Effects of kava (Kava-kava, 'Awa, Yaqona, Piper methysticum) on c-DNA-expressed cytochrome P450 enzymes and human cryopreserved hepatocytes.

The effects of the herbal product kava (Kava kava, 'Awa, Yaqona, Piper methysticum) on human P450 isoforms were studied in vitro using both cDNA-expressed human enzymes and cryopreserved human hepatocytes. Increasing concentrations of an ethanolic extract of dried kava root and three purified kava lactones (methysticin, desmethoxyyangonin, and yangonin) were tested for their ability to inhibit the catalytic activity of a panel of P450 isoforms (1A2, 2A6, 2C9, C2C19, 2D6, 2E1, and 3A4) present as c-DNA expressed-enzymes and in previously cryopreserved human hepatocytes. In addition, the test compounds' effect on hepatocyte viability was evaluated by measuring cellular ATP content. In both models, the kava extract and the three kava lactones were found to be potent inhibitors of CYPs 1A2, 2C9, 2C19, 2E1, and 3A4 with IC50 values of approximately 10 microM. The test compounds were also moderately cytotoxic to human hepatocytes (EC50 values of approximately 50 microM). Methysticin was the most potent enzyme inhibitor as well as the most cytotoxic, followed by (in order of potency:) the kava root extract, desmethoxyyangonin, and yangonin. Our results suggest that the drug interaction and hepatotoxic potential of kava should be further investigated.

Effects of herbal components on cDNA-expressed cytochrome P450 enzyme catalytic activity.

We evaluated the effects of 25 purified 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 (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and their effects on the conversion of specific surrogate substrates measured fluorometrically in a 96-well plate format. For each test substance, the IC50 (the concentration required to inhibit metabolism of surrogate substrates by 50%) was estimated and compared with IC50's for the positive control inhibitory drugs furafylline, sulfaphenazole, tranylcypromine, quinidine, and ketoconazole. 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) significantly inhibited one or more of the cDNA human P450 isoforms at concentrations of less than 10 uM. Some of the test compounds (components of Ginkgo biloba, kava, and St. John's wort) were more potent inhibitors of the isoforms 1A2, 2C19, and 2C19 than the positive controls used in each assay (furafylline, sulfaphenazole, and tranylcypromine, respectively), which are known to produce clinically significant drug interactions. The enzyme most sensitive to the inhibitory of effects of these compounds was CYP2C19, while the isoform least effected was CYP2D6. These data suggest that herbal products containing evening primrose oil, Ginkgo biloba, kava, and St. John's Wort could potentially inhibit the metabolism of co-administered medications whose primary route of elimination is via cytochrome P450.

Effects of intrinsic fluorescence and quenching on fluorescence-based screening of natural products.

To evaluate the effects of intrinsic (natural) fluorescence and quenching as confounding variables in fluorescence-based enzyme inhibition assays of natural products, we measured the fluorescence and quenching properties of 25 components of popular herbal products. The analyses were performed under conditions typically employed in drug-drug interaction studies that use c-DNA-derived P450 isoforms and surrogate fluorogenic substrates. Four of the 25 compounds tested (isorhamnetin, quercetin, vitexin, and yangonin) fluoresced or quenched sufficiently to interfere with these assays. Intrinsic fluorescence had a greater effect on these assays than quenching and for one compound, yangonin, was sufficient to mask inhibition and potentially produce a false negative result. Quenching had less of an effect on these assays, but was significant enough for one compound, quercetin, to mimic "weak" inhibition. Therefore, because intrinsic fluorescence or quenching could render some natural products unsuitable for testing in certain fluorometric assays, it would be prudent to include an evaluation of these properties in experimental protocols.

Quantitative determination of ginsenosides by high-performance liquid chromatography-tandem mass spectrometry.

An HPLC-MS/MS method was developed for the quantitative determination of ginsenosides, which are the marker compounds for herbal products containing Panax ginseng (Korean or Chinese ginseng) and P. quinquefolius (American ginseng). Samples were extracted with BondElut C18 HF extraction columns and the concentrations of seven major ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1) were determined by reversed-phase HPLC-MS/MS employing a quadrupole-ion trap mass spectrometer. Both positive and negative electrospray ionisation techniques were evaluated. Positive ionisation spectra of these compounds gave strong sodium adduct molecular and sodium adduct dimer ions. Negative ionisation yielded the molecular ion primarily and was, therefore, used for analysis: quantitative determination was based on the most abundant product ions for each ginsenoside. The method was used to extract and analyse commercial samples of P. ginseng and P. quinquefolius.

Variability in commercial ginseng products: an analysis of 25 preparations.

