Content of Flavonol Glucosides and Some Properties of Enzymes Metabolizing the Glucosides in Onion (Flavonoid in Fruits and Vegetables, Part II)

642 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. 43, No. 5, 642～649 (1996) 〔Article〕 (180)

Tojiro TSUSHIDA*and Masahiro SUZUKI*

*National Food Research Institute, 2-1-2 Kannondai, Tsukuba-shi, Ibaraki 305

The contents of flavonol glucosides in yellow, red and white onions harvested in Hokkaido were

measured. Seven varieties of yellow onion averaged 16.8mg/100g of quercetin-3, 4'-di-O-β-gluco-

side, 18.5mg/100g of quercetin-4'-O-β-glucoside, 0.8mg/100g of quercetin-3-O-β-glucoside and 2.9

mg/100g of isorhamnetin-4'-O-β-glucoside. The red onion contained about 3-fold the amount of

these glucosides while the white onion contained no detectable amounts of flavonols. The content of each flavonoid was higher in the outer scales than in the inner ones. Flavonol glucosidases and

UDPG: flavonol glucosyltransferases were detected in the onions. The optimum pH of quercetin-3-

O-β-glucosyltransferase, 4'-O-β-glucosyltransferase, 3-O-β-glucosidase and 4'-O-β-glucosidase was

6.0, 8.0, 4.5 and 7.0, respectively. In all the onion varieties, the activity of quercetin-4'-β-

glucosyltransferase was higher than that of 4'-O-β-glucosidase, but the activity of 3-O-β-

glucosyltransferase was lower than that of 3-O-β-glucosidase. Since quercetin-4'-O-β-glucosyltransferase was not able to transglucosylate quercetin-3-O-β-glucoside and rutin, the precursor of

quercetin-3, 4'-di-O-β-glucoside is estimate to be the 4'-O-β-glucoside and not the 3-O-β-glucoside.

Many biological activities of flavonoids in isorhamnetin glucosides and kaempferol gluco- foods, such as antioxidant activity1)2), sides of onion and their chemical structures in antimicrobial3) and antiviral4)5) activities, anti- onion have been carried out by PANISSET et al.13) inflammatory activity6)7), anti-allergic activ- STARKE et al.14), and TISSUT15) have studied the ity8)9), and antitumor activity10)11) have been turnover and changes in the content of reported in recent years. Therefore, the inves- flavonol glucosides in the onion during storage tigation of the compositions and the amount of and clarified the accumulation of the flavonols. flavonoids in foods is important in order to Recently, MIZUNO et al.16) have reported the con- estimate the physiological functionality of tent of free quercetin in onion and its stability foods. against irradiation. We have also inves- Onion has been known to contain flavonoids tigated the flavonol glucosides of onion as a yellow pigment. BRANDWEIN12) reported harvested in Hokkaido and showed the occur- the composition of flavonoids in onion and es- rence of 9 kinds of flavonol glucosides17) includ- timated their chemical structures. He reported ing isorhamnetin glucosides and kaempferol that onion contained quercetin-4'-O-β-gluco- glucosides. side (spiraeoside) and quercetin-3, 4'-di-O-β- In general, the most commonly found glucoside as the main constituents of the flavonoid glycosides are the 3-O-β-glycosides flavonoids. The occurrence of a relatively of flavonols, but the major flavonol glycosides small amount of quercetin-4', 7-di-O-β-gluco- in onion are quercetin-4'-O-β-glucoside and side, and only a trace of quercetin-3-O-β- quercetin-3, 4'-di-O-β-glucoside. Therefore, glucoside (isoquercitrin) or quercetin has also we are very interested in the transglucosyla- been estimated in the report. Details on the tion and glucosidation of quercetin-4'-O-β-glu constituents of flavonol glucosides including coside and 3, 4'-di-O-β-glucoside in onion. As (181) TSUSHIDA et al.: Flavonol Glucoside Metabolism in Onion 643 a preliminary experiment for the clarification 70% in 50min at a flow rate of 1ml/min. of the biosynthesis and biodegradation of Flavonol glucosides eluted from the column quercetin-4'-O-β-glucoside and 3, 4'-di-O-β- were monitored at 360nm and the concentra- glucoside in onion, we report the content of tion of each peak was calculated by comparing flavonol glucosides in various kinds of onions the peak area with that of each flavonoid and the estimation of some properties of standard. flavonol glucosyltransferases and flavonol Preparation of enzyme extract glucosidases. About 20g of onion slices obtained as already mentioned was homogenized with 2g of Materials and Methods polyvinylpolypyrrollidone and 40ml of 25mM Chemicals and plant materials McIlvaine buffer containing 10mM merca-

