2.5. Cellular Antioxidant Activity (CAA) Assay

HepG2 cells (ATCC Co., Manassas, VA, USA) were incubated in DMEM with 1% penicillin-streptomycin and 10% FBS and maintained at 37 °C with 5% CO₂. The modified methylene blue assay was used to determine cytotoxicity [14].

The CAA assay was performed, according to Wolfe et al. [15], with slight modification. In brief, 6 × 10⁴ HepG2 cells were placed in each well of a black-walled 96-microwell plate. 200 μL of PBS was added to the edge of the plate to prevent the edge effect. After adherence, cells were treated with a range of concentrations of benzoic acid derivatives and a standard (quercetin) containing 50 μM DCFH-DA for 1 h under the same culture condition. After removing the supernatant medium, the cells were washed with or without PBS. Then promptly added 600 μM ABAP into each well except blank groups before reading at 485 nm excitation and 538 nm emission using a multifunctional microplate detector (Molecular Devices, Sunnyvale, CA, USA). The fluorescence values were recorded every 5 min for 1 h. The CAA values were obtained by converting the EC₅₀ values and presented as μmol quercetin equivalents (QE)/100 μmol.