Stereoselective Conjugation, Transport and Bioactivity of S- and R-Hesperetin Enantiomers in Vitro

Division of Toxicology, Wageningen University, Tuinlaan 5, PO Box 8000, 6703 HE Wageningen, The Netherlands.
Journal of Agricultural and Food Chemistry (Impact Factor: 2.91). 05/2010; 58(10):6119-25. DOI: 10.1021/jf1008617
Source: PubMed


The flavanone hesperetin ((+/-)-4'-methoxy-3',5,7-trihydroxyflavanone) is the aglycone of hesperidin, which is the major flavonoid present in sweet oranges. Hesperetin contains a chiral C-atom and so can exist as an S- and R-enantiomer, however, in nature 2S-hesperidin and its S-hesperetin aglycone are predominant. The present study reports a chiral HPLC method to separate S- and R-hesperetin on an analytical and semipreparative scale. This allowed characterization of the stereoselective differences in metabolism and transport in the intestine and activity in a selected bioassay of the separated hesperetin enantiomers in in vitro model systems: (1) with human small intestinal fractions containing UDP-glucuronosyl transferases (UGTs) or sulfotransferases (SULTs); (2) with Caco-2 cell monolayers as a model for the intestinal transport barrier; (3) with mouse Hepa-1c1c7 cells transfected with human EpRE-controlled luciferase to test induction of EpRE-mediated gene expression. The results obtained indicate some significant differences in the metabolism and transport characteristics and bioactivity between S- and R-hesperetin, however, these differences are relatively small. This indicates that for these end points, including intestinal metabolism and transport and EpRE-mediated gene induction, experiments performed with racemic hesperetin may adequately reflect what can be expected for the naturally occurring S-enantiomer. This is an important finding since at present hesperetin is only commercially available as a racemic mixture, while it exists in nature mainly as an S-enantiomer.

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    • "Though, in humans, sulfonation of the flavone baicalein occurs mainly in the liver (Zhang et al. 2007b). In intestinal cells, UGTs and SULTs catalyse the conjugation of the flavanone hesperetin into its glucuronidated and sulfonated metabolites (Brand et al. 2010). Colon bacteria posess different deconjugating enzymes able to release flavonoid aglycones from their glycosides and glucuronides, e.g. "
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    ABSTRACT: It has been widely acknowledged that regular consumption of fresh fruits and vegetables is linked with a relatively low incidence of cancers (e.g. breast, cervix, and colon). Notably, dietary polyphenolic compounds that show some structural similarity to human estrogen, e.g. isoflavones, coumestans, lignans, flavones, have been proposed to play a role in cancer prevention. However, at present there is no satisfactory explanation for the cancer preventative properties of this group of compounds. Whereas polyphenolic compounds have been shown to inhibit proliferation of tumour cells in vitro, the results of in vivo tests have mostly been disappointing in this respect. It seems that mammalian phase II detoxification mechanisms make that dietary polyphenols are rapidly and effectively removed from the body, i.e. their concentration in the blood plasma hardly ever reaches levels high enough to have a possible effect on tumour growth. The polymethoxyflavones nobiletin and tangeretin, common constituents of Citrus peel, are better absorbed than polyhydroxy flavonoids, and maintain their biological activity for a longer period of time. The compounds are known to be substrates for the estrogen-converting cytochrome P450 enzymes CYP1A1 and CYP1B1, which are typically over-expressed in a range of tumour tissues. The enzymes catalyse regioselective hydroxylation and dealkylation of the polymethoxyflavones, resulting in reaction products that appear to inhibit cell proliferation via interference with the MAPK/ERK cell signalling pathway.