Uridine 5'-diphosphoglucose (UDPG) and ptoethanol using a Hiscotron at 4℃. The thymidine 5'-diphosphoglucose (TDPG) were homogenate was filtered through nylon mesh purchased from the Sigma Chemical Company. and the filtrate was centrifuged at 15000×g for

Quercetin-4'-O-β-glucoside, quercetin 3, 4'-di- 15min. The supernatant obtained was used

O-β-glucoside and isorhamnetin-4'-O-β-gluco- as the crude enzyme solution. All of the pro- side were purified from onion, while other cedures were carried out at 4℃. flavonoids were purchased from the Funakoshi Measurement of the activities of flavonol Co. Toyopearl HW-40C was from the Tosoh -glucosyltransferases O Co. and Polyclar AT (polyvinylpolypyrroli- The reaction mixtures for routine assay of done) was from the Gokyou Sangyou Co. UDPG: flavonol O-glucosyltransferases con- Seven cultivated varieties of yellow onions sisted of 20μl of 0.8mM quercetin solution dis- (Allium cepa L.) such as Sekihoku, Sapporoki, solved in 20% 2-methoxyethanol, 20μl of 36.4 Kitamomiji, Sorachiki, Kitamiki, Huranui and mM UDPG, 20μl of 0.2M Tricin-KOH buffer

Tsukihikari, one cultivated variety of red type (pH 8.0 for 4'-O-β-glucosyltransferase) or 0.2M onion, SRG, and three cultivated varieties of McIlvaine buffer (pH 6.0 for 3-O-β-glucosyl- white onions, such as BGS, SWG and SWG-HiS, transferase) containing 5mM mercaptoethanol, were harvested in Hokkaido and used for the 20μl water and 20μl of enzyme solution. The experiment. reaction was carried out at 37℃ for 10min and Extraction of flavonol glucoside terminated by the addition of 100μl of ethanol. About 20g of onion slices obtained from the The mixture was centrifuged at 8000×g for 10 middle part of the onion was homogenized min. Quercetin-4'-O-β-glucoside formed by with 80ml of methanol using a Hiscotron at flavonol-4'-O-β-glucosyltransferase, and quer- room temperature. The homogenate was cetin-3-O-β-glucoside formed by flavonol- filtered through No. 2 filter paper, then the 3-O-β-glucosyltransferase in the reaction mix- residue was mixed with 100ml of 80% metha- tures were measured by an HPLC equipped nol and then filtered again. Both filtrates with an ODS-C18 column. were poured into a 200ml volumetric flask and Measurements of the activities of flavonol the flask was filled with 80% methanol. glucosidases Measurement of flavonol glucosides by The reaction mixtures for routine assay of HPLC the flavonol glucosidases consisted of 200μl of The extract was filtered through a 0.45μm 0.2M McIlvaine buffer (pH 5.5 for quercetin- filter. Ten μl of the filtrate was then directly 3-O-β-glucosidase and pH 7.0 for 4'-O-β- injected in an HPLC equipped with ODS-C18 glucosidase), 100μl of 8.5mM quercetin-3, 4'-

(LiChrospher RP100, 4mm×250mm: Merck di-O-β-glucoside (for 3-O-β-glucosidase) or Co.). A gradient elution was carried out with 100μl of 8.5mM quercetin-4'-O-β-glucoside solvent A (containing 0.2% formic acid) and by (for 4'-O-β-glucosidase) and 200μl of enzyme increasing solvent B (methanol), from 25% to solution. The reactions were carried out at 644 日本食品科学工学会誌第43巻第5号 1996年5月 (182)

37℃ for 60min and terminated by the addition in the reaction mixture was measured by of 1.0ml of ethanol. The quercetin-3-O-β- HPLC as already mentioned and the quercetin glucoside produced by flavonol-4'-O-β-glu- produced by flavonol-3-O-β-glucosidase in the cosidase from quercetin-3, 4'-di-O-β-glucoside reaction mixtures was also measured by HPLC

equipped with an ODS-C18 column.

An outline of the measurement of the ac-

tivities for these four enzymes is shown in Fig.

Results and Discussion

Content of quercetin glucosides in various onions

The contents of flavonol glucosides in the

seven kinds of cultivated varieties of yellow

onions, one red onion and three kinds of white

onions were measured using HPLC (Table 1).

