IC50 determination by MTT assay

The cell proliferation inhibitory effect of OA-MVLs on HepG2 cells was evaluated by MTT assay. Briefly, cells were adjusted to a density of 4×10⁵ cells/mL and seeded into 96-well plates (100 μL/well), and cultured for 48 hours at 37°C. Then, the cells were treated with OA-MVLs at different concentrations such as 10, 20, 40, 80, 120, 160, 200, 220, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, and 350 μmol/L, and concentrations of OA solution were maintained consistent with those of OA-MVLs.9 After incubation for 24 hours, 20 μL of MTT reagent (5 mg/mL) was added to each well, and the plate was incubated for another 4 hours at 37°C in the dark. The liquid was then removed, and the remaining crystals were solubilized with 150 μL of dimethyl sulfoxide. The plate was incubated for an additional 10 minutes at 37°C with gentle shaking before the measurement of absorbance at 490 nm using Varioskan Flash instrument (Thermo Fisher Scientific Inc). The rate of cell proliferation inhibition was calculated by the following formula: Inhibition Rate (%) = (1 − A_sample/A_control) ×100. Each assay was carried out in triplicate with the empty wells as the negative control and the well containing cell culture medium alone as the blank control.