Research Article E.Susithra et al. / Journal of Pharmacy Research 2011,4(2),352-355 ISSN: 0974-6943 Available online through http://jprsolutions.info Evaluation of In-vitro Antioxidant activity of isolated compounds of lichen, undulata E. Susithra*1a, K.Mallikarjuna Rao1b, K.V. Ramseshu2, S.Meena2 a1*Department of Phytochemistry, Annamacharya College of Pharmacy, Kadapa, Rajampet-516 126, Andhra Pradesh, India. b1Department of Pharmaceutics, Annamacharya College of Pharmacy, Kadapa, Rajampet-516 126, Andhra Pradesh, India. 2K.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 80µg/ml. At 80µg/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 today’s pharmaceutical industry lies in the discovery and devel- Phenolic constituents from the lichen Parmotrema stuppeum (Nyl.) Hale opment of new pharmacologically active molecules. Metabolites produced by () including methyl orsenillate, orsenillic acid, atranorin and lecanoric micro-organisms and fungi in particular, are a resource for which the therapeutic acid showed moderate antioxidant activity12. An animal study reported antioxi- potential has been recognized, but the one that remains largely unexplored and dant activity of lichen Cetraria islandica13. Stictic acid derivatives from the lichen unexploited is approximately 20% of all the known fungal species, which are Usnea articulata (Ach.) Motyka were reported to have significant activity14. obligate symbionts in lichens. This major group of fungi has been long neglected Concerning inflammation, free radicals are well known to play an important part by mycologists and overlooked. They have been used in traditional medicines for in the inflammatory process. In the cyclooxygenase pathway, oxygen radicals are centuries and still hold considerable interest as alternative treatments in various liberated during the conversion from PGG2 to PGH2, and different reports have parts of the world1, 2. Further, about 40 diseases including atherosclerosis, hyper- suggested that an enhancement of free radical generation may contribute to the tension, ischemic diseases, Alzheimer’s disease, Parkinsonism, cancer and inflam- development of inflammatory processes and particularly to the pathogenesis of matory conditions are being considered as free radical-mediated and have been airway hyperactivity in asthma. In these conditions, the use of drugs with both investigated in detail3, 4, 5. Anti-oxidant principles from natural resources possess scavenging activity and anti-inflammatory properties may be of benefit in the multifacedness in their multitude and magnitude of the activities and provide treatment of airway inflammation and bronchial hyper-responsiveness15. enormous scope in correcting the imbalance. Therefore, much attention is being directed to harness and harvest the anti-oxidant principles from natural resources6. MATERIALS AND METHODS Free radicals are fundamental to any biochemical process and represent an essen- Lichen specimens were collected from Mahendragiri hills, Kanyakumari in the tial part of aerobic life in our metabolism. Reactions of free radicals such as DPPH, year 2008. The sample was dried at room temperature for 48 h. Dr. D.K Upreti, hydroxyl radical (OH), peroxy radical (ROO), nitric oxide (NO) and other reac- Lichen Laboratory, National Botanical Research Institute (CSIR), Lucknow, U.P, tive oxygen and nitrogen species are associated with diseases such as atherosclero- authenticated them as Usnea undulata Stirton (Authenticaton No : NBRI/LICH/ sis, dementia and cancer. Lipids, proteins and DNA are the targets of such species DH-2004-75). 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from drugs and undergo oxidative reactions leading to their degradation. An anti-oxidant is India, Hyderabad. Ascorbic acid, FeCl3, trichloroacetic acid, hydrogen peroxide, any substance that, when present at low concentrations compared to those of an sodium nitroprusside, sulphanilamide, H3PO4, Napthylethylenediamine oxidizable substrate, significantly delays or prevents oxidation of that substrate7. dihydrochloride. All chemicals used including solvents were of analytical grade.

The anti-oxidant activity of lichen compounds is relatively the new area of Preparation of the extracts and isolation of compounds investigation, usnic acid content from Parmelia caperata and Parmelia soredians Air-dried and powdered lichen (250 g) was extracted with 2.5 liters each of sol- has increased when subjected to oxidative stress, which indicates the anti-oxidant vents of increasing polarity starting from diethyl ether, dichloromethane, ac- action. Lichens represent a unique division in the plant kingdom. They have been etone, ethanol and methanol by using Soxhlet extractor for 72 hrs at a tempera- used in traditional systems of medicine including Traditional Indian Medicine ture not exceeding the boiling point of the solvents. The crude extracts from (TIM), Traditional Chinese Medicine (TCM), Homeopathic and Western Medical respective solvents were concentrated under reduced pressure, transfered to a Herbals8. The Lichen Division is comprised of at least 8 orders, 45 families, and watch glass and kept in a dessicator containing fused calcium chloride. The diethyl 6,000 species. Information on the edible and medicinal uses of the lichens is ether, dichloromethane and the acetone fractions were found to be promising16, 17. scattered9. The medicinal utility of lichens is regarded to presence of secondary compounds like of usnic acid and atranorin10. One of the reasons for exploring 1 g of dry residue in each case was passed through a column packed with silica gel biological compounds in lichens is its potential for medical use11. However, much (100 – 200 mesh) and developed according to the following lines. The column work remains to link medical effects with specific lichen species. 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 *Corresponding author. a series of solvent starting with hexane, hexane: ether, diethyl ether, E.Susithra, M.Pharmacy., (Ph.D), dichloromethane and acetone as eluants depending on the polarity of compounds. Department of Phytochemistry, Annamacharya College of Pharmacy, The different ratios with succeeding solvents were fixed as shown in the Table Kadapa, Rajampet-516 126, Andhra Pradesh, India, 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

