Interactions of flavonoids with iron and copper ions: a mechanism for their antioxidant activity

The metal chelating properties of flavonoids suggest that they may play a role in metal-overload diseases and in all oxidative stress conditions involving a transition metal ion. A detailed study has been made of the ability of flavonoids to chelate iron (including Fe 3+ ) and copper ions and its dependence of structure and pH. The acid medium may be important in some pathological conditions. In addition, the ability of flavonoids to reduce iron and copper ions and their activity-structure relationships were also investigated. To fulfil these objectives, flavones (apigenin, luteolin, kaempferol, quercetin, myricetin and rutin), isoflavones (daidzein and genistein), flavanones (taxifolin, naringenin and naringin) and a flavanol (catechin) were investigated. All flavonoids studied show higher reducing capacity for copper ions than for iron ions. The flavonoids with better Fe 3+ reducing activity are those with a 2,3-double bond and possessing both the catechol group in the B-ring and the 3-hydroxyl group. The copper reducing activity seems to depend largely on the number of hydroxyl groups. The chelation studies were carried out by means of ultraviolet spectroscopy and electrospray ionisation mass spectrometry. Only flavones and the flavanol catechin interact with metal ions. At pH 7.4 and pH 5.5 all flavones studied appear to chelate Cu 2+ at the same site, probably between the 5-hydroxyl and the 4-oxo groups. Myricetin and quercetin, however, at pH 7.4, appear to chelate Cu 2+ additionally at the ortho -catechol group, the chelating site for catechin with Cu 2+ at pH 7.4. Chelation studies of Fe 3+ to flavonoids were investigated only at pH 5.5. Only myricetin and quercetin interact strongly with Fe 3+ , complexation probably occurring again between the 5-hydroxyl and the 4-oxo groups. Their behaviour can be explained by their ability to reduce Fe 3+ at pH 5.5, suggesting that flavonoids reduce Fe 3+ to Fe 2+ before association.     

Interactions of Flavonoids with Iron and Copper Ions: A Mechanism for their Antioxidant Activity

The metal chelating properties of flavonoids suggest that they may play a role in metal-overload diseases and in all oxidative stress conditions involving a transition metal ion. A detailed study has been made of the ability of flavonoids to chelate iron (including Fe 3+ ) and copper ions and its dependence of structure and pH. The acid medium may be important in some pathological conditions. In addition, the ability of flavonoids to reduce iron and copper ions and their activity-structure relationships were also investigated. To fulfil these objectives, flavones (apigenin, luteolin, kaempferol, quercetin, myricetin and rutin), isoflavones (daidzein and genistein), flavanones (taxifolin, naringenin and naringin) and a flavanol (catechin) were investigated. All flavonoids studied show higher reducing capacity for copper ions than for iron ions. The flavonoids with better Fe 3+ reducing activity are those with a 2,3-double bond and possessing both the catechol group in the B-ring and the 3-hydroxyl group. The copper reducing activity seems to depend largely on the number of hydroxyl groups. The chelation studies were carried out by means of ultraviolet spectroscopy and electrospray ionisation mass spectrometry. Only flavones and the flavanol catechin interact with metal ions. At pH 7.4 and pH 5.5 all flavones studied appear to chelate Cu 2+ at the same site, probably between the 5-hydroxyl and the 4-oxo groups. Myricetin and quercetin, however, at pH 7.4, appear to chelate Cu 2+ additionally at the ortho -catechol group, the chelating site for catechin with Cu 2+ at pH 7.4. Chelation studies of Fe 3+ to flavonoids were investigated only at pH 5.5. Only myricetin and quercetin interact strongly with Fe 3+ , complexation probably occurring again between the 5-hydroxyl and the 4-oxo groups. Their behaviour can be explained by their ability to reduce Fe 3+ at pH 5.5, suggesting that flavonoids reduce Fe 3+ to Fe 2+ before association.     

