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)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.