ANTIMICROBIAL STUDIES IN ANDROGRAPHIS PANICULATA

PROJECT REPORT submitted in partial fulfillment of the requirements for the award of the degree of BACHELOR OF TECHNOLOGY in BIOTECHNOLOGY

by

PREETY PRIYA (10904215) & K.S.PRASHANSA (10904202)

under the guidance of Mrs. S. Rupachandra, M.Sc., M.Phil., (Lecturer, Department of Biotechnology, School of Bio-engineering, SRM University)

DEPARTMENT OF BIOTECHNOLOGY SCHOOL OF BIOENGINEERING FACULTY OF ENGINEERING AND TECHNOLOGY SRM UNIVERSITY KATTANKULATHUR 603 203
April 2008

CERTIFICATE
Certified that the project report entitled ANTIMICROBIAL STUDIES IN ANDROGRAPHIS PANICULATA submitted by PREETY PRIYA (10904215) is a record of project work done by her under my supervision. This project has not formed the basis for the award of any degree, diploma, associateship or fellowship.

INTERNAL GUIDE

HEAD OF THE DEPARTMENT

For the purpose of viva voce 1. 2.

CERTIFICATE
Certified that the project report entitled ANTIMICROBIAL STUDIES IN ANDROGRAPHIS PANICULATA submitted by K.S.PRASHANSA (10904202) is a record of project work done by her under my supervision. This project has not formed the basis for the award of any degree, diploma, associateship or fellowship.

INTERNAL GUIDE

HEAD OF THE DEPARTMENT

For the purpose of viva voce 1. 2.

DECLARATION
I do hereby declare that the project report entitled ANTIMICROBIAL STUDIES IN ANDROGRAPHIS PANICULATA is a record of original work carried out by me under the supervision of Mrs. Rupachandra, Lecturer, Department of Biotechnology,School of Bio-engineering, SRM University.This project has not been submitted earlier in part or full for the award of any degree, diploma, associateship or fellowship.

Kattankulathur Date

PREETY PRIYA

DECLARATION
I do hereby declare that the project report entitled ANTIMICROBIAL STUDIES IN ANDROGRAPHIS PANICULATA is a record of original work carried out by me under the supervision of Mrs. Rupachandra, Lecturer, Department of Biotechnology,School of Bio-engineering, SRM University.This project has not been submitted earlier in part or full for the award of any degree, diploma, associateship or fellowship.

Kattankulathur Date

K.S.PRASHANSA

ACKNOWLEDGEMENTS

It gives us great pleasure to humbly place on record my heartfelt thanks and gratitude to Dr. K. Ramasamy, Dean, School of Bioengineering, SRM University. Our sincere gratitude and thanks to Dr. D. Kantha Arunachalam, Head of Department of Biotechnology. We would also like to sincerely thank Dr. David Paul Raj, Assistant professor and Head of department of Bio process engineering, SRM University, for his guidance and support. Our special thanks are to our internal guide Mrs. S. Rupa Chandra, Lecturer, Department of Biotechnology, School of Bio-engineering, SRM University, for her constant support and help in the presentation and editing of the thesis. We would also like to thank Dr. Lakshmi Narasu, Head of the Department, Centre for Biotechnology, Institute of Science and Technology, JNT University, for the ample facilities provided in completing the project work. We take this opportunity to respectfully thank our guide Dr. Archana Giri, Assistant professor, Centre for Biotechnology, for her encouragement and her keen interest to carry out this work. We also wish to acknowledge and express our sincere thanks to senior research scholars at JNT University, Ms. Bhuwaneshwari.C.H and Ms. Kiranmayee Rao, for their valuable assistance and encouragement. We would like to extend our appreciation and thanks to the supporting staff members, Ms. Shabana and Ms. Bharati and other lab assistants for their timely help, extended throughout the course of the project. Last but not the least, we would like to thank one and all who have been a part of making this project work a success.

LIST OF TABLES

Table No. 1.0

Title

Antimicrobial activity of Andrographis paniculata Methanol extract.

2.0

Antimicrobial activity of Andrographis paniculata Ethyl acetate extract

LIST OF FIGURES

Fig.No. 1.0 2.0 Structure of Andrographolide

Title

Zone of Inhibition shown by Andrographis paniculata Methanol extract against Bacillus subtilis.

3.0

Zone of Inhibition shown by Andrographis paniculata Methanol extract against Enterococcous faecalis.

4.0

Zone of Inhibition shown by Andrographis paniculata Methanol extract against Salmonella typhimurium

5.0

Zone of Inhibition shown by Andrographis paniculata Ethyl acetate extract against Bacillus subtilis.

6.0

Zone of Inhibition shown by Andrographis paniculata Ethyl acetate extract against Staphylococcous epidermis.

7.0

Zone of Inhibition shown by Andrographis paniculata Ethyl acetate extract against Salmonella typhimurium

10

Fig.No. 8.0 9.0 10.0

Title Chromatogram of the Methanol extract Chromatogram of the Ethyl acetate extract Chromatogram of the authentic Andrographis paniculata sample

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CONTENTS

S.No.

CHAPTER

PAGE No.

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

ABSTRACT INTRODUCTION REVIEW OF LITERATURE MATERIALS AND METHODS RESULTS DISCUSSION SUMMARY CONCLUSION REFERENCES

01 02 10 19 28 30 32 33 I-IV

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1.0 ABSTRACT
Andrographis paniculata, a well known medicinal plant has been used since ancient times in Traditional Chinese medicine and Indian Ayurvedic medicine. It has been used as bitter tonic to treat digestive problems, snake bite, and infections ranging from Malaria to Dysentery. Recent research has thrown light on the anti-microbial activity of the plant. The most significant pharmacological activities of the plant that have been discovered are anti-allergic, anti-cancer and anti HIV effect. In the present study, the antimicrobial activity of the Andrographis paniculata plant extract has been tested against nine common human-affecting bacterial pathogens. Two different solvents - Methanol and Ethyl acetate have been used to prepare the Andrographis paniculata plant extract and the antimicrobial activity of the two extracts has been tested separately. The effectiveness of the extract against the pathogen is taken as the measure of the diameter of zones of inhibition, formed on the bacterial culture plates after a 24 hour Incubation period. The growth medium used is Mueller Hinton Agar.

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2.0 INTRODUCTION
Many higher plants are sources of natural products used as pharmaceuticals, agrochemicals, flavor and fragrance ingredients, food additives, and pesticides (Balandrin and Klocke, 1988). The search for new plant derived chemicals should thus be a priority in current and future efforts towards sustainable conservation and rational utilization of biodiversity (Phillipson, 1990). In search for alternatives to production of desirable medicinal compounds from plants, biotechnological approaches, specifically, plant tissue cultures, are found to have potential as a supplement to traditional agriculture in the industrial production of bioactive plant metabolites (Ramachandra Rao and Ravishankar, 2002). Plants are a tremendous source for the discovery of new products of medicinal value for drug development. Today, several distinct chemicals derived from plants are important drugs currently used in many countries in the world. The evolving commercial importance of secondary metabolites has in recent years resulted in a great interest in secondary metabolism, particularly in the possibility of altering the production of bioactive plant metabolites by means of tissue culture technology. Medicinal plants are the most important source of life saving drugs for the majority of the worlds population. Plants have been an important source of medicine for thousands of years. Even today, the World Health Organization (WHO) estimates that up to 80% of people still rely mainly on traditional remedies such as herbs for their medicines. It is estimated that approximately one quarter of prescribed drugs contain plant extracts or an active ingredient obtained from or modeled on plant substances. Throughout the history, secondary metabolites of plants have been utilized by humanity. There are approximately four major classes of secondary compounds that are significant to humans, viz. Alkaloids, Phenyl propaniods, Flavonoids and Terpenoids (Edwards and Gatehouse, 1999).