Dietary flavonoids interact with trace metals and affect metallothionein level in human intestinal cells

Flavonoids are natural compounds found in food items of plant origin. The study examined systematically the interaction of structurally diverse dietary flavonoids with trace metal ions and the potential impact of dietary flavonoids on the function of intestinal cells. Spectrum analysis was first performed to determine flavonoid-metal interaction in the buffer. Among the flavonoids tested, genistein, biochanin-A, naringin, and naringenin did not interact with any metal ions tested. Members of the flavonol family, quercetin, rutin, kaempferol, flavanol, and catechin, were found to interact with Cu(II) and Fe(III). On prolonged exposure, quercetin also interacted with Mn(II). Quercetin at 1:1 ratio to Cu(II) completely blocked the Cu-dependent color formation from hematoxylin. When quercetin was added to the growth medium of cultured human intestinal cells, Caco-2, the level of metal binding antioxidant protein, metallothionein, decreased. The effect of quercetin on metallothionein was dose and time-dependent. Genistein and biochanin A, on the contrary, increased the level of metallothionein. The interaction between dietary flavonoids and trace minerals and the effect of flavonoids on metallothionein level imply that flavonoids may affect metal homeostasis and cellular oxidative status in a structure-specific fashion.     

Effect of flavonoids of different structure on peroxidation of neutral lipids of animal origin

The effect of flavonoids of different structure (baikalein, baikalin, quercetin, dihydroquercetin, genistein, and daidzein) on the process of formation of lipid hydroperoxides during thermoinduced autooxidation of neutral lipids of animal origin. The minimum inhibitory concentration of isoflavones was found to be equal to 10(-3) M. Effective inhibitory concentration of other flavonoids (except baikalin) was equal to 10(-4) M. Baikalin was an effective promoter of lipid peroxidation. The antioxidant activity of the flavonoids tested was calculated taking ionol as a reference.     

Structure-activity relationships of flavonoids for vascular relaxation in porcine coronary artery

Flavonoids are polyphenolic compounds that are widespread in the plant kingdom, and structure-activity relationships (SAR) for vascular relaxation effects were examined for 17 of them using porcine coronary arteries. Density functional theory was employed to calculate the chemical parameters of these compounds. The order of potency for vascular relaxation was as follows: flavones (apigenin and luteolin) >or= flavonols (kaempferol and quercetin)>isoflavones (genistein and daidzein)>flavanon(ol)es (naringenin)>chalcones (phloretin)>anthocyanidins (pelargonidin)>flavan(ol)es ((+)-catechin and (-)-epicatechin). SAR analysis revealed that for good relaxation activity, the 5-OH, 7-OH, 4'-OH, C2=C3 and C4=O functionalities were essential. Comparison of rutin with quercetin, genistin with genistein, and puerarin with daidzein demonstrated that the presence of a glycosylation group greatly reduced relaxation effect. Total energy and molecular volume were also predictive of their relaxation activities. Our findings indicated that the most effective relaxing agents are apigenin, luteolin, kaempferol and genistein. These flavonoids possess the key chemical structures demonstrated in our SAR analysis.     

Photophysical and phototoxic properties of the antibacterial fluoroquinolones levofloxacin and moxifloxacin

Two antibacterial fluoroquinolones, levofloxacin and moxifloxacin, were investigated to evaluate their photophysical properties and to explore the mechanism of their phototoxicity. Photophysical experiments were carried out in aqueous solution by stationary and time-resolved fluorimetry, and by laser flash photolysis, to obtain information on the various decay pathways of the excited states of the drugs and on transient species formed upon irradiation. The results obtained show that levofloxacin is able to photosensitize red blood cell lysis in an oxygen-independent way and induce a high decrease in cell viability after UVA irradiation, although to a lesser degree than the racemic mixture ofloxacin. Moxifloxacin, which is an 8-MeO-substituted fluoroquinolone, is less phototoxic than the other compounds. Cellular phototoxicity was inhibited by the addition of superoxide dismutase, catalase, and free radical and hydroxyl radical scavengers (BHA, GSH, mannitol, and DMTU), indicating the involvement of superoxide anion and/or a radical mechanism in their cytotoxicity. A good correlation was observed between lipid peroxidation, protein photodamage, and cellular phototoxicity, indicating that test compounds exert their toxic effects mainly in the cellular membrane. Experiments carried out on pBR322 DNA show that these derivatives do not significantly photocleave DNA directly, but single-strand breaks were evidenced after treatment of photosensitized DNA by two base-excision-repair enzymes, and Endo III.     

