Free thiols content was determined by suspending 30 mg of sample in 1.0 ml of reaction buffer (RB1) consisting of 8 mol/L urea, 10 mmol/L 5,50 -dithio-bis(2-nitrobenzoic acid) (DTNB), 3 mmol/L
ethylene-diaminetetraacetic acid (EDTA), 0.2 mol/L Tris-HCl, pH 8.0 and 10 g/L sodium dodecyl sulphate (SDS) for 20 min under N2 in the dark. The suspension was centrifuged at 13,600 g for 10 min at room temperature and 0.1 ml of the supernatant was diluted with 0.9 ml of RB1 without DTNB. The solution was centrifuged at 13,600 g for 10 min at room temperature and absorbance was read at 412 nm against a reagent blank.