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Various medical plants have been used for years in daily life to treat disease all over the world. According to a study performed by the WHO based on publications on pharmacopoeias and medical plants in 91 countries, the number of medicinal plants is nearly 20,000. The characteristics of the plants that inhibit microorganisms and are important for human health have been researched in laboratories since 1926. Many efforts have been made to discover new antimicrobial compounds from various kinds of sources such as soil, microorganisms, animals and plants. One such resource is folk medicine and systematic screening of these many results in the discovery of novel effective compounds (Janovska et al, 2003).

2.1. ABOUT THE PLANT ANDROGRAPHIS PANICULATA

Andrographis paniculata is traditionally known as Kalmegh. The plant belongs to family Acanthaceae and is widely used in Traditional Chinese, Ayurvedic and Homeopathic systems of medicine. The plant grows in waste grounds and prefers moist habitat. The herb is bitter in taste and has weak odour. The whole plant is used in medicine, with leaves and roots being the mostly used parts. It is widely cultivated in southern Asia, where it is used to treat infections and some diseases.

2.1.1. SYSTEMIC POSITION Division: Angiosperms Class: Dicotyledonae Subclass: Gamopetalae Series: Bicarpellatae Order: Personales Tribe: Justicieae Family: Acanthaceae Genus: Andrographis

2.1.2. DESCRIPTION It grows erect to a height of 30-110 cm in moist, shady places with glabrous leaves and white flowers with rose-purple spots on the petals. Stem dark green, 0.3 - 1.0 m in height, 2 - 6 mm in diameter, quadrangular with longitudinal furrows and wings on the angles of the younger parts, slightly enlarged at the nodes; leaves glabrous, up to

15

8.0 cm long and 2.5 cm broad, lanceolate, pinnate; flowers small, in lax spreading axillary and terminal racemes or panicles; capsules linear-oblong, acute at both ends, 1.9 cm x 0.3 cm; seeds numerous, sub quadrate, yellowish brown.

2.1.3. CHARACTERISTICS Since ancient times, A. paniculata is used as a wonderdrug in traditional Siddha and Ayurvedic systems of medicine as well as in tribal medicine in India and some other countries for multiple clinical applications. The therapeutic value of Kalmegh is due to its mechanism of action which is perhaps by enzyme induction. The plant extract exhibits antityphoid and antifungal activities. Kalmegh is also reported to possess antihepatotoxic, antibiotic, antimalarial, antihepatitic, antithrombogenic, antiinflammatory, antisnakevenom, and antipyretic properties to mention a few, besides its general use as an immunostimulant agent. A recent study conducted at Bastyr University, confirms anti-HIV activity of andrographolide.

2.1.4. DISTRIBUTION Andographis paniculata is distributed in tropical Asian countries, often in isolated patches. It can be found in a variety of habitats ie. plains, hill slopes, waste lands, farms, dry or wet lands, sea shore and even road sides. Native populations of A. paniculata are spread throughout south India and Sri Lanka which perhaps represent the centre of origin and diversity of the species. The herb is also available in northern stations of India, Java, Malaysia, Indonesia, West Indies and elsewhere in Americas where it is probably introduced. The species is also available in Hong Kong, Penang, Malacca, Pangkor Island (south of Penang), Malaya, Thailand, West Java, Borneo, Celebes, Brunei, West Indies, Jamaica, Barbados, Bahamas etc. However, precise data are lacking on the introduction and naturalization of the species in these countries. Unlike other species of the genus, A. paniculata is of common occurrence in most of the places in our country including the plains and hilly areas up to 500 m, which accounts for its wide use. Since time immemorial, village and ethnic communities in India have been using this herb for treating a variety of ailments.

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2.1.5. CULTIVATION It prefers a sunny situation. The seeds are sown during May-June. The seedlings are transplanted at a distance of 60 cm x 30 cm.

2.1.6. PHARMACOLOGY Andrographis paniculata plant extract is known to possess a variety of pharmacological activities. Andrographolide (Fig.1.0), the major constituent of the extract is implicated towards its pharmacological activity. A study has been conducted on the cellular processes and targets modulated by andrographolide treatment in human cancer and immune cells. Andrographolide treatment inhibited the in vitro proliferation of different tumor cell lines, representing various types of cancers. The compound exerts direct anticancer activity on cancer cells by cell cycle arrest at G0/G1 phase through induction of cell cycle inhibitory protein p27 and decreased expression of cyclin dependent kinase 4 (CDK4). Immunostimulatory activity of andrographolide is evidenced by increased proliferation of lymphocytes and production of interleukin 2. Andrographolide also enhanced the tumor necrosis factor production and CD marker expression, resulting in increased cytotoxic activity of lymphocytes against cancer cells, which may contribute for its indirect anticancer activity. The in vivo anticancer activity of the compound is further substantiated against B16F0 melanoma syngenic and HT 29 xenograft models. These results suggest that andrographolide is an interesting pharmacophore with anticancer and immunomodulatory activities and hence has the potential for being developed as a cancer therapeutic agent (Rajagopal et al, 2003).

2.1.7. PHYTOCHEMISTRY Andrographolide is the major constituent extracted from the leaves of the plant which is a bicyclic diterpenoid lactone. This bitter principle was isolated in pure form by Gorter (1911). Andrographolide is also attributed with other such activities like liver protection under various experimental conditions of treatment with galactosamine, paracetamol etc. (Saraswat et al, 1996). Systematic studies on chemistry of A. paniculata had been carried out by various researchers during various times.

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Fig. 1.0 Structure of Andrographolide

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2.2. CONSTITUENTS OF ANDROGRAPHIS PANICULATA

Some known constituents are: 14-Deoxy-11-dehydroandrographolide, Plant 14-Deoxy-11-oxoandrographolide, Plant 5-Hydroxy-7,8,2',3'-Tetramethoxyflavone, Plant 5-Hydroxy-7,8,2'-Trimethoxyflavone, Tissue Culture Andrographine, Root Andrographolide, Plant Neoandrographolide, Plant Panicoline, Root Paniculide-A, Plant Paniculide-B, Plant Paniculide-C, Plant

2.3.

SECONDARY METABOLITES FROM ANDROGRAPHIS PANICULATA

The secondary metabolites obtained from Andrographis paniculata are two new flavonoid glycosides, 5-hydroxy-7,8-dimethoxy (2R)-flavanone-5-O--D glucopyranoside and 5-hydroxy-7,8,2,5-tetramethoxy-flavone-5-O--Dglucopyranoside and a new diterpenoid, andrographic acid along with andrographidine A. Their structures were determined on the basis of physicochemical and spectroscopic analysis.

2.4. ABOUT THE PATHOGENS

2.4.1. Bacillus subtilis Bacillus subtilis is a Gram-positive, catalase-positive bacterium commonly found in soil. A member of the genus Bacillus, B. subtilis has the ability to form a tough, protective endospore, allowing the organism to tolerate extreme environmentalconditions. B. subtilis has proven highly amenable to genetic manipulation, and has therefore become widely adopted as a model organism for

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laboratory studies, especially of sporulation, which is a simplified example of cellular differentiation. It is also heavily flagellated, which gives B.subtilis the ability to move quite quickly.

2.4.2. Enterobacter cloacae Enterobacter cloacae is a clinically significant Gram-negative, facultativelyanaerobic, rod-shaped bacterium. The optimum growth temperature for this bacterium is 30C, Appropriate growth media is nutrient agar and nutrient broth. The Genomic sources for the restriction enzymes are Ecl136II, EclHKI, EclXI. The bacteria is Gram Negative and the mode of respiration is Facultatively anaerobic. Motility is by Peritrichous flagella.

