In vitro Free Radical Scavenging Activity of Alcoholic Root Extract of Pseudarthria viscida

Transcription

1 In vitro Free Radical Scavenging Activity of Alcoholic Root Extract of Pseudarthria viscida K. Rajendran 1 *, Annie Shirwaikar 1, Arun Shirwaikar 1, P. Ramalingam 1 and K.K. Srinivasan 2 1College of Pharmacy, Gulf Medical University, Ajman, UAE. 2Department of Medicinal Chemistry, Manipal College of Pharmaceutical Sciences, Manipal, India. *Presenting Author ABSTRACT Objective: This research investigated on in vitro ffree-radical scavenging property for the alcoholic extract from Pseudarthria viscida Linn. (Leguminosae). Different antioxidant models of screening were employed. Material and methods: The alcoholic root extract of P. viscida was screened for free radical scavenging potential using 2, 2- azinobis- (3- ethyl-benzothiazoline- 6- sulphonate) (ABTS), 1, 1- diphenyl, 2- picrylhydrazyl (DPPH), superoxide and nitric oxide radicals. Results: Concentration dependent free radical scavenging activity was observed for the extract in which a concentration of 1000 g/ ml exhibited maximum scavenging activity against the radical cation, 2,2- azinobis- (3- ethylbenzothiazoline- 6- sulphonate) (99.41%), followed by superoxide radical using Riboflavin- Ethylene diamine tetra-acetic acid (97.60%) and the nitric oxide model (82.50%). However, only moderate scavenging activity was observed with the 1, 1- diphenyl, 2- picryl hydrazyl radical (59.61%) at the same concentration. Conclusion: All the results of the in vitro antioxidant models of screening revealed the potent ffree-radical scavenging of the alcoholic root extract of Pseudarthria viscida Linn. Keywords: Free radical scavenging, alcoholic root extract, Pseudarthria viscida Page 117

2 INTRODUCTION Free radicals are essential for biochemical process and signify vital role in aerobic life as well in metabolism. They are formed by oxygen of respiration and cell mediated immune functions. A dynamic equilibrium always exists between the levels of free radical formed in the biological process and antioxidants to scavenge those radicals, and thus this equilibrium protects biological system from their harmful effects. The ideology of a majority of disease conditions like hypertension, cancer, diabetes mellitus, inflammatory conditions, atherosclerosis, Alzhemier s disease and parkinsonism are assumed due to the imbalance that exist between pro-oxidant and antioxidant homeostasis. Constituents with antioxidant property of natural resources provide vast scope in altering the imbalance. The roots of Pseudarthria viscida Linn. (Leguminosae) are used as an astringent, anthelmintic, bitter, anti-inflammatory, digestive, diuretic, aphrodisiac, cardiotonic, and rejuvenating as a tonic 1. There is an increasing significance all over the world for discovering the unexploited reservoir of medicinal plants. Hence, the present study was focused on measuring free radical scavenging property of alcoholic root extract from Pseudarthria viscida. MATERIALS AND METHODS Plant material In the month of August, P. viscida roots were collected was identified by a Botanist from Medicinal Plant Research Unit, Chennai, Tamil Nadu. A voucher specimen (PP 546) of the root was archived at pharmacognosy, department. Manipal College of Pharmaceutical Sciences (MCOPS), Manipal. Preparation of PV alcoholic root extract About 500 g of finely powdered root powder was subjected to extraction using 90% ethanol in a Soxhlet extractor for period of 72 h. The extract for the study was prepared by solvent distillation in vacuo and the residue was stored under refrigeration for further experimental use. Phytochemical screening The alcoholic extract was subjected to preliminary phytochemical screening using standard methods 2,3. In vitro antioxidant studies ABTS radical cation decolorization assay ABTS radical cation (ABTS.+ ) was produced by reacting ABTS solution (7mM) with mm ammonium persulfate. The mixture was allowed to stand in dark at 20 C for hours before use. The different concentrations ( µg/ml) of the alcoholic extract (0.5 ml) were added to 0.3 ml of ABTS solution and the final volume was made up to 1 ml with ethyl alcohol. The absorbance was measured at 745 nm and the percentage inhibition was calculated 4. DPPH free radical scavenging property DPPH scavenging activity was determined by the spectrophotometric method. To an aqueous solution of DPPH (200 µm), 0.05 ml of alcoholic extract were added at different concentrations ( µg/ml) and an equal amount of water and ethyl alcohol was added to the control. After 20 min the decrease in absorbance of test extract (due to quenching of DPPH free radicals) was measured at 517 nm and the percentage of inhibition was calculated 5. Page 118

