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Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Enzymatic Browning in Sugar Beet Leaves (Beta vulgaris L.) : Influence of Caffeic Acid Derivatives, Oxidative Coupling, and Coupled Oxidation
    Vissers, Anne ; Kiskini, Alexandra ; Hilgers, Roelant ; Marinea, Marina ; Wierenga, Peter Alexander ; Gruppen, Harry ; Vincken, Jean Paul - \ 2017
    Journal of Agricultural and Food Chemistry 65 (2017)24. - ISSN 0021-8561 - p. 4911 - 4920.
    Beta vulgaris - flavonoids - oxidative coupling - phenolic acids - PPO activity

    Sugar beet (Beta vulgaris L.) leaves of 8 month (8m) plants showed more enzymatic browning than those of 3 month (3m). Total phenolic content increased from 4.6 to 9.4 mg/g FW in 3m and 8m, respectively, quantitated by reverse-phase-ultrahigh-performance liquid chromatography-ultraviolet-mass spectrometry (RP-UHPLC-UV-MS). The PPO activity was 6.7 times higher in extracts from 8m than from 3m leaves. Substrate content increased from 0.53 to 2.45 mg/g FW in 3m and 8m, respectively, of which caffeic acid glycosyl esters were most important, increasing 10-fold with age. Caffeic acid glycosides and vitexin derivatives were no substrates. In 3m and 8m, nonsubstrate-to-substrate ratios were 8:1 and 3:1, respectively. A model system showed browning at 3:1 ratio due to formation of products with extensive conjugated systems through oxidative coupling and coupled oxidation. The 8:1 ratio did not turn brown as oxidative coupling occurred without much coupled oxidation. We postulate that differences in nonsubstrate-to-substrate ratio and therewith extent of coupled oxidation explain browning.

    Physiologically based kinetic modelling based prediction of oral systemic bioavailability of flavonoids, their metabolites, and their biological effects
    Boonpawa, Rungnapa - \ 2017
    Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Arjen Punt. - Wageningen : Wageningen University - ISBN 9789463430371 - 180
    flavonoids - bioavailability - modeling - metabolites - quercetin - physiology - hesperidin - flavonoïden - biologische beschikbaarheid - modelleren - metabolieten - quercetine - fysiologie - hesperidine

    Flavonoids, abundantly present in fruits and vegetables, have been reported to exert various positive health effects based on in vitro bioassays. However, effects detected in in vitro models cannot be directly correlated to human health as most in vitro studies have been performed using flavonoid aglycones at high concentration ignoring extensive metabolism of flavonoids in the human body. To better understand positive health effects of flavonoids in humans, it is of importance to gain insight in at which form and concentration flavonoids are present in the systemic circulation after consumption. This insight can be obtained using physiologically based kinetic (PBK) computer modeling. The results obtained show that PBK modeling provides a useful additional research tool for studies on the fate of flavonoids in the human body and can reveal at what oral dose levels of flavonoids in vitro positive health effects can be expected to occur in vivo, presenting opportunities that are not easily provided by other methods.

    The influence of phase II conjugation on the biological activity of flavonoids
    Beekmann, K. - \ 2016
    Wageningen University. Promotor(en): Ivonne Rietjens; Peter van Bladeren, co-promotor(en): L. Actis-Goretta. - Wageningen : Wageningen University - ISBN 9789462577640 - 171
    flavonoids - biological activity - in vitro - biosynthesis - peroxisomes - microarrays - daidzein - genistein - oestrogen receptors - isoflavones - quercetin - kaempferol - serine proteinases - threonine - flavonoïden - biologische activiteit - in vitro - biosynthese - peroxisomen - microarrays - daidzin - genisteïne - oestrogeenreceptoren - isoflavonen - quercetine - kaempferol - serine proteïnasen - threonine

    Flavonoid consumption is often correlated with a wide range of health effects, such as the prevention of cardiovascular diseases, neurodegenerative diseases, and diabetes. These effects are usually ascribed to the activity of the parent flavonoid aglycones, even though these forms of the flavonoids generally have a low systemic bioavailability. During uptake, flavonoids undergo phase II metabolism and are present in the systemic circulation nearly exclusively as conjugated metabolites. The aim of this thesis was to study the effect of conjugation on the biological activity of selected flavonoids towards different endpoints relevant for human health. To this end, conjugation with glucuronic acid was taken as the model type of conjugation because this modification is generally observed to be the most important metabolic conjugation reaction for flavonoids in man.

    A review of scientific literature published until early 2012 reveals that metabolic conjugation can affect the biological activity of flavonoids in different ways. Conjugation can increase, decrease, inverse or not affect the biological activity, depending on the flavonoid, the type and position of conjugation, the endpoint studied, and the assay system used. Based on the literature reviewed it is concluded that the effect of conjugation has to be studied on a case-by-case basis.

    As the research on the biological activity of biologically relevant flavonoid conjugates is often hampered by the generally low commercial availability and high prices of these conjugates, a simple and versatile method for the biosynthesis of metabolically relevant flavonoid conjugates is described. Using this method, relevant conjugates can be prepared from different flavonoid substrates in sufficient quantities for in vitro bioassays. Further, an efficient strategy for the identification of these flavonoid conjugates by LC-MS and 1H-NMR using MetIDB (Metabolite Identification Database), a publicly accessible database of predicted and experimental 1H-NMR spectra of flavonoids, is presented.

    To study the effect of conjugation on the biological activities of flavonoids, several different assay systems and endpoints were used to study the activity of different flavonoids and their conjugates. The effects of quercetin, kaempferol, and their main plasma conjugates quercetin-3-O-glucuronide and kaempferol-3-O-glucuronide (K-3G) on different endpoints related to peroxisome proliferator-activated receptor (PPAR)-γ were studied. PPAR-γ activation is reported to have positive health effects related to adipogenesis, insulin resistance and inflammation. The presented results show that the flavonoid aglycones increased PPAR-γ mediated gene expression in a stably transfected reporter gene cell line, and that glucuronidation diminished this effect. These observed increases in reporter gene expression were accompanied by increased PPAR-γ receptor-mRNA expression upon exposure to kaempferol, an effect that was also reduced by glucuronidation. Using the cell-free Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction (MARCoNI) it was demonstrated that, unlike the known PPAR-γ agonist rosiglitazone, neither the flavonoid aglycones nor the conjugates are agonistic ligands of the PPAR-γ receptor. Supporting the hypothesis that the tested compounds have a different mode of action from normal LBD agonism, quercetin appeared to synergistically increase the effect of rosiglitazone in the reporter gene assay. The modes of action behind the observed effects remain to be elucidated and might include effects on protein kinase activities affecting expression of the PPAR-γ receptor, or posttranscriptional modifications of PPAR-γ.

    Another type of nuclear receptor known to be targeted by certain flavonoids are the estrogen receptor (ER)α- and ERβ. ERs are the main targets of estrogenic compounds, and upon their activation different transcriptional responses with opposite effects on cell proliferation and apoptosis are elicited; ERα activation stimulates cell proliferation, while ERβ activation causes apoptosis and reduces ERα mediated induction of cell proliferation. Using the MARCoNI assay, the intrinsic estrogenic effects of the two main dietary isoflavones daidzein and genistein, and their plasma conjugates daidzein-7-O-glucuronide and genistein-7-O-glucuronide on the ligand induced coregulator binding of ERα- and ERβ-LBD were studied and compared to the effect of the positive control 17β-estradiol (E2). The results show that the tested isoflavone compounds are less potent agonists of ERα- and ERβ-LBD than E2, although they modulate the LBD-coregulator interactions in a manner similar to E2. Genistein is shown to be a more potent agonist than daidzein for both receptor subtypes. While in the MARCoNI assay genistein had a strong preference for ERβ-LBD activation over ERα-LBD activation, daidzein had a slight preference for ERα-LBD activation over ERβ-LBD activation. Glucuronidation reduced the intrinsic agonistic activities of both daidzein and genistein to induce ERα-LBD and ERβ-LBD - coregulator interactions and increased their average half maximal effective concentrations (EC50s) by 8 to 4,400 times. The results presented further show that glucuronidation changed the preferential activation of genistein from ERβ-LBD to ERα-LBD and increased the preferential activation of daidzein for ERα-LBD; this is of special interest given that ERβ activation, which is counteracting the possible adverse effects of ERα activation, is considered one of the supposedly beneficial modes of action of isoflavones.

