find out the anti hyperglycaemic potentiality of Asiatic acid in high fat diet induced obese rats.
Obesity was induced by using high fat diet and the body weight of each rats were measured. Hyperglycaemic
markers namely blood glucose, oral glucose tolerance test, insulin, haemoglobin, glycated haemoglobin,
hexokinase, glucose-6-phosphatase, fructose-1, 6-bisphosphatase, glycogen synthase, phosphorylase, and
liver glycogen were assayed by the commercially available kits. Oral administration of Asiatic acid (20 mg/
kg body weight) significantly reduced the levels of glucose and glycated hemoglobin and tissue glucose-
6-phosphatase, fructose1, 6- bis phosphatase activity and glycogen phosphorylase in high fat diet induced
hyperglycaemic rats. The decreased hemoglobin, hepatic glycogen and the activity of hexokinase and
glycogen synthase in the liver of hyperglycaemic state was found to be significantly increased by Asiatic acid
treatments. In conclusion the decreased level of blood glucose accompanied with the changes in the activities
of the glucose metabolizing enzymes showed the anti hyperglycaemic effect of Asiatic acid.
Key Words: Hyperglyceamia; Asiatic acid, High fat diet; Natural medicine
Introduction
Plant phytochemicals have been reported to prevent a number of diseases, including cancer, cardiovascular
disease, infection and inflammation. Traditional medicine has provided important information regarding medicinal
plants. Studying these traditions has led to the discovery of important active substances; the best examples are
taxol, colchicine and cyclopamine extracted from the plants Taxus brevifolia1, Colchicum autumnale2 and Veratrum
californicum3, respectively. All three molecules have been characterized as potential antitumor drugs and have made
an important contribution to modern oncology and cancer therapy. To date, many cytotoxic agents from natural
products have been investigated for the discovery of novel anti-cancer drugs. Alpinia purpurata serve as potential
antioxidant and anticancer agents against ovarian cancer cell lines4.
Cell-based assays are used to detect if the test molecules have effects on cell proliferation or show cytotoxic
effects that eventually lead to cell death5.Tetrazolium reduction is a typical method for detecting viable cells. MTT
is the most reliable compound that can be used. MTT is a water soluble dye that is yellow in colour. It is taken up
by viable cells and reduced when mitochondrial succinate dehydrogenase enzymes act on them. As a result, a water
insoluble product called formazon is formed. It is purple in colour. Formazon has to be dissolved for calorimetric
measurement6.
The amount of formazon product is directly proportional to the number of living cells present during MTT
exposure7. Colorimetric assays are measured using spectrophotometer. MTT assay is no different. The range of
the light is kept between 540-720nm8.When there is a decrease in the cell number, it indicates the inhibition of
cell growth. The concentration of a drug or chemical that inhibits 50% of the growth when compared to that of the
growth of untreated control, it is called as Inhibitory Concentration, IC50.The advantage being that this assay is not
only used to measure cytotoxicity and cell proliferation but also to measure cell activation. The cell activation can
be measured independently of proliferation9, 10.
434
Cell growth inhibition assay (MTT assay)
Principle
The mitochondrial dehydrogenase catalyse the conversion of yellow coloured tetrazolium salt MTT (3- (4, 5
dimethyl thiazole 2yl)-2, 5-diphenyl tetrazolium bromide) into a purple coloured complex (formazon). The intensity
of the colour produced is directly propotional to mitochondrial activity and cell viability.
Procedure
PC3 (human prostatic carcinoma) cell line was initially procured from National Centre for Cell Sciences
(NCCS), Pune, India and maintained Dulbecos modified Eagles medium (Gibco, Invitrogen). The cell line was
cultured in 25 cm2 tissue culture flask with DMEM supplemented with 10% FBS,L-glutamine, sodium bicarbonate
and antibiotic solution containing: Penicillin (100U/ml), Streptomycin (100g/ml), and Amphotericin B (2.5g/ml).
Cultured cell lines were kept at 37C in a humidified 5% CO2 incubator (NBS Eppendorf, Germany). The viability
of cells were evaluated by direct observation of cells by Inverted phase contrast microscope and followed by MTT
assay method.
Cells seeding in 96 well plate
Two days old confluent monolayer of cells were trypsinized and the cells were suspended in 10% growth
medium, 100l cell suspension (5x104 cells/well) was seeded in 96 well tissue culture plate and incubated at 37C
in a humidified 5% CO2 incubator.
Preparation of compound stock
1 mg of Pentatriacontanoic acid was added to 1ml of DMEM and dissolved completely by cyclomixer. After
that the extract solution was filtered through 0.22 m Millipore syringe filter to ensure the sterility.
Antiproliferative effect Evaluation
After attaining sufficient growth of the cells, the growth medium was removed, freshly prepared samples in
5% DMEM were five times serially diluted by two fold dilution (100g, 50g, 25g, 12.5g, 6.25g in 100l of
5% MEM) and each concentration of 100l were added in triplicates to the respective wells and incubated at 37C
in a humidified 5% CO2 incubator.
Antiproliferative effect by Direct Microscopic observation
Entire plate was observed after 24 hours of incubation in an inverted phase contrast tissue culture microscope
(Olympus CKX41 with Optika Pro5 CCD camera) and microscopic observation were recorded as images.
Any detectable changes in the morphology of the cells, such as rounding or shrinking of cells, granulation and
vacuolization in the cytoplasm of the cells were considered as indicators of cytotoxicity.
Antiproliferative effect by MTT Method
Fifteen mg of MTT (Sigma, M-5655) was reconstituted in 3 ml PBS until completely dissolved and sterilized
by filter sterilization. After 24 hours of incubation period, the sample content in wells were removed and 30l of
reconstituted MTT solution was added to all test and cell control wells, the plate was gently shaken well, then
incubated at 37C in a humidified 5% CO2 incubator for 4 hours. After the incubation period, the supernatant was
removed and 100l of MTT Solubilization Solution DMSO was added and the wells were mixed gently by pipetting
up and down in order to solubilize the formazan crystals. The absorbance values were measured by using microplate
reader at a wavelength of 570 nm11.
The percentage of growth inhibition was calculated using the formula:
Mean OD Samples x 100

% of viability = _________________________
Mean OD of control group
435
Result and Discussion
Figure 1: Effect of Pentatriacontanoic acid Treatment on the Viability of Prostate Cancer (PC-3) Cells
after 48 h
Figure 2: Cells were treated with various concentrations of Pentatriacontanoic acid
Table 1 PC-3 Cells Percentage Viability at their Optical Density

Sample concentration (g/ml) Average OD (540nm) Percentage Viability
Control 0.7461 -
6.25 0.63405 84.98191
12.5 0.5758 77.17464
25 0.5509 73.83729
50 0.5073 67.99357
100 0.40401 54.14958
The Pentatriacontanoic acid isolated from Ethyl acetate leaf extract of Alpinia purpurata was found to have
no effect up to 100 g/ml against PC-3 prostate cancer cells, as it was evident from the IC50 value of the first
experiment in which cells were exposed to the test sample in the concentration range of 6.25 to 100 g/ml (6.25,
12.5, 25, 50 & 100 g/ml) (Figure 1). The Figure 2 shows the morphological changes of cells after 48 h treatment
with Pentatriacontanoic acid. The effect of Pentatriacontanoic acid in various concentrations was also depicted in
the figure 2. From the table 1.1 explains upto 100 g/ml the pentatriacontanoic acid was not appears in LD50.i.e. In
436
100 g/ml the cell viability reach 54.14 at 540 nm. The LD50 value obtained in the concentration of 111.17 g/ml,
calculated using ED50 PLUS V1.0 software.
Conclusion
The isolated compound Pentatriacontanoic acid from Ethyl acetate leaf extract of Alpinia purpurata has a good
cytotoxic effect on PC-3 Prostate cancer cell line and the LD50 value may obtained in the concentration of 111.17
g/ml.
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of Sciences,United States of America, 1980, pp.1561-65.
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Oatis JE, Brunsfeld P, Rushing JW, Moeller PD, Bearden DW, Gallien TN (2008) Isolation, purification, and full NMR
assignments of cyclopamine from Veratrum californicum Chem Cent journal 2,12.
Arul raj C, Ragavendran P, Sophia D, Rathi MA, Gopalakrishnan VK (2012) Evaluation of In vitro antioxidant and anticancer
activity of Alpinia purpurata. Chinese J Nat Med 10, 0263-0268.
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Sylvester, PW. Optimization of the tetrazolium dye (MTT) colorimetric assay for cellular growth and viability. Drug Design
and Discovery: Methods and Protocols (2011) Vol.1219, pp.157-168.
Morgan, David ML (1998) Tetrazolium (MTT) assay for cellular viability and activity. Polyamine protocols 179-184.
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Design and Discovery: Methods and Protocols vol.716, Chapt.8, pp.157-168, Humana press, United States.
Patel S, Gheewala N, Suthar A, Shah A (2009) In-vitro cytotoxicity activity of Solanum nigrum extract against Hela cell line and
Vero cell line International J pharmacy and pharm sci 1, 38-46.
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Figure Captions
Figure 1: Effect of Pentatriacontanoic acid Treatment on the Viability of Prostate Cancer (PC-3) Cells after
48 h
Figure 2: Cells were treated with various concentrations of Pentatriacontanoic acid
437
Native seaweeds: Role in mitigating the toxic effects of oxysterols
under in vitro conditions
S.P.Preetha* and H.Devaraj1
*Department of Veterinary Pharmacology & Toxicology, Madras Veterinary College, Chennai - 07
1
Unit of Biochemistry, University of Madras
Abstract
Introduction: 25 hydroxy cholesterol (25-OHC) is a toxic oxysterol involved in the pathogenesis of
cardiovascular diseases. Interestingly, certain coastal societies who consume seaweeds regularly have lower
incidence of this disease, attributed to the presence of sulphated polysaccharides in the seaweeds. Hence this
study was designed to explore the seaweed sulphated polysaccharides in ameliorating 25 OHC induced
toxicity under in vitro conditions.
Methodology: The HepG2 cells were incubated in petridish in the presence or absence of various concentrations
of (a) 25-OHC (b) sulphated polysaccharides from S.wightii (c) sulphated polysaccharides from F. vesiculosis
(d) 25-OHC + sulphated polysaccharides from S.wightii (e) 25-OHC + sulphated polysaccharides from F.
vesiculosis for 24 hours. After 24 hours, the cells were harvested and used for cytotoxicity assays.
Results: 25-OHC induced ROS accumulation, LDH leakage and NO expression in HepG2 cells. These
cytotoxic changes were mitigated with seaweed sulphated polysaccharides.
Conclusion: Seaweed polysaccharides exert a potential role in countering the detrimental changes associated
with oxysterol toxicity.
Introduction
Hyperlipidemia is a major risk factor for atherosclerosis (Fidle et al., 2017; Li et al., 2013; Madubunyi, 2012;
Libby, 2005). Oxysterols are oxygenated derivatives of cholesterol involved in the pathogenesis of several diseases
(Bellanti et al., 2017) including atherosclerosis (Zhang et al., 2014). 25 hydroxy cholesterol (25-OHC) is one of the
most toxic and atherogenic oxysterols (Zhou et al., 1993) detected in macrophage-derived foam cells from human
atherosclerotic tissue (Liu et al., 1997). There is increasing need to control the growing burden of atherosclerosis
and cardiovascular diseases (CVD) in our society, but interestingly in certain coastal populations who consume
seaweeds regularly, the incidence of CVD is considerably less. The relative longevity of the Okinawan Japanese
populations with lower risk of developing CVD has been accounted to some extent to their habit of regular intake
of seaweeds (Yamori et al., 2001).
The beneficial properties of seaweeds have been attributed in part to the presence of sulphated polysaccharides
(SPS) in it. The SPS from the seaweeds are phytochemical analogues of mammalian heparin sulphate and exert
functions more than just the anti-coagulant effect. There are several edible seaweeds with valuable properties on
the Indian coasts. With this informative background, the present study was designed to explore the cytoprotective
role of SPS from native seaweeds Sargassum wightii in ameliorating 25 OHC induced toxicity under in vitro
conditions and compare its efficacy with commercial SPS (Fucoidan, F. vesiculosus) using HepG2 cell lines.
Drugs and Chemicals
25-OHC and fucoidan (SPS, F. vesiculosus) were purchased from Sigma Chemicals (St. Louis, MO, USA).
The Seaweed collection, identification and extraction of SPS from S. wightii has been detailed previously (Preetha
and Devaraj, 2010). The plastic culture wares were bought from TPP, Switzerland. Dulbeccos modified essential
medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were obtained from Seromed-
Biochrom AG, Germany. All other chemicals and solvents used were of highest purity and analytical grade.
438
Culture and Drug Exposure
The human hepatic cell line HepG2 was obtained from the American type culture collection (Manassas, Va)
and cultured in DMEM containing 10% (v/v) FBS, 100g/ml penicillin and 100 U/ml streptomycin. Cells were
maintained in a humidified incubator at 37C with 95% (v/v) air and 5% (v/v) CO2. The cells were replenished with
fresh media every 2 days. 25-OHC, SPS from S. wightii and F. vesiculosus were dissolved in ethanol before adding
to the wells. HepG2 cells were incubated in petridishes in the presence or absence of various concentrations of a) 25-
OHC b) SPS (S. wightii) c) SPS (F. vesiculosus) d) 25-OHC+ SPS (S. wightii) and e) 25-OHC+ SPS (F. vesiculosus)
for 24 h. After 24 h of experimental period the cells were harvested and used for cytotoxicity assay for dose fixation
and the assessment of apoptotic studies.
Determination of cell viability by trypan blue exclusion assay
The viability of cells was assessed by trypan blue exclusion test (Aldred and Cooke, 1983). HepG2 cells were
incubated in petri dishes in the presence or absence of various concentrations of a) 25-OHC b) SPS (S. wightii) c)
SPS (F. vesiculosus) d) 25-OHC+ SPS (S. wightii) and e) 25-OHC+ SPS (F. vesiculosus) for 1 h. The medium was
removed and fresh medium was added. After 24 h, the culture medium was removed and cells were collected by
trypsinisation. The cells were centrifuged and again resuspended in phosphate buffered saline (PBS). Trypan blue
solution (0.5% trypan blue in DMEM containing 0.1% BSA) was added to the cells and incubated for 15-20 min.
The solution was removed and cells were again washed with PBS to remove excess dye solution. The unstained
viable cells and the blue colored non-viable cells were observed under the phase contrast microscope and the
percentages of viability calculated.
Lactate dehydrogenase release assay for membrane damage
HepG2 cells were incubated in 96 wells plate in the presence and absence of various concentrations of a) 25-
OHC b) SPS (S. wightii) c) SPS (F. vesiculosus) d) 25-OHC+ SPS (S. wightii) and e) 25-OHC+ SPS (F. vesiculosus)
for 24 h. LDH activity was determined by the method of King (1965a). The enzyme activity is expressed as
Units/h/106 cells.
Measurement of intracellular ROS accumulation
HepG2 cells were incubated in the presence of optimized concentrations of a) 25-OHC b) SPS (S.
wightii) c) SPS (F. vesiculosus) d) 25-OHC+ SPS (S. wightii) and e) 25-OHC + SPS (F. vesiculosus) for 24 h.
2,7-dichlorodihydrofluorescein diacetate (DCF-DA) was used to detect intracellular ROS levels in HepG2 cells
(Cao et al., 2003). Briefly, HepG2 cells were rinsed once with PBS after 24 h and then incubated with 10 M DCF-
DA in DMEM at 37C for 30 min. Then, the cells were washed once with PBS and lysed in 3 ml of ice-cold 10 mM
TrisHCl buffer (pH 7.4), containing 0.2% sodium dodecyl sulphate. The cell lysates were collected and centrifuged
at 2000 x g for 5 min at 4C. The fluorescence of the supernatants was measured at an excitation wavelength of 495
nm and an emission wavelength of 525 nm.
Assay of nitric oxide production by Griess reaction
NO was measured as nitrite released from HepG2 cells according to the manufacturers protocol (StressXpress
Nitric Oxide detection kit, Catalog Number: EKS-300). Culture was incubated for 24 h at 37C in an atmosphere of
5% CO2 and 95% humidity. Thereafter, 100 ml of media from each well was aspirated and replenished with the same
amount of fresh medium and further incubated for 24 h with optimized concentration of SPS from S. wightii and F.
vesiculosus in the presence or absence of 25-OHC (10 mg/ml). To measure nitrite, 100 ml of supernatant was mixed
with equal volume of Griess reagent (1% sulphanilamide and 0.1% naphthyl ethylenediamine dihydrochloride in
2.5% phosphoric acid) and incubated at room temperature for 10 min. The absorbance at 570 nm was determined in
a micro plate reader. NO estimation was carried out using standard curve plotted against known quantity of sodium
nitrite. Results are presented in nmoles concentrations obtained from the mean optical density of triplicate wells of
each group.
Expression of Bcl2 and Bax by Western blotting
Cells were collected, washed twice with ice cold PBS and lysed with 50 mM Tris HCl (pH 8.0), containing 150
439
mM NaCl, 1% Triton X-100, 100 g/ml phenyl methyl sulfonyl fluoride and 1 mg/ml aprotinin. After incubation on
ice for 30 min the lysate was centrifuged at 14,000 rpm for 10 min at 4C. 50 mg of protein was separated on 10%
sodium dodecyl sulphate polyacrylamide gel. The gel was transferred onto a nitrocellulose membrane (Hybond C+,
Amersham life sciences.) at 20V and 120 mA for 90 min. Membrane was then washed thrice with PBS and blocking
was done with 3% skimmed milk for overnight at 4C. Then the membrane was washed thrice with PBS for 5 min
each and primary antibody (Bcl-2 and Bax) was added with the concentration of 1 mg/ml in PBS containing 1%
BSA and 0.1% Tween 20 and rocked gently at room temperature for 1 h. The blot was washed thrice with PBS for
5 min each. Secondary antibody (1:5000) in PBS containing 1% BSA and 0.1% Tween 20 was allowed to hybridize
for 1 h at room temperature. The bands were detected using chromogenic substrate 3,3-diaminobenzidine
tetrahydrochloride (DAB) in 1 M citrate buffer.
Estimation of cytochrome c
For analysis of cytochrome c, the cells for lysate preparation were washed twice with ice-cold PBS and
collected by centrifugation at 1000 x g for 10 min at 4C. The cell pellets were washed once with ice-cold PBS and
resuspended in lysis buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1.0 mM sodium EDTA, 1.0 mM
sodium EGTA, 1.0 mM DTT, 0.1 mM PMSF, and 250 mM sucrose) supplemented with protease inhibitors (10 g/
ml leupeptin, 10 g/ml aprotinin and pepstatin A). The cells were then homogenized in a glass homogenizer, and the
nuclei and cell debris were removed by centrifugation at 1000 x g for 15 min at 4C. The supernatants were further
centrifuged at 10,000 x g for 15 min at 4C, and the resulting mitochondrial pellets removed. The supernatants
obtained from the 10,000 x g centrifugation were centrifuged once again at 100,000 x g for 1 h at 4C, and the
supernatant was collected and designated as the cytosolic fraction for ELISA of cytochrome c (Kim et al., 2005a).
The primary antibody for cytochrome c raised in rabbit was procured from Cell Signaling Technology, Danvers,
MA, USA.
Propidium iodide staining
Propidium iodide staining was done according to the method of Ekhterae et al. (1999). HepG2 cells were
grown in a 6 well plate and treated with optimized doses of SPS from S. wightii and F. vesiculosus in the presence or
absence of 25-OHC and incubated for 24 h. For morphological analysis of apoptosis, cells were fixed, permeabilized
and stained with a solution of 30% methanol in PBS containing 20 mmoles/l propidium iodide and incubated at
37C for 1 h. Cells were harvested, placed on glass slides and visualized under Nikon fluorescence microscope.
The results are expressed as mean S.D. for three experiments. Differences between groups were assessed by
ANOVA using the SPSS (Statistical Package for Social Sciences) software package for Windows. Post hoc testing
was performed for inter-group comparisons using the Least Significance Difference (LSD) test; significance at p -
values < 0.001, < 0.01, < 0.05 have been given respective symbols in the figures.
The dose dependent cytotoxicity of 25-OHC on HepG2 cell lines was studied. A careful validation of the
viability assay procedures is suggested in experiments where cholesterol is a constituent, to avoid a potential bias in
concluding results of cytotoxicity studies (Ahmad et al., 2006). In the present study, determination of cell viability
by trypan blue exclusion assay revealed that the effective concentration to induce cell death by 25-OHC is 10 g/
ml. 50 g/ml of SPS from S. wightii and F. vesiculosus were found to be protective against cell death after 25-OHC
administration and were used for further experiments. Figure 1 shows dose dependent cytotoxic effect of 25-OHC
on HepG2 cells. 25-OHC at the dose of 10 g/ml induced 54% cytotoxicity in HepG2 cells; hence this dosage was
followed for the further studies of 25-OHC induced apoptosis. SPS from S. wightii and F. vesiculosus significantly
increased the cell viability from 47% to 87.6% and 46% to 88.1% respectively, at 50 g/ml in 25-OHC treated
cells. Hence, this dose (50 g/ml) was chosen to study the influence of SPS from S. wightii and F. vesiculosus on
apoptotic stimuli induced by 25-OHC. Individual inhibitory effects of SPS from S. wightii and F. vesiculosus on
25-OHC induced cytotoxicity at different doses are displayed in Figures 2.a and 2.b. Also, SPS from S. wightii and
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F. vesiculosus did not have any harmful effects on the viability of cells.
DCF-DA is a widely used probe for detecting cellular ROS. DCF-DA reacts with intracellular ROS to form
the fluorescent product, 2,7-dichlorofluorescein (DCF). Studies have shown that 25-OHC induced oxidative stress
is associated with cytotoxic effect in different cell lines (McCluskey et al.,1999). Incubation of HepG2 cells with
25-OHC for 24 h resulted in a dramatic (p < 0.001) intracellular accumulation of ROS (Figure 3). McCluskey et al.
(1999) demonstrated that 25-OHC induces oxidative stress in porcine ovarian granulosa cells. In the present study,
when compared with the control cells, the 25-OHC induced intracellular accumulation of ROS was reduced by SPS
from S. wightii and F. vesiculosus treated HepG2 cells (Figure 3). This can be attributed to their ability to directly
scavenge the free radicals. Several reports proving the free radical scavenging ability of algal polysaccharides are
present (Kuda et al., 2005; Park et al., 2005).
LDH release is a good indication of lysis of cells (Kim et al., 2001). In the present study, 25-OHC administration
caused a marked 54.23% increase in LDH leakage in HepG2 cells confirming the membrane damages (Figure 4).
Permeability of cells to ions has been observed to be increased by cholesterol oxides such as 25-OHC (Boissonneault and
Heiniger, 1985). The increased LDH leakage by oxysterols may be due partly to a cytotoxic effect of oxysterols and partly
to the increased permeability of the cells by oxysterols (Liu et al., 1997). SPS from S. wightii and F. vesiculosus protected
the HepG2 cells from LDH leakage. These results highlight the positive cytoprotective role of SPS from S. wightii and
F. vesiculosus. The cholesterol and the oxysterol induced toxicity and the subsequent altered membrane permeability
are curtailed by the administration of the algal polysaccharides, which may be attributed to its anti-lipidemic and
free radical scavenging ability.
Increased NO production (p < 0.001) was observed in 25-OHC treated HepG2 cells, as against the controls
(Figure 5). Kim et al. (2002) demonstrated that in cultured HepG2 cells, 3 days treatment with 25-OHC induced
iNOS mRNA expression, which suggested that the chronic exposure of hepatocytes to high concentration of
cholesterol or oxysterols may induce iNOS expression and subsequent synthesis of NO, which may be important in
the pathogenesis of atherosclerosis. SPS from S. wightii and F. vesiculosus administration significantly inhibited the
generation of NO species induced by 25-OHC (Figure 5). Bcl-2 family proteins appear to regulate a distal step in
an evolutionarily conserved pathway for physiological cell death and apoptosis, with some members functioning as
suppressors of apoptosis and others as promoters of cell death (Reed, 1995). The relative ratios of these various pro-
and anti-apoptotic members of the Bcl-2 family have been shown to determine the ultimate sensitivity or resistance
of cells to diverse apoptotic stimuli, including chemotherapeutic drugs and radiation, growth factor deprivation, loss
of cell attachment to extracellular matrix proteins, hypoxia, and lysis by cytolytic T cells (Reed, 1994 and 1995).
Bax is a member of the Bcl-2 family of apoptosis-regulating proteins (Reed, 1996). Kobayashi et al. (2002)
suggested that Bax protein, when present above a threshold level, is sufficient to trigger an apoptosis cascade.
Apoptosis induced by chemotherapeutic agents, is enhanced by Bax overexpression (Kobayashi et al., 2000). Mishra
et al. (2006) reported that NO free radical leads to NO mediated altered expression of Bax leading to increased ratio
of pro-apoptotic / anti-apoptotic protein resulting in apoptosis. In agreement with the above reports, treatment of
HepG2 cells with 25-OHC significantly induced the expression of NO species and subsequently the pro-apoptotic
protein, Bax (Figure 6). SPS from S. wightii and F. vesiculosus decreased the expression of Bax in 25-OHC treated
cells. Bcl-2 protein is known to promote cell survival as well as to suppress cell death by various apoptotic stimuli
(Sutton et al., 1997). The modulation of expression of Bcl-2 protein by 25-OHC was studied. Treatment of cells with
25-OHC markedly decreased the expression of anti-apoptotic protein, Bcl-2 (Figure 7). SPS from S. wightii and SPS
from F. vesiculosus prevented the decrease in the expression of Bcl-2 in 25-OHC treated cells.
Figure 8 shows the cytosolic levels of cytochrome c in HepG2 cell lines. The cytochrome c levels in cytosol
were significantly (p < 0.001) increased in 25-OHC treated cells. Cytochrome c is a 13-kDa protein, localized to the
intermembrane space and to the surface of the inner mitochondrial membrane (Yang et al., 1997). It translocates to
the cytosol on mitochondrial alterations and induces apoptosis. However, treatment with SPS from S. wightii and
SPS from F. vesiculosus decreases the cytochrome c levels in the cytosol of 25-OHC treated cells, which confirm
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that SPS from S. wightii and SPS from F. vesiculosus inhibit the opening of MPTP and subsequent release of
cytochrome c.
Hills et al. (2006) have demonstrated that heparin activates multiple anti-apoptotic pathways in human
trophoblast. Matsuki et al. (2003) have reported that prevention of cytochrome c release is of relevance as drugs
targeting to prevent cytochrome c release are efficient in preventing apoptosis. In consonance, the algal SPS may
inhibit apoptosis through multiple anti-apoptotic pathways, which might include induction of the expression of
Bcl-2, the anti-apoptotic protein and suppression of the pro-apoptotic Bax and cytochrome c release from the
mitochondria. Thus the favourable modulatory effect of SPS from S. wightii and F. vesiculosus on the apoptotic
pathways are pointed in this study.
An increased apoptotic stimulus in 25-OHC treated HepG2 cells is further confirmed by assessment of nuclear
morphology. Apoptotic nuclei can be visualized under a fluorescent microscope using DNA-binding dyes such as
propidium iodide (Figure 9). HepG2 cells cultured under normal conditions showed no fragmentation of genomic
DNA. In contrast, HepG2 cells was degraded into nucleosomal fragments when cultured with 25-OHC (Figure 9.a),
characteristic of apoptosis. Cells treated with SPS from S. wightii and F. vesiculosus in the presence of 25-OHC,
prevented the nucleosomal fragmentation induced by 25-OHC (Figures 9.b and c).
Taken together the results of the present study, it is likely to conclude that algal SPS are effective modulators
of apoptotic pathways induced by oxysterols. Both SPS from S. wightii and SPS from F. vesiculosus were almost
equally effective and prove to be promising cytoprotective candidates.
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Figure 1. Dose dependent effect of
25-hydroxycholesterol on HepG2 cells Figure 2a. Dose dependent effect of SPS from
S.wightii on 25-hydroxycholesterol treated HepG2
All experiments were done in triplicates. Average of cells
three values are expressed in the graph.
All experiments were done in triplicates. Average

Figure 2b. Dose dependent effect of SPS from
of three values are expressed in the graph. Units:
F. vesiculosus on 25-hydroxycholesterol treated
Percentage of control. Comparisons are made between
HepG2 cells
(a) - Groups I and groups II, III, IV, V, VI. (b) Group
All experiments were done in triplicates. Average of
II and groups V, VI. The symbols represent statistical
three values are expressed in the graph.
significance at *** - p<0.001, **-p<0.01 and * - p<0.05.
Figure 5. Effect of SPS from S.wightii and Figure 6. Effect of SPS from S.wightii and F.
F. vesiculosus on levels of nitric oxide of vesiculosus on Bax expression in control and
25-hydroxycholesterol treated HepG2 cells experimental HepG2 cells.
All experiments were done in triplicates. Average of three values are expressed in the graph. Units: nmoles of
total nitrate + nitrite/mg protein. Comparisons are made between (a) - Groups I and groups II, III, IV, V, VI. (b)
Group II and groups V, VI. The symbols represent statistical significance at *** - p<0.001, **-p<0.01 and * - p<0.05.
444
Figure 7. Effect of SPS from S.wightii and
F. vesiculosus on Bcl-2 expression in control and
experimental HepG2 cells
Figure 8. Effect of SPS from S.wightii and

F. vesiculosus on cytochrome c level of
25-hydroxycholesterol treated HepG2 cells
All experiments were done in triplicates. Average of three values are expressed in the graph. Units: Percentage
of control. Comparisons are made between (a) - Groups I and groups II, III, IV, V, VI. (b) Group II and groups V,
VI. The symbols represent statistical significance at *** - p<0.001, **-p<0.01 and * - p<0.05.
Figure 9. Propidium iodide staining of HepG2 cells treated with SPS (S.wightii), (F.
vesiculosus) and 25-hydroxycholesterol (200x)
Figure 9b. 25-OHC+ SPS igure 9a. 25-OHC+ SPS (F.

