Staff Publications

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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    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.

    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.

    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.

    Dietary A- and B-type procyanidins : characterization and biofunctional potential of an abundant and diverse group of phenolics
    Appeldoorn, M.M. - \ 2009
    Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Jean-Paul Vincken; Peter Hollman. - [S.l. : S.n. - ISBN 9789085853671 - 168
    fenolverbindingen - flavonoïden - biologische beschikbaarheid - darmabsorptie - bioconversie - phenolic compounds - flavonoids - bioavailability - intestinal absorption - bioconversion
    Procyanidins (PCs) are phenolic compounds that belong to the class of flavonoids and are oligomers of monomeric (epi)catechin units. These monomeric units can be linked to each other by a single C4-C8 or C4-C6 linkage, which is referred to as B-type. Besides these single linkages an additional ether bond can be present, C2-O-C7 or C2-O-C5, which is referred to as A-type. PCs are highly abundant in our diet. Well known PC food sources are cocoa, apple, grape seeds, wine and nuts. After the intake of PC-rich sources health beneficial effects have been detected, which are mainly related to the prevention of cardiovascular diseases such as lowering of blood pressure.
    The aims of this thesis were to study the bioavailability, bioconversion and bioactivity of purified PCs. Therefore, we first developed techniques for the efficient purification of both A- and B-type PCs from peanut skins and grape seeds, respectively. Furthermore, tools were set-up to analyze and characterize individual PCs in complex mixtures.
    We showed that A-type PC dimers were absorbed from the small intestine of rats and that they were better absorbed than B-type PC dimers. The PC dimers were not conjugated or methylated upon absorption in contrast to their monomeric units (epi)catechin. Furthermore, the presence of A-type PC tetramers enhanced the absorption of B-type PC dimers.
    The microbial conversion of B-type PC dimers was studied by exposing them to human microbiota. The main microbial metabolites were 2-(3,4-dihydroxyphenylacetic acid and 5-(3,4-dihydroxyphenyl)-γ-valerolactone. Based on these and other metabolites that were detected, a tentative microbial degradation route was proposed for B-type PC dimers, in which the interflavanic bond does not need to be cleaved upon degradation.
    Subsequently, the vasorelaxing potential of purified PCs and their microbial metabolites was analyzed by measuring their effect on the NO production of endothelial cells. Both A- and B-type PCs showed a tendency (insignificant) to increase NO production with increasing degree of polymerization and several of their human microbial metabolites that were tested were inactive. Besides enhancing NO production, several other mechanisms could be targets of PCs and were also discussed.
    This thesis increased our knowledge on the absorption, biotransformation and bioactivity of A- and B-type PCs. A possible interaction between oligomers with a high and low degree of polymerization, influencing absorption processes has been discussed, which suggests that until now the biofunctional potential of PAs has been underestimated.

