Biochemical assay

The level of malondialdehyde (MDA) was estimated as a lipid peroxidation (LPO) marker in the brain tissue using the method described by Ohkawa et al. [26]. Briefly, a mixture consisting of distilled water, 0.67% thiobarbituric acid, and 0.22% sulphuric acid was added to 500 µl of brain supernatant. The formed mixture was boiled at 95°C for 30 min and then cooled at room temperature and centrifuged for 15 min at 1000 g. Finally, the supernatant was estimated spectrophotometrically 540 nm. The obtained data are expressed as nanomoles MDA per milligram of protein. Nitric oxide (NO) level was measured using the Griess reagent according to Green et al. [27]. Briefly, 100 μl of brain supernatant was mixed for 10 min with Griess reagent at room temperature. The formed reddish purple azo dye was measured spectrophotometrically at 540 nm.

Glutathione (GSH) was estimated by the reduction of Elman’s reagent (5,5′-dithiobis (2-nitrobenzoic acid) (DTNB)) with GSH to produce a yellow compound. The reduced chromogen is directly proportional to the GSH concentration, and its absorbance can be measured at 405 nm [28]. Catalase (CAT) activity was determined as described by Aebi [29]. Briefly, 0.1 ml of the brain homogenate was mixed with mixture containing phosphate buffer (50 mM, pH 7.0) and H₂O₂ (30 mM). The decrease in absorbance was observed for 3 min and the enzyme activity was determined as μM H₂O₂ decomposed/s/mg protein. The activity of superoxide dismutase (SOD) was determined using the method of Nishikimi et al. [30]. Briefly, 0.05 ml of brain homogenate was added to mixture containing phenazine methosulphate (93 µM), reduced NAD (0.47 mM), nitroblue tetrazolium (0.3 mM), and phosphate bufer (0.1 M; pH 8.5). The increase in absorbance was estimated at 560 nm for 5 min. The obtained results were expressed as units per milligram of protein. Meanwhile, glutathione peroxidase activity was measured using the method of Paglia and Valentine [31]. GPx activity was estimated in terms of the decrease in NADH per min using a reaction coupled with glutathione reductase (GR). The decrease in absorbance at 340 nm was recorded, and GPx activity was expressed as U/mg protein. In addition, GR activity was measured by quantitating GSH-dependent oxidation of NADPH at 340 nm and expressed as U/mg protein.