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Soichi Tanabe - One of the best experts on this subject based on the ideXlab platform.
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transepithelial transport of Hesperetin and hesperidin in intestinal caco 2 cell monolayers
2008Co-Authors: Shoko Kobayashi, Soichi Tanabe, Masanori Sugiyama, Yutaka KonishiAbstract:The cell permeability of Hesperetin and hesperidin, anti-allergic compounds from citrus fruits, was measured using Caco-2 monolayers. In the presence of a proton gradient, Hesperetin permeated cells in the apical-to-basolateral direction at the rate (Jap-->bl) of 10.43+/-0.78 nmol/min/mg protein, which was more than 400-fold higher than that of hesperidin (0.023+/-0.008 nmol/min/mg protein). The transepithelial flux of hesperidin, both in the presence or absence of a proton gradient, was nearly the same and was inversely correlated with the transepithelial electrical resistance (TER), indicating that the transport of hesperidin was mainly via paracellular diffusion. In contrast, the transepithelial flux of Hesperetin was almost constant irrespective of the TER. Apically loaded NaN3 or carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the Jap-->bl of Hesperetin, in the presence of proton gradient, by one-half. In the absence of a proton gradient, both Jap-->bl and Jbl-->ap of Hesperetin were almost the same (5.75+/-0.40 and 5.16+/-0.73 nmol/min/mg protein). Jbl-->ap of Hesperetin in the presence of a proton gradient was lower than Jbl-->ap in the absence of a proton gradient. Furthermore, Jbl-->ap in the presence of a proton gradient remarkably increased upon addition of NaN3 specifically to the apical side. These results indicate that Hesperetin is absorbed by transcellular transport, which occurs mainly via proton-coupled active transport, and passive diffusion. Thus, Hesperetin is efficiently absorbed from the intestine, whereas hesperidin is poorly transported via the paracellular pathway and its transport is highly dependent on conversion to Hesperetin via the hydrolytic action of microflora. We have given novel insight to the absorption characteristics of Hesperetin, that is proton-coupled and energy-dependent polarized transport.
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evaluation of the anti allergic activity of citrus unshiu using rat basophilic leukemia rbl 2h3 cells as well as basophils of patients with seasonal allergic rhinitis to pollen
2006Co-Authors: Shoko Kobayashi, Soichi TanabeAbstract:The anti-allergic activity of the 50% methanol extract of Citrus unshiu powder (MEC) was examined. Fifty percent methanol extract of MEC powder showed potent inhibitory activity against histamine release from basophils of patients suffering from seasonal allergic rhinitis to ceder pollen. To examine this anti-allergic mechanism in detail, we next used rat basophlilic leukemia RBL-2H3 cells. MEC significantly inhibited IgE-induced histamine and beta-hexosaminidase release from RBL-2H3 cells. Since MEC contains a variety of flavonoids, we selected nobiletin, Hesperetin, and hesperidin (Hesperetin glycoside) as representative compounds, and further evaluated these inhibitory activities. Among the flavonoids tested, Hesperetin was the most potent, while hesperidin had far less, if any, inhibitory activity. The mechanism by which flavonoids inhibited the degranulation process was then examined. As a result, Hesperetin and nobiletin suppressed the phosphorylation of Akt-1, direct downstream effector of phosphatidylinositol 3-kinase (PI3-K). Thus, it was assumed that these flavonoids suppressed IgE-mediated stimulation of basophils through PI3-K pathway and that proper intake of Citrus unshiu would be favorable for managing seasonal allergic rhinitis to ceder pollen.
Jeremy P E Spencer - One of the best experts on this subject based on the ideXlab platform.
