Interindividual Differences in Human Intestinal Microbial Conversion of (-)-Epicatechin to Bioactive Phenolic Compounds.

To quantify interindividual differences in the human intestinal microbial metabolism of (-)-epicatechin (EC), in vitro anaerobic incubations with fecal inocula from 24 healthy donors were conducted. EC-derived colonic microbial metabolites were qualitatively and quantitively analyzed by liquid chromatography triple quadrupole mass spectrometry (LC-TQ-MS) and liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS). Quantitative microbiota characterization was achieved by 16S rRNA analysis. The results obtained show 1-(3',4'-dihydroxyphenyl)-3-(2″,4″,6″-dihydroxyphenyl)-2-propanol (3,4-diHPP-2-ol) and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone (3,4-diHPV) to be key intermediate microbial metabolites of EC and also revealed the substantial interindividual differences in both the rate of EC conversion and the time-dependent EC metabolite pattern. Furthermore, substantial differences in microbiota composition among different individuals were detected. Correlations between specific microbial phylotypes and formation of certain metabolites were established. It is concluded that interindividual differences in the intestinal microbial metabolism of EC may contribute to interindividual differences in potential health effects of EC-abundant dietary foods or drinks.

Effects of Maerua subcordata (Gilg) DeWolf on electrophile-responsive element (EpRE)-mediated gene expression in vitro.

Plant extracts and phytochemicals may prevent chronic diseases via activation of adaptive cellular stress response pathways including induction of antioxidant and phase II detoxifying enzymes. The regulatory regions of these inducible genes encode the electrophile-response element (EpRE). This study tested the EpRE induction ability of Maerua subcordata (fruit, leaf, root, seed) methanol extracts and selected candidate constituents thereof, identified by liquid chromatography coupled with multistage mass spectroscopy, employing an EpRE luciferase reporter gene assay using hepa-1c1c7 mouse hepatoma cells. A parallel Cytotox CALUX assay using human osteosarcoma U2OS cells was used to monitor any non-specific changes in luciferase activity or cytotoxicity. Results showed that fruit, root, and seed extracts were non-cytotoxic up to a concentration of 30 gram dry weight per litre but the leaf extract exhibited some cytotoxicity and that the leaf (despite some cytotoxicity), fruit, and seed extracts showed strong induction of EpRE mediated gene expression while induction by the root extract was minimal. Selected candidates included glucosinolates, isothiocyanates, and some biogenic amines. Subsequent studies showed that methyl-, ethyl-, isopropyl-, isobutyl- isothiocyanates, and sec-butyl thiocyanate as well as glucobrassicin induced concentration (1-100 µM) dependent EpRE-mediated gene expression while the biogenic amines stachydrine and trigonelline acted as inhibitors of EpRE-mediated gene expression at 100 µM. The identification of glucolepidiin, glucobrassicin, glucocapparin, stachydrine, and trigonelline in all extracts was confirmed using standards and based on multiple reaction monitoring; yet, glucobrassicin level in the root extract was negligible. In conclusion, this study provided a first report on EpRE mediated gene expression effects of M. subcordata; and despite detection of different glucosinolates in all extracts, those containing glucobrassicin particularly displayed high EpRE induction. Because EpRE inducers are cytoprotective and potential chemopreventive agents while inhibitors are suggested adjuvants of chemotherapy, results of this study imply that process manipulation of this plant may result in herbal preparations that may be used as chemopreventive agents or adjuvants of chemotherapies.

Use of physiologically based kinetic modelling-facilitated reverse dosimetry to convert in vitro cytotoxicity data to predicted in vivo liver toxicity of lasiocarpine and riddelliine in rat.

Lasiocarpine and riddelliine are pyrrolizidine alkaloids (PAs) present in food and able to cause liver toxicity. The aim of this study was to investigate whether physiologically based kinetic (PBK) modelling-facilitated reverse dosimetry can adequately translate in vitro concentration-response curves for toxicity of lasiocarpine and riddelliine to in vivo liver toxicity data for the rat. To this purpose, PBK models were developed for lasiocarpine and riddelliine, and predicted blood concentrations were compared to available literature data to evaluate the models. Concentration-response curves obtained from in vitro cytotoxicity assays in primary rat hepatocytes were converted to in vivo dose-response curves from which points of departure (PODs) were derived and that were compared to available literature data on in vivo liver toxicity. The results showed that the predicted PODs fall well within the range of PODs derived from available in vivo toxicity data. To conclude, this study shows the proof-of-principle for a method to predict in vivo liver toxicity for PAs by an alternative testing strategy integrating in vitro cytotoxicity assays with in silico PBK modelling-facilitated reverse dosimetry. The approach may facilitate prediction of acute liver toxicity for the large number of PAs for which in vivo toxicity data are lacking.

Risk assessment of genotoxic and carcinogenic alkenylbenzenes in botanical containing products present on the Chinese market.

In the present study, a risk assessment of plant food supplements (PFS), traditional Chinese medicines (TCM) and herbal teas containing alkenylbenzenes was performed using the Margin of Exposure (MOE) approach. The levels of alkenylbenzenes in botanical preparations collected on the Chinese market were quantified and the combined estimated daily intake (EDI) was determined using dose additivity. The combined EDI values obtained assuming equal potency of all alkenylbenzenes detected in the PFS, TCM and herbal teas were 0.3 to 14.3, 0.05 to 539.4 and 0.04 to 42.5 µg/kg bw/day, respectively. Calculating combined EDI values taking into account the toxic equivalency (TEQ) approach, the values for PFS, TCM and herbal teas were 0.3 to 7.7, 0.05 to 278.0 and 0.02 to 16.5 µg estragole equivalents/kg bw/day, respectively. The MOE values resulting from consumption of these PFS, TCM and one cup of herbal tea per day during life-time were generally lower than 10 000, suggesting a potential priority for risk management. For short-term exposure such as two weeks consumption, applying Haber's rule, only one TCM 6 () still had an MOE value below 10 000. It is concluded that selected consumption of Chinese botanical preparations raise a concern because of exposure to alkenylbenzenes, especially when exposure is for longer periods of time.

Risk assessment for pyrrolizidine alkaloids detected in (herbal) teas and plant food supplements.

Pyrrolizidine alkaloids (PAs) are plant metabolites present in some botanical preparations, with especially 1,2-unsaturated PAs being of concern because they are genotoxic carcinogens. This study presents an overview of tumour data on PAs and points of departure (PODs) derived from them, corroborating that the BMDL10 for lasiocarpine represents a conservative POD for risk assessment. A risk assessment using this BMDL10 and mean levels of PAs reported in literature for (herbal) teas, indicates that consumption of one cup of tea a day would result in MOE values lower than 10 000 for several types of (herbal) teas, indicating a priority for risk management for these products A refined risk assessment using interim relative potency (REP) factors showed that based on the mean PA levels, 7(54%) of 13 types of (herbal) teas and 1 (14%) of 7 types of plant food supplements (PFS) resulted in MOE values lower than 10 000, indicating a priority for risk management also for these products in particular. This includes both preparations containing PA-producing and non-PA-producing plants. Our study provides insight in the current state-of-the art and limitations in the risk assessment of PA-containing food products, especially (herbal) teas and PFS, indicating that PAs in food presents a field of interest for current and future risk management.