Abts method

Intracellular reactive oxygen species generation was determined using dichloro-dihydro-fluorescein diacetate (DCFH-DA) method as described before. T-AOC was detected by commercially kit with ABTS method (Beyotime Biotechnology, China). The contents of intercellular MDA were detected by commercially kit with thibabituric acid method (Beyotime Biotechnology, China). The concentration of GSH and GSSG were analyzed by commercially kit based on the DTNB method (Beyotime Biotechnology, China). Simultaneously, the protein concentrations were determined using a BCA kit to normalize the results. All procedures were carried out according to the manufacturers’ instructions.

The total antioxidant capacity was evaluated using the ABTS method (Beyotime, Shanghai, China). ABTS radical cation (ABTS⁺) solution was produced by reacting ABTS stock solution with 2.45 mmol/L potassium persulfate in the dark at room temperature for 12–16 h before use, then it was diluted with 80% ethanol to adjust the absorbance to 0.70 ± 0.05 at 734 nm. After that, 10 µL of sample or Trolox standard was added into 200 µL of diluted ABTS⁺ solution. The absorbance at 734 nm was measured after 2–6 min in the dark at room temperature. Trolox, a water-soluble analogue of vitamin E, was used as the reference standard to prepare a calibration curve with a concentration range of 0.15–1.5 mM. Results were expressed as mmol/g Trolox equivalent antioxidant capacity.