Hepatoprotective Effect of Apocynum venetum and its Active Constituents

Abstract

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 tetrachloride (CCl₄, 30 μl/mouse) or D-galactosamine (D-GalN, 700 mg/kg)/lipopolysaccharide (LPS, 20 μg/kg)-induced liver injury in mice. Tumor necrosis factor-α (TNF-α) secreted from LPS-stimulated macrophages is the most crucial mediator in the D-GalN/LPS-induced liver injury model. The extract had no significant inhibition on the increase of serum TNF-α (1169 } 132 pg/ml vs. 1595 } 314 pg/ml of control), but exhibited a complete inhibition at the concentration of 100 μg/ml on TNF-α (100 ng/ml)-induced cell 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 Œ-O-acetate (7), isoquercetin-6 Œ-O-acetate (8), catechin-[8,7-e]-4α-(3,4-dihydroxpyhenyl)-dihydro-2(3H)-pyranone (9), apocynin B (10), apocynin A (11), cinchonain Ia (12), apocynin C (13), apocynin D (14) and quercetin (15). All the compounds showed inhibitory effects on TNF-α-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-α-induced cell death with IC₅₀ values of 37.5, 14.5, 31.2, 55.1, 71.9 and 41.2 μM, respectively. In contrast, the clinically used 5 and its analogues 1, 2 and 6 showed apparent activity only at 80 μM. These flavonoids appeared to be the hepatoprotective principles of the leaves of A. venetum. The hepatoprotective effects exhibited by the extract and its constituents suggest a validation of the leaves as a tea material.

Abbreviations

Av:water extract of the leaves of Apocynum venetum

CCl₄:carbon tetrachloride

D-GalN:D-galactosamine

LPS:lipopolysaccharide

TNF-α:tumor necrosis factor-α

ALT:alanine aminotransferase

MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

ROIs:reactive oxygen species

Apocynum venetum - Apocynaceae - flavonoids - hepatoprotective activity - carbon tetrachloride - D-galactosamine - tumor necrosis factor-α

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