INTERNATIONAL RESEARCH JOURNAL OF PHARMACY ISSN Research Article

Transcription

1 INTERNATIONAL RESEARCH JOURNAL OF PHARMACY ISSN Research Article IN VITRO ANTIOXIDANT POTENTIAL OF ETHANOLIC EXTRACT OF ANISOMELES MALABARICA Suresh Subramanian and Suriyavathana Vedanarayanan* Dept of Biochemistry, Periyar University, Salem , Tamilnadu, India Article Received on: 20/02/12 Revised on: 11/04/12 Approved for publication: 28/04/12 *Dr. Suriyavathana Vedanarayanan M.Sc., M.Phil., Ph.D., Assistant Professor, Dept of Biochemistry, Periyar University, Salem , Tamilnadu, India. ABSTRACT The ethanolic extract of leaves from Anisomeles malabarica was tested for in vitro antioxidant using various free radical scavenging assays, such as 2,2- diphenyl-1-picryl hydrazyl (DPPH), chelating ability, reducing power, 2,2 -azinobis-(3-ethyl-benzothiazoline-6-sulfonicacid) (ABTS), hydroxy radical, nitric oxide, superoxide radical and ferric reducing antioxidant power (FRAP) were performed. All the concentrations of leaves extract showed effective free radical scavenging and antioxidant potential. The various antioxidant activities were compared with standard antioxidants such as ascorbic acid, EDTA, mannitol and butylated hydroxyanisole (BHA). Thus, the results obtained in the present study indicate that extract have more potential in vitro antioxidants and nutritional value, thereby this plant can be recommended as a potential natural economical viable and eco-friendly source and can be applied for land reclamation under agricultural sector in future. KEY WORDS: Anisomeles malabarica, Antioxidant, DPPH, FRAP, ABTS. INTRODUCTION Free radicals mediate some of the reactions of oxidants. They are implicated as agents of oxidative stress 1,2. The Reactive oxygen species (ROS) include peroxyl radical (ROOS), hydroxyl radical (HOS), superoxide radical (O - 2 ), hydrogen peroxide(h 2 O 2 ), and hypochlorous acid (HOCl) in addition to the RNS nitric oxide (NO) and peroxynitrite anion (ONOO - ). Oxidative stress, induced by oxygen radicals, is believed to be a primary factor in the development of several degenerative changes in cells and tissues, which ultimately lead to several degenerative disorders such as arthritis and atherosclerosis as well as other ailments viz cancer, diabetes, hepatitis, neurodegeneration and early aging 3,4,5. Bodily defenses are not completely efficient in preventing ongoing oxidative damage to DNA, lipids and proteins 6. Antioxidants are compounds that protect cells against the damaging effects of ROS which can neutralize free radicals before they can do harm and may help undo some damage already caused to specific cells. To determine radical scavenging capacities the measurement of the disappearance of free radicals, such as 2,2'-azino-bis(3-ethylbenzenthiazoline-6-sulphonic) acid radical (ABTS + ), the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH ) or other colored radicals, with a spectrophotometer 7,8. Dietary antioxidants, vitamins, flavonoids, plant phenolics, herbal formulations and Ayurvedic preparations are very essential in protecting against oxidative stress 9. The use of traditional medicine is wide-spread and plant still represent a large source of natural antioxidants that might serve as leads for the development of novel drugs 10,11,12. Plants (fruits, vegetables, medicinal herbs) contain a wide variety of free radical-scavenging molecules, such as phenolic compounds, nitrogen compounds, vitamins, terpenoids, and some other endogenous metabolites, that are rich in antioxidant activity 13. Anisomeles malabarica(l.) (Malabar catmint) is a medicinal plant has been used as a folkloric medicine to treat amentia, anorexia, fevers, swellings, rheumatism 14. The herb is reported to possess anticancer, allergenic, anthelmintic, antiallergic, antianaphylactic, antibacterial, anticarcinomic, antiedemic, antihistaminic, antiinflammatory, antileukemic, antinociceptive, antiplasmodial, antiseptic and antiperotic properties 15. In the present study, we investigated the ethanolic extract of for its free radical scavenging activities. MATERIALS AND METHODS Sample Extraction 10gm of dried powdered plant materials (leaves) were extracted with ethanol using Soxhlet apparatus for 48hrs. The solvent was distilled at lower temperature under reduced pressure and concentrated on water bath to get