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, Usnea 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 (Parmeliaceae) 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 :Ascomycota 50 Subdivision :Pezizomycotina pared with that of ascorbic acid, which was used as standard. IC50 value is the Class :Lecanoromycetes concentration of the sample required to scavenge 50% free radical of sample20, 21. Subclass :Lecanoromycetidae Inhibition (%) = [Abs – Abs /Abs ] × 100. Order :Lecanorales (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