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neuroprotective effects of Hesperetin in mouse primary neurones are independent of creb activation
2008Co-Authors: Stephanie R Raineysmith, Jeremy P E Spencer, Catherine Riceevans, Larswilhelm Schroetke, Parmvir K Bahia, Ahmed Fahmi, Rachel Skilton, Marcus Rattray, Robert J WilliamsAbstract:Dietary flavonoids, including the citrus flavanone Hesperetin, may have stimulatory effects on cytoprotective intracellular signalling pathways. In primary mouse cortical neurone cultures, but not SH-SY5Y human neuroblastoma cells or human primary dermal fibroblasts (Promocells), Hesperetin (100–300 nM, 15 min) caused significant increases in the level of ERK1/2 phosphorylation, but did not increase CREB phosphorylation. Administration of Hesperetin for 18 h did not alter gene expression driven by the cyclic AMP response element (CRE), assessed using a luciferase reporter system, but 300 nM Hesperetin partially reversed staurosporine-induced cell death in primary neurones. Our data show that Hesperetin is a neuroprotective compound at concentrations where antioxidant effects are unlikely to predominate. The effects of Hesperetin are cell-type dependent and, unlike the flavanol (−)epicatechin, neuroprotection in vitro is not associated with enhanced CREB phosphorylation or CRE-mediated gene expression.
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activation of pro survival akt and erk1 2 signalling pathways underlie the anti apoptotic effects of flavanones in cortical neurons
2007Co-Authors: David Vauzour, Katerina Vafeiadou, Robert J Williams, Catherine Riceevans, Jeremy P E SpencerAbstract:There is growing interest in the potential beneficial effects of flavonoids in the aging and diseased brain. We have investigated the potential of the flavanone Hesperetin and two of its metabolites, Hesperetin-7-O-beta-d-glucuronide and 5-nitro-Hesperetin, to inhibit oxidative stress-induced neuronal apoptosis. Exposure of cortical neurons to hydrogen peroxide led to the activation of apoptosis signal-regulating kinase 1 via its de-phosphorylation at Ser963, the phosphorylation of c-jun N-terminal kinase and c-Jun (Ser73) and the activation of caspase 3 and caspase 9. Whilst Hesperetin glucuronide failed to exert protection, both Hesperetin and 5-nitro-Hesperetin were effective at preventing neuronal apoptosis via a mechanism involving the activation/phosphorylation of both Akt/protein kinase B and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Protection against oxidative injury and the activation of Akt and ERK1/2 followed a bell-shaped response and was most apparent at 100 nmol/L concentrations. The activation of ERK1/2 and Akt by flavanones led to the inhibition of the pro-apoptotic proteins, apoptosis signal-regulating kinase 1, by phosphorylation at Ser83 and Bad, by phosphorylation at both Ser136 and Ser112 and to the inhibition of peroxide-induced caspase 9 and caspase 3 activation. Thus, flavanones may protect neurons against oxidative insults via the modulation of neuronal apoptotic machinery.
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activation of pro survival akt and erk1 2 signalling pathways underlie the anti apoptotic effects of flavanones in cortical neurons
2007Co-Authors: David Vauzour, Katerina Vafeiadou, Robert J Williams, Catherine Riceevans, Jeremy P E SpencerAbstract:There is growing interest in the potential beneficial effects of flavonoids in the aging and diseased brain. We have investigated the potential of the flavanone Hesperetin and two of its metabolites, Hesperetin-7-O-beta-d-glucuronide and 5-nitro-Hesperetin, to inhibit oxidative stress-induced neuronal apoptosis. Exposure of cortical neurons to hydrogen peroxide led to the activation of apoptosis signal-regulating kinase 1 via its de-phosphorylation at Ser963, the phosphorylation of c-jun N-terminal kinase and c-Jun (Ser73) and the activation of caspase 3 and caspase 9. Whilst Hesperetin glucuronide failed to exert protection, both Hesperetin and 5-nitro-Hesperetin were effective at preventing neuronal apoptosis via a mechanism involving the activation/phosphorylation of both Akt/protein kinase B and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Protection against oxidative injury and the activation of Akt and ERK1/2 followed a bell-shaped response and was most apparent at 100 nmol/L concentrations. The activation of ERK1/2 and Akt by flavanones led to the inhibition of the pro-apoptotic proteins, apoptosis signal-regulating kinase 1, by phosphorylation at Ser83 and Bad, by phosphorylation at both Ser136 and Ser112 and to the inhibition of peroxide-induced caspase 9 and caspase 3 activation. Thus, flavanones may protect neurons against oxidative insults via the modulation of neuronal apoptotic machinery.