    In vitro and in vivo interplay between NAD(P)H: quinone oxidoreductase 1 and flavonoids
    Lee-Hilz, Y.Y. - \ 2007
    Wageningen University. Promotor(en): Ivonne Rietjens; Sacco de Vries. - Wageningen : - ISBN 9789085047902 - 165
    oxidoreductasen - flavonoïden - in vitro - in vivo experimenten - oxidoreductases - flavonoids - in vitro - in vivo experimentation
    Flavonoids are naturally occurring, health-promoting, bioactive compounds, omnipresent in the human diet. The protective effect of these phytochemicals is accomplished for an important part by modulating the activity of enzyme systems responsible for deactivation of chemical carcinogens, such as NAD(P)H: quinone oxidoreductase 1 (NQO1). Several flavonoids act as NQO1 inducers by increasing the NQO1 gene expression level through the electrophile-responsive element (EpRE). On the other hand certain flavonoids are efficient inhibitors of the NQO1 enzyme activity in vitro. The objective of this thesis is to elucidate the complex interplay between flavonoids and NQO1. First, inhibition of NQO1 by flavonoids, pointing at a mechanism contradicting the proven beneficial properties of these natural compounds was studied. Kinetic and molecular dynamics studies were conducted and a method to monitor NQO1 activity in living cells was developed. These studies revealed that although flavonoids possess the potential to inhibit NQO1 activity, inhibition of NQO1 is not likely to happen in cellular systems due to intracellular physiological conditions. Furthermore, the mechanism by which flavonoids are able to induce the EpRE- mediated expression of NQO1 was studied. Reporter gene assays elucidated that upstream XRE-mediated gene expression is not nessessary to induce EpRE-mediated gene expression and quantum-mechanical calculations revealed that flavonoids with a higher intrinsic potential to generate oxidative stress and redox cycling, are the most potent inducers of NQO1. Radioactive binding studies showed Keap1 modification by the flavonoid quercetin, resulting in switching on of EpRE-mediated gene transcription activation. In addition, in vivo metabolites of quercetin were studied on their ability to induce EpRE-mediated gene expression. The results show, that, although quercetin-derived glucuronides are the major metabolites present in the systemic circulation, the deglucuronidated parent compound and its methylated derivatives are the active compounds responsible for the beneficial EpRE-mediated gene expression effects. Overall, the studies presented in this thesis provide insight in the complex interplay between NQO1 and flavonoids on the protein as well as on the gene expression level.
    Effect of (mixtures of) flavonoids on the in vitro and in vivo bioavailability of 2-mino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) : a biologically based modelling approach
    Schutte, M.E. - \ 2007
    Wageningen University. Promotor(en): Ivonne Rietjens; John Groten, co-promotor(en): Gerrit Alink; J.J.M. van de Sandt. - [S.l.] : S.n. - ISBN 9789085047155 - 165
    biologische beschikbaarheid - flavonoïden - darmslijmvlies - darmabsorptie - bioavailability - flavonoids - intestinal mucosa - intestinal absorption
    The transport of food ingredients across the intestinal epithelium is an important factor determining the absorption upon oral intake. Uptake of compounds in the intestine may be influenced by transport proteins such as the ATP binding cassette transporters (ABC transporters). ABC transporters have been shown to be involved in the efflux of several food related compounds and in the efflux of xenobiotics. The pro-carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was found to be transported by efflux transporters from the intestinal cells back into the intestinal lumen and in this way its uptake into the body could be prevented. Also it has been shown that flavonoids, present in fruits, vegetables, nuts, wine and tea are capable of inhibiting the efflux of substrates by ABC transporters. The aim of this thesis was to investigate the possible effect of flavonoids and mixtures thereof on the transport of PhIP across the intestinal barrier and to predict the absorption of PhIP through kinetically modelling. The results presented in this thesis show that different flavonoids are capable of increasing the apical to basolateral PhIP transport through Caco-2 monolayers, an in vitro model for the intestinal epithelium. It is also shown that the flavonoids can do so by acting as inhibitors of the apical ABC transporters in the intestinal cells. Using typical inhibitors for the ABC transporters experiments in Caco-2 monolayers revealed that especially MRP2 and BCRP are involved in the apical excretion of PhIP and these transporters can be inhibited by the flavonoids. Furthermore, an in silico model describing this process taking into account passive diffusion and active transport of PhIP was developed. Using the in silico model it could be demonstrated that for several flavonoids, including flavone, kaempferol chrysoeriol, myricetin, luteolin, naringenin, quercetin and apigenin, their apparent Ki values for inhibition of the active transport to the apical side are in the 5 to 50 μM range and thus within the physiological concentration range that may be achieved within the intestine upon supplement intake. Additional experiments revealed that several binary flavonoid mixtures and one mixture containing five model flavonoids increased the apical to basolateral PhIP transport through the Caco-2 monolayer. Assuming competitive inhibition of the apparent active transporter by the flavonoids and concentration-additivity for their inhibiting effect, the kinetic model could be extended and thus adequately described the experimental values obtained for the flavonoid mixtures. This illustrates that the effect of different flavonoids present in the diet is additive and from this it can be concluded that the effects of flavonoids on PhIP bioavailability can even be expected at levels achieved upon normal dietary intake. Finally, it was demonstrated that the observation that quercetin increases the transport of PhIP through Caco-2 monolayers in vitro could be confirmed in an in vivo rat model. Co-administration of PhIP and quercetin significantly increased the blood AUC(0-8hr) of PhIP in rats to 131±14% of the AUC(0-8hr) for rats dosed with PhIP alone. Therefore, all together the studies presented in this thesis point to a flavonoid-mediated increase of the bioavailability of PhIP and, thus, a possible adverse effect of these supposed beneficial food ingredients when present in combination with the pro-carcinogen PhIP.
    Genetic engineering of flavonoid biosynthesis in tomato
    Schijlen, E.G.W.M. - \ 2007
    University of Amsterdam. Promotor(en): A.J. van Tunen, co-promotor(en): Arnaud Bovy. - Amsterdam : - 160
    genetische modificatie - biosynthese - flavonoïden - tomaten - solanum lycopersicum - genetic engineering - biosynthesis - flavonoids - tomatoes
    Planten beschikken over een enorme capaciteit om een breed scala aan secundaire metabolieten te produceren waarmee ze kunnen reageren op hun continu veranderende omgeving. Flavonoïden, een van de meest voorkomende soort secundaire metabolieten, zijn laagmoleculaire stoffen die van nature in vrijwel alle planten voorkomen. Ze zijn bij uiteenlopende natuurlijke processen betrokken. Zo ontstaat bijvoorbeeld de rode, blauwe en paarse kleur van veel bloemen door de aanwezigheid van anthocyanen, een specifieke klasse van flavonoïdpigmenten. Ook rijpe vruchten danken hun kleur vaak aan deze klasse van flavonoïden. Daarnaast spelen flavonoïden een rol bij processen zoals bescherming van planten tegen schadelijke UV-straling, afweer tegen infecties, pollenvorming en fertiliteit. Omdat flavonoïden wijdverspreid voorkomen in het plantenrijk, vormen deze stoffen een permanent onderdeel van ons plantaardig voedsel. Een deel van de gezondheidsbevorderende effecten van groenten en fruit wordt toegeschreven aan de aanwezigheid van bepaalde flavonoïden. Hoewel flavonoïden in vrijwel alle planten voorkomen, zijn sommige klassen specifiek voor een bepaalde plantensoort, terwijl deze ondervertegenwoordigd of geheel afwezig kunnen zijn in andere plantensoorten. Vanuit dit oogpunt kan het wenselijk zijn om te selecteren voor gewassen met bepaalde (hoeveelheden van) flavonoïden, dan wel de samenstelling daarvan aan te passen. Onderzoek naar de mogelijkheden om de productie van flavonoïden in planten te veranderen wordt veelal uitgevoerd om de aantrekkelijkheid van sierteeltgewassen óf de voedingswaarde van bepaalde gewassen te verhogen. Elio Schijlen onderzocht de genetische modificatie van de biosyntheseroute van flavonoïden in tomaat. Hiermee wil hij meer inzicht geven in de endogene flavonoïdbiosynthese én de mogelijkheden onderzoeken om nieuwe flavonoïden te produceren die van nature niet voorkomen in tomaten.
    Flavonoiden en preventie van chronische zieketen : waar staan we nu?
    Arts, I.C.W. ; Hollman, P.C.H. - \ 2005
    Voeding Nu 7 (2005)10. - ISSN 1389-7608 - p. 11 - 13.
    flavonen - flavonoïden - chemische samenstelling - voedingsmiddelen - ziektepreventie - hart- en vaatziekten - epidemiologische onderzoeken - flavones - flavonoids - chemical composition - foods - disease prevention - cardiovascular diseases - epidemiological surveys
    Er zijn steeds meer aanwijzingen dat flavonoïden in de voeding, die veel voorkomen in thee, wijn, groente en fruit, beschermen tegen hart- en vaatziekten en kanker. De aanwijzingen zijn het sterkst als het gaat om hart- en vaatziekten, maar zijn indirect. Tot nu toe is met geïsoleerde flavonoïden alleen op ruime schaal in vitro-onderzoek gedaan, maar de tijd lijkt rijp te zijn voor een grote interventiestudie bij mensen
    Modulation of multidrug resistance by flavonoids. Inhibitors of glutathione conjugation and MRP-mediated transport
    Zanden, J.J. van - \ 2005
    Wageningen University. Promotor(en): Ivonne Rietjens; Peter van Bladeren, co-promotor(en): N.H.P. Cnubben. - Wageningen : Ponsen & Looijen BV - ISBN 9789085042259 - 157
    flavonoïden - meervoudige resistentie tegen geneesmiddelen - geneesmiddelmetabolisme - ontgifting - flavonoids - multiple drug resistance - drug metabolism - detoxification
    In this thesis, the use of flavonoids for inhibition of two important players in the glutathione related biotransformation system involved in multidrug resistance was investigated using several in vitro model systems. The enzymes of interest included the phase II glutathione S-transferase enzyme GSTP1-1, able to detoxify anticancer agents through conjugation with glutathione and the two multidrug resistance proteins MRP1 and MRP2 involved in glutathione mediated cellular efflux of, amongst others, anticancer drugs.

