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