Hepatoprotective effect of Apocynum venetum and its active constituents

The leaves of Apocynum venetum L. are used as a tea material in north China and japan. A water extract (500 mg/kg/day, one week administration) of the leaves of A. venetum showed protective effects against carbon tetrachlorid e (CCl4, 30 mu l/mouse) or D-galactosamine (D-GalN, 700 mg/kg)/lipopolysacc haride (LPS, 20 mu g/kg)-induced liver injury in mice. Tumor necrosis facto r-alpha (TNF-alpha) secreted from LPS-stimulated macrophages is the most cr ucial mediator in the D-GalN/LPS-induced liver injury model. The extract ha d no significant inhibition on the increase of serum TNF-alpha (1169 +/- 13 2 pg/ml vs. 1595 +/- 314 pg/ml of control), but exhibited a complete inhibi tion at the concentration of 100 mu g/ml on TNF-alpha (100 ng/ml)-induced c ell death in D-GalN (0.5 mM)-sensitized mouse hepatocytes. Further activity -guided fractionation resulted in the isolation of fifteen flavonoids viz.( -)-epicatechin (1), (-)-epigallocatechin (2), isoquercetin (3), hyperin (4) , (+)-catechin (5), (+)-gallocatechin (6), kaempferol-6'-O-acetate (7), iso quercetin-6'-O-acetate (8), catechin-[8,7-e]-4 alpha-(3,4-dihydroxpyhenyl)- dihydro-2(3H)-pyranone (9), apocynin 8 (10), apocynin A (11), cinchonain 1a (12), apocynin C (13), apocynin D (14) and quercetin (15). All the compoun ds showed inhibitory effects on TNF-alpha-induced cell death with different intensities. The flavonol glycosides 3, 4, 7 and 8 and the phenylpropanoid -substituted flavan-3-ols 11 and 12 showed potent inhibitory effects on TNF -alpha-induced cell death with IC50 values of 37.5, 14.5, 31.2, 55.1, 71.9 and 41.2 mu M, respectively. In contrast, the clinically used 5 and its ana logues 1, 2 and 6 showed apparent activity only at 80 mu M. These flavonoid s appeared to be the hepatoprotective principles of the leaves of A. venetu m. The hepatoprotective effects exhibited by the extract and its constituen ts suggest a validation of the leaves as a tea material.