34s
Journal of Natural P r d c t s Vol. S I , No. 2 , pp. 345-348, Mar-Apr I988
INHIBITION OF COW’S MILK XANTHINE OXIDASE BY FLAVONOIDS TOSHIMIEUHAYASHI; k U K 0 SAWA, I MASARUKAWASAKI, MUNEHISAARISAWA, MINEOSHIMIZU,and NAOKATAMORITA
Faculty of Pharmaceutical Scienca Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan ~
Flavonoids, a large group of naturally occurring pigments, are widely distributed in the plant kingdom. They are known to inhibit a number of enzymes such as phosphodiesterase (1-3), Ca2+ ATPase (4), aldose reductase (5-7), lipoxygenase ( 8 , 9 ) , and cyclooxygenase (IO). The inhibition of xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.2.3.2) by flavonoids has also been reported by several workers (11-15). The major function of this enzyme is to oxidize hypoxanthine and xanthine to uric acid. An inhibitor of xanthine oxidase has been expected to be a therapeutic agent for hyperuricemia that causes gout (16), renal stones (16,17), or ischemic myocardium (18).Allopurinol is a potent inhibitor of xanthine oxidase that has been clinically used (19). The present paper describes inhibition of xanthine oxidase from cow’s milk by 103 flavonoids. Some structure-activity relationships are also discussed. TABLE1.
Inhibitory Activities of Flavones on Xanthine Oxidase. Substituents
Compound” 5
cosmosiin . . . rhoifolin . . . acacetin . . . acaciin . . . . tilianine . . . baicalein . . . baicalein-Me3 . scutellarein . . scutellarin . . cinimaritin . . cinimarin . . pectolinarigenin pectolinarin . .
IN& Sekino.
. . . . . . . .
. . . . .
RESULTS AND DISCUSSION The inhibitory activities of 103 flavonoids against xanthine oxidase at 50 pg/ml were measured. The inhibition percentages of individual flavonoids excluding 19 compounds previously reported (11-15) are presented in Tables 1-5. IC,,’s are also listed with flavonoids indicating more than 50% inhibition at 50 pgiml. Baicalein, robinetin, and 3,6-dimethoxyapigenin were newly found to possess a strong inhibitory effect on xanthine oxidase. Luteolin, diosmetin, quercetin, and naringenin were confirmed to possess strong activity, and 3,6-dimethoxyapigenin showed the highest activity. Generally speaking, flavones and flavonols without a glycosyl group were relatively strong inhibitors. As Iio et a / . reported (14), the presence of a glycosyl group decreased the inhibitory activity. Methylation of an aromatic hydroxyl group also decreased inhibition except for diosmetin
. . . . .
. . . . .
. . . . .
. . . . . . . . . . . .
. . . . . . . .
. . . .
OH OH OH OH OH OH OMe OH OH
6
7
0 - R1 0-R2
OH 0-R2
0-Rl OH OMe OH
OH OMe OH
OH OMe
0-R3
OH OH OH OH
OMe OMe OMe
OMe OMe OH
0-R,
8
3’
-
4‘ OH OH OMe OMe OMe
Inhibition %at 5 0 pglml)
13.9 12.9 55.7 15.1
0 85.5
OH OH OH
0 - R1 OMe OMe
22.0 67.3 16.6 0 12.2 32.1 11.5
346
[Vol. 5 1, No. 2
Journal of Natural Products TABLE1. Continued. Substituents
Compound" 5
6
7
OAc OH OH OH OH OH OH OH OAc OH OH OH OH OH OH OH
OAc
OAc 0-R1 0-R, OH 0-R, 0-R, 0-R I 0-R, OAc OH OMe OMe OMe OMe OH OH
scutellarein-Ac, luteolin-7-R, . lonicerin . . . chrysoeriol . . chrysoeriol-7-R, diosmetin-7-R4 diosmetin-7-R, diosmin . . . luteolin-Ac4 . pedalt i n . . . ciniliol . . . . ciniliol-4'-R, . cirsileneol . . cirsileneol-4'-R, wogonin . . . fukugeti n . . . flavone . . . .
. . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
'R = -CO-CH-CH-Ph-OH, binose-glucose. 0 ''
OMe OMe OMe OMe OMe R
3'
4'
OH OH OMe OMe OH OH OH OAc OH OH OH OMe OMe
OAc OH OH OH OH OMe OMe OMe OAc OH OH 0-R 1 OH 0-R,
OH
OH
8
OMe
tent inhibitory activity ( 15). These findings suggested the presence of free hydroxyl groups at C-5 and C-7 to be important for producing inhibitory activity against xanthine oxidase. In the
Subsrituents
~
,
O-R, O-R, O-R,
OH O-R2 0-R, O-R, 0-R, OR, 0-R,
OH OMe OMe OMe
OH
0 67.2 20.1 61.5 0.3 30.5 28.9 0 47.7 49.0 0.5 0 1.0 0 50.2 97.2 15.4
Inhibitorv Activities of Flavonols on Xanthine Oxidase.
