Agric. Biol. Chem., 55 (9), 2227-2233, 1991 2227

Malonyl Glycosides in Seeds (Glycine max Merrill) Shigemitsu Kudou, Yvette Fleury,* Dieter Welti,* Daniele Magnolato,* Teiji Uchida, Keisuke Kitamura** and Kazuyoshi Okubo*** Kanesa Co., Ltd., 202 Hamada, Tamagawa, Aomori 030, Japan * Research Centre, Nestle Ltd., P.O. Box 44, Vers-Chez-Les-Blanc, CH-1000 Lausanne 26, Switzerland ** National Agriculture Research Center, Tsukuba 305, Japan ** Faculty of Agriculture, Tohoku University, 1-1, Tsutsumitori Amamiya machi, Aoba-ku, Sendai 981, Japan Received January 21, 1991

The isoflavone constituents in soybean seeds were investigated, and 9 kinds of isoflavone glycosides were isolated from the hypocotyls of soybean seeds. Three kinds were proved to be malonylated soybean named 6"-0-malonyldaidzin, 6"-O-malonylglycitin and 6"-0-malonylgenistin by UV, MS, IR and NMR.The malonylated isoflavone glycosides as major isoflavone constituents in soybean seed were thermally unstable, and were converted into their corresponding isoflavone glycosides. All of the isoflavone components produced intensely undesirable taste effects such as bitter, astringent and dry mouth feeling.

Soybeans are known to contain the five also exhibited undesirable bitter and astringent isoflavone glycosides, ,^ ,1} tastes.9'10) In a previous paper,11} the isolation 7-O-jS-D-glucoside (glycitin),2) 6"-O- of a new isoflavone, glycitein l-O-fi-(6"-O- acetylgenistin3) and 6"-(9-acetyldaidzin,4) and acetyl)-D-glucoside (6"-<9-acetylglycitin), was their corresponding aglycones, , glyci- reported, this forming part of one of the tein and (Fig. 1). It has been reported components responsible for the undesirable that these isoflavone compounds possessed taste characteristics in soybean (Fig. 1). antihaemolytic,5) antioxidative,5) antifungal,6) Further investigation suggested that malonyl oestrogenic7) and antitumorial8) activities, and daidzin and malonyl genistin were likely to occur in soybean seed.12) The presence of malonyl genistin has been reported in clover,13) and the use of malony daidzin from Pueraria lobata as an aldose reduetase inhibitor has been patented. 14) In this paper, the isolation and chemical structure of the three malonyl isoflavone glycosides, and their taste characteristics are described.

Results and Discussion Effect of extraction temperature on isoflavone composition Fig. 1. Structures of the Isoflavones in Soybean Seeds. To determine the extraction conditions for 2228 S. Kudou et al.

9 have been identified as daidzin, glycitin, genistin, 6"-0-acetyldaidzin, 6"-6>-acetylglyci- tin and 6"-0-acetylgenistin by comparing their UV, MSand NMRspectra with published data.2~4) The isolation of compounds 4, 5 and 6 was effected and their structures determined. Structures of compounds 4, 5 and 6 Compounds 4, 5 and 6 were isolated from the hypocotyl of soybean seeds (Perikan Co., Ltd.) according to the procedure given in the experimental section. Mass and UVspectral data for compounds 4, 5 and 6 showed them to be daidzin, glycitin and genistin derivatives, respectively.2"4'15) Prominent [M+H-86] ions due to the loss of malonate were observed in the FABmass spectra of the three compounds, indicating that these were malonylated isoflavone glyco- sides.16-18) Assignments from the 1H- and 13C-NMR spectra of daidzin, glycitin, genistin, com- pounds 4, 5 and 6, were established by 13C-1H COSYspectra coupled with ^^H COSY spectra, and are summarized in Tables I and II. The 13C- and ^-NMRspectral data for demalonylated compounds 4, 5 and 6 prepared Fig. 2. High-performance Liquid Chromatogram of with 0.5 n aqueous Na2CO313) were completely 70% Ethanol Extracts Obtained from the Hypocotyls of consistent with those of daidzin, glycitin and Mature Soybean Seeds at RoomTemperature and at 80°C. genistin, respectively. The 13C-NMR spectra 1, daidzin; 2, glycitin; 3, genistin; 4, 6"-0-malonyldaidzin; of compound 4, 5 and 6 showed signals of 3 5, 6"-0-malonylglycitin; 6, 6"-0-malonylgenistin; 7, 6"- 167.9, 166.8 and 41.4 (COOR, COOH and O-acetyldaidzin; 8, 6"-0-acetylglycitin; 9, 6"-0-acetyl- genistin; 10, daidzein; ll, glycitein; 12, genistein. See -CH2- in a malonyl group), neither of which were detected in the spectra of their corre- the experimental section for HPLCconditions. sponding isoflavone glycosides. The low field methylene protons Ha-6" and Hb-6", and the isoflavone compounds from soybean seeds, the chemical shifts ofC-6" and C-5" in compounds isoflavone constituents extracted with 70% 4, 5 and 6 when compared with those of aqueous ethanol at 80°C and at room daidzin, glycitin and genistin indicated acyla- temperature were compared by HPLC. As tion of the sugar moieties at C-6".16~18) shown in Fig. 2, compounds 4, 5 and 6 were Thus, the structures of compounds 4, 5 and detected as the major constituents in the extract 6 were deduced to be 6"-(9-malonyldaidzin, obtained at room temperature, whereas the 6"-<9-malonylglycitin and 6"-0-malonylgenis- major constituents in the extract at 80°C were tin, respectively. As far as we know, this is the compounds 1, 2 and 3. This suggested that first report of 6//-Omalonylglycitin. The compounds 4, 5 and 6 were thermally unstable, presence of 6//-O-malonylgenistin in clover13) and were converted into compounds1, 2 and and of 6"-0-malonyldaidzin in Pueraria 3. Previously,11} compounds 1, 2, 3, 7, 8 and Inhntn1^ has been described before, but not in Malonyl Isoflavone Glycosides in Soybean 2229