Figure 9a. 25-OHC
(S.wightii) vesiculosus)
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Cytoprotective effects of P.ginseng and G.biloba against amnesic shell
fish poison induced toxicity in Caco-2 cell line
E M Ramya, G Phani Kumar *, Y Chandrasekhar, K R Anilakumar
Applied Nutrition Division, Defence Food research Laboratory, DRDO, Mysore-570011-India
Abstract
Domoic acid a potent marine algal toxin produced by diatomic genus of Pseudo-nitzschia causing amnesic
shell fish poisoning. The major cause of domoic acid toxicosis is excitotoxic effect coupled with oxidative
stress. The present study aims to understand the beneficiary effects of saponin rich extract of P.ginseng (PG)
and flavonoid rich extract of G. biloba (GB) against domoic acid induced toxic effects in Caco-2 cell line. In
the present study it was observed that the toxicity induced by domoic acid in Caco-2 cells was mediated by
oxidative stress leading to cellular inflammation and apoptosis. In our study pre-treatment of the cells with
PG (35g/mL) and GB (40g/mL) showed significant protection against domoic acid induced morphological,
oxidative, apoptotic damages and cellular inflammation as evidenced in results of MTT assay, morphological
studies, ROS estimation, mitochondrial membrane potential and western blot studies. However, further clinical
studies are required to consider these extracts as a natural remedy to prevent amnesic shell fish poisoning.
Key words: Apoptosis, Domoic acid, G biloba, Inflammation, Oxidative stress, P ginseng.
Introduction
Domoic acid, a tricarboxylic amino acid, is responsible for the major human food poisoning called amnesic
shell fish poisoning1. This environmental neurotoxin produced by diatomic species of Psuedo-nitzhia is a rigid
analogue of kainic acid, can excite and kill neurons at low doses by activating glutamate receptors2. The global
environmental changes fostering harmful algal blooms producing domoic acid as well as the role of domoic as toxin
to human have made the studies on domoic acid exposure crucial3.
Panax ginseng (PG) and Ginkgo biloba (GB) are two well known nootropic plants that have been used as
herbal drug for several thousand years4. GB has been used for the treatment of several ailments like Alzheimers,
short term memory loss, lack of attention, cerebral vascular damages etc.5 PG has been extensively studied for its
stress attenuating activity and memory enhancing properties. It is known to have multi- therapeutic properties and
is used for treating diseases viz., cerebral ischemia, mood disorders, learning and memory deficit6.
Indigenous plants have been used as traditional medicines and are frequently screened for discovery of drug7.
The presence of antioxidants, lesser side effects and increased effectiveness make them popular in the modern
world. The present study aim to understand beneficiary effects of saponin rich P.ginseng and flavonoid rich G.biloba
against domoic acid induced amnesic shell fish poison in Caco-2 cell line. Cell viability, morphology, mitochondrial
membrane potential and other molecular markers involved in oxidative stress, inflammation and apoptosis were
targeted to study the effect of treatment of PG and GB against domoic acid induced cytotoxicity.
Chemicals and reagents
Domoic acid, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 2,7-Dichloro
dihydrofluorescein diacetate (H2 DCFDA), rhodamine 123, Dulbeccos modified Eagles medium (DMEM),
L-glutamine, trypsin, protease and phosphatase inhibitor cocktail, luminol and p-coumaric acid, primary antibodies
for glyceraldehyde 3-phosphate dehydrogenase, superoxide dismutase, catalase, glutathione peroxidase, cox-2,
bcl-2, caspase-3 anti-mouse polyvalent secondary antibody were obtained from Sigma (USA). Fetal bovine serum
was purchased from HyCloneTM (US).
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Plant Material: 20% saponin rich extract of P.ginseng and 20% flavonoid rich extract of G.biloba were
purchased from Nature and Nurture Health Care Pvt. Ltd, Delhi.
Cell maintenance
Caco-2 cells were obtained from NCCS, Pune, India. The cells were grown using Dulbeccos modified Eagles
medium (DMEM) supplemented with 20% fetal bovine serum (FBS), L-glutamine, and antibiotic solution (Sigma).
The temperature was maintained at 37C in a humidified atmosphere of 5% CO2. The media was changed every
alternate day and 80% confluent cells were used for all the experiments.
MTT assay
The methods of MTT assay was as per previous study with minor changes8. Caco-2 cells were seeded in 96
wells plate. The LD50 of the toxin was studied by treating cells with various concentrations of domoic acid. The cells
pre-treated for 1h with non-toxic concentrations of PG i.e. 35g/ml and GB, 40 g/ml before the domoic acid (LD50)
treatment were incubated for 24h and 100l of MTT (0.7mg/ml) was added and further incubated for 4h at 37C.
100l of DMSO was added to dissolve the formazan crystals and the absorbance was read at 570 nm using VERSA
max Hidex plate chameleonTMV (Finland). Results were expressed as percentage viability, where absorbance of
untreated well (control) was taken as 100%.
Morphological study under bright-field microscope
Cells seeded in 25 cm2 cell culture flasks were exposed with domoic acid (75 ng/ml) for 24 hrs with or without
prior treatment of PG (35 g/ml) and GB (40 g/ml) for 1 hr. The cells without domoic acid or plant extract
treatments were considered as control. After incubation period, image of the cells were taken (Olympus, Tokyo,
Japan) and observed for morphological changes.
Estimation of mitochondrial membrane potential (MMP)

Fluorescent probe rhodamine 123 was used to determine MMP9.
Estimation of reactive oxyg
en species (ROS).
ROS estimation was done as per previous study9
Immunoblotting studies
The methods of SDS polyacrylamide gel electrophoresis (SDS-PAGE) and western blot were according to
previous study10.
The data were expressed as mean SEM for three independent experiments. Statistical differences between
groups were determined by ANOVA test and were considered significant at p < 0.05 using graph pad prism version
5.03
Results
Cytotoxicity assay
Caco-2 cell lines were exposed with different concentration of domoic acid to observe the mortality of cell
line and the LD50 was found to be 75 3 ng/ml (Fig. 1A). Domoic acid exposed cells were significantly protected
by pre-treatment with PG and GB p<0.05 (Fig. 1B).
Morphological study under bright-field microscope
Loss of adherence, shrinkage was observed in domoic acid treated group when compared to control group
cells. However PG and GB treatment showed improvement in the adherence property of Caco-2 cell line (Fig.2).
447
Effect of PG and GB on domoic acid induced oxidative stress
Caco-2 cells treated with domoic acid showed a many folds increase in ROS generation with respect to control
group (Fig.3A). The fluorescence percent of cells pre-treated with PG (35 g/ml) and GB (40 g/ml) showed a
significant reduction of ROS production in when compared to group which was treated with only domoic acid
(p<0.05).
Estimation of mitochondrial membrane potential (MMP)
In the present study, domoic acid exposure resulted in loss of MMP by means of fluorescence intensity (32.2
3.5 %), with respect to untreated cell line. PG (35 g/ml) and GB (40 g/ml) pre-treatment attenuated domoic acid
induced loss of MMP and improved it by 89.5 4.2 % and 85.2 2.1 % respectively (Fig.3B).
Immunoblotting studies
Domoic acid exposure significantly down regulated the levels of antioxidant enzymes (p<0.05), such as, SOD,
CAT and Gpx with respect to control group. Pre-treatment of PG (35 g/ml) and GB (40 g/ml) showed protective
effect domoic acid induced oxidative stress with respect to levels of SOD, CAT and GPx (Fig.4). The inflammatory
as well as the apoptotic molecular marker i.e. Cox-2, caspase-3 were considerably up-regulated and bcl-2 expression
was down-regulated in domoic acid treated group, however the levels were reversed by pre-treatment with plant
extracts i.e. PG (35 g/ml) and GB (40 g/ml) (Fig.4).
Discussion
Domoic acid structurally resembles with glutamic acid and kainic acid hence binds with higher affinity to
glutamate receptors rich organs thereby causing excitotoxicity, oxidative stress and associated molecular cascade
that can lead to neuropathology11. Domoic acid can primary exert its effect on digestive system and can clinical sign
can manifest as gastrointestinal distress. Hence present study is focused to study the protective effect of PG and GB
supplementation against domoic acid toxicity in Caco-2 cells.
Domoic acid exposure considerably decreased cell viability in a concentration dependent manner i.e. from 25-
100 ng/ml indicating the toxic effects of domoic acid on Caco-2 cells. Our results are in line with previous studies
conducted by Carvalho et al12 where domoic acid exerted cytotoxic effect on Caco-2 cells. However, pre-treatment
with PG and GB prevented the domoic acid induced cell death in terms of increased cell viability. Previous studies
of Park et al.13 demonstrated the cytoprotective effect of PG in mouse Leydig cells. Morphological analysis of Caco-
2 exposed with domoic acid without pre-treatment with plant extracts resulted in shrinkage resulting in irregular cell
borders and related structural abnormalities in Caco-2 cells. However, pre-treatment with PG and GB, prevented
domoic acid induced structural abnormalities in the cells. Previous studies reports that flavonoids from GB can
induce beneficial effects on the vascular system and changes in neuronal morphology14.
Mitochondria play a key role in activating apoptosis, a major defence mechanism to remove potentially
dangerous cells. In the present study the effect of domoic acid on activation of programmed cell death was
observed in terms of decline in the mitochondrial membrane potential. However supplementation with plant extracts
significantly increased mitochondrial membrane potential. In the present study domoic acid exposure significantly
up-regulated pro-apototic marker, caspase- 3 and down-regulated anti-apoptotic marker, bcl-2. Our present study
was in line with previous reports of Ananth et al.15 where domoic acid induces apoptosis by the modulation of
gene expressions such as, bcl-2, bax, and caspase-3. However the same were reversed by pre-treatment with plant
extracts. The increase in MMP in PG and GB treated groups may be due to the active regulation of pro-apoptotic and
anti-apoptotic factors. Previous in-vitro and in-vivo studies of GB extract, where it attenuated doxorubicin-induced
apoptotic damage16 as well as anti-apoptotic effects ginseng on hydrogen peroxide in SK-N-SH Cells17 provides
scientific evidences for the observations in the present study.
Mitochondrial damage and accumulation of production of reactive oxygen species is a cyclic process i.e.
mitochondria contribute to apoptosis signalling via the production of reactive oxygen species, accumulation of
reactive oxygen species (ROS) can cause decreased oxidative phosphorylation and damage to mitochondrial DNA
448
resulting mitochondrial dysfunction. Our present results demonstrated that domoic acid exposure has significantly
triggered ROS generation. Our findings were in line with previous literature presented18-19. The increased amount
of ROS produced in the cells exposed to toxin can lead to deficiency in antioxidant enzyme levels challenging
the homeostasis. In the present study revealed a down regulated expression levels of SOD, CAT and GPx in the
toxin exposed group. However, PG and GB supplementation have substantially increased the levels of SOD, CAT
and GPx suggesting the potential modulatory effect of saponin rich PG and flavonoid rich GB in the Nrf2-ARE-
mediated anti-oxidative stress pathway.
Domoic acid induced the expression of inflammatory marker COX-2 in Caco-2 cells. However, the PG and
GB supplementation significantly down-regulated all the studied inflammatory mediators and acted as neuro anti-
inflammatory agent. Earlier studies reported that ginseng saponin Rg3 improve learning and memory impairment
via modulation of inflammatory markers like TNF-, IL-1, and COX-220. Results of the present study are in line
with previous research on anti-inflammatory effects of GB extract on lipopolysaccharide-induced inflammation21.
In the present study the anti-inflammatory effect offered by the plant extracts might be due to their efficacy to
prevent oxidative stress which can initiate inflammation or via direct anti-inflammatory effect.
Till date, however, only a few studies have examined the protective effect of herbal extracts against domoic
acid induced toxicity. In the present study PG and GB prevented domoic acid induced toxic effects in Caco-2 cell
lines via modulation of oxidative stress, inflammatory and apoptotic markers. In the present study pre-treatment
with PG (35g/ml) offered higher protective effect with respect to GB (40g/ml) in terms of increase in anti-oxidant
enzymes, reduced inflammatory marker and bcl-2 level. However with respect to the expression levels of caspase-3
GB was found to be more potent as it decreased the expression of caspases-3 in a significant manner with respect to
domoic acid treated group. However, further clinical studies are required to consider PG and GB as a natural remedy
to prevent amnesic shell fish poisoning.
Conflicts of interest
The authors declare that there is no conflict of interest.
Acknowledgment
Authors are thankful to Director, DFRL, Mysore for providing constant guidance, encouragement and
necessary facilities during this investigation.
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Figures and Legends
Fig. 1- Cytotoxicity assay

Cytotoxicity of domoic acid B) Effect of pre-treatment of GB and PG against domoic acid induced cytotoxicity
in Caco-2 cells. Data expres
s the mean SEM of three independent experiments. Statistically significant differences, *p < 0.05 vs control
and **p < 0.05 vs domoic acid.
Fig.2- Effect of pre-treatment of GB and PG on domoic acid induced structural alterations of Caco-2
cells. i) Control Caco2 cells, ii) Domoic acid (75 ng/ml) exposed cells for 24 h., iii, iv) Pre-treatment of GB (40 g/
ml) PG respectively followed by domoic acid (75 ng/ml) exposure for 24 h.
450
Fig.3- Protective effect of pre-treatment of GB and PG on domoic acid induced ROS generation and loss of
mitochondrial membrane potential.
A) Effect of pre-treatment of GB and PG on domoic acid induced ROS generation. B) Effect of pre-treatment of
GB and PG on domoic acid induced loss of mitochondrial membrane potential. Statistically significant differences,
*p < 0.05 vs control and **p < 0.05 vs domoic acid.
Fig.4- Western blot analysis for SOD, CAT, GPx, COX-2, caspase-3, bcl-2. 1. Control 2. Domoic acid (75 ng/
ml) 3. GB (40 g/ml) and 4. PG (35 g/ml).
451
Modulatory effect of tannin rich extract of Terminalia chebula against
picrotoxin induced anxiety
Y. Chandrasekhar *, G. Phani Kumar, E. M. Ramya, K. R. Anilakumar
Applied Nutrition Division, Defence Food research Laboratory, DRDO, Mysore-570011-India
Abstract
Stress and emotions are associated with several illnesses from headaches to heart disease, and immune
deficiencies to central nervous system. Terminalia chebula (TC) has been referred as traditional Indian
medicine for several ailments. The present study aimed to elucidate the modulatory effect of tannin rich
extract of TC against picrotoxin induced anxiety. Forty two female Balb/c mice were randomly divided
into six experimental groups (n=7): control, diazepam (1.5 mg kg bwt.-1), picrotoxin (1 mg kg bwt.-1) and
three TC treatment groups (25, 50 and 100 mg kg bwt.-1). Behavioural paradigms and RT-PCR studies were
performed to know the positive effect of TC against picrotoxin induced anxiety. The results showed that TC
supplementation increased locomotion towards open arm (EPM) and illuminated area (light-dark box test), and
increased rearing frequency (open field test) compared to picrotoxin (p < 0.05) in a dose dependent manner.
Furthermore, TC increased number of licks and shocks in Vogels conflict. RT-PCR studies showed an up-
regulation of genes such as BDNF, CREB, GABAA and 5-HT1A in TC administered groups. In conclusion,
these results support the hypothesis that the tannin rich extract of TC interacts with the GABAA receptor
complex to induce its anxiolytic-like actions, which might be beneficial in the treatment of stress-induced
anxiety disorders.
Key words: Anxiety, Picrotoxin, Terminalia chebula, GABA, Oxiative stress, Synaptic plasticity
Introduction
Anxiety is a feeling of unease, nervousness, or worry about an event with an uncertain outcome. Due to its
high frequency of occurrence it acts as a main psychiatric problem in public health concerns1. Almost 60-70% of
the anxiety and depressive patients respond to drugs like tricyclic antidepressants. Imbalance of neurotransmitters
is responsible for the etiopathogenesis of several psychosomatic disorders. Out of various neurotransmitters,
serotonergic, noradregic, dopaminergic and GABAergic systems play important role in pathophysiology of anxiety.
Therefore, the development of new anxiolytic medicine with known mechanism is currently the central theme
of modern research in psychiatric disorders3. A recent survey reveals that about 63.9% of the studied population
endorse the use of alternative medicine (including herbal) and this trend is similar throughout the world4. Authors
confirmed that predominant users were willing to try new and alternative therapy without abandoning the
conventional treatment. With this background, we feel that in the quest for drug/s that ameliorate anxiety disorders,
plant selected in the current study tannin rich of Terminalia chebula might play an important role in future owing to
its interesting medicinal properties.
Terminalia chebula Retzius (Combretaceae), commonly has known as haritaki, exhibits a number of medicinal
activities due to the presence of a large number of different phyto-constituents. The major phyto-constituents present
in the fruits are tannins such as gallic acid, chebulic acid, ellagic acid, and gallate esters5. It has been extensively
used in ayurvedic medicine for its cardioprotective, antimicrobial, antioxidant, antidiabetic, gastrointestinal motility
and wound healing activities6.
Picrotoxin is a famous sesquiterpene that affects the central nervous system. It was previously used as an
analeptic in the treatment of barbiturate poisoning. Picrotoxin is a highly toxic CNS stimulant, which acts via
interaction with the convulsant site of the GABA-gated Cl- channel7. Several researchers have studied the mechanism
by which picrotoxin acts on GABAA receptors. Therefore, we designed this study to investigate the in vivo role of
tannin rich extract of TC in protecting against picrotoxin induced anxiety.
452
Plant material
,dTerminalia chebula tannin rich extract (40%) was procured from Nature and Nurture Healthcare Private
Limited, Bangalore.
Animals.
Female Balb/c mice weighing 25-30 g were selected from the stockpile colony, Defence Food Research
Laboratory (DFRL), Mysore, India, housed in an acryl fibre cage in a temperature controlled room (232C).
Animals were maintained in 12 h light/ dark cycle with standard pellet chow feed and pure water were provided
ad- libitum. Ethical clearance for performing the experiments on animals was obtained from the Institutional Animal
Ethics Committee (IAEC) with CPCSEA approval no IAEC-2016/AN/11.
Experimental design.
Female Balb/c mice were randomly divided into the following six experimental groups: control group, positive
control group, negative control group and three treatemt groups. Different doses of tannin rich extract of TC were
orally administered to the mice groups separately such as, TC-25, TC-50 and TC-100 (25 mg/kg b.wt, 50 mg/kg
b.wt. and 100 mg/kg b.wt, respectively) for a period of 21 days. Positive control group was received diazepam 1.5
mg/kg b.wt, p.o. (DIZ) for the above said duration; Control and negative control groups were orally administered
with an equal amount of saline during the experimental period. Except control group, remaining all mice received
picrotoxin (PTX; 1.0 mg/kg b.wt, i.p.) at the 21st day, just before thirty minutes to the behavioural test. Animals
were killed under mild anaesthesia immediately after the behavioural studies. Brain samples were stored at -80 C
until further analysis.
Behavioural models
Open field test
The OFT was applied to assessment the locomotor behavior of animals8 in separated groups.
Elevate plus maze test
EPM is commonly used for the evaluation of anxiety9.
Light-dark paradigm
Light-dark paradig was performed as per previous study10.
Vogels conflict test
The Vogels conflict test is a simple and reliable conflict procedure to verify the anxiolytic activity11.
Histopathological studies
Brain samples were fixed in 10% formalin saline for 24 hrs. Briefly, the tissue was thoroughly washed in tap
water followed by washing with serially diluted alcohol (methyl, ethyl) to achieve dehydration. Specimens were
cleared in xylene, and then embedded in paraffin at 56 C in hot air oven for 24 h. The obtained tissue sections were
collected on glass slides, deparaffinized, stained by hematoxylin and eosin, and examined under a light microscope.
Assay of antioxidant enzyme levels in the brain
Whole brain tissues were homogenized in 50 mM phosphate buffer (pH 7.4) at a concentration of 10% (w/v).
Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidises (GPx) and glutathione reductase (GR) activity
were determined with commercially available kits as per user manual (BioVisoin, USA).
Gene expression analysis
Real-time reverse transcriptase-polymerase chain reaction (RT- PCR)
Total RNA was extracted from 100 mg of brain tissue by using12 The integrity and quality of cDNA was
examined using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primer as housekeeping. Primers were
453
purchased from Sigma Aldrich India and are as follows:
GAPDH F-5-GGGTGAGGCCGGTGCTGAG-3; R-5-TGGGGGTAGGAACACGGAAGG-3
BDNF F-5-GTGACTGAAAAAGTTCCACC-3; R-5-GACGTTTACTTCTTTCATGGG-3
CREB F-5-TCTAATGAAGAACAGGGAGG-3; R-5-GTCCTTAAGTGCTTTTAGCTC-3
GABAA F-5-AAAGCGTGGTTCCAGAAAA-3; R-5-GCTGGTTGCTGTAGGAGCAT-3
5-HT1A F-5-TCGCTCACTTGGCTCATTGGCTTT-3;
R-5 TTCCACCTTCTTGACCGTCTTGCG-3
The relative value of the mRNA expression of CREB, BDNF, GABAA and 5-HT1A genes were calculated by
comparing the cycle thresholds (CTs) of the target gene with that of the housekeeping gene (GAPDH) using the
2-ct method14
The results were expressed as mean SD. Data were analyzed by one-way ANOVA followed by Tukeys
test and Dunnets multiple comparisons using GraphPad Prism version 5.03, statistics software. In all the tests,
differences at p < 0.05 were considered to be significant.
Results
Effect of TC supplementation on PTX induced behavioural paradigms.
Anxiolytic activity was evaluated using elevated plus maze (EPM), light/dark box, open field test and vogels
conflict paradigms. In EPM, DIZ (1 mg/kg, i.p.) increased the time spent in the open arms significantly (p < 0.01)
compared to PTX treated group. However, treatment with TC (50 and 100 mg/kg) showed a significant (p < 0.05)
increase in the time spent open arm compared to PTX treated group (Fig1). In light/dark box model, Diazepam (1
mg/kg, i.p.) increased the time spent in the light chamber significantly (p < 0.01) compared to PTX treated group.
Animals treated with TC at doses of 25, 50 and 100 mg/kg also showed a significant increase in the time spent in
the light chamber (p < 0.01) compared to PTX treated group (Fig. 6). Picrotoxin administration (PTX; 1.0 mg kg
bwt.-1 ; i.p.) had negative affect on the locomotion of the animal in the open field conflict. As shown in (Fig-1),
supplementation of TC and DIZ showed a markedly significant difference in locomotor activity of mice (p < 0.05).
The TC treatment (25100 mg kg bwt.-1 ) produced a dose dependent increase in the total locomotion. To confirm
the anxiolytic like effect of TC, the Vogel conflict test was performed. In this experiment, one-way ANOVA revealed
significant variance among PTX group and the TC treated groups. Supplementation of TC at higher concentrations
(TC-50 and TC-100) significantly (p < 0.05) increased the number of punished licks and shocks in the Vogels
conflict. However, TC-25 treatment did not show any significant change in the number of punished shocks (Fig - 1).
As expected, DIZ significantly increased the number of punished licks and shocks (p < 0.05).
Effect of TC in Antioxidant enzymes
PTX treatment significantly depleted antioxidant enzyme activities like SOD, CAT, GPx and GR (Fig. 2).
Supplementation of TC was eliciting significant increase in the antioxidant enzyme activities in a dose dependent
manner (p < 0.05). However, the efficacy of TC extract (50 mg/kg and 100 mg/kg) with respect to antioxidant
enzyme levels was comparable to that of DIZ (1 mg/kg).
Effect of TC in 0RNA expression levels of CREB, BDNF, 5-HT1A and GABAA in brain by qRT-PCR.
Quantitat00ive RT-PCR was employed to check the mRNA expression of CREB, BDNF, 5-HT1A and GABAA
gene in the brain. Compared to the picrotoxin group, mice from TC treated groups showed up-regulation in the
expression of CREB, BDNF, GABAA and there is a down-regulation in the 5-HT1A. The relative expression of
mRNA of targeted gene was shown in Fig - 3.
454
Effect of TC in Brain histopathology study
In figure 4 shows that PTX, led to marked histopathological alterations in the cerebral cortex, including
neuronal degeneration as cytoplasmic vacuolization, haemorrhage. Administration of TC at 100 mg/kg along with
PTX treated mice showed decreased histopathological abnormalities in comparison to PTX mice, thus portraying
its protective action.
Discussion
The current study demonstrates that various doses of tannin rich extract of TC can suppress picrotoxin induced
anxiety in mice. The main tannins contents of TC were found to ellagic acid, chebulagic acid, quinic acid, gallic
acid, corilagin, ethyl gallate, chebulic acid by HPLC (results not provided). Recent studies have identified that
polyphenols could cross the blood brain barrier (BBB) and thus improve the CNS functions, such as modulation of
monoaminergic responses and antioxidant defence mechanism13. In the present study, tannins rich extract might have
contributed anxiolytic like property of TC in PTX induced mice, and findings are in line with literature presented14.
Furthermore, our study shows that these protective effects of TC are mediated via CREB-BDNF signaling pathway.
We found that picrotoxin administration alters behavioural parameters, such as OFT, EPM, L&D and Vogels
conflict paradigms.
Antioxidant response is the most vulnerable mechanism in the development and progression of anxiety
and other neurological disorders. In previous report suggested that Terminalia arjuna extract can act as a potent
antioxidant agent and eliminates several free radicals such as O2, HO, H2O2 and NO15. The present studies on
antioxidant enzymes revealed that, PTX decreased SOD, CAT, GPx and GR levels; further the study denoted that
the administration of TC down-regulated free radical generation at molecular level and reversed the antioxidant
enzyme levels.
Several reports indicated that sub-convulsive doses of picrotoxin induce anxiogenic effects such as increase
in heart rate, increase plasma catecholamine levels, changes in behavioural conflicts in the elevated plus maze,
open field, light dark and social interaction16-17. Our observations in behaviour models were further substantiated
by the modulation of neurotransmitters levels. Drugs that can interact with postsynaptic monoaminergic receptors
may possess complementary mechanisms of anxiolytic properties. Monoamine receptors modulate the cyclic-AMP
pathway by increasing cAMP levels, which lead to the activation of the gene transcription factor cAMP responsive
elementbinding protein (CREB). Previous studies reported that CREB-dependent BDNF expression was linked to
pathophysiology of anxiety, and demonstrated that the decreased CREB function might be operative in maintaining
high anxiety levels18. Present m-RNA expression data is in line with these previous studies; PTX treatment might
be involved in signal transduction of cAMP pathway; consequently the expression of CREB was down-regulated.
However, known anxiolytic drug (DIZ) and treatment groups reversed this CREB expression.
Brain derived neuro factor (BDNF) is in controlling emotions by regulating the plasticity and integrity of
neurons in brain circuits as well as in the pathogenesis of anxiety 19. The persistent down regulation of BDNF
expression observed in the brain tissue of PTX treated mice in the present study is thus likely related to the anxiety
that was confirmed by the behavioural paradigms like, elevated plus-maze test, lightdark test, and exploratory open
field test. The anxiolytic effect of TC may be mediated by increasing expression of the brain-derived neurotrophic
factor (BDNF), since all three doses of TC were significantly up regulated the BDNF expression. The increased
expression of BDNF might be related to the atrophy caused by PTX induced stress in hippocampal CA3 neurons20.
Clinical and pre-clinical studies have suggested that serotonergic neurotransmission may be involved in the
wide range of psychiatric disorders such as, anxiety, depression, post traumatic stress, schizophrenia, anorexia
nervosa, obsessive-compulsive disorder and attention-deficit disorder 21. There are at least 14 different types of
serotonin receptors have been identified, among them 5HT1A receptor is thought to play an important role in the
etiology of anxiety22. However, there is a contradiction about the exact role of 5-HT neurotransmission; some reports
support both anxiogenic and anxiolytic roles of 5-HT in anxiety/ depression like disorders23. Previous findings on
5-HT action in animal behaviour models revealed that 5-HT reuptake blockers, 5-HT synthesis precursor, 5-HTP,
455
and some 5- HT receptor agonists impair same kind of behaviour 24. In the present study, 5-HT1A receptor expression
was down-regulated by TC supplementation, decreased activation of 5- HT1A may be suppressed anxiety like
behaviour through multiple neurotransmitter pathways.
In the present study, TC treatment and DIZ enhanced the anxiolytic activity via positive modulation of the
GABAA receptor. It has been demonstrated that when the balance between excitatory and inhibitory neurotransmitter
activities is shifted pharmacologically in favour of GABAergic transmission, anxiolytic effect is induced. On the
other hand, an inhibition of the GABAergic system results in anxiety, restlessness, and even epileptic seizures. These
pharmacological findings point to the contribution of inhibitory neurotransmitter GABA to the pathophysiology of
brain disorders like anxiety disorders, epilepsy, and schizophrenia25. These evidences suggest a prominent role of
GABA in anxiety disorder. This made us to find out the involvement of GABA in anxiolytic activity of TC.
Acknowledgments
Authors are thankful to Director, DFRL, DRDO and Mysore for his kind support and keen interest.
Conflicts of interest
All authors have none to declare any conflicts of interest.
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Figures and legends
Fig.1- Effect of TC on Behavioural paradigms