the crude extract which is stored for further use. Chemicals All chemicals used including the solvents were of analytical grade. BHA, Folin Ciocalteau reagent and ascorbic acid were purchased from Merck, Bangalore. All other chemicals and reagents used were of the highest commercially available purity. The solvents were purchased from Mercury Chemical Company, TamilNadu. DPPH, ABTS, trichloroaceticacid, hydrogen peroxide, potassium ferric cyanide, sodium carbonate, sodium phosphate, ammonium molybedate, mannitol and gallic acid from Aldrich chemicals. Phytochemical Screening The Phytochemical screening for the plant extract were carried out using standard chemical procedure 16. DETERMINATION OF ANTIOXIDANT CAPACITY It is important to select and employ a stable and rapid method to assay antioxidant activity, because the determination of many samples is time-consuming. Several methods have been developed to assay free radical scavenging capacity of plant extracts. The most common and reliable method involves the determination of the disappearance of free radicals using a spectrophotometer. DPPH Radical Quenching Activity Various concentrations of ethanolic extract of the sample (4.0ml) were mixed with 1.0ml of solution containing DPPH radicals, resulting in the final concentration of DPPH being 0.2mM 17. The mixture were shaken vigorously and left to stand for 30mins, and the absorbance was measured at Page 394

2 517nm. Ascorbic acid was used as control. The percentage of DPPH decolorization of the sample was calculated according to the equation: % decolorization = [1-(ABS sample /ABS control )] x 100 IC 50 value (µg extract/ml) is the inhibitory concentration at which DPPH radicals were scavenged by 50%. Ascorbic acid was used for comparison. Iron Chelating Activity The reaction mixture contained 1.0ml of various concentrations of the extract, 0.1ml of 2mM FeCl 2 and 3.7ml methanol 18. The control contained all the reaction reagents except sample. The reaction was initiated by the addition of 2.0ml of 5mM ferrozine. After 10min at room temperature, the absorbance of the mixture was determined at 562nm against a blank. A lower absorbance of the reaction mixture indicated a higher Fe 2+ chelating ability. The capacity to chelate the ferrous ion was calculated by % chelation = [1-(ABS sample /ABS control] x 100. Reducing Power The reaction mixture contained 2.5ml of various concentrations of ethanolic extract of the sample 2.5ml of 1% potassium ferricyanide and 2.5ml of 0.2M sodium phosphate buffer 19. The control contained all the reagents except the sample. The mixtures was incubated at 50 C for 20mins and were terminated by the addition of 2.5ml of 10% (W/V) of trichloroaceticacid, followed by centrifugation at 3000rpm for 10mins. 5.0ml of the supernatant upper layer was mixed with 5.0ml distilled water and 0.5ml of 0.1% ferric chloride and then absorbance was measured at 700nm against blanks that contained distilled water and phosphate buffer. Increased absorbance indicates increased reducing power of the sample. Ascorbic acid was used for comparison. ABTS Samples were diluted to produce 0.02 to 0.1µg/ml. The reaction was initiated by the addition of 1.0ml of diluted ABTS to different concentration of ethanolic extract of the sample or methanol as control 20. The absorbance was read at 734nm and the percentage inhibition was calculated. The inhibition was calculated according to the equation I = A 1 /A 0 x 100, where A 0 is the absorbance of control reaction, A 1 is the absorbance of test compound. Hydroxy Radical Activity The reaction mixture 3.0ml contained 1.0 ml of 1.5mM FeSo 4, 0.7ml of 6mM hydrogen peroxide, 0.3ml of 20mM sodium salicylate and varying concentrations of the extract 21. After incubation for 1 hour at 37 C, the absence of the hydroxylated salicylate complex was measured at 562nm. The percentage scavenging effect was calculated as scavenging activity = [1-(A 1 -A 2 )/A 0 ] x 100%. Where A 0 was absorbance of the control (without extract) and A 1 was the absorbance in the presence of the extract, A 2 was the absorbance without sodium salicylate. Nitric Oxide Radical Activity Nitric oxide radical generated from sodium nitroprusside (SNP) was measured according to the method 22. Briefly, the reaction mixture (5.0ml) containing SNP (5mM) in phosphate-buffered saline (ph 7.3), with