Identification of the active components of plants with hepatoprotective properties requires screening of large numbers of samples during fractionation and purification. A screening assay has been developed based on protection of human liver-derived HepG2 cells against toxic damage.
Various hepatotoxins were incubated with HepG2 cells in 96-well microtitre plates (30000 cells well−1) for 1 h and viability was determined by metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS). Bromobenzene (10 mm) and 2,6-dimethyl-N-acetyl-p-quinoneimine (2,6-diMeNAPQI, 200 mm) had greater toxic effects than tert-butyl hydroperoxide (1.8 mm) or galactosamine (10 mm), reducing mean viability to 44.6 ± 1.2% (s.e.m.) and 561 ± 21% of control, respectively. Protection against toxic damage by these agents was tested using a crude extract of a known hepatoprotective Sri Lankan plant, Osbeckia aspera, and two pure established hepatoprotective plant compounds, (+)-catechin and silymarin (1 mg mL−1). Viability was significantly improved by Osbeckia (by 37.7 ± 2.4%, P < 0.05, and 36.5 ± 21%, P < 0.05, for bromobenzene and 2,6-diMeNAPQI toxicity, respectively). Comparable values for (+)-catechin were 68.6 ± 2.9% and 63.5 ±11%, and for silymarin 24.9 ± 1.4% and 25.0 ± 1.6%.
This rapid and reproducible assay should prove useful for the isolation and identification of active hepatoprotective compounds in crude plant extracts.