2.4.3. Staphylococcus epidermidis Staphylococcus epidermidis is a member of the bacterial genus Staphylococcus, consisting of Gram-positive cocci arranged in clusters. It is catalase-positive and coagulase-negative and occurs frequently on the skin of humans and animals and in mucous membranes.

2.4.4. Enterococcus faecalis Enterococcus faecalis is a Gram-positive commensal bacterium inhabiting the gastrointestinal tracts of humans and other mammals. Like other species in the genus Enterococcus, E. faecalis can cause life-threatening infections in humans and monkeys, especially in the nosocomial (hospital) environment.

2.4.5. Salmonella typhimurium S. enterica has an extraordinarily large number of serovars or strainsup to 2000 have been described. Salmonella enterica Serovar Typhi (historically elevated to species status as S. typhi) is the disease agent in typhoid fever. Other serovars such as Typhimurium (also known as S. typhimurium) can lead to a form of human gastroenteritis sometimes referred to as salmonellosis. Salmonella Typhi possesses three main antigenic factors: the O, or somatic antigen; the Vi, or encapsulation antigen; and the H, or flagellar antigen.

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2.4.6. Staphylococcus aureus S. aureus is a Gram-positive coccus, which appears as grape-like clusters when viewed through a microscope and has large, round, golden-yellow colonies, often with hemolysis, when grown on blood agar plates. S. aureus is catalase positive (meaning that it can produce the enzyme "catalase") and able to convert hydrogen peroxide (H2O2) to water and oxygen, which makes the catalase test useful to distinguish staphylococci from enterococci and streptococci.

2.4.7. Escherichia coli Escherichia coli is a bacterium that is commonly found in the lower intestine of warm-blooded animals. Most E. coli strains are harmless, but some, such as serotype O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for costly product recalls. E. coli is Gram-negative, facultative anaerobic and non-sporulating. It can live on a wide variety of substrates. E. coli uses mixedacid fermentation in anaerobic conditions, producing lactate, succinate, ethanol, acetate and carbon dioxide.

2.4.8. Klebsiella pneumoniae Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterium found in the normal flora of the mouth, skin, and intestines. It is clinically the most important member of the Klebsiella genus of Enterobacteriaceae; it is closely related to K. oxytoca from which it is distinguished by being indole-negative and by its ability to grow on both melezitose and 3-hydroxybutyrate. It naturally occurs in the soil and about 30% of strains can fix nitrogen in anaerobic condition.

2.4.9. Pseudomonas aeruginosa Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium with unipolar motility. An opportunistic human pathogen, P. aeruginosa is also an opportunistic pathogen of plants. P. aeruginosa is the type species of the genus Pseudomonas

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2.5. OBJECTIVES
The present study involves the test of the antimicrobial activity of Andrographis paniculata plant extract against nine common human-affecting bacterial pathogens with the following objectives: 1. To check the antimicrobial activity of the plant extract against human pathogens 2. To evaluate the activity of the plant extract in 2 different solvents - Methanol and Ethyl acetate being the solvents used respectively. 3. To perform HPLC of the extract and compare it with the authentic Andrographolide sample

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3.0. REVIEW OF LITERATURE

The use of alternative medical therapy has increased the interest of pharmacologists and herbalists over the past decade. Historically, plants have provided a source of inspiration for novel drug compounds, as plant derived medicines have made large contributions to human health and well being. On the other hand, there is an increase in use of herbal products all over the world; in USA, it reached 380% between 1990 and 1997. The success story of chemotherapy lies in the continuous search for new drugs to counter the challenge posed by resistant strains of microorganisms. The investigation of certain indigenous plants for their antimicrobial properties may yield useful results. A large number of plants indeed were used to combat different diseases and known to possess antimicrobial activity. The antibacterial activities of hot water, methanol and ethanol extracts of five plant extracts utilized in Palestine in popular medicine were studied. The dried extracts of Sygyium aromaticum (Myrtaceae) (seed), Cinnamomum cassia (Lauraceae) (cassia bark, Chinese cinnamon) (bark), Salvia officinalis (Lamiaceaea) (leaf), Thymus vulgaris (Lamiaceaea) (leaf) and Rosmarinus officinalis ( Labiatae) (leaf) were tested in vitro against four bacterial species by disc diffusion and micro-dilution (Bassam Abu-Shanab et al, 2004). The patterns of inhibition varied with the plant extract, the solvent used for extraction, and the organism tested. Methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus subtilis ATCC 6633 were the most inhibited microorganisms S. aromaticum extract was the most active against multi drug resistant Pseudomonas aeruginosa and enterohaemorrahagic E.coli O157 EHEC. The combinations of ethanolic extracts of S.officinalis with R. officinalis and of R. officinalis with T. vulgaris on bacterial species tested exhibited a higher effect than that of any individual extract. Results of this kind herald the interesting promise of designing a potentially active antibacterial synergized agent of plant origin.

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Voravuthikunchai et al., (2006), prepared ethanolic extracts of eight Thai medicinal plants (representing five families) that are used as traditional remedies for treating diarrhea were examined with a salt aggregation test for their ability to modulate cell surface hydrophobicity of enterohemorrhagic Escherichia coli strains, including E. coli O157:H7. Four of these medicinal plants, Acacia catechu, Peltophorum pterocarpum, Punica granatum, and Quercus infectoria, have high bacteriostatic and bactericidal activities. The ethanolic extract of Q. infectoria was the most effective against all strains of E. coli, with MICs of 0.12 to 0.98 mg/ml and MBCs of 0.98 to 3.91 mg/ml. The ethanolic extract of P. granatum had MICs of 0.49 to 1.95 mg/ml and MBCs of 1.95 to 3.91 mg/ml. Ethanolic extracts of Q. infectoria, P. pterocarpum, and P. granatum were among the most effective extracts against the two strains of E. coli O157:H7. The other four plants, Andrographis paniculata, Pluchia indica, Tamarindus indica, and Walsura robusta, did not have high bacteriostatic and bactericidal activities but were able to affect hydrophobicity characteristics on their outermost surface. All plants except Q. infectoria had some ability to increase cell surface hydrophobicity. There appears to be no correlation between antibacterial activity and cell aggregative properties. Poolsup. N et al., (2004) assessed the efficacy of Andrographis paniculata in the symptomatic treatment of uncomplicated upper respiratory tract infection. Methods: Systematic review of the literature and meta-analysis of randomized controlled trials. Mean difference in the reduction in symptom severity scores between treatment and control groups was calculated to obtain an overall estimate of effect. Four studies met our inclusion criteria and were reviewed. A total of 433 patients reported in three trials were included in the statistical analysis. Andrographis paniculata fixed combination with Acanthopanax senticosus was more effective than placebo. . Current evidence suggests that A. paniculata extract alone or in combination with A. senticosus extract may be more effective than placebo and may be an appropriate alternative treatment of uncomplicated acute upper respiratory tract infection. Jada, et al, (2006), identified a new diterpenoid lactone of the plant Andrographis paniculata , known to possess antitumour activity in in vitro and in vivo breast cancer models was subjected to semisynthesis leading to the preparation of a number of novel compounds.

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Zhang. et al.,(2006) investigate diterpenoids from the aerial parts of Andrographis paniculata (Burm. f.) Nees, three new ent-labdane diterpenoids, namely 19norandrographolides A-C (compounds 1-3), were isolated from the ethanolic extract of A. paniculata. Their structures were established by HRESIMS and NMR spectral data in combination with X-ray crystallographic analysis.