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    • "Little previous researches on the biological activities of hesperidin took the differences in configuration into consideration. Recently, Daxin et al. found that both (2S)-and (2R)-hesperidin could prevent the formation of AGEs in vitro and the activities are not affected by differences in configuration[42], but Brand et al. reported that there are some significant differences in the metabolism and transport characteristics between (2S)-and (2R)-hesperetin, although these differences are relatively small[43]. Taken together, these findings implicate that the differences in the in vivo activities of (2S)-and (2R)-hesperidin are worth thorough investigation. Based on the results in our experiment and well established ocular bioactivity of hesperidin[44], further studies are still needed to explore the exact effect of hesperidin on DR as well as the relationship between configurations and in vivo activities. "
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    ABSTRACT: Diabetic retinopathy is a complex disease that potentially involves increased production of advanced glycosylation end products (AGEs) and elevated aldose reductase (AR) activity, which are related with oxidative stress and inflammation. The aim of this study was to investigate the effects of hesperidin on retinal and plasma abnormalities in streptozotocin-induced diabetic rats. Hesperidin (100, 200 mg/kg daily) was given to diabetic rats for 12 weeks. The blood-retina breakdown (BRB) was determined after 2 weeks of treatment followed by the measurement of related physiological parameters with ELISA kits and immunohistochemistry staining at the end of the study. Elevated AR activity and blood glucose, increased retinal levels of vascular endothelial growth factor (VEGF), ICAM-1, TNF-α, IL-1β and AGEs as well as reduced retina thickness were observed in diabetic rats. Hesperidin treatment significantly suppressed BRB breakdown and increased retina thickness, reduced blood glucose, AR activity and retinal TNF-α, ICAM-1, VEGF, IL-1β and AGEs levels. Furthermore, treatment with hesperidin significantly reduced plasma malondialdehyde (MDA) levels and increased SOD activity in diabetic rats. These data demonstrated that hesperidin attenuates retina and plasma abnormalities via anti-angiogenic, anti-inflammatory and antioxidative effects, as well as the inhibitory effect on polyol pathway and AGEs accumulation.
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    ABSTRACT: Flavonoids form a large class of polyphenolic compounds omnipresent in plant derived products, often present in the form of ß-glycosides. They have been frequently proposed to be associated with possible beneficial effects on health. The flavanoid hesperetin ((+/-)-4’-methoxy-3’,5,7-trihydroxyflavanone) is the aglycone of the rutinoside hesperidin, which is present in large amounts in citrus fruits and orange juice and is deglycosylated upon oral uptake. Hesperetin has been proposed to provide anti-carcinogenic effects, and also to reduce the risk of osteoporosis. Although the dietary intake can be considerable, the bioavailability of hesperetin is limited. An important mechanism behind this limited bioavailability is the metabolism and transport in intestinal cells: flavonoids are conjugated by phase II enzymes such as UDP-glucuronosyl transferases (UGTs), sulfotransferases (SULTs) or catechol-O-methyltransferases (COMT), and are subsequently transported from the intestinal cells back into the intestinal lumen by apically located ATP binding cassette (ABC) transporters. Because specific flavonoids are known modulators of phase II metabolism and of ABC transporter activity, in this thesis it was hypothesized that simultaneous exposure to hesperetin and selected other flavonoids may increase hesperetin bioavailability by modulating its intestinal metabolism and transport. To further support this hypothesis the thesis contains a literature overview on the capacity of flavonoids to modulate the oral bioavailability of other compounds. This overview reveals that the transport of compounds across the intestinal epithelial can be highly dependent on the activity of ABC transporters, especially those involved in the transport from the intestinal cells, either to the basolateral blood side, facilitating absorption, or back into the intestinal lumen, opposing bioavailability. The function of the ABC transporters in intestinal transcellular uptake also implies a role for inhibitors of these transporters in modulation of the bioavailability upon oral uptake. The role of flavonoids as important modulators or substrates of intestinal ABC transport proteins and their effect on the intestinal transport and bioavailability of other compounds