*2.8. Antioxidant Enzyme Extraction and Assay*

Enzyme extraction was performed following the method of Ishikawa et al. [30], with certain modifications. Frozen fruit tissue (5 g) was homogenized in a cooled mortar and pestle with 20 mL of cold 50 mM potassium phosphate buffer (pH 7.0) containing 20% sorbitol, 1 mM ascorbate, 1 mM EDTA, and 4% polyvinylpyrrolidone. The homogenate was passed through two layers of Miracloth (Calbiochem, San Diego, CA, USA) and centrifuged at 15,000× *g* for 15 min at 4 ◦C.

The activity of CAT was determined according to the procedure established by Aebi [31]. H2O2 consumption was recorded as the decrease in absorbance at 240 nm (Shimadzu UV-1800, Shimadzu, Japan) at 25 ◦C for 120 s. The calculation of CAT activity used an extinction coefficient of 39.4 mM−<sup>1</sup> cm<sup>−</sup>1.

The activity of APX was estimated according to the procedure of Nakano and Asada [32]. The ascorbate oxidation was recorded as the absorbance decrease at 290 nm (Shimadzu UV-1800, Shimadzu, Japan) at 25 ◦C for 120 s. The calculation of APX activity used an extinction coefficient of 2.8 mM−<sup>1</sup> cm<sup>−</sup>1.

The activity of MDHAR was estimated according to the procedure of Hossain and Asada [33]. The NADH oxidation was recorded as the absorbance decrease at 340 nm (Shimadzu UV-1800, Shimadzu, Japan) at 25 ◦C for 120 s. The calculation of MDHAR activity used an extinction coefficient of 6.2 mM−<sup>1</sup> cm<sup>−</sup>1.

The activity of DHAR was estimated according to the procedure of Hossain and Asada [33]. The generation of ascorbate was recorded as the increase in absorbance at 265 nm (Shimadzu UV-1800, Shimadzu, Japan) at 25 ◦C for 120 s. The calculation of DHAR activity used an extinction coefficient of 14 mM−<sup>1</sup> cm<sup>−</sup>1.

The activity of GR was estimated according to the procedure of Klapheck et al. [34]. The NADPH oxidation was recorded as the absorbance decrease at 340 nm (Shimadzu UV-1800, Shimadzu, Japan) at 25 ◦C for 120 s. The calculation of GR activity used an extinction coefficient of 6.2 mM−<sup>1</sup> cm<sup>−</sup>1.
