Analytical procedure for the in-vial derivatization-extraction of phenolic acids and flavonoids in methanolic and aqueous plant extracts followed by gas chromatography with mass selective detection.

An in-vial simple method for the combined derivatization and extraction of phenolic acids and flavonoids from plant extracts and their direct determination with GC-MS, is described. The method is taking advantage of the beneficial potentials of phase transfer catalysis (PTC). Catalysts in soluble and polymer-bound form were tested with the latter being the format of choice due to its high reaction yield and facile separation from the rest of the reaction system. Optimization of experimental conditions was established. Chromatographic separation of eight phenolic acids and four flavonoids methylated via the PTC derivatization step was achieved in 45 min. The detection limits for the described GC-MS(SIM) method of analysis ranged between 2 and 40ng/ml whereas limits of quantitation fall in the range 5-118 ng/ml, with flavonoids accounting for the lowest sensitivity due to their multiple reaction behavior. Four methanolic extracts from Tilia europea, Urtica dioica, Mentha spicata and Hypericumperforatum grown wild in north-westem Greece and four aquatic infusions from commercially available Mentha spicata, Origanum dictamnus, Rosemarinus officinalis and Sideritis cretica were analyzed. Good trueness of the method was demonstrated as no matrix effects were found for the analytes concerned. (C) 2004 Elsevier B.V. All rights reserved

Human cytochrome p450 enzyme specificity for the bioactivation of estragole and related alkenylbenzenes

Human cytochrome P450 enzymes involved in the bioactivation of estragole to its proximate carcinogen 1 '-hydroxyestragole were identified and compared to the enzymes of importance for 1'-hydroxylation of the related alkenylbenzenes methyleugenol and safrole. Incubations with Supersomes revealed that all enzymes tested, except P450 2C8, are intrinsically able to 1 '-hydroxylate estragole. Experiments with Gentest microsomes, expressing P450 enzymes to roughly average liver levels, indicated that P450 1A2, 2A6, 2C19, 2D6, and 2E1 might contribute to estragole 1 '-hydroxylation in the human liver. Especially P450 1A2 is an important enzyme based on the correlation between P450 1A2 activity and estragole 1 '-hydroxylation in human liver microsomal samples and inhibition of estragole 1 '-hydroxylation by the P450 1A2 inhibitor alpha-naphthoflavone. Kinetic studies revealed that, at physiologically relevant concentrations of estragole, P450 1A2 and 2A6 are the most important enzymes for bioactivation in the human liver showing enzyme efficiencies (k(cat)/K-m) of, respectively, 59 and 341 min(-1) mM(-1). Only at relatively high estragole concentrations, P450 2C19, 2D6, and 2E1 might contribute to some extent. Comparison to results from similar studies for safrole and methyleugenol revealed that competitive interactions between estragole and methyleugenol 1 '-hydroxylation and between estragole and safrole 1'-hydroxylation are to be expected because of the involvement of, respectively, P450 1A2 and P450 2A6 in the bioactivation of these compounds. Furthermore, poor metabolizer phenotypes in P450 2A6 might diminish the chances on bioactivation of estragole and safrole, whereas lifestyle factors increasing P450 1A2 activities such as cigarette smoking and consumption of charbroiled food might increase those chances for estragole and methyleugenol

Human cytochrome P450 enzyme specificity for bioactivation of safrole to the proximate carcinogen 1'-hydroxysafrole

In the present study, the cytochrome P450 mediated bioactivation of safrole to its proximate carcinogenic metabolite, 1'-hydroxysafrole, has been investigated for the purpose of identifying the human P450 enzymes involved. The 1'-hydroxylation of safrole was characterized in a variety of in vitro test systems, including Supersomes, expressing individual human P450 enzymes to a high level, and microsomes derived from cell lines expressing individual human P450 enzymes to a lower, average human liver level. Additionally, a correlation study was performed, in which safrole was incubated with a series of 15 human liver microsomes, and the 1'-hydroxylation rates obtained were correlated with the activities of these microsomes toward specific substrates for nine different isoenzymes. To complete the study, a final experiment was performed in which pooled human liver microsomes were incubated with safrole in the presence and absence of coumarin, a selective P450 2A6 substrate. On the basis of the results of these experiments, important roles for P450 2C9*1, P450 2A6, P450 2D6*1, and P450 2E1 were elucidated. The possible consequences of these results for the effects of genetic polymorphisms and life style factors on the bioactivation of safrole are discussed. Polymorphisms in P450 2C9, P450 2A6, and P450 2D6, leading to poor metabolizer phenotypes, may reduce the relative risk on the harmful effects of safrole, whereas life style factors, such as the use of alcohol, an inducer of P450 2E1, and barbiturates, inducers of P450 2C9, and polymorphisms in P450 2D6 and P450 2A6, leading to ultraextensive metabolizer phenotypes, may increase the relative ris

Human cytochrome P450 enzymes of importance for the bioactivation of methyleugenol to the proximate carcinogen 1′-hydroxymethyleugenol

In vitro studies were performed to elucidate the human cytochrome P450 enzymes involved in the bioactivation of methyleugenol to its proximate carcinogen 1′-hydroxymethyleugenol. Incubations with Supersomes, expressing individual P450 enzymes to a high level, revealed that P450 1A2, 2A6, 2C9, 2C19, and 2D6 are intrinsically able to 1′-hydroxylate methyleugenol. An additional experiment with Gentest microsomes, expressing the same individual enzymes to roughly average liver levels, indicated that P450 1A2, 2C9, 2C19, and 2D6 contribute to methyleugenol 1′-hydroxylation in the human liver. A study, in which correlations between methyleugenol 1′-hydroxylation in human liver microsomes from 15 individuals and the conversion of enzyme specific substrates by the same microsomes were investigated, showed that P450 1A2 and P450 2C9 are important enzymes in the bioactivation of methyleugenol. This was confirmed in an inhibition study in which pooled human liver microsomes were incubated with methyleugenol in the presence and absence of enzyme specific inhibitors. Kinetic studies revealed that at physiologically relevant concentrations of methyleugenol P450 1A2 is the most important enzyme for bioactivation of methyleugenol in the human liver showing an enzyme efficiency (kcat/Km) that is ∼30, 50, and >50 times higher than the enzyme efficiencies of, respectively, P450 2C9, 2C19, and 2D6. Only when relatively higher methyleugenol concentrations are present P450 2C9 and P450 2C19 might contribute as well to the bioactivation of methyleugenol in the human liver. A 5-fold difference in activities was found between the 15 human liver microsomes of the correlation study (range, 0.89-4.30 nmol min-1 nmol P450-1). Therefore, interindividual differences might cause variation in sensitivity toward methyleugenol. Especially lifestyle factors such as smoking (induces P450 1A) and the use of barbiturates (induces P450 2C) can increase the susceptibility for adverse effects of methyleugenol. © 2006 American Chemical Society