Sinha. J, et al., (2000), despite the rapid development in medicinal and pharmaceutical technology, the targeting of drugs to phagocytic cells in macrophagerelated diseases still remains a major unsolved problem. By using the mannosylfucosyl receptors on macrophages, attempts were made to target antileishmanial drugs encapsulated in mannosylated or fucosylated liposomes to treat experimental leishmaniasis in the hamster model. Mannosylated liposomes were found to be more potent in delivering antileishmanial drugs to phagocytic cells. Liposomes loaded with an indigenous drug, andrographolide, a labdane diterpenoid isolated from Indian medicinal plant Andrographis paniculata, were prepared and tested against experimental leishmaniasis in a hamster model. Mannosylated liposomes loaded with the drug were found to be most potent in reducing the parasitic burden in the spleen as well as in reducing the hepatic and renal toxicity. In addition, mannosylated drugloaded liposome-treated animals showed a normal blood picture and splenic tissue histoarchitecture when compared with those treated with free drug or regular liposomal drug. Such a drug-vehicle formulation may be considered for clinical trials.

Tipakorn . N et al., (2004), conducted studies to determine the antibacterial activity of A. paniculata (AP) leaf extracts (at 1:10, 1:100 and 1:1000) diluted with three solvents (distilled water, 70 and 85% alcohol). The extracts were tested against Salmonella typhimurium, Salmonella spp. (from Lampang and Phitsanulok provinces), Escherichia coli (from chicken, pig, deer and duck, and ATCC 25922 as standard), and Pasteurella multocida (from buffalo tissue, buffalo liver, beef tissue and beef heart). Streptomycin 2 mg/ml was used as the control. The concentration 1:10 of 70 and 85% alcoholic extract showed moderate to intermediate activity against S. typhimurium, with inhibition zones of 12 and 10 mm, respectively. The minimum inhibitory concentration of 70 and 85% AP alcohol extracts AP was 1:10.

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Likewise, the 70 and 85% AP alcohol extracts showed antibacterial activity towards P. multocida isolated from buffalo tissue, buffalo liver and beef tissue. E. coli strain ATCC 25922 as well as those isolated from chicken, pig and duck, Salmonella sp. from Phitsanulok, and P. multocida isolated from buffalo liver and beef tissue showed resistance to Streptomycin. Thus, the use of AP leaves as antibacterial agents against bacteria causing diarrhoea is promising.

Youhong Xu,

et al., (2006), prepared

aqueous and two ethanolic extracts of

Andrographis paniculata, used in traditional Chinese, Thai and Indian medicine and andrographolide, an active principle of Andrographis paniculata, were investigated for their antimicrobial activity against nine bacterial species including Salmonella typhimurium, Escherichia coli, Shigella sonnei, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus pyogenes, Legionella pneumophila and Bordetella pertussis, using the disc diffusion method. Of all tested concentrations, direct antimicrobial activity of the two ethanolic Andrographis paniculata extracts was observed for only two human pathogens, Legionella pneumophila and Bordetella pertussis.

P Borgna, et al., (1996), N-Hydroxyalkyl derivatives of 1,2-benzisothiazol-3(2H)one and 1,2-benzisothiazol-3(2H)-thione have been prepared and their antifungal and antibacterial activity evaluated. Several compounds were active against selected fungi and Gram-positive microorganisms. Interesting activity was observed against the anaerobic strain Clostridium perfringens. Generally the more active compounds belong to the class of 1,2-benzisothiazol-3(2H)-ones. The retardation matches RM of the compounds was also evaluated but the results obtained show that lipophilicity has only a minor effect on the antimicrobial activity.

G Daidone, et al., (1996), prepared number of derivatives of new 4-diazopyrazole reaction of 1-R-3-methyl-5(R1-substituted)benzamidopyrazoles with a sevenfold excess of nitrous acid in acetic medium. The compounds were tested for activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus,

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Staphylococcus epidermidis, Streptococcus faecalis, Listeria monocytogenes, Candida albicans, Candida tropicalis and Paecilomyces varioti. The highest microbial susceptibility was shown by Gram-positive bacteria, with minimum inhibitory concentrations (MIC) in the range 0.512.5 g/mL. For S aureus the R1 substituents were screened utilizing the Topliss operational scheme. The 4-nitro group was found to be the best substituent. We also tested the compounds 41,o,p, found to be the most active in the test against S aureus ATCC 25923, on ten clinical S aureus strains, five of which were sensitive and five resistant to methicillin. The above compounds were active in the range 28 g/mL against methicillin-resistant S aureus strains. An X-ray analysis of compounds 4i and 4q is reported. zlem Temiz, , et al., (1998), synthesis of a new series of 5- or 6-methyl-2-(2,4disubstituted phenyl)benzoxazoles (4, 5) is disubstituted phenyl)benzoxazoles (4, 5) is described in order to determine their antimicrobial activities and feasible structureactivity relationships. The synthesized compounds were tested in vitro against three Gram-positive bacteria, three Gram-negative bacteria and the yeast Candida albicans, in comparison with several control drugs. OM Walsh, et al., (1996), synthesized a series of 3-acetoxyazetidin-2-ones 3an and 3-hydroxyazetidin-2-ones 6aj is reported together with the antibacterial and antifungal evaluation of these compounds. An additional series of 3-acetoxyazetidin2-ones 11ah which possess a free carboxylic acid group on the N-1 aryl ring were obtained by treatment of suitably substituted Schiff bases 10ah with acetoxyacetyl chloride. The novel bicyclic structures 7-acetoxy-6-phenyl-5-thia-1azabicyclo[4.2.0]octan-8-one 13 and 7-hydroxy-6-phenyl-5-thia-1azabicyclo[4.2.0]octan-8-one 14 were also obtained. Many of the compounds displayed antifungal activity in vitro when evaluated against the pathogenic fungi Cryptococcus neoformans, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, and Trichosporon cutaneum, while 3acetoxyazetidin-2-ones 11ah containing a free carboxylic acid group on the N-1 aryl ring displayed antibacterial activity against Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, Bacillus subtilis, Klebsiella aerogenes and Escherischia coli

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Prajjal K. Singha, (2003), studied the antimicrobial activity of aqueous extract, andrographolides and arabinogalactan proteins from Andrographis paniculata were evaluated. The aqueous extract showed significant antimicrobial activity, which may be due to the combined effect of the isolated arabinogalactan proteins and andrographolides. Essawi and M. Srour. Of the 15 plants tested, eight showed antibacterial activity. Each plant species has unique against different bacteria. The most active antibacterial plants against both gram-positive and gram-negative bacteria were Thymus vulgaris and Thymus origanium. The organic and aqueous extract from the same plants showed different fecalis. Of the 15 plants tested, eight showed antibacterial activity. Each plant species has unique against different bacteria. The most active antibacterial plants against both gram-positive and gram-negative bacteria were Thymus vulgaris and Thymus origanium. Finally, the holeplate diffusion method showed larger activity than the disc diffusion method. Jonathan E et al., (2000), prepared aqueous, methanolic and ethyl acetate extracts of 14 plants used in traditional Zulu medicine for treatment of ailments of an infectious nature were screened for antibacterial activity. Most of the activity detected was against Gram-positive bacteria. Tuber bark extracts of Dioscorea sylvatica had activity against Gram-negative Escherichia coli and extracts of Dioscorea dregeana, Cheilanthes viridis and Vernonia colorata were active against Pseudomonas aeruginosa. The highest antibacterial activity was found in extracts of C. viridis, D. dregeana, D. silvatica, Melianthus comosus and V. colorata. In general, methanolic extracts exhibited higher activity than aqueous and ethyl acetate extracts.