Table I. ^-NMRData for Isoflavone Glycosides from the Hypocotyl of Soybean Seed (400 MHz, in DMSO-d6, <5H)

^ . ,. Manonyl . .. Malonyl . Malonyl Daidzm ,.,. Glycitm , .. Gemstm . . daidzm glycitin gemstm

AandCrings C-2 8.38s 8.35s 8.37s 8.32s 8.41 s 8.39s C-5 8.05d (8.8) 8.07d (8.8) 7.48s 7.47s C-6 7.14dd (8.8, 2.4) 7.14d (8.8, 2.4) 6.47d (2.5) 6.47d (2.5) C-8 7.23d (2.4) 7.22d (2.4) 7.32s 7.31 s 6.72d (2.5) 6.70d (2.5) OMe 3.88s 3.88s OH 12.92 br 12.94 br Bring C-2' and 6' 7.41d (8.8) 7.40d (8.8) 7.41d (8.8) 7.40d (8.8) 7.40d (8.8) 7.39d (8.8) C-3' and 5' 6.82d (8.8) 6.82d (8.8) 6.82d (8.8) 6.82d (8.8) 6.83d (8.8) 6.83d (8.8) OH 9.57 br 9.57 br 9.57br 9.57br 9.66br 9.64br Glucose moiety C-l" 5.17d (7.3) 5.14d (7.3) 5.18d (7.8) 5.20d (7.8) 5.06d (7.3) 5.12d (7.3) C-2"-5" 1 3.15-3.80m -) 3.15-3.80m -\ 3.15-3.80m C-6"a V3.15-3.85 4.41d(12.0) V3.15-3.85 4.38d(12.2) V3.15-3.85 4.36d(12.0) C-6"b J 4.lldd (12.0, 7.1)3 4.12dd(12.2,6.8) J 4.12dd(12.0,7.1) Malonyl C-2'" 3.40 3.38 3.38

soybean seeds.

Isoflavone accumulation during the development of soybean seeds The accumulation of daidzin, genistin, glycitin and their corresponding malonylated forms in immature seeds during development followed from between 35 and 60 days after flowering (Fig. 3). Malonylgenistin and the genistin content increased during the late development of the beans, whereas malo- nyldaidzin and daidzin accumulated through- out the whole period. Minor isoflavone glycosides, malonylglycitin and glycitin, were Fig. 3. Isoflavone Accumulation during the Maturation also detected. The isoflavone content of the of Maple Arrow Soybean Seeds. mature seed reported in Table III shows that Immature seeds in pods harvested at variuous stages of malonylgenistin and malonyldaidzin com- maturation after flowering were used as the samples for quantitative analyses. Peak area shows the change in prised 66%of the total isoflavones in the beans. relative amounts of isoflavone glycosides for a certain dry weight. O, daidzin; D, glycitin; V, genistin; #. Isoflavone content in mature soybean seeds 6"-0-malonyldaidzin; å , 6"-0-malonylglycitin; y, 6"-O- The isoflavone content of mature soybean malonylgenistin. seeds was compared by using two extraction methods. Whole soybean seeds were divided into the seed coat, cotyledon and hypocotyl of 70% aqueous ethanol. As shown in Table (containing the plumule and radicle), and each IV, the total isoflavone content of the part was extracted at room temperature for hypocotyl part was 5.5 or 6.0 times higher than 24hr or at 80°C for 15hr by a 10-fold volume that of the cotyledons, and glycitin and its 2230 S. Kudou et al.