Broad-spectrum sunscreens prevent the secretion of proinflammatory cytokines in human keratinocytes exposed to ultraviolet A and phototoxic lomefloxacin

The combination of phototoxic drugs and ultraviolet (UV) radiation can trigger the release of proinflammatory cytokines. The present study measured the ability of sunscreens to prevent cytokine secretion in human keratinocytes following cotreatment of these cells with a known photoreactive drug and UVA. Keratinocytes were treated for 1 h with increasing concentrations of lomefloxacin (LOM) or norfloxacin (NOR), exposed to 15 J/cm2 UVA, and incubated for 24 h. NOR, owing to the absence of a fluorine atom in position 8, was non-phototoxic and used as a negative control. Cell viability and the release of 3 cytokines were assessed, namely interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-alpha). The measurement of these cytokines may be a useful tool for detecting photoreactive compounds. To measure their ability to prevent cytokine secretion, various sunscreens were inserted between the UVA source and the cells. Treatment with NOR, NOR plus UVA, or LOM had no effect on the cells. LOM plus UVA, however, had an effect on cell viability and on cytokine secretion. IL-1alpha levels increased with LOM concentration. The release of TNF-alpha and IL-6 followed the same pattern at lower concentrations of LOM but peaked at 15 micromol/L and decreased at higher concentrations. Sunscreens protected the cells from the effects of LOM plus UVA, as cell viability and levels of cytokines remained the same as in the control cells. In conclusion, the application of broad-spectrum sunscreen by individuals exposed to UVA radiation may prevent phototoxic reactions initiated by drugs such as LOM.     

Modulation of MRP1 protein transport by plant, and synthetically modified flavonoids

The influence of novel synthetic and plant origin flavonoids on activity of multidrug resistance-associated protein (MRP1) was investigated in human erythrocytes used as a cell model expressing MRP1 in plasma membrane. The fluorescent probe, BCPCF (2', 7'-bis-(3-carboxy-propyl)-5-(and-6)-carboxyfluorescein), was applied as a substrate for MRP1 multidrug resistance transporter. The effect of compounds belonging to different classes of natural flavonoids: flavone, flavonol, isoflavones and flavanolignan was compared with action of new synthetic derivatives of genistein. Most of the flavonoids showed strong or moderate ability to inhibit transport carried out by MRP1. Inhibitory properties of flavonoids were compared to the effects of indomethacin, probenecid and MK-571 known as MRP1 inhibitors. Studying the influence of new synthetic genistein derivatives on BCPCF transport we have found that the presence of hydrophobic groups substituting hydrogen of hydroxyl group at the position 4' in ring B of isoflavone is more important for inhibitory properties than hydrophobic substitution at the position 7 in ring A. In case of naturally occurring isoflavones the replacement of hydrogen at position 4' by hydrophobic ring structure seems also to be favourable for inhibition potency.     

Effect of flavonoids of various structures on peroxidation of neutral lipids of animal origin

The effect of flavonoids of various structures (baikalein, baikalin, quercetin, dihydroquercetin, genistein, and daidzein) on the process of formation of lipid hydroperoxides during thermoinduced autooxidation of neutral lipids of animal origin is studied. The minimum inhibitory concentration of isoflavones was found to be equal to 10^-3 M. The effective inhibitory concentration of other flavonoids (except baikalin) was equal to 10^-4 M. Baikalin was an effective promoter of lipid peroxidation. The antioxidant activity of the flavonoids tested was calculated, taking ionol as a reference.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 23–28.Original Russian Text Copyright © 2005 by Antoshina, Selishcheva, Sorokoumova, Utkina, Degtyarev, Shvets.     

Role of flavonoids in intestinal tight junction regulation

The gastrointestinal tract provides a physical barrier to the diffusion of foreign materials from the lumen into the circulatory system. Impairment of the intercellular tight junction (TJ) shield, which is the major determinant of intestinal barrier function, is associated with various diseases. Dietary flavonoids demonstrate various beneficial effects on our health; however, the information regarding their effects on TJ function is quite limited. To date, four flavonoids — epigallocatechin gallate (EGCG), genistein, myricetin and quercetin — have been reported to exhibit promotive and protective effects on intestinal TJ barrier functions. Genistein, a major soybean isoflavone, protects TJ barrier function against oxidative stress, acetaldehyde, enteric bacteria and inflammatory cytokines. Genistein blocks the tyrosine phosphorylation of the TJ proteins induced by oxidative stress and acetaldehyde, which results in the disassembly of the proteins from the junctional complex. Quercetin, a flavonol, enhances intestinal TJ barrier function through the assembly and expression of TJ proteins. The change in phosphorylation status is responsible for the quercetin-mediated assembly of TJ proteins. TJ protein induction has an additional role in this effect. This review presents the recent advances in our understanding of the flavonoid-mediated promotive and protective effects on intestinal TJ barrier function with a particular focus on intracellular molecular mechanisms.     

