Elsevier

Free Radical Biology and Medicine

Volume 29, Issue 12, 15 December 2000, Pages 1234-1243
Free Radical Biology and Medicine

Original contribution
Conjugation position of quercetin glucuronides and effect on biological activity

https://doi.org/10.1016/S0891-5849(00)00416-0Get rights and content

Abstract

Quercetin glycosides are common dietary antioxidants. In general, however, potential biological effects of the circulating plasma metabolites (e.g., glucuronide conjugates) have not been measured. We have determined the rate of glucuronidation of quercetin at each position on the polyphenol ring by human liver cell-free extracts containing UDP-glucuronosyltransferases. The apparent affinity of UDP-glucuronosyltransferase followed the order 4′- > 3′- > 7- > 3, although the apparent maximum rate of formation was for the 7-position. The 5-position did not appear to be a site for conjugation. After isolation of individual glucuronides, the inhibition of xanthine oxidase and lipoxygenase were assessed. The Ki for the inhibition of xanthine oxidase by quercetin glucuronides followed the order 4′- > 3′- > 7- > 3-, with quercetin-4′-glucuronide a particularly potent inhibitor (Ki = 0.25 μM). The glucuronides, with the exception of quercetin-3-glucuronide, were also inhibitors of lipoxygenase. Quercetin glucuronides are metabolites of quercetin in humans, and these compounds can retain some biological activity depending on conjugation position at expected plasma concentrations.

Introduction

Flavonols are a group of polyphenolic compounds present in fruit- and vegetable-rich diets. Evidence has accumulated from in vitro studies that flavonols have biological activity that may be beneficial to health in vivo. For example, they are good antioxidants [1], can inhibit platelet aggregation [2], induce phase II detoxification enzymes [3], and have also been shown to inhibit the growth of certain human cancer cells such as those of the colon [4], ovary [5], and gastrointestinal tract [6]. More specifically, quercetin (a major flavonol of dietary origin) is an effective inhibitor of xanthine oxidase [7] and lipoxygenase [8]. Xanthine oxidase catalyzes the oxidation of hypoxanthine and xanthine to uric acid-generating superoxide radicals, which are involved in many pathological processes such as inflammation, atherosclerosis, cancer, and aging [9]. Lipoxygenase induces oxidation of LDL, which has been implicated in the progression of both atherosclerosis and cancer [10].

The biological activity of the flavonols is predicted to be highly dependent on the structure, particularly the availability of hydroxyl groups [11]. Most in vitro biological activity has been assessed by experimentation using the aglycone; however, in nature flavonols are mainly found glycosylated, which greatly affects the antioxidant properties [12], [13]. Although deglycosylation is likely to occur either pre- [14], [15] or postabsorption [16], metabolism of these compounds in vivo leads to conjugation of the hydroxyl groups with sulfate and glucuronic acid. The nature and position of these substitutions will affect subsequent biological activity, possibly reducing or abolishing the activity seen with the aglycone.

Conjugation of xenobiotics with glucuronic acid or sulfate are common detoxification pathways that lead to an increased solubility and a higher molecular weight. Sulfation is a major pathway of metabolism at low concentrations for many xenobiotics, although this pathway can become saturated. Glucuronidation occurs readily due to an abundant supply of UDP-glucuronic acid in tissues and is particularly important for increasing molecular weight, necessary for excretion in the bile. More than 40 different isoforms of UDP-glucuronosyltransferase (UGT) have been identified [17]. Some of these have been shown to be active toward the flavonoids, but isoform specificity for flavonoids has not been thoroughly investigated and it is likely that several isoforms will have activity toward these compounds.

Circulating plasma species of flavonols have been identified as sulfoglucuronide mixed conjugates in rats [18], [19] and this is likely to be the case in humans [20], [21]. The position of conjugation in vivo has not been assessed, as most studies on the absorption and metabolism of flavonols require enzymic or chemical pretreatment to convert quercetin metabolites from urine, plasma, or bile into the parent aglycone for quantification. The position of conjugation, however, is of immense significance in determining potential biological activity of the flavonols. One study on the metabolism of the flavone diosmetin (4′-methyl luteolin) showed that the 3′- and 7, 3′-glucuronides were the major metabolites in rat [22]. The preference for glucuronide conjugation in vitro was determined indirectly for flavones by studying glucuronidation of related compounds with different free hydroxyl groups. The order of glucuronidation was found to be 3′- > 7- > 5-, although the 5-position was glucuronidated if it was the only available hydroxyl group. Flavonols have an additional hydroxyl at the 3-position (Fig. 1), but the ability of UGT to glucuronidate at this site has not been studied.

The aims of this paper were to (1) determine the positions of conjugation of quercetin glucuronides, (2) determine the catalytic efficiency of human UGT activity to form quercetin glucuronides, and (3) assess the ability of potential quercetin metabolites to inhibit xanthine oxidase and lipoxygenase in vitro.

Section snippets

Materials and methods

Quercetin, quercetin glucosides, 3′- and 4′-methylquercetin, and quercetin-3-sulfate were purchased from Extrasynthese (Genay, France), except quercetin-3,4′-diglucoside, which was purified from onion bulb tissue [23] and quercetin-3-glucuronide, purified from green bean tissue [24]. These compounds have previously been confirmed by MS and NMR. All flavonols were checked for purity by HPLC prior to use and were found to be >98% pure. Xanthine oxidase (EC 1.1.3.22, from buttermilk), lipoxygenase

Activity of human UDP-glucuronosyltransferase

Quercetin gave rise to four major glucuronide conjugates (P1, P2, P3, and P4) after incubation with human liver cell-free extracts in the presence of excess UDPGA, as shown by Fig. 2. In a similar experiment using 3′-methylquercetin as a substrate, three products were formed (M1, M2, and M3; retention times: 12.6, 13.7, and 15.1 min, respectively). All peaks disappeared on treatment with β-glucuronidase, with concomitant formation of the parent aglycone, demonstrating that each peak was a

Discussion

Quercetin is consumed from the diet predominantly in the glycosidic form. Absorption of quercetin is dependent on the nature of glycoside [30], with transfer across the small intestine a result of either active transport [23] or luminal deglycosylation by lactase phlorizin hydrolase [15] followed by diffusion of the aglycone. Absorption of quercetin from the colon will occur after hydrolysis of the glycoside by gut microflora [14]. If flavonoid glucosides are absorbed intact, deglycosylation by

Acknowledgements

We would like to thank the Biotechnology and Biological Sciences Research Council for funding (including a studentship to AJD), Dr. Saxon Ridley and Dr. Mike Rhodes at the Norfolk and Norwich Hospital for donating liver biopsy material, and Keith Price for kindly donating quercetin-3-glucuronide as a standard.

This work was supported by a grant from the Biotechnology and Biological Sciences Research Council.

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