SCREENING OF HEPATOPROTECTIVE PLANT-COMPONENTS USING A HEPG2 CELL CYTOTOXICITY ASSAY

Identification of the active components of plants with hepatoprotectiv e properties requires screening of large numbers of samples during fra ctionation and purification. A screening assay has been developed base d on protection of human liver-derived HepG2 cells against toxic damag e. Various hepatotoxins were incubated with HepG2 cells in 96-well mic rotitre plates (30 000 cells well(-1)) for 1 h and viability was deter mined 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-di MeNAPQI, 200 mM) had greater toxic effects than tert-butyl hydroperoxi de (1.8 mM) or galactosamine (10 mM), reducing mean viability to 44.6 +/- 1.2% (s.e.m.) and 56.1 +/- 2.1% of control, respectively. Protecti on against toxic damage by these agents was tested using a crude extra ct of a known hepatoprotective Sri Lankan plant, Osbeckia aspera, and two pure established hepatoprotective plant compounds, (+)-catechin an d silymarin (1 mg mL(-1)). Viability was significantly improved by Osb eckia (by 37.7 +/- 2.4%, P < 0.05, and 36.5 +/- 2.1%, P < 0.05, for br omobenzene and 2,6-diMeNAPQI toxicity, respectively). Comparable value s for (+)-catechin were 68.6 +/- 2.9% and 63.5 +/- 1.1%, and for silym arin 24.9 +/- 1.4% and 25.0 +/- 1.6%. This rapid and reproducible assa y should prove useful for the isolation and identification of active h epatoprotective compounds in crude plant extracts.