2.4.1 MTT assay

The evaluation of cytotoxicity was performed using MTT assay. The K562 cells were plated in a 96-well culture plate with various concentrations (25 µM–160 µM) of the methanol extract and fractions. The cultured plates were incubated for 24, 48, and 72 h at 37°C and 5% CO₂. Following incubation, 20 µL MTT solution in phosphate-buffered saline (PBS) was added to each well at a final concentration of 0.5 mg/mL followed by further incubation for 3 h at 37°C. The medium was then removed, and 100 µL DMSO was added to each well for solubilizing the formazan. The absorbance was measured at 490 nm (630 nm as a reference) using an ELISA reader (SkanIt™ Software, Microplate Readers, Thermo Fisher Scientific). Three independent experiments were carried out, and 8 replicates were taken for each experiment. The concentration of the methanol extract and fractions which resulted in a 50% reduction of cell viability, the half maximal inhibitory concentration (IC50 value), was calculated using the following formula: % inhibition = (control abs - sample abs)/(control abs) × 100. Paclitaxel was used as a positive control at the concentration of 0.2–50 μg/mL (Ghasemi et al., 2021).