植物研究雑誌 J. J. Jpn. Bo t. 72: 72: 337-346 (1997)

Flavonoid Flavonoid Glycosides from of Aucuba japonica and Helwingiajaponica Helwingiajaponica (): Phytochemical Relationship with with the Cornus

Tsukasa Iw ASHINA a，K 吋iKAMENOSONO b and Hiroaki HATTA a

aTsukuba aTsukuba Botanical Garden ，National Science Museum ，4-1-1 Amakubo ，Tsukuba ，Ibaraki ， 305 JAPAN; bTsukuba bTsukuba Research Laboratory ， Nippon Oil & Fats Co. Lt d. ，5-10 Tokodai ，Tsukuba ，Ibaraki ，300-26 JAPAN (Recei (Recei ved on March 17 ，1997)

Seven Seven flavonol and eleven flavone glycosides were isolated from the leaves of Aucub α japonica including var. borealis and including subsp. liukiuensis ，respectively. They were identified orpartially characterized as quercetin 3，7-

di-O-glucoside ，quercetin 3-0-sambubioside ，quercetin 7-0 四 glucoside ，quercetin 3-0-

xyloside-7 哨 0 ・・ glucoside ，quercetin 3-0-xyloside-7-0-xylosylglucoside ， kaempferol 3- 0- xylosylglucoside ， kaempferol 3-0- xyloside-7 -O-glucoside (from Aucuba japonica) ， apigenin apigenin 7 -O-glucoside ，apigenin 7 -O-diglucoside ，apigenin 7 -O-neohesperidoside ，api-

genin genin 7-0-xylosylglucoside ，luteolin 7-0 四 glucoside ，luteolin 7・.O-diglucoside ，luteolin 7- O-neohesperidoside ，luteolin 7-0- xy losylglucoside ，6 ，8-di -C- gl ycosy lapigenin ，6 ，8-di-

C-glycosylluteolin C-glycosylluteolin and vitexin (fromHelwingi αjaponic α)byPC ，FAB-MS ， IH-and 13H 目 NMR etc. The flavonoid characters were chemotaxonomically compared among Aucuba ， Helwingia Helwingia and Cornus. As the results ，it was shown that Helwingia keeps aloof from Cornus Cornus and Aucuba.

Aucuba japonica Thunb. is endemic to Turner 1984). Japan ， but now cultivated as an ornamental As a part of chemotaxonomical studies of plant in the world. On the other hand ， the Cornaceae ，we have observed the flavo- Helwingia japonica (Thunb.) F. G. Dietrich noid compositions of thirty-three Cornus taxa ，