Table II. 13C-NMRData for Isoflavone Glycosides Isolated from the Hypocotyl of Soybean (100MHz, in DMSO-J6, <5C)

Malonyl ^ .. Malonyl . . Daidzin Glycitm . . / Genistm daidzin glycitm A and C rings C-2 153.3 153.3 153.1 154.6 154.6 C-3 123.7 123.7 123.1 122.6 122.6 C-4 174.8 174.8 174.4 180.5 180.5 C-5 127.0 127.1 104.8 161.7 161.7 C-6 115.6 115.4 147.4 99.6 99.5 C-7 161.4 161.1 151.2* 163.0 162.7 C-8 103.4 103.6 103.6 94.6 94.6 C-9 157.0 157.0 151.2" 157.2 157.2 C-10 118.5 118.6 117.9 106.1 106.2 B ring c-r 122.3 122.3 122.5 122.6 121.0 121.0 C-2' and 6' 130.1 130.1 130.0 130.0 130.2 130.2 C-3' and 5' 115.0 115.0 114.9 115.0 115.1 115.1 C-4' 157.3 157.3 157.2 157.2 157.5 157.5 OCH3 55.8 55.9 Glucose moiety C-l" 100.0 99.8 99.7 99.4 99.9 99.5 C-2" 73.2 73.0 73.0 72.9 73.1 73.0 C-3" 76.5 76.2 76.8 76.5 76.4 76.1 C-4" 69.7 69.7 69.6 69.6 69.6 69.6 . C-5" 77.2 73.8 77.2 73.8 77.2 73.7 C-6" 60.7 64.1 60.6 64.0 60.6 64.0 Malonyl COOR 167.9 CH2 41.5 COOH 166.9

Assignments for these signals within the same column may be interchanged.

Table III, Isoflavone Content in Maple constituents in the extract at room temperature, Arrow Soybean Seeds" the contents of malonylated isoflavone gly- Amount*7 cosides in the extract at 80°C decreased Compound (mg) significantly, and an increase in all isoflavone glycosides and acetyl isoflavone glycosides, Daidzin 129.4 Glycitin 20.5 with the exception of 6"-0-acetylgenistin, was Genistin 96.6 observed. The acetyl derivatives maythus have 6"-<9-Malonyldaidzin 251.0 arisen from corresponding malonyl derivatives 6"-0-Malonylglycitin 20.0 6"-0-Malonylgenistin 275.5 as artifacts during the work-up procedures, as was pointed out by Horowitz et al.19) a Wholemature seeds were used. b per lOOgdryweight. Threshold values for the isoflavone components The threshold value and taste character of derivative only occurred in the hypocotyl part each isoflavone componentwere examinedas of the soybean seed. Isoflavones were absent described in the experimental section. All of from the seed coat of soybean. While the isoflavone componentsproduced intensely malonylated isoflavone glycosides were major undesirable taste characteristics such as bit- Malonyl Isoflavone Glycosides in Soybean 2231

Table IV. Content of Isoflavone Compounds in Soybean Seed0 (mg/lOO g) Roomtemperature5 80 °CC Compound Hypocotyl Cotyledon Hypocotyl Cotyledon

Daidzin 320 45 838 145 Glycitin 485 -d 1 004 - Genistin 1 18 80 246 210 6"-O-Malonyl daidzin 423 70 8 3 6"-O-Malonyl glycitin 445 - 11 - 6"-0-Malonyl genistin 144 1 17 4 - 6"-<9-Acetyl daidzin 2 2 57 8 6"-0-Acetyl glycitin 6 - 89 - 6"-<9-Acetyl genistin 105 1 39 1 Daidzein 102 33 35 1 1 Glycitein - - 1 5 - Genistein 35 48 1 6 14

Total 2185 396 2362 392 a Suzuyutaka strain cultivated in Akita Prefecture in 1988. b Extraction at room temperature for 24hr. c Extraction at 80°C for 15hr. d Not detected.

conversion of malonyl isoflavone glycosides into isoflavone glycosides during processing was necessary to produce soymilk with a very low degree of objectionable after taste.