or without the plant extract at different concentrations, was incubated at 25 C for 180mins. The NO thus generated interacted with oxygen to produce the nitrite ion (NO 2 ) which was assayed at 30mins intervals by mixing 1.0ml of incubation mixture with an equal amount of Griess reagent. The absorbance of the chromophore (purple azodye) formed during the diazotisation of nitrite ions with sulphanilamide and subsequent coupling with naphthylethylenediamine dihydrochloride was measured at 546nm. Superoxide Radical Scavenging Activity This assay was based on the reduction of nitro blue tetrazolium (NBT) in the presence of NADH and phenazine methosulphate (PMS) under aerobic condition 23. The 3ml reaction mixture contained 50µl of 1M NBT, 150µl of 1M NADH with or without sample and tris buffer (0.02M, ph 8.0). The reaction was started by adding 15µl of lm PMS to the mixture and the absorbance change was recorded to 560nm after 2mins. Percentage inhibition was calculated against a control without the extract. FRAP The stock solution of 10 mm 2,4,6- tripyridyl-s- triazine (TPTZ) in 40mM HCL, 20mM FeCl 3. 6H 2 O and 0.3M acetate buffer (ph 3.6) were prepared 24. The FRAP reagent contained 2.5ml TPTZ solution, 2.5ml ferric chloride solution and 25ml acetate buffer was freshly prepared and warmed to 37 C. 900µl FRAP reagent were mixed with 90µl water and 30µl test ethanolic extract of the sample and standard antioxidant solution. The reaction mixture was then incubated at 37 C for 30mins and the absorbance was recorded at 595nm. An intense blue colour complex was formed when ferric tripyridyl triazine (Fe 3+ -TPTZ) complex was reduced to ferrous (Fe 2+ ) form. The absorption at 540nm was recorded. RESULTS AND DISCUSSION Plant and plant products are being used as a source of medicine since long. The medicinal properties of plants have been investigated in the recent scientific developments throughout the world, due to their potent antioxidant activities, harmless effect and economic viability. Antioxidants are widely applied to foods and medicine because they can counteract cellular free radicals and reduce metal ion to interrupt the oxidizing chain reaction before they cause damage. PHYTOCHEMICAL SCREENING Phytochemical screening reveals the presence of Alkaloids, Flavonoids, Tannins, Resins, and Phenols. Absence of Saponins, Steroids and Glycosides. IN VITRO ANTIOXIDANT ACTIVITY DPPH Radical Scavenging Activity The ethanolic extract of exhibited a significant dose dependent inhibition of DPPH activity,with a 50% inhibition (IC 50 ) at a concentration of 300µg. The IC 50 value of the extract was found to be close to that of the standard; Compared to standard the extract exhibited a similar curve of antioxidant activity. The data obtained in this study reveal that the tested extract was a significant free radical scavenger which reacts with DPPH radical owing to its electrondonating ability. Comparison of the antioxidant activity of the extract and ascorbic acid is shown in Fig.1. The % activity of standard and the ethanolic extract of at the concentration range of µg/ml showed a good anti-radical activity in scavenging DPPH radical with an inhibition of about 39.11% to 52.19% and 43.21%to 56.26% respectively. Chelating Ability The chelating ability of ethanolic extract of and standard is represented in the Fig.2. The chelating ability of the plant extract exhibits minimum inhibition of 21% at 200µg and the maximum inhibition of 44% at 1000µg and the standard shows the minimum inhibition of 28.17% at 200 µg and maximum inhibition of 46.6% at 1000µg. Chelating Page 395

3 ability of the plant extract investigate in the present work is dose-dependent (i.e) the activity was increased on increasing concentration from 15% to 44.8%. Reducing Power The reducing power of both ethanolic extract of and ascorbic acid is shown in the Fig.3. The reducing power of extract (absorbance at 700nm) correlated well with increasing concentrations. The figure shows that the plant exhibit maximum absorbance of 1.02 at a concentration 500µg and the minimum absorbance of 0.5 at 100µg, whereas the standard shows the minimum absorbance of 0.3 at 100 µg and maximum absorbance of 0.96 at 500µg. The reducing power of extract was relatively more pronounced than that of Ascorbic Acid, it suggests that plant extract has significant