Agric. Biol. Chem., 48 (6), 1559~1563, 1984 1559
Effect of Flavonoids on a-Glucosidase and /?-Fructosidase from Yeast Masayoshi Iio, Ayako Yoshioka, Yumiko Imayoshi, Chikako Koriyama and Ayako Moriyama Departmentof Foodand Nutrition Science, Faculty of Life Sciences, KumamotoWomen's University, Kumamoto862, Japan Received December 9, 1983
The influence of 16 flavonoids on yeast a-glucosidase and /?-fructosidase was investigated. Either />-nitrophenyl-a-D-glucopyranoside or maltose was used as substrate for a-glucosidase, and sucrose for jS-fructosidase. Quercetin, myricetin, fisetin, and quercitrin strongly inhibited a- glucosidase at 0.1 mM.The concentrations which gave 50%inhibition were 4, 8, 8, 20, and 20jum for myricetin, quercetin, fisetin, kaempferol, and quercitrin, respectively. The mode of inhibition was found to be mixed type, close to non-competitive type with quercetin and quercitrin. Fisetin, naringenin, and morin showed mixed type inhibition. Albumin from bovine serum slightly affected the anti-a-glucosidase activity of quercetin. The flavonoids tested exhibited little effect on /?- fructosidase activity at 0.1 mM.
Flavonoids have attracted interest as food kinase, glucose-6-phosphate dehydrogenase, ATPsodium components. They show bitterness in taste, salt, and NADP sodium salt were obtained from Boehringer. Other chemicals were also purchased from the anti-vitamin Bx activity, antioxidative action, commercial sources indicated. Maltose, quercetin, morin, mutagenicity,1* reaction with proteins,2) and so quercitrin, and rutin (Wako); naringenin, and naringin forth. They have been studied from the food (Tokyo Kasei); fisetin, chrysin, and myricetin (Aldrich); chemical point of view. On the other hand, hesperidin (Nakarai); kaempferol (Sigma); /?-nitrophenyl- their biochemical, physiological, and pharma- a-D-glucopyranoside (Koch-Light). Apiin, herbacitrin, cological effects have also been investigated myricitrin, robinin, and gossypitrin were obtained through the courtesy of Professor Toshio Nakabayashi of and various activities were found.3) Some in- Shizuoka University. vestigators consider them to be semi-essential nutrients. Enzymeactivity assay. (1) a-Glucosidase with p-nitrophenyl-
50 fi\ of a-glucosidase solution. The amount of enzyme per had I50 values listed in Table II. Myricetin, reaction mixture was one which liberated 3.3/xmol of quercetin, and fisetin were found to be the glucose per minute. The reaction was initiated by adding the enzymesolution and the mixture was kept at 37°C for most potent in inhibitory action. Whenmal- 4 min. The reaction was terminated by putting the mixture tose was employed as substrate, quercetin into a boiling bath for 3min. An aliquot (10/d) was taken and fisetin showed considerable inhibition, to determine the amount of glucose liberated, by the too (Table III). The degree of inhibition was method described in ref. 6. reduced to some extent, probably because the (3) P-Fructosidase. The reaction mixture contained substrate concentration was much higher 2.50ml of 2him sucrose in 0.2m phosphate buffer, pH 6.8, and 25jjI of 10mMflavonoid in DMSO.The reaction was initiated by adding 0. 10 ml of enzyme solution, which produced 0.46/miol of glucose and fructose in 1 min, and the mixture was kept at 37°C for 20min. An aliquot (100//I) was used to determine the reducing power due to t* 50 glucose and fructose liberated by the modification of Somogyi and Nelson's method described by Hatanaka and Kobara.7)
RESULTS AND DISCUSSION 0 10 20 Effect offlavonoids on a-glucosidase activity Quercetin Concentration (uM) The effect is summarized in Table I when Fig. 1. Inhibition of a-Glucosidase Activity as a NPGwas used as substrate. Quercetin, myri- Function of Concentration. setin, fisetin, quercitrin, and kaempferol The reaction conditions were the same as those described showed strong inhibition, whereas myricitrin, in the legend to Table I except that concentrations of morin, naringenin, gossypitrin, herbacirin, quercetin were varied. Each point indicates the average of and rutin exhibited mediumto weak inhibi- quadruplicate measurements. tion at 0.1 mM.Figure 1 shows inhibition (%) Table II. I50 Values of Flavonoids Inhibitory of the activity as a function of quercetin con- TO a-GLUCOSIDASE centration. The quercetin concentration The reaction condition was the same as that described giving 50%inhibition (I50) was determined to in the legend to Table I except that concentrations of the be 8//M from this result. Other flavonoids flavonoid were varied.