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modulation of peroxynitrite induced fibroblast injury by Hesperetin a role for intracellular scavenging and modulation of erk signalling
2006Co-Authors: Susan E Pollard, Matthew Whiteman, Jeremy P E SpencerAbstract:Peroxynitrite is thought to contribute to the progression of many diseases including cardiovascular disease, cancer, and neurodegenerative disorders. We report that pre-treatment of fibroblasts with the citrus flavanone, Hesperetin, prior to peroxynitrite exposure protects against peroxynitrite-mediated cytotoxicity. This protection was partially mediated by the intracellular scavenging of peroxynitrite by Hesperetin as exposure of fibroblasts to peroxynitrite following Hesperetin loading led to the formation of two intracellular nitro-Hesperetin derivatives. In addition, protection appeared to be mediated by Hesperetin-induced changes in MAP kinase signalling. Exposure of fibroblasts to Hesperetin led to concentration-dependent increases in the phosphorylation of ERK1/2 and was observed to restore peroxynitrite-mediated decreases in ERK1/2 phosphorylation. We propose that the protective potential of Hesperetin in fibroblasts may be mediated both by intracellular scavenging of peroxynitrite and by modulation of fibroblast signalling.
Shoko Kobayashi - One of the best experts on this subject based on the ideXlab platform.
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transepithelial transport of Hesperetin and hesperidin in intestinal caco 2 cell monolayers
2008Co-Authors: Shoko Kobayashi, Soichi Tanabe, Masanori Sugiyama, Yutaka KonishiAbstract:The cell permeability of Hesperetin and hesperidin, anti-allergic compounds from citrus fruits, was measured using Caco-2 monolayers. In the presence of a proton gradient, Hesperetin permeated cells in the apical-to-basolateral direction at the rate (Jap-->bl) of 10.43+/-0.78 nmol/min/mg protein, which was more than 400-fold higher than that of hesperidin (0.023+/-0.008 nmol/min/mg protein). The transepithelial flux of hesperidin, both in the presence or absence of a proton gradient, was nearly the same and was inversely correlated with the transepithelial electrical resistance (TER), indicating that the transport of hesperidin was mainly via paracellular diffusion. In contrast, the transepithelial flux of Hesperetin was almost constant irrespective of the TER. Apically loaded NaN3 or carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the Jap-->bl of Hesperetin, in the presence of proton gradient, by one-half. In the absence of a proton gradient, both Jap-->bl and Jbl-->ap of Hesperetin were almost the same (5.75+/-0.40 and 5.16+/-0.73 nmol/min/mg protein). Jbl-->ap of Hesperetin in the presence of a proton gradient was lower than Jbl-->ap in the absence of a proton gradient. Furthermore, Jbl-->ap in the presence of a proton gradient remarkably increased upon addition of NaN3 specifically to the apical side. These results indicate that Hesperetin is absorbed by transcellular transport, which occurs mainly via proton-coupled active transport, and passive diffusion. Thus, Hesperetin is efficiently absorbed from the intestine, whereas hesperidin is poorly transported via the paracellular pathway and its transport is highly dependent on conversion to Hesperetin via the hydrolytic action of microflora. We have given novel insight to the absorption characteristics of Hesperetin, that is proton-coupled and energy-dependent polarized transport.