    Many flavonoids are reported to be inhibitors of protein kinases. To study the effect of conjugation on the inhibition of serine/threonine protein kinases by flavonoids, kaempferol and its main plasma conjugate K-3G were selected as model compounds. Protein kinases are involved in a wide range of physiological processes by controlling signaling cascades and regulating protein functions; modulation of their activities can have a wide range of biological effects. The inhibitory effects of kaempferol, K-3G, and the broad-specificity protein kinase inhibitor staurosporine on the phosphorylation activity of recombinant protein kinase A (PKA) and of a lysate prepared from the hepatocellular carcinoma cell line HepG2 were studied using a microarray platform that determines the phosphorylation of 141 putative serine/threonine phosphorylation sites derived from human proteins. The results reveal that glucuronidation reduces the intrinsic potency of kaempferol to inhibit the phosphorylation activity of PKA and HepG2 lysate on average about 16 and 3.5 times, respectively. It is shown that the inhibitory activity of K-3G in the experiments conducted was not caused by deconjugation to the aglycone. Furthermore, the results show that kaempferol and K-3G, unlike the broad-specificity protein kinase inhibitor staurosporine, did not appear to inhibit all protein kinases present in the HepG2 lysate to a similar extent, indicating that kaempferol selectively targets protein kinases, a characteristic that appeared not to be affected by glucuronidation. The fact that K-3G appeared to be only a few times less potent than kaempferol implies that K-3G does not necessarily need to be deconjugated to the aglycone to exert potential inhibitory effects on protein kinases.

    The results obtained in the present thesis support the conclusion that glucuronidation of flavonoids does not necessarily abolish their activity and that flavonoid glucuronides may be biologically active themselves, albeit at higher concentrations than the parent aglycones. In line with the conclusions from the earlier literature review, an updated literature review on the effect of conjugation on the biological activity of flavonoids concludes that that the effect of conjugation on the biological activity of flavonoids depends on the type and position of conjugation, the endpoint studied and the assay system used. Based on the results described and the literature reviewed in this thesis, several recommendations and perspectives for future research are formulated. Several methodological considerations are formulated that need to be taken into account when studying the biological activity of flavonoids and their conjugates to avoid confounding results. Further, the relevance of the gut microbiome for flavonoid bioactivity is highlighted, and considerations regarding the pharmacokinetics and pharmacodynamics of flavonoids in vivo are formulated. Altogether, it can be concluded that circulating flavonoid conjugates may exert biological activities themselves, and that understanding these is a prerequisite to successfully elucidate the mechanisms of action behind the biological activities linked to flavonoid consumption.

    Plants4Cosmetics : perspectives for plant ingredients in cosmetics
    Boeriu, C.G. - \ 2015
    Wageningen : Wageningen UR - Food & Biobased Research (Report / Wageningen UR Food & Biobased Research 1603) - 38
    cosmetics - plants - flavonoids - phenols - pigments - plant pigments - polysaccharides - geranium - hyacinthus - chrysanthemum - orchidaceae - skin - hair - oil plants - medicinal plants - natural products - biobased chemicals - biobased economy - cosmetica - planten - flavonoïden - fenolen - pigmenten - plantenpigmenten - polysacchariden - geranium - hyacinthus - chrysanthemum - orchidaceae - huid - haar - olieleverende planten - medicinale planten - natuurlijke producten - chemicaliën uit biologische grondstoffen - biobased economy
    In opdracht van Bio Base Westland en de TKI Tuinbouw Koepel PPS Plantenstoffen, heeft Wageningen UR – Food & Biobased Research een exploratieve desktop studie uitgevoerd gericht op de identificatie van veelbelovende routes voor de valorisatie van plantinhoudstoffen - waaronder ook reststromen uit de tuinbouw - voor de cosmetische industrie. Een uitgebreide analyse van de beschikbare informatie werd uitgevoerd om de mogelijkheden voor de Nederlandse tuinbouwsector te bepalen. Er is gekeken naar marktkansen in de cosmetische industrie met inbegrip van natuurlijke en biologische ingrediënten.
    Estrogenicity and metabolism of prenylated flavonoids and isoflavonoids
    Schans, M.G.M. van de - \ 2015
    Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Jean-Paul Vincken; Toine Bovee. - Wageningen : Wageningen University - ISBN 9789462574748 - 180
    flavonoïden - isoflavonoïden - glycine max - sojabonen - oestrogeenreceptoren - zoethout - glycyrrhiza glabra - in vitro - flavonoids - isoflavonoids - glycine max - soyabeans - oestrogen receptors - liquorice - glycyrrhiza glabra - in vitro

    Binding of (prenylated) flavonoids and isoflavonoids to the human estrogen receptors (hERs) might result in beneficial health effects in vivo. To understand structure-activity relationships of prenylated (iso)flavonoids towards the hERs, prenylated (iso)flavonoids were purified from extracts of licorice roots and elicited soybean seedlings. It was observed that prenylation can modulate estrogenicity. Unprenylated, chain and δ-position pyran prenylated (iso)flavonoids show an agonistic mode of action, whereas α/β-position pyran, α/β-position furan and double chain prenylated (iso)flavonoids show an antagonistic mode of action towards hERα in the yeast bioassay. The mode of estrogenic action of prenylated (iso)flavonoids could be related to structural features of the hER. In particular, the increase in length of α/β-position pyran prenylated compounds was related to indirect antagonism. It was also shown that heat and acid affected the stability of 6a-hydroxy-pterocarpans, converting them into their respective 6a,11a-pterocarpenes and consequently modulate their estrogenicity. Six prenylated isoflavonoids acted as SERMs and eight prenylated isoflavonoids showed ER subtype-selective behavior. The kind of prenylation (chain, furan or pyran) was most important for determining SERM activity, whereas additionally the backbone structure, i.e. the presence of an additional D-ring, was of importance for determining ER subtype-selectivity. To determine structure-metabolism relationships, in vitro conversion of purified prenylated (iso)flavonoids by liver enzymes was studied. These compounds can be extensively metabolized by phase I and II enzymes. A glucuronidation yield between 70-80% was observed. It was also shown that pyran and chain prenylation gave more complex hydroxylation patterns with 4 or more than 6 hydroxyl isomers, respectively, compared to unprenylated compounds (only 1 hydroxyl isomer).

    Induction of prenylated isoflavonoids and stilbenoids in legumes
    Aisyah, S. - \ 2015
    Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Jean-Paul Vincken. - Wageningen : Wageningen University - ISBN 9789462574816 - 154
    flavonoïden - stilbenoïden - isoflavonoïden - peulgewassen - glycine max - sojabonen - arachis hypogaea - fylogenetica - phaseolus - lupinus - rhizopus - aspergillus - kwantitatieve analyse - flavonoids - stilbenoids - isoflavonoids - legumes - glycine max - soyabeans - arachis hypogaea - phylogenetics - phaseolus - lupinus - rhizopus - aspergillus - quantitative analysis

    The germination of legume seeds in the presence or absence of stress factors was studied with respect to compositional changes in prenylated isoflavonoids and stilbenoids. Different strategies were applied using (i) different types of legume seed, (ii) different stress factors i.e. biotic, abiotic and their combination, and (iii) different time point of application of the fungus. Mass spectrometric tools to better characterize the position of prenyl groups in the molecules were optimized. Isoflavonoids and stilbenoids appeared more inducible than flavonoids. Fungus was a more effective stress factor than light and wounding. The impact of fungus might be enhanced by combining it with other stress factors; the combination of fungus and light was more promising than that of fungus and wounding. The seeds of various legume species appeared to respond differently towards elicitation by Rhizopus during germination. The kind of molecules induced followed the phylogenetic relationship of the various species, but their amounts induced during germination, alone or combined with elicitation, did not. In terms of quantities of compounds induced, some species such as Glycine max, Phaseolus spp., Lupinus spp. and Arachis hypogaea were more promising than Vigna spp., Lablab purpureus and Psophocarpus tetragonolobus. Moreover, the fact that Rhizopus and Aspergillus could metabolize the stilbenoids induced during the process of simultaneous germination and elicitation of peanut seedlings showed that the type of fungus was a crucial parameter for optimizing accumulation of potentially bioactive compounds.