    The studies presented in this thesis reveal that the major site for flavonoid mediated interaction with GSH-dependent multidrug resistance processes are the GS-X pumps MRP1 and MRP2 rather than the conjugating GSTP1-1 activity. Whereas flavonoids are unlikely to be efficient cellular or in vivo GSTP1-1 inhibiting agents useful to reverse this aspect of multidrug resistance, they might be useful as inhibitors of MRP1 and MRP2 activity. A model compound used in this thesis able to inhibit both MRP1 and MRP2 activity, the flavonoid myricetin, was shown to effectively inhibit vincristine efflux by these transporters in MRP1- and MRP2-transfected cells, thereby effectively sensitizing the cells towards the anticancer drug. Moreover, phase II metabolism, occurring to a major extent in vivo , of the other model flavonoid used in this thesis, quercetin, resulted in equally potent or even better inhibitors of MRP1 and MRP2. This indicates that phase II metabolism is unlikely to reduce the MRP inhibiting potential of quercetin for use of this flavonoid as an inhibitor to overcome MRP-mediated multidrug resistance. Furthermore, it was shown that the flavonoid myricetin is unlikely to affect MRP-mediated transport of glutathione conjugates to a significant extent, because, in general, glutathione conjugates such as the glutathione conjugates of the endogenous compound prostaglandin A 2 , are high affinity substrates of MRP1 and MRP2. These results provide an argument for the possible absence of specific negative side effects on the kinetics and physiology of endogenous MRP substrates, to be expected upon use of these natural MRP inhibitors in the reversal of multidrug resistance. Testing of the in vitro outcomes of the present study in clinical settings may start with flavonoids that have already a safe history of use in for example food supplements and requires the confirmation of involvement of the MRPs in specific cases of clinical drug resistance prior to therapeutic use of the flavonoids as MRP inhibitors.
    Studies on the pro-oxidant chemistry of flavonoids
    Awad, H.M. - \ 2002
    Wageningen University. Promotor(en): I.M.C.M. Rietjens; P.J. van Bladeren; J. Vervoort. - S.l. : S.n. - ISBN 9789058085887 - 133
    flavonoïden - oxidatiemiddelen - antioxidanten - quercetine - structuuractiviteitsrelaties - hplc - kernmagnetische resonantiespectroscopie - massaspectrometrie - flavonoids - oxidants - antioxidants - quercetin - structure activity relationships - hplc - nuclear magnetic resonance spectroscopy - mass spectrometry

    There is currently much interest in the development of functional foods aiming at the prevention of the development of some diseases, for example cancer, by the introduction of selected natural substances at elevated levels into the diet. The rationale for this approach is based especially on epidemiological data that indicate that food items containing such chemicals may reduce the risk of these diseases in humans. Epidemiological studies indicate, for example, that diets rich in fruit and vegetables protect against a variety of diseases, including heart diseases and certain forms of cancer. However, identification of the actual ingredient in a specific diet responsible for the beneficial health effects remains an important bottleneck for translating observational epidemiology to development of a functional food ingredient. The protection against cancer afforded by fruit and vegetables has been attributed to antioxidant micronutrients such as vitamin C, beta-carotene and vitamin E, which may act at many sites, including the stomach, intestine, lung and bladder. However, present scientific attention is focusing as well on the significance of other minor dietary components, notably the flavonoids as protectants against disease. Flavonoids are widespread in nature and are found in considerable quantities in fruits, vegetables, seeds, peel and tubers. The average Western diet may provide up to 1 g of flavonoids per day. Numerous in vitro studies show that flavonoids are potent antioxidants and metal chelators. Their potential as anti-inflammatory, antiallergic and antiviral compounds has also attracted attention. These studies provide the basis for the present rapidly increasing interest for the use of flavonoids as functional food ingredients. As a result increased human exposure to flavonoids can be expected in the near future. In shops and at the internet, food and food supplements based on (iso)flavonoids as functional ingredients are marketed. This, although hard scientific data supporting the health claims as well as data allowing a balanced risk-benefit evaluation are lacking. For flavonoids increased future human exposure regimens induce the question on their pro-oxidant chemistry. There is considerable evidence that some flavonoids are mutagenic in both bacterial and mammalian experimental systems. A high incidence of gastric cancer in some human populations has been linked to consumption of wine containing potentially mutagenic flavonoids (Tamura et al. , Proc. Natl. Acad. Sci. USA. 77, 4961-4965, 1980, Hoey et al. , Am. J. Epidemiol., 113, 669-974, 1981). Relatively little is understood about either the toxicity or protection afforded by flavonoids in humans.

    Since flavonoid quinone/quinone methides have been suggested as the major metabolites responsible for the possible pro-oxidant toxicity and mutagenicity of flavonoids, characterisation of flavonoid quinone chemistry is of importance. However, little information is available on the structure and reactivity of these flavonoid oxidation products. Therefore, the objective of this thesis was to investigate the pro-oxidant chemistry of flavonoids and to perform structure activity studies on the chemical behaviour of 3',4'-dihydroxyflavonoids with special emphasis on the nature and reactivity of the quinone/quinone methide type metabolites formed. Using the GSH trapping method, HPLC, LC/MS, MALDI-TOF, 1H NMR, 13C NMR and quantum mechanical computer calculations the quinone/quinone methide chemistry of a series of 3',4'-dihydroxyflavonoids could be characterised.

    The results provide insight in structure-activity-relationships for the pro-oxidant chemistry of these electrophilic quinone/quinone methide flavonoid metabolites. The results obtained also reveal an unexpected pH-dependent electrophilic behaviour of B ring catechol flavonoids. Furthermore the results of this thesis also reveal, for the first time, evidence for the pro-oxidative chemistry of quercetin in a cellular in vitro model. The formation of these glutathionyl-flavonoid adducts provides evidence for the actual pro-oxidative formation of reactive quinone type metabolites from B ring catechol flavonoids in the selected cellular in vitro model using melanoma cells. Oxidation of the catechols to quinones and their isomeric quinone methides generates potent electrophiles that could alkylate DNA. Interestingly, the structural requirements essential for good antioxidant activity match the requirements essential for pro-oxidant action and quinone methide formation. Altogether, the pro-oxidant behaviour of flavonoids and their quinone/quinone methides are far from straight forward and need to be re-evaluated especially in the framework of the risk-benefit evaluation of the use of these flavonoids as functional food ingredients and/or food supplements.

