OXIDATION OF THE FLAVONOIDS GALANGIN AND KAEMPFERIDE BY HUMAN LIVER MICROSOMES AND CYP 1 A 1 , CYP 1 A 2 , AND CYP 2

There is very limited information on cytochrome P450 (P450)-mediated oxidative metabolism of dietary flavonoids in humans. In this study, we used human liver microsomes and recombinant P450 isoforms to examine the metabolism of two flavonols, galangin and kaempferide, and one flavone, chrysin. Both galangin and kaempferide, but not chrysin, were oxidized by human liver microsomes to kaempferol, with Km values of 9.5 and 17.8 M, respectively. These oxidations were catalyzed mainly by CYP1A2 but also by CYP2C9. Consistent with these observations, the human liver microsomal metabolism of galangin and kaempferide were inhibited by the P450 inhibitors furafylline and sulfaphenazole. In addition, CYP1A1, although less efficient, was also able to oxidize the two flavonols. Thus, dietary flavonols are likely to undergo oxidative metabolism mainly in the liver but also extrahepatically. Flavonoids are polyphenolic compounds present ubiquitously in fruits, vegetables, and beverages such as tea and red wine (Hertog et al., 1992, 1993b). Flavonoids have been implicated to be protective against coronary heart disease (Hertog et al., 1993a; Knekt et al., 1996), stroke (Keli et al., 1996), and cancer (Dorant et al., 1996; Knekt et al., 1997; Le Marchand et al., 2000). The bioavailability of the dietary flavonoids, however, seems to be low, limited both by poor transport (Walgren et al., 1998) and by extensive metabolism (Walle et al., 1999). The metabolism seems to be mediated by conjugative and oxidative enzymes. Many studies have shown potent inhibition of cytochrome P450 (P450) oxidation by flavonoids, in particular the CYP1A1/1A2 isoforms (Tsyrlov et al., 1994; Lee et al., 1998; Zhai et al., 1998; Moon et al., 2000). Flavonoids have also demonstrated potent interactions with the aryl hydrocarbon receptor (Ciolino and Yeh, 1999; Ciolino et al., 1999; Ashida et al., 2000). Much less is known about the oxidative metabolism of the flavonoids, in particular in humans. Studies with liver microsomes from Aroclor 1254-induced rats demonstrate extensive oxidative metabolism of several flavonoids (Duarte Silva et al., 1997a; Nielsen et al., 1998). This oxidation may be mediated by CYP1A1, as supported by subsequent studies (Duarte Silva et al., 1997b; Doostdar et al., 2000). In a recent study of chrysin (5,7dihydroxyflavone; Fig. 1), we found no evidence of oxidation of this compound either by uninduced rat hepatocytes or in human intestinal and hepatic cell lines with induced CYP1A1 (Galijatovic et al., 1999). Surprisingly, there have been no studies of human liver microsomal metabolism of flavonoids. To gain a better understanding of the role of human P450-mediated metabolism of the flavonoids, we investigated the metabolism of two flavonols (3-hydroxylated flavones) [i.e., galangin and kaempferide (Fig. 1)] and chrysin by human liver microsomes and recombinant P450 isoforms. In contrast to chrysin, both galangin and kaempferide underwent extensive oxidative metabolism to kaempferol (Fig. 1). This oxidation was catalyzed not only by both CYP1A1 and CYP1A2 but also by CYP2C9. Experimental Procedures Materials. Chrysin, furafylline, D-glucose 6-phosphate (G6P), G6P dehydrogenase, kaempferol, and NADP were purchased from Sigma Chemical Co. (St. Louis, MO). Galangin and trifluoroacetic acid (spectrophotometric grade) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Kaempferide was purchased from Indofine Chemical Company, Inc. (Belle Mead, NJ), and sulfaphenazole was obtained from Sigma/RBI (Natick, MA). Pooled human liver microsomes from 11 donors and microsomes containing lymphoblastexpressed human CYP1A1, 1A2, 2C9 (Arg144), and 3A4 and baculovirusexpressed human CYP3A4 (Supersomes) were obtained from GENTEST (Woburn, MA). Flavonoid Incubations with Human Liver Microsomes and Recombinant Enzymes. Galangin, kaempferide, and chrysin (1–100 M) were each incubated with pooled human liver microsomes from 11 donors (50 g of protein) in a final volume of 100 l of 100 mM sodium phosphate buffer, pH 7.6, containing 0.5 mM NADP, 10 mM MgCl2, 10 mM G6P, and G6P dehydrogenase (1 unit) at 37°C for 30 min. Recombinant human CYP1A1, 1A2, 2C9, and 3A4 (25 g of protein) were incubated with the flavonoids under identical conditions, using substrate concentrations of 1 to 100 M. Inhibition of CYP1A2 and 2C9 in the pooled human liver microsomes used identical incubation conditions with 10 M concentrations of the flavonoid substrates. Furafylline (20 M), a selective CYP1A2 inhibitor (Sesardic et al., 1990; Clarke et al., 1994; Clement and Demesmaeker, 1997) was preincubated for 15 min with the enzyme mixture before the addition of substrate. Sulfaphenazole (25 M), a selective inhibitor of CYP2C9 (Eagling et al., 1998; Giancarlo et al., 2001), was added together with the substrates. HPLC Analysis. The incubation mixtures were subjected to solid-phase extraction using Oasis HLB C18 1-ml extraction cartridges (Waters, Milford, MA), as previously described (Galijatovic et al., 1999). The dried methanol eluates were reconstituted in a mobile phase for HPLC analysis. A Symmetry C18 column (3.9 150 mm; Waters) with a Bondapak C18 Guard-Pak This study was supported by the National Institutes of Health Grant GM55561. 1 Abbreviations used are: P450, cytochrome P450; G6P, D-glucose 6-phosphate; HPLC, high-performance liquid chromatography. Address correspondence to: Dr. Thomas Walle, Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Avenue, P.O. Box 250505, Charleston, SC 29425. E-mail: wallet@musc.edu 0090-9556/02/3002-103–105$3.00 DRUG METABOLISM AND DISPOSITION Vol. 30, No. 2 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 525/961689 DMD 30:103–105, 2002 Printed in U.S.A. 103 at A PE T Jornals on A ril 5, 2017 dm d.aspurnals. org D ow nladed from precolumn insert (Waters), and a mobile phase of 60% methanol (galangin and kaempferide) or 55% methanol (chrysin) in 0.3% trifluoroacetic acid at a flow rate of 0.9 ml/min was used. Detection was by photodiode array (Waters model 996 detector, Millennium software), monitoring galangin and kaempferide and their metabolites at 365 nm and chrysin and its metabolites at 268 nm. The identity of possible metabolites was confirmed by a comparison of retention time and UV spectrum with synthetic standards. Data Analysis. All data are reported as means S. E. The apparent enzyme kinetic parameters Km and Vmax were obtained from the Henri-MichaelisMenten equation by nonlinear regression analysis of velocity versus substrate concentration plots, using the Solver function of Microsoft Excel (Microsoft,