Metabolite profiling of natural substances in human: in vitro study from fecal bacteria to colon carcinoma cells (Caco-2).

Flavonoids, including anthocyanins, are polyphenolic compounds present in fruits, vegetables and dietary supplements. They can be absorbed from the intestine to the bloodstream or pass into the large intestine. Various bacterial species and enzymes are present along the entire intestine. The aim of the present work was to investigate the intestinal metabolism of selected dietary polyphenol and polyphenol glycosides (quercetin, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, and delphinidin-3-O-galactoside) by human fecal bacteria. Moreover, the metabolism of metabolites formed from these compounds in human colon carcinoma cells (Caco-2) was also point of the interest. Test compounds were added to fresh human stool in broth or to Caco-2 cells in medium and then incubated for 6 or 20 h at 37°C. After incubation, samples were prepared for LC/MS determination. Main metabolic pathways were deglycosylation, hydrogenation, methylation, hydroxylation, and decomposition. 2,4,5-trihydroxybenzaldehyde, as a metabolite of cyanidin glycosides, was detected after incubation for the first time. Metabolites formed by fecal bacteria were further glucuronidated or methylated by intestinal enzymes. This metabolite profiling of natural compounds has helped to better understand the complex metabolism in the human intestine and this work also has shown the connection of metabolism of natural substances by intestinal bacteria followed by metabolism in intestinal cells.

High-throughput assay for determining enantiomeric excess of chiral diols, amino alcohols, and amines and for direct asymmetric reaction screening.

Determining enantiomeric excess (e.e.) in chiral compounds is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for determining e.e. in mixtures of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcohols, and amino-acid esters. The method is based on dynamic self-assembly of commercially available chiral amines, 2-formylphenylboronic acid, and chiral diols in acetonitrile to form fluorescent diastereomeric complexes. Each analyte enantiomer engenders a diastereomer with distinct fluorescence wavelength/intensity originating from enantiopure fluorescent ligands. In this assay, enantiomers of amines and amine derivatives assemble with diol-type ligands containing a binaphthol moiety (BINOL and VANOL), whereas diol enantiomers form complexes with the enantiopure amine-type fluorescent ligand tryptophanol. The differential fluorescence is utilized to determine the amount of each enantiomer in the mixture with an error of <1% e.e. This method enables high-throughput real-time evaluation of enantiomeric/diastereomeric excess (e.e./d.e.) and product yield of crude asymmetric reaction products. The procedure comprises high-throughput liquid dispensing of three components into 384-well plates and recording of fluorescence using an automated plate reader. The approach enables scaling up the screening of combinatorial libraries and, together with parallel synthesis, creates a robust platform for discovering chiral catalysts or auxiliaries for asymmetric transformations and chiral drug development. The procedure takes ~4-6 h and requires 10-20 ng of substrate per well. Our fluorescence-based assay offers distinct advantages over existing methods because it is not sensitive to the presence of common additives/impurities or unreacted/incompletely utilized reagents or catalysts.

Effect of bilberry extract (Vaccinium myrtillus L.) on drug-metabolizing enzymes in rats.

Vaccinium myrtillus L. (bilberry) fruit is a blue-colored berry with a high content of anthocyanins. These bioactive secondary metabolites are considered to play a major role in the health-promoting properties of bilberries. Our in vivo study was designed to assess the possible influence of bilberry extract on drug-metabolizing enzymes (DMEs). Rats were exposed to bilberry extract in drinking water at two concentrations (0.15 and 1.5 g/L). Selected DMEs were determined (mRNA expression and enzymatic activity) after 29 and 58 days in rat liver. In addition, a panel of antioxidant, physiological, biochemical and hematological parameters was studied; these parameters did not demonstrate any impact of bilberry extract on the health status of rats. A significant increase in activity was observed in cytochrome P450 (CYP) 2C11 (131% of control) and CYP2E1 (122% of control) after a 29-day administration, while the consumption of a higher concentration for a longer time led to a mild activity decrease. Slight changes were observed in some other DMEs, but they remained insignificant from a physiological perspective. According to our results, we conclude that the consumption of bilberries as a food supplement should not pose a risk of interacting with co-administered drugs based on their metabolism.

Isothiocyanate from Broccoli, Sulforaphane, and Its Properties.

Sulforaphane is an isothiocyanate occurring in stored form as glucoraphanin in cruciferous vegetables such as cabbage, cauliflower, and kale, and at high levels in broccoli especially in broccoli sprouts. Glucoraphanin requires the plant enzyme myrosinase for converting it into sulforaphane. Sulforaphane is metabolized through mercapturic acid pathway, being conjugated with glutathione and undergoes further biotransformation, yielding metabolites. Sulforaphane is extensively investigated and is in the interest in medicine for its health benefits. It has been shown that sulforaphane may protect against various types of cancer, may also decrease the risk of cardiovascular disease, and help in autism and osteoporosis. Our review offers a short summary of interesting properties of sulforaphane. Both the in vitro and in vivo methods/models and clinical studies are mentioned.