    Samenvatting

    Er is momenteel veel interesse voor de ontwikkeling van functionele voedingsmiddelen (functional foods), met als doel het voorkomen van het ontstaan van ziekten zoals bijvoorbeeld kanker, via het in verhoogde mate introduceren van geselecteerde natuurlijke bestanddelen in het dieet. De basis voor deze aanpak wordt momenteel met name gevonden in epidemiologische studies die laten zien dat diëten rijk aan specifieke voedselcomponenten of ingrediënten de kans op bepaalde ziekten bij de mens verlagen. Zo geven epidemiologische studies bijvoorbeeld aan dat diëten die rijk zijn aan fruit en groenten beschermen tegen een aantal ziekten zoals hartziekten en bepaalde vormen van kanker. Echter, het identificeren van de belangrijke ingrediënten in het betreffende dieet die het gezondheidsbevorderende effect tot stand brengen is een knelpunt voor het vertalen van de resultaten uit de epidemiologie naar de ontwikkeling van een functioneel voedingsingrediënt.

    De bescherming tegen kanker door groenten en fruit is toegeschreven aan antioxidanten zoals vitamine C, beta-caroteen en vitamine E, die op vele plaatsen in het lichaam, zoals de maag, darmen, long en de blaas actief zijn. Wetenschappelijk wordt momenteel veel aandacht besteed aan het mogelijke belang van andere belangrijke dieet componenten, zoals flavonoïden, als beschermende ingrediënten tegen ziekte. Flavonoïden komen in de natuur veel voor, en worden met name in hoge concentraties gevonden in fruit, groenten, knollen en zaden. Het gemiddelde Westerse dieet bevat ongeveer 1 gram aan flavonoïden per dag.

    Vele in vitro studies tonen aan dat flavonoïden goede antioxidanten en metaal chelatoren zijn. Daarnaast hebben ze anti-inflammatoire, anti-allergische en anti-virale eigenschappen die van belang worden geacht. Deze bevindingen verschaffen de basis voor de momenteel snel groeiende interesse om flavonoïden te gebruiken als functionele voedingsingrediënten. Als gevolg hiervan zou er in de nabije toekomst een toename in de opname van flavonoïden via het dieet verwacht kunnen worden. In winkels en via het internet worden voedingsmiddelen en voedingssupplementen gebaseerd op (iso)flavonoïden als functionele voedingsingrediënten verkocht. Dit, terwijl zowel de wetenschappelijke onderbouwing voor de gezondheidsclaims als gegevens die een gebalanceerde "risk-benefit" analyse mogelijk maken, nog ontbreken. In het geval van verhoogde toekomstige blootstelling van mensen aan flavonoïden worden voor de risk-benefit evaluatie vragen van belang rond hun mogelijk pro-oxidatieve chemisch gedrag. Er zijn aanwijzingen dat sommige flavonoïden mutageen zijn in zowel bacteriële als zoogdier in vitro test systemen. Een verhoogde mate aan maagkanker in bepaalde humane populaties is in verband gebracht met de consumptie van wijn met daarin mogelijk mutagene flavonoïden (Tamura et al. , Proc. Natl. Acad. Sci. USA. 77, 4961-4965, 1980, Hoey et al. , Am. J. Epidem., 113, 669-974, 1981). Alles samenvattend is er eigenlijk weinig bekend van de schadelijke maar ook van de gezondheidsbevorderende effecten van flavonoïden.

    Omdat flavonoid chinon/chinon methides genoemd zijn als de belangrijkste metabolieten die verantwoordelijk zouden zijn voor de mogelijke pro-oxidatieve toxiciteit en mutageniteit van flavonoïden, is karakterisering van deze pro-oxidant chemie van flavonoïden van belang. Echter er is weinig bekend over de structuur en de reactiviteit van deze flavonoid oxidatie producten. Daarom was het doel van deze studie de pro-oxidant chemie van flavonoïden te onderzoeken en een structuur-activiteits studie uit te voeren naar het chemische gedrag van 3',4'-dihydroxyflavonoïden. Daarbij werd speciale aandacht besteed aan de aard en reactiviteit van de gevormde chinon/chinon methide metabolieten. Met behulp van de GSH-trapping methode, HPLC, LC/MS, MALDI-TOF, 1H-NMR, 13C-NMR en kwantum-chemische computerberekeningen kon de chinon/chinon methide chemie van een serie 3',4'-dihydroxyflavonoiden gekarakteriseerd worden.

    De verkregen resultaten geven inzicht in de structuur-activteits relaties voor de pro-oxidatieve chemie van de electrofiele chinon /chinon methides metabolieten van de flavonoïden. De resultaten laten ook een onverwacht effect zien van de pH op het electrofiele gedrag van de B-ring catechol flavonoïden. Bovendien laten de resultaten van het proefschrift zien dat zelfs onder reducerende omstandigheden in een cellulair in vitro model (melanoma cellen) de pro-oxidatieve chemie van quercetine van belang kan zijn. Met name de vorming van glutathion-flavonoid conjugaten is een bewijs dat in het gekozen cellulaire model de pro-oxidatieve vorming van reactieve flavonoid chinon/ chinon methide metabolieten is opgetreden. Oxidatie van de catecholen naar chinonen en hun isomere chinon methides genereert electrofielen die DNA kunnen alkyleren. Van belang is dat de structurele randvoorwaarden die een flavonoid een goede antioxidant maken gelijk blijken te zijn aan de structurele kenmerken die essentieel zijn voor pro-oxidant gedrag en chinon methide vorming.

    Al met al is de pro-oxidant chemie van flavonoïden en van hun chinon /chinon methides verre van recht toe recht aan gebleken en zou de pro-oxidatieve chemie en de toxiciteit van de flavonoïden in het kader van hun gebruik als functional food ingredienten beter onderzocht en afgewogen moeten worden, rekening houdend met hun mogelijk gezondheidsbevorderende effecten.

