Biochemical and Molecular Action of Nutrients

Quercetin-3-Glucoside Is Transported by the Glucose Carrier SGLT1 across the Brush Border Membrane of Rat Small Intestine S. Wolffram,1 M. Blo¨ck and P. Ader Institute of Animal Nutrition, Physiology and Metabolism, Christian-Albrechts-University Kiel, D-24098 Kiel, Germany ABSTRACT In the present study we investigated a possible involvement of the intestinal sodium-dependent glucose transporter (SGLT)1 in the absorption of quercetin-3-glucoside (Q3G). Pieces of rat jejunum or proximal colon were mounted in Ussing-type chambers and incubated under short-circuited conditions. Test flavonols were added to the mucosal or serosal bathing solution (initial concentration, 100 ␮mol/L) and disappearance from the donor compartment was monitored for 2 h. With jejunal tissue, only 13.6 ⫾ 3.5% of the initial dose of Q3G was found in the mucosal compartment 2 h after mucosal addition. Simultaneous addition of D-glucose (10 mmol/L) significantly reduced the disappearance of Q3G (remaining concentration, 33.4 ⫾ 6.9%) as did a Na⫹-free buffer solution containing phloridzin (final mucosal concentration of Q3G, 54.2 ⫾ 7.7%). In these experiments, disappearance of Q3G was paralleled by the appearance of quercetin in the mucosal solutions. In contrast, D-fructose (10 mmol/L) did not influence the disappearance of Q3G (Na⫹-free conditions). With proximal colon, 78.2 ⫾ 11.5% of the initial concentration of Q3G was still present in the mucosal solution after 2 h. When added to the serosal side, the concentration of Q3G decreased only slightly (jejunum, 96.1 ⫾ 2.1%; proximal colon, 90.7 ⫾ 1.2%). The concentration of rutin did not change after mucosal or serosal addition. Neither transport of intact glycosides nor of free quercetin from the donor into the acceptor compartment was observed under our experimental conditions. Taken together, the results clearly indicate a role of SGLT1 in mucosal uptake of the Q3G. J. Nutr. 132: 630 – 635, 2002. KEY WORDS:

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flavonoids

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quercetin

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isoquercitrin

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intestine

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SGLT1

effect of plant flavonoids against coronary heart disease appears to be justified (11–13) by most of those studies, there is presently no epidemiologic study showing a clear effect of flavonols against various forms of cancer (14,15). It should be mentioned, however, that many in vitro and in vivo studies indicate a strong anticarcinogenic potential of flavonoids (1). Apart from local effects within the gastrointestinal tract, absorption of the flavonoids and/or their biologically active metabolites is a prerequisite for any systemic effect. Some publications indicate a higher bioavailability of quercetin derived from quercetin-glucosides as present in onions compared with the free aglycon or the quercetin-glucorhamnoside rutin (16 –19). From these findings, an involvement of the intestinal sodium-dependent glucose transporter-1 (SGLT1)2 in the absorption of quercetin glucosides has been deduced. Although experimental evidence for the absorption of intact glucosides into the systemic circulation is scanty (20,21), several studies have demonstrated interactions between SGLT1 and quercetin glucosides at the intestinal brush border membrane (BBM) (22–24). In accordance with those studies, we recently demonstrated a competitive inhibition of quercetin-3-glucoside

The flavonoids are a large group of natural polyphenols; they are nearly ubiquitous in plants (1) and thus are ingested by humans and animals with their regular diet. In plants and most plant-derived foods, flavonoids are largely present as conjugates with the flavonoid aglycon linked to a variable sugar moiety by a ␤-glycosidic bond (2). More than 6000 chemically different flavonoids have been identified (3) whereby variations in the sugar moiety are mainly responsible for the large number of different flavonoids (1,3,4). A number of in vitro studies have revealed a multitude of effects of flavonoids, including pronounced antioxidant activities in various biological systems (1,5). It has been repeatedly shown that flavonoids, including quercetin, can inhibit several key enzymes, e.g., phospholipases A2 and C, tyrosine protein kinases, lipoxygenase, cyclooxygenase, cyclic nucleotide phosphodiesterase, and cytochrome P450 systems (1,4). Independent of the effect investigated and the experimental model used, quercetin seems to be among the most potent naturally occurring flavonoids. High concentrations of quercetin are present in tea, apples and onions (6 – 8) and human daily intake ranges between 10 and 20 mg (9 –11). On the basis of epidemiologic studies, several health-promoting effects of dietary flavonoids have been claimed. Although the conclusion of a protective

2 Abbreviations used: BBM, brush border membrane; DMSO, dimethyl sulfoxide; KHPB, Krebs-Henseleit phosphate buffer; LPH, lactase phloridzin hydrolase; MRP, multidrug resistance-associated protein; Q3G, quercetin-3-O-glucoside; SGLT, sodium-dependent glucose transporter.

