International Journal of Molecular Sciences Article Inhibitory Effects of Quercetin and Its Human and Microbial Metabolites on Xanthine Oxidase Enzyme Violetta Mohos 1,2, Attila Pánovics 1, Eszter Fliszár-Nyúl 1,2, Gabriella Schilli 3, Csaba Hetényi 3 , PˇremyslMladˇenka 4 , Paul W. Needs 5, Paul A. Kroon 5,Gábor Peth˝o 1,3 and Miklós Poór 1,2,* 1 Department of Pharmacology, University of Pécs, Faculty of Pharmacy, Szigeti út 12, H-7624 Pécs, Hungary; [email protected] (V.M.); [email protected] (A.P.); [email protected] (E.F.-N.); [email protected] (G.P.) 2 János Szentágothai Research Center, University of Pécs, Ifjúság útja 20, H-7624 Pécs, Hungary 3 Department of Pharmacology and Pharmacotherapy, University of Pécs, Medical School, Szigeti út 12, H-7624 Pécs, Hungary; [email protected] (G.S.); [email protected] (C.H.) 4 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; [email protected] 5 Quadram Institute Bioscience, Norwich Research Park, Norwich NR4 7UA, UK; [email protected] (P.W.N.); [email protected] (P.A.K.) * Correspondence: [email protected]; Tel.: +36-536-000 (ext. 34646) Received: 10 May 2019; Accepted: 29 May 2019; Published: 31 May 2019 Abstract: Quercetin is an abundant flavonoid in nature and is used in several dietary supplements. Although quercetin is extensively metabolized by human enzymes and the colonic microflora, we have only few data regarding the pharmacokinetic interactions of its metabolites. Therefore, we investigated the interaction of human and microbial metabolites of quercetin with the xanthine oxidase enzyme. Inhibitory effects of five conjugates and 23 microbial metabolites were examined with 6-mercaptopurine and xanthine substrates (both at 5 µM), employing allopurinol as a positive control. Quercetin-30-sulfate, isorhamnetin, tamarixetin, and pyrogallol proved to be strong inhibitors of xanthine oxidase. Sulfate and methyl conjugates were similarly strong inhibitors of both 6-mercaptopurine and xanthine oxidations (IC50 = 0.2–0.7 µM); however, pyrogallol inhibited xanthine oxidation (IC50 = 1.8 µM) with higher potency vs. 6-MP oxidation (IC50 = 10.1 µM). Sulfate and methyl conjugates were approximately ten-fold stronger inhibitors (IC50 = 0.2–0.6 µM) of 6-mercaptopurine oxidation than allopurinol (IC50 = 7.0 µM), and induced more potent inhibition compared to quercetin (IC50 = 1.4 µM). These observations highlight that some quercetin metabolites can exert similar or even a stronger inhibitory effect on xanthine oxidase than the parent compound, which may lead to the development of quercetin–drug interactions (e.g., with 6-mercaptopurin or azathioprine). Keywords: xanthine oxidase; quercetin metabolites; 6-mercaptopurine; xanthine; enzyme inhibition; pharmacokinetic interactions 1. Introduction Quercetin (Q) is a flavonoid; it is found in many commonly consumed foods and beverages including apples, onions, tea, and red wine [1]. It is included in dietary supplements in high doses (from several hundreds to thousands of milligrams). These supplements are advertised and freely available through the Internet for several purported uses. The oral bioavailability of Q is low, due to its poor aqueous solubility and significant presystemic elimination [2,3]. As a result of the metabolism, Q is known to form methyl, sulfate, and glucuronic acid conjugates, such as 30-O-methylquercetin (isorhamnetin, IR), 40-O-methylquercetin (tamarixetin, TAM), quercetin-30-sulfate Int. J. Mol. Sci. 2019, 20, 2681; doi:10.3390/ijms20112681 www.mdpi.com/journal/ijms Int. J. Mol. Sci. Sci. 20192019,, 2020,, x 2681 FOR PEER REVIEW 2 of 16 methylquercetin (isorhamnetin, IR), 4′-O-methylquercetin (tamarixetin, TAM), quercetin-3′-sulfate (Q3(Q30′S), quercetin-3-glucuronide (Q3G), andand isorhamnetin-3-glucuronide (I3G) (Figure1 1))[ [3–6].3–6]. Q3 ′0S, Q3G, and I3G are the main circulating metabolites of Q with preserved preserved flavonoid flavonoid core in humans humans [7]. [7]. Even after high dietary intake of Q, total plasma Q (the parent compound and its metabolites) commonly only reaches nanomolarnanomolar concentrationsconcentrations [[33,,6].6]. Micromolar total plasma QQ concentrationsconcentrations were detecteddetected only only after after the the administration administration of of dietary dietary supplements supplements with with exceedingly exceedingly high high Q content Q content (e.g., 1000(e.g., mg 1000/day) mg/day) [8]. Furthermore, [8]. Furthermore, Q is biotransformed Q is biot byransformed the colonic microfloraby the colonic through microflora degradation through of the flavonoiddegradation ring(s) of the into flavonoid smaller products. ring(s) into These smaller can be products. classified These in four can major be groups:classified hydroxybenzoic, in four major hydroxyacetic,groups: hydroxybenzoic, and hydroxycinnamic hydroxyacetic, acids, and and hydrox hydroxybenzenesycinnamic acids, [6, 9and–11 ].hydroxybenzenes [6,9–11]. Figure 1. 