Procedure:
Cells were seeded at a density of 1×105cells/ in each well of the 24 well plates and incubated overnight. After 24h, the media was flicked off, the cells were treated with eupalitin 3-O- β-D-galactopyranoside (100 and 500 µg/ml) in separate wells of a 24-well plate and incubated for 2 h. Silymarin (250, 500µg/ml) was used as a reference standard After incubation, the cells were treated with D-galactosamine at a concentration of 40mM (Quintero et al 2002) and was allowed to incubate for 2h. After incubation , the cells were washed and incubated for 1 h with MTT (20 μl of 5mg/ml of MTT in PBS) was added to each well. Formation of formazan crystal was observed under a microscope. In case crystal, formation was not proper, incubation was continued for an hour more. Media was removed and the remaining formazan crystals were dissolved in 200 μl of DMSO in each well. The cell culture plate was kept on a shaker for 15 min; The absorbance was recorded by ELISA reader at 540 nm.
The % hepatoprotection was obtained by the following formula:
% hepatoprotection =100 - [((absorbance of control - absorbance of test)/absorbance of
control)×100