Owais .M, et al, (2005), evaluated the antibacterial activity of ashwagandha (Withania somnifera L. Dunal (Solanaceae; root and leaves), an Indian traditional medicinal plant against pathogenic bacteria. Both aqueous as well as alcoholic extracts of the plant (root as well as leaves) were found to possess strong antibacterial activity against a range of bacteria, as revealed by in vitro Agar Well Diffusion Method. The methanolic extract was further sub-fractionated using various solvents and the butanolic sub-fraction was found to possess maximum inhibitory activity against a spectrum of bacteria including Salmonella typhimurium. Moreover, in

28

contrast to the synthetic antibiotic (viz. chloramphenicol), these extracts did not induce lysis on incubation with human erythrocytes, advocating their safety to the living cells. Finally, the antibacterial efficacy of the extracts isolated from plant (both root and leaves) was determined against experimental salmonellosis in Balb/C mice. Oral administration of the aqueous extracts successfully obliterated salmonella infection in Balb/C mice as revealed by increased survival rate as well as less bacterial load in various vital organs of the treated animals. Mice studies have shown that Andrographis paniculata is a potent stimulator of the immune system in two ways: 1. Antigen-specific response: antibodies are made to counteract invading microbes, and 2. Nonspecific immune response: macrophage cells scavenge and destroy invaders. A. paniculata activated both responses-making it effective against a variety of infectious and oncogenic (cancer-causing) agents. AP has a record of effective treatment rooted in its mechanism of immune boosting. Cancer results when cells do not respond to signals that are intended to limit growth. If a cancer cell can be made to mature (or differentiate), it will not have the ability to grow out of control. AP was chosen because it contained substances (Terpenes) which were known to cause differentiation of cancer cells. AP extracts from the leaves of the plant are also cytotoxic (Cell killing) against cancer cells. This cancer cell-killing ability was demonstrated against human epidermoid carcinoma (Squamous cell carcinoma) of the skin lining of the Nasopharynx and against lymphocytic leukemia cells. It was the andrographolide component that was found to have the cancer cell-killing ability. It was recommended by National Cancer Institute as a cytotoxic substance. Japanese researchers have reported that AP stopped stomach cancer cells from multiplying. After 3 days, there were less than 8 cancer cells growing in the presence of AP, while the untreated cancer cells numbered 120. Another group of Japanese researchers tested AP on sarcoma cells. These very malignant cancers affect muscles, connective tissue and bones. When tumor cells were examined under the microscope, AP was found to inhibit the growth of tumors.

29

Laboratory tests conducted in Buffalo, New York, demonstrated that AP inhibited the growth of human breast cancer cells. Extracts of AP are much less toxic than most chemotherapeutic agents used to fight cancer. In 1977, a human study was conducted using AP in 60 cancer patients, including 41 with confirmed metastasis (the cancer was spreading). As reported in the Journal of Chinese Medicine, 12 patients given AP and its compounds alone, recovered. All other patients were given AP along with standard drugs; there was no tumor regrowth in 47 of these patients. In 1996, early trials showed that the extract safely and effectively blocked growth of both prostate and breast cancer cells, grown in laboratory. Researchers believed that AP probably inhibits synthesis of cancer cell DNA. Immune deficiency is at the root of susceptibility to a variety of infections, and its is the basis of the Acquired Immune Deficiency Syndrome (AIDS). HIV, like all virus, cannot reproduce by itself or even live, without using the resources of other cells. When HIV virus finds a suitable cell, it attaches to the cell, using proteins on its cell surface. In the case of human cells, the HIV virus enters the cells by binding to molecules on the cell surface. The first of these to be identified was CD4; other more recently identified molecules are CCR5 and CXCR4. The HIV virus actually subverts the cells messengers, tricking them into producing more viral particles. Using signal transduction technology (methods to investigate the cell message systems), scientists found that AP contains substances that destroyed the virus communications mechanism. One component of the herd Andrographolide prevented transmission of the virus to other cells and stopped the progress of the disease by modifying cellular signal transduction. Andrographolide probably does this by inhibiting enzymes that facilitate the transfer of phosphates. AP can thus interfere with key enzymes that result in viral reproduction. HIV alters the action of central information -processing enzyme, Cyclin dependent Kinase (CDK), particularly CDK1, that coordinates all events relating to cell division. Agents that can prevent this phosphorylation can lessen the severity of AIDS. The new class of anti viral compounds with this ability is called Tyrosin Kinase inhibitors which includes Andrographolides. An extract of AP can, in fact, inhibit CDK1 that has been altered by HIV. Cooperative research at National Cancer Institute has shown that Andrographolide can also inhibit HIVs toxic effect on cells. Testing of AP done 30

at Frederick Research Centre, demonstrated that extracts of AP increased AZTs ability to inhibit replication of HIV. An added benefit is that lower doses of AZT could be used. Some researchers believe that AP extracts may also be useful in combating other viruses, including the Ebola virus and the viruses associated with Herpes, Hepatitis, and Influenza.

31

4.0. MATERIALS AND METHODS

4.1. Materials 1. Conical flasks 2. Petri plates 3. Test tubes 4. Micropipettes 5. Mueller Hinton Broth 6. Bacteriological Agar 7. Plant extract 8. Methanol 9. Ethyl acetate. 10. Distilled water 11. Cotton 12. Ethanol 13. Para film 14. Glass markers 15. Rubber bands 16. Aluminum foil

4.1.1. Equipment 1. Laminar Air Flow 2. Decontaminator 3. Autoclave 4. Ultra sonicator 5. High Pressure/Performance Liquid Chromatography (HPLC) column and detector

32

4.2. Methods

4.2.1. Sterilization of culture vessels and instruments Culture vessels and instruments were sterilized by exposure to hot dry air (160 C) for 2-4 hr in a hot air oven. All the vessels were properly sealed with aluminum foil before sterilization.

4.2.2. Preparation of the Media 21gms of the Mueller Hinton broth was taken and added to 1000ml of distilled water. The pH of the media was then adjusted to 7, and then 7 gms of agar was added. The media was then sterilized in an autoclave at 121 C, at 15 lbs pressure for 15 minutes.

4.2.3. Media composition Beef Infusion - 300 g/l (from Caesin Hydrolysate- 17.50 g/l and Starch 1.50 g/l) Final pH at 25 C is 7.4 (plus or minus) 0.2

4.2.4. Sterilization of Mueller Hinton Agar Media The media used in the present investigation was sterilized in an autoclave at 121 C under 15 lbs for 15-20 minutes. Distilled water, micro-nutrients, macro-nutrients and other stable mixtures were autoclaved. Culture media in glass containers were sealed with cotton plugs and autoclaved at 14 lbs at 121 C for 15-20 minutes.

4.2.5. Plant Material In the present study, the plant material used is Andrographis paniculata, belonging to the family Acanthaceae. Extracts were prepared using two different solvents Methanol and Ethyl acetate, using leaves of the plant.

4.2.6. Preparation of the Plant extract In the present study, the Methanol and Ethyl acetate extracts of leaves of Andrographis paniculata were used for evaluation of their antimicrobial activity against human pathogens.