Elevated plus maze ; B) Vogels conflict test; C) Open field test
Data represent the means SD (n = 6). Significance was determined by one way ANOVA followed by Tukeys
test, # p < 0.05 versus control, *p < 0.05 versus PTX
457
Fig.2- Effect of TC on A) Behavioural paradigms (Light and dark box test) and
Antioxidant enzymes in PTX induced mice.
Data are presented as mean values (SD) from group of six mice. # indicates P <0.05 compared with control,
*indicates p < 0.05 ** indicates p < 0.01*** indicates p<0.001 compared with PTX group
Fig.3- Effect of TC on mRNA expression in PTX induced mice. A), Gamma-Aminobutyric acid (GABAA)
B) 5-HT1A receptor, C), Cyclic AMP reactive element binding protein (CREB) and D) Brain derived neuro factor
(BDNF).
Data represent the means SD (n = 6). Significance was determined by one way ANOVA followed by Tukeys
test, # p < 0.05 versus control, *p < 0.05 versus PTX.
458
Fig.4- Histopathology section depicting the effect of extract of tannin rich extract of Terminalia chebula in
PTX-induced changes in brain regions of mice. () indicates the damage
Photomicrograph of a - control group depicting normal cellular morphology. b- PTX group with predominantly
shows chromosome condensation and fragmentation (arrows). Many neurons appear shrunken and eosinophilic,
while others demonstrate chromatin compaction and segregation, which is characteristic of apoptosis, c-DIZ group
depicting normal cellular morphology, d-25mg/kg body weight TC + PTX group showing abnormal and some
unhealthy neurons, e-50mg/kg body weight of TC + PTX group showing depicting normal cellular morphology with
some damage, f-100mg/kg body weight TC+ PTX group showing normal and healthy neurons with less damage
459
Extraction of Oil from Cucurbita maxima seeds and Evaluation of its
Anti Obesity potentiality in High Fat Diet induced Obese Rats
A. Kalaivani, V. V. Sathibabu Uddandrao, Ganapathy Saravanan and
S. Vadivukkarsi*
Department of Biochemistry, Centre for Biological Sciences, K. S. Rangasamy College of Arts and Science
(Autonomous), Tiruchengode, Namakkal Dt, Tamilnadu-637215.
Abstract
Regulation of energy homeostasis to overcome metabolic disorders is one of the most rapidly advancing
subjects in biomedical research today. Obesity, a complex metabolic disorder, is considered a killer lifestyle
disease over the past few decades due to sedentary lifestyle, caloric- rich diets and genetic make-up. In this
study, we made an attempt to found that potentiality of Cucurbita maxima seeds oil against high fat diet
(HFD) induced obesity in rats. We investigated the effect of Cucurbita seed oil supplementation over 30
days on the changes of HFD induced obese rats in body weight, markers of lipid metabolism such as LDL,
HDL, triglycirides, total cholesterol, adiponectin, leptin, amylase and lipase. The rats were treated orally with
Cucurbita seed oil (100 mg/kg body weight) once daily for 30 days with a Orlistat (10 mg/kg body weight)
treated group included for comparison. Oral administration with Cucurbita seed oil significantly reduced the
increase in body weight when compared to the HFD control group. The level of plasma lipids were significantly
increased in experimental obese rats as compared to the normal rats. Oral treatment with Cucurbita seed oil
reduced the concentrations of plasma lipids in obese rats to near normal level and also significantly reduced
the activity of amylase and pancreatic lipase. In conclusion, this study confirmed that Cucurbita seed oil
prevents the HFD induced obesity by altering the markers important to lipid metabolism.
Key words: Cucurbita seed oil, Obesity, Metabolic disorders, Lipid metabolism
Introduction
Obesity is a chronic metabolic disorder caused by an imbalance between energy intake and expenditure.
Obesity is one of the greatest health threats of this century1. Obesity is defined as a disease in which excess body fat
has accumulated to an extent that health is adversely affected. It is also known to be caused by altered lipid metabolic
processes, including lipogenesis and lipolysis. The exact etiology of this disease in the majority of humans is not
known. It is however, widely accepted that obesity results from a chronic imbalance between energy intake and
energy expenditure2. The addition of extra calories in the diet induces fat accumulation, leading to overweight and
obesity3.
Obesity is associated with multiple macro and micro environmental factors. It is manageable by several different
approaches, including pharmaceutical drugs, traditional medications, and surgery. Of these approaches, the use of
medicinal plants is increasingly popular and is preferable to conventional chemotherapeutic methods4. Moreover,
medicinal plants are naturally available sources with potentially beneficial biological and pharmacological effects
and are easy to consume, whereas there is still doubt about the other more invasive therapeutic modalities for obesity,
such as surgery5. Specific phytochemical constituents present in medicinal plants may assist in regulating weight
and body fat through modifying metabolic pathways at the molecular level which are responsible for signalling
adipogenesis and lipolysis6.
The pumpkin (Cucurbita maxima) of the Cucurbitaceae family is widely grown and consumed in many
countries around the world7. The oil from the pumpkin seed is claimed to be useful in the management of various
disorders. Pumpkin seed oil has been implicated in providing many health benefits8. There is no report of anti
obesity capability of these seeds; hence the present study was designed to evaluate the anti obesity potentiality of
Cucurbita seed oil against high fat diet induced obesity in rats.
460
Sample preparation
Cucurbita maxima seeds were washed, cleaned and dried at 50C for 12 h in an oven, and then crushed into
powder in a grinder with a size range of 0.55-1.0 mm. The resulted powder was kept in a vacuum dryer until use.
Pumpkin seed powder was mixed with water (1:6, m/V) at (100C) and oil was extracted by using a Clevenger
apparatus and extraction was repeated for 6 h and extracted oil was used for the study. All chemicals used in this
study were analytical grade.
Animals
Male Wistar rats were obtained from K.S.R. College of Technology, Tiruchengode, Namakkal District, and
Tamilnadu, India. Experimental animals were kept up under standard laboratory conditions (temperature; 222C:
moistness; 40-60%), and permitted food and water ad libitum. Rats, at first, weighing 150 - 180g were separated into
four groups of six each (n6). All procedures involving laboratory animals were in accordance with the institutional
animal ethical committee of K.S.R. College of Technology, Tiruchengode, Namakkal District, and Tamilnadu, India.
High Fat Diet formula
High fat diet was obtained from National centre for laboratory animal sciences, National institute of nutrition
(NIN-ICMR), Hyderabad. High fat diet was composed by Corn starch-15%, Sugar-27.5%, Lard oil-17.6%, Vitamin
mixture-1%, Mineral mixture-3.5%, Casein-20%, Cellulose powder-5%, Corn oil-9.9%, Choline bitartrate- 0.2%.
Experimental design
In the experiment, a total of 24 animals (18 HFD, 6 normal rats) were used for this study. The animals were
grouped into 4 with 6 animals in each group.
Group 1: Normal control rats
Group 2: HFD control rats
Group 3: HFD rats treated with seed oil (100 mg/kg body weight) for 30 days.
Group 4: HFD rats treated orally with Orlistat (10 mg/kg body weight) for 30 days (suspended in 0.5%
carboxy methylcellulose).
Measurement of Body weight
The body weight of each rats were measured. Toward the end of the experiment, blood was collected from
overnight fasted animals under inhalation of anaesthesia by retro-orbital puncture strategy. Blood was collected in
anticoagulant coated vials and permitted for 15 minutes at room temperature. Plasma was separated by centrifugation
at 2500 rpm for 15 minutes.
Blood samples were centrifuged at 4000 rpm/min for 10 min to separate plasma for analyzing lipid profile. The
plasma levels of blood glucose, LDL, HDL, Triglycirides, Total cholesterol, adiponectin, leptin, amylase and lipase
were determined using commercially available kits.
One-way ANOVA method was used and results were expressed as mean S. D followed by
Least Significant Difference (LSD) test. The level of statistically significance was set at P<0.05.
Results
Figure 1 showed revolutionizes in body weight in control and HFD group of animals during the experiment.
Intake of HFD for 30 days educed a momentous (p < 0.05) rise in body weight, compared to the normal control
group. Oral administration with Cucurbita seed oil (100 mg/kg body weight) or Orlistat significantly (P < 0.05)
condensed the rise in body weight when compared to the HFD control group.
461
Fig 1: Effect of Cucurbita seed oil treatment on body weight of control and experimental rats, Values are
mean SD, n = 6, Values are statistically significant at p<0.05
Table 1 show the levels of lipid profile in control and HFD induced obese rats correspondingly. The level of
plasma lipids were considerably enhanced in experimental obese rats as compared to the normal rats. Oral treatment
with Cucurbita seed oil or Orlistat significantly (p < 0.05) decreased the concentrations of plasma lipids in obese
rats to near normal level.
Table 1: Effect of Cucurbita seed oil on plasma lipid profile in control and experimental rats
Control HFD HFD + Cucurbita seed oil HFD + Orlistat
LDL (mg/dL) 17.361.09 35.891.99 25.641.56 22.891.09

HDL (mg/dL) 32.01 1.89 9.971.25 23.261.90 27.901.87
Triglycirides (mg/dL) 15.491.43 32.151.90 26.781.67 22.121.25
Total cholesterol (mg/dL) 87.901.76 167.892.67 100.211.67 95.241.32
Figure 2 represents the level of leptin and adiponectin in control and experimental obese rats. There was a
noteworthy (p<0.05) altitude in leptin levels and reduction in adiponectin in HFD induced obese rats compared with
control rats. Oral supplementation with Cucurbita seed oil or Orlistat brought back towards near normal levels.
Fig 2: Effect of Cucurbita seed oil on the levels of leptin and adiponeptin in control and experimental
rats, Values are mean SD, n = 6, Values are statistically significant at p<0.05
462
The activities of the amylase and lipase of normal and experimental obese rats were demonstrated in Figure 3.
HFD induced obese rats showed considerable increase (p<0.05) in amylase and lipase activity. Oral supplementation
of Cucurbita seed oil or Orlistat to obese rats drastically condensed the activity of amylase and lipase.
Fig 3: Effect of Cucurbita seed oil on activities of the amylase and lipase of normal and experimental
obese rats, Values are mean SD, n = 6, Values are statistically significant at p<0.05
Discussion
In the current study, improved body weight and fat was observed in HFD fed rats. Long-standing HFD fed to
rats exhibited with enhanced bodyweight and plasma co morbidity features9 and it might be due to the utilization
of a diet rich in energy in the form of saturated fats and its accumulation in different body fat pads10 directs to
unnecessary progress of adipose tissue and also the ratio of calorie ingestion to energy consumption concludes
bodyweight. Addition of Cucurbita seed oil demonstrated an extra momentous reduction in body weight increase
compared with that of HFD control rats.
HFD induced obesity is linked with dyslipidaemia characterized by increasing triglycerides, very low density
lipoprotein, total cholesterol, phospholipids and low density lipoprotein and a diminish in plasma level of high
density lipoprotein11. In the present study, dyslipidaemic changes like elevated triglycirides, total cholesterol,
and LDL, and turn downed plasma level of HDL has been noticed. These results were in line with Park et al12.
Triglycirides increase was for the reason that of dietary cholesterol that had been appeared to reduce unsaturated
fat oxidation, which thus prolonged the levels of hepatic and plasma triglycirides and the excessive accretion of
triglycirides in the lipid stores13. Conspicuous total cholesterol levels strengthen the menace of increasing coronary
heart disease by elevating LDL. In addition, high HDL is accommodating in transporting surplus cholesterol to
the liver for emission in the bile14. Oral administration of Cucurbita seed oil considerably lowered total cholestrol,
triglycirides, and LDL levels in plasma of rats with HFD-induced obesity.
In the body, adipose tissue is a vital part of energy storage and is decisive for energy homeostasis10.
It is well recognized that insulin can eventually activate leptin secretion during the metabolism
of nutrients, predominantly on glucose utilization in adipocytes15. Gathering of superfluous fat in
adipocytes is the elementary apparent truth for obesity. Leptin and adiponectin concealed by adipocyte
are hypothetical to play crucial roles in the controlling of metabolic homeostasis. A reduced plasma
adiponectin levels were found in obese animals and it furthermore confirmed a positive connection
with HDL16. During the progression of obesity, lofty level of leptin and affiliated reduced level of
adiponectin were found17. In this study, enhanced leptin and reduced adiponectin levels were noticed in
plasma of HFD control group compared with the control group. Oral supplementation with Cucurbita
seed oil diminished leptin and increased adiponectin in plasma of rats. These annotations recommend
that the reduced leptin and enhanced adiponectin levels after Cucurbita seed oil administration may
463
have restored insulin resistance in obese rats, resulting in reduced plasma lipid levels and weight
loss18.
Activating lipase or inhibiting pancreatic lipase would have an anti-obesity efficiency. Lowered pancreatic
amylase release narrowed the absorption of carbohydrates by suppressing their dispensation, prompting cut energy
intake19. In this study, pancreatic lipase and amylase level was observed to be improved in HFD induced rats. Oral
treatment of Cucurbita seed oil restrained the activity of pancreatic lipase and -amylase in treated rats compared
with HFD control rats. It is hopeful that the diminished levels of pancreatic amylase and lipase may represent a
constituent that would obstruct the incorporation and intake of starches and lipids which can lesser the caloric
admittance in Cucurbita seed oil treated HFD-fed obese rats, in order to urge weight diminution.
Conclusion
In conclusion, these results recommended that dietary supplementation of Cucurbita seed oil possibly will
hold back the augmentation in body weight and fat percentage in HFD induced obese rats. This strongly implies that
Cucurbita seed oil has beneficial prospective for the supervision of obesity. Further detailed study is necessary to
find out the exact mechanism of Cucurbita seed oil against the obesity.
Acknowledgement
The authors thank to the Management of K. S. Rangasamy College of Arts and Science (Autonomous),
Tiruchengode, Tamilnadu, India for their encouragement and for providing facilities to do experiments.
References
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Kazemipoor M, Radzi, Cordell G A & Yaze I (2012) Int J Che Eng and Appl 3 288-292
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464
Protective effect of green tea extract against carbofuran induced
toxicity in Wistar rats
Binitha P P and Ramadasan Kuttan
Amala Cancer research Centre, Amala Nagar Thrissur-680 555, Kerala
Abstract
Regulation of energy homeostasis to overcome metabolic disorders is one of the most rapidly advancing
subjects in biomedical research today. Obesity, a complex metabolic disorder, is considered a killer lifestyle
disease over the past few decades due to sedentary lifestyle, caloric- rich diets and genetic make-up. In this
study, we made an attempt to found that potentiality of Cucurbita maxima seeds oil against high fat diet
(HFD) induced obesity in rats. We investigated the effect of Cucurbita seed oil supplementation over 30
days on the changes of HFD induced obese rats in body weight, markers of lipid metabolism such as LDL,
HDL, triglycirides, total cholesterol, adiponectin, leptin, amylase and lipase. The rats were treated orally with
Cucurbita seed oil (100 mg/kg body weight) once daily for 30 days with a Orlistat (10 mg/kg body weight)
treated group included for comparison. Oral administration with Cucurbita seed oil significantly reduced the
increase in body weight when compared to the HFD control group. The level of plasma lipids were significantly
increased in experimental obese rats as compared to the normal rats. Oral treatment with Cucurbita seed oil
reduced the concentrations of plasma lipids in obese rats to near normal level and also significantly reduced
the activity of amylase and pancreatic lipase. In conclusion, this study confirmed that Cucurbita seed oil
prevents the HFD induced obesity by altering the markers important to lipid metabolism.
Key words: Cucurbita seed oil, Obesity, Metabolic disorders, Lipid metabolism
Introduction
Carbofuran (2, 3- dihydro-2, 2- dimethyl- 7- benzofuranyl methyl carbamate) is a broad spectrum, non-
cumulative carbamate insecticide. It is applied in granular form or aerially, on a variety of crops, corn, grapes,
banana and wheat in India and other countries. Their primary mechanism of toxicity is the inhibition of serine group
of acetylcholinesterase via carbamoylation at the nerve terminals. Tea plant (Camellia sinensis) belongs to the
family theacaceae, green tea extracts are widely used as dietary supplements. The increasing interest in the health
properties of tea extract and its main catechin polyphenols have led to a significant rise in scientific investigation for
prevention and therapy in several diseases.
The present study focuss on the effect of green tea to reduce carbofuran induced neurotoxicity and tissue
toxicity in Wistar rats. We have further investigated the effect of green tea to restore mitochondrial enzymes,
carbohydrate metabolizing enzymes, antioxidant enzymes as well as oxidative stress induced by carbofuran.
Rats were randomly divided into 5 groups each containing 6 animals (6 rats/group). Rats in Group I served as
normal vehicle control and was given pea nut oil (0.5 ml/rat). Rats in Group II, control were given carbofuran in pea
nut oil at a dose of 5 mg /kg b.wt in pea nut oil for 30 days. Rats in Group III, IV and V were given the same dose
of carbofuran along with different doses of green tea , 500, 250 and 100 mg/kg b.wt respectively dissolved in warm
water. The animals were treated with respective drug doses through oral gavage for 30 days. 10 days before sacrifice
carbofuran induced behavioral problems were tested by grip strength test, rota rod test and pain threshold test.
At the end of experiments animals of each group were sacrificed. Serum and tissue marker enzymes such
as acetylcholinesterase (AchE), creatine kinase (CK), lactate dehydrogenase (LDH) and gamma glutamyl
transferase (- GT) were analysed using commercially available kits. Liver homogenate was used for assessing
the activities of the following assays, superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase
465
(GPX), catalase (CAT), lipid peroxidation, tissue hydroperoxides and conjugated dienes. More over hexokinase,
glucose-6-phosphate dehydrogenase, glucose-6-phosphatase and fructose 1,6 bisphosphatase were also analysed.
Isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and NADH-
dehydrogenase were analysed from liver mitochondria.
Results
Effect of green tea on serum and tissue marker enzymes
The carbofuran administration showed lowered levels of serum and tissue AchE when compared to normal
animals. Green tea treatment elevated the AchE significantly (p0.01) in carbofuran treated groups both in serum
as well as in tissues when compared to carbofuran alone treated group (Table 1) .The levels of CK (Table 2), LDH
(Table 3) and -GT (Table 4) in serum and tissues were found to be increased in the group of animals treated with
carbofuran when compared to the normal animals.
Effect of green tea extract on carbofuran induced neurobehavioural changes
Carbofuran treated animals exhibited significant increase in pain threshold test, rota rod test and pain threshold
test as compared with normal rats (p0.01). Carbofuran with green tea significantly improved these tests as
compared with carbofuran treated animals. Carbofuran with green tea 500 mg/kg b.wt was the most effective
group when compared to other treated groups like 250 and 100 mg/kg b.wt (Table 5).
Effect of green tea on antioxidant enzymes, GSH levels and oxidative stress
The levels of SOD, CAT, GPX and GSH in the liver tissue were decreased in the carbofuran treated group of
animals when compared to the normal animals and the levels were significantly (p0.05) enhanced by green tea
administration. Markers of oxidative stress were conjugated dienes, tissue hydroperoxides as well as lipid peroxide
formation were high in the carbofuran alone treated animals compared with normal rats, and these parameters were
significantly (p0.05) reduced to normal levels in green tea treated groups .
Effect of green tea administration on mitochondrial enzymes and carbohydrate metabolizing enzymes
The levels of SDH, ICDH, MDH and NADH dehydrogenase were decreased in the control group animals,
treated with carbofuran alone when compared to the normal animals and the levels were significantly (p0.05)
enhanced after green tea administration. Hexokinase, glucose-6-phosphate dehydrogenase, glucose -6-phosphatase
and fructose-1, 6-bis phosphatase levels were altered in carbofuran treated group and green tea administration
restored these enzymes in a significant manner (p0.05).
Discussion
Administration of green tea (500, 250 and 100 mg/kg) for a period of 30 days, significantly prevented the
carbofuran induced elevation in the activities of the diagnostic marker enzymes in serum, and tissues. Pesticide
Carbofuran has been shown to produce severe neurotoxicity in mammals as seen by its inhibition of the activity of
acetylcholine esterase. Administration of green tea extract significantly reduced the neurotoxicity as seen from the
increased levels of acetylcholinesterase levels in serum and tissue.Green tea extract also reduced the tissue toxicity
as seen from the decreased levels of creatine kinase, lactate dehydrogenase, -glutamyl transferase which were
increased by the administration of carbofuran. Administration of green tea extract normalized the mitochondrial and
carbohydrate metabolizing enzymes which were decreased by carbofuran on administration. Carbofuran induced
oxidative stress in rats was also reduced by the administration of green tea extract. Moreover reduced levels of
antioxidant enzymes in the tissues and serum produced by carbofuran were significantly enhanced by administration
of green tea extract. The biochemical alterations accompanied by histopathological changes resulted from carbofuran
exposures were alleviated following green tea administration. These results indicate the ameliorating effect of green
tea extract during carbofuran induced toxicity.
466
Table 1 Effect of green tea on carbofuran induced changes in acetylcholinesterase activity in serum and
various tissues
Group Serum Brain Liver Intestine Kidney
Normal 5380.08214 2264.67300 2271.67211 2944.33711 2741.30211
Carb1 5 mg/kg 1723.96322** 1434.33265** 1392.00301** 1283.33345** 1565.65133**
Carb1 5 mg/kg+GT 2
4587.23423** 2491.33310** 2859.7590** 2566.67211** 2942.78134**
500 mg/kg
Table 2 Effect of green tea on carbofuran induced changes in creatine kinase activity in serum and
various tissues
Group Serum Brain Liver Kidney Intestine
Normal 90.806.7 12.001.92 79.3312.9 145.005.23 64.008.1
1
Carb 5 mg/kg b. wt 124.679.3** 197.675.7** 201.676.2** 224.674.1** 34.008.5**
1 2
Carb 5 mg/kg + GT
101.335.1** 66.674.7** 93.003.12** 110.3310.2** 78.015.3**
500 mg/kg
Table 3 Effect of green tea on carbofuran induced changes in lactate dehydrogenase activity in serum
and various tissues
Group Serum Brain Intestine Kidney Liver
Normal 95.2311.4 145.3318.4 188.3312.6 84.00 13.9 160.338.3
1 ns ns
Carb 5 mg/kg b.wt 233.529.4** 197.667.1** 207.0013.4 104.3312.0 295.0010.1**
1 2
Carb 5 mg/kg + GT ns ns
85.4612.4** 143.837.3** 196.04 10.4 76.008.9 124.679.4**
500mg/kg
Table 6.4. Effect of green tea on carbofuran induced changes in gamma glutamyl transferase (-GT)
activity in serum and various tissue
Group Serum Brain Liver Kidney Intestine
Normal 34.007.1 120.115.6 110.0910.4 88.012.5 64.53 3.1
1 ns ns ns
Carb 5 mg/kg 165.46.9** 110.838.9 291.074.2** 80.01 2.9 51.015.5
1 2
Carb 5 mg/kg + GT ns ns ns
44.009.1** 168.336.3 97.018.5** 75.617.3 69.083.9
500 mg/kg
Values are mean SD (n=6). **P<0.01, ns; non significant, significance was calculated against the normal and
carbofuran group as well as green tea and carbofuran treated groups. Unit for tissues (U/L/mg protein) and for serum
1 2
was (U/L). Carbofuran. Green tea.
Table 5. Effect of green tea administration on carbofuran induced changes in neurobehavioural study
Pain threshold Grip strength method Rota rod

