Twelve hours after evaluation of learning and memory, the rats were sacrificed through chloroform anesthesia and transcardial perfusion with ice-cold normal saline (10 mL/10 g BW). The entire intact brain was carefully removed and placed on an ice-chilled dish for cleaning. The hippocampus was rapidly removed and weighed. Then the hippocampus were homogenized in 4 volumes of 0.1 mol/L ice phosphate buffer (pH 7.4) and centrifuged at 20,000 × g for 30 min at 4 °C. SOD activity in the hippocampus was determined by the method of Misra and Fridovich [
29] based on auto oxidation of epinephrine. Briefly, an aliquot of 0.25 ml ice cold chloroform was added to 0.1 ml of supernatant followed by adding 0.15 ml ice cold ethanol. The mixture was centrifuged at 3000 rpm for 10 min at 4 °C. Then 0.2 ml of supernatant was taken and 1.5 ml carbonate buffer, 0.5CC EDTA, and 0.8 ml distilled water was added. Reaction was started by adding 0.4 ml epinephrine. Change in absorbance ΔOD/min at 480 nm was read for 3 min. The results were expressed in terms of nmol/min/mg protein. CAT activity in the hippocampus was measured at 37 °C by following the rate of disappearance of H
2O
2 at 240 nm [
30]. To initiate the CAT activity, 0.2 ml of tissue homogenate/0.2 ml of serum was added to the reaction mixture containing 1.9 mL of 100 mM Phosphate buffer (pH 7.0), 0.1 mL of enzyme extract, and 1.0 mL of 0.075 % H
2O
2 solution. CAT activity was assayed in 1.0 min at 240 nm. GSH-Px activity in the brain was estimated according to the method described by Ellman [
31]. The reaction mixture contained 0.1 M phosphate buffer (pH 7.0), 1 mM EDTA, 10 mM glutathione (GSH), 1 mM NaN3, 1 unit of glutathione reductase, 1.5 mM NADPH and 0.1 ml of homogenate/serum. After incubation for 10 min at 37o C, H2O2 was added to each sample at a final concentration of 1 mM. GPX activity was measured as the rate of NADPH oxidation at 340 nm. The MDA content, a quantitative measurement of lipid peroxidation, was assayed in the form of thiobarbituric acid reactive substances by the method of Wills [
32]. Collected supernatant was incubated in a solution containing 8.1 % SDS, 20 % acetic acid (pH 3.5), 0.8 % TBA (Thiobarbituric acid) for 1 h at 95 °C. After cooling at RT, the samples were well mixed with butanol/pyridine mixture (15:1, v/v), and the aqueous phase was carefully collected by aspiration. Absorbance of the aqueous phase was found out at 532 nm. MDA content was expressed as nmol/mg protein. Advanced protein oxidation product (APOP) was estimated by the method followed by Salde et al. [
33]. Briefly, two mL of plasma was diluted 1 : 5 in PBS: 0.1 mL of 1.16 M potassium iodide was then added to each tube, followed by 0.2 mL acetic acid after 2 min. The absorbance of the reaction mixture was immediately read at 340 nm against a blank containing 2 mL of PBS, 0.1 mL of KI, and 0.2 mL of acetic acid. The chloramine-T absorbance at 340 nm being linear within the range of 0–100 mmol/L, AOPP concentrations were expressed as
μmol·L
−1 chloramine-T equivalents The accumulation of nitrite, an indicator of nitric oxide (NO) production was determined by a colorimetric assay as described by Green et al. [
34]. Aliquots of 50 μL of supernatants were incubated with 50 μL of Griess reagent, consisting of 1 % sulfanilamide, 0.1 % N-(1-naphthylethylenediamine) dihydrochloride and 2.5 % ortho-phosphoric acid. After 10 min at room temperature, the optical density was measured at 540 nm. Nitrite concentrations were calculated by comparison to a calibration curve obtained with sodium nitrite standard solutions.