is illustrated by several examples from in vitro and some in vivo studies. Subsequently, the concept of increasing the bioavailability of a compound by co-administration of inhibitors of phase II metabolism and/or ABC transporters was tested for hesperetin using different in vitro and in vivo model systems. The in vitro models used to study intestinal metabolism and transport included a two-compartment transwell model with Caco-2 cell monolayers, simulating the intestinal transport barrier. After differentiation, Caco-2 cells are known to display morphological and biochemical characteristics of human enterocytes, including the expression of ABC transporters and phase II metabolizing enzymes, and to form a tight layer of polarized intestinal cells. Grown on a membrane separating an apical compartment (simulating the intestinal lumen side) and a basolateral compartment (simulating the blood/plasma side) they form a well accepted in vitro model to study intestinal transport. This Caco-2 cell monolayer in a two-compartment transwell model system was used to define the characteristics of the intestinal transport and metabolism of hesperetin in vitro. The metabolites of hesperetin formed by the Caco-2 cells were identified using HPLC-DAD and uPLC-DAD-MS-MS techniques and available reference compounds combined with specific enzymatic deconjugation reactions. The role of the apically located ABC transporters P-glycoprotein (Pgp), Multidrug Resistance Protein 2 (MRP2) and Breast Cancer Resistance Protein (BCRP) in the efflux of hesperetin and its metabolites was studied by co-administration of compounds known to inhibit several classes of ABC transporters. Furthermore, the cellular expression of these ABC transporters in Caco-2 cells was confirmed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. It was shown that apically-applied hesperetin was metabolized into hesperetin 7-O-glucuronide and hesperetin 7-O-sulfate, which were transported predominantly to the apical side of the Caco-2 cell monolayer. The inhibition studies showed that efflux of hesperetin conjugates to the apical side involved active transport, which from the pattern of inhibition, appeared to be mediated mainly by BCRP. Upon inhibition by the specific BCRP inhibitor Ko143, the apical efflux of hesperetin conjugates was significantly reduced by 1.9-fold and transport to the basolateral side was significantly increased by 3.1-fold. Because flavonoids can be expected to be converted by the same phase II enzymes and thus to interfere with hesperetin metabolism, and because specific flavonoids can also inhibit the apically located ABC transporter BCRP, it was further studied to what extent flavonoids can modulate the metabolism and transport of hesperetin. To this end the effect of their co-administration with hesperetin on hesperetin metabolism and transport was studied using the Caco-2 cell monolayers two-compartment transwell model system. It was shown that co-administration of hesperetin with specific flavonoids reduced the ratio of apical to basolateral transport of hesperetin metabolites, and in some cases, also reduced the amount of hesperetin metabolites detected extracellularly. Flavonols and flavones were more potent inhibitors than isoflavones, with quercetin being the most potent compound, while co-administration of flavanols or glycosylated derivatives, reported not to inhibit BCRP, did not modulate the transport of hesperetin metabolites. Because intracellular accumulation of hesperetin metabolites did not account for the decrease in hesperetin metabolites detected extracellularly, inhibition of metabolism of hesperetin is likely the underlying mechanism for the reduced metabolite formation and excretion. In spite of the reduction in metabolism the amount of hesperetin metabolites transported to the basolateral side significantly increased upon co-exposure with specific flavonoids. Whether this in vitro observation, showing that co-administration of quercetin may increase the basolateral transport of hesperetin through Caco-2 cell monolayers at the cost of its apical transport, could be confirmed in an in vivo rat model, hesperetin 7-O-glucoside, a monosaccharide derivative of hesperidin, was administrated by oral gavage to Sprague-Dawley rats in the presence or absence of quercetin. In this in vivo study hesperetin 7-O-glucoside was used instead of hesperetin itself, because previous studies in rats revealed hesperetin to be already absorbed to a significant extent in the stomach excluding the possibility to study the effects of co-administering quercetin on intestinal uptake, while hesperetin 7-O-glucoside was found to be absorbed in the small intestine and thus provided a better model compound to study the effect of quercetin on its