Group
(In seconds) (In seconds) (In seconds)
Normal 1.011.43 40.651.70 31.450.43
1
Carb 5 mg/kg 20.120.56** 3.114.87** 13.172.34**
1 2
Carb +GT 100 mg/kg 5.010.98** 20.11 0.23** 20.070.21**
1 2
Carb +GT 250 mg/kg 5.110.88** 24.82 3.11** 22.113.56**
1 2
Carb +GT 500 mg/kg 9.132.40** 30.21 5.12** 22.041.89**
467
Study to assess the effectiveness of acupressure on menstrual pain
perception among adolescent girls with primary dysmenorrhea at
selected schools in Coimbatore
Mrs. Poornima Sriram
Asst.Professor, Department of Physician assistant,
Avinashilingam Deemed University, Mettupalayam Road, Coimbatore, Tamilnadu
Abstract
Objectives: Determine the effectiveness of acupressure on menstrual pain perception among adolescent in
experimental and control group and assess the problems associated with menstruation among adolescent
girls with primary dysmenorrhea. Design: Quasi experimental, pre test and post test non equivalent control
group design. Setting: CSI girls higher secondary school, Avinashi road and Girls higher secondary school,
Raja street, Coimbatore, Tamilnadu. Sample: 60 adolescent girls during menstruation were selected for the
study, out of which 30 were assigned to the experimental group, and 30 to the control group. Conceptual
Frame Work :Modified Ernestine Wiedenbachs Helping Art of Clinical Nursing Theory (1970) was framed.
Outcome Measures: Menstrual Pain perception level was measured of by using visual analogue pain scale and
problems associated menstruation was assessed by menstrual problem rating scale. Intervention: Acupressure
at SP6 point (Located four fingers with above the tip of medial malleous) was given to the experimental group
during contraction. Control group was allowed to take rest in the school. Results: Adolescent girls who had
acupressure during menstruation reported significant reduction on pain perception of 0.80 than 4.30 in control
group. Conclusion: The result supported that acupressure application is a very suitable and practicable therapy
of non pharmacological measure of reduction in pain perception during menstruation. The findings suggest
that acupressure at Sanyinjiao can be an effective, cost-free intervention for reducing pain and anxiety during
dysmenorrhoea, and recommend its use for self-care of primary dysmenorrhoea.
Keywords: Sanyinjiao (SP6), primary dysmenorrhoea, anxiety, acupressure, menstrual pain.
INTRODUCTION:
Reproductive health of women is considered as vital important and one that has wide spread implications
on health, well being and developments of the entire population. Women who fall in the reproductive age group
especially adolescent girls are neglected in reality. There is a consensus among health care providers and researcher
that reproductive age group is a period of marked physical, social and cognitive changes. (Robert, et al.2001)
Dysmenorrhea is the leading cause of recurrent short term school absent in adolescent girls and a common
problem for women of reproductive age. Primary dysmenorrhea is defined as painful menses in women with normal
pelvic anatomy began during adolescence characterized by pelvic pain begins shortly before or an onset of menses
and lasting one to three days. [51]
In world, Primary dysmenorrhea may affect up to 75% of women and 5 6% may have in capacitating pain
.The extent of pain incapacitating from her daily activity the pain is usually experienced in lower abdomen but
may extend to back and thighs. In United States, it was found that 91% of surveyed high school adolescents had
dysmenorrhea. Among respondents, symptoms affected academic work in 55% of and some times resulted in
missed classes (26%) it express the burden of disease in the country . [26]
The Quasi-experimental, pretest and post test non equivalent control group design was adopted for this study
468
VARIABLES UNDER STUDY
In this study Acupressure at Sp6 point was the independent variable and Pain perception during menstruation
was the dependent variable.
SETTING OF THE STUDY
The study was conducted among adolescent girls in two selected schools. Those schools are Church of South
India (CSI) Girls Higher Secondary School at Uppipalayam, Coimbatore and Training Girls Higher Secondary
School, Raja street, Coimbatore. CSI Girls Higher Secondary School is located 10 km away from KMCH College
of Nursing. It has seven sections in ninth standard and each section has about 50 students totally 350 students .This
was considered as a school for conducting the study for experimental group.Girls Higher Secondary School, Raja
Street is located 12 km away from KMCH College of Nursing. It has six sections in ninth standard and each section
has about 40 student totally 240 students. This was considered as a center for conducting the study for control group.
POPULATION
The study population comprises adolescent girls who had pain during menstruation and studying at selected
schools between the age group of 13 15 years.
SAMPLE SIZE
The sample size consists of 60 adolescent school girls. Among this, 30 adolescent girls studying in CSI Girls
Higher Secondary School were assigned to the experimental group and 30 adolescent girls who were studying in
Training Girls Higher Secondary School were assigned to the control group.
SAMPLING TECHNIQUE
Non probability convenient sampling technique was adopted for the selection of samples for the study. In CSI
Girls Higher Secondary School, total school children are 2500. In that 600 students are in the age group of 13 -15
years. the survey was conducted regarding the discomfort during menstruation among 350 students of ninth standard
girls. About sixty seven girls complained pain during menstruation. The 30 girls who had menstrual pain during the
data collection period were recruited for the experimental group of the study. In Training Girls Higher Secondary
School the total numbers of children are 2000. Among them 550 girls were from 13 15 years of age. The survey
was conducted among 240 students of ninth standard and 59 students had complained painful menstruation. During
data collection period, the 30 girls complained painful menstruations were recruited for control group samples.
CRITERIA FOR THE SAMPLE SELECTION
Inclusion criteria
yy Adolescent girls those who have painful menstruation for first 3 days of menstruation.
yy Adolescent girls between the age group of 13 15 years studying in selected schools and attained menarche.
yy Those who can communicate in Tamil and English.
Exclusion criteria
yy Adolescent girls who have gynecological and associated problems.
yy Those who had drugs during menstruation.
yy Those who are absent from the school during data collection period.
DESCRIPTION OF INTERVENTION
The primary purpose of acupressure application on SP6 was to reduce the menstrual pain perception. It is
acting by decreasing the number of pain impulses recognized by adolescent girls, releases neurotransmitters and
increasing blood supply to the uterus.
Step1: Explain the procedure to the girls. Make Adolescent girls to lie-down or sit in a comfortable position.
469
Step2:Locate SP6 point using four fingers with above the tip of medial malleous. This point is found by sliding
off finger at the edge of the tibia bone towards inside of the leg and mark the point.
Step3: Apply gentle pressure with the tip of the finger on the SP6 point of the leg for a minute continuously.
Repeat the same procedure for another leg.
Step4:Alternatively apply pressure in both SP6 points of the legs for about 20 minutes and assess the pain
perception.
Step.2 Step.3
DEVELOPMENT AND DESCRIPTION OF THE TOOL

Section 1: Demographic Data
Demographic data consisted of four parts. First part includes general information. It contains sample number,
age, religion, type of family, monthly income of the family and food habits. Second part was menstrual information
that is age at menarche, the present day of menstruation and last day of menstruation. Third section is physical
information that contains height, weight and Body Mass Index. Forth part has mothers educational status.
Section 2: Menstrual Problem questionnaire
This tool was prepared by the investigator after reviewing many books and literatures to under stand the problems
associated with menstruation. It consists of twelve statements in various aspects of problems of menstruation. It
helps to know the frequency of menstruation, bleeding, abdominal pain, back pain, head ache, vomiting, emotional
disturbances, consuming medications for pain, and other problems associated with menstruation. . The scoring was
given as below. Final scoring of the statements:
Final score Type of problem

> 28 Very severe problem
19 27 severe problem
10 18 moderate problem
0 9 not severe problem
Section 3: Visual Analogue Pain Scale
Visual analog pain scale was framed by Chris Adams (2004). It consists of a 10 cm horizontal scale with
descriptors from no distress to unbearable distress. It is a useful device for accurately determine the level of pain
perception and indicate the intensity of pain on a colored gradient and graduated line ranges from green ,yellow,
orange and red .Subjects were asked to place a mark on the 10 cm line at a point that corresponded to the level of
pain intensity they felt.
PROCEDURE FOR DATA COLLECTION
470
The study was conducted for a period of six weeks. The investigator selected the samples that fulfilled the
inclusion criteria in the selected schools .The purpose of the study was explained to the adolescent girls and consent
was obtained.
A survey was conducted adolescent girls regarding menstrual pain perception for selecting samples. The
investigator established rapport with the girls and interviewed them privately. As a pre test, pain intensity was
assessed before applying acupressure for both control and experimental group with visual pain scale. Menstrual
Problem rating scale was administered and was filled by themselves. In experimental group, gentle pressure was
applied with the tip of the finger on the SP6 point of the leg for a minute continuously. The same procedure was
repeated for another leg. Alternatively the pressure was applied in both SP6 point of the leg for about 20 minutes
and assessed the pain perception. For the control group without applying acupressure pain perception was assessed.
It last for 40 minutes For each sample.
STATISTICAL ANALYSIS
The collected data was analyzed by descriptive and inferential statistics. The descriptive statistics included
Mean, Standard deviation and Percentage to assess the menstrual pain of perception of adolescent girls. Inferential
statistical analysis such as Independentt test and Paired t test were used for the effectiveness of acupressure on
menstrual pain perception in control and experimental group. Chi-square were used to associate the pain perception
with demographic variables.
SUMMARY:
The researcher aimed to conduct a study to assess the effectiveness of acupressure application on pain
perception among adolescent girls during menstruation
Major findings of the study:
yy The mean score on menstrual problem of control group was 14.77 and experimental group was 15.77. It shows
that experimental and control group had suffered more or less equally with problems of menstruation.
yy According to the pre test score, 13 (43.3%) in control group and 14(46.7%) in experimental group were
between dreadful to horrible pain (5 7).
yy Mean difference of pre test and post test score in control group was 1.26. (p<0.01., t=8.69)
yy Mean difference of pre test and post test score in experimental group was 1.98. (p<0.01., t=15.13)
yy Mean score of pre test pain perception in control group was 6.33and in experimental group was 6.26.there is
no difference between control and experimental group in pre test score. (p<0.01., t=0.16)
yy According to the post test score in control group 17(56.7) between 1-3 range and in experimental group
17(56.7%) had no pain. Experimental group had significant pain reduction than control group. (p<0.01.,
t=13.56)
yy There was no association between score of menstrual pain perception with demographic variables.
CONCLUSION:
The following conclusions are made based on the above finding that Most of the subjects suffer from
moderate to sever dysmenorrhea and discomforts during menstruation. Acupressure is an effective, simple, non-
pharmacological measure to reduce dysmenorrhea. Dysmenorrhea affects the regular classes, studies and daily
activities of the subjects. The study can create awareness regarding menstrual pain management among adolescent
girls, school teachers and parents Acupressure is an effective and safe form of therapy for adolescents with primary
dysmenorrhea. Single Acu point pressure at Sanyinjiao (SP6) is cost-free and easy to learn. It can be integrated
into clinical practice and health education in order to enhance the quality of life for adolescents with primary
471
dysmenorrhea.
IMPLICATIONS:
Society has tremendous technological advancement in day to day life. The natural method of pain reduction
is acceptable and accessible to everyone in the world. T Therefore the nurse practitioners in the field of community
health can help in supporting the adolescent girls to provide comfort during menstruation in order to assure the best
possible outcome.
LIMITATIONS:
yy The study was limited to adolescent girls of 13-15 years of age.
yy The study samples were taken from selected schools.
yy 3. The immediate effects of acupressure after 20 minutes only assessed.
RECOMMENDATIONS:
yy A similar study can be conducted in larger group to generalize the findings.
yy A long term study to reinforce the effectiveness of acupressure can be undertaken
yy An extensive descriptive study to assess the knowledge attitude and practice of menstrual pain among
adolescent girls can be conducted.
yy A study can be conducted to assess the prevalence of dysmenorrhea.
yy A comparative study can be conducted between acupressure and pharmacological treatment.
TABLES
SECTION A:
Table 1: Distribution of the subjects according to their Demographic variables:
No. of Subjects
SI.NO Demographic variables Control Experimental

Percentage Percentage
Group Group
% %
(n=15) (n=15)
1. Age in years
5 16.7 6 20.0
13 Yrs
23 76.7 17 56.7
14 Yrs
2 6.7 7 23.3
15 Yrs
2. Religion
Hindu 26 86.7 21 70.0
Christian 1 3.3 6 20.0
Muslim 3 10.0 3 10.0
Type of family
23 76.7 22 73.3
3. Nuclear
7 23.3 8 26.7
Joint
4. Monthly income of the family
22 73.3 14 46.7
Up to Rs. 2500
2 6.7 9 30.0
Rs.2501- 3500
6 20.0 7 23.3
Rs.3501& above
5. Food habits
9 30 9 30
Vegetarian
21 70 21 70
Non-vegetarian
472
6. Age at menarche
Up to 11yrs 10 33.3 5 16.6
12 yrs 1 3.3 7 23.3
13 yrs & above 19 63.1 18 60.1
7. Body mass index

13 43.3 9 30.0
Up to 17
13 43.3 10 33.3
18 - 20
4 13.4 11 36.7
21 & above
8. Mothers educational status
No formal education 9 30.0 6 20.0
Primary 4 46.7 12 40.0
Secondary 4 13.3 4 13.3
Higher secondary & above 3 10.0 8 26.7
SECTION B:
Table 2: Description of Subjects According to pre test menstrual pain perception in control and
experimental group.
n = 30 n = 30
Control group Experimental group
S.NO Level Of Pain Perception
n % n %
1. Pre-test score 6 20.0 6 20.0
a)0 - 4 13 43.3 14 46.7
b)5 7 11 36.7 10 33.3
c)8 & above
Table 3 Comparison of Pre and Post test pain perception of subjects in Control Group:
Sl. No Group Mean pain perception S.D df Paired t value
1. Pre test 6.33
1.26 29 8.69**
2. Post test 4.30
Table 4 Comparison of Pre and Post test pain perception of subjects in Experimental Group:
Sl. No Group Mean pain perception S.D df Paired t value
1. Pre test 6.26
1.98 29 15.13**
2. Post test 0.80
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Phytochemical analysis of Freshwater algae Chlorella pyrenoidosa,
Chlorella vulgaris and Spirulina platensis and its Antibiofilm activity
N.S.Sridevi, Rajeswari Murugasan* and N.Santhi*
Research scholar, Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home
Science and Higher Education for Women, Coimbatore, Tamil Nadu, India
*Assistant Professor, Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for
Home Science and Higher Education for Women, Coimbatore, Tamil Nadu, India
Abstract
Algae are aquatic unicellular and multicellular organisms in both fresh and marine water environment having
unique potential to synthesize valuable natural products. In this paper the antibiofilm activity of the different
extracts from fresh water algae Chlorella vulgaris and Spirulina platensis was tested against biofilm forming
bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, and
Streptococcus mitis and discussed.
INTRODUCTION
Chlorella vulgaris is a unicellular; green algae found in many aquatic systems and are widely used as a health
food, food supplement, as well as in the pharmaceutical and cosmetics industry. Extracts of Chlorella were found to
have diverse antitumor, antioxidant, anti-inflammatory and antimicrobial activities. [1,2,3].
Spirulina, a blue green alga is now becoming a health food worldwide. It is an edible, microscopic,
multicellular, filamentous, alkalophilic, photoautotrophic cyanobacterium belonging to the class of Cyanophyta,
known for its rich source of protein, vitamins, and minerals.[4] Spirulina platensis is a prokaryotic organisms that
produce a variety of industrially important secondary metabolites such as antibiotic, cytotoxic, immunosuppressive
and enzyme inhibiting agents [5].
Biofilms are group or micro-organisms in which microbes encased in an extracellular polymeric substances
(EPS) such as proteins (<1-2%) including enzymes), DNA (<1%), polysaccharides (1-2%) and RNA (<1%), and
in addition to these components, water (up to 97%) is the major part of biofilm which is responsible for the flow of
nutrients inside biofilm matrix. Bacterial biofilms are normally resistant to antibiotics and human immune system
[6]. Microbial biofilms are at the root of various persistent infections such as those associated with prosthetic joints,
cystic fibrosis pneumonia, urinary and central venous catheters, periodontitis, and chronic dermal wound.[7]
Freshwater algae are rich source of novel compounds and biologically important secondary metabolites..So
considering these points, the aim of the study is to isolate a compound from the freshwater algae which is used to
prevent the bioflim forming bacterial infections.
Culture and growth conditions: The culture of green alga Chlorella vulgaris and Spirulina platensis were
grown in 1000 ml conical flasks with 500 mL medium. The medium used was BG-11and BBM medium. The
cultures were performed in a temperature controlled incubator at 25C providing 24 h fluorescent illumination (40
watt, white tube light). The cultures were hand shaken two to three times daily to avoid sticking.
Preparation of extract: The algal cultures were centrifuged at 2500 rpm for 10 minutes to harvest the algal
biomass. After centrifugation, algal pastes were air dried in oven at 60C. Chlorella vulgaris, Chlorella pyrenoidosa
and Spirulina platensis samples were finely powdered and extracted using sequential extraction method depending
using the solvents of increasing polarity of the selected solvents. 10 g of the sample was dissolved in 100 ml of
petroleum ether and it was kept in the shaker for 24 hours at 37C. The extract was filtered using Whatman No.1 filter
477
paper and the filtrate was kept for evaporation. The collected residue was again extracted using different solvents
like dichloromethane, chloroform, ethyl acetate, methanol, acetone by following above mentioned procedure. The
crude substance obtained after the evaporation was dissolved in DMSO and this is taken as the sample for the further
experiments.
Collection of microbial strains: The microbial strains selected for the antibiofilm activity are Pseudomonas
aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, and Streptococcus mitis. The strains were
collected from the Micro lab, Coimbatore and maintained in a nutrient agar medium.
Qualitative analysis of phytoconstituents (Raaman, 2006) : Various chemical tests were carried out to
screen for the phytochemicals namely carbohydrates, proteins, phenols, flavonoids, alkaloids, saponins, phenolic
content, phytosterol present in the petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol, and
acetone following standard procedure.
TLC analysis of the algal extracts: For the identification of compounds, the stationary phase was the pre-
coated silica sheets and was cut into strips of 2 cm X 7.5 cm. The mobile phase was chloroform/methanol/water in
ratio of 10/1.35/1. 10 l of Algal extracts were added in the spot of TLC sheet (stationary phase) then placed into
the TLC chamber which contain solvents (mobile phase). The TLC strips were kept in the chromatography chamber
for 40 min. Then strips were air dried till the solvent evaporates completely. The bands were detected under UV to
observe the bands present in the extracts. Compounds were identified and Rf (Retention factor) value was calculated.
Inhibition of biofilm formation by Algae extracts: Biofilm inhibition was carried out in tube method with
some modification (Karthick et al., 2013). The 100l of cell suspension of isolates was added into tube containing
5 ml of Muller Hinton broth and algae extract (1000 g /ml) was added and incubated at 37 C for 3 days. After the
incubation, the liquid suspension was removed and 100 l of 1% w/v aqueous solution of crystal violet was added.
Following staining at room temperature for 30 minutes the dye was removed and the tubes were washed thoroughly,
95% ethanol was added and incubated for 15 minutes. The reaction mixture was read spectrophotometrically at 570
nm. Inhibition mediated reduction of biofilm formation was calculated by the following formula.
% of inhibition = OD in control OD in treatment 100
OD in control
Phytochemicals have a great deal of attraction due to their preventive role in various diseases. The promising
pharmacological properties of medicinal plants are due to the presence of phytoconstituents such as alkaloids,
flavonoids, phenols and proteins (Thilagavathi et al., 2015). The various extracts of Chlorella vulgaris and Spirulina
platensis were screened for the phytoconstituents and the results are presented in Table 1.
Table-1
Phytochemical screening of various solvent extracts of Chlorella vulgaris and Spirulina platensis
Phytochemical Chlorella vulgaris Spirulina platensis
S. No
Constituents PE DI CF EA MN AE PE DI CF EA MN AE
1 Phenols + + + + + + + + + + + +
2 Flavanoids + + + + + + + + + + + +
3 Alkaloids - + + - + + + + + + + +
4 Glycosides - - - - - - - - - - - -
5 Saponins - + + - + + + + + + + +
6 Terpenoids - - - - - - - - - - - -
7 Steroids - + + - + - - + + - + -
+ denotes presence; - denotes absence
478
Phytoconstituents such as carbohydrates, proteins, aminoacids were found to be present in the samples.
Phenols and flavanoids were present in all extracts of both Chlorella and Spirulina sp. ethanol, ethyl acetate and
acetone extracts in Chlorella vulgaris and Spirulina platensis. Alkaloids and saponins are absent in petroleum ether
and ethyl acetate extract of Chlorella vulgaris. Steroids are absent in petroleum extract, ethyl acetate, and acetone
in Chlorella vulgaris and Spirulina platensis. Terpenoids and Glycosides were absent in Chlorella vulgaris and
Spirulina platensis.
TLC analysis of the algal extracts
The active principles in the methanol extract of the algae were separated using TLC. Three bands were
visualized under UV light.
1. Methanol extract of Spirulina

