Research Article ISSN: 0974-6943

E.Susithra et al. / Journal of Pharmacy Research 2011,4(2),352-355

Available online through www.jpronline.info Evaluation of In-vitro Antioxidant activity of isolated compounds of lichen, Usnea undulata
E. Susithra* 1a, K.Mallikarjuna Rao 1b, K.V. Ramseshu2, S.Meena 2 a1* Department of Phytochemistry, Annamacharya College of Pharmacy, Kadapa, Rajampet-516 126, Andhra Pradesh, India. b1 Department of Pharmaceutics, Annamacharya College of Pharmacy, Kadapa, Rajampet-516 126, Andhra Pradesh, India. 2 K.M College of Pharmacy, Uthangudi, Madurai 625 107, Tamilnadu, India .Received on: 15-11-2010; Revised on: 10-12-2010; Accepted on:13-01-2011

ABSTRACT
The aim of the present study was to assess the In-vitro potential anti-oxidant and anti- inflammatory activities of isolated compounds (SU-I, SU-II, SU-III) of lichen, Usnea undulata, by different methods viz. DPPH radical scavenging assay, Hydroxyl radical scavenging assay Nitric oxide scavenging assay and Hydrogen peroxide assay. The Lichen material was extracted by continuous hot percolation method using solvents of increasing polarity and the obtained extracts were column chromatographed using silica gel (100-200 mesh) pooled and processed further. Accordingly, three compounds were isolated and characterized. The free radical scavenging activity of the isolated compounds was estimated by IC50 values at various concentrations of 20 to 80g/ml. At 80g/ml, DPPH radical scavenging assay, Hydroxyl radical scavenging assay, Nitric oxide assay and Hydrogen peroxide assay showed maximum inhibition of 80.31%, 81.20%, 83.83% and 84.31% respectively for the compound SU-I. These results clearly indicate that SU-I is effective in scavenging free radicals, thus proving its potential for powerful antioxidant activity. Key words:Hydroxyl scavenging assay, DPPH radical Scavenging, Usnea undulata, Usnic acid, Lecanoric acid, Atranorin and Salazinic acid