Experimental Spectroscopy. UVspectra (in methanol) were measured Fig. 4. Threshold Values of the Isoflavones in Soybean by a JASCOU-30 spectrometer, and NMRspectra were Seeds. recorded on a JEOLGSX-400spectrometer (*H-NMRat 400MHz, and 13C-NMR at 100MHz in DMSO-J6, with SiMe4 as an internal standard). IR spectra (in KBr) were recorded with a JASCOA-202 instrument and mass spectra terness, astringency and dry mouth feeling. were recorded with a JEOL JMS HX-105. The threshold values for daidzin and its relat- ed compoundswere in the order of daidzin> Isolation of the isoflavone compounds.Soybean seed daidzein = acetyl daidzin > malonyldaidzin (Fig. hypocotyls (500g), which had been kindly supplied by Perikan Co., Ltd., were milled and extracted with a 10-fold 4). Those of the related compoundsof glycitin volume of 70%aqueous ethanol at room temperature for and genistin were in the order of glycoside> 30 min. After filtering, the residue was repeatedly extracted aglycone^6"-0-acyl glycoside, confirming three times. After the combined filtrate was evaporated to the results by Francis for genistin and malonyl- dryness under reduced pressure, the extract was partitioned genistin.20) Matsuura et al.21) have demon- between water and 1-butanol (1 : 1, v/v). After again evaporating to dryness, the butanol layer was repeatedly strated that an increase in daidzein and genis- fractionated by Sephadex LH-20 column chromatography tein by the action of /?-glucosidase in (5 x 74cm, methanol). TLCanalysis showed successive during soymilk manufacturing process result- elution of the soybean saponin Agroup, soybean saponin ed in an increase in the objectionable after- B group, glycitin, daidzin, and genistin fractions. Glycitin, taste. It was, therefore, considered that the compound5, daidzin and compound4 were isolated from 2232 S. Kudou et al. the glycitin and daidzin fraction by HPLC (YMC-Pack Compound4. mp 169°C. UV 2max (nm) MeOH: 231 sh, ODSAM-323-7 column, 10x250mm; mobile phase of 248sh, 258, 303 (log6 4.37, 4.47, 4.50, 4.00); +MeONa: 45% methanol). Genistin and compound 6 were obtained 237sh, 245sh, 279, 300sh; +A1C13: 231sh, 247sh, 258, from the genistin fraction by HPLC (YMC-Pack ODS 303 sh; +AICI3-HCI: 231 sh, 247sh, 258, 303 sh; +NaOAc: AM-323-7 column, 10x250mm; mobile phase of 50% 248sh, 259, 304sh. IR vmax (KBr): 1735, 1695cm"1. methanol). 6"-0-Acetyldaidzin, 6"-0-acetylglycitin and High-resolution FAB-MS: found, m/z 503.1 181 [M +H]+; 6"-0-acetylgenistin were isolated according to procedure calcd. for C24H23O12, 503.1188. FAB-MS: m/z 503 reported in the previous paper.11} [M+H]+, 417 [M+H-86]+, 255 [M+H-86-162]+. EI-MS: m/z 254 [M-86-162]+, 137, 118. Quantitative analyses of the isoflavones. Whole mature soybean seeds were lyophilized in a freeze dryer (FD-550, Compound 5. mp 145°C. UV Xmax (nm) NeOH: 227sh, Tokyo Rikakikai Co., Ltd.) and divided into the seed coat, 260, 318 (log6 4.25, 4.42, 3.95); +MeONa: 284, 322sh; cotyledon and hypocotyl (containing the plumule and +A1C13: 227sh, 260, 317; +A1C13-HC1: 227sh, 260, 318; radicle) parts. After each part had been milled, 0.5 g of the +NaOAc: 260.317. IR vmax (KBr): 1735, 1695cm"1. milled sample was extracted in screw-capped test tubes High-resolution FAB-MS: found, m/z 533.1285 [M +H] +; with 10 volumes of 70% ethanol for 24hr at room calcd. for C25H25O13, 533.1293. FAB-MS: m/z 533 [M temperature or for 15 hr at 80°C. After centrifugation, the +H]+, 447 [M+H-86]+, 285 [M+H-86-162]+. supernatant was used directly in quantitative HPLC EI-MS: m/z 