Table I. Effects of Flavonoids on oc-Glucosidase Flavonoid (fiM) Activity (Substrate =/?-Nitrophenyl- (X-D-GLUCOPYRANOSIDE)
The enzyme activity was estimated by measuring p- nitrophenol liberated. Experimental details are described
KaempferolMyricitrin NaringeninMorin 20 20 in the text. 3040 50
. å. , Inhibition r, .. Inhibition Flavonoid ( O/,\ /o) Flavonoid (O/.\ /o) Table III. Effects of Quercetin and Fisetin ON a-GLUCOSIDASE ACTIVITY Quercetin 96 Gossypitrin 48 (Substrate = Maltose) Myricetin 94 Herbacitrin 3 8 The enzyme activity was estimated by measuring glu- Fisetin 89 Rutin 27 cose formed with enzymatic assay method. For the Quercitrin 8 7 Robinin 7 details, see Materials and Methods. Kaempferol 78 Hesperidin 2 Myricitrin 69 Apiin 0 _f ., Inhibition M orin 68 N aringin 0 Flavonoid { 0/, Naringenin 66 Chrysina 0 \/o) Quercetin 5 8 The concentration was 0.01mM owing to low Fisetin 46 solubility. IsoFlavonoidI50 Flavonoids Inhibit a-Glucosidase 1561
the presence and absence of quercetin, indicat- ing that the modewas mixed type very close to -r 30 " non-competitive type. The results of kinetic studies with a numberof flavonoids are sum- o JF marized in Table IV. In all the cases studied, both slopes and intercepts were changed by the presence of each flavonoid tested. This sug- gests that flavonoids can bind both free en- zyme and enzyme-substrate complex. Quer- citrin appears to inhibit in the enzyme in a
0 , 0.01 similar manner as quercetin, although the Ki 1/ S (pM-1) and Ki' for quercitrin were one order larger than those for quercetin. The Ki and Ki' are Fig. 2. >rDouble-reciprocal Plots of a-Glucosidase dissociation constants of the enzyme-inhibitor Reaction in the Presence and Absence of Quercetin. The reaction conditions were the same as those described complex and enzyme-substrate-inhibitor com- in the legend to Table I except that concentrations of plex, respectively, as shown in Chart 1. Two substrate were varied. Minus quercetin ( ) and plus +s quercetin ( ). Quercetin concentration was 5 fiM. Each El' Ki Ks ESå point indicates the average of quintuplicate measurements. \Ks' Kiy E + P Table IV. Kinetic Constants of a-GnjcosiDASE Inhibition by Flavonoids possibilities have been considered from the The reaction conditions were the same as those de- structure of flavonoids. Oneis that someflavo- scribed in the legend to Table I except that concen- noids like rutin and quercitrin have glycosyl trations ofNPG were varied. Each set ofKi and Ki' were determined from slopes and intercepts in double- substituents, which may cause competitive in- reciprocal plots. hibition of a-glucosidase through the glycosyl groups. The other is that the product, p- Ki KV Flavonoid - Modeof inhibition nitrophenol has, at least some structural re- (m) semblance to aglycons, which may cause bind- ing of the aglycons to the substrate binding site Quercetin 6.2 8.2 Mixed type, close to of the enzyme. But the result suggests that non-competitive flavonoids can bind both free enzyme and Quercitrin 13 22 Mixed type, close to non-competitive enzyme-substrate complex. This means that Fisetin 3.2 24 Mixed type flavonoids are able to combine with the en- Naringenin 41 120 Mixed type zymeat site(s) other than the substrate-binding Morin 47 230 Mixed type site.
Effect ofalbumin on inhibition by quercetin ofa- in this case in comparison with the case glucos idase where NPGwas used, because the analyzing Inhibition of a-glucosidase caused by flavo- method needs a higher concentration of noids mayseemnon-specific in nature, because maltose (see Materials and Methods). polyphenol compounds often bind to protein non-specifically and eventually deactivate en- Modeof inhibition of oc-glucosidase activity by zymes by denaturation. To examine the speci- fla vono ids ficity of quercetin inhibition to a-glucosidase, The mode of inhibition was estimated by bovine serum albumin was added to the re- kinetic analysis. Figure 2 shows double- action mixture. The amount of albumin added reciprocal plots of a-glucosidase reaction in ranged from 1-fold to 100-fold the concen- 1562 M. Iio etal tration of protein originally present in the tion by flavonoids of a-glucosidase and glyoxa- enzyme preparation. The presence of added lase I.5) Fairly good correlation can be seen albumin did not much affect the inhibitory in Fig. 4, the correlation coefficient being 0.86. action of quercetin, indicating specificity of the Therefore flavonoids which inhibit a-gluco- flavonoid compoundto a-glucosidase (Fig. 3). sidase activity also usually hinder the action of glyoxalase I. Nevertheless the modes of in- Relationship of the influence offlavonoids on a- hibition by quercetin were different in the two glucosidase and glyoxalase I enzymereactions. The modeof a-glucosidase Comparison was made between the inhibi- inhibition by quercetin was mixed type, while it inhibited glyoxalase I competitively.5)
