Hydrogen peroxide and superoxide radical

The hydrogen peroxide (H₂O₂) content was measured using Wu et al.’s method [33], albeit with some modifications. Peel fruit tissue (0.5 g) was mixed with 8 mL of 5% cold trichloroacetic acid (w/v) and was subsequently homogenized and centrifuged at 10,000×g at 4 °C for 10 min. The reaction mixture was prepared using 0.5 mL of the supernatant, 4 mL of 5% trichloroacetic, and 0.5 mL of assay reagent containing 500 µM ferrous ammonium sulfate, 50 mM sulfuric acid (H₂SO₄), 200 µM xylenol orange, and 200 mM sorbitol. The reaction mixture was incubated at 25 ± 2 °C for 45 min. During the incubation period, the hydrogen peroxide molecule oxidized Fe²⁺ to Fe³⁺ ion, which was determined by measuring the absorbance of the ferric-xylenol orange complex at 560 nm. The absorbance values were calibrated to a standard curve, generated using the known concentrations of H₂O₂.

The production rate of superoxide radical (O^−.₂) was analyzed using the method given by Elstner and Heupel [34]. Peel tissue samples (1 g) were homogenized with 8 mL of 65 mM phosphate buffer (pH 7.8) containing 1 mM ethylenediaminetetraacetic acid (EDTA), 1% polyvinylpolypyrrolidone (PVPP w/v), and 0.3% Triton X-100. The extraction mixture was centrifuged at 5000×g for 15 min at 4 °C. After centrifugation, 0.5 mL of the supernatant was mixed with 1 mL of 50 mM phosphate buffer (pH 7.8) and 0.5 mL of 10 mM hydroxylamine hydrochloride and subsequently incubated at 25 ± 2 °C for 20 min in dark conditions. A 1 mL sample of the above reaction mixture was added to 1 mL of 19 mM p-aminobenzene sulfonic acid and 1 mL of 7 mM α-naphthylamine to formulate a new mixture, which was then incubated at 25 ± 2 °C for 20 min in dark conditions. The O^−.₂ content was calculated based on a comparison of absorbance with a standard curve (using sodium nitrite as the standard) at 530 nm. The O^−.₂ production rate was expressed as µmol NO₂ kg⁻¹ s⁻¹ of fresh weight.