2.4.3. Cellular Antioxidant Activity assay

Hepatocarcinoma cell line, HepG2, was obtained from American Type Culture Collection (Rockville, MD, USA), cultured in RPMI supplemented with 5% (v/v) FBS, 2 mM L-glutamine, 50 IU/mL penicillin, and 50 µg/mL streptomycin, and maintained in a humidified atmosphere with 5% CO₂ at 37 °C [6]. Cells were mostly cultured in 75 cm² culture flasks and were trypsinized using trypsin-EDTA before the confluence was reached.

The fruit extract was employed for cellular antioxidant activity (CAA) assay [16], which was performed as previously described [13]. Briefly, HepG2 were seeded in 96-well plates at a density equal to 6.0 × 10⁴ cells/well in RPMI medium. After 24 h, the medium was removed and 25 µM DCFH-DA was added in each well along with different concentration of the fruit extract for two hours. In order to ensure that the observed effect depended on the tested sample, the same amount of EtOH contained in the extracts was added to the culture medium of the control cells. However, in all experimental conditions, EtOH never exceeded 0.25% (v/v). After 2 h of incubation, cells were washed twice with PBS, and then incubated 600 μM ABAP dissolved in HBSS was added. The plates were then placed into a plate-reader thermostat at 37 °C, and emission at 538 nm was measured for one hour, during which an excitation at 485 nm every 5 min was made. Each plate included control and blank wells. Control wells were preincubated with 25 µM DCFH-DA and then treated with 600 μM ABAP. Blank wells were pretreated with 25 µM DCFH-DA and then incubated in HBSS without the oxidant agent. The area under the curve of fluorescence units versus minutes was used to calculate the CAA value for each extract concentration using the following equation (Equation (3)):

where CAA is the cellular antioxidant activity; ∫SA is the integrated area under the curve of fluorescence obtained for samples and normalized for blanks; ∫CA is the integrated area under the curve of fluorescence obtained for controls and normalized for blanks.

Finally, the concentration necessary to inhibit 50% of 2′,7′-dichlorofluorescin (DCF) formation (CAA₅₀) for each fruit extract was calculated from concentration/response curves using linear regression analysis. Data were expressed as CAA₅₀ (mg of FW per mL cell medium). The experiments were repeated three times.