Journal of Pharmacy Research Vol.4.Issue 2. February 2011 352-355 E.Susithra et al. / Journal of Pharmacy Research 2011,4(2),352-355 Table No. 01, Plant Profile lichen, Usnea undulata was assessed by the In-Vitro method by 1,1 - Diphenyl-2- Scientific classification picryl-hydrazyl (DPPH) assay. About 20, 40, 60 and 80 µg/ml each ml of extract or standard was added to 3 ml of DPPH in methanol in a test tube. After mixing of Superregnum :Eukaryota o Supergroup :Unikonta this solution to standard and test, which was incubated at 37 C for 30 minutes, the Cladus :Opisthokonta absorbance of each solution was determined at 517 nm using spectrometer against Regnum :Fungi the corresponding test and standard blanks. IC values were calculated and com- Division : 50 Subdivision :Pezizomycotina pared with that of ascorbic acid, which was used as standard. IC50 value is the Class : concentration of the sample required to scavenge 50% free radical of sample20, 21. Subclass :Lecanoromycetidae Inhibition (%) = [Abs – Abs /Abs ] × 100. Order : (control) (stdandard) (control) Family :Parmeliaceae Genus :Usnea Where, Abs : Absorbance of DPPH radical + methanol Species :undulata (control) Common names : Kalpasi, Marapasi Abs (stdandard) : Absorbance of DPPH radical + extract/standard. Binomial name : Usnea undulate Synonym : Usnea dasceae Hydroxyl Radical Scavenging Activity The scavenging capacity for hydroxyl radical was measured according to the Table No:02, Results of Different fractions of Thin Layer Chromatogra- modified method of Halliwell. Stock solutions of EDTA (1mM), FeCl (10 mM), phy technique. 3 ascorbic acid (1mM), H2O2 (10mM) and deoxyribose (10mM) were expressed in Fraction Elutes Nature of residue TLC distilled deionized water. The assay was performed by adding 0.1 ml of EDTA ,

0.01 ml of FeCl3, 0.1 ml of H202, 0.36 ml of deoxyribose,1.0 ml of isolated 1-3 n-hexane Colourless No distinctSpot compounds (concentrations used were 20, 40, 60 and 80µg/ml ) in distilled water, 4-7 n-hexane:ether 8 : 2 Colourless Single bluefluorescence spot 0.33 ml of phosphate buffer ( 50mM, pH 7.4) and 0.1ml of ascorbic acid in 8-14 Ether Yellow colour Single blue fluorescence spot 0 15-20 Dichloromethane Light yellow colour One red Spot sequence. The mixture was then incubated at 37 C for 1hr. A 1.0 ml portion of the 20-25 Acetone Pale yellow colour Red spot not clear. 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 – Abs / Abs ] × 100. Table No-03, Different fractions of eluates TLC profile. (control) (standard) (control) Fraction Elutes Nature of residue T. L. C Where, Abs (control): Absorbance of the control reaction , Abs : Absorbance of the extract/standard. 1 Methylene chloride Colourless No spot (standard) 2 – 6 Methylene chloride Colourless No spot 7-11 Methylene chloride: Chloroform. 9.5:0.5 Colourless No spot Nitric Oxide Radical Scavenging Activity 12-14 Methylen chloride: Chloroform. 7:3 Colourless No spot Sodium Nitroprusside spontaneously generates nitric oxide in aqueous solution. 15, 16 Chloroform : Acetone7:3 Light yellow colour Two spots Nitric oxide generated in this manner is converted into nitric acid and nitrous acid 17, 18 Chloroform : Acetone5:5 Colourless Two spots (UV) in contact with dissolved oxygen and water. The liberated nitrous acid is estimated 19 – 29 Chloroform: Acetone 2:8 Colourless Single spot (Red fluorescence in UV) 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. Table No: 04, Spectral data’s of isolated compounds of lichen, Usnea The degree of decrease in the formation of purple azo dye will reflect the degree undulata of scavenging. Sodium nitroprusside (5mM) in phosphate–buffered saline (PBS) was mixed with 3.0 ml of different concentrations (20, 40, 80, 160 and 320 µg / Compounds d ppm l, cm-1 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% SU-I (diethyl 1.3-1.5 (CH3 proton), 2.3 (aryl CH3 2900 (C-H stretching), 1750 (ester ether fraction) hydrogen), 4.3 (OH proton), 7.2-7.8 carbonyl), 1663 (C-O stretching), 1579 sulphanilamide, 2% H3PO4 and 0.1% napthylethylenediamine dihydrochloride). (aryl hydrogen), 8.6 (aromatic (aromatic), 1200 ( aromatic ester), 850 The absorbance of the chromophore formed during the diazotization of nitrite protons) (aliphatic, alcoholic ester) with sulphanilamide and subsequent coupling with napthylethylenediamine was SU-II (DCM 2.4 (aryl CH proton), 3.9 (aryl 3500 (OH stretching), 2850 (CH 3 read at 546 nm and referred to the absorbance of standard solutions of potassium columnfraction) CH2OH proton), 6.2, 6.8 (aryl H stretching), 1734 (ester carbonyl), 1650 ( 23, 24 proton), 10.4 (CHO). C=O ketonic), 1464 (aromatic proton), nitrite treated in the same way with Greiss reagent . 1213 (ester). scavenged (%) = [Abs (control) – Abs (standard) / Abs (control)] × 100.