including including subsp.liukiuensis (Hatusima) Hara and isolated and identified variously 3-0 時 is is distributed in Japan and rarely in China glycosylated flavonols based on kaempferol ， (Hara 1989). Iridoid ，aucubin has been re- quercetin and rarely myricetin ，i.e. ，glucoside ， ported ported from Aucuba japonica as a secondary galactoside ，rhamnoside ，glucuronide ，arabi- product product (Bate-Smith et al. 1975). noside ，xyloside ，rutinoside etc. (I washina and The Cornaceae has chemotaxonomically Hatta 1990 ，1992 ，1993 ， 1994). been discussed in relation to various taxo- In this paper ，we describe the f1 avonoid nomic treatments based on non- f1 avonoid sub- compositions of Aucuba japonica including stances ，iridoids and ellagitannins (Jensen et va r. borealis and Helwingia japonica includ- l. al. 1975 ，Bate-Smith et al. 1975) ， but not ing subsp. liukiuensis ，which are other genera studied studied by fI avonoid components ，which in the Cornaceae ， for chemotaxonomical elu- have already been used in many as cidation of the systematics among Cornus ， chemotaxonomic markers (Harborne and Aucuba and Helwingi α，and compare the f1 a- 338 植物研究雑誌第72 巻第6号 平成9年12 月 vonoid vonoid characters of these species with those 7 was applied to polyamide column chroma- of of Cornus species which we have previously tography using 70% MeOH. Flavonoids 6 and reported. reported. 7 were separated by PPC using BA W and 15% AcOH. Finally ， they were purified by Sephadex Materials Materials and Methods LH -20 column chromatography (70% MeOH) Plant Plant materials and obtained as pale yellow powders. Aucubajaponica Aucubajaponica Thunb. var.japonic α(TBG Aqueous residue was also applied to PPC 49968) 49968) and v訂 . borealis Miyabe & Kudo using BA W and/or 15% AcOH. The separated (TBG 7283) were cultivated in Tsukuba Bo- seven flavonoids and some organic acids were tanical tanical Garden ， National Science Museum ， purified by Sephadex LH-20 column (70% Tsukuba ， Ibaraki Pre f. Two samples of MeOH). Flavonoids 8，9 ，10 and 13 were Helwingia Helwingia japonica (Thunb.) F.G. Dietrich obtained as pale yellow powders and 11， 12 were collected in Botanic Garden ， Faculty of and 14 as pure solutions. Agriculture ，Hokkaido University ，Sapporo ， High pe ゆrmance liquid chromatography Hokkaido ，and Hakone ，Kanagawa Pre f. (HPLC) Voucher specimens of H. japonica subsp. HPLC separation was performed with japonica japonica and subsp. liukiuensis were depos- JASCO HPLC systems. Multi channel UV- ited ited in TNS. visible detector Multi -330 coupled with a com- Isolation Isolation offla ν onoids puter was used to record chromatograms and Aucuba japonica Fresh leaves (104 g) UV spectra. A TSKgel ODS-80TM column were extracted with MeOH ， evaporated to (I. D. 4.6 x 150 mm) was used. Crude MeOH dryness dryness and dissolved in water. Crops were extracts or isolated flavonoid solutions were chromatographed chromatographed on a polyamide column and filtrated through Toyopak ODS M (Tosoh) eluted eluted with 70% MeOH. The fractions con- and then Maisyoridisc ，0 .4 5μm (Tosoh) ， and taining taining same flavonoids were combined and eluted with CH 3CN-H 20 ・・ H 3P0 4 (22:78:0.2 ， applied applied to preparative paper chromatography So l. 1) and (18:82:0.2 ，So l. 11). Detection was (PPC) (PPC) using solvent systems ，BA W (n-BuO Hl made at 200-350 nm and the flow-rate was 1.0

AcOH/H 20 = 4:1:5 ， upper phase) and 15% ml/min AcOH. Flavonoids were purified by Sephadex Identification of flavonoids LH-20 column (70% MeOH). Ofisolated five The flavonoid glycosides were identified flavonoids ， four were obtained as pale yellow by UV spectral analysis according to Mabry powders (1 and 2) or pure solution (3 and 4). It et al. (1970) ，fast atom bombardment mass was proved by HPLC analysis that remaining spectra (FAB-MS) using nitrobenzyl alcohol flavonoid flavonoid 5 was a mixture which consists of (NBA) ， and lH_ and 13C-nuclear magnetic three three flavonoids. A mixture was applied to resonance ct H- and 13C_ NMR) spectra using

PPC using solvent system ，BEW (n-BuOHI dimethyl-sulfoxide-d 6 (DMSO-d 6) and direct EtOHIH 20 = 4: 1:2.2) and divided into three PC and HPLC comparisons of original flavonoids flavonoids (5a ，5b and 5c). glycosides with authentic specimens. Helwingia Helwingia japonica Fresh leaves (1 10 g) Identification of their hydrolysates which were extracted with MeOH and concentrated were obtained by complete (1 29 も aq. HCl ， to to aqueous residue in vacuo. The aq. extract 100 0 C ，30 min) and partial acid hydrolysis was washed with petroleum ether and ex- (1. 2% HCl:MeOH (1:1)， 100 o C ，10 -4 0 min) tracted tracted with ethyl acetate (EtOAc). EtOAc was also performed by PC ，TLC and HPLC layer layer which mainly contains flavonoids 6 and comparisons with authentic specimens. December 1997 Joumal of Japanese Botany Vo l. 72 No. 6 339