Compound*
trifolin . . . . . . . . kaernpferirrin . . . . . juglvlin . . . . . . . kaempfrrol-7-R, . . . krmpferol-3-R, . . . hyperin . . . . . . . . quercetin-3-R, . . . . reynourrin . . . . . . ivicdarin . . . . . . . quercetin-3-RI-2"-% . quercimerirrin . . . . quercecin-3-Me . . . . quercerin-3-Me-4'-Rl . quercerin-3-Me-7-RI . irorhamncrin . . . . . persicarin . . . . . . . narcissin . . . . . . . qucrccrin-Ac, . . . . . myricetin-3-R1,, . . . . myricerin-Ac6 . . . . . robinecin . . . . . . . apigenin-3,6-OMe2 . .
Inhibition % at 50 pg/ml:
R,=glucose, R, = rhamnose-glucose, R3=glucuronic acid, R*=ara-
and 3,6-dimethoxyapigenin. All acetates, isoflavones, and C-glycosylflavonoids tested showed less inhibitory activity. Recently, Nishibe et al. found 4'-O-glycosylated flavonoids with poTABLE2.
-
OH OH OH OH OH OH
OH OH OH OH OH OH OH OH OH
0-KSO, 0-R, OAc 0-R,,, OAc OH
OH OH OAc OH OAc
OMe
OH
-7 3' -OH 0-R,
OH O-R I
OH OH OH OH OH OH O-R 1
OH OH O-R, OH OH OH OAc OH OAc OH OH
'R =glucose, R2 = I nnose-glucose, R,=glucuronic acid, gallic acid, R ,,,=rhmnore-galactose.
OH OH OH OH OH
OH OH OH OH OMe OMe OMe OAc OH OAc OH
Inhibition 4'
(%at 50 Fg/ml)
5'
OH OH OH OH OH OH OH OH OH OH OH OH 0-R, OH OH OH OH OAc OH OAc OH OH
C-%
OH OAc OH
16.1 15.5 0 0 14.4 43.0 36.5 33.1 30.8 21.0 0 98.5 69.1 39.5 49.0 90.3 22.2 0 88.6 13.6 82.9 95.0
, =galactose, Re= rhamnose, R,=arabinose,
R,=xylose,
%=
Mar-Apr 19881
347
Hayashi etal. : Inhibition of Xanthine Oxidase
Inhibitory Activities of Flavanones, Flavanonols, and Isoflavones on Xanthine Oxidase.
TABLE3 .
Subrtituents'
-
Compound'
3
6
5
7
3'
4'
-
Inhibition R at 50 &nl
5'
-
F ~ M U C
liquiritigenin . liquiritin . . . prunin . . . . saktuanetin . . iroukuranetin poncirin . . . ericdicryol . .
. . . .
OH OH O-R I OMe OH
OH OH OH OH OH
. .
OH
100 18.0 4.2 0 0 0 47.8
O-R I
OR2
OH
OH
OH OH OH OH O-Ri
OH OH OH
OH OH OMe OMe OH
Fiavanonol
mmadendrin fustin . . taxifolin . . artilbin . . phellamurin
. . . . , . . . . .
OH OH OH
OH
OH OH OH
0-% OH
OH OH OH OH OH
66.9
OH OH OH
OH OH OMe OH OH O-R 1 OH OH OMe OMe OMe
OMe
0 0 17.6 0 0.8 15.3 0 0 0 0 0
OMe
OH OMe
OMe
0 0
27.2 17.8 0 0
IJO&Wne
dudzein . . . . daidzin . . . . . formononetin . . genirtein . . . , genisrin . . . . rophoricoside . . rectorigenin . . rectoridin . . . irisrectorigeninA irigenin . . . . iridin . . . . .
OH OH OH OH OH OH OH OH
irirolone . irisflorcncin
OMe OMe
OH 0 - RI OH OH 0-R I OH OH 0-R I OH OH 0-RI
-O-CH,-O-OCH,-0-
'R, =glucose, R,= rhamnose-glucose, %=rhamnose.
Rl I=4CH2)2-C(OHXCH,)2
-
be due to the steric factor of the bulky group at C - 3 . The low activity of flavanones and flavanonols suggested the presence of a double bond between C-2 and C-3 to be an important factor. The high activity of diosmetin (IC5,0.07 p,M) and 3,6-dimethoxyapigenin (IC?,
cases of vitexin and orientin possessing free OH at C-5 and C-7, the C-glycosyl group might be tcm bulky and prevent the flavonoids from combining with the enzyme. The lack of activity of isoflavones and 3-0-glycosylated flavonols with a free OH at C-5 and C-7 seemed to
TABLE4 .