1. ChemicalChemical structures structures of quercetin of quercetin and its and conj itsugated conjugated metabolites metabolites with preserved with preservedflavonoid flavonoidstructure. structure. Xanthine oxidase oxidase (XO) (XO) is isa molybdenum-containin a molybdenum-containingg enzyme enzyme playing playing an important an important role in rolepurine in purinecatabolism catabolism [12]. The [12 antitumor]. The antitumor drug 6-mercaptopurine drug 6-mercaptopurine (6-MP) is (6-MP) also biotransformed is also biotransformed by XO, yielding by XO, yieldingpharmacologically pharmacologically inactive inactive6-thiouric 6-thiouric acid (6-TU) acid (6-TU)[13]. Allopurinol [13]. Allopurinol (APU) (APU) is a well-known is a well-known XO XOinhibitor inhibitor used used in the in thetreatment treatment of hyperuricemia of hyperuricemia or gout or gout [12].[ 12Besides]. Besides the inhibition the inhibition of uric of acid uric acidformation, formation, the inhibition the inhibition of XO of may XO also may be also benefi be beneficialcial by decreasing by decreasing superoxide superoxide radicals radicals under under some somepathological pathological conditions conditions [14,15]. [14 However,,15]. However, the simultaneous the simultaneous administration administration of APU of and APU 6-MP and slows 6-MP slowselimination elimination of the of latter the latter compound, compound, which which may may cause cause toxic toxic effects, effects, i.e., i.e., severe severe bone bone marrow depression [[16].16]. Based on previous studies, Q is a strong competitive inhibitor of XO [[17–19],17–19], while other authors suggest the the involvement involvement of of noncompetitive noncompetitive mechanis mechanismsms as aswell well [20,21]. [20,21 The]. The effect eff ofect Q of on Q the on XO- the XO-catalyzedcatalyzed oxidation oxidation of xanthine of xanthine has has been been described; described; however, however, the the in influencefluence of of Q Q on on 6-MP 6-MP oxidation has notnot beenbeen reported.reported. Furthermore, as as mentio mentionedned above, Q undergoes significantsignificant presystemicpresystemic elimination; therefore, therefore, its its metabolites metabolites reach reach higher higher concentrations concentrations in blood in blood (and (and likely likely in tissues) in tissues) than thanthe parent the parent compound, compound, Q. Considering Q. Considering the thefact fact that that some some Q Qmetabolites metabolites produce produce similar similar or or even stronger interaction withwith somesome proteins thanthan QQ itself [[22,23],22,23], it is reasonable to hypothesize that some Q metabolites maymay influenceinfluence the the XO-catalyzed XO-catalyzed xanthine xanthine and and/or/or 6-MP 6-MP oxidation. oxidation. Since Since the the inhibition inhibition of 6-MPof 6-MP elimination elimination may may have have serious serious adverse adverse consequences, consequences, we we aimed aimed to investigateto investigate the the interactions interactions of Qof asQ wellas well as its as human its human and microbial and microbial metabolites metabo (includinglites (including also other also flavonoid other microbial flavonoid metabolites) microbial withmetabolites) XO enzyme with employing XO enzyme xanthine employing and 6-MP xanthine substrates andas 6-MP well assubstrates APU and as oxipurinol well as (theAPU active and metaboliteoxipurinol of(the APU) active as metabolite positive controls. of APU) as positive controls. 2. Results 2.1. Inhibitory Effects of Q and Its Human Metabolites on XO-Catalyzed 6-MP Oxidation 2.1. Inhibitory Effects of Q and Its Human Metabolites on XO-Catalyzed 6-MP Oxidation First, the effects of Q and its conjugated metabolites on 6-MP oxidation were examined. Figure2 First, the effects of Q and its conjugated metabolites on 6-MP oxidation were examined. Figure demonstrates the time-dependence of 6-TU formation in the absence and presence of Q metabolites, 2 demonstrates the time-dependence of 6-TU formation in the absence and presence of Q metabolites, where % conversion of 6-MP to 6-TU is expressed. Even relatively high concentrations (20 µM four-fold where % conversion of 6-MP to 6-TU is expressed. Even relatively high concentrations (20 μ=M = four- excessfold excess vs. the vs. substrate) the substrate) of the glucuronideof the glucuronid conjugatese conjugates (Q3G and (Q3G I3G) didand not I3G) inhibit did thenot formation inhibit the of 6-TU.formation However,