    Quercetin tests negative for genotoxicity in transcriptome analyses of liver and small intestine of mice
    Hil, E.F. van den; Schothorst, E.M. van; Stelt, I. van der; Keijer, J. ; Rietjens, I. - \ 2015
    Food and Chemical Toxicology 81 (2015). - ISSN 0278-6915 - p. 34 - 39.
    in-vivo - reverse mutation - bone-marrow - dna-damage - flavonoids - rats - cells - carcinogenicity - mutagenicity - polyphenols
    Given the positive results of quercetin in in vitro genotoxicity studies, the in vivo genotoxic properties of this important dietary flavonoid warrant testing, especially considering possible high intake via widely available food supplements. Here, this was done by transcriptome analyses of the most relevant tissues, liver and small intestine, of quercetin supplemented mice. Quercetin (0.33%) supplemented to a high-fat diet was administered to mice during 12 weeks. Serum alanine aminotransferase and aspartate aminotransferase levels revealed no indications for hepatotoxicity. Microarray pathway analysis of liver and small intestine showed no regulation of genotoxicity related pathways. Analysis of DNA damage related genes also did not point at genotoxicity. Furthermore, a published classifier set of transcripts for identifying genotoxic compounds did not indicate genotoxicity. Only two transcripts of the classifier set were regulated, but in the opposite direction compared with the genotoxic compounds 2-acetylaminofluorene (2-AAF) and aflatoxin B1 (AFB1). Based on the weight of evidence of three different types of analysis, we conclude that supplementation with quercetin at ~350¿mg/kg bw/day for 12 weeks in mice showed no up-regulation of genotoxicity related pathways in liver and small intestine.
    Unravelling mechanisms of dietary flavonoid-mediated health effects: effects on lipid metabolism and genotoxicity
    Hoek-van den Hil, E.F. - \ 2015
    Wageningen University. Promotor(en): Ivonne Rietjens; Jaap Keijer, co-promotor(en): Peter Hollman. - Wageningen : Wageningen University - ISBN 9789462573031 - 157
    flavanoïden - flavonoïden - vetzuren - quercetine - flavonolen - lichaamsgewicht - lipidenmetabolisme - hart- en vaatziekten - lever - vetweefsel - gezondheid - genotoxiciteit - voeding - muizen - flavanoids - flavonoids - fatty acids - quercetin - flavonols - body weight - lipid metabolism - cardiovascular diseases - liver - adipose tissue - health - genotoxicity - nutrition - mice

    Summary

    Consumption of foods containing flavonoids is associated with a reduced risk of cardiovascular diseases (CVD), possibly by lipid-lowering effects. On the other hand, for one of these flavonoids, quercetin, also genotoxicity was shown especially in in vitro bioassays. Therefore, the first aim of this thesis was to identify mechanisms underlying potential beneficial health effects of flavonoids. The focus was on hepatic lipid metabolism and circulating lipids and a molecular and physiological approach was used. Secondly, we aimed to study the potential in vivo genotoxic effects of quercetin by transcriptome analyses in liver and small intestine, since these represent the tissues of first contact exposed to relatively high levels upon oral intake of flavonoids.

    Circulating lipids are important CVD-related risk markers, which are in general determined with commercially available enzyme-based assays. However, the usual enzyme in these assays, peroxidase, has previously been reported to be inhibited by flavonoids. Therefore, we have studied in chapter 2 whether these assays can adequately be used in flavonoid research. We observed that various flavonoid aglycones interfere with peroxidase used in triglycerides (TG) and free fatty acids (FFA) enzymatic assays, reporting incorrect lower TG and FFA levels than actually present. Furthermore, addition of metabolites such as isorhamnetin or quercetin-3-O-glucuronide, the major metabolite of quercetin in human and rat plasma, to murine serum also resulted in a significant reduction of the detected TG levels, while a trend was seen towards reduced FFA levels. It can be concluded that when applying these biochemical assays, vigilance is needed and alternative analytical methods assessing FFA or TG levels should preferably be applied for studying the biological effects of flavonoids on TG and FFA levels.

    In chapter 3 mechanistic and physiological effects of quercetin on hepatic lipid metabolism were studied. C57BL/6JOlaHsd male adult mice received a mild high-fat (30 en%) diet without or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Gas chromatography and 1H-NMR were used to quantitatively measure serum lipid profiles. Whole genome microarray analysis of liver tissue was used to identify potential mechanisms underlying altered circulating lipid levels by quercetin supplementation. Body weight, energy intake and hepatic lipid accumulation did not differ significantly between the quercetin and the control group. In serum of quercetin-fed mice, TG levels were decreased by 14% (p<0.001) and total poly unsaturated fatty acids (PUFA) levels were increased by 13% (p<0.01). Levels of palmitic acid, oleic acid, and linoleic acid were all decreased by 9-15% (p<0.05) in quercetin-fed mice. Both palmitic acid and oleic acid can be oxidized by omega-oxidation. Gene expression profiling showed indeed that quercetin increased hepatic lipid metabolism, especially omega-oxidation. At the gene level, this was reflected by the up-regulation of cytochrome P450 (Cyp) 4a10, Cyp4a14, Cyp4a31 and Acyl-CoA thioesterase 3 (Acot3). Two relevant regulators, cytochrome P450 oxidoreductase (Por, rate limiting for cytochrome P450 activities) and the transcription factor constitutive androstane receptor (Car; official symbol Nr1i3) were also up- regulated in the quercetin-fed mice. We concluded that quercetin intake increased hepatic lipid omega-oxidation and lowered corresponding circulating lipid levels, which may contribute to potential beneficial effects of quercetin on CVD.

    Subsequently, in chapter 4 effects of quercetin supplementation were studied in mice given a high-fat (40 en%) background diet. The set-up of the experiment was the same as in chapter 3, with the exception of the background diet that was used, which was different in fat content and composition. This high-fat diet-induced body weight gain, and serum and hepatic lipid accumulation, which are all known risk factors for CVD. The aim of this study was to investigate the effects and underlying molecular mechanisms of the effects of the flavonoid quercetin on hepatic lipid metabolism in mice given this high-fat diet background. C57BL/6JOlaHsd male adult mice received the high-fat diet without or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Body weight gain was 29% lower in quercetin fed mice versus control mice (p<0.01), while the energy intake was not significantly different. Quercetin supplementation lowered high-fat diet-induced hepatic lipid accumulation to 29% of the amount present in the control mice (p<0.01). 1H-NMR serum lipid profiling revealed that the supplementation also significantly lowered high-fat diet-induced increases in serum lipid levels. Global gene expression profiling of liver showed that cytochrome P450 2b (Cyp2b) genes, key target genes of the transcription factor Car, were down-regulated. However, the induction of omega-oxidation observed by quercetin supplementation to a mild high-fat (30en%) diet (chapter 3), was not observed this time with the high-fat (40en%) diet. Cumulatively, quercetin decreased high-fat diet-induced body weight gain, hepatic lipid accumulation and serum lipid levels. This was accompanied by regulation of cytochrome P450 2b genes in liver, which are considered to be under transcriptional control of CAR. The quercetin effects are likely dependent on the fat content and composition of the diet.