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evaluation of the anti allergic activity of citrus unshiu using rat basophilic leukemia rbl 2h3 cells as well as basophils of patients with seasonal allergic rhinitis to pollen
2006Co-Authors: Shoko Kobayashi, Soichi TanabeAbstract:The anti-allergic activity of the 50% methanol extract of Citrus unshiu powder (MEC) was examined. Fifty percent methanol extract of MEC powder showed potent inhibitory activity against histamine release from basophils of patients suffering from seasonal allergic rhinitis to ceder pollen. To examine this anti-allergic mechanism in detail, we next used rat basophlilic leukemia RBL-2H3 cells. MEC significantly inhibited IgE-induced histamine and beta-hexosaminidase release from RBL-2H3 cells. Since MEC contains a variety of flavonoids, we selected nobiletin, Hesperetin, and hesperidin (Hesperetin glycoside) as representative compounds, and further evaluated these inhibitory activities. Among the flavonoids tested, Hesperetin was the most potent, while hesperidin had far less, if any, inhibitory activity. The mechanism by which flavonoids inhibited the degranulation process was then examined. As a result, Hesperetin and nobiletin suppressed the phosphorylation of Akt-1, direct downstream effector of phosphatidylinositol 3-kinase (PI3-K). Thus, it was assumed that these flavonoids suppressed IgE-mediated stimulation of basophils through PI3-K pathway and that proper intake of Citrus unshiu would be favorable for managing seasonal allergic rhinitis to ceder pollen.
Gary Williamson - One of the best experts on this subject based on the ideXlab platform.
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uplc ms ms quantification of total Hesperetin and Hesperetin enantiomers in biological matrices
2012Co-Authors: Antoine Leveques, Gary Williamson, Lucas Actisgoretta, Maarit Rein, Fabiola Dionisi, Francesca GiuffridaAbstract:Abstract Hesperidin (Hesperetin-7- O -rutinoside), a flavonoid affecting vascular function, is abundant in citrus fruits and derived products such as juices. After oral administration, hesperidin is hydrolyzed by the colonic microbiota producing Hesperetin-7- O -glucoside, the glucoside group is further cleaved and the resulting Hesperetin is absorbed and metabolized. Flavanones have a chiral carbon generating (R)- and (S)-enantiomers, with potentially different biological activities. A rapid UPLC–MS/MS method for the analysis of (R)- and (S)-Hesperetin enantiomers in human plasma and urine was developed and validated. Biological matrices were incubated with β-glucuronidase/sulfatase, and Hesperetin was isolated by solid-phase extraction using 96-well plate mixed-mode cartridges having reversed-phase and anion-exchange functionalities. Racemic Hesperetin was analyzed with a UPLC HSS T3 reversed phase column and Hesperetin enantiomers with a HPLC Chiralpak IA-3 column using H 2 O with 0.1% CHOOH as solvent A and acetonitrile with 0.1% CHOOH as solvent B. The method was linear between 50 and 5000 nM for racemic Hesperetin in plasma and between 25 and 2500 nM for (S)- and (R)-Hesperetin in plasma. Linearity was achieved between 100 and 10,000 nM for racemic Hesperetin in urine and between 50 and 5000 nM for (S)- and (R)-Hesperetin in urine. Values of repeatability and intermediate reproducibility for racemic Hesperetin and enantiomers in plasma and urine were below 15% of deviation in general, and maximum 20% for the lowest concentrations. In addition, the method was applied for the quantification of total Hesperetin and of Hesperetin enantiomers in human plasma and urine samples, obtained after oral ingestion of purified Hesperetin-7- O -glucoside. In conclusion, the developed and validated method was sensitive, accurate and precise for the quantification of enantiomers of Hesperetin in biological fluids.
