Inhibition of Xanthine Oxidase by Flavonoids

Agric. Biol. Chem., 49 (7), 2173-2176, 1985 2173

Inhibition of Xanthine Oxidase by Flavonoids Masayoshi Iio, Ayako Moriyama, Yoshiko Matsumoto, Nahoko Takaki and Michi Fukumoto Department of Food and Nutrition Science, KumamotoWomen's University, Kenguncho, Kumamoto862, Japan Received February 1, 1985

The influence of nineteen flavonoids on cow's milk xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.2.3.2) was investigated. The enzyme activity was estimated by measuring the increase in absorbance at 290nmdue to uricate formation as well as by a colorimetric method assaying hydrogen peroxide generated from uricate by uricase. Amongthe flavonoids tested, myricetin, kaempferol, quercetin, fisetin, quercitrin, and morin inhibited the enzyme strongly at 50/im; the concentrations which gave 50% inhibition (ID50) were 2, 2, 3, 7, 15, and 19jim, respectively. The inhibition modeof the former three compounds was of mixed type and the kinetic parameters were determined. Chrysin and naringenin were moderately inhibitory, while other flavonoids showed weak to no inhibition.

Flavonoids have various biochemical ef- MATERIALS AND METHODS fects.1} They inhibit a number of hydrolytic Reagents. The sources of the flavonoids tested were enzymes such as alkaline phosphatase,2) yeast described elsewhere.3) Newly tested commercial flavonoids a-glucosidase3) and Streptococcus mutans dex- in this study were flavone and 3-hydroxyflavone (Tokyo transucrases.4) They were also reported to Kasei). Baicalin and wogoninglucuronide were obtained inhibit glyoxalase I,5) which is probably in- through the courtesy ofDr. T. Yamagishi of the Hokkaido volved in inflammation. They were described Institute of Public Health. Other chemicals were pur- as inhibiting oxidation-reduction enzymes chased from the commercial sources indicated and used without further purification. Xanthine oxidase from cow such as cyclooxygenase6) and lipoxygenase7) in milk (20 U/ml), beef liver catalase (1.3 x 106 U/20mg/ml) addition to aldose reductase.8) Some flavo- (Boehringer); xanthine (Nakarai); and allopurinol noids also suppressed non-enzymatic collagen (Wako). degradation caused by superoxide.9) Other flavonoids were antiinflammatory.10) Quer- Xanthine oxidase activity. The increase in absorbance at cetin and morin inhibit carcinogenesis.n'12) 290nmwas measured after terminating the reaction in 30 min by adding hydrochloric acid to the reaction mixture We happened to find that some flavonoids as described by Noro et al.,13) who modified the original inhibit cytochrome c reduction by super- method reported by Kalckar.14) DMSOwas used instead oxide generated by a xanthine-xanthine oxi- of ethanol and Tween 80 in our study. The rate of dase system. We soon noticed that the in- inhibition (%) was calculated as follows: (l-AAteJ ^control) x 100, where AAindicates the increase in absor- hibition was caused by suppression of xan- bance in 30 min. thine oxidase activity by flavonoids. This study In a second method, uricate concentration was de- deals with the inhibition of xanthine oxidase termined with a commercial kit, the Uric Acid B-Test (xanthine : oxygen oxidoreductase, EC 1.2.3.2) (Wako). The principle of the measurement involves con- by some flavonoids, as one of the studies on version of uricate into allantoin and hydrogen peroxide, biochemical effects of flavonoids being per- and condensation of 4-aminoantipyrine and 3-methyl-TV- ethyl-A^-(j5-hydroxyethyl)-aniline into a purple pigment in formed in our laboratory. the presence of hydrogen peroxide by the help of per- oxidase. To eliminate hydrogen peroxide formed in the xanthine oxidase reaction, the reaction mixture was 2174 M. Iio et al. treated with catalase in advance. The mixture contained RESULTS 1.56ml of50mM sodium phosphate buffer, pH 7.8, 0.20ml of 1mMdisodium EDTA, 0.20ml of 10mMxanthine, 0.02ml of 10mMflavonoid in DMSO.After preincubation Ejfects of flavonoids on xanthine oxidase at 30°C for lOmin, 0.04ml ofxanthine oxidase, diluted 15- activity fold with 1mMEDTAin 2m ammoniumsulfate, was Table I gives the effects of flavonoids on added. The mixture was incubated at 30°C for lOmin and xanthine oxidase activity at 50jim concen- placed in a boiling water bath for 3min. One ml of the supernatant (3,000 rpm x 5 min) of the heated mixture was tration. ID50's are also listed with flavonoids treated with 0.1 ml of catalase, diluted 5-fold with 50mM inhibiting more than 70% at 50piM. One of the sodium phosphate buffer pH 7.8 at 30°C for 3min. The results is illustrated in Fig. 1, when quercetin mixture was heated in a boiling water bath and the was used as an inhibitor. Myricetin, kaemp- supernatant (3,000 rpm x 5min) was used as a sample for ferol, quercetin, fisetin, quercitrin, and morin uricate determination. The procedure thereafter was ac- were strong inhibitors. Table I also contained cording to the manual attached to the kit. The suppression the result with allopurinol, a potent inhibitor of coloration was observed with someflavonoids. Hence the correction was made with these flavonoids in the of xanthine oxidase. estimation of the amount of uricate formed. Inhibition of uricate formation by some