    In chapter 5 we investigated whether flavonoids from other flavonoid subclasses can exert the same effects as we observed for quercetin. Effects of quercetin, hesperetin, epicatechin, apigenin and anthocyanins, in C57BL/6JOlaHsd male adult mice fed a high-fat diet for 12 weeks were compared, relative to a normal-fat diet. High-fat diet-induced body weight gain was significantly lowered by all flavonoids (17-29%), but most by quercetin. Quercetin significantly lowered high-fat diet-induced hepatic lipid accumulation (by 71%). High-fat diet-induced increases of mesenteric adipose tissue weight and serum leptin levels were significantly lowered by quercetin, hesperetin, and anthocyanins. Adipocyte cell size and adipose tissue inflammation were not affected.

    The effects on body weight and adiposity could not be explained by individual significant differences in energy intake, energy expenditure, nor by differences in activity. Lipid metabolism was not changed as measured by indirect calorimetry or expression of known lipid metabolic genes in liver and white adipose tissue. Hepatic expression of Cyp2b9 was strongly down-regulated by all flavonoids. Overall, all five flavonoids lowered parameters of high-fat diet-induced adiposity, with quercetin being most effective.

    Next to the beneficial health effects of flavonoids, the safety of flavonoids is under discussion, mainly because of potential genotoxic effects found for quercetin in vitro. Therefore, in chapter 6 the in vivo genotoxicity of this flavonoid was studied by transcriptome analyses in two tissues, small intestine and liver, where the highest exposure to quercetin is expected. This is especially of interest in view of high intake by widely available food supplements. Quercetin (0.33%) supplemented to a high-fat diet was administered to C57BL/6JOlaHsd male adult mice during 12 weeks. Serum alanine aminotransferase and aspartate aminotransferase levels revealed no indications for hepatotoxicity. General microarray pathway analysis of liver and small intestinal tissue samples showed no regulation of genotoxicity related pathways. In addition, analysis of DNA damage pathways in these tissues did also not point at genotoxicity. Furthermore, comparison with a published classifier set of transcripts for identifying genotoxic compounds did not reveal any similarities in the regulation of these classifier set by quercetin. Available microarray datasets of known genotoxic liver carcinogens, 2-acetylaminofluorene and aflatoxin B1 in mice were taken along as positive controls for comparison, and indeed showed genotoxic properties (regulation of genotoxic related genes) in the analyses. This transcriptomic analysis showed that supplementation with quercetin at ~350 mg/kg bw/day for 12 weeks did not induce genotoxicity in liver and small intestine.

    In conclusion, we have shown in vivo efficacy of flavonoids reflected by effects on metabolic health parameters, including hepatic lipid metabolism. These effects on hepatic lipid metabolism seemed to be related or influenced by the transcription factor CAR. The dietary contexts appeared to modify the health effects. The five studied flavonoids in general showed the same effects, with quercetin being the most effective. No genotoxicity of quercetin was found by transcriptome analyses in liver and small intestine. Overall, we have obtained indications for beneficial health effects of flavonoids in mice, which makes it interesting to study if these effects can be extrapolated to humans to further explore their potential as functional compounds of dietary flavonoid intake.