2. Standard
3. Methanol exract of Chlorella
ANTIBIOFILM ACTIVITY
The percent inhibition of biofilm formation by different extracts of Spirulina platensis and Chlorella vulgaris
against different microorganisms were presented in the Table-2.
Figure-1
Percent inhibition of bioflim formation of Spirulina extract in different microorganism
Figure -1 depicts that the different extracts of Spirulina platensis showed greater than 50 % reduction in cell
attachment against Pseudomonas aeruginosa , B. sublitis, E. coli and S. aureus in Chloroform and acetone extract.
The highest inhibition was shown by Chloroform & acetone extracts of Pseudomonas aeruginosa (greater than
80%). In case of Bacillus sublitis, 50 % reduction in biofilm formation was observed in extract of Chloroform.
Figure-2
479
Percent inhibition of bioflim formation of Chlorella vulgaris in different microorganism
The figure-2 portrays the highest percent biofilm inhibition for Pseudomonas aeruginosa strain was exhibited
by methanol extract (80%) whereas petroleum ether extract showed (79%)percent of inhibition. The methanol
extract showed (75%) percentage of biofilm inhibition against the pathogen B. sublitis, E. coli whereas acetone
extract exhibited more than 70% per cent of inhibition. In case of S. aureus greater than 60% reduction in biofilm
formation was observed in extract of methanol and acetone.
From the results of the present study, it was known that Chlorella and Spirulina sp. showed the rich sources
for bio active compounds especially Carbohydrates, Proteins, Flavonoids, Phenols, Alkaloids and Saponins. The
Chloroform and Methanol extract gave better inhibition of biofilm formation in the case of Pseudomonas aeruginosa
followed by B. sublitis, E. coli and S. aureus. The Chloroform and Methanol extract performed good antibiofilm
activity against Pseudomonas aeruginosa. This indicates the presence of antibiofilm substances in chlorella and
spirulina which triggers and stimulates against pathogens. So these bio active compounds will need further studies
to identify the chemical nature and to examine their beneficial effects for inhibition of pathogenic bacteria.
REFERENCES
Ashish G. Waghmare Manoj K. Salve, Jean Guy LeBlanc and Shalini S. Arya (2016) Concentration and characterization of
microalgae proteins from Chlorella pyrenoidosa.Bioresources and Bioprocessing:3:16
Viegas CV, Hachemi I, Maki-Arvela P, Smeds A, Aho A, Murzin DY (2015) Algal products beyond lipids: comprehensive
characterization of different products in direct saponification of green alga Chlorella sp. Algal Res 11:156164
Sharma, R., G.P. Singh and V.K. Sharma. 2011. Comparison of different media formulations on growth,morphology and
chlorophyll content of green algaeChlorella vulgaris. International Journal of Pharmacyand Biological Sciences 2(2):
509-516.
Usharani,G.,Srinivasan.G.,Sivasakthi.S..Saranraj.A(2015)Antimicrobial Activity of Spirulina platensis Solvent Extracts
Against Pathogenic Bacteria and Fungi. Advances in Biological Research 9 (5): 292-298
Shaieb,F., Issa, A. and Meragaa, A (2014) Antimicrobial activity of crude extracts of cyanobacteria Nostoc commune and
Spirulina platensis. Archives of Biomedical Sciences 2 (2): 34-41
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480
Evaluation of Antioxidant Status and Anticancer Activity of Thespesia
populnea on Daltons Lymphoma Ascites Induced Swiss Albino Mice
*Dr. Santhi, R. and **Dr. Annapurani, S.
*WOS - A Project Staff, **Professor and Head
Department of Biochemistry,Biotechnology and Bioinformatics, Avinashilingam Institute for Home Sciences and
Higher Education for Women,Coimbatore, Tamil Nadu,India.
santhiakash@yahoo.com,9942483393
Abstract
Nowadays there is an increasing centre of attention on the magnitude of medicinal plants and traditional
health system to prevent and cure various diseases. Plants have been an essential part of human society since
the civilization started. A medicinal plant is one in which one or more of its parts contain substances that can
be used for therapeutic purposes or which are precursors for the synthesis of useful drugs. The present work
was carried out to find out the antioxidant status and antitumor activity of flavonoid fractions of Thespesia
populnea (TpFf) was evaluated against Dalton s Lymphoma Ascites (DLA) in mice. After tumor induction
the TpFf was administered for the period of 15th, 30th, 45th and 60th days. After the last dose, mice were
sacrificed for the observation of antioxidant and antitumor activity for each treatment period. The activity
of enzymic antioxidants such as Catalase (CAT), Superoxide dismutase (SOD) and glutathione peroxidase
(GPX) and the levels of non enzymic antioxidants such as Vitamin A, Vitamin E and Reduced glutathione
(GSH) and lipid peroxide (MDA) in the liver homogenate of control and experimental mice were determined.
The activities of enzymic antioxidants and the levels of non enzymic antioxidants were decreased in DLA
control mice and MDA level was increased. Administration of TpFf significantly altered the antioxidants
level and MDA to normal level. The histopathological study also supports the same. The result suggests that
flavonoid fractions of Thespesia populnea exhibited antitumor effect by modulating lipid peroxidation and
augmenting antioxidant defense system in DLA bearing mice.
Keywords: Thespesia populnea, DLA, antioxidants, lipid peroxidation
Recent awareness of therapeutic potential of several traditionally used plants has opened a new dimension
for the study and research of medicinal plants1. Cancer is a class of diseases or disorders characterized by
uncontrolled division of cells. These cells can spread either by direct growth into adjacent tissue through invasion
or by implantation into distant sites by metastasis. It is the second most common cause of death. This disease
annually affects 9 million people and causes 5 million deaths2. Cancer is not a single disease but a wide spectrum of
conditions which are biologically different and even the causation and carcinogenic pathways are different. The
only common feature is the uncontrolled proliferation of cells which ultimately ends in death unless successfully
treated3.
Many important bioactive compounds have been discovered from natural sources using bioactivity directed
fractionation and isolation 4. The various parts of medicinal plants and their exudates, gums and resins are used
for the treatment of different ailments in Ayurvedic and other traditional systems of medicine5.
Thespesia populnea (L.) Linn. (Fam. Malvaceae), a fast growing, medium-sized evergreen tree, up to 10 m tall
with yellow, cup-shaped flowers having maroon centre and distributed throughout coastal forests of India and also
largely grown as a roadside tree6. The tree well grows under full sunlight and tolerates drought conditions.
The tree is valuable as coastal windbreak because it is it highly resistant to wind. It propagates easily and grows
rapidly. In the present study the flavonoids extracted from the plant to find out its anticancer activities.
Collection of plant materials and preparation of different extracts:
Fresh leaves of Thespesia populnea (Tp) were collected in an area free of pesticides and other contaminants
481
from the surrounding of Coimbatore. The collected leaves were washed thoroughly and blotted dry with filter paper,
dried, powdered and used to prepare the aqueous, ethanol, chloroform and ethyl acetate extracts individually7.
Preparation of Flavonoid fractions of selected extracts and Estimation of flavonoids:
Flavonoid fractions of selected extracts were prepared by a small modification8. Total flavonoid contents of
the residue of extracts were estimated by Aluminium chloride method 9. The flavonoid fraction which showed
maximum flavonoid content was selected for the further studies and referred as TpFf.
Animals:
Swiss albino mice weighing 25-30g of either sex were used in this study. They were procured from Amala
Cancer Research centre, Thrissur, Kerala. The animals were acclimatized for 15 days under laboratory conditions.
They were housed in polypropylene cages and maintained at 27C 2C. They were fed with standard mice feed
and water ad libitum was provided. The litter in the cages was renewed thrice a week to ensure hygeinicity
and maximum comfort for animals. Ethical clearance for handling the animals was obtained from the Institutional
Animals Ethical Committee prior to the beginning of the project work (IAEC.2015.BC:02).
Tumor cell lines maintenance:
The mice were acclimatized for two weeks and cells were propagated by intraperitoneal transplantation of 1
x 106 cells in 100 l of PBS. The tumor cell lines were procured from Amala Cancer Research centre, Thrissur,
Kerala. After 10-15 days, the cells were drawn from the intraperitoneal cavity and used for the in vitro studies.
In vitro cytotoxic studies:
In vitro cytotoxic studies were carried out by trypan blue exclusion method10. The fraction which showed
minimum concentration of flavonoid as EC50 was selected for the in vivo studies.
In vivo Studies:
In vivo studies were carried out by the intraperitoneal administration of EC50 of TpFf to examine their
antitumorigenic effect. The mice were divided into seven groups with 6 mice in each. The serum and liver was used
for the study.
Assessment of liver marker enzymes in serum
Glutamic Oxaloacetic Transaminase and Glutamic Pyruvic Transaminase were assayed11 and Alkaline
Phosphatase12.
Assessment of enzymic and non-enzymic antioxidants in the liver
Enzymic antioxidants such as Catalase (CAT)13, Superoxide dismutase (SOD)14 and Glutathione Peroxidase
(GPx)15 were assessed in the liver. The levels of the non-enzymic antioxidants such as Vitamin A16, E17 and
reduced glutathione 18 were also assessed in the liver.
Assessment of lipid peroxidation in the liver
The level of TBARS was assessed19.
Assessment of antitumor effect of TpFf by percentage of mortality rate in in vivo cytotoxic studies
The percentage of increase in life span was calculated20.
Assessment of histological status of liver
Histopathological observation was done21.
Results were expressed as mean SD. Statistical analysis was performed with one way and two way
analysis of variance (ANOVA).
482
Result and Discussion:
Phytochemical analysis was carried out in four different extracts namely aqueous, ethanol, ethyl acetate and
chloroform. It shows that most of the constituents are present in ethyl acetate extracts of Tp. Flavonoid was
absent in chloroform extract so that the quantification was carried out only for the three leaf extracts namely ethanol,
ethyl acetate and aqueous extracts.
Figure 1 shows the flavonoid content for the three extracts. In this, the highest flavonoid content was
present in ethyl acetate extract of Tp leaves.
Figure 1: Flavonoid content of Tp

In vitro cytotoxic studies:
It is based on the principle that live cells possess intact cell membrane that exclude the dye while the
dead cells do not and have blue colored cytoplasm under light microscope22. The extract killed 50 percent of DLA
tumor cells at a concentration of 70 g of TpFf (Fig 2). This concentration was designated as fifty percent effective
concentration (EC50) and was used in the following in vivo studies.
Figure 2: Cytotoxic effect of TPFf to DLA tumor cells

In vivo Studies:
In vivo studies were carried out by the intraperitoneal administration of 70 g (EC50) of TpFf to examine their
antioxidative and antitumorigenic effect.
Liver marker enzymes activity in the serum of Swiss albino mice:
The decrease in liver marker enzymes in the serum of mice treated with TpFf and silymarin are shown
in Figure 3. The liver marker enzymes activity of TpFf administered mice showed significant decrease on all
treatment periods when compared to 15 days treatment period. Co administration of TpFf to DLA tumor induced
mice showed significant decrease in liver marker enzymes in all treatment periods when compared to 15 days
treatment period of DLA tumor induced mice. The levels of liver marker enzymes was found to be significantly
increased in DLA tumor cells induced mice and indicated the membrane damage whereas TpFf treatment in tumor
cells induced mice showed a significant decrease in the liver marker enzymes activity and indicated their protective
483
role against DLA tumor induced toxicity. The significant decrease in the levels of biochemical marker enzymes
of liver like ALT, AST, ALP and bilirubin in plant extract administered animals might be due to decreased
leakage of the enzymes in liver cells23.
Figure 3: Activities of Liver Marker Enzymes in

Controls and Experimental Groups
Enzymic and non enzymic antioxidants activities in the serum of Swiss albino mice:
The enzymic and non enzymic antioxidants activities were found to be significantly increased in the
mice administered with TpFf when compared to PBS control mice in 15, 30, 45 and 60 days treatment
periods. Compared to standard antioxidant silymarin, TpFf administration showed a significant increase in the
enzyme activities on all the experimental periods. In DLA induced mice, the flavonoid fractions showed significant
enhanced activity in all the treatment periods. As the treatment period increased, the activity of them was also found
to be increased (Table 1 and 2). The activities of GPx, SOD and CAT were significantly increased in the liver of
EAC induced mice by the administration of flavonoid fraction of Terminalia catappa24. Antitumor activity of these
antioxidants is either through induction of apoptosis25 or by inhibition of neovascularisation26.
Lipid peroxidation activity in the liver of Swiss albino mice:
The level of lipid peroxides in the standard antioxidant silymarin treated mice was found to be significantly
decreased when compared to paraffin oil treated mice on all treatment periods (Figure 4).
Figure 4: Activities of Lipid peroxides