PC ，UV ，HPLC ，FAB-MS ， lH_ and 13C_ J = 8.6 Hz ，H-5') ， 6.61 (1 H ，d ，J = 2.0 Hz ，H- NMR data were as follows. 8) ，-6 .4 0 (1 H ，d ，J = 2.0 Hz ，H-6) ， 5.91 (IH ，d ， Quercetin Quercetin 3-0-xylosyl (l→ 2) glucoside (1). J = 7.6 Hz ，galactosyl anomer) ， 5.68 (1 H ，s ， PC: Rf 0.4 2 (BAW) ， 0.56 (BEW) ， 0.54 xylosyl anomer) ，5.2-3.8 (1 1H ，m ，galactosyl (l 5%AcOH) ，0 .4 2 (5%AcOH); UV - dark and xylosyl protons). i3 C-NMR (67 MHz ， purple ，UVINH 3 - dark yellow. UV:λ 詑? DMSO-d 6 ): (aglycone) O 155.3 (C-2) ， 133.1 (nm) (nm) 256 ，265sh ， 356; +NaOMe 272 ，329 ，407 (C-3) ， 177.5 (C-4) ，16 1. 3 (C-5) ，98 .4 (C-6) ，

(inc.); (inc.); +AIC1 3 275 ， 432; +AIC1 3/HC1270 ，297 ， 164.1 (C-7) ，93 .4 (C-8) ， (C-9) 156.3 ， 104.7 (C- 360 ， 400; +NaOAc 273 ，324 ， 388; +NaOAc/ 10) ，122 .4 (C-l ')， 115.3 (C-2') ， 145.0 (C-3') ，

H 3B0 3 261 ， 377. HPLC: Retention time (Rt) 148.6 (C-4') ， 115.8 (C-5') ，12 1. 3 (C 自 6'); 4.71 4.71 min (So 1. 1). FAB-MS (NBA): [M+Ht at (galactosyl) O 98.6 (C-l ")， 79.9 (C-2") ， 73.7 m セ597 ，calcd. for C26H28016 ，[M-xylos)ι (C-3") ， 67.8 (C-4") ， 74.0 (C-5") ， 60.0 (C-6"); Ht at m/ z 465 ，calcd. for C21H20012' [M- (xylosyl) O 103.9 (C-l "')， 76.0 (C-2"') ， 76.3 xylog1ucosyl+Ht at m/z 303 ， calcd. for (C-3"') ， 69.5 (C-4"') ， 65.7 (C-5"').

C 1s H lO 0 7・lH-NMR (270 MHz ，DMSO-d 6 ): O Kaempfero13-0-xylosylglucoside (2). PC: 12.80 12.80 (IH ，s， 5-0H) ， 7.79 (2H ，d ，J = 8.6 Hz ， Rf 0.53 (BAW) ， 0.71 (BEW) ， 0.58 (1 5% H-2' ，6') ， 6.96 (l H ，d ，J = 8.6 Hz ，H-5') ， 6.52 AcOH) ，0 .4 4 (5%AcOH); UV - dark purple ， (IH ，s， H-8) ， 6.31 (l H ，s， H-6) ， 5.84 (l H ，d ，J UV INH 3- dark greenish yellow. UV: 入詑 ?H = 6.9 Hz ，glucosyl anomer) ， 5.53 (l H ，s ， xylosyl (nm) 266 ， 349; +NaOMe 275 ，325 ，397 (inc.); anomer) ，5.1-3.6 (11 H ，m ，glucosy 1 and xy losy 1 +AIC1 3 274 ，304 ，351 ， 394; +AICliHC1275 ， protons). protons). 13C-NMR (67 MHz ，DMSO-d 6 ): 302 ，346 ， 395; +NaOAc 274 ，307 ， 383; (aglycone) (aglycone) O 155 .4 (C-2) ， 133.1 (C-3) ， 177.5 +NaOAc/H 3B0 3 267 ， 352. HPLC: Rt 7.14 (C-4) ，16 1. 3 (C-5) ， 98.7 (C-6) ， 164.1 (C-7) ， min (So 1. 1). 93.5 93.5 (C-8) ， 156.3 (C-9) ， 104.6 (C-I0) ， 122.0 Quercetin 3，7-di-O-glucoside (3). PC: Rf (C-l (C-l ')， 115.3 (C-2') ， 145.0 (C-3') ， 148.6 0.24 (BA W) ， 0.20 (BEW) ， 0.56 (1 5%AcOH) ，

(C-4') ， 116.1 (C-5') ， 121.3 (C-6'); (glucosyl) O 0.4 3 (5%AcOH); UV - dark pu 叩le ，UVINH 3 98.0 98.0 (C-l") ，8 1. 9 (C-2") ， 74.0 (C-3") ， 69.5 (C- - yellow. UV:入 ~~~H (nm) 256 ，266sh ， 359;