Role of flavonoids in controlling the phototoxicity of Hypericum perforatum extracts.

Hypericum perforatum extracts are used mainly as oral antidepressants. Depending on source the extracts contain various amounts of phenylpropanes, flavonol derivates, biflavones, proathocyanidines, xanthones, phloroglucinoles, some amino acids, naphtodianthrones (hypericines) and essential oil constituents. The therapeutic use of Hypericum perforatum extracts however is limited by their phototoxic potential. It was the aim of the present study to investigate the phototoxic potential of 3 Hypericum perforatum extracts from different sources as well as some of its main constituents. In order to systematically study the phototoxic potential we established a modified neutral red assay utilizing an immortalized human keratinocyte cell line (HaCaT cells) as substrate and UVA irradiation. This modified neutral red assay was found to be a simple and reliable method for detecting phototoxic effects of reference agents and plant extracts. The validity of this method was demonstrated with known phototoxic compounds like chloropromazine and psoralenes like 5-MOP. Hypericum perforatum extracts demonstrated cytotoxicity and photocytotoxicity in a dose and UVA-dose dependent manner. Hypericine itself also evoked severe phototoxic effects and was thus identified as the main phototoxic constituent. Among the tested flavonoids quercitrin was found to be cytotoxic, while rutin unexpectedly demonstrated phototoxicity whereas quercitrin was effective to control the phototoxic activity of Hypericum perforatum extracts.     

Differential mechanisms for the inhibition of human cytochrome P450 1A2 by apigenin and genistein

The inhibitory effects of flavonoids on the human cytochrome P450 1A2 (CYP1A2) were examined. Among flavonoids tested, galangin, kaempferol, chrysin, and apigenin were potent inhibitors. Although apigenin belonging to flavones and genistein belonging to isoflavones are similar in the chemical structures, the inhibitory potencies for CYP1A2 were distinguished markedly between these two flavonoids. In computer‐docking simulation, apigenin interacted with the same mode of cocrystallized α‐naphthoflavone in the active site of CYP1A2, and then the B ring of apigenin was placed close to the heme iron of the enzyme with a single orientation. In contrast, the docked genistein conformation showed two different binding modes, and the A ring of genistein was oriented to the heme iron of CYP1A2. Furthermore, the binding free energy of apigenin was lower than that of genistein. These results demonstrate a possible mechanism that causes the differential inhibitory potencies of apigenin and genistein for CYP1A2. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:230–234, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20328     

Anti- and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with alpha-linolenic acid

Lipid hydroperoxide (LOOH)-dependent lipid peroxidation was induced in alpha-linolenic acid (LNA)-loaded hepatocytes by adding Fe, Cu, V, or Cd ions at concentrations from 20 to 500 microM. The effects of structurally related flavonoids at concentrations from 10 to 500 microM on the lipid peroxidation were examined. The results with regard to each flavonoid subclass are as follows: (i) Flavonols such as myricetin, quercetin, fisetin, and kaempferol, but not morin, showed dose-dependent antioxidative activity against metal-induced lipid peroxidation at all metal concentrations. Myricetin, quercetin, and fisetin were the most effective antioxidants, although their efficacies depended on the metal ion. Kaempferol and morin had antioxidative activity equal to the other flavonols in the presence of Cu ions, but were much less effective for the other three metal ions. (ii) Flavones, luteolin, apigenin, and chrysin were antioxidative at low Fe concentrations, but were pro-oxidative at high Fe concentrations. Luteolin exhibited antioxidative activity similar to that of catechol-containing flavonols in the presence of the other three metal ions. Apigenin and chrysin also acted as pro-oxidants with V or with all metal ions, respectively. (iii) Taxifolin, a flavanone, also showed both anti- and prooxidative activity, depending on Fe concentrations, but with other metal showed only antioxidative activity ions. (iv) Epigallocatechin, a flavanol, was antioxidative with all metal ions, and its activity was similar to that of catechol-containing flavonols. The various effects of flavonoids on metal-induced lipid peroxidation in LNA-loaded hepatocytes is discussed with regard to the change in redox potential of flavonoid-metal complexes.     