The mice transplanted with DLA tumor cells showed a significant increase in lipid peroxides when compared
to the controls and experimental groups in 15 days treatment period. Coadministration of TpFf to DLA tumor
induced mice showed significant decreased levels of lipid peroxide in liver when compared to the tumor induced
mice on 15 days treatment period. Flavonoid fractions administration showed more significantly decreased levels
of lipid peroxide in all treatment periods than that of silymarin administered mice.
484
An increase in lipid peroxide indicates serious damage to cell membranes, inhibition of several important
enzymes, reduced cellular function, and cell death27. Lipid peroxidation plays an important role in carcinogenesis28
and may lead to the formation of several toxic products such as malondialdehyde (MDA) and4-hydroxynonenal29.
These products can attack cellular targets including DNA, thereby inducing mutagenicity and carcinogenicity.
Percentage of mortality rate in in vivo cytotoxic studies:
The tumor bearing mice life span was found to be 15-25 days with the average life span of 20 days because
of tumor burden with tumor cell proliferation. Coadministration of TpFf inhibited the growth and proliferation of
tumor cells and hence the life span increased which indicated their antitumorigenic effect (Table 3). In vivo
antitumour activity of crude methanolic extract of the leaves of Leea indica (L. indica) was reported against EAC
cells in Swiss albino mice30.
Histopathological status of liver
Plate I illustrates the section of the liver of all the experimental groups. Results indicated the malignant
changes observed in the DLA tumor induced liver sections (H & E, 100X).
Plate I: Histopathological Status of Liver
Table 1: Activities of enzymic antioxidants in controls and experimental groups
485
Table 2 Levels of nonenzymic antioxidants in controls and experimental groups
Average number of mice surviving after
EC50 in g/100
Groups transplantation of tumor cells (days) Average life span
l/g.b.wt/ 1x106 cells
10 20 30 40 50 60
DLA - 6/6 4/6 0/6 0/6 0/6 0/6 20
TpFf+DLA 70 g 6/6 6/6 6/6 6/6 6/6 6/6 60
Table 3 Average Life span of DLA tumor induced mice treated with Thepesia populnea
Conclusion
It may be concluded that the flavonoid fractions of T. populnea possesses considerable antioxidative and
antitumorigenic activity against the tested DLA which offer a future study for the development of drug designing
and also justifies the medicinal values of the plants.
Acknowledgement
The authors are gratefully acknowledging the financial support of Department of Science and Technology,
New Delhi.
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487
In-silico prediction of binding sites of certain phytoconstituents to
select human olfactoryreceptors (ORs) for intranasal drug designing
Balasubramaniam Nagarathnam
Department of Biotechnology, K.S.Rangasamy College of Technology, Tiruchengode-637 215. Tamilnadu.
Abstract
Drug designing is an intense, laborious and multidisciplinary approach. It mostly depicted as a linear,consecutive
process that starts with receptor ligand (odor) discovery, followed by drug optimisation, pre-clinical in-
vitroandin-vivostudies. As GPCRs are significant drug targets, in the current study olfactory receptors (Class
A type GPCRs) are considered for computational approaches to design intranasal drug further to promote
aromatherapy. In-silico approaches on ORs such as topology prediction,conserved motif identification
(TM-MOTIF tool), phylogeny, databases (example: DOR-Database of Olfactory receptors), molecular
modelling and docking play a vital role to identify right drug (odor) for right receptor. Since human ORs are
discriminated to class I (sense water-borne odors) and class II type (sense air-borne odors), OR subclusters
have been establised in the current study using MEGA 7 and 100 representative ORs have been proposed for
secondary structural prediction (Pyre2 server). A pilot study was performed using Autodock vina on select
class I (HS51E2) and class II type (HS5G6) receptors against 15 ligands/phytoconsituents of anti-chronic
obstructive pulmonary disease (COPD) in particular asthma. By observing significant binding affinity, dock
scores, contact residues, best OR-ligand pairs have been documented and proposed for further analysis. Thus,
to conclude computational prediction of binding sites of therapeutically important drugs (odors) provide a
strong background to understand OR-ligand specificity, in turn to carryout in-vitro and in-vivo studies to
design intranasal drugs.
Introduction:
All living organisms sense diverse odors and odor recognisation is mediated by olfactory receptors (ORs).
ORs belong to GPCRs- i.e., membrane proteins and human ORs are discriminated into class I (sense water-born
odors), class II (sense air-born odors) type and are embedded in cell membrane of the olfactory neurons located
on the olfactory epithelium. The role of olfactory receptors and organization of olfactory system in humans have
been explained by the landmark paper published by Nobel Laureates Buck and Axel [1] . And It is understood
from prior literatures [2,3] that one OR recognizes multiple or various odorants, also different odorants are sensed
by different combinations of ORs. As per recent data, odorants could act both as agonist for some ORs and as
antagonist for others [4-6]. This reflects the counteraction phenomenon [7,8] and around 650 OR genes were found
in human in which 350 ORs were found potentially functional genes [9] . Class I ORs (fish-like ORs), and class II
(terrestrial type) ORs together as olfactory sub genome contribute >0.1% to human genome [10,11]. For the current
study, DOR (Database of olfactory receptors) http://caps.ncbs.res.in/DOR [12] is used to retrieve class I and II type
sequences of human olfactory receptors.
As we know, drug designing is a multi-disciplinary approach; the current objective of the study is to provide a
basic knowledge to predict/identify the binding sites of select ORs to certain phytoconstituents. As per prior literature,
annotated class I and II OR clusters have been sub clustered (data unpublished), and few representative ORs (100 in
nos) were predicted for secondary structural details. A ligand library for anti-chronic obstructive pulmonary disease
(COPD) in particular asthma disease was prepared and a pilot docking study with HS5E1 (Class I type) and HS5G6
(Class II type) receptors against 15 ligands were performed to predict binding sites. So, the outcome of the study
could be useful effectively further for drug optimisation, pre-clinical in-vitroandin-vivostudies in particular intra
nasal drug delivery.
Methodology:
1.1. Collection of OR sequences from DOR :
For the current study, class I (54 sequences) and II type (517 sequences) OR sequences were collected from
DOR database [12] and the established human OR clusters were named as HSC1 to HSC10 for 10 human OR
488
clusters as in DOR database, in which a distinct cluster namely HSC1 represents class I type, rest of the clusters
from 2-9 belongs to class II type ORs. Collected OR sequences of cluster1 were saved in a file called HSC1.fasta,
and OR sequences of cluster 2 were saved in other file called HSC2.fasta and so on (refer figure 1).
1.2. Alignment Procedure using MAFFT :
MAFFT online alignment server [13] was used to align the collected OR sequences of cluster 1 (HSC1) to
cluster 10 (HSC10). The multiple sequence alignment (MSA) was performed using parameters such as JTT 200
scoring matrix with gap opening penalty as 1.53. The initial alignment was exported to MEGA 7 software [14] and
at this stage; a careful editing was done to refine the quality of alignment. The obtained final alignment session was
saved (file.mas) in MEGA 7 to construct OR phylogeny.
1.3. Phylogeny to construct OR subclusters using MEGA 7 :
MSA of human OR clusters were used to construct (Nj) method of phylogeny by fixing 1000 bootstrap
replicates. The generated tree session files were saved with file extension .mts and radial, rectangular displays
were used to analyze tree topology and to classify subgroups. At this stage, we could obtain OR sub clusters (data
unpublished).
1.4. Representative ORs and molecular modeling using Phyre 2
Among the established 10 OR subclusters, 100 representative OR sequences (data unpublished) were
recommended to predict secondary structure using Phyre 2 server [15]. For a pilot study, representative ORs such as
HS51E2 (NCBI Reference Sequence: NP_689643.2) and HS5G6 (UniProtKB/Swiss-Prot: P0C626) of class I and
II type were selected to undergo docking studies.
1.5. Docking study using Autodock :
Prior, 50 ligands were collected under Lipinskis rule of drug likeness. Around 15 phytoconstituents were
considered for the study and downloaded from NCBI-pubchem database in .sdf file. The.sdf format was converted
into .pdb format (using open Babel) and used for docking procedure. The active sites of target proteins were
predicted using Database of Enzyme Catalytic Mechanisms EzCatDB and the predicted active site residues were
used to generate grid file (.gpf) in Autodock 4.0 software for molecular docking. GA (Genetic algorithm) was
applied to generate docking file (.dpf). Minimum binding energy, Estimated inhibition constant were noted from
the created .dlg file. The docked protein-ligand complex was visualized by using UCSF chimera software and
interacting residues were identified by LIGPLOT+ academic software (refer figure 2, 3 in results).
Figure 1: Flow chart for methodology
Note : Flow chart illustrates the steps involved to construct OR subclusters and predict the binding sites of
select human ORs such HS51E2 (class I type) and HS5G6 (class I type) to select 15 ligands .
489
Results and Discussion:
In-silico prediction of binding sites of certain ligands (refer table 1 and 2 ) to select human olfactory receptors
play a crucial role in providing preliminary knowledge on identifying interacting amino acids of ORs for select
phytoconstituents. Since, phytoconsitutents were collected from various medicinally important herbs such as
B.diffusa, S.trilobatum, V.negundo,, O.tenuiflorum, the predicted binding sites could propose the possible binding
affinity of select ligands for human ORs and the outcome could further facilitate to study in detail for drug dose
optimization, pre-clinical in-vitroandin-vivostudies. Also the results could viably useful for various practical
implications such as preparation of herbal drugs particularly for intra nasal drug delivery in future.
Table 1 : Docking results of class I type OR (HS51E2) with select 15 phytoconstituents :
Mol. Minimum Inhibition
S. H-bond H-bond LogP Pubchem
Compound Weight (g/ Binding Constant Plant Source
No donor Acceptor Value ID
mol) Energy (Ki)
1 Rotenone * 394.423 g/mol 0 6 4.1 -5.9 47 M 6758 B.diffusa
2 Boeravinone J * 298.048 g/mol 3 6 3.5 -5.72 64.40 M 16203335 B.diffusa
3 Abronisoflavone * 286.28 g/mol 4 5 1.54 -5.29 131.96 M N/A B.diffusa
4 Esculetin 178.143 g/mol 2 4 1.2 -5.13 174.15 M 5281416 S.trilobatum
5 Scopoletin 192.17 g/mol 1 4 1.5 -4.98 222.38 M 5280460 S.trilobatum
6 Alpha guaiene 204.357 g/mol 0 0 4.6 -4.95 235.60 M 5317844 V.negundo
7 Rosmarinic acid 360.318 g/mol 5 8 2.4 -4.79 307.53 M 5281792 O.tenuiflorum
8 Kaempferol 286.239 g/mol 4 6 1.9 -4.73 342.16 M 5280863 B.diffusa
9 Carvacrol 150.221 g/mol 1 1 3.1 -4.69 363.82 M 10364 O.tenuiflorum
10 Germacrene D 204.357 g/mol 0 0 4.7 -4.65 388.04 M 5373727 O.tenuiflorum
11 Luteolin 286.239 g/mol 4 6 1.4 -4.52 488.74 M 5280445 V.negundo
12 Quercetin 302.238 g/mol 5 7 1.5 -4.24 786.28 M 5280343 B.diffusa
13 Linalool 154.253 g/mol 1 1 2.7 -4.01 1.16 mM 6549 S.trilobatum
14 Methyl caffeate 194.186 g/mol 2 4 1.5 -3.74 1.82 mM 689075 S.trilobatum
15 Casticin 374.345 g/mol 2 8 3.1 -3.67 2.04 mM 5315263 V.negundo
Note : Table 1 shows the docking results of select 15 phytoconsituents of various herbs to class I type receptor
(HS51E2) with respective minimum binding energy, inhibition constant (Ki ). * denotes the top ranked ligands and
discussed..
Table 2 : Docking results of class II type OR (HS5G6) with 15 phytoconstituents
Minimum Inhibition
H-bond H-bond LogP Pubchem
S.No Compound Mol.weight Binding Constant Plant Source
donor Acceptor Value ID
Energy [Ki]
1 Esculetin 178.143 g/mol 2 4 1.2 -5.48 95.55 M 5281416 S.trilobatum
2 Rosmarinic acid 360.318 g/mol 5 8 2.4 -5.45 100.57 M 5281792 O.tenuiflorum
3 Boeravinone J 298.048 g/mol 3 6 3.5 -4.66 384.51 M 16203335 B.diffusa
4 Alpha guaiene 204.357 g/mol 0 0 4.6 -4.63 403.74 M 5317844 V.negundo
5 Scopoletin 192.17 g/mol 1 4 1.5 -4.27 737.38 M 5280460 S.trilobatum
6 Casticin 374.345 g/mol 2 8 3.1 -4.24 776.91 M 5315263 V.negundo
7 Luteolin 286.239 g/mol 4 6 1.4 -4.22 810.18 M 5280445 V.negundo
8 Linalool 154.253 g/mol 1 1 2.7 -4.12 961.35 mM 6549 S.trilobatum
9 Germacrene D 204.357 g/mol 0 0 4.7 -4.1 987.70 M 5373727 O.tenuiflorum
10 Quercetin 302.238 g/mol 5 7 1.5 -4.01 1.15 mM 5280343 B.diffusa
11 Carvacrol 150.221 g/mol 1 1 3.1 -3.99 1.19 mM 10364 O.tenuiflorum
12 Kaempferol 286.239 g/mol 4 6 1.9 -3.86 1.9 mM 5280863 B.diffusa
13 Methyl caffeate 194.186 g/mol 2 4 1.5 -3.51 2.70 mM 689075 S.trilobatum
490
14 Abronisoflavone 286.28 g/mol 4 5 1.54 -2.79 8.9 mM N/A B.diffusa
15 Rotenone 394.423 g/mol 0 6 4.1 0.07 0 6758 B.diffusa
Note : Table 2 shows the docking results of select 15 phytoconsituents of various herbs to class II type
receptor (HS5G6) respective minimum binding energy, inhibition constant (Ki). *denotes the top ranked ligands
and discussed.
Compounds of B.Diffusa show higher affinity towards Class I type ORs than class II ORs:
The given result tables (refer table 1 and 2) illustrate the list of collected phytoconstituents with respective
molecular weight, H-bond donor, H-bond acceptor, Log P value, minimum binding energy (Kcal/mol), inhibition
constant (Ki), pubchem ID and plant source. These results clearly illustrate the favorable binding affinity of select
ligands towards class I and II type ORs.When minimum binding energy is lower than -6 kcal/mol, a better binding
affinity is achieved, by referring to this statement, all the selected ligands showed the values for minimum binding
energy lesser than -6 kcal/ mol (refer table 1 and 2) which reflects the significant molecular interaction between
select ligands and receptor. Among the 15 ligands, three top ranked ligands for class I and class II were identified
through Autodock protocol. Interestingly, the identified top three compounds for class I type OR were exclusively
from the herb B.diffusa, and a compound from B.diffusa observed as ranked among the top three ligands for class
II type receptor also. This is very evident from the results (refer table 1,2) that compounds from B.diffusa shows
significant binding affinity than all other ligands, in which top three compounds favours to bind to class I type OR
than to class II type OR..
Interestingly, compounds such as, rotenone, boeravinone J, abronisoflavone derived from B.diffusa , showed
the minimum binding energy values -5.9 Kcal/mol, -5.72 Kcal/mol, -5.29 Kcal /mol and inhibition constant [ki]
as 47 M, 64.40 M, 131.96 M respectively (refer table 1). Notably, all the top ranked ligands showed the
values of minimum binding energy below than -6 Kcal/mol, and as mentioned earlier, all the three compounds are
derived only from B.diffusa. This shows the molecular attraction/ affinity of select ligands towards class I type
OR (HS51E2) and can be further explored to compound purification and drug formulation particularly for the
application of intranasal drug delivery. The selected herb B.diffusa is medicinally important and fall on rasayana
category as per ayurvedic claim also involve in disease prevention, providing hepatoprotection, immunomodulation
and can be explored further for anti-asthmatic studies ([16-18].
Ligands of various plant sources show affinity towards Class II type OR:
The phytoconstituents namely esculetin, rosmeric acid, boeravinone J from various plant sources such as
S.trilobatum O.tenuiflorum, B.diffusa showed minimum binding energy -5.48 k cal/mol, -5.45 k cal/mol, -4.66 k
cal/ mol, inhibition constant [ki] 95.55 M, 100.57 M, 384.51 M to class II type OR (HS5G6) respectively (refer
table 2). Here, we could understand that ligands from diverse plant sources interacting to class II type ORs, but it is
not the same to class I type OR in the prior observation (refer table 1).
In-silico prediction of binding sites of boervinone J with class I type OR (HS 51E2) and
class II type OR (HS 5G6)
As mentioned, the phytocompound boervinone J derived from B.diffusa showed significant molecular
interactions with both class I and II type receptors (refer figure 2, 3). Boervinone J is observed as second ranked
ligand to class I OR, and third ranked ligand to class II type OR among the top ranked ligands (refer table 1, 2).
Table 3 : The table illustrates the list of interacting residues of class I (HS51E2) and II type OR (HS5G6)
for three top ranked ligands.
Interaction with HS51E2
Compounds Interacting residues x-y-z coordinates
Thr 236, Arg 118,Typ 288, Ile 287, Lys 291, Ser 239, His 240, Gly 235,
Rotenone 36.699, -116.950, -64.771
Lys 232
Boeravinone J Gly 235, Thr 236, ser 239, His 240, Tyr 288, Ile 287, Lys 291, Thr 292 36.699, -116.950, -64.771
491
Abronisoflavone Gly-235, Thr 236, Ser 239, His 240, Ile 297, lys 291, Thr 292 36.699, -116.950, -64.771
Interaction with HS5G6
Esculetin Leu290, Gly39, Asn40, Pro285, Ile35, Met282, 57.386, 86.944, 96.553
Rosmarinic acid Phe101, Cys70, Phe105, Ser73, Ile69, Phe66, Ile44 57.386, 86.944, 96.553
Boeravinone J Leu 41, Ile 44, Phe 66, Ile 69, Cys 70, Ser 73, Phe 106. 57.386, 86.944, 96.553
Figure 2 : In-silico prediction of binding sites of boervinone J with class I type OR (HS 51E2)
Note : Figure 2. A shows the molecular visualization of boervinone J Complex with HS51E2 and 2.B shows
the ligplot of interacting residues of HS51E2 with boervinone J
Amino acid residues such as Gly 235, Thr 236, Ser 239, His 240, Tyr 288, Ile 287, Lys 291, Thr 292 were
observed as interacting residues for Boervinone J in class I type OR, the residues such as Leu 41, Ile 44, Phe 66, Ile
69, Cys 70, Ser 73, Phe 106 were contributed for molecular interaction with class II type OR in the current study
(refer table 3). As noted in earlier studies , B.diffusa shows remarkable medicinal properties in different solvent
extracts[18], and the current observation clearly infers the preference of solubility of compounds play a crucial
role in establishing interactions /ligand specificity with class I type OR (which sense water soluble odors) , class II
type OR ( sense air soluble odors). Here, compounds of B.diffusa prefers to interact with class I than class II type
OR due to preference of solubility.
Figure 3 : In-silico prediction of binding sites of boervinone J with class II type OR (HS5G6)
Note : Figure 3.A shows the molecular visualization of boervinone J Complex with HS5G6 and 3.B shows the
H-bond of boervinone J interaction with HS5G6
Conclusion
The preliminary study on prediction of binding sites of select phytoconstituents to class I (HS5E1) and class II
(HS5G6) OR showed significant molecular interactions. Though 15 ligands of various plant sources given chance,
the compound namely boeravinone J derived from B.Diffusa exhibited favorable interaction with both class I and
II type ORs. In that, boeravinone J prefers to interact class I OR (sense water soluble compounds) than class II
OR (sense air- soluble compounds) which denotes the preference of solubility of select compounds. The attempts
of identifying interacting residues of class I and II type OR with medicinally important compounds provide a
preliminary knowledge on molecular interaction of receptor-ligand complex and the type of interaction could guide
further to perform pre-clinical in-vitroandin-vivoassays and ultimately to design a drug for pulmonary diseases
and particularly for the utility of intra nasal drug delivery.
492
Acknowledgement:
I would like to acknowledge DST (WOS-B) scheme for the grant and K.S.Rangasamy College of Technology
for infrastructure and other facilities.
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493
Medicinal Plant Drugs for Asthma
Naveen.S1 , Prathap.M1 and Ananthalakshmi .R1*
(1Department of Biochemistry, Sacred Heart college, Tirupattur, Tamilnadu, India)
*ananthalakshmi@shctpt.edu
Abstract
Asthma is a chronic condition that causes inflammation and narrowing of the bronchial tubes, the passageways
that allow air to enter and leave the lungs. The major drug that is used for the asthma disease is Xopenex. It
has many possible side effects. They are Chest pain or discomfort, dizziness, fainting, fast, slow or irregular
heartbeat, increase in urine volume, light headedness, persistent vomiting, pounding or rapid pulse, shakiness,
seizures. These are the major causes or side effects that can cause while taking this drug. We can use some
medicinal plants like Adhatoda, Ginkgo biloba, coltsfoot, Maritime Pine Bark, Turmeric, Chinese Skull cap,
Grindelia, Coleus Forskohlii, Licorice, Garlic, Red clove, Yellow Dock and so many for the treatment of
asthma. It can cure asthma without any side effects. Hence, we can use these plants to prepare a medicine to
cure asthma. By using these we can get good results without getting any side effects. By using these plants we
can produce drugs for asthma or we can use them as such as medicine.
Key words: Medicinal plants, Xopenex, Side effects, Asthma.
Introduction:
Asthma is one of the old disease in earliest recorded reference to respiratory distress a disorder characterized
by noisy breathing (wheezing?) is found in China in 2600 BC.The Babylonian Code of Hammurabi recorded
symptoms of breathlessness: If a mans lungs pant with his work.(1792-1750 BC).Hippocrates (~400 BC) was
the first to use the term Asthma (Greek for wind or to blow) for panting and respiratory distress. He is
considered to be the physician who identified the relationship between the environment and respiratory disease
correlating climate and location with illness. Some suggest he was the first allergist.
When Alexander the Great invaded India, smoking the herb stramonium (an anticholinergic agent related to
ipratropium and tiotropium currently used in inhalers) was used to relax the lungs.Roman doctors described asthma
as gasping and the inability to breathe without making noise.They noted if from running or any other work,
the breath becomes difficult, it is called asthma.Pliny the elder (~ 50 AD) observed that pollen was a source of
respiratory difficulty and recommended the use of ephedra (forerunner of ephedrine) in red wine as an asthma
remedy. Unfortunately, he also suggested that drinking the blood of wild horses and eating 21 millipedes soaked in
honey could help.[1]
The Jewish Talmud (200-500 AD) counseled drinking three weights of hiltith, a resin of the carrot family as
a therapy for asthma. Maimonides (1135-1204 AD), Jewish scholar and Saladins physician treated the Egyptians
son for asthma. His Treatise on Asthma prescribed rest, good personal hygiene and environment, avoidance of
opium, a small quantity of wine and a special diet. Nuts, fruit, milk, cool vegetables and legumes (peanuts are a
member of this family) were forbidden, while The soup of fat hens was considered beneficial.[2]
Tobacco introduced from the Americas to Europe (1500s), was used to induce coughing and expectorate
mucus. In Central America, Aztecs ingested an ephedra containing plant to clear mucus and, in South America,
Incas treated asthma with a cocaine-like dried leaf. In the 1800s, Arsenic was prescribed for respiratory conditions.
In the early 1900s, allergy immunotherapy was first introduced to treat asthma.[3]
Asthma medicines of the 1940s and 1950s consisted of epinephrine injections (adrenaline) and aminophylline
tablets or suppositories. In the 1960s oral combinations were the staples of chronic therapy. Inhalation of
epinephrine (Primatene) and isoproterenol (Isuprel) were used as rescue agents. Oral prednisone was and continues
494
to be prescribed for severe disease.Since the Allergy and Asthma Medical Group & Research Center was founded
in 1969, many therapeutic advances have occurred. Inhaled bronchodilator medications are less likely to stimulate
the heart and are available in both short and long acting formulations. Inhaled corticosteroids target the underlying
inflammation and minimize the potential cortisone side effects seen with the tablet and liquid products. Our clinical
research department is currently actively evaluating new asthma therapies that promise to further benefit patients.[4]
Though asthma has been a known entity for over two and a half millennia, nearly 25 million people in the United
States still suffer from this condition. However, we have come a long way in understanding its causes and triggers
and have made large strides in our ability to treat and control it. We pledge to continue to give our best efforts to
expertly and compassionately care for our patients with asthma. [5]
Main causative factors of Asthma:
Asthma is caused by environmental and genetic factor which can influence how severe asthma is and how well
it responds to medication. Some environmental and genetic factors have been confirmed by further research, while
others have not been.[6]
Xopenex:
Asthma can be cured by a specialized medications one of that major drug is xopenex. Xopenex (levalbuterol)
is a short-acting bronchodilator that relaxes muscles in the airways and increases air flow to the lungs.Xopenex
inhalation is used to treat or prevent bronchospasm in people with reversible obstructive airway disease.Xopenex
solution is for use in adults and children 6 years of age and older. Xopenex HFA aerosol is for use in adults and
children 4 years of age and older.[7]
Common Xopenex side effects may include:
yy Dizziness, nervousness, tremors
yy Runny nose, Sore throat
yy Chest pain or tightness, irregular heartbeats
yy Pain; or
yy Vomiting. [8]
Mechanism of Action
Activation of 2-adrenergic receptors on airway smooth muscle leads to the activation of adenylate cyclase and
to an increase in the intracellular concentration of cyclic-3, 5-adenosinemonophosphate (cyclic AMP). The increase
in cyclic AMP is associated with the activation of protein kinase A, which in turn inhibits the phosphorylation of
myosin and lowers intracellular ionic calcium concentrations, resulting in muscle relaxation. Levalbuterol relaxes
the smooth muscles of all airways, from the trachea to the terminal bronchioles. Increased cyclic AMP concentrations
are also associated with the inhibition of release of mediators from mast cells in the airway. Levalbuterol acts
as a functional antagonist to relax the airway irrespective of the spasmogen involved, thus protecting against all
bronchoconstrictor challenges. While it is recognized that beta2-adrenergic receptors are the predominant receptors
on bronchial smooth muscle, data indicate that there are beta-receptors in the human heart, 10% to 50% of which
are beta2-adrenergic receptors. The precise function of these receptors has not been established. However, all beta-
adrenergic agonist drugs can produce a significant cardio vasculareffect in some patients, as measured by pulse rate,
blood pressure, symptoms, and/or electrocardiographic changes. [9]
Ingredients
Active ingredient: Levalbuterol hydrochloride
Inactive ingredients: Sodium chloride, sulfuric acid, water and nitrogen
Drugs affect xopenex:
yy Digoxin (Lanoxin, Lanoxicaps);
yy Epinephrine (Adrenaclick, Epipen, Twinject);
495
yy Another inhaled bronchodilator;
yy An antidepressant such as Amitriptyline (Elavil, Vanatrip, Limbitrol), Doxepin (Sinequan, Silenor),
Nortriptyline (Pamelor), and others;
yy A beta blocker such as Atenolol (Tenormin, Tenoretic), Carvedilol (Coreg), Labetalol (Normodyne, Trandate),
Metoprolol (Dutoprol, Lopressor, Toprol), Nadolol (Corgard), Nebivolol (Bystolic), Propranolol (Inderal,
InnoPran), Sotalol (Betapace), and others;
yy A diuretic (water pill) such as Bumetanide (Bumex), Chlorothiazide (Diuril), Chlorthalidone (Hygroton,
Thalitone), Ethacrynic acid (Edecrin), Furosemide (Lasix), Hydrochlorothiazide (HCTZ, HydroDiuril, Hyzaar,
Lopressor HCT, Vaseretic, Zestoretic), Indapamide (Lozol), Metolazone (Mykrox, Zaroxolyn), Torsemide
(Demadex), and others; or
yy An MAO inhibitor such as Furazolidone (Furoxone), Isocarboxazid (Marplan), Phenelzine (Nardil), Rasagiline
(Azilect), Selegiline (Eldepryl, Emsam, Zelapar), or Tranylcypromine (Parnate). [10]
When we use the drugs like xopenex we can face so many defects and side effects. If we use our natural herbs
or meedicinal plants we can get more benefits and too healthier.
Adhatoda vasica: (Family-Acanthaceae; Common name: Adusa).
The traditional healers are using this herb for the treatment of chronic Asthma. Adusa is known as Vasa or
Vasak in Sanskrit and is a reputed drug for Asthma mentioned in Ayurveda. Adhatoda vasica is considered in the
east to be the best possible treatment for all chest diseases and used in India as an expectorant, antitussive and in
other respiratory disease. It is also used widely to relieve asthma. Adhatoda vasica has been traditionally used in the
management of allergic disorders and bronchial asthma. Research performed over the last three decades revealed
that the alkaloids present in the leaves, vasicine and vasicinone, possess powerful respiratory stimulant activity . Its
essential oil exhibited Antitussive (cats), Expectorant (rats and rabbit), and Antiasthmatic (guinea pig) activity in
in-vivo experiments. [11]
Grindelia camporum: (Family-Asteracea; Common name: Great valley gum plant).
This is an expectorant herb with broncho spasmolytic activity. It is traditionally recommended for the treatment
of spasmodic respiratory conditions such as asthma and bronchitis. The British Herbal Pharmacopoeia 1983 lists
the specific indication as bronchial asthma with tachycardia. Californian Native Americans used grindelia not only
for skin infections but also for bronchial conditions where grindelia eventually gained the attention of the Catholic
missionaries. The dried leaf and flowering tops of grindelia were official in the United States Pharmacopoeia 1882-
1926, and have been in the National Formulary, 1926-1960.[12]
Curcuma longa: (Family-Zingiberaceae ; Common name: Turmeric/Haldi).
Curcuma longa has been known to Indians since centuries. It has been purported to have anti-inflammatory
actions. Anti-asthmatic property of Curcuma longa has been tested in experimental animal model of airway
hyperresponsiveness and has been documented to be effective in improving the impaired airways features. A study
from Journal of Alternative and Complementary Medicine confirms that curcumin is safe in several human trials and
inhibits a number of pro-inflammatory mediators that play an important role in asthma. [13]
Ginkgo biloba: (Family: Ginkgoaceae ; Common name: Maidenhair Tree).
Ginkgo biloba is a widely used complementary and alternative medicine option that most people associate
with combating memory loss. However, some may consider using it for asthma, too.Nearly 60% of patients with
asthma report using alternative asthma treatment and have tried complementary and alternative medicine (CAM)
practices to improve their asthma, though theres a lack of definitive evidence that CAM works for this condition.
In terms of treating Asthma, Ginkgo is thought to:
yy Decrease inflammation
yy Reduce hyper responsiveness of the airway.
yy Diminish Bronchospasm [14]
496
Tussilago farfara: (Family: Asteraceae ; Common name: Coltsfoot)
Tussilago has a soothing expectorant and antispasmodic action which is effective in the treatment of acute or
chronic bronchitis, irritating coughs, whooping cough and asthma. It reduces non-productive coughs and soothes
dry, irritable airways. Its has a role in most conditions of the respiratory tract, including the chronic states of
emphysema and silicosis. The mucilages supply the soothing action while the sesquiterpenes are spasmolytic. The
triterpene saponins in the flowers provide a stimulating expectorant action. [15]
Tussilago is a mild diuretic and has been used in cystitis. It contains appreciable levels of zinc which may
be responsible for the herbs anti-inflammatory and healing properties; the fresh, bruised leaves can be applied to
boils, abscesses and ulcers while compresses made from the fresh leaves may help to relieve joint pain. Antibacterial
activity has been documented against various Gram-negative bacteria including Staphylococcus aureus, Proteus
hauseri, Proteus vulgarisand Pseudomonas aeruginosa. [16]
Rumex crispus: (Family: Polygonaceae; common name: Yellow dock)
Drinking yellow dock tea may help you treat coughing by reducing the irritation in your throat and upper
airways. You may also find it useful in treating bronchitis and asthma. Yellow dock may help relieve the symptoms
of arthritis, rheumatism, gout, cystitis, water retention, urinary stones, ulcers and headaches. Historically, people
have used yellow dock root to treat venereal diseases, such as syphilis. Women have used it to help heal unbalanced
menstrual cycles, reduce menstrual pain and heavy menstrual bleeding and treat benign uterine tumors called
fibroids. [17]
Pinus maritima: (family: Pinaceae; common name: Maritime pine)
The pine bark extract, 55% was able to reduce inhaler doses over the treatment period and none of the patients
had to increase their inhaler dosage. Among the second, no-supplemented group, 6% was able to reduce inhaler dose
while nearly 19% had to increase their inhaler doses.The researchers measured cough, wheezing and other asthmatic
symptoms during the treatment period. The inhaler-only groups symptoms did not improve while the pine bark-
groups symptoms improved considerably. The researchers also found that the Pycnogenol groups average IgE
titers (measures allergic response in the bloodstream) decreased by 15% while the inhaler-only groups IgE titers
increased by 13%. [18]
Scutellaria baicalensis: (family: Lamiaceae; common name: Chinese skull cap)
The baical skullcap is now also suggested to treat all kinds of allergic conditions, with the evidence from
recent research. Thus this remedy is suggested for use in the treatment of conditions like asthma, conditions such
as hay fever, skin disorders such as eczema, and in the treatment of nettle rash. The remedy is thus mainly used for
the treatment of topical disorders of the skin, even if the anti-inflammatory properties will no doubt be much more
effective and useful in treating all sorts of digestive infections and problems associated with the gastrointestinal
tract. [19]
Plectranthus barbatus: (family: Lamiaceae; common name: Coleous)
Coleus forskohlii preparations used as eye drops are known to reduce eye pressure in glaucoma. Like all
good tonics, coleus directly stimulates digestion and is thought to assist in the absorption of nutrients in the small
intestine. Increased cellular cyclic AMP reduces histamine, making coleus beneficial in the treatment of allergies.
Coleus is also a bronchodilator with an anti-histamine action, making it useful in treating asthma . Conditions such
as hypothyroidism, eczema, psoriasis are also improved by using coleus, largely due to its ability to increase cyclic
AMP. It is a popular herb for angina and for the health of the heart.[20]
Conclusion:
As a conclusion , when we use these types of medicinal plants we can treat and cure the asthma. Also many
health benefits we can get by taking these drugs. When compare to the chemical or synthetic drugs, it contains very
little or no side effects so we can take these type of herbs and live a happier life
497
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Plectranthus barbatus. Natural Resources Conservation Service PLANTS Database. USDA. Retrieved 7 October 2015.
Dubey MP, Srimal RC, Nityanand S, et al. (1981). Pharmacological studies on coleonol, a hypotensive diterpene from Coleus
forskohlii. J Ethnopharmacol. 3: 113. doi:10.1016/0378-8741(81)90010-6.
Fal, P.L., Borges, C., Madeira, P.J.A., Ascenso, L.; et al. (2009). Rosmarinic acid, scutellarein 4-methyl ether 7-O-glucuronide
and (16S)-coleon E are the main compounds responsible for the antiacetylcholinesterase and antioxidant activity in herbal
tea of Plectranthus barbatus (falso boldo). Food Chem. 114: 798805. doi:10.1016/j.foodchem.2008.10.015.
Pizzorno, Joseph E.; Murray, Michael T. (2012). Textbook of Natural Medicine (4th ed.). Edinburgh: Churchill Livingstone.
p. 686. ISBN 9781437723335.
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499
An Ayurvedic Approach to Improve Quality of Life in Children with
Duchenne Muscular Dystrophy
M. Preethika, B.R.Lakshmi*, Shalini H. Kumar, A. Kalpana and N. Agila
A collaborative initiative between MDCRC, Coimbatore and Arya Vaidya Pharmacy, Coimbatore
Abstract
Duchenne Muscular Dystrophy (DMD) is a most common, severe neuromuscular and an X- linked recessive
disorder caused by mutations in the dystrophin gene which leads to physical disability. It is a progressive,
incurable disorder. The patients become wheelchair bound by early teens and they succumb to the disease
by late teens. Clinical suspicion of the disease is best to be followed up and confirmed by molecular (DNA)
diagnosis. Corticosteroids are prescribed as gold standard towards management of the disorder; however,
possible side effects are a major concern. It is imperative to find an alternative system of medicine to manage
DMD with less or no side effects and provide a better quality of life. In this attempt, PedsQL(Pediatric Quality
of Life Inventory) was used to understand if Ayurveda had a possible role in improving the Quality of Life in
children with DMD. The results are encouraging after a 2 years follow up.
Keywords - Duchenne Muscular Dystrophy, corticosteroids, Ayurveda, Quality of Life.
INTRODUCTION:
Duchenne Muscular Dystrophy (DMD) is an X-linked recessive, hereditary disorder caused by mutations in
the DMD gene, encoding dystrophin. It is a severe, progressive and incurable disorder manifested with muscular
weakness and hypertrophy of the calf muscles. Patients are usually wheelchair-bound before 14 years of age and
develop cardiac and respiratory complications which lead to death in the third decade of life. So far only early
introduction of corticosteroids allows for some years delay in the loss of independent ambulation, in the development
of skeletal deformities and cardiac and respiratory impairment (1).
However, none of these approaches so far has been effective in slowing disease progression and reducing
complications. For this reason new therapeutic approaches are strongly needed. In recent years Ayurveda, the Indian
traditional system of medicine, has been taken into account as a potentially complementary approach to conventional
strategies, particularly in chronic and degenerative diseases.
The fundamental principle of Ayurveda is that individual health is the outcome of different components,
including physical, psychological, emotional and social dimensions. Health is considered as the balance between
these different components, and disease is understood as a state of imbalance. Interventions can be aimed at
prevention as well as disease treatment. They are based on the use of natural remedies administered orally, and
physical therapies with medicated oils that are applied on the entire body or specific parts of it (2). Treatments are
usually personalized according to the specific psychophysical constitution (Prakriti) of the single patient and to
contextual features such as climate, demographics and social characteristics. Many remedies and treatments used
in Ayurveda acts on inflammation, a process which is altered in Duchenne Muscular Dystrophy (3). Ayurvedic
intervention was chosen to improve the overall quality of life of the patients in terms of their general health,
briskness, appetite and bowel movement amidst slowing the progressive nature of the disorder.
In this study we present our assessment to evaluate the role of Ayurvedic therapy in improving the Quality of
Life in a small group of patients with Duchenne muscular dystrophy.
Participants: Thirty males who were clinically suspected and confirmed by molecular analysis for DMD of
age groups 6-32 years, registered their willingness to avail the therapeutic advantages of Ayurveda offered by Arya
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Vaidya Pharmacy (AVP), Coimbatore through the coordination of Molecular Diagnostics, Counseling, Care and
Research Centre (MDCRC), Coimbatore. Informed consent was obtained from the parent of the patients and the
patient himself (who are older than 18 years of age) for availing the same. The base-line assessments were done
at the start of the medication. Customized medications were provided for the patients by AVP on a monthly basis,
for which assessments were made by an Arya Vaidyan (Ayurvedic assessment) and specialist doctors (modern
medicine), once in 3 months in MDCRC. After a period of 6 to 8 months, there were 18 children who were regular
in their participation to receive the Ayurveda medicine and to attend the multi disciplinary clinical care (MD care)
sessions. The dropouts were mainly due to difficulty in consuming the medicine, the distance to reach AVP/MDCRC
for assessments.
Study Design: The children were categorized into 3 groups based on their age 6 to 10 years (Group I 2
No.s), 11 to 15 years (Group II 9 No.s) and 16 years and above (Group III 7 No.s).
Outcome Measures - PedsQL 3.0 Neuromuscular Module: The Quality of Life of children affected
with DMD was assessed using the PedsQL (4), after modification and translation in the local language (Tamil) for
the ease of parents understanding. The original tool comprised of 25 items. The PedsQL was revised based on the
socio-cultural differences and for better clarity and content. The revised 59 item PedsQL comprises of questions
addressing a) About My/My childs Neuromuscular disease 33 No.s b) Communication (5 No.s related to the
patients ability to communicate with health care providers and others about his/ her illness) c) Family Resources (6
No.s related to family financial and social support systems) and d) Activities of Daily living (15 No.s). PedsQL was
administered to all the patients and their parents to understand the quality of life of the patients. The scoring was
then categorized under various domains and results consolidated. This structured questionnaire administered to the
parents and kids throws light on the health status of the children covering the physical, mental and emotional plane.
Procedure: The PedsQL was administered in the waiting room or in the clinical examination room. Team
members were trained in administering the tool to the patient/parent. The member was on hand to answer any study
subject questions about the PedsQL. All the patients and the parents of the patients responded to the questionnaire.
For each question, respondents are asked to indicate to what degree a statement has been true during the previous
assessment (five response categories, ranging from never to almost always). It was then scored and grouped
according to the specialties in which they addressed namely General health, Gastrointestinal function, pulmonology,
cardiology, psychosocial well being, motor functions, pain index, trunk control, fine motor skills and gross motor
skills. The outcomes have been recorded, scored and values compared to the baseline data throughout the study
period and are presented as improved, status-quo or deteriorated.
RESULTS:
A total of 18 patients including both ambulatory and non ambulatory with DMD responded to the questionnaire
together with one of their parents. Irrespective of current ambulatory status most patients general health, gastro
intestinal and pulmonary functions, motor functions was perceived as improved by their parents.
Group 1- Patients between 6 to 10 years
In this group, the parents felt the patients general health had a significant improvement (figure 1). The gastro
intestinal function had markedly improved for about 38% with regular bowel movements. Also the psychosocial
well being and motor function had improved. Patients view for the first group was not obtained because they were
too young to respond. The parents didnt report any ill health or problems regarding pain and cardiac health before
the intervention which was maintained throughout the study. There was a 25 % improvement with regard to the
pulmonary health in this group.
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Figure 1: Parents ratings of patients health and psychosocial well being (group I)
G.Health- General health, G.I function- Gastro intestinal function, Pulmo- Pulmonology,
Cardio- Cardiology, PSWB- Psychosocial well being
In addition, parents of ambulant patients indicated that frequent falls had decreased substantially in their
children and episodes of cold and cough has become a rare event.
Group 2- Patients between 11 to 15 years

