## 3 Materials and Methods
### Antioxidative Enzymes  
The enzymatic extract was homogenized in the fresh sample in 100 mM sodium phosphate buffer, pH 7.5, and 1 mM EDTA. Superoxide dismutase was assayed by measuring its ability to inhibit the photochemical reduction of nitro-blue tetrazolium (NBT) [95]. The 3.0 mL reaction mixture contained 2.5 mL of 60 mM Tris-HCl (pH 7.8) and 0.1 mL each of 420 mM L-methionine, 1.80 mM NBT, 90 \(\mu\)M riboflavin, 3.0 mM EDTA, and enzyme extract. The tubes were thoroughly shaken and placed 30 cm below a light source of three 20 W fluorescent lamps (Philips Global Business Services LLP, Calcutta, India). The absorbance was recorded at 560 nm. One enzyme unit was defined as the amount of enzyme required to inhibit the photoreduction of 1 \(\mu\)mol of NBT, which was expressed as U g\({}^{-1}\) fresh weight and converted to U mg protein\({}^{-1}\) by estimating the sample total soluble proteins. The percentage inhibition was calculated following the formula of [PERSON] et al. [96]. Percentage inhibition = V - v/v \(\times\) 100, where V = rate of assay reaction in the absence of SOD and v = rate of assay reaction in the presence of SOD. Catalase activity was measured according to the method of [PERSON] [97]. The reaction mixture (1.0 mL) consisted of 0.5 mL of 0.2 M phosphate buffer (pH 7.0), 0.4 mL of 0.2 M hydrogen peroxide, and 0.1 mL of enzyme extract. The reaction mixture was incubated at 37 \({}^{\circ}\)C. The reaction was terminated by adding a 3 mL mixture of 5% (\(w/v\)) potassium dichromate and glacial acetic acid (1:3 \(v/v\)) and heating for 10 min. The absorbance of the test sample and control was measured at 570 nm. The catalase activity (CAT; EC 1.11.6) was measured by observing the dissociation of H\({}_{2}\)O\({}_{2}\) at a wavelength of 240 nm. Peroxidase was assayed according to the method of [PERSON] et al. [98]. The reaction mixture, with a final volume of 2.9 mL, contained 2.5 mL of 50 mM phosphate buffer (pH 6.5), 0.1 mL of 0.5% hydrogen peroxide, 0.1 mL of 0.2% \(o\)-dianisidine, and 50 \(\mu\)L of enzyme extract. The change in absorbance was recorded at 430 nm for 3 min at 15 s intervals. One unit ofperoxidase represents a change of 1.0 O.D. in 1 min. The enzyme activity was measured using the method of [PERSON] [96], which is based on the spectrophotometric monitoring of ascorbic acid oxidation. The reaction mixture for the APX activity assay consisted of 2.5 mL of 0.1 M phosphate buffer (pH 7.0), 0.25 mL of 1.2 mM EDTA, 0.1 mL of 15 mM ascorbic acid, and 0.1 mL of enzyme extract. The reaction was initiated by adding 0.05 mL of 35 mM H\({}_{2}\)O\({}_{2}\). One enzyme unit is defined as the enzyme required to oxidize 1 nmol of ascorbic acid per min at 290 nm. The glutathione peroxidase activity was determined using a method based on the continuous regeneration of oxidized glutathione produced by the action of glutathione peroxidase [99]. The reaction mixture consisted of 2.0 mL of 0.1 M phosphate buffer (pH 8.0), 0.1 mL of 1 M NaCl, 75 \(\upmu\)L of 10 mM EDTA, 0.3 mL of 10 mM GSH, 0.15 mL of 8 mM NADPH, 25 \(\upmu\)L of GR (100 U mL\({}^{-1}\)), 0.25 mL of enzyme extract, and 0.1 mL of 25 mM H\({}_{2}\)O\({}_{2}\). The kinetic changes in the absorbance were measured by the decrease in the absorbance at 340 nm for 15 min at 30 \({}^{\circ}\)C. The GPX activity was measured by evaluating the non-enzymatic oxidation of NADPH with an extinction coefficient [\(\varepsilon\) = 6.22 mM\({}^{-1}\) cm\({}^{-1}\)]. The reaction mixture consisted of 2 mL of 0.1 M phosphate buffer (pH 7.5), 0.1 mL of 5 mM oxidized glutathione (GSSG), 0.1 mL of 3.5 mM NADPH, and 0.1 mL of enzyme extract for a final volume of 2.3 mL. The decrease in absorbance at 340 nm due to the oxidation of NADPH was monitored. Non-enzymatic oxidation of NADPH was recorded and subtracted. One enzyme unit was defined as the amount of enzyme required to oxidize 0.1 \(\upmu\)mol of NADPH oxidized per min. According to [PERSON] et al.