1 To whom correspondence should be addressed. E-mail: [email protected].

0022-3166/02 $3.00 © 2002 American Society for Nutritional Sciences. Manuscript received 24 September 2001. Initial review completed 8 November 2001. Revision accepted 3 January 2002. 630

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(Q3G) and quercetin-4⬘-glucoside on mucosal glucose uptake (25). Although inhibition by quercetin glucosides of glucose or galactose transport across the BBM clearly indicates an interaction with SGLT1, it does not necessarily mean that these glucosides are transported by this carrier mechanism (25). To our knowledge, there is only one study to date demonstrating glucose-inhibitable uptake of quercetin-4⬘-␤glucoside into Caco-2 cells and Chinese hamster ovary cells stably transfected with rabbit SGLT1 (24). Interestingly, quercetin-4⬘-␤-glucoside is secreted across the luminal membrane of Caco-2 cells by the multidrug resistance-associated protein (MRP)2 (26), explaining the lack of net absorption of this glucoside across Caco-2 cell monolayers (26,27). The aim of the present study was to obtain some more direct evidence for an involvement of the glucose carrier SGLT1 in the transport of Q3G across the intestinal BBM. For this purpose, rat jejunum and proximal colon were mounted in Ussing-type chambers and the disappearance of Q3G as influenced by D-glucose, D-fructose, Na⫹ and phloridzin was monitored. MATERIALS AND METHODS Animals and tissue preparation. The following protocol was approved by the Animal Subjects Committee of the Christian-Albrechts-University, Kiel, Germany. Intestinal tissues (mid-jejunum, proximal colon) were obtained from male Wistar rats (Wistar Hannover, Institute of Physiology, University Kiel) with a body weight of 350 –380 g. Rats had free access to a pelleted commercial standard diet (ssniff R/M-H, Spezialdia¨ ten GmbH; Soest; Germany; crude nutrients: 19% protein, 66.1% carbohydrates, 3.3% fat, 4.9% fiber, 6.7% ash, supplemented with vitamins and minerals; 12.2 MJ metabolizable energy) and to tap water and were deprived of food 12 h before the experiment. The rats were anesthetized with diethylether and subsequently killed by exsanguination after removal of the intestinal segments, which were immediately transferred into chilled oxygenated Krebs-Henseleit phosphate buffer (KHPB, in mmol/L: 120.8 NaCl, 4.8 KCl, 1.2 MgSO4, 16.5 Na2HPO4, pH adjusted to 6.8 with HCl and NaOH) and opened longitudinally along the mesenteric border. Intestinal contents were thoroughly removed by rinsing with the buffer solution taking special care not to damage the mucosa. Experimental set-up. Small intestinal pieces (⬃1.5 cm) were mounted in modified Ussing chambers and bathed with a volume of 4 mL of KHPB with the addition of 10 mmol/L of D-mannose or ⫹ D-glucose on the mucosal and serosal side, respectively. Na -free buffer solutions were prepared by isomolar replacement of NaCl and Na2HPO4 by choline chloride and H3PO4 (pH adjusted to 6.8 with tetramethylammoniumhydroxide). Each chamber was equipped with a water jacket, allowing a constant temperature of 37°C during the course of the experiments. Tissues were continuously gassed with pure oxygen and short-circuited by an automatic voltage clamp device (Aachen Microclamp, AC Copy Datentechnik, Aachen, Germany) with correction for solution resistance. The exposed surface of the tissue was 1 cm2. A current of ⫾ 100 ␮A was applied to the tissue at 1-min intervals and the change in voltage measured. The tissue conductance (Gt) was calculated from these values according to Ohm’s law. Values for Gt and the continuously applied short-circuit current (Isc) were registered in 6-s intervals. After an equilibration period of at least 30 min, the test substances (Fig. 1) quercetin, rutin (purity ⱖ 98.5%, Roth, Karlsruhe, Germany), and Q3G (purity ⱖ 98.5%, Extraysynthese, Genay, France) at a final concentration of 100 ␮mol/L [dissolved in dimethyl sulfoxide (DMSO), final concentration of DMSO 0.25%] were added either to the mucosal or serosal compartment. Samples (150 ␮L) for HPLC analysis of flavonoids were removed immediately after the addition of the test substance and after 1 and 2 h from the mucosal and serosal compartment, respectively. The viability of the tissue was routinely checked by means of the Gt (tissues with a Gt ⬎ 50 mS/cm2 were excluded) and by the Isc response to D-Glucose (20 mmol/L, mucosal, mid-jejunum) or to forskolin (1 ␮mol/L, serosal, colon) at the end of the experi-