Table I. Effects of Flavonoids on Xanthine Oxidase Activity as Measured by Uricate Formation The enzyme activity was estimated either by measuring the absorbance at 290 nmdue to uricate formed or by determining the amount of hydrogen peroxide after uricase treatment. Experimental details are described in the text. In the class column, A, B, and C mean flavonol, flavone, and flavanone, respectively; G indicates glycoside. The number in the parenthesis means the numberof aromatic hydroxyl groups present in each flavonoid. The flavonoid concentration was 50 fiu except that those of chrysin and 3-hydroxyflavone were 25 and 5 jWM,respectively, because of their low solubilities. Activity assay method

t-1Flavonoidà"j Class ^i UVMeasurement Uricase method* Inhibition ID50 Inhibition (%) (m) (%)

Myricetin A (6) 96 2 63 Kaempferol A (4) 90 2 80 Quercetin A (5) 86 3 76 Fisetin A (4) 86 7 Quercitrin A ~ G (4) 85 15 Morin A (5) 74 19 Chrysin B (2) 64 Naringenin C (3) 49 Myricitrin A ~ G (5) 30 Apiin B~G (2) 25 Baicalin B ~G (2) 16 Gossypitrin A ~G (5) 14 Naringin C ~G (2) 12 Herbacitrin A ~ G (4) 6 Wogonin glucuronide B ~ G (0) 5 Rutin A-G (4) 4 Robinin A~G (3) 3 Hesperidin C ~G (2) - 7 3-Hydroxyflavone A (1) - 9 Flavone B (0) - 15 (Allopurinol) 1 00 0. 3 * Final flavonoid concentration= IOO/^m. 2175 Inhibition of Xanthine Oxidase by Flavonoids

à"5 0.4

3 0.2

5 10 Quercetin Concentration (jjM) 1/S (pM"1)

Fig. 1. Inhibition of Xanthine Oxidase Activity as a Fig. 2. Double-reciprocal Plots of Xanthine Oxidase Function of Quercetin Concentration. Reaction in the Presence and Absence of Quercetin. The reaction was followed by measuring absorbance at The enzyme activity was estimated by measuring ab- 290 nmdue to uricate formation. The experimental con- sorbance at 290nmdue to uricate formation. The re- ditions were the same as described in the legend to Table I action conditions were the same as those described in the except that concentrations of quercetin were varied. Each legend to Table I except that concentrations of xanthine point indicates the average of triplicate measurements. were varied. Minus quercetin <#-#) and plus quercetin (A-A)- Quercetin concentration was 3/iM. Each point indicates the average of triplicate measurements. flavonoids was also confirmed by a colorimetric method with a different principle using Table II. Kinetic Constants of Xanthine Oxidase uricase. Table I shows the result. Myricetin, Inhibition by Some Flavonoids quercetin, and kaempferol were strong in- The reaction conditions were the same as those de- hibitors as they were in the experiments with scribed in the legend to Fig. 2. KEl and KEIS mean the dissociation constant of enzyme-substrate complex and the activity assay measuring UVabsorbance. that of enzyme-substrate-inhibitor complex, respectively. Each set of KEl and KEls were determined from Effects of albumin on xanthine oxidase in- slopes and intercepts in double-reciprocal plots. hibition by flavonoids _ . , ID50 Mode of Km Kms To confirm the specificity of flavonoid in- Flavonoid ^ inhibition ^ ^ hibition against xanthine oxidase, albuminwas added to the reaction mixture. The degree of Myricetin 2 Mixed type, 5 1 0.55 xanthine oxidase inhibition by quercetin de- very close to uncompetitive creased slightly, as the albumin concentration Kaempferol 2 Mixed type 3. 1 0.55 increased, although the data are not shown Quercetin 3 Mixed type 3.0 2.3 here. Yet in the presence ofa large amount of albumin (50-fold), quercetin still inhibited con- siderably, suggesting that specific binding of flavonoids. quercetin with xanthine oxidase occurs. DISCUSSION Inhibition mechanism The inhibition mechanisms of the three Kinetic analysis (Table II) revealed that flavonoids that are potent inhibitors of xan- myricetin inhibited xanthine oxidase largely thine oxidase were studied by kinetic analysis imcompetitively with respect to xanthine as a with double-reciprocal plotting. Figure 2 substrate. Thus myricetin is supposed to bind shows the Lineweaver-Burk plots of reactions to the enzyme-substrate complex rather than in the presence and absence of quercetin in a to free enzyme. The inhibitions by kaempferol xanthine oxidase reaction mixture. Table II and quercetin were a mixture of the uncom- gives the types of inhibition and the dis- petitive and non-competitive modes, with re- sociation constants with quercetin and other spect to xanthine as a substrate. 2176 M. Iio et al.

