title: Screening of hepatoprotective plant components using a HepG2 cell cytotoxicity assay.
abstract: 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 (30,000 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 56.1 +/- 2.1% 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 +/- 2.1%, P < 0.05, for bromobenzene and 2,6-diMeNAPQI toxicity, respectively). Comparable values for (+)-catechin were 68.6 +/- 2.9% and 63.5 +/- 1.1%, 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.
authors.affiliation: Institute of Liver Studies, King's College School of Medicine and Dentistry, London, UK.
authors.forename: M I
authors.initials: MI
authors.lastname: Thabrew
authors.forename: R D
authors.initials: RD
authors.lastname: Hughes
authors.forename: I G
authors.initials: IG
authors.lastname: McFarlane
mesh_terms: D001969:Bromobenzenes / Q000037:antagonists & inhibitors / Q000633:toxicity*; D002392:Catechin / Q000627:therapeutic use; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D008099:Liver / Q000166:cytology / Q000187:drug effects*; D010936:Plant Extracts / Q000627:therapeutic use*; D012838:Silymarin / Q000627:therapeutic use; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles
chemical_list: D001969:Bromobenzenes; D010936:Plant Extracts; D012838:Silymarin; D013778:Tetrazolium Salts; D013844:Thiazoles; D002392:Catechin; C032036:bromobenzene; C022616:thiazolyl blue