Pro-oxidative effect of beta-carotene and the interaction with flavonoids on UVA-induced DNA strand breaks in mouse fibroblast C3H10T1/2 cells

It has been suggested that β-carotene itself is unstable under certain conditions and that a combination of antioxidants may prevent the pro-oxidative effects of β-carotene. Thus, the present study aimed to investigate the interaction of β-carotene with three flavonoids—naringin, rutin and quercetin—on DNA damage induced by ultraviolet A (UVA) in C3H10T1/2 cells, a mouse embryo fibroblast. The cells were preincubated with β-carotene and/or flavonoid for 1 h followed by UVA irradiation, and DNA damage was measured using comet assay. We showed that β-carotene at 20 μM enhanced DNA damage (by 35%; P<.05) induced by UVA (7.6 kJ/m²), whereas naringin, rutin and quercetin significantly decreased UVA-induced DNA damage. When each flavonoid was combined with β-carotene during preincubation, UVA-induced cellular DNA damage was significantly suppressed and the effects were in the order of naringin≥rutin>quercetin. The flavonoids decreased UVA-induced oxidation of preincorporated β-carotene in the same order. Using electron spin resonance spectroscopy, we showed that the ability of these flavonoids to quench singlet oxygen was consistent with protection against DNA damage and β-carotene oxidation. All three flavonoids had some absorption at the UVA range (320–380 nm), but the effects were opposite to those on DNA damage and β-carotene oxidation. Taken together, this cell culture study demonstrates an interaction between flavonoids and β-carotene in UVA-induced DNA damage, and the results suggest that a combination of β-carotene with naringin, rutin or quercetin may increase the safety of β-carotene.     

Inhibition of apoptosis by menadione on exposure to UVA

Quinones are widely distributed in the environment, both as natural products and as pollutants. This paper reports that one of the simplest quinones, 2-methyl-1,4-naphthoquinone (menadione), effectively inhibited apoptosis in the presence of UVA. Menadione suppressed the apoptosis induced by serum depletion and cell detachment. This effect was significantly enhanced by UVA irradiation. An antioxidant, N-acetylcysteine, completely inhibited the antiapoptotic effects of both menadione itself and menadione plus UVA, and peroxidation of the cells after treatment was observed using a probe to detect the intracellular production of peroxides. By contrast, 2-hydroxy-1,4-naphtoquinone (lawsone) showed no antiapoptotic effect in the presence or absence of UVA. Lawsone is reported not to undergo the redox process that produces reactive oxygen species. These results indicated that intracellular peroxidation contributed to the antiapoptotic effects of both menadione itself and menadione plus UVA. Dysregulation of the apoptotic process is critical to carcinogenesis. The photosensitization of quinone compounds as it relates to the inhibition of apoptosis should be examined in the future.     

Flavonoid systematics of the genus Perideridia (Umbelliferae)

A survey of flavonoids in sixteen of the seventeen taxa in the genusPerideridia (Umbelliferae) showed the presence of thirteen glycosides of the flavonols kaempferol, quercetin, and isorhamnetin, and seven glycosides of the flavones apigenin, luteolin and chrysoeriol. An anthocyanin and four other flavonoids also occur, but remain unidentified dueto their low concentration. Several species characteristically produce speciesspecific compounds. The majority of species, however, produce flavonoids common to one or more taxa, but each taxon can be distinguished by its own specific complement of these flavonoids. Based on classes of flavonoids the genus can be divided into three groups: (1) those species which produce only flavonols; (2) those which produce mainly flavonols and a few flavones; and (3) those which produce predominantly flavones with flavonols absent or present only in trace amounts. Geographically, the flavonol-producing species are centered in California, extending northeastward to Idaho and eastward into Arizona. The flavonol/flavone producers are concentrated more towards the Pacific Northwest and eastward through the Rocky Mountains to the midwestern United States.     