intestinal uptake and subsequent bioavailability. Rats were orally administered hesperetin 7-O-glucoside, in the presence or absence of quercetin and systemic blood was taken on 8 time points after dosing from 15 min up to 8 hr in order to determine the area under the concentration time curve (AUC) of plasma hesperetin and its demethylated and remethylated metabolites eriodictyol and homoeriodictyol after treatment of blood samples with beta-glucuronidase/sulfatase. Hesperetin, eriodictyol and homoeriodictyol were determined and quantified by uPLC-DAD. Co-administration of quercetin did especially increase hesperetin bioavailability in an early phase of the concentration time curve (at 15 min) when elimination was not yet dominating over uptake, but did not significantly increase the AUC from time zero to 8 hr. It is concluded that the effect of co-administration of quercetin as an inhibitor of the intestinal BCRP mediated transport might result in increased bioavailability, especially during the early phase of exposure when absorption processes still dominate over elimination processes. Additionally the phase II metabolism of hesperetin was further studied by determining the kinetics of hesperetin conversion by human or rat small intestinal, colonic and hepatic microsomal and cytosolic fractions. Furthermore, the kinetics for glucuronidation and sulfonation of hesperetin by, respectively, 12 individual UGT and 12 individual SULT enzymes were determined in order to identify the responsible UGT and SULT isoforms, of which the expression levels in different tissues are relatively well documented in literature. Results obtained demonstrate that hesperetin is conjugated at positions 7 and 3', and that major enzyme-specific differences in kinetics and regioselectivity for the UGT and SULT catalyzed conjugations exist. UGT1A9, UGT1A1, UGT1A7, UGT1A8 and UGT1A3 are the major enzymes catalyzing hesperetin glucuronidation, the latter only producing 7-O-glucuronide, while UGT1A7 mainly produced 3'-O-glucuronide. Furthermore, UGT1A6 and UGT2B4 only produce hesperetin 7-O-glucuronide, while UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B7 and UGT2B15 conjugate both positions. SULT1A2 and SULT1A1 catalyze preferably and most efficiently the formation of hesperetin 3'-O-sulfate, and SULT1C4 preferably and most efficiently the formation of hesperetin 7-O-sulfate. Based on expression levels SULT1A3 and SULT1B1 will likely play a role in the sulfo-conjugation of hesperetin in vivo. These results help to explain discrepancies in metabolite patterns determined in tissues or systems with different expression levels of UGTs and SULTs, e.g. hepatic and intestinal fractions or Caco-2 cells. The incubations with rat and human tissue samples support an important role for the intestinal cells during first pass metabolism in the formation of hesperetin 3'-O-glucuronide and 7-O-glucuronide, which appear to be the major hesperetin metabolites found in vivo, as compared to metabolism by liver tissue. In addition, the thesis pays attention to the stereochemistry of hesperetin which contains a chiral C-atom and therefore can exist as an S- and R-enantiomer. This is of importance because in nature 2S-hesperidin and its S-hesperetin aglycone are the predominant chemical forms. However, in spite of this, many studies have been performed with the commercially available racemates of hesperidin and hesperetin. The transport and metabolism characteristics so far studied for the commercially available racemic mixture of hesperetin were investigated for the S- and R-hesperetin enantiomers. To this end a chiral separation method on an analytical and semi-preparative scale was developed allowing characterization of the stereoselective differences in metabolism and transport in the intestine and activity in a selected bioassay of the separated hesperetin enantiomers in in vitro model systems. S- or R-hesperetin were compared in several assays: (1) in incubations with human small intestinal fractions containing UGTs or SULTs and their cofactors; (2) with Caco-2 cell monolayers as a model for the intestinal transport barrier; and (3) with mouse Hepa-1c1c7 cells transfected with human EpRE-controlled luciferase to test induction of EpRE-mediated gene expression. The results obtained indicate that although there are some significant differences in metabolism and transport characteristics between S- and R-hesperetin, these differences are relatively small. This indicates that for these endpoints, including intestinal metabolism and transport, experiments performed with racemic hesperetin may adequately reflect what can be expected for the naturally occurring S-enantiomer.
    Full-text · Thesis · May 2010
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