The yellow onions and red onion contained not

Fig. 1 Outline of enzymatic reaction in routine only quercetin-3, 4'-di-O-β-glucoside and quer-

measurement of activities for quercetin cetin-4'-O-β-glucoside as the main constitu-

β-glucosyltransferase and β-glucosi- ents of the flavonoids but also quercetin-3-O- dases. β-glucoside and isorhamnetin-4'-O-β-gluco-

side as minor flavonoids. The amounts of

quercetin-3, 4'-di-O-β-glucoside, quercetin-4'- O-β-glucoside, quercetin-3-β-glucoside and

isorhamnetin-4'-β-glucoside in the yellow

Table 1 Content of flavonol glucosides in various onions (183) TSUSHIDA et al.: Flavonol Glucoside Metabolism in Onion 645

onions were 13～20.6mg/100g, 14.9～23.5mg/

100g, 0.4～1.5mg/100g and 1.9～3.8mg/100g

on a fresh weight basis, respectively. On the

other hand, red onion contained about three-

fold the amount of flavonol glucosides of

yellow onion. But white onions contained no detectable amounts of the flavonol glucosides.

KIVIRANTA et al.18) have reported that the con-

tents of quercetin-3, 4'-di-O-β-glucoside and

quercetin-4'-O-β-glucoside of the yellow onion

harvested in Finland were 17mg/100g and 13

mg/100g, and those of red onion were 41mg/

100g and 35mg/100g in fresh tissue, respec- Fig.2 Optimum pHs of quercetin-3-O-gluco-

tively. Therefore, the content of the major syltransferaseand 4'-O-glucosyltrans-

flavonol glucosides of onions harvested in ferase

Hokkaido approximate those flavonol gluco-

sides in Finland. But KIVIRANTA et al. have not

reported the content of quercetin-3-O-β-gluco-

side and isorhamnetin-4'-O-β-glucoside, therefore, this study appears to be the first report about the content of these two in onion. Yellow onion contained about 39mg/100g of total flavonol-glucosides on a fresh weight basis. Red onion contained more, but the white onion contained no detectable amounts of flavonols. The difference in the levels of the flavonol contents among the various types of onions suggests differences in enzymes and enzymatic activities related to the metabolism of the flavonol glucosides. Enzymes for the metabolism of flavonol in onion and some of their properties There have been no reports on the enzymes of flavonol glucoside metabolism in onion as far as we know. Since onion contained

quercetin-3-O-β-glucoside and quercetin-4'-

β-glucoside, the activities of quercetin-3- O-β-glucosyltransferase, quercetin-4'-O-β- Fig.3 Optimum pHs of quercetin-3-O-gluco-

glucosyltransferase, quercetin-3-O-β-gluco- sidaseand 4'-O-glucosidase sidase and quercetin-4'-O-β-glucosidase in

yellow onion were examined. Figs. 2 and 3 show that the optimum pHs of quercetin-

3-O-β-glucosyltransferase and 4'-O-β-glucosyltransferase were 6.0 and 8.0, respectively of quercetin are estimated to be active under and those of quercetin-3-O-β-glucosidase and acidic conditions in onion cells, On the con- quercetin-4'-O-β-glucosidase were 4.5 and 7.0, trary, those reactions for 4'-O-β-glucoside are respectively. Therefore, the transglucosyla- estimated to be active under neutral and rela-

tively alkaline conditions. In both cases tion and glucosidation of the 3-O-β-glucoside , the 646 日本食品科学工学会誌第43巻第5号 1996年5月 (184)

optimum pHs for transglucosylation were few reports on the glucosyltransferase of

higher than that of glucosidation. The sepa- flavonol-ring B are available. BAJAJ et al.22) rate pH optimums of the four enzymes support have reported the 2' and 5'-O-β-glucosyl-

the fact that the transglucosylation and transferase in Chrysosplenium americanum as glucosidation of 3-O- position and 4'-O- posi- the flavonol-ring B glucosyltransferase. But tion of quercetin seem to be controlled by the there has been no report on flavonol-4'-O-β-

corresponding enzymes in onion. glucosyltransferase. So, we studied the sub-

There has been much research on flavonol- strate specificity and glucose donor specificity

3-O-β-glucosyltransferase. In general, flavo- of the enzyme (Table 2). The flavonol-4'-

noid-3-O-β-glucosyltransferases have been β-glucosyltransferase of onion transgluco- reported to have an optimum pH between pH 8 sylated fisetin but the activities toward

to 9. But that of 3-O-β-glucosyltransferase in kaempferol and taxifolin were very weak.

onion was in relatively low pH region. Morin (3, 5, 7, 2', 4'-pentahydroxyflavon), iso-

Glycosyltransferases having an acidic pH quercitrin (quercetin-3-O-β-glucoside), rutin,

optima were reported by KAMSTEEG et al.19), naringenin, eriodictyol, and (-)-epicatechin

TEUSCH20) and LARSON21). On the other hand, were not transglucosylated. TDPG was also

useful as a glucose donor, but the efficiency

was 22% of UDPG. Since the quercetin-3-

O-glucoside was not transglucosylated to 3,

4'-di-O-β-glucoside by this enzyme, 3, 4'-O-β-

glucoside is estimated to be synthesized from 4'-O-β-glucoside in onion. We propose the

pathway for quercetin-3, 4'-di-O-β-gucoside

synthesis as depicted in Fig. 4.

