2.3. MTT Cytotoxicity Assay

MCF-7, MDA-MB-231, and 3T3-NIH cells were seeded at a concentration of 1 × 10³ cells/ml in a 96-well plate and incubated for 24 h in an incubator at 37°C with 5% CO₂. After incubation, the old medium was replaced with the extract diluted with the respective complete growth medium (200, 100, 50, 25, 12.5, 6.5, and 0.0 µg/ml) and followed by incubation for 72 h. After the incubation, each well was added with 20 µl of 5 mg/ml MTT solution and incubated in dark condition for another 3 h [18]. Then, the medium with the MTT solution was removed, and 100 µl of absolute dimethyl sulfoxide (DMSO) was added to each well to dissolve formazan crystal that has been formed by viable cells. Each well was measured at 540 nm using an ELISA plate reader (FLUOstar Omega, BMG LABTECH, Germany). The percentage of cell viability was calculated as follows:

where OD = optical density, control = without treatment of extract, and sample = with treatment of extract. The IC₅₀ values were determined as the concentration of the extracts that caused 50% inhibition or cell death.