à" à"/
0 5 10 15 Albumin Concentration (ug/ml Table V. Substituent Groups of Flavonoids Tested Name 3 5 7 8 2' 3' 4' 5' Flavonol Kaempferol OH OH OH OH Robinin O-Glya OH OH OH Quercetin OH OH OH OH OH Quercitrin O-Gly* OH OH OH OH Rutin O-Glyb OH OH OH OH Myricetin OH OH OH OH OH OH Myricitrin O-Glyfl OH OH OH OH OH Morin OH OH OH OH OH Fisetin OH OH OH OH Herbacitrin OH OH O-Glyc OH OH Gossypitrin OH OH O-Glyc OH OH OH Flavone Apiin OH O-Glyd O H Chrysin OH OH Flavanone Naringenin OH OH OH N aringin OH O-Glye OH Hesperidin OH O-Glye OH O-Me a Rhamnosyl. b Rhamnoglucosyl. c Glucosyl. d Glucoapiosyl. e Glucorhamnosyl. Flavonoids Inhibit a-Glucosidase 1563 Table VI. Effects of Flavonoids on oxalase I,5) and lipoxygenase.10) Thus flavo- /?-Fructosidase Activity noids show physiological effects like anti- The enzyme activity was estimated by measuring glu- inflammatory and anti-allergic action,n) anti- cose and fructose with a modification of Somogyi and promotor activity in carcinogenesis,12) and Nelson's method. Experimental details are described in the text. suppression of platelet aggregation13) through inhibition of the enzymes mentioned above. Inhibition The authors aim at investigating influence of (%) Flavonoid flavonoids on the digestion, absorption, and (%) Inhibition Morin metabolism of nutrients. The yeast enzymes Naringenin were used as preliminary tests for ones from Hesperidin mammalian sources. Experiment with disac- charide-hydrolyzing enzymes from porcine intestinal mucosaare nowin progress. Structure-activity relationship Table V summarizes substituent groups on Acknowledgment. We express our thanks to the phenylbenzopyrone ring of the flavonoids Professor Toshio Nakabayashi of Shizuoka University tested. A requisite for the inhibitory action for his generous gift of certain flavonoids. seems to be the presence ofa certain numberof REFERENCES hydroxyl groups. Those flavonoids whose number of hydroxyl groups are less than three 1) Y. Seino, M. Nagao, T. Yahagi, T. Sugimura, T. (e.g. naringin and hesperidin) have no in- Yasuda and S. Nishimura, Mutation Res., 58, 225 (1978). hibitory activity. The idea that the inactivity of 2) K. Igarashi, T. Tsunekuni and T. Yasui, /. Nutr. Sci. naringin and hesperidin results from their Vitaminol, 29, 227 (1983). saturated bond between C2 and C3 is reversed 3) B. Havsteen, Biochem. Pharmacol, 32, 1141 (1983). by the fact that fairly strong inhibition was ob- 4) M. Iio, K. Ushijima, M. Fujita, M. Matsuura and S. served with naringenin which has the same Miyatake, Nippon Nogeikagaku Kaishi, 54, 171 (1980). structure at this position. A hydroxyl group 5) M. Iio, S. Himeno, K. Miyauchi, K. Mikumo and N. at the 5-position does not seemessential, be- Ohta, Nippon Nogeikagaku Kaishi, 57, 765 (1983). cause fisetin showed very strong inhibition in 6) H. U. Bergmeyer, E. Bernt, F. Schmidt and H. Stork, spite of the absence of a 5-OH. Glycosylation "Methods of Enzymatic Analysis," Vol. Ill, ed. by of hydroxyl groups decreases the inhibitory H. U. Bergmeyer, Verlag Chemie, Weinheim/ Academic Press Inc., NewYork and London, 1974, activity; kaempferol to robinin, quercetin to pp. 1196-1201. quercitrin and to rutin, naringenin to narin- 7) C. Hatanaka and Y. Kobara, Agric. Biol. Chem., gin, myricetin to myricitrin, although the de- 44, 2943 (1980). gree of the regression varies from flavonoid 8) M. Ruckstuhl, A. Beretz, R. Anton and Y. Landry, to flavonoid. Biochem. Pharmacol., 28, 535 (1979). 9) C. Cochet, J. J. Feige, F. Pirollet, M. Keramidas and E. M. Chambaz, Biochem. Pharmacol., 31, 1357 Effects offlavonoids on P-fructosidase activity (1982). As can be seen from Table VI, the flavo- 10) K. Sekiya and H. Okuda, Biochem. Biophys. Res. noids tested showed only weak or no in- Commun., 105, 1090 (1982). hibition to /?-fructosidase in contrast to a- ll) W, C. Hope, A. F. Welton, C. Fiedler-Nagy, C. Batula-Bernardo and J. W. Coffey, Biochem. glucosidase. The result would indirectly sup- Pharmacol., 32, 367 (1983). port the idea of specific action of flavonoids on 12) H. Okamoto, D. Yoshida and S. Mizusaki, Cancer a-glucosidase conversely. Lett., 19, 47 (1983). Flavonoids are known to inhibit various 13) A. Beretz, J.-P. Cazenave and R. Anton, Agents and kinds of enzymes, including cyclic nucleotide Actions, 12, 383 (1982). phosphodiesterase,8) protein kinase,9) gly-