*2.6. Hydrogen Peroxide Content*

The content of hydrogen peroxide (H2O2) was determined by the chromogenic peroxidase-coupling method following the procedures of Veljovic-Jovanovic et al. [27]. Three grams of frozen sample were extracted in 12 mL of cold 1 M HClO4, then passed through filter paper. After centrifugation at 12,000× *g* for 10 min at 4 ◦C, the supernatant of extract was neutralized to pH 5.6 by 5 M K2CO3 and then centrifuged at 12,000× *g* for 10 min at 4 ◦C to eliminate insoluble KClO4. To oxidize ascorbate before incubation, the supernatant was incubated with 1-unit ascorbate oxidase for 10 min. Then, 1 mL of neutralized supernatant was reacted with the reaction mixture solution of 0.1 M phosphate buffer (pH 6.5) containing 16.5 mM 3-(dimethylamino) benzoic acid, 0.35 mM 3-methyl-2-benzothiazoline hydrazine, and 250 ng horseradish peroxidase. The absorbance was measured spectrophotometrically at 590 nm (Jasco V-530, Jasco, Tokyo, Japan) and monitored at 25 ◦C. The content of hydrogen peroxide was determined from a 25–100 μM H2O2 standard curve.