4") ， 77.7 (C-5") ， 60.7 (C 国 6"); (xylosyl) O 104.0 +NaOMe 275 ，415 (inc.); +AIC1 3 274 ， 438; (C-l 川)， 76.2 (C-2 川)， 76.9 (C-3"') ， 69.6 +AICliHCI269 ，295sh ，363 ， 398sh; +NaOAc

(C-4"') ， 65.7 (C-5"'). 263 ， 405; +NaOAc/H 3B0 3 261 ， 382. HPLC: Quercetin Quercetin 3-0-xylosyl (l→ 2) galactoside Rt 2.24 min (So 1. 1). from the leaves of Cornus capitata. PC: Rf Kaempferol 3-0-xyloside-7-0-g1ucoside 0.51 0.51 (BA W) ， 0.53 (BEW) ， 0.58 (l 5%AcOH) ， (4). PC: Rf 0.27 (BAW) ， 0.21 (BEW) ， 0.76

0.4 3 (5%AcOH); UV - dark purple ，UV INH 3 (1 5% もAcOH) ， 0.73 (σ5%AcOH); UV 一 dark H - yellow. UV:入 ~~?H (nm) 257 ，264sh ， 356; p卯u町r叩ple ，UV/ 別NH 3 一- gre 閃en 凶 h y戸う屯fellow.UV 恥: λ臼詑:?? +NaOMe 272 ，326 ，405 (inc.); +AIC1 3 275 ， (何 nm) 267 ， 349; +NaOMe 274 ，386 (inc.); 434; 434; +AIC1 3/HCl 270 ，297sh ，360 ， 401; +AIC1 3 269 ，300 ，351 ， 398sh; +AICliHCl +NaOAc 274 ，326 ， 388; +NaOAc/H 3B0 3 262 ， 272 ，298sh ，347 ，395sh; +NaOAc and +NaOA c/ 375. 375. FAB-MS (NBA): [M+H]+ at m/z 597 ， H 3B0 3 267 ， 352. HPLC: Rt 2.21 min (So 1. 1). calcd. calcd. for C26H28016 ，[M-xylosyl+Ht at m/ z Quercetin 7-0-g1ucoside (5a). PC: RfO.35 465 ，calcd. forC 21 H 20 0 12 ，[M-xylogalactosyl+ (BA W) ， 0.04 (1 5%AcOH); UV and UV INH 3 Ht at m/ z 303 ，calcd. for C 1s H lO 0 7・lH-NMR -ye l1 ow. UV:λrH(nm)256 ，375;+NaOMe (270 (270 MHz ，DMSO-d 6 ): O 12.91 (l H ，s， 5-0H) ， decomp.; +AIC1 3 272 ， 460; +AICI 3/HCl 266 ， 7.98 7.98 (2H ，d ，J = 8.6 Hz ，H-2' ，6') ， 7.03 (l H ，d ， 300sh ，365 ， 430; +NaOAc 259 ， 410; +NaOAc/ 340 植物研究雑誌第72 巻第6号 平成9年12 月

H 3B0 3 260 ，39 1. HPLC: Rt 9.00 min (So l. 1). 0.08 (5%AcOH); UV - dark purple ，UV INH 3 Quercetin Quercetin 3-0- xyloside-7 -0- xylosylgluco- -b 均 ht yellow. UV:λrH(nm)251264sh ， side? side? (5b). PC: Rf 0.23 (BA W) ， 0.11 (BEW) ， 349; +NaOMe 266 ，388 (inc.); +AIC1 3 273 ， 0.69 0.69 (1 59 もAcOH) ， 0.66 (5%AcOH); UV - 426; +AIC1 3/HC1274 ，294 ，359 ， 385; +NaOAc d紅 kpurple ，UVINH 3 -darkyellow. UV:λ 盟?H 259 ， 403; +NaOAc/H 3B0 3 260 ， 373. HPLC: (nm) (nm) 256 ，266sh ， 357; +NaOMe 273 ， 398 Rt 14.18 min (9b) and 18.57 min (9a) (So l. 11).