The activity of flavonol glucosyltransferase

and flavonol glucosidase in various onions

The activities on the four enzymes in various

types of onions are shown in Table 3. The

white onion contained the four enzymes in

Fig. 4 Proposed reactions involved in the spite of no detected flavonols (Table 1). More- formation of quercetin-3, 4'-di-O-gluco- over, the activity of quercetin-3-O-β-gluco-

side sidase was higher on white onion than in

yellow onion. The activity of quercetin-4'-O-

β-glucosyltransferase was high in yellow

Table 2 Substrate specificity of UDPG: quercetin-4'-O-glucosyltransferase in onion (185) TSUSHIDA et al.: Flavonol Glucoside Metabolism in Onion 647 onion and red onion and higher than the 4'-β- Comparison of flavonol glucosides and glucosidase activity. These results support enzyme activities in each scale of the onion the high levels of quercetin-4'-O-β-glucoside The scales of the yellow onion Sapporoki found in onion bulbs. On the contrary, 3-β- were separated and the content of quercetin glucosidase activity was higher than 3-β- glucosides and quercetin, and the activities of glucosyltransferase, and low levels of quer- the four enzymes in each scale were measured. cetin-3-O-β-glucoside in onion bulbs is proba- The separated edible scales were numbered in bly caused by the higher enzymatic activity. order from the outside. The contents of

From the results of these enzymatic studies, quercetin-3, 4'-di-O-β-glucoside (A), quer- the difference in the content of each quercetin cetin-4'-O-β-glucoside (B) and quercetin were

glucoside was thought to be brought about by lower in the inner scales than in the outer ones

the balance of the activities of transglucosyla- (Table 4). However, the ratios of (A)/(B) of

tion and glucosidation. outer edible scales were higher in the outer

Table 3 The activities of flavonol glucosyltransferases and flavonol glucosidases in various cultivars of onion

Table 4 Activities of enzymes and the content of flavonoids in each scale of onion 648 日本食品科学工学会誌第43巻第5号 1996年5月 (186) ones than in the inner ones. These results 8) FEWTRELL,C.M.S. and GOMPERTS,B.D.: Nature, suggest that the glucosidation of quercetin glu- 265, 17 (1977). coside is predominant in the outer scales of the 9) KIMURA, Y., KUBO, M., TANI, T., ARICHI, S., onion. The non-edible dried brown peels con- OHMINAMI,H. and OKUDA, H.: Chem. Pharm. Bull., 29, 2308 (1981). tained a large amount of quercetin and the 10) MATSUDA,H., YANO,M., KUBO, M., IINUMA,M., ratio (A)/(B) was very low. Therefore, the OYAMA,M. and MIZUNO, M: Yakugakuzasshi, flavonol glucosides seem to be hydrolyzed 111, 193 (1991). during the formation of the outer non-edible 11) SOULININA,E.M., BUCHSBAUM,R.N. and RACKER, brown peel. E.: Cancer Res., 35, 1865 (1975). Table 4 shows the differences in activities of 12) BRANDWEIN,B.J.: J. Food Sci., 30, 680 (1965). the four enzymes in the onion scales. The 13) PANISSET,B. and TISSUT,M.: Physiol. Veg., 21, 49 glucosyltransferase showed higher activities in (1983). scale 3 to scale 6 than the outer ones. The 14) STARKE,H. and HERRMANN,K.: Z. Lebensum. glucosidases of the outside scales showed Unter. Forsch., 161, 137 (1976). 15) TISSUT,M.: Phytochemistry, 12, 2155 (1973). higher activities than those of the inner ones, 16) MIZONO, M., TSUCHIDA,H., KOZUKUE,N. and as was expected. From these results and the MIZUNO,S.: Nippon Shokuhin Kogyo Gakkaishi, higher ratios of (A)/(B) in the outer scales, it is 39, 88 (1992). clear that the glucosidation was predominant 17) TSUSHDIA,T. and SUZUKI,M.: Nippon Shokuhin in the outer scales. This results in the accu- Kogyo Gakkaishi, 41, 100 (1994). mulation of quercetin in the outer scales by 18) KIVIRANTA,J., HUOVINEN,K. and HILTUNEN,R.: glucosidation. STARKE et al.14) also has es- Acta Pharm. Fennica, 97, 67 (1988). timated that the ratio of quercetin-4'-O-β- 19) KAMSTEEG,J., Von BREDERODE,J. and Von

glucoside to quercetin-3, 4'-di-O-β-glucoside NIGTEVECHT, G.: Z. Pflanzenphysiol, 96, 87 increases during the growing and maturation (1980).

