Cytotoxicity assay

The cytotoxicity of each compound on Vero cells was determined using MTT assay. Vero cell is a non-tumorigenic cell from the kidney tissue of African green monkeys [59]. Cells will proliferate by expressing the NADPH-dependent oxidoreductase in mitochondria that reduces the MTT reagent into the reduction state of formazan crystal [60]. Cells (1 × 10⁴/well) were seeded in 96-well flat-bottomed plates and incubated with each sample at various concentrations for 24 h. Compound solutions were prepared in the following concentrations: 10, 20, 40, 80, 160, 320, 640 and 1280 µg/mL. 30 μL of MTT solution (5 mg/mL in PBS) was added to each well and the plate was incubated at 37 °C for another 4 h. Then, the medium was discarded and 150 µl of DMSO was added to dissolve the formazan crystals. The absorbance of each sample was read at 595 nm using a microplate reader. Results were expressed as a percentage of cell viability with respect to untreated control cells (as 100%) [61]. The drug-dose dependent curve and its statistical analysis (95% confident interval) were generated using GraphPad Prism 5.0 (https://graphpad-prism.software.informer.com/5.0/).