Figure 3: Parents ratings of patients health and
Figure 2: Patients ratings of general health and psychosocial well being (group II)
psychosocial well being - (group II)
G.Health- General health, G.I function- Gastro intestinal G.Health- General health, G.I function- Gastro intestinal
function, Pulmo- Pulmonology, Cardio- Cardiology, PSWB- function,Pulmo- Pulmonology, Cardio- Cardiology, PSWB-
Psychosocial well being Psychosocial well being
Figure 2 depicts the patients view of age 11 to 15, in which the gastro intestinal function, motor functions of
hands and legs has markedly improved. There was a 20% improvement in cardiac health whereas least improvement
seen in pulmonary health. Only 8% of improvement was observed in psychosocial well being of the patients. The
fine motor skills has improved in both hands and legs compared to the gross motor skills which was maintained
throughout the study. The patient perceived a decline in the trunk control at this stage. The pain has reduced after
the first year but a slight increase was noted after the second year.
Figure 3 depicts the health status percent from the parents scoring which shows a marked improvement
in general health and Gastro intestinal function (30% improvement) which correlates with that of the patients
perception. There was an improvement in motor functions of hands of about 30% and cardiac function improved
nearly by 40% after 2 years of ayurvedic intervention. There was a minimal improvement in pulmonary health and
psychosocial well being in this group. The parents also perceived a decline in the trunk control similar to that of the
patients view. There is a decrease in pain noted after 2 years.
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Group 3- Patients above 16 years
Figure 5: Parents ratings of patients health and
Figure 4: Patients ratings of general health and psychosocial well being (group III)
psychosocial well being- (group III)
G.Health- General health, G.I function- Gastro intestinal G.Health- General health, G.I function- Gastro intestinal
function, Pulmo- Pulmonology, Cardio- Cardiology, PSWB- function, Pulmo- Pulmonology, Cardio- Cardiology, PSWB-
Psychosocial well being Psychosocial well being
In group III, the general health had a significant improvement in terms of the quality of life. Also there was
a gross improvement with regard to gastro intestinal and pulmonary functions with less frequent cold and chest
congestions even in cold weather in all the 7 patients in this group. Interestingly in both the early and late non
ambulatory patient group, the patients reported notable and gradual improvement in hand motor functions along
with the fine motor skills. There was a betterment followed by a steep decline in motor functions of legs and pain
relief. The trunk control was perceived as improved by the patients
In both the early (Group II) and late non ambulatory (Group III) patient group, the parents reported notable
improvement of cardiac functions. Initially there was a decline noted, later followed by a gradual improvement in
both general health and gastro intestinal function. In the late non ambulatory stage (Group III), the parents reported
a 19 % improvement in terms of psychosocial well being of the patients in contrast with a 4 % improvement
perceived by the patient himself. The motor function in legs was improved when compared to a decline in the hand
motor function and gross motor skills as perceived by the parents.
DISCUSSION:
Children with DMD are born into a life of chronic illness, inexorably increasing physical disability and
dependence, and knowledge of an inevitably premature death. Compared with the reference points of a healthy
peer, this life would appear bleak (5). There is no cure as of date except corticosteroids and International guidelines
for care and hence the quality of life in patients with DMD is compromised. To this date, there are very few studies
on participants with Ayurveda intervention. With these premises in mind, the present study was designed to measure
the HRQOL assessment in children, adolescents, and young adults with DMD in the age group of 6 to 32 years
with an Ayurvedic intervention. PedsQL that measures the general health, gastro intestinal function, pulmonology,
cardiology, psycho social well being, motor functions of hands and legs, pain, trunk control, fine and gross motor
skills of the patients was used for the assessment of Quality of life in children with DMD.
The direct analysis shows that the quality of life of children is improved in terms of general health for
the participants of all age groups irrespective of their ambulation status. The significant improvement in the
gastrointestinal function in terms of appetite, bowel movements, dysphagia could be attributed to the ayurvedic
intervention. Ayurveda based solutions for the improvement of gastrointestinal problems has been extensively
studied and reported by Metri et al., 2013 and Patel et al., 2011(6&7). The decreased cold and cough in the
participants, reflected in the pulmonary and cardiac health could be connected to the medication that is in agreement
with the findings of Meghwani et al., 2017 who have reported that an ayurvedic drug could reduce the incidence
of cold and cough (8). Landfeldt et al., in 2016 has reported that most of the patients are perceived as happy by the
caregivers indicating that influential domains of HRQOL remain intact through the disease progression which is
reflected in the present study where there was an improved psychosocial well being irrespective of the progression of
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the disease(5). This is further emphasized by the results of Uzark et al. (9) who have reported that older boys seem
to perceive better psychosocial QoL than perceived by their parents. In contrast, a negative association between
HRQOL and wheelchair use and ventilation support was also found in a study of 27 Italian patients with DMD
(10). In the study with Australian DMD patients it was observed that patient HRQOL is significantly negatively
associated with physical functioning but uncorrelated with psychosocial well-being (11).
The nature of the disease progression could be blamed for the reduced trunk control and gross motor function
in the early non ambulatory (Group II) and the late non ambulatory (Group III) participants amidst ayurvedic
intervention. A mild discrepancy was observed in the perception between the parent and the participant in group III.
There are several possible reasons for the observed discrepancy between patient and the parent proxy score. The
perspective of the affected individuals, who do not know any other life, may perceive their well-being differently as
they do not have the reference as a healthy individual because DMD is a genetic disorder (5).
The PedsQL is positively associated with age, the disease stage, number of days spent outside home, and
the attitude of the local community. Affordable and easy access to medical devices and aids is crucial to maintain
better quality of life in patients with DMD as their physical functioning deteriorates. Studies are needed to further
elucidate changes in QoL over time and to evaluate the impact of targeted interventions to improve outcomes.
Overall, children with DMD perceived high quality of life inspite of the disease progression across all age groups.
REFERENCES:
Bushby K, Muntoni F, Urtizberea A , Hughes R & Griggs R (2004) Neuromuscular Disorders 14, 526534
Sharma H, Chandola H M,Singh G & Basisht G(2007) The Journal of Alternative and Complementary Medicine. 13, 1135-
1150.
Basnyat S & Kolasinski S L Complementary and Alternative Medicine 10.1007/s11926-014-0435-6
Iannaccone S T, Hynan L S, Morton A, Buchanan R, Limbers C A, Varni J W & the AmSMART Group (2009) Neuromuscul
Disord. 19, 805-812.
Landfeldt E, Lindgren P, Bell C F, Guglieri M, Straub V, Uller H L & Bushby K (2015) Developmental science and child
neurology DOI: 10.1111/dmcn.12938
Metri K, Bhargav H, Chowdhury P & Koka PS (2013) J Stem Cells. 8, 115-29.
Patel K, Gupta S N & Shah N (2011) AYU 32, 55-8.
Meghwani H, Prabhakar P & Mohammed S A (2016) J Ethnopharmacol 197, 184-194.
Uzark K, King E & Cripe L (2012) Pediatrics 130, 55966.
Baiardini I, Minetti C & Bonifacino S (2011) J Child Neurol 26, 70713.
Bray P, Bundy AC, Ryan MM, North KN & Burns J (2011) J Paediatr Child Health 47, 55762.
504
Effect of supplementation of Hibiscus rosasenensis leaves and flowers
in Swiss albino mice
Ramadevi, A1, Shyama, K1, Jasmine Rani. K1, Ally .K1, And Koorse, K.G2
Department of Animal Nutrition, College of Veterinary and Animal Science, Mannuthy, Kerala
1
2
Department of veterinary pharmacology and toxicology, College Of Veterinary and Animal Science, Mannuthy, Kerala
Abstract
Hibiscus (Hibiscus rosasenesis) is one of the well-known medicinal plant in sub-tropic countries like India.
It is often used in herbal preparation in veterinary medicine due to its positive effect.The present study was
carried to study theeffect of hibiscus on glucose and cholesterol level in mice at 1 and 2 %. Thirty Swiss albino
mice of 15 4 gram were used as experimental group. Animals were divided into 5 groups of 6 mice in each
treatment. Mice ofT1fed on basal diet kept as control, T2and T3 on hibiscus flower at 1 and 2% respectively,
T4 and T5 on hibiscus leaves at 1 and 2% respectively for a period of 21 days. The result reveals that mice
supplemented with 2 % driedhibiscus flowers and leaves powder shown significant (p < 0.05)decrease in
serum cholesterol level.
Introduction:
Diabetics and hyperlipidaemia is one of the most common prevailing condition in sub-tropic countries like
India. Due to improper food habits, lack of exercise and side effects of synthetic compound used against these
conditions necessitates an alternative source. Hibiscus is a perennial shrub common in sub tropic countries,
which Belongs to family Malvaceae, genus Hibiscus, species rosasinensis proved to have many medicinal values.
Thiamine, riboflavin, niacin and ascorbic acids, calcium oxalate, cyaniding, quercetin, hentriacontane were some
of the compounds isolated from hibiscus plants. Many proved that the plant exhibited anticonvulsant, antitumor6,
antispasmodic, antihyperlipidiaemia5, anti diabetic 7 and antioxidant2 potential activities. Due to its positive effect
it is commonly used in many pharmacological preparation. The present study was carried to investigate the effect of
supplementing their leaves and flowers in the diet of Swiss albino mice on glucose and total cholesterol.
Materials and method
Experiment design:
The present study was carried out for a period of 21 days. Thirty Two week old male Swiss albino mice
weighed around 19 4 grams were selected and maintained at laboratory animal house at college of veterinary and
animal science, Mannuthy. Animal were divided into 5 groups of 6 mice each. Each group of animal were kept in
individual polypropylene cage under standard management condition prevalent in the house. All the mice belong
to all the groups were fed with basal diet (ICAR, 2013). Animal belong to T1 group fed with basal diet alone, while
those of T2, T3, T4 and T5 were supplemented with dried hibiscus flower and leaves at 1 % and 2 % respectively. All
the animals were fed ad libitum and clean drinking water was provided all the time till the end of the experiment.
Collection of sample:
Flowers and leaves of Hibiscus rosasenensis were collected and were dried at 20 25 C and finely powdered.
Packed in a sterile polyethylene bags and were used later.
Biochemical analysis:
Blood was collected by retro- orbital sinus puncture using anticoagulant coated capillary tube in a sterile
Eppendorf tubes and centrifuged at 2500rpm for 10 mins for serum separation. Serum samples were analysed
for total cholesterol and glucose concentration estimated by using Semi Automated Biochemical Analyser
(Master T). The standard biochemical kits used for these assays were purchased from M/s. Agappe Diagnostics
Limited,Ernakulam,and Kerala. Experiments were conducted by following ethical norms suggested by CPCSEA,
Ministry of Social Justice and Empowerment, Government of India.
505
Data were statically evaluated by using one way ANOVA. Wherever the ANOVA values were found to be
significant Duncan method was applied (SPSS computer software). The value were considered significant when P
< 0.05.
Result and discussion:
Effect on body weight:
After 21 days of feeding trail no significant difference (P> 0.05) was obtained in body weight of mice (table1)
Effect on blood glucose:
After 21 days, 72.14 4.81 mg/ dl,69.90 6.13mg/ dl,65.65 2.04mg/dl, 50.93 3.43 mg/ dlof T2, T4, T5
and T3 respectively showed similar glucoselevel with that of control (T1) 96.64 4.20 mg /dl. All the values under
normal glucose respect to that species (table 1)
Effect on total cholesterol:
Total serum cholesterol decreased significantly 70.54 0.43,70.13 1.17, 64.34 1.31 under the influence of
T3, T4, and T5respectively compared with that of control (T1) 132.34 4.35 and T282.09 2.10 were noted. (Table 1)
Means with different superscript differs significantly, P < 0.05, Body weight and glucose doesnt differ significantly.
Table 1. Effect of dried hibiscus leaves and flowers on change in body weight, glucose and total cholesterol
of experiment groups of mice after 21 days
(Values are mean SE from 6 animal in each group)
Treatment Body weight (grams)
Glucose (mg/ dl) Total cholesterol(mg/ dl)
Groups Initial Final
T1Basal diet 15.8 16.3 96.64 4.20 132.34 4.35b
T2Basal diet +1 % flower 18.2 18.37 72.14 4.81 82.09 2.10c
T3Basal diet + 2 % flower 15.4 20.1 50.93 3.43 70.54 0.43a
T4Basal diet + 1 % leaves 20.8 21.83 69.90 6.13 70.13 1.17a
T5Basal diet + 2 % leaves 19.1 18.9 65.65 2.04 64.34 1.31a
Conclusions:
The result of the present study indicated that diet supplemented with 2 % of dried hibiscus flowers and leaves
powder has shown decreased serum total cholesterol level in Swiss albino mice. Hence, 2 % of dried hibiscus flower
and leaves powder can be used in diet to reduce total cholesterol level.Future research required to know other effect.
Reference:
Kasture, V.S., Chopde, C.T., Deshmukh, V.K. Anticonvulsive activity of Albizzialebbeck, Hibiscus rosasinensisand Butea
monosperma in experimental animals. J. of Ethnopharmacol, 71(2000) 6575.
Kumar, V., Mahdi, F., Khanna, A. K., Singh, R., Chander, R., Saxena, J. K, Mahdi, A. A., Singh, R.K. Antidyslipidemic
and Antioxidant Activities of Hibiscus rosasinensisRoot Extract in Alloxan Induced Diabetic Rats. Indian. J. of Clin.
Biochem.2012.
Moqbel, F.S., Naik, P.R., Najma, H. M. and Selvaraj. S. antidiabetic properties of Hibiscus rosasinensisL. leaf extact fraction on
non- obese diabetic (NOD) mouse. Indian .J. Exp. Biol, 49 (2011) 24.
Sachdewa, A. and khemani, L.K. A preliminary investigation of the possible hypoglycemic activity of Hibiscus rosasinensis,
Biomed Environ Sci,12 (999) 222.
Sachdewa, A. and khemani, L.K. Effect of Hibiscus rosasinensisLinn. Ethanol flower extract on blood glucose and lipid profile
in streptozotocin induced diabetes in rats, J. ethanopharmacol, 89 (2003) 61.
Serrame, E., Lim Sylianco, C.Y. Anti-tumour promoting activity of decoctions and expressed juices from Philippine medicinal
plants. Philippine J. of Sci, 124 (1995) 275281.
Venkatesh, S., Thilagavath, J. and Shyamsundar, D. Anti-diabetic activity of flowers of Hibiscus rosasinensis. Fitoterapia79
(2008) 79.
506
Phytochemical Screening, Total Phenolic Content and Antioxidant
Activity of Ungerminated and Germinated Sunflower Seeds
SA.Thamilovia, L.Uthira, M.Malathi
PSG College of Arts and Science, Affiliated to Bharathiar University, Coimbatore, Tamil Nadu, India.
ovia2611@gmail.com
Abstract
Aim: Sunflower seeds have a long history for use in traditional system of medicine. Sunflower seeds, a
nutrient dense food has been found to have a potential therapeutic property. The purpose of this study is
to assess phytochemical components, total phenol content and antioxidant activity of raw and germinated
sunflower seeds.
Methodology: The aqueous extracts of raw and germinated seeds were screened for the presence of
phytochemical. The total phenol content and antioxidant activity was determined by method of Folin-Denis
and 2,2-diphenyl-1-picryl-hydrazyl-hydrate radical (DPPH) respectively.
Results: Phytochemical screening of the extracts showed the presence of flavonoids, alkaloids, phenols,
tannins, terpenoids and steroids in both the extracts. The total phenol content was 1920g pyrogallolEq/100g
in germinated
While it was 1700g pyrogallolEq/100g in raw sunflower seeds sample. DPPH assay showed a maximum
inhibition of 75% and 70.8% in the germinated and raw sunflower seed extract respectively at a concentration
of 500g/ml.
Conclusion:Ungerminated Sunflower seeds had higher concentration of flavonoids, phenols, tannins,
terpenoids and steroids than alkaloids when compared to germinated seeds. However germinated seeds
showed relatively higher amount of phenols and inhibition of DPPH radical scavenging than ungerminated.
Keywords: Sunflower Seeds, Phytochemical Screening, Total Phenolic Content, Antioxidant Activity.
1. Introduction:
Demand for oil seeds has been increasing owing to their rich phytochemistry (1).Sunflower (Helianthus
annuus L.) is one of the most important oilseed crop grown in the world (2).Sunflower plant is a miraculous oilseed
crop which seeds, a nutrient dense food has been found to have a potential role in chronic inflammatory conditions,
bacterial and fungal infections, cardiovascular diseases and even cancers (3). The objective of the present study
is to analyze the phytochemicals, total phenolic content and antioxidant activity of ungerminated and germinated
sunflower seeds.
2. Materials and Methods:
The methodology pertaining to the study Phytochemical Screening, Total PhenolicContent and Antioxidant
Activity of Ungerminated and Germinated Sunflower Seedsis discussed as follows.
2.1. Collection of sunflower seeds:
The sunflower seed (Helianthus annuus L.) were purchased from the local market. Sunflower seed was selected
due because of a long history for use in traditional system of medicine (4). The seeds were then cleaned, sorted to
remove the impurities present in them. The seed were grounded to a fine powder using a mixer. The resulting seed
powder was then stored at room temperature in air tight container and utilized.
2.2 Germination of sunflower seeds:
The ungerminatedseedswere made into a powder as such. For the germinated seed powder, the seeds were
washed twice to remove the dirt. The sunflower seeds were soaked overnight for 8 hours and in the next morning it
was filtered and washed again. Then the seeds was tied in a muslin cloth and kept in an air tight container for 10-12
hours. After the germination of the sunflower seeds, it was dried and then powdered to prepare the extract.
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2.3 Preparation of aqueous extract of ungerminated and germinated sunflower seeds:
Five gram of the ungerminated powder of the sunflower seeds was weighed. It was dissolved in 100ml of
distilled water which was kept in the boiling water bath for 5-10 mins. This was then filtered through Whatmanfilter
paper No:1 in a
2.4 Phytochemical screening and Determination of Total Phenols Content of ungerminated and
germinated sunflower seeds:
Phytochemical screenings were performed to investigate the presence of various chemical components present
in the seeds. Different types of tests were performed for the screening of flavonoids, alkaloids, phenols, tannins,
terpenoids, steroids and saponins.Following the standard procedure(6).
2.5 Determination of total phenols:
The total phenols content was determined by adopting the method of Folin-Denis described in the (7). In
test tubes was added 1ml of ungerminated and germinated sunflower seeds extract andthe volume was made to 3
mL with distilled water. To it Folin-Denis 0.5ml and after 3 min 2 ml of 20% sodium carbonate was added. The
absorbance reading was measuredafter keeping the test tubes in warm water bath for 5 mins at 715 nm using a
spectrophotometer. The calibration curve was obtained in a similar manner to that described for samples using
standards solutions of pyrogallol. The results were expressed as mg of pyrogallol equivalents per gram of dry
material.
2.6 Antioxidant activity of ungerminated and germinated selected seeds:
DPPH Radical scavenging activity:
Buchner funnel and centrifuged at 1500 rpm for 10 mins. The supernatant was collected and filtered again for
the clear solution. The aqueous extract was stored at 18 degree C until it was used for further study. The germinated
sunflower seeds extract was also prepared in the same way (5).
The antioxidant activity of seed extract was determined in vitro method (DPPH free radical scavenging assay)
by the method proposed by (8). The free radical scavenging capacity of the 1ml aqueous extract of ungerminated and
germinated sunflower seedswas determined using DPPH. To each ofungerminated and germinated sunflower seeds
sample (1 ml), freshly prepared DPPH (0.4Mm) and ethanol were added in the amount of 0.5 and 1 ml, respectively.
The mixture was shaken and allowed to stand at room temperature for 30 mins. The color from deep violet color
in solution changed to colourless or pale yellow when neutralized. Ascorbic acid was used as a reference standard.
Control sample was prepared without adding any extract and ethanol was used as blank.Antioxidant capacity was
measured by recording the absorbance at 517 nm using a spectrophotometer.
3. Results and Discussion:
3.1 Phytochemical screening of ungerminated and germinated sunflower seeds:
The results for phytochemical screening of the seed extract is in Table I
Table I: Preliminary Phytochemical analysis ofaqueous extract of seeds of Helianthus annuus (+Present,
- Not Present):
Phytoconstituents Ungerminated sunflower seeds Germinated sunflower seeds
Flavonoids +++ +++
Phenols +++ ++
Tannins +++ +++
Alkaloids ++ +++
Saponins - -
Steroids +++ +
Terpenoids +++ ++
508
The results of the phytochemical analysis showed that Flavonoids and tannins were of the same level in
ungerminated and germinated sunflower seeds. In the case of Phenols and terpenoids, the colour intensity was
less in germinated than ungerminated sunflower seeds. In steroids the intensity of colourwas much lower in
germinated than ungerminatedsunflower seeds. With regard to alkaloids the intensity was higher in germinated than
ungerminated sunflower seed.Saponins was absent in both the ungerminated and germinated sunflower seeds extract
(Table I). The variedphytochemical constituents present in theseed extractsuch as flavonoids, alkaloids, saponins
possess biological activity against microbes(9). The results of the phytochemical analysis showed in a study by
(10) alkaloids, flavonoids, tannins, saponins,phytosterol, steroids, fixed oils and fats were reported to be present in
theseeds of Helianthus annuusextract .
3.2 Determination of Total Phenols Content of ungerminated and germinated sunflower seeds:
Phenolic compounds are widely distributed in the plant kingdom. These compounds serve as importantantioxidants
because of their ability to donate a hydrogen atom or an electron in order to form stable radical intermediates.
Hence, they prevent the oxidation of various biological molecules (11).The total phenol content results showed that
the aqueous extract of germinated sunflower had higher content (1920g pyrogallolEq/100g)than ungerminated
sunflower seeds (1700g pyrogallolEq/100g).
3.3 Antioxidant activity of ungerminated and germinated sunflower seeds:DPPH Radical scavenging
activity:
The scavenging activity (%) on DPPH radical of the aqueous extracts ofungerminated and
FigureI:DPPH Scavenging Activity of ungerminated and germinated sunflower seeds:
S- standard, US-ungerminated sunflower seeds, GS-germinated sunflower seeds germinated sunflower seeds
samples on the DPPH radical are shown in Figure I. The results showed that theaqueous extract of germinated
sunflower seeds possessedhigher scavenging ability from 55.16% to75% with increasing extract concentration from
100 to 500g/ml on DPPH radical. Meanwhile, the ungerminated sunflower seeds posses scavenging ability from
45% to 70.8% with same concentration.
Conclusion:
Aqueousextracts of seeds of Helianthus annuuswere found tohave high phytochemicals. These seeds extract
possess abroad spectrum of DPPH scavenging activity and total phenol content ere more in germinated than
ungerminated. Owing to these phytochemicals and antioxidant properties, the above plantcan be considered as
natural source of dietary antioxidants. Sunflower seeds can be germinated before the usage to increase the nutrient
content.
509
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extractions using solvent and aqueous enzymatic methods.International Journal of Food Science and Technology, Vol. 44
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thecanadianencyclopedia.com
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International Journal of Research and Development in Pharmacy and Life Sciences, Vol. 3 (3), 967-972
ShivaniSaini, Sunil Sharma (2013) Antidiabetic effect of Helianthus annuusL., seeds ethanolic extract in streptozotocin-
nicotinamide induced type 2 diabetes mellitus. International Journal of Pharmacy and Pharmaceutical Sciences, Vol 5 ( 2),
2013
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Vol 5(3)
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its In Vitro Antioxidant Activity. World Journal of Pharmacy and Pharmaceutical Sciences,Vol 4 (08), 952-958.
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effects and therapeuticpotential Indian Journal Pharm, Vol 33: 2-16
RajakannuSubashini&SritharanUmamaheswariRakshitha (2012),Phytochemical Screening, Antimicrobial Activity and In
Vitro Antioxidant Investigation of Methanolic Extract of Seeds from Helianthus annuus L. Chemical Science Review and
Letters, Vol 1(1), 30-34
Igbinosa O O, Igbinosa E O &Aiyegoro O A (2009) African Journal of Pharmacy and Pharmacology, Antimicrobial activity
and phytochemical screening of stem bark extracts from Jatrophacurcas (Linn). AfricanJournal of Pharmacy and
Pharmacology.Vol 3 (2):058-062
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510
Identification of Bioactive Compounds and Synthesis of Silver
Nanoprticles From Avicennia Marina (Forssk.)Vierh and Its Anti-
inflammatory Properties - In vitro Studies
R.Vijayaraj*, N. Sri Kumaran, and M. Jayaprakashvel
Department of Marine Biotechnology, AMET Deemed to be University, Kanathur, Chennai 603112, Tamilnadu, India.
Abstract
The Avicennia marina (forssk.) vierh, commonly known as grey mangrove, it has been used as traditional
medicine for decades with multifunctional biological activity. In the present study, the anti-inflammatory
properties were comparatively studied by crude extract and synthesized of silver nanoprticles from A. Marina.
The bioactive compounds from A. marina were identified by GC-MS. The synthesize silver nanoparticles
characterized by using UV- spectrophotometry, SEM, FTIR. The in vitro anti-inflammatory & anti-oxidant
assays were followed by the standard methods. In GC-MS analysis, the A. marina leaves revealed the existence
of Squalene (41.30), Phytol (28.03) and Dodecanoicacid (18.55), D-Allose (18.33). The AgNPs A. marina
was characterized by UV-Vis spectral analysis shows a maximum absorption peak at 460.00 nm. The FTIR
spectra of AgNPs exhibited prominent peaks at 3697cm-1,1761cm-1,1390cm-1, 831cm-1 which are associated
with OH stretching, C=C stretching, CH stretching respectively. The SEM analysis of AgNPs clearly showed
the clustered and irregular shapes, mostly aggregated and having the size of 25-80 nm. The in vitro anti-
inflammatory properties, the crude and their synthesized AgNPs showed the protein denaturation inhibition of
68.92% and 72.1% and Proteinase activity inhibition 68.9% and 72.9%. The plant extract exhibited significant
DPPH radical scavenging activity value 62.7 - 98.5 g/mL. In conclusion, this study exhibit that the A.
marina contains various bioactive compounds and recommended as anti-inflammatory and pharmaceutical
importance.
KEYWORDS: Avicennia marina, GC-MS analysis, UV-Spectroscopic analysis, SEM, FTIR analysis.
INTRODUCTION
Medicinal plants are widely used by the traditional medicinal practitioners to cure different diseases due to
their world-wide availability and fewer side effects. The herbal medicines occupy distinct position right from the
primitive period to present day. Medicines that are used today are not definitely the same as those that were used in
ancient times. India has a wealth of medicinal plants most of which have been traditionally used in Ayurveda, Unani
systems of medicine and by tribal healers for generations. The medicinal value of this plant lies in the bioactive
phytochemical constituents that produce definite physiological effect on human body. These natural compounds
signify the base of modern drugs as we use today. Phyto components are the natural bioactive compounds found
in the plants. These phyto components work with nutrients and fibers to form an integrated part of human defense
system against various diseases and stress condition.
The systematic study of mangrove species has revealed that crude extracts of different plantParts in different
solvents and compounds isolated from them exerted potential antibacterial, antifungal, antiviral and antioxidant
activities. The Avicennia marina is commonly known as gray mangrove tree classified in the plant family Acanthaceae
and is commonly used for the treatment of ulcers, rheumatism, smallpox, and other ailments1. Some studies were
already reported for A. marina against parasites, fungi, and bacteria.
Silver is well known for possessing an inhibitory effect toward many bacterial strains and microorganisms
commonly present in medical and industrial processes 2. In medicines, silver and silver nanoparticles have a
ample application including skin ointments and creams containing silver to prevent infection of burns and open
wounds3. Silver nanoparticles are reported to have Anti inflammatory activity 4. The added advantage of using
plants is phytoconstituents which act as capping agents, thereby conferring the silver nanoparticles with additional
pharmacological properties.
511
Hence, the present study was made to investigate identification of bioactive compounds and synthesis of silver
nanoprticles from Avicennia marina (forssk.) vierh and its in vitro anti inflammatory properties.
Collection and Authentication of Plant
The leaves of Avicennia marina were collected from the Muthupet mangrove forest which is located at the
southern end of the Cauvery delta, Tamilnadu, India. It was taxonomically identified and authenticated by Dr. S. John
Britto, Director, The Rapinat Herbarium and Centre for Molecular Systematics, St. Josephs College (Autonomous),
Tiruchirappalli, Tamilnadu, India.
Preparation of Extract
The coarse powder plant material was extracted with ethanol by using soxhlet apparatus. The solvent were
removed under reduced pressure to get crude extract. Standard methods were used for preliminary phytochemical
screening of the extract, which was performed to know the phytoconstituents in the extract5.
Gas Chromatography-Mass Spectrometry
GC-MS analysis of the sample was performed using a Shimadzu GCMS-QP2010 gas chromatograph mass
spectrometer interfaced with a Turbo Mass quadrupole mass spectrometer, fitted with an Rtx-5 fused silica capillary
column (30 X 0.25 mm, with 1 Cm film thickness). The oven temperature was programmed from 100C to 320 C
at 100C/min and a hold for 10 min. Helium was used as carrier gas at flow 1.0 mL/min. The injector temperature
was 250 C, injection size 1 L neat, with split ratio 1:10. The interface and MS ion source were maintained at 3200
Cand 2000 C respectively and the mass spectra were taken at 70eV with a mass scan range of 40-700 amu (atomic
mass unit).
Identification of Compounds
Interpretation of mass spectrum of GC-MS was conducted using the mass spectral database of National Institute
of Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component
was compared with the spectrum of the known components stored in the NIST library.
Preparation OF 1mM Silver Nitrate Aqueous Solution
An accurately weighed 0.017g of silver nitrate was dissolved in 100 mL of double distilled water and
stored in amber colour bottle for further use.
Synthesis of silver nanoparticles from ethanolic extraction of A.marina
The synthesis of silver nanoparticle was performed by Rajasekar et al.,6. 5 mL of the ethanolic leaf extract
of A. marina was taken in the conical flask separately and placed on a magnetic stirrer with hot plate. To
this 50 mL of 1 mM AgNO3 solution was added drop wise with constant stirring of 120 rpm at 50-60C. The
colour change of the solution was checked periodically. The colour change of the medium from colour less to brown
after 5 hours was observed which indicated the formation of silver nanoparticles. It showed that aqueous silver
ions could be reduced by the ethanolic extract of A. marina to generate extremely stable silver nanoparticles.
Characterization Techniques
UV-VIS Spectroscopy
The silver nanoparticles were characterized in a Shimadzu-1800 UV-VIS Spectrophotometer. The optical
properties (absorbance) of the sample were evaluated at the wavelength range of 300-600 nm. The double distilled
water used as a blank reference.
Scanning Electron Microscope
Thin films of the sample were prepared on a carbon coated copper grid by dropping a very small amount of the
sample on the grid. Extra solution was removed using a blotting paper and then the films on the scanning electron
microscopic grid were allowed to dry by putting it under a mercury lamp for 5 min.
512
Fourier Transform Infra-Red Spectroscopy
To remove any free biomass residue or compound that is not the capping ligand of the nanoparticles, after
complete reduction, silver nanoparticles were concentrated by repeated centrifugation (3 times) of the reaction
mixture at 15,000 rpm for 20 min. The supernatant was replaced by distilled water each time. Thereafter, the purified
suspension was freeze dried to obtain dried powder. Finally, the dried nanoparticles were analyzed by ALPHA FT-
IR Spectrometer (from Bruker, Germany) for the detection of different functional groups by showing peaks from
the region of 4000 cm-1to 500 cm-1.
Inhibition of Protein Denaturation
Inhibition of protein denaturation was evaluated by the method of Mizushima et al., (1968)7
Protease Inhibition Assay
Inhibition of trypsin was evaluated by the method of Sakat et al. (2010)8.
Determination of Antioxidant Efficacy
DPPH radical assay the DPPH free radical scavenging assay was performed by Kikuzaki (1993)9
Medicinal plants are the major source of therapeutic agents to cure human diseases. Recent researches in
medicinal and aromatic plants made the health-care enhancement for the purpose of humankind. The vast floral
resources of mangrove forest are best known for their medicinal properties. Vast studies have been made on mangrove
forest plants and their bioactive compounds during these days due to the medical importance. The mangrove herbal
extracts have been practiced as a common method for the treatment of health disorders for many centuries. The
bioactive compounds of mangrove plants are unique in their actions. Since they possess competence in many
bioactive principles against disease producing microbial organisms 10, secondary metabolites such as alkaloids,
steroids, phenols, and terpenoids have been chemically characterized from mangroves which have toxicological,
pharmacological, and ecological importance11.
The GC-MS analysis supports the presence of important bioactive compounds. The relative concentrations
of various compounds were calculated by the use of gas chromatogram which gives many peaks. The height of the
peak corresponds to the relative concentration of compound. The compounds which are eluted at different timings
through gas chromatogram are picked up by the mass analyzer and produce particular fragmentation pattern. This
fragmentation pattern is compared to the compounds present in reference library (NIST) in which the structure of
compounds is determined. This provides the unique chemical fingerprint that shows the importance of plant under
study.
GC-MS chromatogram of ethanolic extract of A. marina was presented in (Fig.1). The relative retention time
(Rt) and mass spectra of the extract components were compared with those of authentic samples and with mass
spectra from a data library. As shown in (Table-1), GC-MS analysis of A. marina extract at the Retention time (Rt)
14.87, 22.18 and 24. 88 resulted in the identification of 15 major different compounds. The compound identified
after comparison of the mass spectra with NIST library (Table-1), indicating the presence of various bioactive
compounds.
Table. 1 GC-MS analysis of Avicennia marina
Retention
S.No. PeakName Pharmacological Properties Reference
time
Cytotoxic, anti- inflammation.
Name: Triquinacene Phenylbutenoid, as antioxidant and
11.59 12-13
Formula:C10H10 MW: 130 anticancer dimmer proved to have
cytotoxic activity
Name: Nonanoicacid Cytotoxicity and Antimicrobial
13.75 35
Formula:C9H18O2 MW: 158 activity, Anti fungal
513
Name: D-Allose Formula:C6H12O6
18.33 Antioxidant activity 35
MW: 180
Name: Dodecanoicacid Hyperglycemic and hypoglycemic,
Formula:C12H24O2 18.55 anti-inflammatory, Hepatoprotective 21-27
MW: 200 activity
Name: Nonanoicacid,3-
Antimicrobial,
methylbutylester Formula:C14H28O2 18.85
Anti-inflammatory 16
MW: 228
Hypocholesterolemic, antiandrogenic,
Name: Heptanoicacid,3,5,5-triethyl-
19.20 flavor, hemolytic 5-alpha reductase
Formula:C13H26O2 MW: 214 36
inhibitor
Name: Benzonitrile,4-ethenyl-
20.47 Anti-Inflammatory,
Formula: C9HN MW:129 37
Name: Phenol,2,6-dimethoxy-4-(2- Antiviral, anti-inflammatory,
propenyl)- Formula:C11H14O3 20.71 cytotoxic activity, ant mutagenic and
MW: 194 ant carcinogenic activities. 37
Name: Benzoicacid,3,4,5-trimethoxy- Anti-Microbial, Hypoglycemic,
21.78
Formula:C10H12O5 MW: 212 antioxidants 19
Name: n-Hexadecanoicacid antioxidants, hypochloresterolenic,
24.88
Formula:C16H32O2 MW: 256 and hemolytic 17-18
Name: Phytol Formula: C20H4O Antimicrobial and
28.03
MW: 296 anti-inflammatory 14
Name: (E)-9-
Octadecenoicacidethylester 29.04 Hypocholesterolemic
36
Formula:C20H38O2 MW: 310
Name:Octadecanoicacid,2-methyl-, Antioxidant, Antimicrobial,
methylester Formula:C20H40O2 29.50 Hypocholesterolemic, antiarthritic,
15
MW: 312 anti-inflammatory
Name:cis-9-Hexadecenal
32.96 Anti-Microbial, Cyotoxicity activity
Formula: C16H3O MW: 238 19
Name:Squalene Formula:C30H50 Antitumor, immunostimulant, chemo
41.30
MW: 410 preventive, lipoxygenase inhibitor 36
Figure.1 GC-MS Chromatogram of Ethanolic Extraction of Avicennia marina
Biological methods of nanoparticles synthesis using plant or plant extract have been suggested as possible
eco-friendly alternatives to chemical and physical methods. Biological synthesis process provides a wide range of
environmentally acceptable methodology, low cost production and minimum time required. At the same time the
biologically synthesized silver nanoparticles has many applications in the field of medicine and agriculture 28.
The Biosynthesis of silver nanoparticles through plant extracts were carried out. It is well known that silver
nanoparticles exhibit yellowish brown colour in aqueous solution due to excitation of surface Plasmon vibration
in silver nanoparticles. The appearances of brown colour in the reaction vessels suggest the formation of silver
nanoparticles (Fig.2).
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Figure 2: Synthesis of silver nanoparticles from ethanolic extraction of A.marina
1mM Silver Nitrate solution 2. 1mM Silver Nitrate solution and ethanolic leaf extraction of A.marina 3. Silver
Nanoparticle synthesized A.marina after 5 hrs of inhibition (Bown colour) 4. Synthesized silver Nanoparticles
The UV absorption peak of silver nanoparticles range from 400 - 450 nm according to Ramteke et al 29. Fig. 3
shows the UV absorption peaks of A.marina. UV-Vis spectra shows the peaks approximately at 460.00nm, clearly
indicating the formation of spherical AgNPs in the plants extracts. The occurrence of the peak at 460 nm is due to
the phenomenon of surface Plasmon resonance, which occurs due to the excitation of the surface plasmon present
on the outer surface of the silver nanoparticles which gets excited due to the applied electromagnetic field30.
Figure 3: UV-Vis spectra of AgNPs in A.marina
The silver nanoparticles are cubical, rectangular, triangular and spherical in shape with uniform distribution.
However, on most occasions, agglomeration of the particles was observed probably due to the presence of a weak
capping agent which moderately stabilizes the nanoparticles31.The measured sizes of the agglomerated nanoparticles
were in the range 287.5 - 293.2 nm. However, the average size of an individual particle is estimated to be 70 nm.
In the present study SEM analysis provides the morphology and size details of the nanoparticles. The high density
AgNPs synthesized by the plant extract of A.marina confirms the presence of AgNPs of size ranging from 20-35nm
(fig 4). Particle size, size distribution and shape of silver nanoparticles are the important parameters that govern the
properties and hence it has wide applications in medicinal fields.
Fig.4: Scanning Electron Microscope analysis of AgNPs in A.marina
FTIR is an important tool which enables us to understand the involvement of functional groups in the
interactions between metal particles and biomolecules 32. The inorganic AgNO3 to elemental silver by the action
of the different phytochemicals which would act simultaneously as reducing, stabilizing and capping agent. FTIR
spectrum clearly illustrates the biofabrication of silver nanoparticles mediated by the plant extracts. Fig. 5 shows
515
the FTIR spectrum of A.marina mediaited synthesized AgNPs, the silver nitrate salt and dried A.marina extract,
in AgNO3 peaks were observed at 3697cm-1,1761cm-1,1390cm-1,831cm-1which are associated OH stretching, C=C
stretching, CH stretching, CH stretching respectively.
Fig.5: FTIR analysis of A.marina
There are certain problems in using animals in experimental pharmacological research, such as ethical issues
and the lack of rationale for their use when other suitable methods are available or could be investigated. Hence,
in the present study the protein denaturation bioassay was selected for in vitro assessment of anti-inflammatory
property of Silver nanoparticles synthesized A.marina. The Denaturation of proteins is a well documented cause of
inflammation. Most biological proteins lose their biological functions when denatured. Production of autoantigen
in certain arthritic disease is due to denaturation of protein. The mechanism of denaturation involves alteration in
electrostatic hydrogen, hydrophobic and disulphide bonding.33
In the presence study denaturation of proteins is the main cause of inflammation. As part of the investigation
on the mechanism of the anti-inflammatory activity, ability of the extract to inhibit protein denaturation was studied.
Selected extracts were effective in inhibiting heat induced albumin denaturation. A.marina was observed as cured
extraction was 68.92% and AgNPs synthesized A.marina 72.1% respectively. Aspirin was used as a standard anti-
inflammation drug. Fig. 6.
Figure 6: Inhibition of protein denaturation activity
The A.marina exhibited significant antiproteinase activity. The percentage of inhibition was observed in A.
marina extract. The standard aspirin 92.87% drug showed maximum proteinase inhibitory action. The A.marina
were observed as crude extraction 68.9% g/mL and AgNPs synthesized A.marina 72.9% g/mL respectively.
Results are shown in Fig.7.
Figure 7: Inhibition of proteinase activity
516
Free radicals are chemical species containing one or more unpaired electrons that makes them highly unstable
and cause damage to other molecules by extracting electrons from them in order to attain stability. The Free
radicals contribute to more than one hundred disorders in humans including atherosclerosis, arthritis, ischemia and
reperfusion injury of many tissues, central nervous system, injury, gastritis, cancer and AIDS. In recent years much
attention has been devoted to natural antioxidant and their association with health benefits34.
There are several methods available to assess antioxidant activity of compounds. DPPH free radical scavenging
assay is an easy, rapid and sensitive method for the antioxidant screening of plant extracts. In presence of an
antioxidant, DPPH radical obtain one more electron and the absorbance decreases 35.
Figure 8: DPPH assay of A.marina
In the present study the mangrove extracts has high DPPH scavenging capacity, which increased with increasing
concentration (Fig. 8) The DPPH assay was carried out at different concentrations of mangrove samples, namely
200g/ml, 400g/ml, 600g/ml and 800g/ml. DPPH assay did not show any significant difference at 200g/ml and
400g/ml concentrations in Avicennia marina sample; however, it was significant for 600g/ml and 800g/ml for
the extracts DPPH is a relatively stable free radical. DPPH radical react with suitable reducing agents, the electrons
become paired off, and the solution losses colour stoichiometrically depending on the number of electrons taken up.
Hence this assay provided information on reactivity of test samples with a stable free radical. The decrease in the
absorbance of the DPPH radical caused by test samples was due to the scavenging of radical by electron donation.
CONCLUSION
In the present study, 15 phytochemical constituents have been identified from ethanol leaf extract of A. marina
by Gas Chromatogram-Mass spectrometry (GCMS) analysis. The presence of phytoconstituents such as alkaloids,
flavonoids, saponin and phytosterols in A. marina which might be responsible for their therapeutic effects. The
biosynthesis of silver nanoparticles with leaf of ethanolic extract of A. marina provides potential source for the Anti
inflammatory properties. It has been reported that one of the features of several non-steroidal anti-inflammatory
drugs is their ability to stabilize and prevent denaturation. Hence, this study gives an idea that the compound of
A.marina can be used as a lead compound for designing a potent anti-inflammatory drug which can be used to cure
inflammation.
ACKNOWLEDGEMENT
We sincerely acknowledged AMET University management for facilities and financial support to carry this
research.
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519
A Cross Sectional Study on Consumption of Indian Herbal
Hypoglycaemic Agents Among Type 2 Diabetics in Coimbatore
District
1
Bhavani. S and 2Jemima Beryl Mohankumar
Department of Nutrition and Dietetics, PSG College of Arts and Science, Coimbatore 641014, TN, India.
Email: bhavani14pndf02@gmail.com
Abstract
Background: The prevalence of diabetes is predicted to double globally in India by 2030. This survey focuses
on the usage of Indian herbals in the management of diabetes.
Methods: A cross sectional study was designed to investigate the usage of herbs to control hyperglycaemia
along with allopath medicines in diabetics. Ethical clearance was obtained from PSG IMSR& H, Coimbatore,
India. Participants (n=329) were aged 45years. A structured questionnaire was used to collect baseline data
on personal, socio economic, anthropometric, biochemical, clinical, medical and dietary information. The
blood parameters like Fasting Blood Glucose [FBG], Postprandial Blood Glucose [PPBG], and Glycosylated
Haemoglobin [HbA1C] were determined by standard laboratory procedures.
Results: Mean age of the participants was 59.2967.0398. Majority of the participants were from urban area
n=203. Only 18.24% used herbals along with allopathic medicines. Fenugreek (Trigonella foenum graecum)
was the widely used (7.90%), followed by 2.13% and 1.52% of Amla (Emblica officinalis) and Bitter gourd
(Momordica charantia) respectively. Other herbs were used in negligible amounts. Significant difference was
seen in the FBG but not in the PPBG between those using herbals along with allopathic and only allopathic
medicines.
Conclusion: We suggest that clinical trials are needed to determine the use of herbal to support allopathic
medicines in treating hyperglycaemia in T2DM.
Keywords: Type 2 diabetes, herbals, hypoglycaemic agents
Introduction
The term diabetes mellitus describes a metabolic disorder with heterogeneous aetiologies which is characterized
by chronic hyperglycaemia and disturbances of carbohydrate, fat and protein metabolism resulting from defects in
insulin secretion, insulin action, or both (WHO, 1999). The prevalence of type 2 diabetes is estimated in people
aged between 20-79 years in South East Asia in the year 2015 to be 78.3 million and projected for year 2040 as
140.2 million (IDF, 2015).
Public, academic and government interest in herbal medicines is growing exponentially due to the increased
incidence of the adverse drug reactions and economic burden of the modern system of medicine (Dubey, 2004). In
1980, WHO recommended the investigation of traditional methods of treatment for diabetes. In recent years, herbal
medicines have started to gain importance as a source of hypoglycemic agents. More than 1000 plant species are
being used as folk medicine for diabetes (Marles and Farnsworth, 1995).
India is the largest producer of medicinal herbs and is appropriately called the botanical garden of the world
(Ahmedullah, 1999). Herbs include crude plant material such as leaves, flowers, fruit, seeds, stems, wood, bark,
roots, rhizomes or other plant parts, which may be entire, fragmented or powdered (WHO, 2000).
Biological actions of the plant products used as herbal medicines to treat diabetes are related to their chemical
composition such as terpenoids, alkaloids, flavonoids, phenolics, and some others, have shown anti diabetic potential
(Jung et al., 2006). So this survey focuses on Indian herbs and its awareness and usage in the treatment of diabetes,
especially in Coimbatore district.
520
Method
A cross sectional study was designed to investigate the use of herbs to control hyperglycaemia along with
allopath medicines. Ethical clearance was obtained from Institutional Human Ethics Committee, PSG Institute of
Medical Sciences & Research, Coimbatore, India. Informed consent was obtained from all the study Participants
(n=329) those who are aged 45years. Patients with known hepatic dysfunction, hypertension, cardio vascular
diseases, diabetic nephropathy, and diabetic neuropathy were excluded from this study. The participants were
grouped into two, based on their mode of treatment viz., participants taking herbals along with allopath (Group A)
and only allopath (Group B).
A structured questionnaire was used to collect baseline data on personal, socio economic, anthropometric,
biochemical, clinical, medical and dietary information. The blood parameters like Fasting Blood Glucose [FBG],
Postprandial Blood Glucose [PPBG], and Glycosylated Haemoglobin [HbA1C] were determined by standard
laboratory procedures. All values were expressed as descriptive statistics and mean SD. To evaluate the differences
between variables t test was used.
Results
Several drugs such as biguanides and sulfonylureas are presently available to reduce hyperglycaemia in
diabetes mellitus. These drugs have side effects and thus searching for a new class of compounds is essential to
overcome these problems. Management of diabetes without any side effects is still a challenge to the medical
community. There is continuous search for alternative drugs. Therefore, it is prudent to look for options in herbal
medicine for diabetes.
Table 1- Mean Age and Area Based Distribution of Participants
Criteria Group A - n=60 (18.24%) Group B - n=269 (81.76%)
Mean age (years) 58.6156.595 59.5437.084
Area Number Percentage Number Percentage
Urban 35 59.4 168 62.5
Rural 16 27.2 68 25.3
Semi-urban 9 13.4 33 12.2
The mean age and area based distribution of participants are shown in table 1. The results indicated that the
mean age of the participants were 59.2967.0398. Majority of the participants were from urban area (Group A-
59.4% and Group B - 62.5%). Though the percentage of selected participants using herbals in addition to allopathic
medicines was low (18.24%), there were no differences pertaining to the area from which they came. This shows
that diabetics are well informed about herbals but there is some apprehension about using them.
A detail of the Indian herbals used by the participants (Group A) is given in Table 2.
Table 2 Indian Herbals Used By the Participants (Group A)
Herbals as Hypoglycaemic Agents No of participants Percentage
Scientific Name Common name Part and Form of usage No = 60 (%) n = 329
Trigonella foenum-graecum Fenugreek Dry powder of seed 26 7.902
Emblica officinalis Amla Raw ground thin slurry of fruit 7 2.127
Momordica charantia Bitter gourd Raw juice extract of vegetable 5 1.519
Andrographis paniculata Nilavembu Dry powder of leaf 3 0.912
Gymnema sylvestre Sirukurinjan Dry powder of leaf 3 0.912
Senna auriculata Avaram poo Dry powder of flower 3 0.912
Eugenia jambolana Jamun, Naval Raw fruit, dry powder of seed 3 0.912
Azadirachta indica Neem leaves Raw extract of leaves 3 0.912
Murraya koenigii Curry leaves Raw extract of leaves 2 0.608
521
Cynodon dactylon Arugampul Raw extract of leaves 2 0.608
Allium sativum Garlic Cloves cooked with milk 1 0.304
Musa acuminate Banana stem Raw extract of stem 1 0.304
Phyllanthus niruri Keezhan nelli Raw extract of leaves 1 0.304
Abelmoschus esculentus Ladies finger Cut soaked overnight 1 0.304
Coriandrum sativum Dhaniya powder Dry powder of seed 1 0.304
Zingiber officinale Ginger Dry powder 1 0.304
Most of the participants were used Fenugreek (Trigonella foenum graecum) (7.902%); while a smaller
percentage of 2.127% were used Amla (Emblica officinalis); 1.519% were used Bitter gourd (Momordica charantia)
as hypoglycaemic agents along with allopathic medicines.
Similar results were observed in a study conducted in Jordan were 31% of interviewed patients have used herbal
products. The most commonly used herbs by diabetic patients wereTrigonella foenumgraecum(22.9%),Alliumm
sativum(11.5%) and Coriandrum sativum(10.4%) (Otoom et al., 2006).
In the context of using traditional medicinal plants for treating diabetes, extensive screening has been performed
in many ethnomedical systems within the Indian subcontinent (Grover, et al., 2002; Mukerjee et al., 2006). Almost
all of them report the herb that was used and the parts and form in which it was used. Most tests have demonstrated
the benefits of medicinal plants containing hypoglycemic properties in diabetes management. The most common
herbal active ingredients used in treating diabetes are flavonoids, tannins, phenolic, and alkaloids (Mamun-or-
Rashid et al., 2014). The mechanisms of actions for hypoglycemic plants include: increasing of insulin secretion,
increasing of glucoses absorption by muscle and fat tissues, prevention of glucose absorption from the intestine, and
prevention of glucose production from liver cells (Hegazy et al., 2013).
We tried to see if there were any differences in the fasting and post prandial glucose levels of those using the
herbals. There was a significant difference in the fasting but not the PPBG. Glycaemic control was not as per the
recommendations of IDF (2007).
Table 3 - Comparison of Biochemical Parameters between Group A and Group B
Biochemical Group A Group B Total participants
t test
parameters n=52 n=256 n=308
FBG 142.71159.618 158.76564.31 0.04236* 156.055263.739
PPBG 236.44288.802 241.2695.899 0.36272** 240.454594.616
Note: *- significant difference: **-not significant difference
Many traditional plant treatments for diabetes are used throughout the world. Plant drugs and herbal
formulations are frequently considered to be less toxic and free from side effects than synthetic ones. Based on the
WHO recommendations, hypoglycemic agents of plant origin used in traditional medicine are important. However
these could be systematically developed into drugs of specific dosage, to enable monitoring of their action.
Conclusion
The traditionally used medicinal plants possessing potent anti-diabetic properties have not been explored
scientifically (Mondal et al., 2013). The research for alternate remedies (from the plant kingdom) for diabetes
mellitus will continue all over the world as the disease poses many challenges not only to the physician but also
to the researcher. This was due to lack of knowledge of the quantum of active components and their molecular
interactions in the diabetics. Clinical trials are needed to determine the use of herbal to support allopathic medicines
in treating hyperglycaemia in T2DM.
522
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Avinashilingam