(inc.); (inc.); +AIC1 3 275 ， 437; +AIC1 3/HCl 270 ， Apigenin 7 -O-diglucoside (10). PC: RfO.31 296sh ，365 ， 401; +NaOAc 262 ， 407; +NaOAc/ (BA W) ， 0.35 (BEW) ， 0.32 (1 5%AcOH) ， 0.17

H 3B0 3 261 ，38 1. HPLC: Rt 1. 70 min (So l. 1). (5%AcOH); UV - dark purple ，UV/NH 3 - Quercetin Quercetin 3-0- xyloside- 7-・・ 0-glucoside(5c). yellow. UV: 入rrH(nm)268 ，334;+NaOMe

PC: Rf 0.23 (BA W) ， 0.16 (BEW) ， 0.69 273 ，378 (inc.); +AIC1 3 275 ，299 ，349 ， 381; (15%AcOH) ， 0.66 (5%AcOH); UV - dark +AICliHC1276 ，299 ，341 ， 380; +NaOAc267 ， pu 叩le ，UV INH 3 - dark yellow. UV:λ 詑?H 389; +NaOAc/H 3B0 3 268 ， 340. HPLC: Rt (nm) (nm) 256 ，266sh ， 359; +NaOMe 273 ，402 13.83 min (So l. 11).

(inc.); (inc.); +AIC1 3 275 ， 439; +AIC1 3/HCl 270 ， Apigenin 7-0-neohesperidoside (1Ia) and 297sh ，365 ， 402; +NaOAc 262 ， 406; +NaOAc/ apigenin 7 -O-xylosylglucoside (1Ib). PC: Rf

H 3B0 3 261 ， 383. HPLC: Rt 2.4 1 min (So l. 1). 0.4 6 (BAW) ，0 .4 7 (BEW) ， 0.32 (1 59 もAcOH) ， Luteolin Luteolin 7-0-g1ucoside (6). PC: Rf 0.31 0.17 (5%AcOH); UV -darkpurple ，UVINH 3 (BA W and BEW) ， 0.08 (1 59 もAcOH) ， 0.03 -yellow. UV:λな~H (nm) 268 ， 334; +NaOMe

(5%AcOH); UV - dark purple ，UV INH 3 - 271 ，379 (inc.); +AIC1 3 274 ，299 ，349 ， 377; bright bright yellow. UV: 入rH(nm)255 ，265sh ， +AICliHCl 277 ，298 ，341 ， 374sh; +NaOAc

348; 348; +NaOMe 266 ，389 (inc.); +AIC1 3 273 ， 268 ， 389; +NaOAc/H 3B0 3 268 ， 339. HPLC: 427; 427; +AICliHC1275 ，295 ，358 ， 386; +NaOAc Rt 9.67 min (1Ib) and 12.33 min (1Ia) (So l. 1).

260 ， 404; +NaOAc/H 3B0 3 260 ， 373. HPLC: 6，8-Di-C-glycosylluteolin (12). PC: RfO.10 Rt 14.61 min (So l. 11). (BAW) ， 0.14 (BEW) ， 0.33 (1 5%AcOH) ， 0.23

Apigenin Apigenin 7-0-g1ucoside (7). PC: Rf 0.50 (5%AcOH); UV - dark pu 中 le ，UVINH 3 - (BAW and BEW) ， 0.16 (1 5%AcOH) ， 0.06 da 比 yellow. UV: 入むご H (nm) 259 ，272 ， 348; (5%AcOH); UV - dark purple ，UV INH 3 - +NaOMe 271 ，337sh ， 411 (inc.); +AIC1 3 278 ， yellow. yellow. UV: 入詑~H (nm) 268 ， 333; +NaOMe 425; +AICliHCl 279 ，297sh ，357 ， 383sh; 272 ，378 (inc.); +AIC1 3 275 ，299 ，348 ， 381; +NaOAc281 ，401;+NaOAc/H 3B0 3 268 ，378 ， +AICliHC1276 ，298 ，340 ， 381; +NaOAc 267 ， 425sh. HPLC: Rt 3.02 min (So l. 11).