antioxidant effect. ABTS Radical Scavenging Activity In this study, the ABTS ability of ethanolic extract was compared with BHA, BHA shows more pronounced activity in a dose-dependent manner. The % activity of shows minimum inhibition of about 22% at 0.02µg and the maximum inhibition of 46% at 0.1µg, where as the standard shows minimum inhibition of 24% at 0.02µg and the maximum inhi8bition of 49% at 0.1µg. Hydroxyl Radicals Scavenging Assay Hydroxyl radical scavenging is concentration dependent, the % of inhibition increase with the increase in concentration, the inhibition of ethanolic extract of ranges from 16% to 53% at a concentration ranging from 100µg to 500µg and the standard mannitol ranges from 18% to 58% at a concentration ranging from 100µg to 500µg. Nitric Oxide Scavenging Activity Nitric oxide scavenging activity of were studied and compared with ascorbic acid. The % activity of shows minimum inhibition of about 28% at 100µg and the maximum inhibition of 64% at 500µg, where as the % activity of standard shows minimum inhibition of 30% at 100µg and the maximum inhibition of 67% at 500µg. Superoxide Radical Scavenging Activity Ethanolic extract of leaves extracts showed a dose dependent inhibition of superoxide radicals. The superoxide anion scavenging ability of extract has been presented in Fig.7.The minimum in vitro superoxide scavenging activity of the ethanolic extract of is 11% at 200µg, whereas the maximum in vitro ability as 53% at 1000µg concentration. FRAP FRAP concentration, dependents on the absorbance; it increases with the increase in concentration, the absorbance of ethanolic extract of ranges from to at a concentration ranging from 20µg to 100µg and the standard ascorbic acid ranges from to at a concentration ranging from 20µg to 100µg. The extract shown to exhibit significant inhibitory activity against free radicals but there is less activity compared to that of standard. DISCUSSION The present study was conducted to investigate the antioxidant potential of. DPPH radicals are widely used in the model system to investigate the scavenging activities of several natural compounds 25. The scavenging DPPH radical assay is extensively used as a basic screening method for testing the antiradical activity of different plant materials such as leaf shoots, calli, leaves, inflorescences and fruits, among others. The DPPH radical contains an odd electron, which is responsible for the absorbance at 517nm and also for visible deep purple color 26. Chelation property may afford protection against oxidative damage and iron-overload 27. The chelating activity was measured by monitoring the colour reduction of the red Fe 2+ /ferrozine complex 28. Ferrozine can quantitatively form complexes with Fe 2+, in the presence of other chelating agents; the complex formation is disrupted with the result that the red colour of the complexes decreases. Measurement of the rate of colour reduction therefore allows estimation of the chelating activity of the coexisting chelator 29. The ethanolic extract of was estimated using potassium ferric cyanide reduction method. In this assay, the yellow reducing power is one mechanism for action of antioxidants and may serve as a significant indicator of potential antioxidant activity 30. The ABTS radical action is reactive towards most antioxidants including phenolics, thiols and Vitamin C. During this reaction, the blue ABTS radical cation is converted back to its colorless neutral form. The reaction may be monitored spectrophotometrically. The scavenging activity of ABTS + radical by the plant extract was found to be appreciable, this implies that the plant extract may be useful for treating radical related pathological damage especially at higher concentration 31. The highly reactive hydroxyl radical can cause oxidative damage to DNA, lipids and proteins 32. Hydroxyl radicals are highly strong reactive oxygen species, and there is no specific enzyme to defense against them in human body 33. Therefore, it is important to discover some chemicals with good scavenging capacity on these reactive oxygen species. In this study, the hydroxyl radical scavenging ability of ethanolic extract was compared with mannitol showed more pronounced hydroxyl radical scavenging activity in a dosedependent manner. Nitric oxide (NO) is a potent pleiotropic inhibitor of