3.2.1. In Vitro Cytotoxicity Evaluation HepG2

The HepG2 cell line was maintained at 37 °C, 5% CO₂, at 90% humidity in MEM supplemented with 10% fetal bovine serum, 1% L-glutamine (200 mM), and penicillin (100 U/mL)/streptomycin (100 μg/mL) (complete RPMI medium). The cytotoxicity of the tested molecules on the HepG2 (hepatocarcinoma cell line purchased from ATCC, ref HB-8065) cell line was assessed according to the method of Mosmann [37] with slight modifications. Briefly, 5.10³ cells in 100 μL of complete medium were inoculated into each well of 96-well plates and incubated at 37 °C in humidified 5% CO₂. After 24 h incubation, 100 μL of medium with various product concentrations dissolved in DMSO (final concentration less than 0.5% v/v) were added and the plates were incubated for 72 h at 37 °C. Triplicate assays were performed for each sample. Each plate-well was then microscopically examined for possible precipitate formation before the medium was aspirated from the wells. Next, 100 µL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl -2H-tetrazolium bromide) solution (0.5 mg/mL in medium without FBS) was added to each well. Cells were incubated for 2 h at 37 °C. After this time, the MTT solution was removed and DMSO (100 μL) was added to dissolve the resulting blue formazan crystals. Plates were shaken vigorously (700 rpm) for 10 min. The absorbance was measured at 570 nm with 630 nm as reference wavelength using a BIO-TEK ELx808 Absorbance Microplate Reader (LabX, Midland, ON, Canada). DMSO was used as blank and doxorubicin (purchased from Sigma Aldrich) as positive control. Cell viability was calculated as a percentage of control (cells incubated without compound). The 50% cytotoxic concentration (CC₅₀) was determined from the dose–response curve, using TableCurve software 2D v.5.0. CC₅₀ values to represent the mean value calculated from three independent experiments.