387; 387; +NaOAc/H 3B0 3 268 ， 339. HPLC: Rt 6，8-Di-C-glycosylapigenin(13). PC: RfO.18 25.89 25.89 min (So l. 11). (BA W) ， 0.23 (BEW) ，0 .4 4 (1 5%AcOH) ， 0.35

Luteolin Luteolin 7 -O-diglucoside (8). PC: Rf 0.16 (5%AcOH); UV - dark purple ，UV/NH 3 - (BA W) ， 0.24 (BEW) ， 0.20 (l 5%AcOH) ， 0.08 dark yellow. UV: 入rH(nm)273 ，332;

(5%AcOH); UV - dark purple ，UV INH 3 - +NaOMe 283 ，334 ，400 (inc.); +AIC1 3 279 ， bright bright yellow. UV: 入YT(nm)258 ，263sh ， 305 ，352 ， 382sh; +AICliHCl 280 ，304 ，345 ，

354; 354; +NaOMe 270 ，394 (inc.); +AIC1 3 273 ， 378sh; +NaOAc 282 ， 393; +NaOAc/H 3B0 3 424; 424; +AIC1 3/HCl 273 ，293sh ，355 ， 381sh; 285 ，322 ， 405sh. HPLC: Rt 3.96 min (So l. 11). +NaOAc 259 ， 374; +NaOAc/H 3B0 3 259 ， 369. 8-C-glucosylapigenin (vitexin ，14). PC: Rf HPLC: Rt 8.22 min (So l. 11). 0.4 0 (BA W) ， 0.38 (BEW) ， 0.17 (1 5%AcOH) ，

Luteolin Luteolin 7-0-neohesperidoside (9a) and 0.09 (5%AcOH); UV -darkpurple ，UVINH 3 luteolin luteolin 7-0 ・xylosylglucoside (9b). PC: Rf - dark yellow. UV:λ 詑~H (nm) 269 ， 336;

0.29 0.29 (BA W) ， 0.35 (BEW) ， 0.22 (1 5%AcOH) ， +NaO 民1e 279 ，332 ，394 (inc.); +AIC1 3 276 ， December 1997 Joumal of Japanese Botany Vo l. 72 No. 6 341

303 ，350 ， 389; +AIC1iHC1277 ，302 ，343 ， 379; glucopyranoside.

+NaOAc 278 ，307sh ， 386; +NaOAc/H 3B0 3 Quercetin 3-0-xylosylglucoside have been 269 ， 348. HPLC: Rt 10.14 min (So l. II). isolated as 3-0-s-D-xylopyranosyl (1→ 2)-0- Chlorogenic Chlorogenic acid. PC: RfO.33 (BA W) ， 0.30 s-D-glucopyranoside ，i.e. ， 3-0-sambubioside (BEW) ， 0.71 (1 5%AcOH) ， 0.69 (5%AcOH); from 也e flowers of Hibiscus mutabilis L. f.

UV - dark blue ，UV INH 3 - dark green. UV: versicolor Makino (Malvaceae) (l shikura

λ~~~H (nm) 280 ， 323; +NaOMe 293sh ，379 1982) and the leaves of Actinidia argut α，

(inc.); (inc.); +AIC1 3 267 ，314 ， 354sh; +AICliHCl (Siebold & Zucc.) Planch. ex Miq. va r. giraldii 281 ，32 1. HPLC: Rt 2.70 min (So l. 1). (Diels) Voroshilov (Actinidiaceae) (Webby 1991). 1991). In this survey ， xylose was reported as Results Results xylofuranose but not xylopyranose ， for the Flavonoids Flavonoids from Aucuba japonica first time. Seven flavonoid glycosides were isolated In relation to above flavonol glycoside ，we from the leaves of A. japonica. In this experi- have isolated quercetin 3-0- xy losy 19a1actoside ment ， the flavonoid profile of A. japonica v訂. from the leaves of Cornus capitata Wall. borealis borealis was also surveyed ， but it could not be (I washina and Hatta 1993). In this survey ， the essentially essentially distinguished with that of A. flavonol glycoside was also proved to be quer-