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FIGURE 1 Chemical structures of quercetin, quercetin-3-glucoside (Q3G; isoquercitrin) and quercetin-3-glucorhamnoside (rutin).

ments. Data from tissues that did not respond to D-glucose or forskolin, respectively, with an increase in Isc of at least 30% were discarded. Figure 2 shows a representative example of the Isc response of jejunal and colonic tissue after an experimental period of 2 h. Sample preparation and HPLC analysis. Samples drawn from the mucosal and serosal compartments of the Ussing chambers were analyzed directly for their flavonoid content. Some samples were treated with 5 ⫻ 105 U/L ␤-glucuronidase and 2.5 ⫻ 104 U/L sulfatase (crude extract from Helix pomatia, Sigma-Aldrich AG, Deisenhofen, Germany) and incubated for 30 min at 37°C before analysis to liberate quercetin from glucuronides and sulfates possibly formed within the intestinal epithelium and secreted into the incubation medium (28 –32). This enzymatic treatment also cleaves the ␤-glycosidic bond present in Q3G. For HPLC analysis, 30- or 80-␮L aliquots of the final samples were injected by an autosampler (950AS) connected to a C18-Kromasil column (dimension: 250 ⫻ 4 mm, particle size 5 ␮m) protected by a C-18 Inertsil ODS-2 precolumn (10 ⫻ 4 mm, 5 ␮m particle size). The columns were placed in a column oven set at 30°C. The eluent was composed of 0.025 mmol/L NaH2PO4 solution, pH 2.4, acetonitrile and methanol (68:27:5 v/v/v) delivered at a rate of 1 mL/min (980-PU-ND pump). Flavonoids were detected using a UV-detector (MD 1510) at a wave length of 254 nm. The HPLC equipment was purchased from Jasco, Gro␤-Umstadt, Germany. HPLC chromatograms were evaluated using the Borwin chromatography software (version 1.22.03 B, JMBS Developments, Grenoble, France). Flavonoids were identified by their retention times compared with authentic substances. Concentrations were calculated from the peak areas using calibration curves for Q3G, quercetin and the methylated quercetin metabolites isorhamnetin and tamarixetin, which might be formed within the intestinal epithelium during absorption (30). Statistics. Data are presented as mean values ⫾ SEM; n indicates the total number of preparations with 2–3 preparations from each rat. The unpaired two-tailed Student’s t test (33) was used to compare means. Calculations were performed on a personal computer using the program GraphPad Instat (34). Differences with P ⱕ 0.05 were considered significant.

RESULTS In the present study, the disappearance of the quercetin glycosides Q3G and rutin from the mucosal or serosal compartment, respectively, was considered to represent tissue uptake. Because autolysis of the parent compounds could contribute to their disappearance, we first tested the stability of the test substances in the continuously oxygenated Ussingchamber system in the absence of intestinal tissue. The concentrations of the glycosides were stable for 3 h, whereas the concentration of the aglycon quercetin rapidly decreased, and

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FIGURE 3 Influence of D-glucose (10 mmol/L) on the disappearance of quercetin-3-glucoside (Q3G; 100 ␮mol/L) from the mucosal side of rat jejunum. Values are means ⫾ SEM, n ⫽ 8 –9 (4 rats). *Different from D-glucose, P ⬍ 0.05.