The inhibitory effects of ß-caryophyllene, ß-caryophyllene oxide and α-humulene on the activities of the main drug-metabolizing enzymes in rat and human liver in vitro.

Sesquiterpenes, the main components of plant essential oils, are often taken in the form of folk medicines and dietary supplements. Several sesquiterpenes possess interesting biological activities but they could interact with concurrently administered drugs via inhibition of drug-metabolizing enzymes. Therefore, the present study was designed to test the potential inhibitory effect of tree structurally relative sesquiterpenes ß-caryophyllene (CAR), ß-caryophyllene oxide (CAO) and α-humulene (HUM) on the activities of the main drug-metabolizing enzymes. For this purpose, rat and human hepatic subcellular fractions were incubated with CAR, CAO or HUM together with specific substrates for oxidation, reduction and conjugation enzymes and their coenzymes. HPLC, spectrophotometric and spectrofluorimetric analyses of product formations were used. All tested sesquiterpenes significantly inhibited cytochromes P4503A (CYP3A) activities in rats as well as in human hepatic microsomes, with CAO being the strongest inhibitor. A non-competitive type of inhibition was found. On the other hand, none of the tested sesquiterpenes significantly affected the activities of carbonyl-reducing enzymes (CBR1, AKRs, NQO1) or conjugation enzymes (UGTs, GSTs, SULTs, COMT). As CYP3A enzymes metabolize many drugs, their inhibition by CAO, CAR and HUM might affect the pharmacokinetics of concurrently administered drugs. Similar results obtained in rat and human hepatic microsomes indicate that rats could be used for further testing of possible drug-sesquiterpenes interactions in vivo.

Nerolidol and Farnesol Inhibit Some Cytochrome P450 Activities but Did Not Affect Other Xenobiotic-Metabolizing Enzymes in Rat and Human Hepatic Subcellular Fractions.

Sesquiterpenes, 15-carbon compounds formed from three isoprenoid units, are the main components of plant essential oils. Sesquiterpenes occur in human food, but they are principally taken as components of many folk medicines and dietary supplements. The aim of our study was to test and compare the potential inhibitory effect of acyclic sesquiterpenes, trans-nerolidol, cis-nerolidol and farnesol, on the activities of the main xenobiotic-metabolizing enzymes in rat and human liver in vitro. Rat and human subcellular fractions, relatively specific substrates, corresponding coenzymes and HPLC, spectrophotometric or spectrofluorometric analysis of product formation were used. The results showed significant inhibition of cytochromes P450 (namely CYP1A, CYP2B and CYP3A subfamilies) activities by all tested sesquiterpenes in rat as well as in human hepatic microsomes. On the other hand, all tested sesquiterpenes did not significantly affect the activities of carbonyl-reducing enzymes and conjugation enzymes. The results indicate that acyclic sesquiterpenes might affect CYP1A, CYP2B and CYP3A mediated metabolism of concurrently administered drugs and other xenobiotics. The possible drug-sesquiterpene interactions should be verified in in vivo experiments.

Influence of Sulforaphane Metabolites on Activities of Human Drug-Metabolizing Cytochrome P450 and Determination of Sulforaphane in Human Liver Cells.

The influence of metabolites of sulforaphane, natural compounds present in broccoli (Brassica oleracea var. botrytis italica) and in other cruciferous vegetables, on drug-metabolizing cytochrome P450 (CYP) enzymes in human liver microsomes and possible entry of sulforaphane into human hepatic cells were investigated. Metabolites studied are compounds derived from sulforaphane by the mercapturic acid pathway (conjugation with glutathione and by following reactions), namely sulforaphane glutathione and sulforaphane cysteine conjugates and sulforaphane-N-acetylcysteine. Their possible effect on four drug-metabolizing CYP enzymes, CYP3A4 (midazolam 1'-hydroxylation), CYP2D6 (bufuralol 1'-hydroxylation), CYP1A2 (7-ethoxyresorufin O-deethylation), and CYP2B6 (7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation), was tested. Inhibition of four prototypical CYP activities by sulforaphane metabolites was studied in pooled human liver microsomes. Sulforaphane metabolites did not considerably affect biological function of drug-metabolizing CYPs in human liver microsomes except for CYP2D6, which was found to be inhibited down to 73-78% of the original activity. Analysis of the entry of sulforaphane into human hepatocytes was done by cell disruption by sonication, methylene chloride extraction, and modified high-performance liquid chromatography method. The results have shown penetration of sulforaphane into the human hepatic cells.

Influence of diet supplementation with green tea extract on drug-metabolizing enzymes in a mouse model of monosodium glutamate-induced obesity.