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interaction of Hesperetin glucuronide conjugates with human bcrp mrp2 and mrp3 as detected in membrane vesicles of overexpressing baculovirus infected sf9 cells
2011Co-Authors: Denis Barron, Walter Brand, Fabiola Dionisi, Peter J Van Bladeren, Ivonne M C M Rietjens, Berend Oosterhuis, Peter Krajcsi, Gary WilliamsonAbstract:The citrus flavonoid Hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone) is the agly- cone of hesperidin, the major flavonoid present in sweet oranges. Hesperetin 7-O-glucuronide (H7G) and Hesperetin 3'-O-glucuronide (H3'G) are the two most abundant metabolites of Hesperetin in vivo. In this study, their interaction with specific ABC transporters, believed to play a role in the disposition and bioavailability of Hesperetin, was studied using Sf9 membranes from cells overex- pressing human BCRP (ABCG2), MRP2 (ABCC2) and MRP3 (ABCC3). Both H7G and H3'G were tested for their potential to activate and inhibit ATPase activity, and to inhibit vesicular transport by these transporters. Both H7G and H3'G demonstrated interaction with all tested ABC transpor- ters, especially with BCRP and MRP3. An interesting difference between H7G and H3'G was seen with respect to the interaction with BCRP: H7G stimulated the ATPase activity of BCRP up to 76% of the maximal effect generated by the reference activator sulfasalazine, with an EC50 of 0.45mM ,s ug- gesting that H7G is a high affinity substrate of BCRP, whereas H3'G did not stimulate BCRP ATPase activity. Only moderate inhibition of BCRPATPase activity at high H3'G concentrations was observed. This study provides information on the potential of Hesperetin glucuronide conjugates to act as specific ABC transporter substrates or inhibitors and indicates that regio-specific glucuronidation could affect the disposition of Hesperetin. Copyright © 2011 John Wiley & Sons, Ltd.
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the effect of co administered flavonoids on the metabolism of Hesperetin and the disposition of its metabolites in caco 2 cell monolayers
2010Co-Authors: Walter Brand, Gary Williamson, Beatriz Padilla, Peter J Van Bladeren, Ivonne M C M RietjensAbstract:Metabolism by phase II enzymes and transport from intestinal cells back into the lumen by ATP binding cassette (ABC) transporters limits the bioavailability of the flavanone Hesperetin, the aglycone of hesperidin. This study investigates to what extent other flavonoids modulate the metabolism and transport of Hesperetin by characterizing the effect of co-administrating a series of flavonoids using Caco-2 cell monolayers in a two-compartment transwell system. Flavonoids may interfere with Hesperetin metabolism and can also inhibit the apically located ABC transporter breast cancer resistance protein (ABCG2) which was previously shown to be responsible for the apical transport of Hesperetin metabolites. Co-exposure of Caco-2 cell monolayers to Hesperetin with specific flavonoids reduced the ratio of apical efflux to basolateral transport of Hesperetin metabolites, and in some cases, also reduced the amount of Hesperetin metabolites detected extracellularly. As intracellular accumulation of Hesperetin metabolites did not account for this decrease, 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 and therefore co-administration of specific flavonoids could be a strategy to improve the bioavailability of Hesperetin.
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stereoselective conjugation transport and bioactivity of s and r Hesperetin enantiomers in vitro
2010Co-Authors: Walter Brand, Gary Williamson, Maarit Rein, Fabiola Dionisi, Jia Shao, Elisabeth Hoekvan Den F Hil, Kathelijn N Van Elk, Bert Spenkelink, Laura H J De HaanAbstract: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...