FIGURE 2 Time course of the short-circuit current (Isc) across rat jejunum and proximal colon mounted in Ussing chambers. Data (single determinations) are derived from representative experiments. Numbered arrows indicate the addition of 100 ␮mol/L quercetin-3-glucoside (Q3G) (1), 20 mmol/L D-glucose (2), and 100 ␮mol/L carbachol (3), respectively.

only ⬃10% of the initial amount was present after 2 h (results not shown). Spontaneous decomposition of quercetin, which can be prevented by protein binding, may occur in neutral or slightly alkaline buffer solutions containing phosphate (35). Bacterial degradation of quercetin could be excluded under our experimental conditions because the experiments on the stability of the test substances were performed in the absence of intestinal tissue. Due to the obvious instability of quercetin under our experimental conditions, we decided not to include quercetin as a test substance for transport experiments. In the absence of D-glucose from the mucosal medium (KHPB), the concentration of Q3G decreased continuously with time. After 1 and 2 h only 43 and 14% of the initial concentration, respectively, were still present in the mucosal medium (Fig. 3). Addition of 10 mmol/L D-glucose, however, significantly lessened the disappearance of Q3G, with 35% of the initial concentration remaining after 2 h (Fig. 3). The disappearance of Q3G was paralleled by the appearance of free quercetin in the mucosal medium, whereas no quercetin conjugates (glucuronides or sulfates) or metabolites of quercetin (kaempferol, isorhamnetin, tamarixetin) or conjugated forms thereof were detected. After 2 h, quercetin concentrations of 67.4 ⫾ 3.4 and 46.0 ⫾ 6.5 ␮mol/L (n ⫽ 8 –9) were measured in the absence or presence of D-glucose, respectively. In the serosal compartment, no intact Q3G, quercetin, quercetin metabolites (tamarixetin, kaempferol, isorhamnetin) or conjugated forms thereof were detected after mucosal addition of Q3G. Because the experiments on the disappearance of Q3G described above suggested the involvement of a glucose-inhibitable mechanism, e.g., SGLT1, we further investigated the disappearance of Q3G under conditions with SGLT1 maximally silenced by omission of Na⫹ from the mucosal medium and addition of the specific SGLT1 inhibitor phloridzin (0.1 mmol/L). Under these conditions, 54% of the initial Q3G dose remained in the mucosal compartment after 2 h (Fig. 4)

compared with 14% under control conditions (with Na⫹, no phloridzin). Because the initial Q3G concentration declined by 46% after 2 h even in the presence of phloridzin and the absence of Na⫹, we sought to determine whether the Na⫹-independent fructose transporter GLUT-5 could be involved in the disappearance of Q3G. In these experiments a Na⫹-free KHPB was used at the mucosal side to reduce the activity of SGLT1. Addition of 10 mmol/L D-fructose was without effect on the disappearance of Q3G (Fig. 5). Interestingly, the remaining concentration of Q3G in the presence of D-mannitol after 1 and 2 h (Fig. 5) was considerably higher than the respective values in the presence of Na⫹ (Figs. 3, 4), indicating a clear Na⫹ dependence on the disappearance of Q3G from the mucosal solution. Because expression of SGLT1 in the intestine is largely restricted to the BBM of the small intestine, we also compared the disappearance of Q3G from the jejunum and proximal colon after mucosal as well as after serosal addition. In contrast to the results obtained with jejunal tissue, the concentration of

FIGURE 4 Influence of phloridzin (100 ␮mol/L) and simultaneous omission of Na⫹ from the mucosal bathing solution on the disappearance of quercetin-3-glucoside (Q3G; 100 ␮mol/L) from the mucosal side of rat jejunum. Values are means ⫾ SEM, n ⫽ 6 –7 (3 rats). ***Different from Na⫹-free ⫹ phloridzin, P ⬍ 0.001.

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FIGURE 5 Influence of D-fructose (10 mmol/L) on the disappearance of quercetin-3-glucoside (Q3G; 100 ␮mol/L) from the mucosal side of rat jejunum. Values are means ⫾ SEM, n ⫽ 6 (3 rats). In these experiments a Na⫹-free buffer solution was present at the mucosal side.

