2.7.1. Lipid peroxidation assay
LPO was measured according to CC0651 method described by Ohkawa et al. (1978). The reaction mixture consisted of 5 μl of 10 mM butyl-hydroxytoluene (BHT), 200 μl of 0.67% thiobarbituric acid, 600 μl of 1% O-phosphoric acid, 105 μl of distilled water and 90 μl of supernatant. The resultant mixture was incubated at 90 °C for 45 min and the OD was measured at 535 nm. The results were expressed as μmol of TBARS formed/h/g tissue.
2.7.2. Protein estimation
The protein content in all the treated as well as untreated groups were estimated according to the method of Bradford (1976) using bovine serum albumin (BSA) as a standard.
2.7.3. Estimation of glutathione (GSH) content
The glutathione (GSH) content was estimated colorimetrically using Ellman’s reagent (DTNB) according to the procedure described by Jollow et al. (1974). The supernatant was precipitated with 4% sulphosalicyclic acid (4%) in the ratio of 1:1. The samples were kept at 4 °C for 1 h and then subjected to centrifugation at 5000 rpm for 10 min at 4 °C. The assay mixture consisted of 550 μl of 0.1 M phosphate buffer, 100 μl of supernatant and 100 μl of DTNB. The OD was read at 412 nm and the results were expressed as μ moles of GSH/gram tissue.