Cytotoxicity determination of carvacrol on BSC-1 cells

The cytotoxicity of carvacrol was determinated by CCK-8 assay [24]. BSC-1 cells were seeded in 96-well plates and cultured in 10% DMEM for 24 h at 37 °C in an atmosphere containing 5% CO₂. The medium was then removed and carvacrol (with dose of 1, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.025 and 0 mmol/L) and 2% carvacrol real solution (with dose of 4, 3.5, 3, 2.5, 2, 1.8, 1.6, 1.4, 1.2, 1, 0.8 and 0 mmol/L) were severally added to individual wells of BSC-1 cells in plates with 3 wells in parallel for each dose and the plates were incubated for 24 h. Cells treated without the carvacrol were used as a control. After that, the supernatant medium of each well was replaced with 100 μL DMEM, and 10 μL CCK-8 solution was added to the cells, and cells were cultured for 4 h avoiding light. The absorbance (A) of each well was measured at 450 nm using BioTek synergy2 microplate reader. The cell viability was calculated using the following formula: Cell viability (%) = (A_s - A_b) / (A_c - A_b) × 100%, where A_s and A_c refer to the absorbance in the presence and absence of carvacrol, respectively, and A_b stands for the blank control. Subsequently, the half-maximal inhibitory concentration (IC₅₀) of carvacrol and 2% carvacrol real solution on BSC-1 was automatically calculated using Bliss principle according to the cell viability values obtained above.