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Phase II Metabolism of Hesperetin by Individual UDP-Glucuronosyltransferases and Sulfotransferases and Rat and Human Tissue Samples
2010Co-Authors: Walter Brand, Marelle G. Boersma, Walter Meinl, Denis Barron, Gary Williamson, Jacques Vervoort, Hansruedi Glatt, Peter J Van BladerenAbstract:Phase II metabolism by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) is the predominant metabolic pathway during the first-pass metabolism of Hesperetin (4′-methoxy-3′,5,7-trihydroxyflavanone). In the present study, we have determined the kinetics for glucuronidation and sulfonation of Hesperetin by 12 individual UGT and 12 individual SULT enzymes as well as by human or rat small intestinal, colonic, and hepatic microsomal and cytosolic fractions. Results 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, whereas UGT1A7 produced mainly 3′- O -glucuronide. Furthermore, UGT1A6 and UGT2B4 only produce Hesperetin 7- O -glucuronide, whereas 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 catalyzes preferably and most efficiently the formation of Hesperetin 7- O -sulfate. Based on expression levels SULT1A3 and SULT1B1 also will probably play a role in the sulfo-conjugation of Hesperetin in vivo. The results help to explain discrepancies in metabolite patterns determined in tissues or systems with different expression 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 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.
Walter Brand - One of the best experts on this subject based on the ideXlab platform.
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interaction of Hesperetin glucuronide conjugates with human bcrp mrp2 and mrp3 as detected in membrane vesicles of overexpressing baculovirus infected sf9 cells
2011Co-Authors: Denis Barron, Walter Brand, Fabiola Dionisi, Peter J Van Bladeren, Ivonne M C M Rietjens, Berend Oosterhuis, Peter Krajcsi, Gary WilliamsonAbstract:The citrus flavonoid Hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone) is the agly- cone of hesperidin, the major flavonoid present in sweet oranges. Hesperetin 7-O-glucuronide (H7G) and Hesperetin 3'-O-glucuronide (H3'G) are the two most abundant metabolites of Hesperetin in vivo. In this study, their interaction with specific ABC transporters, believed to play a role in the disposition and bioavailability of Hesperetin, was studied using Sf9 membranes from cells overex- pressing human BCRP (ABCG2), MRP2 (ABCC2) and MRP3 (ABCC3). Both H7G and H3'G were tested for their potential to activate and inhibit ATPase activity, and to inhibit vesicular transport by these transporters. Both H7G and H3'G demonstrated interaction with all tested ABC transpor- ters, especially with BCRP and MRP3. An interesting difference between H7G and H3'G was seen with respect to the interaction with BCRP: H7G stimulated the ATPase activity of BCRP up to 76% of the maximal effect generated by the reference activator sulfasalazine, with an EC50 of 0.45mM ,s ug- gesting that H7G is a high affinity substrate of BCRP, whereas H3'G did not stimulate BCRP ATPase activity. Only moderate inhibition of BCRPATPase activity at high H3'G concentrations was observed. This study provides information on the potential of Hesperetin glucuronide conjugates to act as specific ABC transporter substrates or inhibitors and indicates that regio-specific glucuronidation could affect the disposition of Hesperetin. Copyright © 2011 John Wiley & Sons, Ltd.
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the effect of co administered flavonoids on the metabolism of Hesperetin and the disposition of its metabolites in caco 2 cell monolayers
2010Co-Authors: Walter Brand, Gary Williamson, Beatriz Padilla, Peter J Van Bladeren, Ivonne M C M RietjensAbstract:Metabolism by phase II enzymes and transport from intestinal cells back into the lumen by ATP binding cassette (ABC) transporters limits the bioavailability of the flavanone Hesperetin, the aglycone of hesperidin. This study investigates to what extent other flavonoids modulate the metabolism and transport of Hesperetin by characterizing the effect of co-administrating a series of flavonoids using Caco-2 cell monolayers in a two-compartment transwell system. Flavonoids may interfere with Hesperetin metabolism and can also inhibit the apically located ABC transporter breast cancer resistance protein (ABCG2) which was previously shown to be responsible for the apical transport of Hesperetin metabolites. Co-exposure of Caco-2 cell monolayers to Hesperetin with specific flavonoids reduced the ratio of apical efflux to basolateral transport of Hesperetin metabolites, and in some cases, also reduced the amount of Hesperetin metabolites detected extracellularly. As intracellular accumulation of Hesperetin metabolites did not account for this decrease, 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 and therefore co-administration of specific flavonoids could be a strategy to improve the bioavailability of Hesperetin.
