2.4.2. Ascorbate Peroxidase (APX) Activity

The APX activity measurements were performed as described by Chen and Liu (2012) [26], with slight modifications. A total of 10 mL extraction solvent (containing 100 mM KH₂PO₄ (J.T. Baker Chemical Inc., Oklahoma, PA, USA), pH 7.8; 1% Triton X-100 (Sigma Chemical Inc., St. Louis, MO, USA); 1 mM EDTA-Na₂ (Sigma Chemical Inc., St. Louis, MO, USA) was added to 0.5 g broccoli powder and the mixture was centrifuged at 10,000× g and 4 °C for 20 min. Then, the supernatant was collected, with the enzyme extract at 0.047–0.077 mg/g protein, and the protein content was determined by the Bradford method, with the standard curves prepared using BSA. A 0.5 mL KH₂PO₄ (250 mM, pH 7), 0.05 mL EDTA-Na₂ (0.5 mM), 0.2 mL H₂O₂ (10 mM), 0.2 mL ascorbic acid (Honeywell Riedel-de Haen, Seelze, Germany), and 0.05 mL enzyme extract was sequentially added to the quartz tube, and the changes in absorbance were measured immediately after mixing at a wavelength of 290 nm within 5 min. The APX activity was calculated using the extinction coefficient of H₂O₂ (2.8 mM⁻¹ cm⁻¹) and the enzyme activity was expressed in units of mmol ascorbate min⁻¹ mg⁻¹ protein.