33

4.2.6.1. Methanol extract: 250 gms of powdered AP plant material was carefully weighed and it was carefully transferred into a round bottomed flask of soxhlet extractor. Then 2 liters of Methanol was added and the plant material was soaked in Methanol for 24 hours at room temperature. Then the methanol extract of the plant was extracted using the soxhlet extractor. The methanol present in the methanol extract was evaporated under reduced pressure to yield the residue. 4.2.6.2. Ethyl acetate extract: 250 gms. of powdered AP plant material was carefully weighed and it was carefully transferred into a round bottomed flask of soxhlet extractor. Then 2 liters of Ethyl acetate was added and the plant material was soaked in Ethyl acetate for 24 hours at room temperature. Then the Ethyl acetate extract of the plant was extracted using the soxhlet extractor. The Ethyl acetate present in the Ethyl acetate extract was evaporated under reduced pressure to yield the residue.

4.2.7. Preparation of pure cultures To prepare pure cultures, Mueller Hinton Agar was prepared. Then, a loopful of culture was taken from stock culture and it was inoculated in the test tubes containing about 25 ml of the medium. These tubes were incubated at 37 C for 24 hours and used for further experimental procedure. The following bacterial strains for the antimicrobial assay have been collected from microbial type culture collection (MTCC) of IMTECH, Chandogarh. The microorganisms that were used in the antimicrobial assay were:

1. Bacillus subtilis 2. Enterobacter cloacae 3. Staphylococcous epidermis 4. Enterococcus faecalis 5. Salmonella typhimurium 34

6. Staphylococcous aureus 7. Escherichia coli 8. Klebsiella pneumoniae

9. Pseudomonas aeroginosa

4.2.8. Procedure The following steps were performed under sterile conditions in the laminar air flow: 1. 25ml of hot Mueller Hinton Agar was poured into 9 test tubes respectively numbered 1-9. 2. These tubes were then placed in a beaker containing hot water, to prevent the agar from solidifying. 3. In each of the test tube, the respective pathogen was added. (10 micro liter vol. for Bacillus subtilis, and 20 micro liters for each of the other pathogen), and mixed properly. 4. The culture was then poured into the respective petri plates (numbered 1-9) and kept aside for about 20 mins, for solidification. 5. Four wells were then punched, using the blue micropipette tip, in the four quadrants of every petri plate. 6. Next, increasing concentrations of the extract was poured into each of the four wells. 7. In the case of the Methanol extract, the concentrations used were 25, 50, 75 and 100 micrograms of the extract. 8. In the case of the Ethyl acetate extract, the concentrations used were 20, 30, 40, 50 micrograms of the extract.

35

9. The volume in each well was made up using the respective solvent (Methanol (Table 1.0, Fig 2.0, 3.0, 4.0) and Ethyl acetate (Table 2, Fig 5.0, 6.0, 7.0)). 10. The plates were then sealed with parafilm, and placed in the freezer for 5-10 minutes. 11. The plates are then removed from the freezer and placed in the bacterial incubator at 37 C, for 24 hours. 12. The zone of inhibition for each well is checked and its diameter is measured. In case of negative activity of the extract against the pathogen, there is no zone of inhibition formed.

36

Table 1.0 - Antimicrobial activity of Andrographis paniculata Methanol extract

S.No.

Name of the organism

Diameter of zone in 25 micro gm of extract(cm)

Diameter of zone in 50 micro gm of extract(cm) 0.6/0.8 1.4/0.8 1.6/0.8 1.6/0.8 -

Diameter of zone in 75 micro gm of extract(cm) 0.6/0.8 1.6/0.8 1.8/0.8 1.2/0.8 -

Diameter of zone in 100 micro gm of extract(cm) 0.2/0.8 1.6/0.8 1.8/0.8 1.6/0.8 -

1.0 2.0 3.0 4.0 5.0 6.0 7.0

Bacillus subtilis 0.4/0.8 Enterobacter cloacae 1.4/0.8

Staphylococcus epidermis Enterococcus faecalis Salmonella typhimurium 1.6/0.8 1.2/0.8

Staphylococcus aureus Escherichia coli -

37

Fig.2.0. Zone of Inhibition shown by Andrographis paniculata Methanol extract against Bacillus subtilis.

38

Fig.3.0. Zone of Inhibition shown by Andrographis paniculata Methanol extract against Enterococcous faecalis.

39

Fig.4.0. Zone of Inhibition shown by Andrographis paniculata Methanol extract against Salmonella typhimurium

40

Table 2.0.Antimicrobial Activity of Andrographis paniculata -Ethyl acetate extract

S.No.

Name of the organism

Diameter of zone in 20 micro gm of extract 2.0/0.8 1.4/0.8 1.4/0.8 1.2/0.8 2.0/0.8 1.6/0.8 1.2/0.8

Diameter of zone in 30 micro gm of extract 2.0/0.8 1.4/0.8 1.4/0.8 1.4/0.8 2.0/0.8 1.3/0.8 1.3/0.8

Diameter of zone in 40 micro gm of extract 2.0/0.8 1.6/0.8 1.5/0.8 1.2/0.8 2.2/0.8 1.6/0.8 1.2/0.8

Diameter of zone in 50 micro gm of extract 1.8/0.8 1.8/0.8 1.5/0.8 1.4/0.8 2.2/0.8 1.4/0.8 1.3/0.8

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

Bacillus subtilis Enterobacter cloacae Staphylococcus epidermis Enterococcus faecalis Salmonella typhimurium Staphylococcus aureus Escherichia coli Klebsiella pneumoniae Pseudomonas aeroginosa

41

Fig.5.0. Zone of Inhibition shown by Andrographis paniculata Ethyl acetate extract against Bacillus subtilis.

42

Fig.6.0. Zone of Inhibition shown by Andrographis paniculata Ethyl acetate extract against Staphylococcous epidermis.

43

Fig.7.0. Zone of Inhibition shown by Andrographis paniculata Ethyl acetate extract against Salmonella typhimurium

44

4.2.9. High Performance Liquid Chromatography (HPLC): (or

High-performance

liquid

chromatography

High

pressure

liquid

chromatography, HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry. HPLC is used to separate components of a mixture by using a variety of chemical interactions between the substance being analyzed (analyte) and the chromatography column.

4.2.9.1 Operation The sample to be analyzed is introduced in small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time and is considered a reasonably unique identifying characteristic of a given analyte. The use of pressure increases the linear velocity (speed) giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. Common solvents used include any miscible combinations of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as Trifluoroacetic acid which acts as an ion pairing agent.

4.2.9.2 Reversed phase chromatography Reversed phase HPLC (RP-HPLC or RPC) consists of a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. The retention time is therefore longer for molecules which are more non-polar in nature, allowing polar molecules to elute more readily.

45

Retention Time (RT) is increased by the addition of polar solvent to the mobile phase and decreased by the addition of more hydrophobic solvent.

RPC operates on the principle of hydrophobic interactions, which result from repulsive forces between a polar eluent, the relatively non-polar analyte, and the nonpolar stationary phase. The binding of the analyte to the stationary phase is proportional to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand in the aqueous eluent. This solvophobic effect is dominated by the force of water for "cavity-reduction" around the analyte and the C18-chain versus the complex of both. The energy released in this process is proportional to the surface tension of the eluent (water: 73 erg/cm, methanol: 22 erg/cm) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased by adding less-polar solvent (MeOH, ACN) into the mobile phase to reduce the surface tension of water.

Structural properties of the analyte molecule play an important role in its retention characteristics. In general, an analyte with a larger hydrophobic surface area (C-H, CC, and generally non-polar atomic bonds, such as S-S and others) results in a longer retention time because it increases the molecule's non-polar surface area, which is non-interacting with the water structure. On the other hand, polar groups, such as OH, -NH2, COO- or -NH3+ reduce retention as they are well integrated into water. Very large molecules, however, can result in an incomplete interaction between the large analyte surface and the ligands alkyl chains and can have problems entering the pores of the stationary phase.