    Discrimination of Polish unifloral honeys using overall PTR-MS and HPLC fingerprints combined with chemometrics
    Kus, P.M. ; Ruth, S.M. van - \ 2015
    Food Science and Technology = Lebensmittel-Wissenschaft und Technologie 62 (2015)1. - ISSN 0023-6438 - p. 69 - 75.
    reaction-mass spectrometry - origin determination - botanical origin - electronic nose - floral markers - l. honey - volatile - classification - identification - flavonoids
    A total of 62 honey samples of six floral origins (rapeseed, lime, heather, cornflower, buckwheat and black locust) were analysed by means of proton transfer reaction mass spectrometry (PTR-MS) and HPLC-DAD. The data were evaluated by principal component analysis and k-nearest neighbours classification in order to examine consistent differences in analytical fingerprints between various honeys allowing their discrimination. The study revealed, that both techniques were able to distinguish the floral origins, however the HPLC shows advantage over PTR-MS providing substantially better differentiation of all analysed honey types. Especially HPLC fingerprints recorded at 210 nm were most suitable for discrimination of botanical origin with the use of chemometric analysis. The obtained classification rates were: 100%, 93%, 100%, 83%, 100%, 100% (HPLC) and 69%, 67%, 78%, 67%, 100%, 88% (PTR-MS) for rapeseed, lime, heather, cornflower, buckwheat and black locust, respectively. Even if performance of PTR-MS in general was lower than HPLC, it might be useful for fast on-line screening of buckwheat honey.
    Combining an in vitro reporter gene assay with metabolomics to identify tomato phytochemicals responsible for inducing electrophile-responsive element (EpRE)-mediated gene transcription
    Eekelen, H.D.L.M. van; Gijsbers, L. ; Maliepaard, C.A. ; Vreeburg, R.A.M. ; Finkers, H.J. ; Tikunov, Y.M. ; Gomez Roldan, M.V. ; Haan, L.H.J. de; Vos, R.C.H. de; Aarts, J.M.M.J.G. ; Rietjens, I. ; Bovy, A.G. - \ 2015
    Metabolomics 11 (2015)2. - ISSN 1573-3882 - p. 302 - 311.
    solanum-lycopersicon - mass-spectrometry - fruit - expression - health - metabolism - flavonoids - lycopene - deglycosylation - polyphenols
    The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression effects of certain dietary compounds. In this study we measured the ability of extracts of glycosidase treated tomato fruits from 97 different accessions to induce EpRE-mediated luciferase expression using EpRE-LUX reporter cells and analyzed the same extracts using LC–MSbased untargeted metabolomics profiling. We were able to pinpoint those tomato compounds that were most correlated with EpRE-mediated luciferase induction, by combining reporter gene assay data with the metabolic profiles of the same extracts. Flavonoids were the compounds showing the strongest positive correlation with EpRE-LUX activity. These results were validated using a transgenic tomato line accumulating high levels of flavonoids. Results obtained corroborated that flavonoids are an important determinant of the ability of tomato fruit extracts to induce EpRE-mediated beneficial health effects. Overall, these results indicate that combining untargeted metabolomics with reporter gene assays provides a powerful tool for nutritionists, plant breeders and food chemists towards identification of potential health-beneficial constituents of tomato fruits, as well as of other crops and products derived thereof.
    Comparison of the chemical composition of three species of smartweed (genus Persicaria) with a focus on drimane sesquiterpenoids
    Prota, N. ; Mumm, R. ; Bouwmeester, H.J. ; Jongsma, M.A. - \ 2014
    Phytochemistry 108 (2014). - ISSN 0031-9422 - p. 129 - 136.
    polygonum genus - antifeedant - flavonoids - humans
    The genus Persicaria is known to include species accumulating drimane sesquiterpenoids, but a comparative analysis highlighting the compositional differences has not been done. In this study, the secondary metabolites of both flowers and leaves of Persicaria hydropiper, Persicaria maculosa and Persicaria minor, three species which occur in the same habitat, were compared. Using gas chromatography–mass spectrometry (GC–MS) analysis of extracts, overall 21/29 identified compounds in extracts were sesquiterpenoids and 5/29 were drimanes. Polygodial was detected in all species, though not in every sample of P. maculosa. On average, P. hydropiper flowers contained about 6.2 mg g FW 1 of polygodial, but P. minor flowers had 200-fold, and P. maculosa 100,000 fold lower concentrations. Comparatively, also other sesquiterpenes were much lower in those species, suggesting the fitness benefit to depend on either investing a lot or not at all in terpenoid-based secondary defences. For P. hydropiper, effects of flower and leaf development and headspace volatiles were analysed as well. The flower stage immediately after fertilisation was the one with the highest content of drimane sesquiterpenoids and leaves contained about 10- fold less of these compounds compared to flowers. The headspace of P. hydropiper contained 8 compounds: one monoterpene, one alkyl aldehyde and six sesquiterpenes, but none were drimanes. The potential ecological significance of the presence or absence of drimane sesquiterpenoids and other metabolites for these plant species are discussed.
    Structural Changes of 6a-Hydroxy-Pterocarpans Upon Heating Modulate Their Estrogenicity
    Schans, M.G.M. van de; Vincken, J.P. ; Bovee, T.F.H. ; Cervantes, A.D. ; Logtenberg, M.J. ; Gruppen, H. - \ 2014
    Journal of Agricultural and Food Chemistry 62 (2014). - ISSN 0021-8561 - p. 10475 - 10484.
    ionization mass-spectrometry - isoflavonoid composition - phytoestrogens - flavonoids - aglycones - genistein - seedlings - daidzein - soy
    The isoflavonoid composition of an ethanolic extract of fungus-treated soybean sprouts was strongly altered by a combined acid/heat treatment. UHPLC-MS analysis showed that 6a-hydroxy-pterocarpans were completely converted to their respective, more stable, 6a,11a-pterocarpenes, whereas other isoflavonoids, from the isoflavone and coumestan subclasses, were affected to a much lesser extent (loss of ~15%). Subsequently, mixtures enriched in prenylated 6a-hydroxy-pterocarpans (pools of glyceollin I/II/III and glyceollin IV/VI) or prenylated 6a,11a-pterocarpenes (pools of dehydroglyceollin I/II/III and dehydroglyceollin IV/VI) were purified, and tested for activity on both human estrogen receptors (ERa and ERß). In particular, the response toward ERa changed, from agonistic for glyceollins to antagonistic for dehydroglyceollins. Toward ERß a decrease in agonistic activity was observed. These results indicate that the introduction of a double bond with the concomitant loss of a hydroxyl group in 6a-hydroxy-pterocarpans extensively modulates their estrogenic activity.
    Quercetin decreases high-fat diet induced body weight gain and accumulation of hepatic and circulating lipids in mice
    Hoek-van den Hil, E.F. ; Schothorst, E.M. van; Stelt, I. van der; Swarts, J.J.M. ; Venema, D.P. ; Sailer, M. ; Vervoort, J.J.M. ; Hollman, P.C.H. ; Rietjens, I. ; Keijer, J. - \ 2014
    Genes & Nutrition 9 (2014). - ISSN 1555-8932 - 8 p.
    cardiovascular-disease - gene-expression - c57bl/6j mice - acid - risk - hepatocytes - metabolism - flavonoids - obesity - women
    Dietary flavonoids may protect against cardiovascular diseases (CVD). Increased circulating lipid levels and hepatic lipid accumulation are known risk factors for CVD. The aim of this study was to investigate the effects and underlying molecular mechanisms of the flavonoid quercetin on hepatic lipid metabolism in mice with high-fat diet induced body weight gain and hepatic lipid accumulation. Adult male mice received a 40 energy% high-fat diet without or with supplementation of 0.33 % (w/w) quercetin for 12 weeks. Body weight gain was 29 % lower in quercetin fed mice (p <0.01), while the energy intake was not significantly different. Quercetin supplementation lowered hepatic lipid accumulation to 29 % of the amount present in the control mice (p <0.01). 1H nuclear magnetic resonance serum lipid profiling revealed that the supplementation significantly lowered serum lipid levels. Global gene expression profiling of liver showed that cytochrome P450 2b (Cyp2b) genes, key target genes of the transcription factor constitutive androstane receptor (Car; official symbol Nr1i3), were downregulated. Quercetin decreased high-fat diet induced body weight gain, hepatic lipid accumulation and serum lipid levels. This was accompanied by regulation of cytochrome P450 2b genes in liver, which are possibly under transcriptional control of CAR. The quercetin effects are likely dependent on the fat content of the diet.
    Direct comparison of health effects by dietary polyphenols at equimolar doses in wildtype moderate high-fat fed C57BL/6JOlaHsd mice
    Schothorst, E.M. van; Bunschoten, A. ; Hoevenaars, F.P.M. ; Stelt, I. van der; Janovska, P. ; Venema, D.P. ; Kopecky, J. ; Hollman, P.C.H. ; Keijer, J. - \ 2014
    Food Research International 65 (2014)Part A. - ISSN 0963-9969 - p. 95 - 102.
    adipose-tissue - body-weight - induced obesity - disease risk - quercetin - bioavailability - expression - flavonoids - leptin - (-)-epigallocatechin-3-gallate
    Polyphenols generally show beneficial health effects upon supplementation in diet-induced obese rodent models, including reduced body weight gain and reduced levels of markers for cardiovascular diseases (CVD). However, there appear to be large differences between studies, which might be due to differences in models, strains, dietary background, or even concentration of polyphenol that is used. Therefore, we performed a systematic phenotypic evaluation of the effects of selected polyphenols in wildtype C57BL/6JOlaHsd mice. Epigallocatechin-gallate, quercetin, and resveratrol, representing three different phenolic classes, were each added in equimolar amounts (0.50% (w/w), 0.33%, and 0.25%, respectively) to a purified moderate high fat (30energy%) diet for 12 weeks. We studied the polyphenol-induced physiological and molecular effects between them and relative to the nonsupplemented control group during and at the end of the nutritional intervention. Results showed that these polyphenols were present in circulation, but did not induce beneficial health effects as analysed by oral glucose tolerance testing or serum adipokines and CVD-markers such as vascular adhesion molecules. Remarkably, transcriptomics of white adipose tissue showed overlapping sets of significantly differential transcript levels between these polyphenols; AMPK and Notch signalling were affected by these polyphenols. However, mitochondrial processes and mitochondrial density in this tissue did not differ between the polyphenols, which suggested that there was no direct effect on adipose tissue.
    Metabolomics as a Potential Chemotaxonomical Tool: Application in the Genus Vernonia Schreb
    Martucci, M.E.P. ; Vos, R.C.H. de; Carollo, C.A. - \ 2014
    PLoS ONE 9 (2014)4. - ISSN 1932-6203 - 8 p.
    ericoides mart. asteraceae - mass-spectrometry - sesquiterpene lactones - chlorogenic acids - flavonoids - identification - compositae - amygdalina - saponins - leaves
    The taxonomic classification of the genus Vernonia Schreb is complex and, as yet, unclear. We here report the use of untargeted metabolomics approaches, followed by multivariate analyses methods and a phytochemical characterization of ten Vernonia species. Metabolic fingerprints were obtained by accurate mass measurements and used to determine the phytochemical similarities and differences between species through multivariate analyses approaches. Principal component analysis based on the relative levels of 528 metabolites, indicated that the ten species could be clustered into four groups. Thereby, V. polyanthes was the only species with presence of flavones chrysoeriol-7-O-glycuronyl, acacetin-7-O-glycuronyl and sesquiterpenes lactones piptocarphin A and piptocarphin B, while glaucolide A was detected in both V. brasiliana and V. polyanthes, separating these species from the two other species of the Vernonanthura group. Species from the Lessingianthus group were unique in showing a positive response in the foam test, suggesting the presence of saponins, which could be confirmed by metabolite annotation. V. rufogrisea showed a great variety of sesquiterpene lactones, placing this species into a separate group. Species within the Chrysolaena group were unique in accumulating clovamide. Our results of LC-MS-based profiling combined with multivariate analyses suggest that metabolomics approaches, such as untargeted LC-MS, may be potentially used as a large-scale chemotaxonomical tool, in addition to classical morphological and cytotaxonomical approaches, in order to facilitate taxonomical classifications.
    Intrinsic bitterness of flavonoids and isoflavonoids and masking of their taste activity
    Roland, W.S.U. - \ 2014
    Wageningen University. Promotor(en): Harry Gruppen; Gerrit Smit, co-promotor(en): Jean-Paul Vincken. - Wageningen : Wageningen University - ISBN 9789461738530 - 188
    flavonoïden - isoflavonoïden - bitterheid - receptoren - chemische structuur - flavonoids - isoflavonoids - bitterness - receptors - chemical structure

    Many flavonoids and isoflavonoids have been associated with beneficial health effects. Therefore, consumption of (iso)flavonoid-rich food products, and enrichment of foods with (iso)flavonoids is becoming increasingly popular. However, several (iso)flavonoids have been reported as bitter. Consequently, their incorporation in (or fortification of) foods can introduce (or enhance) bitterness. Hence, debittering strategies are demanded.

