The absorbance was measured at 532 nm and results were expressed as MDA equivalents formed by Fe2+ and
H2O2. Nitric oxide was generated from spontaneous decomposition of sodium nitroprusside in 20 mM phosphate buffer (pH 7.4). Once generated NO interacts with oxygen to produce nitrite ions, which were measured by the Griess reaction (Basu and Hazra, 2006). The reaction mixture (1 ml) containing 10 mM sodium nitroprusside (SNP) in phosphate buffer and ATR at different concentrations were incubated at 37 °C for 1 h. A 0.5 ml aliquot was taken and homogenized with 0.5 ml Griess reagent. The absorbance of chromophore was measured at 540 nm. Percent inhibition of nitric oxide generated was measured by comparing the absorbance values of negative controls (only 10 mM sodium nitroprusside and vehicle) learn more and assay preparations. Results were expressed as percentage SB203580 mouse of nitrite formed by ATR alone. The ability
of ATR to scavenge H2O2 (“catalase-like activity” or “CAT-like activity”) was measured as described previously (Aebi, 1984). Briefly, H2O2 diluted in 0.02 M phosphate buffer (pH 7.0) to obtain a 5 mM final concentration was added to microplate wells in which different concentrations was placed. The microplate was immediately placed to monitor the rate of H2O2 decomposition in the microplate reader set at 240 nm. The ability of ATR to scavenge superoxide anion (“superoxide dismutase-like activity” or “SOD-like activity”) was measured as previously described. ATR was mixed to native purified catalase (100 U/ml stock solution) in glycine buffer (50 mM, pH 10.2). Superoxide generation was initiated by addition of adrenaline 2 mM and adrenochrome formation was monitored at 480 nm for 5 min at 32 °C. Superoxide production was determined by monitoring the reaction curves of samples and measured as percentage of the rate of adrenaline auto-oxidation into adrenochrome (Bannister and Calabrese, 1987). SH-SY5Y cells were cultured in 10% FBS DMEM/F12 medium. Cells were used for cytotoxicity measurements when reached 70–90% confluence. Cells were treated with different concentrations
of ATR alone or in the presence of H2O2 400 μM for 3 h, and cell viability mafosfamide was assessed by the MTT assay. This method is based on the ability of viable cells to reduce MTT (3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide) and form a blue formazan product. MTT solution (sterile stock solution of 5 mg/ml) was added to the incubation medium in the wells at a final concentration of 0.2 mg/ml. The cells were left for 45 min at 37° C in a humidified 5% CO2 atmosphere. The medium was then removed and plates were shaken with DMSO for 30 min. The optical density of each well was measured at 550 nm (test) and 690 nm (reference). Data are expressed as mean ± SEM. The obtained data was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s test. All tests were performed in triplicate. Data analyses were performed using the GraphPad Prism 5.