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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
Current Trends in Biotechnology and PharmacyVol. 5 (1) 1073-1082 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1078in the human erythrocyte model by measuringdifferent parameters such as lipid peroxidation,SOD, catalase and GSH levels.Lipid peroxidation is an autocatalytic process,which may lead to an oxidative stress,inflammation, cancer, tissue damage, DNAdamage and aging (26). Since, MDA is an endproduct of the lipid peroxidation, we evaluatedthe effect of F2-3 on MDA as an index for thelipid peroxidation. When erythrocytes werechallenged with t-BHP, all the oxidative stressparameters, GSH, SOD, and CAT weresignificantly reduced but MDA levels wereincreased drastically (Fig.4, Fig5, Fig6 and Fig.3,respectively) indicating t-BHP induces oxidativestress in the erythrocyte. In experiments, whereerythrocytes were pre-incubated with fraction F2-3 for one hour and following exposure to t-BHP,MDA levels were significantly reduced in a doseand time dependant manner. MDA levels in 12.5µg/ml treated group at the time intervals of 0, 30,60, 90 min and t-BHP-control and control were68.1, 51.8, 46.5, 40.4, 70.34 and 21.2 nmol/gHb,at the concentration of 25 µg/ml MDA levels were70.6, 46.7, 42.6 and 30.8 and at the concentrationof 50 µg/ml MDA levels were 75.5, 40.6, 29.9and 27.9 nmol/g Hb, respectively (Fig.3). Thesedata suggested that the fraction F2-3 of L.acutangula fruit possesses lipid peroxidationinhibition properties.GSH, one of the most potent biologicalmolecules, it ’ s can prevent occupation includingfree radicals reactions in the erythrocytes. Themain defensive roles of glutathione againstoxidative stress are: glutathione is a cofactor ofseveral detoxifying enzymes against oxidativestress, e.g. glutathione peroxidase (GPx),glutathionetransferase etc. (27). GSH scavengeshydroxyl radical and singlet oxygen directly,detoxifying hydrogen peroxide and lipid peroxidesby the catalytic action of glutathionperoxidase.In this study effects of F2-3 fraction on cellularGSH levels in t-BHP-treated cells wererepresented in Fig.4. GSH levels in 12.5 µg/mltreated group at the time intervals of 0, 30, 60, 90min, t-BHP-control and control were 6.7, 9.0,13.2, 13.6, 7.8 and 28.77 µmol/g Hb, at theconcentration of 25 µg/ml GSH levels were 7.2,12.3, 19.4 and 19.5 and at the concentration of50 µg/ml GSH levels were 7.1, 13.2, 20.6 and21.3 µmol/g Hb, respectively (Fig.4). The obtainedresults clearly indicating that fraction F2-3 hasability to replenish cellular redox buffer with GSH.Since GSH levels are increased, the co-factorfor glutathione related enzymes, glutathioneFig. 3. Effect of methanolic fraction of L.acutangulafruit (F2-3) on t-BHP induced lipid peroxidation inerythrocyte was monitored by measuring themalonaldehyde (MDA). Values are mean ± SD.Fig. 4. Effect of methanloic fraction F2-3 on cellar redoxbuffer (GSH) was monitored in t-BHP treatederythrocytes. Values are mean ± SD.Purushotham Reddy et al.
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