RT increases with hydrophobic - non-polar - surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. Similarly organic compounds with single C-C-bonds elute later than

46

the ones with a C=C or C-C-triple bond, as the double or triple bond is shorter than a single C-C-bond.

Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (ca. 1.5 erg/cm pro Mol for NaCl, 2.5 erg/cm pro Mol for (NH4)2SO4), and because the entropy of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time. This technique is used for mild separation and recovery of proteins and protection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC).

Another important component is the influence of the pH since this can change the hydrophobicity of the analyte. For this reason most methods use a buffering agent, such as sodium phosphate, to control the pH. A volatile organic acid such as formic acid or most commonly trifluoroacetic acid is often added to the mobile phase, if mass spectrometry is applied to the eluent fractions. The buffers serve multiple purposes: they control pH, neutralize the charge on any residual exposed silica on the stationary phase and act as ion pairing agents to neutralize charge on the analyte. The effect varies depending on use but generally improve the chromatography.

4.2.9.3 HPLC Technique 1. The HPLC technique (using Reverse phase adsorptive C18 column) was performed for the Andrographis paniculata aunthentic sample and for both the extracts of the Andrographis paniculata plant. 2. HPLC was performed on Shimadzu LC 10 AT VP series using a supelco column (250 x 46 mm, C18, ODS with particle size of 5 um).

47

3. The sample was injected into the sample injection port at regular time intervals. 4. The mobile phase used was the mixture of water: acetonitrile: Methanol55:30:15/100ml at a flow rate of 1 ml /min. 5. Andrographis paniculata authentic sample and the Methanol and Ethyl acetate extracts were monitored at 223 nm with an UV Vis detector (shumadzu uv visible SPD LC 10 AVP Series) (Tikhomiroff and Jolicoeur 2002) 6. The chromatograms of both the extracts were compared respectively to the chromatogram of the authentic sample.

48

Fig.8.0 Chromatogram of the Methanol extract

49

Fig. 9.0 Chromatogram of the Ethyl acetate extract

50

Fig.10.0 Chromatogram of the Authentic Andrographolide paniculata Sample

51

5.0. RESULTS
5.1. Antimicrobial activity of Andrographis plant

5.1.1. Methanol extract The methanol extract of the plant showed antimicrobial activity against 4 out of the 9 pathogens tested (Table 1). Varying concentrations of 25, 50, 75 and 100 microgram of the Methanol extract was used in each of the four wells of the petri plate cultures. The zones of inhibition of growth were formed in all the 4 wells containing the varying concentrations of the Methanol extract. The 4 microorganisms, against which the Methanol extract was effective were Bacillus subtilis (Fig.2.0), Enterobacter cloacae, Enterococcous faecalis(Fig.3.0) and Salmonella typhimurium(Fig.4.0).

5.1.2. Ethyl acetate extract The ethyl acetate extract of the plant showed antimicrobial activity against 7 out of the total 9 pathogens tested (Table 2). Varying concentrations of 20,40,60 and 80 microgram of the Ethyl acetate extract was used in each of the four wells of the petri plate cultures. The zones of inhibition of growth were formed in all the 4 wells containing the varying concentrations of the Methanol extract. The 7 microorganisms, against which the Ethyl acetate extract was effective were Bacillus subtilis (Fig.5.0), Staphylococcous epidermis (Fig.6.0), Enterococcous faecalis, Salmonella typhimurium (Fig.7.0), Staphylococcous aureus, Escherichia coli, Pseudomonas aerogenus.

52

5.2. HPLC The Andrographis paniculata authentic sample and both the extracts were monitored at 223 nm with an UV Vis detector (shumadzu uv visible SPD LC 10 AVP Series) (Tikhomiroff and Jolicoeur 2002).

5.2.1. Chromatogram of Methanol extract (Fig.8.0) The active constituent Andrographolide is the second compound to elute out in the methanol extract at a retention time of 3.460 minutes. The first compound eluted out at a retention time of 1.380 minutes and the third compound eluted out at a retention time of 4.493 minutes.

5.2.2. Chromatogram of the Ethyl acetate extract (Fig.9.0) The active constituent Andrographolide is the second compound to elute out in the Ethyl acetate extract at a retention time of 3.460 minutes. The first compound eluted out at a retention time of 2.870 minutes and the third, fourth, fifth and sixth compound eluted out at retention times of 3.997, 4.207, 4.497 and 4.753 minutes respectively.

5.2.3. Chromatogram of the Authentic Andrographis paniculata sample (Fig.10.0) The Andrographolide is the first compound to elute out in the authentic sample at a retention time of 3.460 minutes, and the second compound eluted out at a retention time of 4.490 minutes.

53

6.0. DISCUSSION
6.1. Antimicrobial activity of Andrographis paniculata In the present study, varying concentrations of 25, 50, 75 and 100 microgram of the Methanol extract was used in each of the four wells of the nine petri plate cultures. The zones of inhibition of growth were formed in all the four wells containing the varying concentrations of the Methanol extract. The methanol extract of the plant showed antimicrobial activity against four out of the nine pathogens tested. Bacillus subtilis, Enterobacter cloacae, Enterococcous faecalis and Salmonella typhimurium were the microorganisms against which the antimicrobial activity of the Methanol extract was detected. For the Ethy acetate extract, varying concentrations of 20, 30, 40 and 50 micrograms was used in each of the four wells of the nine petri plate cultures. The zones of inhibition of growth were formed in all the four wells containing the varying concentrations of the Methanol extract. The Ethyl acetate extract of the plant showed antimicrobial activity against seven out of the nine pathogens tested.

Bacillus subtilis, Staphylococcous epidermis, Enterococcous faecalis, Salmonella typhimurium, Staphylococcous aureus, Escherichia coli, Pseudomonas aerogenus were the microorganisms against which the antimicrobial activity of the Ethyl acetate extract was detected.

54

The Ethyl acetate extract is inferred to have higher antimicrobial activity when compared to the extract prepared using Methanol as the solvent. This is inferred from the fact that the Ethyl acetate extract is effective against seven of the nine pathogens, when compared to the effectiveness of the Methanol extract, which was effective against only four of the nine pathogens tested. The reason for the higher effectiveness of the Ethyl acetate can be due to a higher content of active Andrographolide being present in the Ethyl acetate extract, in comparison with that present in the Methanol extract. Andrographolide, the active compound confers the antimicrobial property to the Andrographis paniculata plant and hence the higher content of the Andrographolide present in the Ethyl acetate extract, makes it more effective against the bacterial pathogens.

6.2. HPLC HPLC technique (using Reverse phase adsorptive C18 column) was performed for the Andrographis paniculata aunthentic sample and for both the extracts of the Andrographis paniculata plant. The chromatograms of both the extracts were compared respectively to the chromatogram of the authentic sample. It is inferred that the peak of the active constituent, Andrographolide present in both Methanol and Ethyl acetate extracts respectively was obtained at a Retention time of 3.460 minutes, which was same as the retention time of the Andrographolide compound present in the authentic sample of Andrographis paniculata.