Q3G decreased only slightly in the mucosal compartment of Ussing chambers prepared with colonic tissue (Fig. 6). Furthermore, when added to the serosal side, Q3G concentration remained high irrespective of the intestinal segment used. In contrast to the results obtained with Q3G, the concentration of the quercetin glucorhamnoside rutin (100 ␮mol/L) did not decline after addition to the mucosal or serosal compartment of Ussing chambers with either jejunum or with proximal colon (results not shown). DISCUSSION Plant-derived flavonoids are gaining interest due to their putative health-promoting effects linked to their antioxidant, antiproliferative and other properties, including inhibition of key enzymes such as cyclooxygenase and protein kinases involved in cell proliferation and apoptosis (1,5). Quercetin and its glycosides not only are the most abundant dietary flavonoids in Western diets (10) but also rank among the most potent flavonoids in terms of their antioxidant and other putative health-promoting effects (5). Although our knowledge about the bioavailability of quercetin is incomplete, several studies indicate a substantially higher bioavailability of quercetin from glucosides, e.g., quercetin-4⬘-glucoside or Q3G, compared with free quercetin or the quercetin glucorhamnoside rutin (16 –18). According to Olthoff et al. (19), the bioavailability of quercetin in humans from Q3G and quercetin-4⬘-glucoside is not different. One possible explanation for these findings could be transport of quercetin glucosides by the intestinal glucose carrier SGLT1, resulting in an improved intestinal absorption of quercetin. Indeed, several studies indicate that quercetin glucosides interact with the SGLT1 (22,23,25). This indirect evidence is based on inhibition of glucose or galactose transport by quercetin glucosides, e.g., qercetin-3-glucoside or quercetin-4⬘-glucoside. It should be mentioned, however, that inhibition of glucose uptake across the BBM by quercetin glucosides does not necessarily mean that the inhibitor itself is transported across the membrane. To our knowledge there is only one study demonstrating glucoseinhibitable uptake of quercetin 4⬘-␤-glucoside into Caco-2 cells and Chinese hamster ovary cells stably transfected with rabbit SGLT1 (24). The present study was aimed to gain additional evidence for the involvement of SGLT1 in mucosal

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uptake of quercetin glucosides. We therefore used the disappearance of Q3G as a measure of mucosal Q3G uptake in Ussing chamber experiments under various incubation conditions. With jejunal tissues mounted into Ussing chambers, the disappearance of Q3G from the mucosal solution as well as the parallel appearance of the free aglycone quercetin was significantly reduced in the presence of D-glucose. Furthermore, reducing the transport activity of SGLT1 by omitting Na⫹ from the mucosal bathing solution and simultaneously adding phloridzin, a potent inhibitor of SGLT1, drastically reduced the concentration decline of Q3G observed under control conditions. Because autolysis of Q3G was excluded in preliminary experiments, these findings can be explained either by an extracellular enzymatic cleavage of the glucose moiety from Q3G or by uptake of intact Q3G and intracellular cleavage with subsequent diffusion of quercetin back into the mucosal medium. Extracellular hydrolysis of Q3G can be catalyzed by the BBM enzyme lactase phloridzin hydrolase (LPH) with the majority of the activity stemming from the lactase domain rather than from the phloridzin hydrolase domain of LPH (36). LPH purified from sheep jejunum is capable of hydrolyzing a range of flavonol and isoflavone glucosides, including Q3G and quercetin-4⬘-glucoside (36). Although extracellular hydrolysis of Q3G could explain the disappearance of Q3G as well as the appearance of free quercetin in the mucosal compartment, the inhibitory influence of D-glucose, the Na⫹ dependence and the effect of phloridzin observed in our experiments are difficult to reconcile with extracellular hydrolysis of Q3G by LPH. Therefore, our results are best explained by uptake of intact Q3G via the SGLT1 and subsequent intracellular hydrolysis. Because the aglycone is more lipophilic, quercetin might diffuse across the BBM back into the mucosal solution. Although we found quercetin to be rather unstable in the absence of intestinal tissue, a spontaneous decomposition of quercetin might have been prevented by binding to proteins in the presence of intestinal tissue. Intracellular ␤-glucosidases have been found in human and rat small intestine (37,38). The enzymes derived from human and rat small intestine both hydrolyze quercetin-4⬘-glucoside, whereas the rat enzyme also cleaves Q3G although at a considerably lower rate than the 4⬘-glucoside (38). The quercetin glucorhamnoside rutin is not hydrolyzed by intracellular ␤-glu-

FIGURE 6 Disappearance of quercetin-3-glucoside (Q3G; 100 ␮mol/L) after application to the mucosal or serosal side of rat jejunum (solid bars) and proximal colon (open bars). Values are means ⫾ SEM, n ⫽ 4 – 6 (2 rats). *,***Different from colon, P ⬍ 0.05 or P ⬍ 0.001, respectively.