OMe
Inhibitory Activities of C-glycosylflavonoids on Xanthine Oxidase. Substituent'
lnhibirion
Compound'
(aat 50 Fglml) vitexin . . . . , . . . orientin . . . . . . . isoorientin . . . . . . isoorientin-7-R, . . . . swenisin . . . . . . . embigenin . . , . . . embinin . . . . . . . keyakinin . , . , . . . OH lR,=glucose, R,=rhamnose.
OH OH OH
OH OH
OH OH OH
C-R I C-R C-R C-R, C-R C-R
, , ,
OH OH OH 0 - RI OMe OMe OMe OMe
C-R, C-R,
OH OH OH
OH OH OH OH OH OMe OMe OH
0
15.3 25.2 0
24.9
33.5
0-R,
22.8 0
Wol. 5 1, No. 2
Journal of Natural Products
348
TABLE5. Inhibitory Activities of Biflavones on Xanthine Oxidase.
6H Compound amentoflavone . sciadopitysin .
, ,
. , . .
,
.
.
.
.
,
,
.
.
.
H Me
H Me
0.03 pM)might be attributable to their surface activity, as Iio et a / . suggested (14). EXPERIMENTAL MATERIAL.-we obtained most of the flavonoids tested from plant sources; 3 , 6 d i methoxyapigenin was obtained through the courtesy of professor T.J. Mabry, University ofTexas at Austin. Other chemicals were purchased from commercial sources and used without further purification: xanthine oxidase (Boehringer Mannheim Co. Ltd.), xanthine (ICN Pharmaceuticals Inc.), sodium phosphate dibasic 12 hydrate, potassium phosphate monobasic, and Tween 80 (Wako Pure Chemical Industries, Ltd.). ASSAY OF XANTHINE OXIDASE ACTIVITY.Xanthine oxidase activity was measured by the method reported by Noro et af. (13). The inhibitory activity (96)was calculated as follows: (1-B/ A)X 100, where A is the activity of enzyme without test material, and B is the activity of enzyme with test material. LITERATURE CITED M. Ruckstuhl, A. Beretz, R. Anton, and Y. Landry, Biocbem. Pbamwol., 20, 535 (1979). 2. E. Petkov, N . Nikolov, and P. Uzunov, Planta M e d . , 43, 183 (1981). 3. T. Nikaido, T . Ohmoto, T . Nomura, T . Fukai, and U. Sankawa, Cbem. P b a n . Buff., 32,4929 (1984). 4. C.M.S. Fewtrell and B.D. Gomperts, Nature, 265, 635 (1977). 5. S.D. Varma and J.H. Kinoshita, Biocbem. Pba-ol., 25,2505 (1976). 6. J . Okuda, I. Miwa, K. Inagaki, T. Horie, and M. Nakayama, Cbem. Pbann. Buff., 32,767 (1984). 7. M. Shimizu, T . Ito, S. Terashima, T.
H H
H Me
8. 9.
10.
11. 12.
13.
14.
15.
1.
16.
17. 18.
19.
100 0
35.3
-
Hayashi, M. Arisawa, N . Morita, S. Kurokawa, K. Ito, and Y. Hashimoto, Pbyrochernistty, 23, 1885 (1984). S. Sekiya and H . Okuda, Biocbem. Biopbys. Res. Commun., 105, 1090 (1982). T . Yoshimoto, M. Furukawa, S. Yamamoto, T. Horie, andS.K. Watanabe, Biocbem. Biopbys. Res. Cwnmun., 116, 612 (1983). J. Baumann, G . Wurm, and F.V. Bruchhausen, Arch, Pbann. (Weinbeim, Ger.), 313,330 (1980). J.M. Beiler and G.J. Martin, /. Biof. Cbem., 192,8 3 1 (195 1). K. Otsuka, Y. Ogata, H . Shiojiri, Y. Wakabayashi, and K. Kagei, “Abstracts of Papers,” The 103rd Annual Meeting of the Pharmaceutical Society of Japan, Tokyo, 1983, p. 221. T . Noro, Y. Oda, T. Miyase, A. Ueno, and S. Fukushima, Chern. Pbann. Bufi., 31, 3984 (1983). M. Iio, A. Moriyama, Y.Matsumoto, N . Takaki, and M. Fukumoto, Agrir. Biof. Chern., 49,2173 (1985). S. Nishibe, A. Sakushima, T. Noro, and S. Fukushima, Sboyakugaku Zasshi, 41, 116 (1987). A.P. Hall, P.E. Barry, T.R. Dawber, and P.M. McNamara, Am. /. M e d . , 42, 27 (1967). T.F. Yii and A.B. Gutman, Ann. Int. M e d . , 67, 1133 (1967). R.A. DeWall, K.A. Vasko, E.L. Stanley, and P. Kezdi, Am. Hurt J., 82, 362 (197 1). A.G. Gilman, L.S. Goodman, T . W . Rall, and F. M u d , “Goodman and Gilman’s The Pharmacological Basis of Therapeutics,” Macmillan, New York, 7th ed., 1985, p. 712.
Received 28 August 1987