3 Scavenging of nitric oxide radical Nitric oxide was generated from sodium nitroprusside and measured by Griess reaction as described. Sodium nitroprusside (5mM) in standard phosphate buffer solution was incubated with different concentrations ( µg/ml) of the alcoholic extract dissolved in phosphate buffer (0.025 M, ph: 7.4) and the tubes were incubated at 25º C for 5 hr. Control experiments without the test extract but with equivalent amounts of buffer were conducted in an identical manner. After 5 hr, 0.5 ml of incubated solution was removed and diluted with 0.5 ml of Griess reagent (1 % sulphanilamide, 2 % O- phosphoric acid and 0.1 % napthyl ethylene diamine dihydrochloride). The absorbance of the chromophore formed during diazotization of nitrite with sulphanilamide and its subsequent coupling with napthyl ethylene diamine was measured at 546 nm and the experiment was repeated in triplicate 6. Scavenging of superoxide radical The scavenging activity towards the superoxide radical (O 2. - ) was measured in terms of inhibition of generation of O 2.-. The method was performed by using Riboflavin EDTA method. To 1.3 ml of different concentrations of the alcoholic extract added to a mixture containing 0.2 ml EDTA 60 mm (4.47 mg in 10ml water), 0.25 ml riboflavin 53 m (31.92 mg in 100ml distilled water), 0.25 ml Hydroxylamine HCl 10 mm (0.114g in 100ml distilled water) and 2 ml phosphate buffer ph 7.4, riboflavin was added at the end after the tubes had been brought to a standard temperature C. The above solutions were incubated for 30 min at room temperature. Then, 1ml of Griess reagent was added to all test tubes. After 20 minutes, the absorbance was measured at 540 nm 7. RESULTS A concentration range from 2 to 1000 µg/ml of alcoholic extract of P. visida was screened for free radical scavenging potential in various in vitro models. The effect of the extract to scavenge different free radicals was shown in Fig. 1. Results from all models, revealed that free radicals scavenging potential of the extract was found to be as a concentration dependent activity. The maximum % inhibition in all models viz, ABTS, Superoxide, DPPH and Nitric oxide were found to be %, 97.60% and 59.61% respectively. The results revealed that test extract was most active in the ABTS, superoxide and nitric oxide radicals with respective of IC 50 values 3.11 g/ml, g/ml and g/ml when compared to the standard ascorbic acid (IC 50 values of g/ml, g/ml, g/ml). Page 119

4 Figure 1: Effect of alcoholic extract of P. viscida on different antioxidant models DISCUSSION It was evident that an oxidative stress is being associated in pathophysiology of several diseases and disorders such as hypertension, diabetes, cirrhosis, inflammatory conditions, cancer and ageing. Antioxidants are agents that produce resistance against the oxidative stress via scavenging of free radicals, and thus prevent diseases 8. The ABTS assay is performed on the basis of % inhibition on the absorbance value for ABTS.+ radical cation 7. The result as % inhibition on absorbance value by the presence of extract is considered as scavenging potential against the radical cation ABTS.+ radical cation 9. Nitric oxide (NO) is an another free radical predominantly found in mammalian cells, known to be involved in the regulation of several physiological processes. The excess in production of NO is associated with many existing diseases 10. In this attempt, we found that nitrite formed during incubation of sodium nitroprusside in standard phosphate buffer at 25º C was reduced by the alcoholic extract of P. viscida. This is possible only in the presence of antioxidant principles in the extract that could have competed with oxygen to react with nitric oxide and thus inhibit the generation of nitrite 11. DPPH assay depends on the quantification of scavenging ability of an antioxidant against the stable radical DPPH. It may be due to reduction potential of P. viscida roots on radical to the corresponding hydrazine. The reduction in intensity of colour is due to the reaction between DPPH radicals and reducing agents, in which the electrons become paired off. The reduction in intensity is stoichometrically depends on the number of electrons involved 12. Superoxide dismutase is an enzyme that catalyses the dismutation of reactive superoxide anions to oxygen atoms and hydrogen peroxides 13. Superoxide anions are the prime reduction product of oxygen 14 which are measured as of inhibition of generation of O 2. CONCLUSION The results from various in vitro free radicals scavenging methods reveal that alcoholic root extract of P. viscida has significant antioxidant activity. The preliminary phytochemical studies revealed that the root contains phenolic compounds like tannins and flavonoids. Hence, the free radical scavenging potential exerted by the alcoholic root extract from Pseudarthria viscida may be due to the presence of tannins and flavonoids. Page 120

5 REFERENCES 1. Warrier PK, Nambiar VPK and Ramankutty C. Indian Medicinal Plants a compendium of 500 species, Madras: Orient Long Man Ltd. 1995;144(4). 2. Kokate CK. Practical Pharmacognosy, 1 st ed., New Delhi: Vallabh Prakashan, 1994; Harborne JB. Phytochemical Methods. London: Chapman and Hall; 1998: Pellegrini RE, Proteggente N, Pannala A, Yang A and Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation assay. Free Rad Biol Med. 1999; 26: Annie S, Rajendran K and Dinesh KC. In vitro antioxidant studies of Annona squamosa Linn. Leaves. Indian J Exp Biol. 2004;42: Sreejayan N & Rao M N A, Nitric oxide scavenging by curcuminoids, J Pharm Pharmacol.1997;49: Sanchez- Moreno C, Methods used to evaluate the free radical scavenging activity in foods and biological systems. Food Sci Tech Int. 2002;8: Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacognosy Reviews. 2010;4(8): Re R, Pellegrini N, Proteggente A, Pannala A, Yang M and Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 1999; 26: Rice-Evans C and Miller NJ. Factors influencing the antioxidant activity determined by the ABTS.+ radical cation assay. Free Rad Res. 1997;26: Alisi CS and Onyeze GOC. Nitric oxide scavenging ability of ethyl acetate fraction of methanolic leaf extracts of Chromolaena odorata(linn.), Afr J Biochem Res 2008;2 7); Marcocci L, Packer L, Droy- Lefaiz MT, Sekaki A and Gardes- Albert M, Antioxidant action of Ginko biloba extract Egb 761. Meth Enzymol. 1994;234: Perry JJP, Shin DS, Getzoff ED, Tainer JA. The structural biochemistry of the superoxide dismutases. Biochimica et biophysica acta. 2010;1804(2): Kamalakkannan N and Prince PSM, Effect of Aegle marmelos fruit extract on tissue antioxidants in streptozotocin diabetic rats. Indian J Exp Biol. 2003;41: Gibanananda R and Syed AH. Oxidents, antioxidants and carcinogenesis, Indian J Exp Biol. 2002;40; Page 121