    Some (iso)flavonoids have unknown taste properties, as they have never been incorporated in high levels in food products. For other (iso)flavonoids, contradictory findings on bitterness have been made in sensory tests. Therefore, objective tests are necessary to identify which (iso)flavonoids contribute to bitterness of a food product. An objective tool to study bitterness is a cell-based bitter taste receptor assay. Twenty-five different bitter taste receptors (hTAS2Rs) occur on the human tongue, each of which has been introduced in a separate human embryonic kidney (HEK)293 cell line. With these, the “intrinsic bitterness” of a compound can be investigatedin vitro. Intrinsic bitterness is the capacity of a compound to activate bitter taste receptors, uncoupled from cross-modal interactions and interactions with salivary proteins and oral mucosa. The aim of this research was to study the intrinsic bitterness of a large set of (iso)flavonoids and to investigate structural requirements for (iso)flavonoids to activate the bitter receptors identified. A subsequent aim was the investigation of different debittering strategies by the use of the bitter receptor assay.

    Chapter 1provides an overview of flavonoids and isoflavonoids with respect to their structural classification, sensorial properties and occurrence as dietary compounds. Taste perception and the mode of action of bitter taste receptors are introduced. The measurement of bitter receptor activation in vitro is explained, as well as strategies to reduce bitter receptor activation, and bitter taste in general. A state-of-the-art overview of all 25 bitter taste receptors is given with respect to known agonists and antagonists.

    The aim of Chapter 2was to identify the bitter receptor(s) that recognize the bitter taste of the soy isoflavone genistein. Screening of all 25 human bitter receptors revealed genistein as agonist of hTAS2R14 and hTAS2R39. Genistein displayed threshold values of 4 and 8 µM on hTAS2R14 and hTAS2R39, and EC50 values of 29 and 49 µM, respectively. Besides, the behavior of structurally similar isoflavonoids was investigated. Although the two receptors are not closely related, the results for hTAS2R14 and hTAS2R39 were similar towards most isoflavonoid aglycones. Glucosylation of isoflavones seemed to inhibit activation of hTAS2R14, whereas four of five glucosylated isoflavones were agonists of hTAS2R39, namely glycitin, genistin, acetyl genistin, and malonyl genistin. A total of three hydroxyl substitutions of the A- and B-rings of the isoflavonoids seemed to be more favorable for receptor activation than less hydroxyl groups. The concentration of the trihydroxylated genistein in several soy foods exceeds the bitter receptor threshold values determined, whereas those of other soy isoflavones are around or below their respective threshold values. Despite its low concentration, genistein might be one of the main contributors to the bitterness of soy products. Furthermore, the bioactive isoflavonoids equol and coumestrol activated both receptors, indicating that their sensory impact should be considered when used as food ingredients.

    In Chapter 3, the intrinstic bitterness of (iso)flavonoids, which can hamper their use as food bioactives, was investigated further.The effect of a large set of structurally similar (iso)flavonoids on the activation of bitter receptors hTAS2R14 and hTAS2R39 was tested, and their structural requirements to activate these receptors were predicted. In total, 68 compounds activated hTAS2R14 and 70 compounds activated hTAS2R39, amongst which 58 ligands were overlapping. Their activation threshold values varied over a range of three log units between 0.12 and 500 μM. Ligand-based 2D-fingerprint and 3D-pharmacophore models were created to detect structure activity relationships. The 2D-models demonstrated excellent predictive power in identifying bitter (iso)flavonoids and discrimination from inactive ones. The structural characteristics for an (iso)flavonoid to activate hTAS2R14 and hTAS2R39 were determined by 3D-pharmacophore models to be composed of two (for hTAS2R14) or three (for hTAS2R39) hydrogen bond donor sites, one hydrogen bond acceptor site, and two aromatic ring structures, of which one had to be hydrophobic. An additional hydrogen bond donor feature for hTAS2R39 ligands indicated the possible presence of another complementary acceptor site in the binding pocket, compared to hTAS2R14. Hydrophobic interaction of the aromatic feature with the binding site might be of higher importance in hTAS2R14 than in hTAS2R39. Together, this might explain why OH-rich compounds showed different behavior towards the two bitter receptors. The combination of in vitro data and different in silico methods created a good insight in activation of hTAS2R14 and hTAS2R39 by (iso)flavonoids and provided a powerful tool in prediction of their potential bitterness. By understanding the “bitter motif”, introduction of bitter taste in functional foods enriched in (iso)flavonoid bioactives might be avoided.

    Bitter receptor hTAS2R39 is activated by many different classes of bitter compounds, amongst which (iso)flavonoids. Nevertheless, several flavanones are known to mask bitter taste sensorially, andnot all flavanones reported in Chapter 3 activated hTAS2R39. For that reason, in Chapter 4, fourteen flavanones were investigated for their potential to reduce activation of hTAS2R39 by epicatechin gallate (ECG), one of the main bitter compounds present in green tea.Three compounds showed inhibitory behavior towards the activation of hTAS2R39 by ECG: 4’-fluoro-6-methoxyflavanone, 6,3’-dimethoxyflavanone, and 6-methoxyflavanone (in order of decreasing potency). The 6-methoxyflavanones also inhibited activation of hTAS2R14 (another bitter receptor activated by ECG), though to a lesser extent. Dose-response curves of ECG at various concentrations of the most potent antagonist 4’-fluoro-6-methoxyflavanone and wash-out experiments indicated reversible insurmountable antagonism. The same effect was observed for the structurally different agonist denatonium benzoate, suggesting a non-competitive orthosteric mechanism. The bitter receptor blockers identified might not be applicable to food products. Nevertheless, they create insight into structural requirements, which might lead to other, more suitable, blockers.

    Chapter 5investigates another strategy to reduce bitterness, namely complexation of bitter flavonoids with food proteins. The binding characteristics of the bitter tea compound epigallocatechin gallate (EGCG) to purified food proteins, and their equivalent food-grade preparations, were related to their effects on reducing bitter receptor activation by EGCG in vitro and their bitter-masking potential in vivo. β-Casein, in particular, and several gelatins, are known as strong binders of EGCG, contrary to β-lactoglobulin. Also in the bitter receptor assay, β-casein showed the strongest effect, with a maximum reduction of hTAS2R39 activation of about 93%. A similar potency was observed for Na-caseinate, which was applied as food-grade alternative for β-casein. β-Lactoglobulin had little effect on bitter receptor activation, as expected based on its low binding affinity for EGCG. The bitter-masking potential of Na-caseinate was confirmed in vivo using a trained sensory panel. β-Lactoglobulin also slightly reduced EGCG bitter perception, which could not be directly related to its binding capacity. The bitter receptor assay appeared to be a valid tool to evaluate in vitro the efficacy of food proteins as complexing agents for bitter-masking.

    Chapter 6discusses the findings presented in this thesis, addresses prospects and limitations of the bitter receptor cell assay, presents additional results on testing (iso)flavonoids for possible antagonistic properties, and compares taste evaluation by sensory tests, receptor assays and modeling. Furthermore, it evaluates strategies for bitter taste reduction, and applies the findings to soy products and tea.