    Flavonoids from cabbage are feeding stimulants for diamondback moth larvae additional to glucosinolates : chemoreception and behaviour
    Loon, J.J.A. van; Wang, C.Z. ; Nielsen, J.K. ; Gols, R. ; Qiu, Y.T. - \ 2002
    Entomologia Experimentalis et Applicata 104 (2002). - ISSN 0013-8703 - p. 27 - 34.
    brassica oleracea - flavonoïden - waardplanten - plutella xylostella - stimulerende middelen - brassica oleracea - flavonoids - host plants - plutella xylostella - stimulants
    In caterpillars two styloconic contact chemoreceptors on the maxillary galea are assumed to contain the main taste receptors involved in host plant selection. The diamondback moth, Plutella xylostella L. is a specialist feeder of plants in the Brassicaceae, a plant family characterized by the biosynthesis of glucosinolates. We used pea (Pisum sativum L., Leguminosae) as a neutral non-host for a dual-choice leaf disc assay to quantify feeding stimulation by glucosinolates and flavonoids. Increasing concentrations of sinigrin resulted in significant preferences for sinigrin-treated leaf discs, with a threshold between 1 and 3 μM. Millimolar concentrations of four of the five flavonol triglucosides likewise elicited a significant preference for flavonoid-treated leaf discs. A mixture of four flavonoids and sinigrin was significantly preferred over sinigrin-treated leaf discs alone. Vigorous unicellular electrophysiological responses of medial maxillary styloconic taste sensilla were observed in response to five glucosinolates (glucocapparin, sinigrin, glucobrassicin, glucoiberin, and gluconasturtiin). This medial taste neuron responded in a dose-dependent manner to a concentration series of sinigrin, with a threshold of response of ca. 1 μM. The lateral sensillum styloconicum contained a neuron sensitive to sucrose, glucose, and fructose. However, no responses in the two types of maxillary styloconic sensilla to the phagostimulatory flavonoids could be detected, suggesting that other taste organs mediate chemoreception of flavonoids. We conclude that diamondback moth larvae employ a combination of biosynthetically distinct categories of feeding stimulants which allows for a higher degree of discriminatory ability than when this would be based on glucosinolates alone.
    The apple skin: colourful healthiness : developmental and environmental regulation of flavonoids and chlorogenic acid in apples
    Awad, M.A.G. - \ 2001
    Wageningen University. Promotor(en): W.M.F. Jongen; L.H.W. van der Plas; A. de Jager. - S.l. : S.n. - ISBN 9789058084255 - 146
    malus - appels - chemische samenstelling - plantenpigmenten - flavonoïden - chlorogeenzuur - rijp worden - plantenontwikkeling - houdbaarheid (kwaliteit) - gasbewaring - malus - apples - chemical composition - plant pigments - flavonoids - chlorogenic acid - ripening - plant development - keeping quality - controlled atmosphere storage

    The ultimate objective of the production, handling and distribution of fresh fruits and vegetables is to satisfy consumers requirements. In general the attractiveness of fruits and vegetables to consumers is determined both by visible (e.g. colour) and invisible (e.g. healthiness) quality attributes. Flavonoids and hydroxycinnamic acid derivatives, secondary metabolites, contribute largely to both fruit colour and, through fruit consumption, to human health and it is, therefore, very useful to study factors that affect these substances with the aim of further improving the relevant fruit attributes. Flavonoids and hydroxycinnamic acid derivatives are widespread in the plant kingdom, comprise a large group of naturally occurring antioxidants that form part of the human diet. There is considerable evidence for the role of antioxidant constituents of fruits and vegetables in the maintenance of health and disease prevention. Recent studies have shown that the majority of the antioxidant activity of a fruit or vegetable may originate from the flavonoids and other phenolic compounds. Apple fruit are rich in flavonoids such as flavonols (quercetin 3-glycosides), flavanols (catechin, epicatechin, gallocatechin, procyanidins and its polymers), dihydrochalcone glycosides (phloritin glucoside (phloridzin) and phloritin xyloglucoside), and cyanidin 3-glycoside (anthocyanins). Apple fruits also contain considerable amounts of hydroxycinnamic acid derivatives mainly represented by chlorogenic acid. The red colour of apples is primarily a consequence of the flavonoid pigments anthocyanins which are located in the vacuole. Despite the importance of flavonoids for the intrinsic quality of apples very little is known of their regulation in fruit. The aim of the work described in this thesis was therefore, to obtain knowledge on the extent to which the contents of flavonoids and chlorogenic acid in the skin of apples varies, how they develop during fruit growth phase, ripening phase and post harvest phase and how they can be manipulated.

    Chapter 1 contains a review of the literature. It appears that the accumulation of flavonoids and phenolic acids in plants is under control of many internal and external factors.

    In Chapter 2 the extent of natural variation in flavonoids and chlorogenic acid concentration due to within fruit, within tree, between orchards, between cultivars and among mutants was determined. Considerable variation was observed among these variables. Individual flavonoids and chlorogenic acid concentrations were not equally distributed within the fruit. Quercetin 3-glycosides and anthocyanin were almost exclusively found in the skin. The sun-exposed skin of individual fruit had much higher cyanidin 3-galactoside (anthocyanin) and quercetin 3-glycoside concentrations than the shaded skin, while phloridzin, catechins and chlorogenic acid were similar in the skin of both sides (Chapter 2). Significant genotypic variation was observed for the concentration of flavonoids and chlorogenic acid. 'Jonagold' apples contain significant higher concentrations (about 30% higher) (Chapters 2 and 6) and amounts (about 2-fold higher) (Chapter 6) of the total flavonoids than 'Elstar' apples. Chlorogenic acid concentration was about 3-fold higher in 'Jonagold' than in 'Elstar' apples. However 'Elstar' apples contained significant higher concentrations of some quercetin glycosides types as quercetin 3-rhamnoglucoside (about 2-fold higher) and quercetin 3-glucosides (about 30% higher) than 'Jonagold' apples. This might be relevant with respect to differences in bio-activity and antioxidant capacity of various flavonoid compounds. As far as the potential maximum concentration of flavonoids in apple is genetically determined breeding would be an important tool for increasing healthiness of apples. The differences between basic forms and coloured mutants within a given cultivar (for example Jonagold-Jonaprince; Elstar-Elshof) show that the potential anthocyanin accumulation (but only that) may increase several fold without influencing the concentrations of other flavonoid classes (Chapter 2). Microscopic study showed that the most blushed mutants had a higher number of red cells per cell layer and more cell layers containing red cells than the standard cultivar and the less blushed mutants. It is striking to observe coloured and completely uncoloured cells as neighbours. Since the selection of coloured mutants is inherently based on the amount of red coloration, selection of mutants for higher levels of other potential healthy flavonoid classes e.g. quercetin 3-glycosides could be considered, providing that such characteristics can be relatively easily determined. The concentrations of anthocyanin, quercetin 3-glycosides and total flavonoids were highest in fruit borne in the top of the tree followed by fruit from the outer tree parts, whereas the lowest concentrations were found in fruit from the inner tree. Terminal fruit contained the highest concentrations of these compounds, including catechins, compared to lateral and spur fruit. Phloridzin and chlorogenic acid were not affected by the position of the fruit in the tree nor by the bearing wood type. The maximum possible difference in flavonoid concentrations, based on difference between top fruit (optimal light conditions) and inner fruit (minimal light conditions) may be 3-fold for quercetin 3-glycosides and 2-fold for total flavonoids (Chapters 2 and 3). There were significant differences in flavonoid and chlorogenic acid concentrations in 'Elstar' fruit between two normally productive orchards differing mainly in growth vigour and internal shading. All these results show that light conditions are a main regulatory factor in the biosynthesis of flavonoids in apples.

