Isolation and identification of compounds with dioxin-induced AhR transformation inhibitory activity from the leaves of Mallotus japonicus (Thunb.) Muell. Arg.

The solvent extracts of 368 plant parts from 191 different species cultivated in Tsukuba (Ibaraki Prefecture, Japan) were previously screened for aryl hydrocarbon receptor (AhR) transformation inhibitory activity. It was observed that the leaf extract of Mallotus japonicus had strong activity. To clarify the antidioxin phytochemical, a 70% acetone extraction from the leaves was fractionated with three kinds of solvent (n-hexane, ethyl acetate, 1-butanol) in order of increasing polarity. The fractions of ethyl acetate and 1-butanol with strong antidioxin activity were purified by preparative high performance liquid chromatography in combination with AhR transformation assays by a cell-free system using a rat hepatic cytosolic fraction. The structures of isolated compounds were elucidated by nuclear magnetic resonance (NMR) techniques and fast atom bombardment mass spectra. As a result, six compounds (phyllanthusiin D, quercetin, quercitrin, isoquercitrin, rutin, and kaempferol-3-O-rutinoside) as the active components were isolated. The compounds suppressed AhR transformation in a concentration-dependent manner, with 50% inhibitory concentration (IC50) values of 0.12 µM (phyllanthusiin D), 0.45 µM (quercitrin), 0.97 µM (isoquercitrin), and 16 µM (rutin) against 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced AhR transformation. Quercetin and kaempferol-3-rutinoside exhibited very weak activity. Thus, phyllanthusiin D, which is a hydrolysable tannin, exhibited the greatest activity. This is the first report to demonstrate the antidioxin activity of a tannin. Key words: Mallotus japonicus, aryl hydrocarbon receptor (AhR) transformation, flavonol, tannin.