    The systematic investigation of (iso)flavonoid aglycones showed that the substitution pattern of (iso)flavonoids is of higher importance for bitter receptor activation than the backbone structure. In case of bitter receptor antagonists, the substitution pattern as well as backbone structure revealed to be crucial for functionality. The bitter receptor assay was shown to be an appropriate tool not only for identification of bitter receptor agonists and antagonists, but also for identification of reduced receptor activation by complexing agents. Based on the findings of this thesis, it was concluded that complexation with food proteins is the most promising strategy to reduce bitter taste of flavonoids in tea. On the other hand, for soybean isoflavones, debittering by use of bitter receptor blockers seemed to be a promising debittering strategy. Alternatively to the use of receptor blockers, processing conditions (leading to low isoflavone aglycone formation) or raw material choice (i.e. cultivars low in genistein forms) were recommended. In conclusion, the choice of debittering strategies depends on the molecular structure of the bitter food compounds, as exemplified for soybean products and tea. Therefore, each food product seems to require its own tailor-made debittering solution.

    Matrix modulation of the toxicity of alkenylbenzenes, studied by an integrated approach using in vitro, in vivo, and physiologically based biokinetic models
    Al-Husainy, W.A.A.M. - \ 2013
    Wageningen University. Promotor(en): Ivonne Rietjens; Peter van Bladeren, co-promotor(en): Ans Punt. - Wageningen : Wageningen UR - ISBN 9789461738066 - 199
    methyleugenol - toxiciteit - keukenkruiden - flavonoïden - methyl eugenol - toxicity - culinary herbs - flavonoids

    Alkenylbenzenes such as estragole and methyleugenol are common components of spices and herbs such as tarragon, basil, fennel, mace, allspice, star anise and anise and their essential oils (Smithet al., 2002). There is an interest in the safety evaluation of alkenylbenzenes because these compounds can induce hepatic tumours in rodents when dosed orally at high dose levels (Milleret al., 1983; NTP, 2000). Based on the rodent studies with estragole, methyleugenoland structurally related alkenylbenzenes like safrole the hepatocarcinogenicity of alkenylbenzenes is ascribed to their bioactivation by cytochrome P450 enzymes leading to the formation of the proximate carcinogenen, the 1′-hydroxy metabolite, which is further bioactivated to the ultimate carcinogenen, the 1′-sulfooxy metabolite (Milleret al., 1983; Phillipset al., 1984; Randerathet al., 1984; Smithet al., 2010). The 1′-sulfooxy metabolite is unstable and binds via a presumed reactive carbocation intermediate covalently to different endogenous nucleophiles including DNA (Phillipset al., 1981; Boberget al., 1983; Milleret al., 1983; Phillipset al., 1984; Randerathet al., 1984; Fennellet al., 1985; Wisemanet al., 1987; Smithet al., 2002).

    Because of their genotoxicity and carcinogenicity, the addition of estragole and methyleugenolas pure substances to foodstuffs has been prohibited within the European Union since September 2008 (European Commission, 2008). In 2008, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) re-evaluated the safety of alkenylbenzenes and indicated that although evidence of carcinogenicity to rodents given high doses of alkenylbenzenes exists, further research is needed to assess the potential risk to human health at relevant dietary exposure levels (JECFA, 2008).

    A significant difficulty in evaluating the toxicological data for alkenylbenzenes is that human exposure to these substances results from exposure to a complex mixture of food, spice, and spice oil constituents which may influence the biochemical fate and toxicological risk of the alkenylbenzenes. In this regard, it was shown that a methanolic extract of basil inhibited the formation of estragole DNA adducts in human HepG2 cells exposed to the proximate carcinogen 1′-hydroxyestragole (Jeurissenet al., 2008). This inhibition occurred at the level of sulfotransferase (SULT)-mediated bioactivation of 1′-hydroxyestragole into 1′-sulfooxyestragole (Jeurissenet al., 2008).

    The objective of this PhD research was to study the inhibitory action of components in alkenylbenzene-containing herbs and spices on SULT-mediated alkenylbenzene DNA adduct formation and the consequences of this combination effect for risk assessment using estragole and methyleugenol as the model alkenylbenzenes. To achieve this objective, an integrated approach of in vitro, in vivo and physiologically based biokinetic (PBBK) models was applied to investigate how the SULT inhibition influences the bioactivation and thus potentially also the toxicity and risk assessment of estragole and methyleugenol.

    Chapter 1of the thesis presents an introduction to the bioactivation, detoxification, genotoxicity and carcinogenicity of the alkenylbenzenes estragole and methyleugenol as well as a short introduction to PBBK modeling and the state-of-the-art knowledge on risk assessment strategies and regulatory status for alkenylbenzenes.

    Chapter 2of the thesis identifies nevadensin as a basil constituent able to inhibit SULT-mediated DNA adduct formation in rat hepatocytes exposed to the proximate carcinogen 1′-hydroxyestragole and nevadensin. The type of inhibition by nevadensin was shown to be non-competitive with an inhibition constant (Ki) of 4 nM. Furthermore, nevadensin up to 20 μM did not inhibit 1′-hydroxyestragole detoxification by glucuronidation and oxidation. The inhibition of SULT by nevadensin was incorporated into the PBBK models describing bioactivation and detoxification of estragole in male rat and human. The models thus obtained predict that co-administration of estragole at a level inducing hepatic tumours in vivo (50 mg/kg bw) with nevadensin at a molar ratio to estragole representing the molar ratio of their occurrence in basil, results in more than 83% inhibition of the formation of the carcinogenic metabolite, 1ʹ-sulfooxyestragole, inthe liver of male rat and human even at 1% uptake of nevadensin.

    To extend the work to other alkenylbenzene-containing herbs and spices than basil chapter 3 presents data showing that methanolic extracts from different alkenylbenzene-containing herbs and spices such as nutmeg, mace, anise and others are able to inhibit the SULT enzyme activity. Flavonoids including nevadensin, quercetin, kaempferol, myricetin, luteolin and apigenin were the major constituents responsible for this inhibition of SULT activity with Kivalues in the nano to sub-micromolar range. Also, the various flavonoids individually or in mixtures were able to inhibit estragole DNA adduct formation in human HepG2 cells exposed to the proximate carcinogen 1ʹ-hydroxyestragole, and to shift metabolism in favour of detoxification (e.g. glucuronidation) at the cost of bioactivation (e.g. sulfonation).

    In a next step, the kinetics for SULT inhibition were incorporated in PBBK models for estragole in rat and human to predict the effect of co-exposure to estragole and (mixtures of) the different flavonoids on the bioactivation in vivo. The PBBK-model-based predictions indicate that the reduction of estragole bioactivation in rat and human by co-administration of the flavonoids is dependent on whether the intracellular liver concentrations of the flavonoids can reach their Ki values. Finally, we concluded that it is expected that this is most easily achieved for nevadensin which has a Kivalue in the nanomolar range and is, due to its methylation, more metabolically stable and bioavailable than the other flavonoids.

    Chapter 4of the thesis investigates whether the previous observation that nevadensin is able to inhibit SULT-mediated estragole DNA adduct formation in primary rat hepatocytes could be validated in vivo. Moreover, the previously developed PBBK models to study this inhibition in rat and in human liver was refined by including a sub-model describing nevadensin kinetics. Nevadensin resulted in a significant reduction in the levels of estragole DNA adducts formed in the liver of Sprague–Dawley rats orally dosed with estragole and nevadensin simultaneously at a ratio reflecting their presence in basil. Moreover, the refined PBBK model predicted the formation of estragole DNA adducts in the liver of rat with less than 2-fold difference compared to in vivo data and suggests more potent inhibition in the liver of human compared to rat due to less efficient metabolism of nevadensin in human liver and intestine.

    Also, an updated risk assessment for estragole was presented taking into account the matrix effect and this revealed that the BMDL10 and the resulting MOE for estragole increase substantially when they would be derived from rodent bioassays in which the animals would be exposed to estragole in the presence of nevadensin instead of to pure estragole.