    In Chapter 3 the natural distribution of light within the tree canopy in relation to the concentration of flavonoids and chlorogenic acid in fruit skin was analysed. The concentrations of cyanidin 3-galactoside and quercetin 3-glycosides and the percentage of blush in the fruit skin were directly related to light level in the direct vicinity of the fruit. Light in the interior of the canopy was poorer in UV-A, blue, green and red (R) but richer in far-red (FR) light than at all other positions. Consequently, the FR/R ratio (with large influence on formative processes) was much larger at the interior of the canopy than at all other positions. There was a critical FR/R ratio of about 1 above which no anthocyanin and only low amounts of quercetin 3-glycosides were formed.

    In Chapter 4 the relationships between the fruit nutrients N, P, K, Ca and Mg and concentrations of flavonoids and chlorogenic acid in fruit skin were studied with two types of 'Elstar'. In an experiment with the mutant 'Elshof' with the 5 nutrients applied at 5 rates in 4 replications, only N and Ca applications resulted in higher concentration of these nutrients in the fruit, but sufficient variation was present among treatments to correlate the concentration of the other nutrients with those of flavonoids and chlorogenic acid. Negative correlations were frequently found between the concentration of N and Mg and the N/Ca ratio in fruit during growth, and anthocyanin and total flavonoids concentration at maturity in 1996, 1997 and 1998. In 1997, these correlations were weakest but still significant. In that season, P and K concentration were frequently negatively correlated with the concentration of anthocyanin and total flavonoids. The concentration of Ca was not related to the concentration of anthocyanin and total flavonoids. In a study in 1996 with standard 'Elstar', we used the variation in nutrient concentration due to differences in fruit position on tree. The concentrations of N and K and the N/Ca ratio in fruit at maturity were negatively and that of Ca was positively correlated with the concentration of anthocyanin and total flavonoids. Magnesium concentration was negatively correlated with anthocyanin concentration but not with total flavonoids. As a consequence of the relation with position of the fruit in the tree an interaction with the influence of light may, however, be expected. Multiple regression models mainly containing N as factor accounted for up to 40% and 30% of the variance in anthocyanin and total flavonoids concentration of 'Elshof' mutant apples, and for up to 70% and 65% of the variance in anthocyanin and total flavonoids concentration of standard 'Elstar' apples. The relationships between plant nutrients and chlorogenic acid concentration in apples were not consistent and further study is required. It is concluded that, in addition to improving light conditions, the concentration of flavonoids in fruit skin could be further increased by optimising fertilization especially that of N, directed at preventing excess N accumulation.

    In Chapter 5 we tested the concept that under condition of high carbon supply, plants may increase the formation of their secondary metabolites, like phenolic compounds. In field experiments crop load was manipulated by applying flower or fruit thinning at different stages of development and at different severity. At a low crop load, fruit weight, soluble solids, acidity and firmness were significantly higher than at high and moderate loads. However, the concentrations of flavonoid and chlorogenic acid were similar at the different levels of crop load. Time of thinning had no significant influence on the concentration of flavonoids and chlorogenic acid in fruit skin and had no further effect on fruit quality characteristics such as weight, soluble solids, acidity and firmness. Removal of only the interior fruits (about one-third of total fruit) at about 4 weeks before expected commercial harvest had no influence on the concentration of flavonoids and chlorogenic acid or on the quality characteristics of the remaining exterior fruits of either 'Elstar' or 'Jonagold'. The results indicate that, within the 'normal' range of conditions, assimilate availability is not a major regulatory factor in flavonoids and chlorogenic acid formation in apples. These results are in agreement with the lack of any influence of the supply of precursors in the orchard (Chapter 7).

    In Chapter 6 the changes that take place in the concentration and the amount of individual flavonoids and chlorogenic acid in the skin of 'Elstar' and 'Jonagold' apples during development and ripening were investigated. In both cultivars, the concentration on a dry weight basis of quercetin glycosides, phloridzin and chlorogenic acid was highest early in the season but decreased at different rates during fruit development to reach a steady level during maturation and ripening. Catechins (catechin plus epicatechin) concentration showed a similar pattern, but a temporary increase was observed in an early stage of development. The concentration of cyanidin 3-galactoside (anthocyanin) was relatively high early in the season, gradually decreased to a very low steady level during growth, but started to increase near maturation, especially in the outer fruit. On a fruit basis the amount of quercetin glycosides increased during development and was about two times higher in 'Jonagold' compared to 'Elstar', both in outer and inner fruit. These compounds were the most abundant flavonoids in the skin of both cultivars and their accumulation showed a strong dependency on fruit position on tree. The amount of the second most abundant flavonoid type, catechins, increased during development to a maximum and then showed some decrease by mid season which was independent of fruit position on tree. The amount of phloridzin increased only early in the season reaching a steady level during development and ripening, and was independent of fruit position on tree. The amount of chlorogenic acid in both cultivars initially increased, but subsequently decreased to reach a low steady level and was slightly higher in outer than in inner fruit. The latter phenomenon is the only direct evidence for (net) breakdown of any of the studied phenolics. Although anthocyanin concentration was relatively high at early stages of development, significant accumulation on a fruit basis only occurred during maturation and ripening. The accumulation of anthocyanin, similar to that of quercetin glycosides, showed a strong dependency on fruit position on tree. The results indicate that, in general, the overall production of total flavonoids, with the exception of anthocyanin, and chlorogenic acid in apple skin is completed during fruit development before the onset of maturation.