INTRODUCTION
The challenge for todays pharmaceutical industry lies in the discovery and development of new pharmacologically active molecules. Metabolites produced by micro-organisms and fungi in particular, are a resource for which the therapeutic potential has been recognized, but the one that remains largely unexplored and unexploited is approximately 20% of all the known fungal species, which are obligate symbionts in lichens. This major group of fungi has been long neglected by mycologists and overlooked. They have been used in traditional medicines for centuries and still hold considerable interest as alternative treatments in various parts of the world1, 2. Further, about 40 diseases including atherosclerosis, hypertension, ischemic diseases, Alzheimers disease, Parkinsonism, cancer and inflammatory conditions are being considered as free radical-mediated and have been investigated in detail3, 4, 5. Anti-oxidant principles from natural resources possess multifacedness in their multitude and magnitude of the activities and provide enormous scope in correcting the imbalance. Therefore, much attention is being directed to harness and harvest the anti-oxidant principles from natural resources6. Free radicals are fundamental to any biochemical process and represent an essential part of aerobic life in our metabolism. Reactions of free radicals such as DPPH, hydroxyl radical (OH), peroxy radical (ROO), nitric oxide (NO) and other reactive oxygen and nitrogen species are associated with diseases such as atherosclerosis, dementia and cancer. Lipids, proteins and DNA are the targets of such species and undergo oxidative reactions leading to their degradation. An anti-oxidant is any substance that, when present at low concentrations compared to those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate7. The anti-oxidant activity of lichen compounds is relatively the new area of investigation, usnic acid content from Parmelia caperata and Parmelia soredians has increased when subjected to oxidative stress, which indicates the anti-oxidant action. Lichens represent a unique division in the plant kingdom. They have been used in traditional systems of medicine including Traditional Indian Medicine (TIM), Traditional Chinese Medicine (TCM), Homeopathic and Western Medical Herbals8. The Lichen Division is comprised of at least 8 orders, 45 families, and 6,000 species. Information on the edible and medicinal uses of the lichens is scattered9. The medicinal utility of lichens is regarded to presence of secondary compounds like of usnic acid and atranorin 10. One of the reasons for exploring biological compounds in lichens is its potential for medical use11. However, much work remains to link medical effects with specific lichen species. Phenolic constituents from the lichen Parmotrema stuppeum (Nyl.) Hale (Parmeliaceae) including methyl orsenillate, orsenillic acid, atranorin and lecanoric acid showed moderate antioxidant activity12. An animal study reported antioxidant activity of lichen Cetraria islandica13. Stictic acid derivatives from the lichen Usnea articulata (Ach.) Motyka were reported to have significant activity14 . Concerning inflammation, free radicals are well known to play an important part in the inflammatory process. In the cyclooxygenase pathway, oxygen radicals are liberated during the conversion from PGG2 to PGH2, and different reports have suggested that an enhancement of free radical generation may contribute to the development of inflammatory processes and particularly to the pathogenesis of airway hyperactivity in asthma. In these conditions, the use of drugs with both scavenging activity and anti-inflammatory properties may be of benefit in the treatment of airway inflammation and bronchial hyper-responsiveness15. MATERIALS AND METHODS Lichen specimens were collected from Mahendragiri hills, Kanyakumari in the year 2008. The sample was dried at room temperature for 48 h. Dr. D.K Upreti, Lichen Laboratory, National Botanical Research Institute (CSIR), Lucknow, U.P, authenticated them as Usnea undulata Stirton (Authenticaton No : NBRI/LICH/ DH-2004-75). 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from drugs India, Hyderabad. Ascorbic acid, FeCl3, trichloroacetic acid, hydrogen peroxide, sodium nitroprusside, sulphanilamide, H PO 4, Napthylethylenediamine 3 dihydrochloride. All chemicals used including solvents were of analytical grade. Preparation of the extracts and isolation of compounds Air-dried and powdered lichen (250 g) was extracted with 2.5 liters each of solvents of increasing polarity starting from diethyl ether, dichloromethane, acetone, ethanol and methanol by using Soxhlet extractor for 72 hrs at a temperature not exceeding the boiling point of the solvents. The crude extracts from respective solvents were concentrated under reduced pressure, transfered to a watch glass and kept in a dessicator containing fused calcium chloride. The diethyl ether, dichloromethane and the acetone fractions were found to be promising16, 17. 1 g of dry residue in each case was passed through a column packed with silica gel (100 200 mesh) and developed according to the following lines. The column was built up by passing two column volumes of hexane before the residue was loaded. The solvent was kept 5 cm above the bed and the residue was carefully loaded in the form of petroleum ether slurry. The column was then developed with a series of solvent starting with hexane, hexane: ether, diethyl ether, dichloromethane and acetone as eluants depending on the polarity of compounds. The different ratios with succeeding solvents were fixed as shown in the Table No.1. Fractions of 40 ml were collected upto hexane: ether system and thereafter fractions in smaller volumes ( 20 ml ether fractions and 25 ml each of dichloro

*Corresponding author.
E.Susithra, M.Pharmacy., (Ph.D), Department of Phytochemistry, Annamacharya College of Pharmacy, Kadapa, Rajampet-516 126, Andhra Pradesh, India, Tel.: +91 9444628048. +919908299531 E-mail:suchitra303@yahoo.com, malli_pharmacist@yahoo.co.in

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Table No. 01, Plant Profile
Scientific classification Superregnum :Eukaryota Supergroup :Unikonta Cladus :Opisthokonta Regnum :Fungi Division :Ascomycota Subdivision :Pezizomycotina Class :Lecanoromycetes Subclass :Lecanoromycetidae Order :Lecanorales Family :Parmeliaceae Genus :Usnea Species :undulata Common names : Kalpasi, Marapasi Binomial name : Usnea undulate Synonym : Usnea dasceae

lichen, Usnea undulata was assessed by the In-Vitro method by 1,1 - Diphenyl-2picryl-hydrazyl (DPPH) assay. About 20, 40, 60 and 80 g/ml each ml of extract or standard was added to 3 ml of DPPH in methanol in a test tube. After mixing of this solution to standard and test, which was incubated at 37oC for 30 minutes, the absorbance of each solution was determined at 517 nm using spectrometer against the corresponding test and standard blanks. IC50 values were calculated and compared with that of ascorbic acid, which was used as standard. IC50 value is the concentration of the sample required to scavenge 50% free radical of sample20, 21. Inhibition (%) = [Abs (control) Abs (stdandard) /Abs (control)] 100. Where, Abs (control): Absorbance of DPPH radical + methanol Abs (stdandard) : Absorbance of DPPH radical + extract/standard.