    To extend the work to other alkenylbenzenes than estragole chapter 5 of the thesis investigates the potential of nevadensin to inhibit the SULT-mediated bioactivation and subsequent DNA adduct formation of methyleugenolusing human HepG2 cells as an in vitro model. Nevadensin was able to inhibit SULT-mediated DNA adduct formation in HepG2 cells exposed to the proximate carcinogen 1′-hydroxymethyleugenol in the presence of nevadensin.To investigate possible in vivo implications for SULT inhibition by nevadensin on methyleugenolbioactivation, the rat PBBK model developed in our previous work to describe the dose-dependent bioactivation and detoxification of methyleugenolin male rat was combined with the recently developed PBBK model describing the dose-dependent kinetics of nevadensin in male rat. Similar to what was presented for estragole in chapter 4, chapter 5 presents an updated risk assessment for methyleugenoltaking the matrix effect into account. This revealed that the BMDL10 and the resulting MOE for methyleugenolincrease substantially when they would be derived from rodent bioassays in which the animals would be exposed to methyleugenolin the presence of nevadensin instead of to pure methyleugenol.

    In a next step, we aimed at moving one step forward towards endpoints that are closer to initiation of carcinogenesis than DNA adduct formation, namely, formation of hepatocellular altered foci (HAF). Chapter 6 presents data showing that the potent in vivo inhibitory activity of nevadensin on SULT enzyme activity and on alkenylbenzene DNA adduct formation is accompanied by a potent in vivo reduction in early markers of carcinogenesis such as HAF. This also suggests that a reduction in the incidence of hepatocarcinogenicity is expected in liver of rodents when alkenylbenzenes would be dosed simultaneously with nevadensin.

    Chapter 7presents a discussion on the in vitro and in vivo activity of dietary SULT inhibitors and their potential in reducing the cancer risk associated with alkenylbenzene consumption. This chapter also presents some future perspectives based on the major issues raised by our research.

    Altogether, the results of the present thesis indicate that the likelihood of bioactivation and subsequent adverse effects may be lower when alkenylbenzenes are consumed in a matrix containing SULT inhibitors such as nevadensincompared to experiments using pure alkenylbenzenes as single compounds. Also,the consequences of the in vivo matrix effect were shown to be significant when estragole or methyleugenolwas tested in rodent bioassays in the presence of nevadensin at ratios detected in basil, thereby likely increasing BMDL10 and resulting MOE values substantially in a subsequent risk assessment. However, the results also indicate that matrix effects may be lower at daily human dietary exposure levels of estragole or methyleugenoland nevadensin resulting from basil consumption. Also, matrix effects seem to be limited in the presence of other SULT inhibiting dietary flavonoids even at high exposure levels of these flavonoids coming from supplements. This indicates that the importance of a matrix effect for risk assessment of individual compounds requires analysis of dose dependent effects on the interactions detected, an objective that can be achieved by using PBBK modeling.

    Overall, the present study provides an example of an approach that can be used to characterise dose- species- and inter-individual differences as well as matrix effects in the risk assessment of food-borne toxicants present (e.g. alkenylbenzenes). In this approach the most important toxicokinetic interactions are addressed using an integrated strategy of in vitro, in vivo and PBBK modeling approaches.

    Quercetin induces hepatic lipid omega-oxidation and lowers serum lipid levels in mice
    Hoek-van den Hil, E.F. ; Keijer, J. ; Bunschoten, A. ; Vervoort, Jacques ; Stankova, B. ; Bekkenkamp-Grovestein, M. ; Herreman, L. ; Venema, D.P. ; Hollman, P.C.H. ; Tvrzicka, E. ; Rietjens, I. ; Schothorst, E.M. van - \ 2013
    PLoS ONE 8 (2013)1. - ISSN 1932-6203 - 10 p.
    fatty-acid oxidation - cytochrome-p450 reductase - liver-disease - receptor car - metabolism - dietary - rat - flavonoids - risk - hydroxylases
    Elevated circulating lipid levels are known risk factors for cardiovascular diseases (CVD). In order to examine the effects of quercetin on lipid metabolism, mice received a mild-high-fat diet without (control) or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Gas chromatography and 1H nuclear magnetic resonance were used to quantitatively measure serum lipid profiles. Whole genome microarray analysis of liver tissue was used to identify possible mechanisms underlying altered circulating lipid levels. Body weight, energy intake and hepatic lipid accumulation did not differ significantly between the quercetin and the control group. In serum of quercetin-fed mice, triglycerides (TG) were decreased with 14% (p
    Matrix Modulation of the Bioactivation of Estragole by Constituents of Different Alkenylbenzene-containing Herbs and Spices and Physiologically Based Biokinetic Modeling of Possible In Vivo Effects
    Al-Husainy, W.A.A.M. ; Berg, S.J.P.L. van den; Paini, A. ; Campana, A. ; Asselman, M. ; Spenkelink, A. ; Punt, A. ; Scholz, G. ; Schilter, B. ; Adams, T.B. ; Bladeren, P.J. van; Rietjens, I. - \ 2012
    Toxicological sciences 129 (2012)1. - ISSN 1096-6080 - p. 174 - 187.
    p-form phenolsulfotransferase - potent inhibitors - interaction threshold - drug-metabolism - dna-adducts - mouse-liver - flavonoids - rats - 1'-hydroxyestragole - quercetin
    The alkenylbenzene estragole is a constituent of several herbs and spices. It induces hepatomas in rodents at high doses following bioactivation by cytochrome P450s and sulfotransferases (SULTs) giving rise to the ultimate carcinogenic metabolite 1'-sulfooxyestragole which forms DNA adducts. Methanolic extracts from different alkenylbenzene-containing herbs and spices were able to inhibit SULT activity. Flavonoids including quercetin, kaempferol, myricetin, apigenin, and nevadensin were the major constituents responsible for this inhibition with Ki values in the nano to micromolar range. In human HepG2 cells exposed to the proximate carcinogen 1'-hydroxyestragole, the various flavonoids were able to inhibit estragole DNA adduct formation and shift metabolism in favor of glucuronidation which is a detoxification pathway for 1'-hydroxyestragole. In a next step, the kinetics for SULT inhibition were incorporated in physiologically based biokinetic (PBBK) models for estragole in rat and human to predict the effect of co-exposure to estragole and (mixtures of) the different flavonoids on the bioactivation in vivo. The PBBK-model-based predictions indicate that the reduction of estragole bioactivation in rat and human by co-administration of the flavonoids is dependent on whether the intracellular liver concentrations of the flavonoids can reach their Ki values. It is expected that this is most easily achieved for nevadensin which has a Ki value in the nanomolar range and is, due to its methyl ation, more metabolically stable than the other flavonoids.
    Proctection by Flavonal-Rich foods against vascular dysfunction and oxidative damage: 27th Hohenheim consensus conference
    Sies, H. ; Hollman, P.C.H. ; Grune, T. ; Stahl, W. - \ 2012
    Advances in Nutrition 3 (2012). - ISSN 2161-8313 - p. 217 - 221.
    antioxidant capacity assays - arterial stiffness - protein oxidation - dietary-intake - health claims - comet assay - polyphenols - disease - flavonoids - consumption
    Criteria for assessing the purported protection by flavanol-rich foods against vascular dysfunction and oxidative damage to biomolecules was the subject of the 27th Hohenheim Consensus Conference held on July 11, 2011. State-of-the-art evidence was put into perspective, focusing on several questions that were followed by a consensus answer. Among the topics addressed were the major sources of flavanols in the human diet, the bioavailability of flavanols, biomarkers for “health benefit,” and the biological function of flavanols. Consensus was reached on these topics. No conclusion was reached on the design of randomized, controlled trials for substantiation of health claims for flavanol-rich foods as to the necessity of a study arm with an isolated pharmacologically active compound, e.g., (-)-epicatechin.
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