Institute for Home Science and Higher Education for Women

Patron Padma Shri Dr. P.R. Krishnakumar

Co-ordinator Dr. Premavathy Vijayan

Dr. G.P. Jeyanthi

Padma Shri Dr. P. Pushpangadan

Convenor Dr. S. Annapurani

Organizing Secretary Dr. P.R. Padma

1. Elephantopus scaber and Sauropus androginus Herbal Supplement for 1

3. Maintaining and Improving Sustainability of Jamu, a Traditional Local 29

6. Identification, Characterization and Immunomodulatory Role of Leaf Protein 44

24. Anti Inflammatory Assay of Herbal Extracts of Leaves of 141

73. Modulation of GLUT2 expression by aqueous extract of Erythrina variegata L.

MATERIALS AND METHODS

Preparation of Elephantopus scaber and Sauropus androgynus leaf ethanol extract

Treatment 1 (K-) : healthy mice without extract (control)

Experimental Animal Dissection

Preparation of liver histological section

Influence of E.scaber modulated the concentration fo FSH

Influence of E. scaber and S. androgynus modulated decreasing necrotic hepatocyte cell

There is no conflict of interest in this research.

2. Antunes L C M, Arena E T, Menendez, Han J, Ferreira R B R, Buckner M M C, Lolic P, Madilao L L,

5. Obryan M K, Schlatt S, Phillips D J, Kretser D M, Hedger MP (2000) Endocrinology 141(1), 238-246.

11. Merchant, FW (2004) Am J Anat 139, 245-268.

15. Giuliano A, Meiring T, Grant A J, Watson P J (2015) J ClinMicrobiol 53(11), 3674-3676.

19. Ba X &Garg N J (2011) Am J Pathol 178(3), 946-55.

Keywords: Biopiracy, Benefit Sharing, Traditional Knowledge/Medicine, Indian Systems of Medicine,

Biodiversity and traditional knowledge

Ethnobotany and India

Bioactive leads from ethnobotany

Table 1 : Some examples of drugs derived through ethnobotanical leads

Drug Medical use Plant species Family

Atropine Anticholinergic Atropa belladonna Solanaceae

Biological Diversity Act (2002)

Criteria for benefit sharing

The Patents (Amendment) Act (2005)

Access and benefit-sharing

Nagoya protocol on ABS

Nagoya protocol and India

Kani model or TBGRI model of benefit sharing a case study

Outcome and lessons learnt

Keywords : sustainability,jamu, efficacy, safety, fresh preparation and usage.

Jamu, astraditionalmedicinearisingfromexperiencesofthe past and embedded in the culture of society, can

Preparation and Serving of Jamu

e. Jamu in the form of pills, tablets and capsules

[1]. Elfahmi, HermanJ.Woerdenbagb, OliverKayserc, (2014) Journal of herbal medicine 4 ( 2 0 1 4 ) 5173.

[3]. Riswan S, &Roemantyo HS (2002)South Pacific Study 2002;23:210.

[5]. Torri M C (2013) European Journal of Medicinal Plants3(1): 25-39

[6]. Limyati DA &Juniar BLL (1998)Journal of Ethnopharmacology63:2018.

[10]. KeawpradubN,KirbyGC,SteeleJCP,HoughtonPJ (2016), PlantaMedica1999;65:6904.

Keyword : antimicrobial, antioxidant, Chili Pepper, diseases, medicine.

Chili Pepper Effect and Mortality

Chili pepper variability

Future Research and Prospect of Chili as medicinal ingredient

[12]. Yoshioka M, Doucet E &Drapeau V (2001) Br J Nutr 85, 203-211.

[42]. Turnbaugh PJ, Ley RE &Mahowald MA (2006) Nature 444,1027-31

[48]. KusnadiJ Arumingtyas E L Mastuti R Tirtosari D R (2016)Proceedings of theTaipei 9th International

[49]. Zamora A G, Campos E S& Morales R P (2015)J Chem

[50]. Al Othman Z A, Ahmed Y B, Habila M A &Ghafar A (2011) JMol 16, 8919-8929

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[54]. Molina-Torres J, Garca-Chvez A &Ramrez-Chvez E (1999) J Ethnopharmacol 64, 241-248

Keywords: Leucophyllum frutescens, Phytochemicals, qualitative, FTIR and UV-Visible