Table No:02, Results of Different fractions of Thin Layer Chromatography technique.

Fraction 1-3 4-7 8-14 15-20 20-25 Elutes n-hexane n-hexane:ether 8 : 2 Ether Dichloromethane Acetone Nature of residue Colourless Colourless Yellow colour Light yellow colour Pale yellow colour TLC No distinctSpot Single bluefluorescence spot Single blue fluorescence spot One red Spot Red spot not clear.

Table No-03, Different fractions of eluates TLC profile.

Fraction 1 26 7-11 12-14 15, 16 17, 18 19 29 Elutes Methylene chloride Methylene chloride Methylene chloride: Chloroform. 9.5:0.5 Methylen chloride: Chloroform. 7:3 Chloroform : Acetone7:3 Chloroform : Acetone5:5 Chloroform: Acetone 2:8 Nature of residue Colourless Colourless Colourless Colourless Light yellow colour Colourless Colourless T. L. C No spot No spot No spot No spot Two spots Two spots (UV) Single spot (Red fluorescence in UV)

Hydroxyl Radical Scavenging Activity The scavenging capacity for hydroxyl radical was measured according to the modified method of Halliwell. Stock solutions of EDTA (1mM), FeCl3 (10 mM), ascorbic acid (1mM), H2O2 (10mM) and deoxyribose (10mM) were expressed in distilled deionized water. The assay was performed by adding 0.1 ml of EDTA , 0.01 ml of FeCl3, 0.1 ml of H20 2, 0.36 ml of deoxyribose,1.0 ml of isolated compounds (concentrations used were 20, 40, 60 and 80g/ml ) in distilled water, 0.33 ml of phosphate buffer ( 50mM, pH 7.4) and 0.1ml of ascorbic acid in sequence. The mixture was then incubated at 370C for 1hr. A 1.0 ml portion of the incubated mixture was mixed with 1.0 ml of 10% TCA and 1.0 ml of 0.5% TBA to develop the pink chromogen measured spectrophotometrically at 532nm22. OH- scavenged (%) = [Abs (control) Abs (standard) / Abs (control) ] 100. Where, Abs (control): Absorbance of the control reaction , Abs (standard): Absorbance of the extract/standard.

Table No: 04, Spectral datas of isolated compounds of lichen, Usnea undulata
Compounds SU-I (diethyl ether fraction) ppm 1.3-1.5 (CH 3 proton), 2.3 (aryl CH 3 hydrogen), 4.3 (OH proton), 7.2-7.8 (aryl hydrogen), 8.6 (aromatic protons) 2.4 (aryl CH 3 proton), 3.9 (aryl CH 2 OH proton), 6.2, 6.8 (aryl H proton), 10.4 (CHO). 1.2-1.4 (CH 3 and CH 2 protons), 2.12.6 (aryl methyl proton), 4.5 (OCH 3 stretching), 7.2-7.4 (aryl protons). , cm-1 2900 (C-H stretching), 1750 (ester carbonyl), 1663 (C-O stretching), 1579 (aromatic), 1200 ( aromatic ester), 850 (aliphatic, alcoholic ester) 3500 (OH stretching), 2850 (CH stretching), 1734 (ester carbonyl), 1650 ( C=O ketonic), 1464 (aromatic proton), 1213 (ester). 3400 (OH stretching), 2800 (CH stretching), 1769-1644 (C=O carbonyl), 1560-1438 (aromatic stretching), 1003 (Out of plane).

SU-II (DCM columnfraction)

Nitric Oxide Radical Scavenging Activity Sodium Nitroprusside spontaneously generates nitric oxide in aqueous solution. Nitric oxide generated in this manner is converted into nitric acid and nitrous acid in contact with dissolved oxygen and water. The liberated nitrous acid is estimated using Griess reagent which forms a purple azo dye in the presence of a test compound likely to be the scavenger and the amount of nitrous acid will decrease. The degree of decrease in the formation of purple azo dye will reflect the degree of scavenging. Sodium nitroprusside (5mM) in phosphatebuffered saline (PBS) was mixed with 3.0 ml of different concentrations (20, 40, 80, 160 and 320 g / ml) of the drugs dissolved in the suitable solvent systems and incubated at 25oC for 2hrs. The samples from the above were reacted with Griess reagent (1% sulphanilamide, 2% H3PO4 and 0.1% napthylethylenediamine dihydrochloride). The absorbance of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine was read at 546 nm and referred to the absorbance of standard solutions of potassium nitrite treated in the same way with Greiss reagent23, 24. scavenged (%) = [Abs (control) Abs (standard) / Abs (control)] 100. Where, Abs (control) : Absorbance of the control reaction , Abs (standard) : Absorbance of the extract/standard.

