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Current Trends in Biotechnology and PharmacyVol. 5 (1) 1073-1082 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)hydroperoxide(t-BHP), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), GSH, SOD, xanthine,xanthine oxidase, nitroblue tetrazolium, GSH-Px,reduced nicotinamide adenin dinucleotidephosphate (NADPH), bovine serum albumine andglutathione reductase (GR) were purchased fromSigma Aldrich Corp. (St. Louis, MO, USA. )Extraction : Fruit hexane (FHE), fruit methanolic(FME) and fruit aqueous (FAE) extractionprocedure was performed as described by Reddyet al. (19). Briefly around 300g of fresh plantmaterial (fruit) was washed with tap water, airdried and then chopped into small fragments whichwere shade dried and reduced to coarse powderwith mortar and pestle. The powdered materialswere extracted thrice times with hexane (2.5 l),then extracted three times with methanol (2.5 l)and followed by distilled and deionised water (1l) at room temperature in cycle of 48 h each onorbital shaker. The combined methanolic extractswere then concentrated in a rotavapour atreduced pressure, below 40 o C and pooled waterextracts were concentrated by lyophilization.Partial purification : Since methanolic extracthad shown significant antioxidant activity, thisextract was further fractionatedchromatographically as described by Reddy etal., 2009(20). Initially, 15 g of FME was loadedon a silica column (column height and diameterwere 24 and 2 inches, respectively) and was elutedwith stepwise gradient elution of ethyl acetatemethanol(4:1→0:1, v/v). Four fractions werecollected at a regular interval and named as F1,F2, F3 and F4. Antioxidant activity of thesefractions was evaluated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay and found that onlyfraction F2 was showing significant activity.Hence, this fraction was further fractionated withthe solvent system methanol-water (4:1→0:1, v/v). Four fractions collected at a regular intervalwere named as F2-1, F2-2, F2-3 and F2-4.Role of Luffa acutangula in Oxidative damage1075HPTLC (High Performance Thin LayerChromatography) fingerprinting :Fingerprinting of Luffa acutangula wasperformed on 5x10 cm HPTLC plates coated with0.25 mm layer of silica gel 60F254. Before using,the plates were washed with methanol andactivated at 110 0 C for 5min. Samples wereapplied as 4mm wide bands and 6mm apart byusing a Camag (Muttenz, Switzerland) LinomatIV sample applicator equipped with a 100 µlsyringe. A constant application rate of 6 µl/secwas used. The chromatograms were monitoredat 600nm using benzene : acetone (60:40) asmobile phase and vanillin H 2SO 4as reducingagent.DPPH free radical scavenging activity : TheDPPH free radical scavenging activity wasmeasured using a method described previouslyby Reddy et al. (19). Briefly, the stock testextracts of hexane, methanolic and aqueous fruitwere dissolved in Dimethyl sulfoxide (DMSO) ata concentration of 1 mg/ml, the methanolicfractions, (F2-1, F2-2, F2-3 & F2-4) weredissolved in DMSO at concentration of 25 µg/mland ascorbic acid was dissolved in DMSO at aconcentration of 100 µg/ml. DPPH was preparedfreshly in absolute alcohol at a concentration of4.9 mg/25ml. The reaction mixture consisting of125 µl of DPPH, 100µl of freshly prepared 0.5mMtris buffer (pH 7.2) and 25 µl test extracts orstandard was added to 96 well plates. The plateswere incubated at room temperature for 10 minand then absorbance was measured at 517 nmby a UV–visible spectrophotometer (SPECTRAmax PLUS ® , Molecular Devices, USA). Thepercentage of free radical scavenging activity wasdetermined from the following formula:(CONTROL - SAMPLE)Radical scavenging (%) = × 100.CONTROLErythrocytes preparation : Erythrocytes wereprepared as described in Betul et al. (21) Human