Generally speaking, flavonols were strong could be used as a preventive and therapeutic inhibitors. In addition, the greater the number agent for gout. In addition, they might be of aromatic hydroxyl groups in a flavonoid, useful as a biochemical probe, taking advan- the stronger the inhibition of the flavonoid tage of their potent inhibitory activities. against xanthine oxidase. The increase of the numberof hydroxyl groups means the increase Acknowledgment.Weexpress our thanks to Professor of hydrophilicity. Flavonoids having many Toshio Nakabayashi of Shizuoka University and Dr. hydroxyl groups such as myricetin and quer- Takashi Yamagishi for their generous gifts of certain cetin are yet insoluble in water, indicating that valuable flavonoids. they can be surfactants because of the concom- itant presence of hydrophilicity and hydropho- REFERENCES bicity in each molecule. Their inhibition might 1) B. Havsteen, Biochem. Pharmacol, 32, 1141 (1983). be attributable to their surface activity. 2) M. Iio, K. Ushijima, M. Fujita, M. Matsumoto, and Myricetin, and quercetin might act as specific S. Miyatake, Nippon Nogeikagaku Kaishi, 54, 171 surfactants which bind to the enzyme with (1980). special affinity. 3) M. Iio, A. Yoshioka, Y. Imayoshi, C. Koriyama, and and A. Moriyama, Agric. Biol. Chem., 48, 1559 The presence of glycosyl groups decreased (1984). the inhibition as could be seen in the cases of 4) M. Iio, M. Uyeda, T. Iwanami, and Y. Nakagawa, quercitrin, rutin, naringin, and myricitrin, Agric. Biol. Chem., 48, 2143 (1984). compared to their aglycons. Glycosyl groups 5) M. Iio, S. Himeno, K. Miyauchi, K. Mikumo, andN. Ohta, Nippon Nogeikagaku Kaishi, 57, 765 (1983). seemed to be too bulky or too hydrophilic and 6) J. Baumann, G. Wurm, and F. V. Bruchhausen, prevent the glycosidic flavonoids from coming Arch. Pharm. (Weinheim) 313, 330 (1980). into contact with the enzyme. The determi- 7) S. Sekiya and H. Okuda, Biochem. Biophys. Res. nation of partition coefficients of flavonoids Commun., 105, 1090 (1982). between water and organic solvents as a mea- 8) J. Okuda, I. Miwa, K. Inagaki, T. Horie and M. Nakayama, Biochem. Pharmacol, 31, 3807 (1982). sure of surfactant activity and correlating them 9) J. Monboisse, P. Braquet, A. Randoux, and J. P. with the inhibitory activity is nowin progress. Borel, Biochem. Pharmacol, 32, 53 (1983). Xanthine oxidase was reported to be in- 10) W. C. Hope, A. F. Welton, C. Fiedler-Nagy, C. hibited by folic acid,15) amethopterin15) and Batula-Bernardo, and J. M. Coffey, Biochem. aldehydes.16) Beiler et al.11] reported the in- Pharmacol, 32, 367 (1983). hibitory activities of quercetin and rutin ll) H. Nishino, A. Iwashima, H. Fujiki, and T. Sugimura, Gann, 75, 113 (1984). against the enzyme. A study by Noro et 12) T. Nakadate, S. Yamato, E. Aizu, and R. Kato, al.13) on inhibition of xanthine oxidase by Gann, 75, 214 (1984). Daphne genkwa flavonoids showed that api- 13) T. Noro, Y. Oda, T. Miyase, A. Ueno, and S. genin (ID5O=0.74/iM) and luteolin (0.59jim) Fukushima, Chem. Pharm. Bull, 31, 3984 (1983). strongly inhibited the enzyme. The mode 14) H. M. Kalckar, /. Biol Chem., 167, 429 (1947). of their inhibitions were of mixed type 15) A. S. Lewis, L. Murphy, C. McCalla, M. Fleary, and S. Purcell, /. Biol Chem., 259, 12 (1984). with respect to xanthine as a substrate. 16) F. F. Moreth and R. C. Bray, Biochemistry, 23, 1332 Xanthine oxidase produces uricate, the ex- (1984). cess of which is considered to cause hyper- 17) J. M. Beiler and G. J. Martin, J. Biol Chem., 192, uricemia in human gout. Thus flavonoids 831 (1951).