    Chapter 7 reports the influence of exogenous application of a number of chemicals that are precursors of flavonoids or are known to affect ripening on the accumulation of flavonoids and chlorogenic acid in 'Jonagold' apple skin with emphasis on anthocyanin. One aim was to identify a possible substrate limitation and another to separate the formation of anthocyanin from other related maturity/ripening events. Since the occurrence of the second peak in anthocyanin formation more or less parallels the maturation and ripening phase (like starch degradation and aroma production), anthocyanin formation itself is often considered as a ripening phenomenon triggered by ethylene. Our results suggest, however, that there is no simple relation to ripening and consequently to ethylene production (though we did not measure ethylene). This is concluded from the promotion of anthocyanin formation by ethephon (an ethylene releasing compound) and the retardation of anthocyanin formation by ABG and GA 3 (known to lower or counteract endogenous ethylene), without significantly altering starch degradation and changes in streif index (combination of starch index, firmness and sugar concentration). Our results have also shown that the other flavonoid classes quercetin 3-glycosides, catechins and phloridzin and chlorogenic acid do not respond to any of the applied chemicals. It is concluded that anthocyanin formation is dependent on developmental signals and independent of both fruit maturity/ripening and of the synthesis of other flavonoid classes and responds in a complicated way to ethylene.

    In Chapter 8 the changes in individual flavonoids and chlorogenic acid during regular (RS) or ultra low oxygen (ULO) storage conditions at 1°C are reported in both 'Jonagold' and 'Elstar' apples. It could convincingly be shown that during storage of both 'Jonagold' (3, 6 and 8 months) and of 'Elstar' (2, 4 and 6 months) and during 1 or 2 weeks shelf life, the concentrations of cyanidin 3-galactoside and quercetin glycosides were relatively constant, while the concentrations of catechins, phloridzin and chlorogenic acid showed only minor decreases. Moreover there were no significant differences in the concentration of flavonoids and chlorogenic acid between fruits stored under ULO compared to RS conditions. It is concluded that, following harvest, flavonoids present in apples are stable. There is no direct or indirect proof for breakdown (net metabolic turnover) during storage and shelf life.

    In Chapter 9 the practical applications of the findings made in this study were discussed. Our results show that there is much room for increasing the level of potential health phytochemicals in apples. The first step would be cultivar selection either from already available genotypes or by developing new cultivars through classical breeding or molecular biology and gene technology. We showed that light has a significant impact on the final level of flavonoids in fruit. Therefore, the second and more proximate option would be the optimisation of light conditions within tree canopy by measures such as choice of root stocks, planting system, row orientation and training and pruning systems or covering the orchard floor with reflecting films (though the latter is not promoting the visual aspect of the orchard). A third step could be optimisation of the fertilization programme especially avoiding excess N and better timing of N-application. A further possibility is to sort fruit in healthiness classes. As long as a simple method to detect non-destructively quercetin 3-glycosides is lacking, sorting of fruit based on their blush might be a way to make healthiness classes, since blush is a good marker for exposure to light during growth and thus to some extent for the quercetin 3-glycosides level. Even when cultivar choice and cultivation methods succeed in getting high levels of flavonoids in fruit still the treatment by the consumer determines how much of these substances will be consumed. Many consumers still peel the fruit before consumption thereby removing almost all anthocyanin and quercetin 3-glycosides (Chapter 2). Promotion of fruit on the basis of healthiness is, in our opinion, however, only useful if it is accompanied with a guarantee of absence of pesticides, as is most credible, at least to the public, in organic farming.

    Because of the large influence of a number of factors at several steps of the production chain, a quantitative model e.g. integrated with light distribution models, would offer a practical and effective tool for estimating the effect of certain measures and to predict and maximise the final level of healthy compounds in apples enabling the development of more accurate intake data and dietary recommendations.

    Bioavailability of flavonoids and cinnamic acids and their effect on plasma homosysteine in humans
    Olthof, M.R. - \ 2001
    Wageningen University. Promotor(en): M.B. Katan; P.C.H. Hollman. - S.l. : S.n. - ISBN 9789058084170 - 135
    flavonoïden - kaneelzuur - homocysteïne - bloedplasma - biologische beschikbaarheid - mens - quercetine - theaflavine - chlorogeenzuur - hart- en vaatziekten - flavonoids - cinnamic acid - homocysteine - blood plasma - bioavailability - man - quercetin - theaflavine - chlorogenic acid - cardiovascular diseases

    Dietary antioxidants might prevent oxidative damage to tissues and therefore protect against cardiovascular disease and cancer. Dietary phenols are strong antioxidants in vitro but their role in vivo is uncertain. Furthermore, there are only limited data on their bioavailability in humans. The aim of this thesis was to investigate whether bioavailability data on flavonoids and cinnamic acids support the hypothesis that they can affect health in humans . Because the group of phenols in foods is huge, we focussed our research on major phenols in foods; the flavonol quercetin, black tea phenols and chlorogenic acid (5-caffeoylquinic acid). We studied their bioavailability and effect on plasma homocysteine in humans, a potential risk factor for cardiovascular disease.

    The bioavailability of quercetin and chlorogenic acid depends upon their conjugated moieties. Hollman et al. found that the bioavailability of quercetin-3-rutinoside, a major flavonol in tea, was only 20% of that of quercetin-4'-glucoside. We found that transformation of quercetin-3-rutinoside into quercetin-3-glucoside will improve its bioavailability because the 3-glucoside had the same high bioavailability as the 4'-glucoside. Caffeic acid is a major phenol in coffee, but it is present as a conjugate with quinic acid, called chlorogenic acid. We found that the conjugation of caffeic acid with quinic acid hinders absorption in humans: absorption of chlorogenic acid was only 30% of that of its caffeic acid moiety.

    Furthermore, we found that chlorogenic acid, black tea solids and quercetin-3-rutinoside are extensively metabolized in the human body, mainly before they reach the circulation. Their metabolites have no, or less, antioxidant activity in vitro than their parent phenols. Therefore the role of dietary phenols as antioxidants in vivo might be less important than suggested by their in vitro antioxidant activity.

    Coffee consumption increases plasma homocysteine, a potential risk factor for cardiovascular disease. Chlorogenic acid from coffee is partly responsible for the homocysteine-raising effect of coffee, because we found that it increased plasma homocysteine. Black tea solids also raised plasma homocysteine, whereas quercetin-3-rutinoside did not. Furthermore, we found that glycination of metabolites of phenols in the body is not involved in the homocysteine-raising effect of phenols.

    In conclusion, chlorogenic acid, tea phenols and quercetin are available in the human body, but their effects on health are uncertain. Further research on bioavailability and health effects of dietary phenols is needed.

