*2.4. Determination of Oxidative Damage and Enzymatic Antioxidant Activities*

To determine the degree of oxidative damage and different antioxidant activity, 0.2 g fresh leaf was extracted with 5% thiobarbituric acid (TBA) dissolved in 5% trichloroacetic acid (TCA) for MDA content detection and homogenized in 0.2 M phosphate buffers (pH 7.0~7.5) for SOD, POD, APx, and CAT activity detection and GSH content. The assessed activities of MDA, H2O2, GSH, SOD, POD, CAT, and APx using the assay test kits were purchased from Nanjing Jian Cheng Bioengineering Institute in Nanjing, China. Protein content was determined using the Bradford protein colorimetry method with bovine serum albumin (BSA) as a protein standard [24]. Briefly, in a 50 mL 95% ethanol solution, 100 mg Coomassie Brilliant Blue G-250 was dissolved (C2H5OH). Following that, 100 mL of 85% phosphoric acid (H3PO4) was carefully added while stirring and then diluted with distilled water to a total volume of 1 L. The solution was filtered and maintained at a temperature of 4 ◦C. For the measurements, 20 μL extract and 200 μL Bradford solution were combined and incubated for 5 min before determining the absorbance at 595 nm using a UV/Vis spectrophotometer (UV 2700, Shimadzu, Japan).