}apomca v紅 .}apomca. cetin 3-0-s-D-xylofuranosyl (l→ 2)-0-s-D- Major flavonoid 11iberated quercetin ，glu- galactopyranoside based on PC ，FAB-MS ， cose cose and xylose by complete acid hydrolysis. 1H - and 13C_ NMR data (see Materials and UV spec 仕a of original glycoside showed the Methods). Quercetin 3-0-xylosylgalactoside presence presence offree 5- ，7- ，3' ーand 4'-hydroxyl and have been isolated as 3-0-s-D-xylopyranosyl substituted a substituted 3-hydroxyl group. FAB-MS ex- (1 → 2)-0-s-D-galactopyranoside from the hibited hibited [M+H]+ at m/ z597 and [M-xylosyl+Ht leaves of Armoracia rusticana G. M. & Sch. at at m/ z465 showing the presence of each 1 mol (Cruciferae) (Larsen et al. 1982). quercetin ， glucose and xylose ，and direct at- UV spectra of flavonoid 2 showed the pres- tachment tachment of glucose to quercetin. lH-NMR ence of free 5- ，7- and 4'-hydroxyl and a spec 住a of original glycoside indicated five substituted 3-hydroxyl group. Kaempferol ， aromatic aromatic protons (H-6. -8. -2'. -5' and -6') and glucose and xy lose were liberated by complete s-D-glucopyranosyl s-D-glucopyranosyl anomeric proton (8 5.84 ， acid hydrolysis. From the results desribed d，J = 6.9 Hz). Since xylosyl anomeric proton ， above ，flavonoid 2 was identified as kaempferol moreover ，appe 紅 ed as a singlet (8 5.53) ，xy- 3-0-xylosylglucoside ， probabry 3-0- lose lose was regarded as s-D-xylof l町 anosyl but sambubioside as flavonoid 1. not not xylopyranosyl (Markham and Geiger Minor flavonoid 3 produced quercetin and 1994). 1994). It was shown that xylose was on 2-0H glucose ，and quercetin 3-0-g1ucoside and 7- of3-0-g1ucosyl of3-0-g1ucosyl group ，by 13C-NMR compari- O-glucoside by complete 叩 d partial acid hy- sons sons of the gl ycoside with those of isorhamnetin drolysis ，respectively. In addition ，UV spectra 3-0-rhamnosyl(1 → 2) glucoside (Agrawal and of original glycoside showed the presence of

Bansal1989) ，quercetin 7 ，3'-dimethyl ether 3- free 5，3' ，4 '-triOH and substituted 3，7 回 diOH.

0 ・rhamnosyl (1→ 2) glucoside (It okawa et al. Thus ， flavonoid 3 was identified as querce-

1981) and isorhamnetin 3-0-rhamnosyl tin 3，7-di-0 問 glucoside. (1 → 6) glucoside (Markham et al. 1978). Flavonoid 4 showed the lower Rf values in Thus ， flavonoid 1 was identified as querce- alcoholic solvent systems ，BA W and BEW ， tin tin 3-0-s-D-xylofuranosyl (1→ 2)-0-s-D- and higher in aqueous ones ，15%AcOH and 342 植物研究雑誌第72 巻第6号 平成9 年12 月

5%AcOH. Kaempferol ， glucose and xylose M t. Nago ，OkinawaPre f. of αsubsp. H.japonic were liberated by complete acid hydrolysis. liukiuensis were used as plant materials. Their UV spectra of original glycoside showed the flavonoid compositions which were surveyed presence presence offree 5- and4'-hydroxyl and substi- by 2D-PC and HPLC were essentially the tuted tuted 3- and 7-hydroxyl groups. Since the same.