SU-III (Acetone column fraction)

Table No: 05, Anti-oxidant IC 50 values in different In-vitro models.

S.No 1 2 3 4 ASSAY DPPH Scavenging assay Hydroxyl radical scavenging assay Nitric oxide assay Hydrogen peroxide assay SU-I 54.70 52.76 51.65 50.82 SU-II 68.52 67.24 68.35 64.46 SU-III 69.41 68.45 70.78 65.86 ASCORBIC ACID 50.6 49.54 49.25 46.83

-methane and acetone fractions) were collected, checked with T.L.C. and accordingly pooled, concentrated and processed further. The column ether fraction and the dichloromethane fractions were labeled as SU I & SU II respectively. Similarly, the acetone extracts was developed with chloroform: dichloromethane, acetone: ethyl acetate in different ratios. The results are presented in table No. 2. Fractions 19-29 were pooled and processed further (SU-III). All the lichen substances isolated by precipitation or preparative TLC 16, 17 were further characterized by the standardized methods such as spectroscopy techniques UV (Perkin-Elmer Lambda 3 spectrophotometer), NMR, IR and melting point18, 19. ANTIOXIDANT ACTIVITY DPPH Radical Scavenging Activity The antioxidant activity of the isolated compounds (SU-I, SU-II, SU-III) of

Scavenging Of Hydrogen Peroxide Hydrogen peroxide is the least reactive molecule among reactive oxygen species and is stable under physiological pH and temperature in the absence of metal ions. It can be generated through a dismutation reaction from superoxide anion by superoxide dismutase It can generate the hydroxyl radical ion in the presence of metal ions and superoxide anion25, 26. A solution of hydrogen peroxide was prepared in phosphate buffer (pH 7.4). The different concentrations of isolated compounds were prepared (20, 40, 60 and 80g/ml) and added to hydrogen peroxide solution (0.6 ml). Absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. IC50 value is the concentration of the sample required to scavenge 50% free radical of sample. All the assay similarly performed using ascorbic acid standard compound. % scavenging activity [H2O 2] =[Abs (control) Abs (standard) / Abs (control)] 100. Where, Abs (control): Absorbance of the control , Abs (standard): Absorbance of the extract/standard. RESULTS AND DISCUSSION Lichens, symbiotic organisms of fungi and algae, synthesize numerous metabolites, the lichen substances, which comprise phenolic compounds such as depsides, depsidones, dibenzofurans, usnic acids, depsones and others. These substances, as well as their derivatives obtained by structural modification, have a manifold biological activity: antiviral, antibiotic, antitumor, allergenic, plant growth in-