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cosidases (37,38). In addition, recirculation of the intact glucoside might occur at the BBM, consisting of uptake by the SGLT1 and subsequent secretion by MRP2 as demonstrated in Caco-2 cells (24,26). Although neither Q3G nor free quercetin was detected in the serosal compartment, this does not necessarily mean that Q3G, or more likely, free quercetin does not cross the basolateral membrane of the epithelial cells; rather, it indicates that the adjacent tissue layers (subepithelial tissue, muscle tissue), which were still present in our preparations, hindered diffusion into the serosal bathing solution. In in vivo studies on the bioavailability of quercetin in humans, rats and pigs after oral application of quercetin or quercetin glucosides, it was consistently found that the vast majority of quercetin absorbed into the circulation was present as glucuronides and sulfates of quercetin and methylated forms of quercetin such as isorhamnetin and tamarixetin (32,39 – 42). Conjugation can already occur in the intestinal mucosa subsequent to the cleavage of the glucose moiety (41). Quercetin glucuronides and sulfates are not only absorbed into the blood but may also be secreted into the intestinal lumen across the BBM (41). In our study we did not detect glucuronides in either the serosal or the mucosal compartment of the Ussing chambers. In addition, in some preliminary experiments on the retention of quercetin, Q3G and quercetin conjugates within the intestinal wall after incubation of small intestinal preparations in the presence of Q3G in the mucosal bathing solution, we found only free quercetin, not the intact glucoside or conjugates of quercetin (results not shown). We do not know the exact reasons for this discrepancy from the in vivo situation, but factors such as an insufficient energy supply in the intestinal mucosa for formation of glucuronides/sulfates, or an instability of such conjugates under our experimental conditions might have contributed. Even in the absence of Na⫹ and simultaneous presence of phloridzin, incubation conditions in which SGLT1 should not substantially contribute to the disappearance of Q3G from the mucosal bathing solution, Q3G concentration declined considerably over the 2-h incubation period. Because fructose had no effect under these conditions, a contribution of Na⫹independent uptake of Q3G by the intestinal fructose carrier GLUT5 appears to be unlikely. For intestinal glucose transport across the BBM, however, recent data indicate a substantial role of GLUT2 in this process, at least under in vivo conditions (43). Transport of Q3G by facilitated diffusion mediated by GLUT2 together with simple diffusion could explain the Na⫹-independent component of the disappearance of Q3G in our experiments. Taken together, our results demonstrate that SGLT1 is involved in the uptake of Q3G across the small intestinal BBM. This conclusion is supported by the inhibitory effect of ⫹ D-glucose, phloridzin and Na -free medium on the disappearance of Q3G from the mucosal bathing solution. Furthermore, Q3G was not taken up by the proximal colon, a tissue lacking SGLT1. LITERATURE CITED 1. Middleton, E., Jr., Kandaswami, C. & Theoharides, T. C. (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 52: 673–751. 2. Ku¨ hnau, J. (1976) The flavonoids. A class of semi-essential food components: their role in human nutrition. World Rev. Nutr. Diet. 24: 117–191. 3. Harborne, J. B. & Williams, C. A. (2000) Advances in flavonoid research since 1992. Phytochemistry 55: 481–504. 4. Middleton, E. & Kandaswami. C. (1994) The impact of plant flavonoids on mammalian biology: implications for immunity, inflammation and cancer. In:

The Flavonoids: Advances in Research Since 1986 (Harborne, J. B., ed.), pp. 619 – 652, Chapman & Hall, London, UK. 5. Formica, J. V. & Regelson, W. (1995) Review of the biology of quercetin and related bioflavonoids. Food Chem. Toxicol. 33: 1061–1080. 6. Hertog, M.G.L., Hollman, P.C.H. & van de Putte, B. (1993) Content of potentially anticarcinogenic flavonoids in tea infusions, wines and fruit juices. J. Agric. Food Chem. 41: 1242–1246. 7. Picinelli, A., Sua´ rez, B. & Mangas, J. J. (1997) Analysis of polyphenols in apple products. Z. Lebensm.-Unters. -Forsch. A 204: 48 –51. 8. Price, K. R. & Rhodes, M.J.C. (1997) Analysis of the major flavonol glycosides present in four varieties of onion (Allium cepa) and changes in composition resulting from autolysis. J. Sci. Food Agric. 74: 331–339. 9. Linseisen, J., Radtke, J. & Wolfram, G. (1997) Flavonoid intake of adults in a Bavarian subgroup of the national food consumption survey. Z. Ernaehrwiss. 36: 403– 412. 10. de Vries, J.H.M., Janssen, P.L.T.M.K., Hollman, P.C.H., van Staveren, W. A. & Katan, M. B. (1997) Consumption of quercetin and kaempferol in free-living subjects eating a variety of diets. Can. Lett. 114: 141–144. 11. Hertog, M.G.L., Feskens, E.J.M., Hollman, P.C.H., Katan, M. B. & Kromhout, D. (1993) Dietary antioxidant flavonoids and risk of coronary heart disease: The Zutphen Elderly Study. Lancet 342: 1007–1011. 12. Hertog, M.G.L., Sweetman, P. M., Fehily, A. M., Elwood, P. C. & Kromhout, D. (1997) Antioxidant flavonols and ischemic heart disease in a Welsh population of men: The Caerphilly Study. Am. J. Clin. Nutr. 65: 1489 –1494. 13. Rimm, E. B., Katan, M. B., Ascherio, A., Stampfer, M. J. & Willett, W. C. (1996) Relation between intake of flavonoids and risk for coronary heart disease in male health professionals. Ann. Intern. Med. 125: 384 –389. 14. Hertog, M.G.L., Kromhout, D., Aravanis, C., Blackburn, H., Buzina, R., Fidanza, F., Giampoli, S., Jansen, A., Menotti, A., Nedeljkovic, S., Pekkarinen, M., Simic, B. S., Toshima, H., Feskens, E.J.M., Hollman, P.C.H. & Katan, M. B. (1995) Flavonoid intake and long term risk of coronary heart disease and cancer in The Seven Countries Study. Arch. Intern. Med. 155: 381–386. 15. Hertog, M.G.L. & Hollman, P.C.H. (1996) Potential health effects of the dietary flavonol quercetin. Eur. J. Clin. Nutr. 50: 63–71. 16. Hollman, P.C.H., De Vries, J.H.M., Van Leeuwen, S. D., Mengelers, M.J.B. & Katan, M. B. (1995) Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Am. J. Clin. Nutr. 62: 1276 –1282. 17. Hollman, P.C.H., Van Trijp, J.M.B., Buysman, M.N.C.P., van der Gaag, M. S., Mengelers, M.J.B., de Vries, J.H.M. & Katan, M. B. (1997) Relative bioavailability of the antioxidant flavonoid quercetin from various foods in man. FEBS Lett. 418: 152–156. 18. Hollman, P.C.H., Bijsman, M.N.C.P., van Gameren, Y., Cnossen, E.P.J., De Vries, J.H.M. & Katan, M. B. (1999) The sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man. Free Radic. Res. 31: 569 –573. 19. Olthof, M. R., Hollman, P.C.H., Vree, T. B. & Katan, M. B. (2000) Bioavailabilities of quercetin-3-glucoside and quercetin-4⬘-glucoside do not differ in humans. J. Nutr. 130: 1200 –1203. 20. Paganga, G. & Rice-Evans, C. A. (1997) The identification of flavonoids as glycosides in human plasma. FEBS Lett. 401: 78 – 82. 21. Aziz, A. A., Edwards, C. A., Lean, M.E.J. & Crozier, A. (1998) Absorption and excretion of conjugated flavonols, including quercetin-4⬘-O-␤-glucoside and isorhamnetin-4⬘-O-␤-glucoside by human volunteers after the consumption of onions. Free Radic. Res. 29: 257–269. 22. Gee, J. M., DuPont, M. S., Rhodes, M.J.C. & Johnson, I. T. (1998) Quercetin glucosides interact with the intestinal glucose transport pathway. Free Radic. Biol. Med. 25: 19 –25. 23. Gee, J. M., DuPont, M. S., Day, A. J., Plumb, G. W., Willisamson, G. & Johnson, I. T. (2000) Intestinal transport of quercetin glycosides in rats involves both deglycosylation and interaction with the hexose transport pathway. J. Nutr. 130: 2765–2771. 24. Walgren, R. A., Lin, J.-T., Kinne, R.K.-H. & Walle, T. (2000) Cellular uptake of dietary flavonoid quercetin 4⬘-␤-glucoside by sodium-dependent glucose transporter SGLT1. J. Pharmacol. Exp. Ther. 294: 837– 843. 25. Ader, P., Blo¨ ck, M., Pietzsch, S. & Wolffram, S. (2001) Interaction of quercetin glucosides with the Na⫹-dependent glucose carrier (SGLT1) in rat small intestine. Cancer Lett. 162: 175–180. 26. Walgren, R. A., Karnkaky, K. J., Jr., Lindenmayer, G. E. & Walle, T. (2000) Efflux of dietary flavonoid quercetin 4⬘-␤-glucoside across human intestinal Caco-2 cell monolayers by apical multidrug resistance-associated protein-2. J. Pharmacol. Exp. Ther. 294: 830 – 836. 27. Walgren, R., Walle, U. K. & Walle, T. (1998) Transport of quercetin and its glucosides across human intestinal epithelial Caco-2 cells. Biochem. Pharmacol. 55: 1721–1727. 28. Boutin, J. A., Meunier, F., Lambert, P.-H., Hennig, P., Bertin, D., Serkiz, B. & Volland, J.-P. (1993) In vivo and in vitro glucuronidation of the flavonoid diosmetin in rats. Drug Metab. Dispos. 21: 1157–1166. 29. Crespy, V., Morand, C., Manach, C., Besson, C., Demigne´ , C. & Re´ me´ sy, C. (1999) Part of quercetin absorbed in the small intestine is conjugated and further secreted in the intestinal lumen. Am. J. Physiol. 277: G120 –G126. 30. Sfakianos, J., Coward, L., Kirk, M. & Barnes, S. (1997) Intestinal uptake and biliary excretion of the isoflavone genistein in rats. J. Nutr. 127: 1260 –1268. 31. Walle, U. K., Galijatovic, A. & Walle, T. (1999) Transport of the fla-

