Purification of citrus limonoids and their differential inhibitory effects on human cytochrome P450 enzymes

Recent studies demonstrated that citrus limonoids and flavonoids possess numerous health promoting properties. In the present study, glucosides of limonoids and flavonoids were purified from citrus molasses and limonoid aglycones from citrus seeds. Glucosides were separated on styrene (divinylbenzene), Q-sepharose resins with increasing concentration of sodium chloride. A pH-dependent cold precipitation was carried out for the isolation of naringin in large quantity. Major aglycones such as limonin and nomilin were isolated from seeds by direct crystallization and minor limonoids were purified by vacuum liquid chromatography. The structures of the isolated compounds were confirmed by NMR spectra. Individual limonoids were tested for O-dealkylase and hydroxylase activities of human cytochrome P450 (CYP) isoenzymes such as CYP1A2, CYP1B1, CYP3A4 and CYP19, using ethoxyresorufin, methoxyresorufin and dibenzylfluorescein as substrates. Partial to high inhibition of CYPs was observed in dose-dependent assays. Significant (P < 0.001) reductions in enzyme activities were observed with purified compounds above 2 mu mol. Kinetic analyses indicated that limonin glucoside inhibited CYP19 competitively (IC50, 7.1 mu mol L-1), whereas Nomilinic acid glucoside inhibited it noncompetitively (IC50, 9.4 mu mol(-1)). Nomilinic acid glucoside was the most potent limonoid, with an overall IC50 of < 10 mu mol, for all the enzymes tested. The differential inhibition of CYPs can be ascribed to structural variations of the limonoid nucleus. Limonoid inhibition of key CYPs involved in carcinogenesis supports growing evidence that citrus limonoids act as anticancer agents. (c) 2007 Society of Chemical Industry.