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Graph No:- 01, DPPH Assay scavenging profile Lichens were collected from Mahendragiri hills for the purpose of the present study. Air-dried lichen material was extracted by soxhletion using solvents of increasing polarity and three compounds were isolated and characterized as SU-I, SU-II and SU-II from the ether, dichloromethane and acetone fractions respectively. As the introduction of new analytical methods (Thin layer chromatography, high-performance liquid chromatography, ultra violet, infra red, nuclear magnetic resonance spectroscopy) has led to the isolation of many new lichen substances, the different lichen substances were identified by TLC and standardized by spectral analysis (Table no 4). As the physio-chemical properties and spectral datas suggests, compound I (SU-I) might be assumed as usnic acid, a dibenzfuran, compound II (SU-II) as salazinic acid or atranorin, a depsidone, an aromatic aldehyde and compound III (SU-III) as lecanoric acid, a depside. These novel bioactive compounds were screened for potent anti-oxidant activity. The various concentrations ranging from 20-80g/ml of isolated compounds of Usnea undulata were tested for their free radical scavenging activity. It was observed for free radical scavenging by in dose dependent manner. DPPH is one of the free radical generally used for testing preliminary radical scavenging activity of a compound or a plant extract. DPPH radical is a stable free radical in an aqueous or methanol solution. It accepts an electron or hydrogen radical to become stable diamagnetic molecule. Because of the odd electron of DPPH, it gives a strong absorption maximum at 517 nm by visible spectroscopy. The antioxidant activity measured by the capacity of odd electron of the radical becomes paired off in the presence of an extract (hydrogen donor). When it becomes paired off, the absorption strength is decreased, and the resulting decolorization is stoichiometric with respect to the number of electrons captured. Substances which are able to perform this reaction can be considered as antioxidants and hence radical scavengers. The DPPH Scavenging results were shown graph No: 01. Hydroxyl radical is highly reactive species formed in biological damaging almost every molecule found in living cells. In addition, this species is one of the quick initiators of lipid peroxidation process, causing cell damage. Its very important to scavenge hydroxyl radical. The hydroxyl radical scavenging values of the isolated lichen compounds were shown in the graph No: 02. The Nitric oxide radical scavenging assay procedure is based on the principle that, sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of nitric oxide compete with oxygen, leading to reduced production of nitrite ions. The Nitric oxide radical scavenging assay values were shown in Graph No: 03. The hydrogen peroxide radical scavenging values also were shown in graph No: 04. All the methods of anti-oxidant activity were also calculated in the form of IC50, these values were shown in the table No: 05. CONCLUSION The isolated compounds of Lichen (SU-I, SU-II, SU-III) showed different levels of antioxidant activity in all models studied. It possess good activity in scavenging DPPH radical, Hydroxyl radical, Nitric oxide radical and scavenging of H2O2 . By the comparison of all results of above mentioned methods the scavenging inhibition of the isolated compound, SU-I is found to possess better activity compared with other compounds, when compared with that of standard (Ascorbic acid). Graph No: 04, Hydrogen peroxide assay scavenging profile ACKNOWLEDGEMENT We are thankful to Honorable Secretary and Principal of Annamacharya College of Pharmacy, New Boyanapally, Rajampeta for providing all the facilities for carrying out this research work and also thankful to Dr. D.K Upreti, Lichen Laboratory, National Botanical Research Institute (CSIR), Lucknow, U.P, who authenticated them as Usnea undulata. REFERENCES
1) 2) 3) 4) 5) Crittenden P.D and Porter N, Lichen forming fungi, potential sources of novel metabolites, Trends Biotechnol, 1991, 9, 409-414. Richardson D.H.S., Lichens and Man. In frontiers in mycology, honorary and general lectures from the fourth international mycological congress, Regensberg, Germany, C.A.B International, 1991, 187-210. Tripathy Y.B and Upadhyay A.K, Antioxidant property of mucuna pruriens linn, Current Science 2001, 80, 1377 78. Niki E, Anti-oxidants free radicals and biological defense, Eds: Niki E, Shimasaki H and Mino M, Japan Scientific press Tokyo, 1995, 3 - 5. Toda S, Kumura M, Ohnishi M. Effects phenol carboxylic acid on superoxide anion an lipid peroxidation induced by superoxide anion, Planta medica, 1991, 57, 8 10. Tiwari A.K, Imbalence in antioxidant defense and human diseases. Multiple approach of natural Anti-oxidants therapy, Current Science, 2001, 81, 1179 1187. Katja M. Fisch, Volker Bohm, Antony D, Wright and Gabriele M. Konig, Anti-oxidative meroterpenoids from the brown algae cystoseira crinite, Journal of natural products, 2003, 66, 968 975. Bown D, Encyclopedia of Herbs and their Uses. Dorling Kindersley, London. 2001. Chevallier A, The Encyclopedia of Medicinal Plants Dorling Kindersley. London, 1996. Boustie J, Grube M. Lichens-a promising source of bioactive secondary metabolites. Plant Genetic Resources, 2005, 3 : 273-278.

Graph No: 02, Hydroxyl Radical Scavenging assay profile

Graph No: 03, Nitric oxide assay scavenging profile

hibitory, antiherbivore, anti-inflammatory, anti-oxidant and enzyme inhibitory. Numerous studies on the biological activities of phenols have indicated that these substances are also potent antioxidants and free radical scavengers. As different lichens produce chemically different lichen metabolites, our present focus is on the investigation of lichen metabolites from the lichen, Usnea undulata and synthesis of new and possibly more novel bioactive compounds for their wide application as potent anti-oxidant and anti-inflammatory agents.

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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 2. February 2011

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