    Assessment of flavonoid and fatty acid intake by chemical analysis of biomarkers and the duplicate diets
    Vries, J.H.M. de - \ 1998
    Agricultural University. Promotor(en): M.B. Katan; W.A. van Staveren. - S.l. : De Vries - ISBN 9789090113036 - 127
    voedselhygiëne - voedingstoestand - consumptiepatronen - indicatoren - biologische indicatoren - flavonen - flavonolen - flavonoïden - food hygiene - nutritional state - consumption patterns - indicators - biological indicators - flavones - flavonols - flavonoids
    Dietary intake is important to investigate the relationship between diet and the occurrence of disease. However, it is difficult to assess the intake of nutrients such as flavonoids, minor fatty acids and plant sterols because the data on these nutrients in food composition tables are insufficient or because the bioavailability of these nutrients differs between foods. The results of studies investigating the relationship of these nutrients to disease are inconsistent, perhaps because of errors in the methods used to assess nutrient intake. The aim of this thesis was to evaluate physically and chemically based methods of measuring the intake of flavonoids, fatty acids, sterols and energy.

    We found differences of up to 80 mg per day in the intake of flavonols and flavones in subjects eating a variety of diets. The ratio of the within- to the between-subject variation in the intake of flavonols and flavones was lower than one, indicating that it is possible to study the relationship between flavonoid intake and disease. The food frequency questionnaire used in this study was suitable for classifying subjects by their flavonol intakes.

    We also found that the bioavailability of the flavonol quercetin differs between the major dietary sources. The bioavailability of quercetin from red wine was 75% of that from onions and from tea 50% of that from onions. Therefore, flavonols from red wine can probably not explain the lower incidence of coronary heart disease in France compared to other western countries. Concentrations of quercetin in plasma can be used as biomarkers to distinguish between subjects with a low and with a high flavonol intake. This is possible because of a relatively small variation of plasma quercetin and a linear relationship between quercetin intake to its concentrations in plasma and excretions in urine.

    Chemical analysis of food composites is a suitable method to assess the intake of a large number of fatty acids and sterols for subsamples of populations. We found large differences in the amount of these nutrients in the diets of middle-aged men living in 16 cohorts in seven countries. Three-day records are not suitable to measure individual energy intake, but they can be used to classify subjects by their energy intakes.

    In conclusion, the most feasible method to assess flavonol intake in epidemiological studies appears to be a food frequency questionnaire specially devised to assess flavonol intake and validated in a sub-population by biomarkers of flavonol intake. In addition, chemical analysis of food composites is a good tool to assess specific fatty acids and sterols in the diet of a population. Finally, when using 3-day food records, even from a motivated, lean, well-educated population, underestimation of intakes has to be taken into account.

    Dietary non-nutrients and haemostasis in humans : effects of salicylates, flavonoids and ginger
    Janssen, P.L.T.M.K. - \ 1997
    Agricultural University. Promotor(en): M.B. Katan; W.A. van Staveren; R.P. Mensink. - S.l. : S.n. - ISBN 9789054857037 - 70
    vaatziekten - bloedstoornissen - hart- en vaatziekten - hart- en vaatstoornissen - bloedsomloopstoornissen - bloedstolling - aspirine - salicylzuur - flavonen - flavonolen - flavonoïden - specerijen - kruiderijen - vascular diseases - blood disorders - cardiovascular diseases - cardiovascular disorders - circulatory disorders - blood coagulation - aspirin - salicylic acid - flavones - flavonols - flavonoids - spices - condiments

    In this thesis we studied the content of acetylsalicylate and total salicylates in foods, and we studied the effects of the dietary non-nutrients salicylates and flavonoids and of certain foods on haemostatic parameters in humans.

    Acetylsalicylic acid -aspirin- irreversibly inhibits platelet cyclo-oxygenase, leading to decreased platelet thromboxane A 2 production and decreased aggregation. Therefore it is effective as an anti-thrombotic drug in doses as low as 30 mg/d. Qualitative analyses by Swain et al suggested the presence of acetylsalicylate in foods. It was estimated that a normal mixed Western diet provides 10-200 mg/d of total salicylate and 3 mg/d of acetylsalicylate. We showed in 10 healthy subjects that 3 mg/d of acetylsalicylic acid decreased mean platelet thromboxane production by 39±8% (±sd). Thus, quantitative data on dietary acetylsalicylate deserved closer investigation. We determined acetylsalicylate and total salicylates in 30 foods using HPLC with fluorescence detection. Acetylsalicylate was lower than the detection limit (0.02 mg/kg for fresh and 0.2 mg/kg for dried products) in all foods. Total salicylates were 0-1 mg/kg in vegetables and fruits, and 3-28 mg/kg in herbs and spices. We showed that urinary excretion was a valid indicator for intake of pure (acetyl)salicylic acid (recovery 77-80%). We then studied urinary salicylate excretion in 17 subjects eating a variety of diets to estimate the content of bio-available salicylates of diets. Median excretion was 1.4 mg/24 h (range 0.8-1.6). Our data suggest that even purely vegetable diets provide less than 6 mg/d of salicylates, and no measurable acetylsalicylate. These amounts are probably too low to affect coronary heart disease risk, and worries about adverse effects of dietary salicylates on the behaviour of children may be unfounded.

    Others found that dietary flavonoids were associated with a reduced risk of coronary heart disease and stroke. This might be due to effects on haemostasis, because flavonoids have been reported to inhibit platelet aggregation in vitro . We found that concentrations of 2.5 μM of the flavone apigenin inhibited collagen- and ADP-induced platelet aggregation in vitro by about 26%, whereas the flavonols quercetin and quercetin-3-glucoside had no effect. No effects were found on platelet aggregation, thromboxane production, or other haemostatic parameters in 18 healthy subjects after they had consumed large amounts of quercetin- (onions) and apigenin-rich (parsley) foods daily for 7 d each. We conclude that claims for anti-aggregatory effects of flavonoids are based on the in vitro use of concentrations that cannot be attained in vivo . Our findings suggest that it is unlikely that reported effects of dietary flavonoids on coronary vascular disease risk are mediated through platelet aggregation or cyclo-oxygenase activity. Possible effects on known risk indicators for coronary heart disease from the coagulation cascade or the fibrinolytic system should be examined in a larger study.

    It has been claimed that ginger consumption exerts an anti-thrombotic effect by inhibiting platelet thromboxane production. We, however, found no effects on platelet thromboxane production in a placebo-controlled cross-over study in 18 healthy subjects after consumption of raw (-1±9%, mean±sd) or cooked ginger (1±8%).

    We conclude that contents of (acetyl)salicylate in foods are too low to affect disease risk. We could not confirm the putative anti-thrombotic effect of ginger, onions and parsley on haemostatic parameters in humans.

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