BACKGROUND: Because dietary supplements are not subject to the same regulations that pharmaceuticals are, there is concern among medical professionals that these products may lack purity or potency. OBJECTIVE: To determine the variability in a range of ginseng herbal products available in the United States, we identified and measured the concentration of marker compounds by using HPLC and liquid chromatography-tandem mass spectrometry. DESIGN: Twenty-five commercial ginseng preparations from the genera Panax or Eleutherococcus were obtained from a local health food store and analyzed for 7 ginsenosides (marker compounds for Panax species, which include Asian and American ginseng) and 2 eleutherosides (marker compounds for Eleutherococcus senticosus, also known as Siberian ginseng). RESULTS: All plant products were correctly identified by botanical plant species (ie, Panax species or E. senticosus); however, concentrations of marker compounds differed significantly from labeled amounts. There was also significant product-to-product variability: concentrations of ginsenosides varied by 15- and 36-fold in capsules and liquids, respectively, and concentrations of eleutherosides varied by 43- and 200-fold in capsules and liquids, respectively. Although a systematic search for adulterants was not conducted, review of the HPLC and liquid chromatography-tandem mass spectrometry data suggest that no substances other than ginsenosides or eleutherosides were extracted from the plant material. CONCLUSION: Our data suggest that US ginseng products are correctly labeled as to plant genus; however, variability in concentrations of marker compounds suggests that standardization may be necessary for quality assurance and that characterization of herbal products should be considered in the design and evaluation of studies on herbal products.

Shosaiko-to and other Kampo (Japanese herbal) medicines: a review of their immunomodulatory activities.

The use of alternative medicine, including consumption of herbal products and dietary supplements, has been increasing substantially both in the United States and in Western Europe. One area that is garnering increased attention is the use of Oriental Medicine including Kampo, or Japanese herbal medicine. Herein, we review representative examples of research available on the most common use of Kampo medicinals, namely to improve the immune response. We also provide an extensive background on the history of Kampo. There are more than 210 different Kampo formulae used in Japan and most uses of Kampo are to modulate the immune response, i.e. to improve immunity. We have extracted data on seven common Kampo medicinals, and the data are reviewed with respect to in vitro and in vivo activities for both humans and experimental animals; the ingredients as well as the problems with classification of these materials are presented. Research suggests that Kampo herbals are biologically active and may have therapeutic potential. While it is believed that Kampo medicines have few side effects, there is a paucity of data on their toxicity as well as a relative lack of knowledge of the bioactive constituents and potential drug interactions of these agents.

Effects of ginseng components on c-DNA-expressed cytochrome P450 enzyme catalytic activity.

Because little is known about the interactions between herbal products and standard medications, the effects of seven ginsenosides and two eleutherosides (active components of the ginseng root) on the catalytic activity of c-DNA expressed cytochrome P450 isoforms were studied in in vitro experiments. Increasing concentrations of ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 and eleutherosides B and E were incubated with a panel of recombinant human CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and their effects on the conversion of specific surrogate substrates measured fluorometrically in a 96-well plate format. For each test substance, the IC50 (the concentration required to inhibit the metabolism of the surrogate substrates by 50%) was estimated and this value compared with that obtained for positive control inhibitory drugs furafylline, sulfaphenazole, tryanylcypromine, quinidine, and ketoconizole. Of the components tested, three ginsenosides (Rd, Rc, and Rf) modified the activity of the recombinant enzymes. Ginsenoside Rd produced weak inhibitory activity against the surrogate substrates for CYP3A4 and CYP2D6 and even weaker inhibitory activity against the surrogate substrates for CYP2C19 and CYP2C9. The IC50 values of 58 and 74 uM for the two substrates for CYP3A4 are orders of magnitude higher than that for the potent inhibitor ketoconazole used as a positive control. Ginsenoside Rc produced an increase in the activity of CYP2C9 (70% at 200 uM) and ginsenoside Rf produced an increase in the activity of CYP3A4 (54% at 200 uM). The biological significance of this is unclear at this time. Enzyme "activation", the process by which direct addition of one compound to an enzyme enhances the rate of reaction of the substrate, has been observed in a number of cases with P450 enzymes; however, a matrix effect caused by the test compound fluorescing at the same wavelength as the metabolite of the marker substrate cannot be ruled out. In summary, these studies suggest that the ginsenosides and eleutherosides tested are not likely to inhibit the metabolism of coadministered medications in which the primary route of elimination is via cytochrome P450; the potential of ginsenosides to enhance the catalysis of certain substrates requires further investigation.

Cocaine and metabolite concentrations in the hair of South American coca chewers.

Hair samples obtained from South American Indians who were identified as daily chewers of coca leaves were analyzed by a sensitive gas chromatography/mass spectrometry (GC/MS) method for cocaine, benzoylecognine (BE), and ecognine methyl ester (EME). The mean cocaine concentration in the hair of these five subjects was 15.2 ng/mg hair +/- 11.0 (range = 1.0-28.9 ng/mg), mean BE concentration was 2.8 +/- 1.6 ng/mg hair (range = 0.3-4.4 ng/mg hair), and mean EME concentration was 1.6 +/- 1.7 (range = 0.0-4.4 ng/mg hair). The finding that cocaine was present at approximately 5 times higher concentration than BE and approximately 12 times higher than EME is surprising in light of the much longer plasma half lives of these metabolites. Washing the hair before analysis with 1% dodecyl sulfate, methanol, and distilled water reduced the concentration of cocaine in the hair but also reduced the concentrations of the metabolites. These data suggest that factors other than the drug concentration in blood may be important in determining the amount of drug incorporated into hair.