284 [M-86-162]+, 166, 118. analyses. Compound 6. mp 162°C. UV 2max (nm) MeOH: 260, To examine the change in isoflavone content during the 323sh (logs 4.51, 3.59); +MeONa: 271, 322sh, 357sh; development of the beans, the Maple Arrow variety was used. Plants were grown from seed under controlled +A1C13: 272, 308sh, 380; +A1C13-HC1: 272, 308sh, 379; conditions in a phytotron. The flowers were tagged every +NaOAc: 261,329sh. IR vmax (KBr): 1735, 1695cm-1. day, and the pods were harvested at various stages of High-resolution FAB-MS: found, m/z 519.1 169 [M +H]+; maturation between 25 and 60 days after flowering. The calcd. for C24H23O13, 519.1137. FAB-MS: m/z 519 immature seeds in the pods were lyophilized, milled and [M+H]+, 433 [M+H-86]+, 271 [M+H-86-162]+. extracted with 70%aqueous ethanol at room temperature. After centrifugation, the supernatant was used directly in Acknowledgments. We thank Mr. Takayanagi and quantitative HPLCanalyses. Mrs. Sugiyama of Tohoku University in the Faculty of Agriculture for their measurements of NMRand mass TLCand HPLCanalyses. TLCwas carried out on a spectra. We thank Dr. G.R. Fenwick of the AFRCInstitute Kieselgel 60 F254 plate (Merck), using chloroform-metha- of Food Research for his critical reading of this manuscript. nol-2% acetic acid (7:3:1, v/v lower layer). The components on TLCplate were visualized by heating a References 120°C for lOmin after sprayingwith 10% H2SO4. ^/values of compounds 1-9 were 0.41, 0.45, 0.43, 0.08, 0.12, 0.10, 1) E. Waltz, Ann., 489, 118 (1931). 0.56, 0.61 and 0.59, respectively. 2) M. Nairn, B. Gestetner, I. Kirson, Y. Birk and A. Quantitative HPLC analyses were performed on a Bondi, Phytochemistry, 12, 169 (1973). YMC-pack ODS-AM-303 column (250 x 4.6mm), using a 3) N. Ohta, G. Kuwata, H. Akahori and T. Watanabe, linear gradient of acetonitrile from 15 to 35% containing Agric. Biol. Chem., 44, 469 (1980). constant 0.1% acetic acid in 50min. The solvent flow rate 4) N. Ohta, G. Kuwata, H. Akahori and T. Watanabe, was 1 ml/min and the absorption was measured at 260nm. Agric. Biol. Chem., 43, 1415 (1979). The instrument used was a Waters HPLC system, 5) M. Nairn, B. Gestetner, A. Bondi and Y. Birk, J. comprising a Model 600E multi-solvent delivery system, Agric. Food Chem., 24, 1174 (1976). Model 484 UV detector and Model 741 data module. 6) M. Nairn, B. Gestetner, S. Zilkah, Y. Birk and A. Standard solutions used for quantitative analysis were Bondi, /. Agr. Food Chem., 22, 806 (1976). prepared from 9 kinds of isoflavone glycosides isolated by 7) E. Farmakalidis, J. N. Hathcock and P. A. Murphy, HPLC and 3 kinds of aglycones obtained from the Food Chem. Toxic, 23, 741 (1985). corresponding isoflavone glycosides by 5% H2SO4 8) T. Akiyama, J. Ishida, S. Nakagawa, H. Ogawara, hydrolysis in 50%ethanol. S. Watanabe, N. Itoh, M. Shibuya and Y. Fukami, /. Biol. Chem., 262, 5592 (1987). Threshold value. Each sample solution (10"* mM)was 9) A. Huang, O. A. L. HsiehandS. S. Chang, /. Food diluted stepwise in tenths to a final concentration of ScL, 47, 19 (1981). 10"7mM. The tests were carried out initially with the 10) M. Iijima, K. Okubo, F. Yamauchi, H. Hirono and 10~7 mMsample solution until the panelists detected the M. Yoshikoshi: Proc. Int. Symp. on New Technology taste of the sample. This lowest detectable concentration of Vegetable Proteins, Oils and Starch Processing, is defined as the threshold value. Beijing, 1987, pp. 2-109. Malonyl Isoflavone Glycosides in Soybean 2233

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