Thiazolyl blue tetrazolium bromide (MTT) assay

MTT was used to determine the effect of B. spergulifolius extracts on 3T3-L1 cell viability and the dosage of the extracts for further studies [23]. 3T3-L1 adipocytes were cultured in a 96-well plate (2x10³ cells/well) and incubated overnight at 37°C under 5% CO². After 24 h, the medium was removed and 3T3-L1 adipocytes were treated with fresh medium containing different concentrations of plant extracts (for MeOH extract, 0–1000 μg/mL; for EA extract; 0–2000 μg/mL; for Aqueous extract, 0–1000 μg/mL) or 0.1% (v/v) DMSO for 24 h at 37°C. Then, the medium was removed and cells were washed twice with PBS. MTT solution (0.5 g/mL in PBS) was supplemented in each well at 100 μL and the cells were incubated for 4 h at 37°C. After MTT solution was suspended and 100 μL of DMSO was supplemented to each well. 96-well plates wrapped with aluminum foil and placed in a shaker (Everlast Rocker 247, Benchmark, USA) for about 1 h. The color change observed in the wells and the relative cell viability were calculated by determining with a spectrophotometer (Uvmini-1240, Shimadzu, Kyoto, Japan) absorbance at 570 nm. The results of cytotoxicity were presented as the relative reduction of the OD₅₇₀, which correlated with the number of viable cells in relation to the percentage of control cells (as taken 100%) using the following formula. The cells half-proliferation inhibitory concentration (IC₅₀) for MeOH, EA, Aqueous extracts of B. spergulifolius were calculated from the linear regression equation. The relative cell-viability and IC₅₀ were plotted in graphs.