2.7. Lipid Peroxidation (MDA) and Hydrogen Peroxide (H2O2) Determination

The method followed by Faizan et al. [37] was followed to determine the lipid peroxidation as expressed by MDA content. Fresh leaves were homogenized in 0.1% trichloroacetic acid (TCA) and centrifuged at 10,000× g for 15 min. A 20% TCA solution of 0.5% thiobarbituric acid was mixed in with the supernatant. The final mixture was warmed at 95 °C for 30 min. After cooling, the supernatant was centrifuged at 1000× g for 15 min at 4 °C. The absorbance of the mixture was noted at 532 nm.

The amount of H₂O₂ in leaves was determined by the method adopted by Faizan et al. [37]. Leaf tissues were homogenized in 10 mL cold acetone with a mortar and pestle. The homogenate was centrifuged at 5000× g for 15 min and the supernatant was kept. Residue was again extracted with acetone. About 1 mL of mixture was taken in a test tube and 2 mL of 17 M ammonia and 2 mL of 20% titanium chloride were mixed. The supernatant was again extracted with acetone, accompanied by an infusion of 10 mL 2 N H₂SO₄ to absorb it properly. The optical density was measured at 410 nm on a spectrophotometer. The amount of H₂O₂ in the samples was measured in relation to the standard curve adopted from the known concentration of H₂O₂.