of onions in the field. The change in the ratio 20) TEUSCH,M.: Planta, 169, 559 (1986). 21) LARSON,R.L.: Phytochemistry, 10, 3073 (1971). seems to be also caused by the balance of ac- 22) BAJAJ,K.L., DELUCA,V., KHOURI,H. and IBRAHIM, tivities of these glucosyltransferases and R.L.: Plant Physiol., 72, 891 (1983). glucosidases. (Received Dec. 5, 1994) References

1) FAURE, M., LISSI, E., TORRES,R. and VIDELA,L. A.: Phytochem., 29, 3773 (1990). タマネギのフラボノール配糖体含量およびそれらの 2) De VALLEY, C.V., RANKINS, S., HAULT, J.R., 代謝に関与する酵素の性質 JESSUP, W. and LEAKE,D.S.: Biochem. Pharm., (果実・野菜のフラボノイドに関する研究第2報) 39, 1743 (1990). 3) ONISHI,E.: Seikatsu Eisei, 36, 179 (1992). 津志田藤二郎・鈴木雅弘 4) ONO,K., NAKANE,H., FUKUSHIMA,M., CHERMANN, 農林水産省食品総合研究所 J-C. and BARRE-SINOUSSI,F.: Eur. J. Biochem., (〒305 茨城県つくば市観音台2-1-2) 190, 469 (1990). 北海道産の黄色タマネギ,赤色タマネギ及び白色タマ 5) NAGAI,T., MIYAICHI,Y., TOMIMORI,T., SUZUKI,Y. ネギのフラボノール含量を測定したところ,生鮮重当た and YAMADA,H.: Chem. Pharm. Bull., 38, 1329 (1990). り黄色タマネギではケルセチン-3,4'-ジグルコシドが 6) KUBO, M., MATSUDA,H., TANKA,M., KIMURA,Y., 16.8mg/100g,ケルセチン-4'-グルコシドが18.5mg/ OKUDA, H., HIGASHINO,M., TANI, T., HOMBA,K. 100g,セルセチン-3-グルコシドが0.8g/100g,イソラ and ARICHI, S.: Chem. Pharm. Bull., 32, 2724 ムネチン-4'-グルコシドが2.9mg/100g存在していた. (1984). 赤色タマネギでは黄色タマネギに比べて3倍量のフラボ 7) IIO, M., ISHIMOTO,S., NISHIDA,Y., SHIRAMIZU,T. ノール配糖体が検出されたが,白色タマネギには検出さ and YUMOKI, H. : Agric. Biol. Chem., 50, 1073 (1986). れなかった.また,フラボノール配糖体は外側の鱗茎に (187) TSUSHIDA et al.: Flavonol Glucoside metabolism in Onion 649

多く存在した. セチン-4'-β-グルコシドは蓄積される方向にあることが一方 ,フラボノール配糖体の代謝に関与する酵素とし分かった.一方,ケルセチンの3位においては逆にグルては,2種のフラボノールグルコシダーゼと2種のUDP コース転移酵素がグルコシダーゼの活性より弱いため,

-グルコース:フラボノールグルコース転移酵素が検出ケルセチン-3-β-グルコシドは蓄積しにくいことが分

された.これらはそれぞれ至適pHが異なり,3-β-グルかった.

コシダーゼでは4.5, 4'-β-グルコシダーゼでは7.0であまた,ケルセチンの3位に糖が結合したフラボノール

り,3-β-グルコース転移酵素では6.0, 4'-β-グルコース配糖体には4'-β-グルコース転移酵素が作用できないた転移酵素では8.0であった.使用した全ての品種においめ,タマネギのケルセチン-3, 4'-ジグルコシドは,ケル

て,ケルセチン-4'-β-グルコース転移酵素の活性が4'- セチン-4'-β-グルコシドに糖転移が起こることにより

β-グルコシダーゼの活性に勝っていることから,ケル合成されることが推定できた.