Current Trends in Biotechnology and PharmacyVol. 5 (1) 1073-1082 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1076blood was collected from healthy donors (we don’thave any institutional review board protocol todraw the blood, it is done by our technician) andcentrifuged at 600 g for 5 min. The clear plasmaand buffy coat were discarded. Erythrocyteswere washed thrice with phosphate- bufferedsaline (PBS; pH 7.4) by 600 g for 30 sec. Thewashed erythrocytes were used for subsequentantioxidant activity assay of Luffa acutangula.Erythrocyte treatment : Stock solutions of fruitmethanolic fraction (F2-3) were prepared inDMSO then diluted with distilled water to obtain12.5, 25 and 50 µg/ml (DMSO concentrationlower than 1%). Erythrocytes at 5% hematocritin PBS which are treated by 1.5 mM t-BHP for 1h were incubated for different intervals (30, 60and 90 min) at 37 0 C (with continuous mixing)with fruit sub fraction (F2-3) at different dosesfrom 12.5 –50 µg/ml. Samples of erythrocytes awithout fruit sub fraction (F2-3) is used ascontrols. After incubation, the mixtures werehaemolysed in -20 0 C. The mixtures were thawedthe following day and then centrifuged at 3600rpm for 15 min. All assays were performed inthese supernatants. The concentration ofhaemoglobin (Hb) was determined using themethod described by (22). All experimentsdescribed in these studies were reproduced withat least three separate isolates.Lipid peroxidation : The concentration of themalondialdehyde (MDA) in blood was estimatedas described by Stocks and Dormandy (23). Theprinciple of this method is spectrophotometricmeasurement of pink color produced by thereaction of thiobarbituric acid with MDA. Theresults were expressed as nmol/ml. Briefly 1 mlof 28% trichloroacetic acid–sodium meta arsenitesolution was added to 2 ml supernatan andcentrifuged at 2000 rpm for 15 min. Then 0.5 mlof 1% thiobarbituric acid (TBA) was added tosupernatants and placed in a boiling water bathfor 15 min. After reaching room temperature,absorbance was measured using aspectrophotometer at 532 nm. MDA values inerythrocytes were determined from the standardcurve using 1,1,3,3,tetraethoxypropane (TEP) asstandard.Total glutathione (GSH) : Determination of totalgluathione content in incubation solutions wasdone according to the method of Betul (21). Theprinciple of this method is that the oxidizedglutathione (GSSG) is converted into GSH in thepresence of NADPH and glutathione reductase,The chromophoric product 2-nitro-5-thiobenzoicacid, resulting from reaction of the 5, 5 ’ dithiobis-(2-nitrobenzoic acid) (Ellman reagent) with GSHpossesses a molar absorption at 412 nm. 25 µl ofthe supernatant was added to standard assaymixture containing 0.6 µmol DTNB, 10 µgglutathione reductase and 0.2 µmol NADPH. Thereaction was initiated by the addition of NADPHand the color development at 412 nm was followedfor 6 min. The concentrations of total glutathionewere calculated from a standard curve preparedwith GSSG and were expressed as µmolglutathione/g Hb.Superoxide Dismutase (SOD) : Activity of theSOD enzyme in incubation solution wasdetermined according to the method describedby Sun et al. (24). Xanthine reacts with xanthineoxidase and generates superoxide radicals thatreact with nitrobluetetrazolium to form formazandye. To analyze this, 400 µl of incubation solutionwas haemolysed by four times cold water andHb was removed by adding mixture containing0.6 ml chloroform (CHCl 3) and 1 ml ethanol(EtOH) to haemolysates, mixed vigorously andcentrifuged. A 600 µl of reaction mixturecontaining 0.1 mmol of xanthine, 0.1 mmol ofEDTA, 50 mg of bovine serum albumin, 25 µmolof nitrobluetetrazolium per liter was added to 125µl supernatant or 125 µl SOD standard solutions,Purushotham Reddy et al.
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