2.13. Analysis of antioxidant enzymes (CAT, SOD and APX) from leaf and root of the plants

CAT, SOD and APX enzymes in the leaves and roots were investigated as earlier described [25]. Briefly, the leaves and roots sample were ground added phosphate buffer (100 mM, pH 7.0) using a mortar and pestle. Then centrifuged at 12000 rpm for 10 min. The CAT was investigated in a reaction mixture contained 100 μl of leaves and roots extract, 100mM potassium phosphate buffer (pH 7.0) and 6% H₂O₂. The absorbance was taken at 240nm in a spectrophotometer (Genesys 10S, Thermo Scientific, MA, USA) at 30 s intervals up to 1 min where the standard coefficient of the extinction was maintained 0.036 mM⁻¹ cm⁻¹. For the estimation of SOD, 100μl leaves and roots extract mixed with 0.1 mM EDTA, 50mM sodium carbonate buffer (pH 9.8), and 0.6 mM epinephrine [26]. The absorbance was taken at 475nm after 4 min intervals using a spectrophotometer (Genesys 10S, Thermo Scientific, MA, USA). The APX was estimatesd as earlier described [27]. The mixture having 0.1 ml of leaves and roots extract, 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM ascorbic acid, 0.1 mM H₂O₂. The absorbance was taken at 290nm in UV-VIS spectrophotometer (Genesys 10S, Thermo Scientific, MA, USA) at 30 s intervals. Where, the coefficient of the extinction was maintained 2.8 mM⁻¹ cm⁻¹. The GR was estimatesd as earlier described [27]. The mixture having 100 μl of leaves and roots extract, 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.75 ml distilled water, 0.1 ml of 20 mM oxidized glutathione (NADPH) and 0.1 ml of 2mM NADPH. The absorbance was taken at 340nm in UV-VIS spectrophotometer (Genesys 10S, Thermo Scientific, MA, USA) at 30 s intervals.