Effects of herbal components on cDNA-expressed cytochrome P450 enzyme catalytic activity
Introduction
Because little is known about the metabolic interactions between herbal products and pharmaceuticals, we conducted a series of in vitro experiments to evaluate the effects of components of commonly used herbal products on the catalytic activity of cDNA-expressed cytochrome P450 isoforms in in vitro experiments. Increasing concentrations of the compounds were incubated with a panel of recombinant human CYP isoforms and their effects on the conversion of specific surrogate substrates measured fluorometrically in a 96-well plate format. We chose this methodology because inhibition of cytochrome P450 mediated metabolism is often the mechanism of drug-drug interactions and the use of recombinant human cytochrome P450 enzymes with specific surrogate substrates is recognized as a cost-effective technique for predicting such interactions [1]. Our laboratory and others have extended the use of these assays to evaluate metabolic interactions between herbal products and standard medications [2], [3]. We chose to test purified components of commonly used herbs rather than commercial herbal products or whole plant material because the IC50's of purified components can be expressed in uM concentration and, therefore, can be compared with known CYP inhibitors; because the purity and potency of herbal products have been shown to vary considerably [4]; and because some of these extracts exhibit native fluorescence or quenching which can interfere with these tests [5]. Many of the compounds tested are, however, “marker” compounds for herbal products, i.e., constituents of the plant thought to be associated with its biological activity or used to standardize herbal products for the purpose of quality control.
Twenty-five compounds, components of some of the most commonly used herbal products [6], were tested for their ability to inhibit the catalytic activity of cDNA-derived human P450 enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. These isoforms are involved in the majority of clinically important drug metabolizing reactions [7]. Results of these studies are reported herein.
Section snippets
Standards, samples, and reagents
cDNA-derived CYP450 isoforms, substrates, positive controls (furafylline, sulfaphenazole, tranylcypromine, quinidine, and ketoconazole) and fluorescent products AHMC (3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4-methylcoumarin) and 7-HFC (the 7-hydroxy-3-cyanocoumarin) were obtained from Gentest Corporation, Woburn, MA. Resorufin benzyl ether and CHC (7-hydroxy-3-cyanocoumarin) were obtained from Molecular Probes, Eugene, OR. Resorufin and ginkgolides A and B were obtained from Sigma-Aldrich, St.
Results
Data for the recombinant CYP450 enzyme inhibition studies are shown in Table 2.
Discussion
These data show that components of a number of commonly used herbal products inhibit human drug metabolizing enzymes in vitro. Constituents of Ginkgo biloba (ginkgolic acids I and II), kava (desmethoxyyangonin, dihydromethysticin, and methysticin), garlic (allicin), evening primrose oil (cis-linoleic acid), and St. John's wort (hyperforin and quercetin) produced a dose-dependent inhibition of one or more of the cDNA human P450 isoforms at concentrations of less than 10 uM. In these assays,
Conclusions
Our findings suggest that herbal products containing Kava kava, Ginkgo biloba, garlic, or St. John's wort could potentially inhibit the metabolism of co-administered medications whose primary route of elimination is via cytochrome P450. When administered acutely, constituents in these popular herbs may inhibit drug metabolizing enzymes, especially 2C9, 2C19, and 3A4. These effects are similar in magnitude to those produced by medications (sulfaphenazole, tranylcypromine, and ketoconazole) and
Acknowledgements
Supported in part by grant RO1 AT000636 from the National Center for Complementary and Alternative Medicine, National Institutes of Health. We thank Dr. David Stresser, Gentest Corporation, for his comments.
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