full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (1) 1073-1082 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1077then 25 µl of 9.9 nmol xanthine oxidase solutionswas added to each tube at 30 s intervals. Eachtube was incubated for 20 min at roomtemperature and the reaction was terminated byadding 0.5 ml of 0.8 mM CuCl 2solution per tubeevery 30 s. The production of formazan wasdetermined at 560 nm. Under these conditions,the absorbance at 560 nm of the blank tube wasabout 0.25. SOD enzyme concentrations weredetermined from the standard inhibition curve withthe x-axis being the logarithmic SODconcentrations and the Y-axis represent percentinhibition and expressed as IU/g Hb.Catalase (CAT) : CAT activity was measuredaccording to the method described by Aebi (25).The principle of the assay is based on thedetermination of the rate constant of hydrogenperoxide decomposition by CAT enzyme. Amixture containing 50 mM phosphate buffer (pH7.0), 20 mM H 2O 2and 500 ìl of supernatant wasincubated at room temperature for 2 min. Thechange in absorbance at 240 nm in 2 min wascalculated and the decrease in H 2O 2wasmeasured spectrophotometrically at 240 nm, for3 min at 25 0 C. The catalase activity wasexpressed as mmole H 2O 2consumed /min/g Hb.Statistical Methods : All the data were expressedas mean ± S.D. using Microsoft XL 2007software.Results and DiscussionThe percentage extraction yield of hexane,methanol and aqueous extract of L. acutangulafruit was 3.2 %, 15.02 % and 9.98 %, respectively.HPTLC fingerprinting of L. acutangula fruitmethanolic extract is presented in Figure 1 (20)In this study, all the three crude extracts, FruitHexane Extract (FHE), Fruit methanolic Extract(FME) and Fruit Aqueous Extract (FAE), wereinitially tested for their antioxidant activity, FMEwas showed higher antioxidant activity comparedto FHE and FAE (Fig. 2). Hence this extract wasFig. 1. HPTLC chromatogram of fruit methanolic extract(mobile phase of benzene : acetone at (60:40);chromatogram was monitored at 600 nm; Y-axis legend,Rf; X-axis legend, AU) (20).Fig. 2. Antioxidant activity of the extracts (hexane,methanolic, aqueous and methanloic fractions (F1, F2,F3, F4 and F2-1, F2-2, F2-3, F2-4) was estimated usingthe DPPH method. Results were shown as mean ± SD.selected for further purification using columnchromatography. Fractions were collected atregular intervals and named as F1, F2, F3, and F4and these fractions were also subjected toantioxidant activity. F2 demonstrated higherantioxidant activity (Fig. 2) than the other fractions(F1, F3 and F4). Fraction F2 was furtherfractionated chromatographically and named asF2-1, F2-2, F2-3 and F2-4. Out of these fourfractions, F2-3 was found to possess antioxidantactivity (Fig. 2). Hence, fraction F2-3 was selectedto evaluate the oxidative stress inhibitory activityRole of Luffa acutangula in Oxidative damage