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stereoselective conjugation transport and bioactivity of s and r Hesperetin enantiomers in vitro
2010Co-Authors: Walter Brand, Gary Williamson, Maarit Rein, Fabiola Dionisi, Jia Shao, Elisabeth Hoekvan Den F Hil, Kathelijn N Van Elk, Bert Spenkelink, Laura H J De HaanAbstract: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...
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Phase II Metabolism of Hesperetin by Individual UDP-Glucuronosyltransferases and Sulfotransferases and Rat and Human Tissue Samples
2010Co-Authors: Walter Brand, Marelle G. Boersma, Walter Meinl, Denis Barron, Gary Williamson, Jacques Vervoort, Hansruedi Glatt, Peter J Van BladerenAbstract:Phase II metabolism by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) is the predominant metabolic pathway during the first-pass metabolism of Hesperetin (4′-methoxy-3′,5,7-trihydroxyflavanone). In the present study, we have determined the kinetics for glucuronidation and sulfonation of Hesperetin by 12 individual UGT and 12 individual SULT enzymes as well as by human or rat small intestinal, colonic, and hepatic microsomal and cytosolic fractions. Results 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, whereas UGT1A7 produced mainly 3′- O -glucuronide. Furthermore, UGT1A6 and UGT2B4 only produce Hesperetin 7- O -glucuronide, whereas 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 catalyzes preferably and most efficiently the formation of Hesperetin 7- O -sulfate. Based on expression levels SULT1A3 and SULT1B1 also will probably play a role in the sulfo-conjugation of Hesperetin in vivo. The results help to explain discrepancies in metabolite patterns determined in tissues or systems with different expression 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 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.
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metabolism and transport of the citrus flavonoid Hesperetin in caco 2 cell monolayers
2008Co-Authors: Walter Brand, Denis Barron, Gary Williamson, Maarit Rein, Peter J Van Bladeren, Petronella A I Van Der Wel, Ivonne M C M RietjensAbstract:Metabolism and transport from intestinal cells back into the lumen by ATP-binding cassette (ABC) transporters is believed to limit the bioavailability of flavonoids. We studied metabolism and transport of the citrus flavonoid Hesperetin, the aglycone of hesperidin, using a two-compartment transwell Caco-2 cell monolayer system, simulating the intestinal barrier. The role of apically located ABC transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance protein 2 (ABCC2), and breast cancer resistance protein (BCRP/ ABCG2) in the efflux of Hesperetin and its metabolites was studied by coadministration of compounds known to inhibit several classes of ABC transporters, including cyclosporin A, GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide], Ko143 [3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester], MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), and PSC-833 (Valspodar). Apically applied Hesperetin (10 μM) was metabolized into Hesperetin 7-O-glucuronide and Hesperetin 7-O-sulfate, identified using high-performance liquid chromatographydiode array detector (DAD), ultraperformance liquid chromatography-DAD-tandem mass spectrometry, and authentic standards, which were transported predominantly to the apical side of the Caco-2 cell monolayer (1.12 cm2), at average (S.D.) rates of 14.3 (3.7) and 2.1 (0.8) pmol/min/monolayer, respectively. Hesperetin aglycone also permeated to the basolateral side, and this process was unaffected by the inhibitors used, possibly implying a passive diffusion process. Inhibition studies, however, showed that efflux of Hesperetin conjugates to the apical side involved active transport, which from the pattern of inhibition appeared to involve mainly BCRP. Upon inhibition by the BCRP inhibitor Ko143 (5 μM), the apical efflux of Hesperetin conjugates was 1.9-fold reduced (p ≤ 0.01), and transport to the basolateral side was 3.1-fold increased (p ≤ 0.001). These findings elucidate a novel pathway of Hesperetin metabolism and transport and show that BCRP-mediated transport could be a limiting step for Hesperetin bioavailability.