55

7.0 SUMMARY
In the present study, the antimicrobial activity of Andrographis paniculata plant extract was tested against nine common human-affecting bacterial pathogens. The evaluation was carried out using two different solvents - Methanol and Ethyl acetate. Pure stock cultures of the nine bacterial pathogens were prepared and maintained throughout the study. 10 micro liter volume for Bacillus subtilis, and 20 micro liters for each of the other pathogens, were taken from the pure stock culture and were then added to 25 ml of the Mueller Hinton Agar present in nine test tubes numbered 1-9. This mixture was then poured into the Petri plates and left for solidification. Next, four wells were punched into each Petri plate and the extract was added in increasing concentrations in each of the four wells. The plates were sealed and then kept for Incubation in a bacterial incubator for 24 hours at 37 C. The same procedure is performed for both the extracts. After the 24 hour incubation period, each of the nine plates was checked for the zones of inhibition of growth. The diameter of the zones were measured and noted down. For microorganisms that showed zones of inhibition of growth, it is inferred that the plant extract is effective against the respective microorganisms. The evaluation of both the extracts is done based on its effectiveness against higher number of microorganisms. Hence, it is concluded that Ethyl acetate is more effective against the bacterial pathogens, in comparison to the Methanol extract. The HPLC of the authentic Andrographis paniculata sample and both the extracts was performed and their respective chromatograms were compared. In this, it is found that the active compound Andrographolide, is present in all three samples and therefore has the same retention time of 3.460 minutes. It is further noted that the antimicrobial property of the Andrographis paniculata plant is conferred by its active component, Andrographolide.

56

8.0. CONCLUSION
Based on the results of the present study, it can be concluded that Andrographis paniculata plant extract shows significant antibacterial activity against most of the human- affecting pathogens. Extracts made from solvents that are more effective against the pathogens can be used to develop drugs that can be taken along with other Antibiotics. It is of common knowledge that the Andrographis paniculata plant can be used to treat a wide range of ailments. Hence, the advantage of such a combinatorial drug therapy would be the absence of side-effects and also increased effectiveness, in comparison with the present day anti-biotic based drug therapy.

57

REFERENCES
1. Balandrin, M.F. and Klocke J.A. 1988. Medicinal, Aromatic and Industrials from plants. Medicinal and Aromatic plants. 33(1): 15-25 2. Bassam Abu- Shanab, Ghaleb Adwan, Dawood Abu-Safiya, Naser Jarrar, Kamel Adwan; 2004.Antibacterial activities of some plant extracts utilized in popular medicine in Palestine 3. Borgna .P, ML Carmellino, M Natangelo, G Pagani, F Pastoni, M Pregnolato and M Terreni, 2003. Antimicrobial activity of N-hydroxyalkyl 1,2-benzisothi. European Journal of Medicinal Chemistry.Volume 74, Issues 7-8, 692-694. 4. Daidone. G , ML Bajardi, S Plescia, D Raffa, D Schillaci, B Maggio, F Benetollo and G Bombieri, 1996. One-step synthesis, crystallographic studies and antimicrobial activity of new 4-diazopyrazole derivatives, European Journal of Medicinal Chemistry, Volume 31, Issue 6, 461-468. 5. Edwards, R., Gatehouse, J.A., 1999. Secondary Metabolism. In: P.Lea and R.C. Leegood (Eds.), Plant biochemistry and Molecular Biology. New York: John Wiley and Sons. 193-218. 6. Essawi .T and M. Srour, 2000. Screening of some Palestinian medicinal plants for antibacterial activity. Journal of Ethnopharmacology, Volume 70, Issue 3, 343-349.

7. Jada, Srinivasa Rao; Hamzah, Ahmad Sazali; Lajis, Nordin Haji; Saad, Mohammad Said; Stevens, Malcolm F.G.; Stanslas, Johnson. 2006. Semisynthesis and cytotoxic activities of andrographolide analogues, Journal of Enzyme Inhibition and Medicinal Chemistry, 327-327(1)

58

8. Janovska, D., Kubikova, K., Kokoska, L. 2000, Screening for antimicrobial activity of some medicinal plant species of traditional Chinese medicine. Czech. J. Food Sci. 21: 107-111.

9. Jonathan E. Kelmanson, Anna K. Jger and Johannes van Staden ,2000, Zulu medicinal plants with antibacterial activity. Journal of Ethnopharmacology, Volume 69, 241-246. 10. OM Walsh, MJ Meegan, RM Prendergast and T Al Nakib, 1996. Synthesis of 3acetoxyazetidin-2-ones and 3-hydroxyazetidin-2-ones with antifungal and antibacterial activity, European Journal of Medicinal Chemistry, Volume 31, Issue 12, 989-1000.

11. Owais .M , K.S. Sharad , A. Shehbaz and M. Saleemuddin , 2005. Antibacterial efficacy of Withania somnifera (ashwagandha) an indigenous medicinal plant against experimental murine salmonellosis, Phytomedicine, 229-235

12. zlem Temiz, lkay ren, Esin ener, smail Yalin and Nejat Uartrk, 1998. Synthesis and microbiological activity of some novel 5- or 6-methyl-2-(2,4disubstituted phenyl) benzoxazole derivatives Il Farmaco, European Journal of Medicinal Chemistry, Volume 53, Issue 5, 30 337-341

13. Phillipson JD (1990) Natural products and the development of selective antiprotozoal drugs. Phytother.

14. Poolsup N, Suthisisang C, Prathanturarug S, Asawamekin A, Chanchareon U. 2004 Andrographis paniculata in the symptomatic treatment of uncomplicated upper respiratory tract infection:systematic review of randomized controlled trails.

59

15. Prajjal K. Singha, 2003. Antimicrobial activity of Andrographis paniculata , Fitoterapia, Volume 74, Issues 7-8, 692-694.

16. Rajagopal S, Kumar RA, Deevi DS, Satyanarayana C, Rajagopalan R. 2003 May-Jun;3(3):147-58. Andrographolide, a potential cancer therapeutic agent isolated from Andrographis paniculata.

17. S.
a

Ramachandra

Rao

and

G.

A.

Ravishankarb

Laboratory of Biofunctional Materials, School of Materials Science, Japan 923-1292, Japan

Advanced Institute of Science and Technology, 1-1, Asahidai, Tatsunokuchi, Ishikawa

b

Plant Cell Biotechnology Department, Central Food Technological Research

Institute, Mysore 570 013, India

18. Sarsawat B, Visen PKS, Dayal R, Agarwal DP, Patnaik GK. 1996. Protective action of Ursolic acid against chemical induced hepatotoxicity in rats. Indian Journal of Pharmacology;28:232-39.

19. Sinha J.; Mukhopadhyay S.; Das N.; Basu M.K. 2000. Targeting of Liposomal Andrographolide to L.donovani-Infected Macrophages in Vivo Drug Delivery, Volume 7, Number 4, 1. 209-213(5).

20. Tipakorn, N., Tartrakoon, W., Thinggaard, G., Meulen, U. ter, 2004. Antibacterial activity of Andrographis paniculata leaf extracts. Journal of Agriculture and Rural Development in the Tropics and Subtropics, (Supplement 80) 187-194

60

21. Voravuthikunchai, Supayang Piyawan; Limsuwan, Surasak 2006 ; Medicinal Plant Extracts as Anti-Escherichia coli O157:H7 Agents and Their Effects on Bacterial Cell Aggregation. Journal of Food Protection, Volume 69, Number 10, 2336-2341(6).

22. Xiang, Y.., Liu, Y and Lee M.L.., (2006) . Journal of Chromatography A 1104 (1-2) 198-202.

23. Youhong Xu ; Raymond L. Marshall ; Trilochan K.S. Mukkur. 2006. An Investigation on the Antimicrobial Activity of Andrographis paniculata Extracts and Andrographolide in vitro, Asian Journal of Plant Sciences, Volume: 5 Issue: 3.

24. Zhang, Xiao-Qi; Wang, Guo-Cai; Ye, Wen-Cai; Li, Qian; Zhou, GuangXiong; Yao, Xin-Sheng , 2006. New Diterpenoids from Andrographis paniculata (Burm. f.) Nees, Journal of Integrative Plant Biology, Volume 48, Number 9, 1122-1125(4).

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