PURPOSE: Consumption of dietary supplements with green tea extract (GTE) is popular for weight management, but it may be accompanied by various side effects, including interactions with drugs. The aim of the present in vivo study was to evaluate the effect of defined GTE (Polyphenon 60) in three dosage schemes on insulin, leptin and drug-metabolizing enzymes in obese mice. METHODS: Experimental obesity was induced by repeated s.c. application of monosodium glutamate to newborn mice. Green tea extract was administered in three dosage schemes in chow diet. The plasmatic levels of insulin and leptin were assayed using enzyme-linked immunosorbent assay. Enzyme activities and mRNA expressions of drug-metabolizing enzymes (totally 13) were analyzed in liver and small intestine using spectrophotometric and HPLC assays and RT-PCR, respectively. RESULTS: GTE-treatment decreased insulin and leptin levels. Eleven enzymes were significantly affected by GTE-treatment. Long-term administration of 0.01% GTE caused increase in the activity and mRNA level of cytochrome P450 3A4 (CYP3A4) ortholog in the liver as well as in the small intestine. Interestingly, short-term overdose by GTE (0.1%) had more pronounced effects on enzyme activities and mRNA expressions than long-term overdose. CONCLUSIONS: GTE-mediated induction of CYP3A4 ortholog, the main drug-metabolizing enzyme, could result in decreased efficacy of simultaneously or subsequently administered drug in obese individuals.

Effects of anthocyanidins and anthocyanins on the expression and catalytic activities of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in primary human hepatocytes and human liver microsomes.

Anthocyanidins and anthocyanins are pharmacologically active constituents of various berry fruits, such as blueberry and cranberry. These compounds are also contained in massively used nutritional supplements based on extracts or dry matter from berry fruits. The current study evaluated the effects of anthocyanidins and anthocyanins on the expression and catalytic activity of major drug-metabolizing enzymes CYP2C9, CYP2A6, CYP2B6, and CYP3A4 in primary cultures of human hepatocytes and human liver microsomes. Expression of mRNA was quantified by qRT-PCR. Expression of proteins was evaluated by Western blotting and immunochemiluminescence. The catalytic activity of CYP enzymes was measured by HPLC using specific enzyme substrates. Tested anthocyanidins (6) and anthocyanins (21) did not induce the expression of mRNA and protein of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 genes in human hepatocytes. Catalytic activities of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 enzymes were inhibited by all anthocyanidins to different extents (e.g., delphinidin inhibits CYP3A4 by >90% at 100 µM with IC50 = 32 µM). Of 21 anthocyanins tested, only cyanidin-3-O-rhamnoside (CYP3A4 by >75% at 100 µM with IC50 = 44 µM) and two glycosides of delphinidin significantly inhibited examined cytochromes P450. It may be concluded that in the ranges of common ingestion of either food or dietary supplement an induction or significant inhibition of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 activity is most probably not expected.

Effects of anthocyanins on the AhR-CYP1A1 signaling pathway in human hepatocytes and human cancer cell lines.

Anthocyanins are plant pigments occurring in flowers and berry fruits. Since a phenomenon of food-drug interactions is increasingly emerging, we examined the effects of 21 major anthocyanins and the extracts from 3 food supplements containing anthocyanins on the aryl hydrocarbon receptor (AhR)-cytochrome P450 CYP1A1 signaling pathway in human hepatocytes and human hepatic HepG2 and intestinal LS174T cancer cells. Pelargonidin-3-O-rutinoside (PEL-2) and cyanidin-3,5-O-diglucoside (CYA-3) dose-dependently activated AhR, as revealed by gene reporter assay. PEL-2 and CYA-3 induced CYP1A1 mRNA but not protein in HepG2 and LS174T cells. Neither compounds induced CYP1A1 mRNA and protein in four different primary human hepatocytes cultures. The effects of PEL-2 and CYA-3 on AhR occurred by ligand-dependent and ligand-independent mechanisms, respectively, as demonstrated by ligand binding assay. In a direct enzyme inhibition assay, none of the antocyanins tested inhibited the CYP1A1 marker activity to less than 50% even at 100 µM concentration. PEL-2 and CYA-3 at 100 µM inhibited CYP1A1 to 79% and 65%, respectively. In conclusion, with exception of PEL-2 and CYA-3, there were no effects of 19 major anthocyanins and 3 food supplements containing anthocyanins on AhR-CYP1A1 signaling, implying zero potential of these compounds for food-drug interactions with respect to AhR-CYP1A1 pathway.

Effect of silybin and its congeners on human liver microsomal cytochrome P450 activities.

Silybin and related flavonolignans form a major part of the Silybum marianum extract, silymarin, which has been used to treat liver diseases for hundreds of years. Although regarded as safe, many of the extract constituents remain thus far untested for their possible effects on liver biotransformation enzymes. Cytochromes P450 (CYP) are very important in this regard. We tested the effect of four flavonolignans: silybin, its hemisynthetic derivative dehydrosilybin, silydianin, and silycristin on three specific CYP activities: bufuralol 1'-hydroxylation (CYP2D6), p-nitrophenol hydroxylation (CYP2E1), and nifedipine oxidation (CYP3A4). All flavonolignans displayed dose-dependent inhibition of these activities with IC(50) values in the micromolar range. The inhibition was competitive or mixed as revealed by double reciprocal plots of kinetic experiments. However, the inhibition is not considered to be relevant for therapy because physiological concentrations of the individual flavonolignans do not exceed 0.5 microM. The data support the use of the extract as a dietary supplement.