physiological processes such as smooth muscle relaxation, neuronal signaling, inhibition of platelet aggregation and regulation of cell mediated toxicity 34. In addition to reactive oxygen species, nitric oxide is also implicated in inflammation, cancer and other pathological conditions 35. The plant/plant products may have the property to counteract the effect of NO formation and in turn may be of considerable interest in preventing the ill effects of excessive NO generation in vivo. Superoxide anions are indirectly initiating the lipid oxidation 36. In cellular oxidation reactions, superoxide radicals are normally formed first, and their effects can be magnified because they produce other kinds of cell-damaging free radicals and oxidizing agents 37,38. Inhibitory effect of extract on superoxide radicals in a dosedependent manner, high inhibitions are observed at high concentrations. FRAP assay is quick and simple to perform and reaction is reproducible and linearly related to the molar concentration of the antioxidants present 39. In this present study leaves extract of results showed the ability to reduce free radicals which may stop the free radical initiation or retard free radical chain reaction in the propagation of the oxidation mechanism. CONCLUSION Recent years, we have seen an exponential increase in search of antioxidant properties in medicinal plants. Therefore it is time for us, to explore and identify our traditional therapeutic Page 396

4 knowledge and plant sources and interpret it according to the recent advancements to fight against oxidative stress. The results from this study indicate that the leaves of possess antioxidant properties and could serve as free radical inhibitor or scavenger or act possibly as primary antioxidant. The results of antioxidant activity in this study are in justification with the medicinal importance of plants as naturally occurring antioxidants. ACKNOWLEDGEMENT The authors are grateful to Periyar University, Salem, Tamilnadu, India for the financial support. REFERENCES 1. Eze MO. The oxygen paradox and the place of oxygen in our understanding of life, aging, and death. Ultimate Reality and Meaning (URAM): Studies in Medicine and Health 2006;29: (1 2), Eze MO, Yuan L, Crawford RM, Paranavitana JA, Hadfield TL, Bhattacharjee AK. Effects of opsonization and gamma-interferon on growth of Brucella melitensis 16M in mouse peritoneal Macrophages in vitro. Infection and Immunity 2000;68: Halliwell B, Gutteridge JMC, Cross CE. Free radicals, antioxidants and human disease: where are we now?journal of Laboratory and Clinical Medicine 1992;119: Halliwell B. Free radicals, antioxidants and human diseases: curiosity, cause or consequences. Lancet 1994; 334: Aviram M. Review of human studies on oxidative damage and antioxidant protection related to cardiovascular diseases. Free Radical Research 2000; 33: S85 S Weiss JF, Landauer MR. Radioprotection by antioxidants. Annals of the New York Academy of Sciences 2000; 899: Miller NJ, Rice-Evans CA. Factors influencing the antioxidantactivity determined by the ABTS radical cation assay. Free Radic, Res 1997; 26: Sanchez-Moreno C, Larrauri JA, Saura-Calixto F. A procedure to measure the antiradical efficiency of polyphenols. J. Sci. Food Agric 1998; 76: Weiss JF, Landauer MR. Radioprotection by antioxidants. Annals of the New York Academy of Sciences 2000; 899: Perry EK, Pickering AT, Wang WW, Houghton PJ, Perru NS. Medicinal plants and Alzheimer s disease: from ethnobotany to phytotherapy. Journal of Pharmacy and Pharmacology 1999; 51: Lin CC, Huang PC. Antioxidant and hepatoprotective effects of Acanthopanax senticosus. Phytotherapy Research 2002; 14: Repetto MG., Llesuy SF. Antioxidant properties of natural compounds used in popularmedicine for gastric ulcers. Brazilian Journal of Medicine and Biological Research 2002; 35: Cai YZ, Sun M, Corke H. Antioxidant activity of betalains from plants of the Amaranthaceae. Journal of Agricultural and Food Chemistry,2003;51: Chopra RN., Nayar SL., and Chopra IC., (1956). Glossary of Indian Medicinal Plants. Counsil of Scientific and Industrial Research, New Delhi Jeyachandran R., Mahesh A., and Cindrella L.,(2007). Int J Can Res; 3 (4): Harborne JB Phytochemical methods, Chapmann and Hall, London; 1973; Shimada K, Fujikawa K, Yahara Nakamura T. Antioxidative 505 properties of xanthin on autoxidation of soybeanoil in cyclodextrin emulsion. J. Agric. Food Chem 1992; 40: Dinnis TCP, Maderia VM, Almeida LM. Action of phenolic derivaties (acetaminophen, salicylate and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys 1994; 315: Oyaizu M. Studies on product of browning reaction prepared from glucose amine. Japanese Journal of Nutrition 1986; 44: Re R, Pellegrini N, Proteggente A, Pannala A, Yang, M and Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolrization assay. Free Radical Biology and Medicine 1999; Smirnoff N, Cumbes QJ. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 1989; 28: Garret DC. The quantitative analysis of drugs, champman and Hall, Japan 1964;3: Nishikimi M, Rao NA, Yagi K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophys. Res. Com 1972; 46 (2): Benzie FF, Strain JJ. Ferric Reducing/ Antioxidant Power Assay: Direct Measure of Total antioxidant Activity of Biological Fluids and Modified Version for Simultaneou s Mea surement of Total Antioxidant Power and Ascorbic Acid Concentration. Methods in enzymology 1999; 299: Singh, R.P., K.N. Chidambara Murthy and G.K. Jayaprakash, Studies on antioxidant activity of pomegranate peel and seed extracts using in vitro models. J.Agric. Food Chem., 50: Ferreres F, Sousa C, Valentao P, Seabra RM, Pereira JA, Andrade PB. Tronchuda cabbage (Brassica oleracea L. var. costata DC) seeds: phytochemical characterization and antioxidant potential. Food Che 2007; 101: Lai LS, Chan ST, Chao WW. Studies on the antioxidative activities of mesona procumbers Hems/leaf gum. J.Agric. Food Chem 2001;19: Elmastas M, Gulcin I, Isildak O, Kufrevioglu OI, Ibaoglu K, Aboul- Enein HY. Radical scavenging activity and antioxidant capacity of bay leaf extracts. Journal of the Iranian Chemical Society 2006; 3(3): Yamaguchi F, Ariga T, Yoshimira Y, Nakazawa H. Antioxidant and anti-glycation of carcinol from Garcinia indica fruit rind. Journal of Agricultural and Food Chemistry 2000; 48: Jayaprakasha GK, Negi PS, Sikder S, Mohanrao LJ, Sakariah KK. Antibacterial activity of Citrus reticulate peel extracts. Zeitschrift für Naturforschung C-A Journal of Biosciences 2000;55: Wang M, Li J, Rangarajan M, Shao Y, La Voie EJ, Huang T. Ho C:Antioxidative phenolic compounds from sage (Salvia officinalis)j Agric Food Chem 1998;46: Chatgilialoglu C, O Neill P. Free radicals associated with DNA damage. Exp Gerontol. 2001; 36: Liu CZ, Yu JC, Zhang XZ, Wang T, Han JX. On changes of activity of antioxidases in hippocampus of rats with multi-infarct dementia and the intervention effects of acupuncture China Journal of Traditional Chinese Medicine and Pharmacy,2005; 20: Hagerman AE, Riedl KM, Jones GA, Sovik KN, Ritchard NT, Hartzfeld PW. High molecular weight plant polyphenolics(tannins) as biological antioxidants. J Agric and Food Chem 1998; 46: Moncada A, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacological Reviews 1991;43: Gulcin I, Tel AZ, Kirecci E (2008). Antioxidant, antimicrobial, antifungal, and antiradical activities of Cyclotrichiumniveum (Boiss.) Manden and Scheng. Internat. J. Food Properties 11: Aruoma O. Assessment of potential prooxidant and antioxidant actions. J. Am. Oil Chem. Soc. 1996a; 73: Aruoma O. Assessment of potential prooxidant and antioxidant actions. J. Am. Oil Chem. Soc. 1996b; 73: Benzie IFF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of Antioxidant power the FRAP assay. Anal Biochem. 1999; 239: TABLE.1 PRELIMINARY PHYTOCHEMICAL EVALUATION OF ETHANOLIC EXTRACT OF ANISOMELES MALABARICA S.NO Phytochemical Constituents Anisomeles malabarica 1. Alkaloids + 2. Flavonoids + 3. Tannins + 4. Steroids - 5. Glycosides - 6. Resins + 7. Saponins - 8. Phenols + (+) Presence; (-) Absence Figure 1 DPPH Radical Scavenging Activity of Ethanolic extract of Page 397

5 Suriyavathana Vedanarayanan et al. IRJP 2012, 3 (5) Figure 2 Chelating Ability of Ethanolic extract of Figure 3 Reducing power of Ethanolic extract of Figure 4 ABTS Scavenging Activity of Ethanolic extract of Figure 5 Hydroxy Radical Scavenging Activity of Ethanolic extract of Figure 6 Nitric oxide Scavenging Activity of Ethanolic extract of Figure 7 Superoxide Radical Scavenging Activity of Ethanolic extract of Figure 8 FRAP Radical Scavenging Activity of Ethanolic extract of Source of support: Nil, Conflict of interest: None Declared Page 398