Evolution des flavonoides au cours de la croissance des bractees de Poinsettia

The changes in flavonoids were studies during the growth of Poinsettia bracts. Pelargonidin glycosides appeared after cyanidin glycosides; the 3-rutinoside after the 3-monoglucoside. The 3-mono-glucosides were predominant at all times. The bracts contained kaempferol and quercetin, both as aglycones and glycosides. There was no direct relationship between the pathways of flavonol and anthocyanin biosynthesis, but two separate pathways corresponding to the 4′-monohydroxylated- and 3′,4′-dihydroxylated-, flavonoids. Flavanonols and isoflavones were detected but not characterized.     

Flavonoid inhibition of overexpressed human 3beta-hydroxysteroid dehydrogenase type II

The inhibitory effects of various flavonoids on human 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase type II (3beta-HSD type II), overexpressed in baculovirus, were investigated, and the structure-inhibition relationship was examined. The isoflavone derivatives daidzein, genistein, formononetin and biochanin A inhibited 3beta-HSD type II activity at a concentration of 10 microM and of these, genistein was the most potent inhibitor. 6-Hydroxyflavone (6-HF), a synthetic flavone, also strongly inhibited 3beta-HSD activity but 5-HF, 7-HF and other natural flavones were less potent. Energy minimization structures of the flavonoids, as produced using MOE software, showed that isoflavones and flavones have an almost flat A-C ring structure, and that flavonoids that acted as inhibitors had similar steric structures to DHEA. Genistein, 6-HF and cyanoketone, which is known as a typical 3beta-HSD inhibitor, were found to act as competitive inhibitors with K(i) values of 0.12 microM, 0.19 microM and 0.67 nM, respectively. Furthermore, the LUMO (lowest unoccupied molecular orbital (LUMO)) values, as calculated using WinMOPAC (Fujitsu, Japan), of the inhibitors were correlated with the IC(50) values (r2 = 0.84). From these results, it appears that inhibitory effects of flavonoids are due to the combination of steric structure and electron affinity between the active center of 3beta-HSD type II and the flavonoid molecule.     

Genistein as a potential inducer of the anti-atherogenic enzyme paraoxonase-1: studies in cultured hepatocytes in vitro and in rat liver in vivo

A number of cardioprotective effects, including the reduced oxidation of the low‐density lipoprotein (LDL) particles, have been attributed to dietary soy isoflavones. Paraoxonase 1 (PON1), an enzyme mainly synthesized in the liver, may exhibit anti‐atherogenic activity by protecting LDL from oxidation. Thus, dietary and pharmacological inducers of PON1 may decrease cardiovascular disease risk. Using a luciferase reporter gene assay we screened different flavonoids for their ability to induce PON1 in Huh7 hepatocytes in culture. Genistein was the most potent flavonoid with regard to its PON1‐inducing activity, followed by daidzein, luteolin, isorhamnetin and quercetin. Other flavonoids such as naringenin, cyanidin, malvidin and catechin showed only little or no PON1‐inducing activity. Genistein‐mediated PON1 transactivation was partly inhibited by the oestrogen‐receptor antagonist fulvestrant as well as by the aryl hydrocarbon receptor antagonist 7‐ketocholesterol. In contrast to genistein, the conjugated genistein metabolites genistein‐7‐glucuronide, genistein‐7‐sulfate and genistein‐7,4′‐disulfate were only weak inducers of PON1 transactivation. Accordingly, dietary genistein supplementation (2 g/kg diet over three weeks) in growing rats did not increase hepatic PON1 mRNA and protein levels as well as plasma PON1 activity. Thus, genistein may be a PON1 inducer in cultured hepatocytes in vitro, but not in rats in vivo.     

Inhibition of xanthine oxidase by flavonoids

Various dietary flavonoids were evaluated in vitro for their inhibitory effect on xanthine oxidase, which has been implicated in oxidative injury to tissue by ischemia-reperfusion. Xanthine oxidase activity was determined by directly measuring uric acid formation by HPLC. The structure-activity relationship revealed that the planar flavones and flavonols with a 7-hydroxyl group such as chrysin, luteolin, kaempferol, quercetin, myricetin, and isorhamnetin inhibited xanthine oxidase activity at low concentrations (IC50 values from 0.40 to 5.02 microM) in a mixed-type mode, while the nonplanar flavonoids, isoflavones and anthocyanidins were less inhibitory. These results suggest that certain flavonoids might suppress in vivo the formation of active oxygen species and urate by xanthine oxidase.