SU-III (Acetone 1.2-1.4 (CH3 and CH2 protons), 2.1- 3400 (OH stretching), 2800 (CH column 2.6 (aryl methyl proton), 4.5 (OCH stretching), 1769-1644 (C=O carbonyl), 3 Where, Abs (control) : Absorbance of the control reaction , fraction) stretching), 7.2-7.4 (aryl protons). 1560-1438 (aromatic stretching), 1003 (Out Abs : Absorbance of the extract/standard. of plane). (standard) Scavenging Of Hydrogen Peroxide Table No: 05, Anti-oxidant IC50 values in different In-vitro models. Hydrogen peroxide is the least reactive molecule among reactive oxygen species S.No ASSAY SU-I SU-II SU-III ASCORBIC ACID 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 1 DPPH Scavenging assay 54.70 68.52 69.41 50.6 superoxide dismutase It can generate the hydroxyl radical ion in the presence of 2 Hydroxyl radical scavenging assay 52.76 67.24 68.45 49.54 metal ions and superoxide anion25, 26. A solution of hydrogen peroxide was pre- 3 Nitric oxide assay 51.65 68.35 70.78 49.25 4 Hydrogen peroxide assay 50.82 64.46 65.86 46.83 pared in phosphate buffer (pH 7.4). The different concentrations of isolated compounds were prepared (20, 40, 60 and 80µg/ml) and added to hydrogen perox- ide solution (0.6 ml). Absorbance of hydrogen peroxide at 230 nm was determined -methane and acetone fractions) were collected, checked with T.L.C. and accord- after 10 min against a blank solution containing phosphate buffer without hydro- ingly pooled, concentrated and processed further. The column ether fraction and gen peroxide. IC50 value is the concentration of the sample required to scavenge the dichloromethane fractions were labeled as SU –I & SU – II respectively. 50% free radical of sample. All the assay similarly performed using ascorbic acid Similarly, the acetone extracts was developed with chloroform: dichloromethane, standard compound. acetone: ethyl acetate in different ratios. The results are presented in table No. 2. % scavenging activity [H O ] =[Abs – Abs / Abs ] × 100. Fractions 19-29 were pooled and processed further (SU-III). 2 2 (control) (standard) (control)

16, 17 Where, Abs (control): Absorbance of the control , All the lichen substances isolated by precipitation or preparative TLC were Abs : Absorbance of the extract/standard. further characterized by the standardized methods such as spectroscopy tech- (standard) niques UV (Perkin-Elmer Lambda 3 spectrophotometer), NMR, IR and melting 18, 19 RESULTS AND DISCUSSION point . Lichens, symbiotic organisms of fungi and algae, synthesize numerous metabo- lites, the “lichen substances,” which comprise phenolic compounds such as depsides, ANTIOXIDANT ACTIVITY depsidones, dibenzofurans, usnic acids, depsones and others. These substances, as DPPH Radical Scavenging Activity well as their derivatives obtained by structural modification, have a manifold The antioxidant activity of the isolated compounds (SU-I, SU-II, SU-III) of biological activity: antiviral, antibiotic, antitumor, allergenic, plant growth in- Journal of Pharmacy Research Vol.4.Issue 2. February 2011 352-355 E.Susithra et al. / Journal of Pharmacy Research 2011,4(2),352-355 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 respec- tively. As the introduction of new analytical methods (Thin layer chromatogra- phy, 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 standard- ized by spectral analysis (Table no 4). As the physio-chemical properties and spectral data’s 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 20-80µg/ml of isolated compounds of Usnea undulata were tested for their free radical scavenging activity. It was Graph No: 02, Hydroxyl Radical Scavenging assay profile 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 decol- orization is stoichiometric with respect to the number of electrons captured. Substances which are able to perform this reaction can be considered as antioxi- dants 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. It’s very important to scavenge hydroxyl radical. The hydroxyl radical scavenging values of the isolated lichen compounds were shown in the graph No: 02. The Graph No: 03, Nitric oxide assay scavenging profile Nitric oxide radical scavenging assay procedure is based on the principle that, sodium nitroprusside in aqueous solution at physiological pH spontaneously gen- erates 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 scav- enging 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 inhibi- tion 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.

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

Journal of Pharmacy Research Vol.4.Issue 2. February 2011 352-355