MUCOSAL UPTAKE OF QUERCETIN GLUCOSIDES vonoid chrysin and its conjugated metabolites by the human intestinal cell line Caco-2. Biochem. Pharmacol. 58: 431– 438. 32. Ader, P., We␤mann, A. & Wolffram, S. (2000) Bioavailability and metabolism of the flavonol quercetin in the pig. Free Radic. Biol. Med. 28: 1056 – 1067. 33. Sachs, L. (1992) Angewandte Statistik. Springer-Verlag, Berlin, Germany. 34. GraphPad Software (1998) GraphPad Instat, V 2.05a, and GraphPad Inplot V4.04⫹, San Diego, CA. 35. Dangles, O., Dufour, C., Manach, C., Morand, C. & Re´ me´ sy, C. (2001) Binding of flavonoids to plasma proteins. Methods Enzymol. 335: 319 –333. 36. Day, A. J., Canada, F. J., Diaz, J. C., Kroon, P. A., Mclauchlan, R., Faulds, C. B., Plumb, G. F., Morgan, M.R.A. & Williamson, G. (2000) Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase. FEBS Lett. 468: 166 –170. 37. Day, A. J., DuPont, M. S., Ridley, S., Rhodes, M., Rhodes, M.J.C., Morgan, M.R.A. & Williamson, G. (1998) Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glucosidase activity. FEBS Lett. 436: 71–75.

635

38. Ioku, K., Pongpiriyadacha, Y., Konishi, Y., Takei, Y., Nakatani, N. & Terao, N. (1998) Beta-glucosidase activity in the rat small intestine toward quercetin monoglucosides. Biosci. Biotechnol. Biochem. 62: 1428 –1431. 39. Manach, C., Morand, C., Texier, O., Favier, M. L., Agullo, G., Demigne´ , C., Re´ ge´ rat, F. & Re´ me´ sy, C. (1995) Quercetin metabolites in plasma of rats fed diets containing rutin or quercetin. J. Nutr. 125: 1911–1922. 40. Manach, C., Texier, O., Re´ ge´ rat, F., Agullo, G., Demigne´ , C. & Re´ me´ sy, C. (1996) Dietary quercetin is recovered in rat plasma as conjugated derivatives of isorhamnetin and quercetin. J. Nutr. Biochem. 7: 375–380. 41. Crespy, V., Morand, C., Manach, C., Besson, C., Demigne´ , C. & Re´ me´ sy, C. (1999) Part of quercetin absorbed in the small intestine is conjugated and further secreted in the intestinal lumen. Am. J. Physiol. 277: G120 –G126. 42. Graefe, E. U., Wittig, J., Mueller, S., Riethling, A. K., Uehleke, B., Drewelow, B., Pforte, H., Jacobasch, G., Derendorf, H., & Veit, M. (2001) Pharmacokinetics and bioavailability of quercetin glycosides in humans. J. Clin. Pharmacol. 41: 492– 499. 43. Kellett, G. L. (2001) The facilitated component of intestinal glucose absorption. J. Physiol. (Lond.) 531: 585–595.