flavonoid flavonoid produced kaempferol 7-0 四 gluco- Eleven flavonoids were isolated from the side side and a 仕ace of unknown intermediate ， leaves. probabry probabry labile kaempferol 3-0-xyloside ， Major flavonoids 6 and 7 were shown to which were monitored by HPLC ，by partial possess of free 5，3' ，4 '-triOH and 5，4 '-diOH ， acid acid hydrolysis ， flavonoid 4 is regarded as respectively ，by UV spec 位a. The aglycones kaempferol 3-0-xyloside-7 -O-glucoside. and sugars obtained by acid hydrolysis were Kaempferol Kaempferol 3-0-xyloside-7-0-g1ucoside has identified as luteolin and glucose (6) ， and api- been found in Zinnia elegans Jacq. genin and glucose (7) by direct PC compari- (Compositae) (Compositae) (Harborne and Williams 1988). sons with authentic specimens ，respectively. Flavonoid Flavonoid 5a was visuarized to yellow by Finally ，flavonoids 6 and 7 were identified UV lamp without exposure to fuming ammo- as luteolin 7-0-g1ucoside (6) and apigenin 7- nia ， showing to be a flavonol glycoside or an O “glucoside (7) by direct PC and HPLC com- aglycone aglycone having free 3- and 4'-hydroxyl parisons with authentic specimens. groups. groups. Quercetin and glucose were liberated It was shown by UV spectra and complete as as the hydrolysates. UV spectra of original acid hydrolysis that flavonoids 8 and 10 were glycoside glycoside showed the presence of free 3，5 ，3' ，4'- also luteolin and apigenin glycosides which tetraOH. tetraOH. attached glucose to each 7 -hydroxyl group. From the results described above ，flavo- Each one intermediate which was obtained by noid noid 5a was identified as quercetin 7 -O-gluco- partial acid hydrolysis of flavonoids 8 and 10 side. side. was identified as 7-0-monoglucosides of Complete acid of hydrolysis flavonoids 5b luteolin and apigenin. and 5c gave quercetin ，glucose and xylose. UV Accordingly ，flavonoids 8 and 10 were re- spectra spectra of original glycosides showed the pres- garded as luteolin 7 -O-diglucoside and api- ence ence offree 5- ，3' ー and 4'-hydroxyl and substi- genin 7-0'-diglucoside ，respectively. tuted tuted 3- and 7 -hydroxyl groups. Partial acid Flavonoid 9 appeared as single spot on the hydrolysis hydrolysis of 5c gave quercetin 7 -O-glucoside paper chromatogram ， but it divided into two (5a) (5a) and a trace of quercetin 3-0-xyloside as peaks (9a and 9b) by HPLC analysis. Com- intermediates. intermediates. On the other hand ，partial acid plete acid hydrolysis of the mixture produced hydrolysis hydrolysis of 5b gave 5c and 5a as intermedi- luteolin ，glucose ，rhamnose and xylose. ates. ates. Luteolin 7 -O-glucoside was obtained as an From the results described above ， 5c was intermediate by partial acid hydrolysis of the identified identified as quercetin 3-0-xyloside-7-0-g1u- mixture. Since the presence offree 5- ，3'- and coside coside and 5b was presumed as quercetin 3-0 ・ 4'-hydroxyl groups was shown by UV spectral xyloside-7-0-xylosylglucoside. xyloside-7-0-xylosylglucoside. analysis of original glycoside ，it is clear that sugars sugars are on 7 -position of luteolin. Of two Flavonoids Flavonoids jヤom Helwingia japonica peaks on HPLC ，retention time of 9a agreed Two samples from The Botanic Garden ， with that of authentic luteolin 7-0-rhamnosyl Hokkaido Univ. and Hakone ，Kanagawa Pre f. (l→ 2) glucoside (l uteolin 7-0-neohesperido- of of H. japonica subsp. japonica and one from side). Accordingly ， another one (9b) is re- December 1997 Joumal of Japanese Botany Vo l. 72 No. 6 343 garded garded as luteolin 7-0-xylosylglucoside. caffeoylquinic acid) by characterization of Flavonoid Flavonoid 11 also appeared as single spot hydrolysates ，UV spectra ， and PC and HPLC on paper chromatogram ， but it was divided comparison with authentic specimen. Other into into two peaks (11a and 11b) by HPLC sepa- components were characterized as chlorogenic ration. ration. Apigenin ，glucose ，rhamnose and xy- derivative acid (one kind) and caffeoyl sugar lose ，and apigenin 7 -O-glucoside were ob- esters (four kinds). tained tained by complete and partial acid hydrolysis ， respecti respecti vel y. UV spectra of the mixture showed Discussion

the the presence of free 5，4 '-diOH and a substi- The flavonoids of Aucuba japonica includ 山 tuted tuted 7 -OH. Since retention time of peak 11a ing va r. borealis and Helwingia japonica in- agreed agreed with that of authentic apigenin 7-0- cluding subsp. liukiuensis were reported in neohesperidoside neohesperidoside (rhoifolin) ，anotherone (11b) this paper for the first time. In spite of both was characterized as apigenin 7-0-xylosyl- genera belonging to the family Cornaceae ， glucoside. glucoside. their flavonoid characters were clearly differ- Flavonoids Flavonoids 12 ， 13 and 14 could not be ent ，i.e. ， the former flavonoids consist of flavo- hydrolyzed hydrolyzed by hot acid treatment showing that no13-0-di- and 3，7-di-0-glycosides based on the the compounds are C-glycosylflavonoids. UV kaempferol and quercetin ， while the latter ones spectra spectra of 13 and 14 showed the presence of are constituted with flavone 7 -O-glycosides free free 5- ，7- and 4'-hydroxyl groups (apigenin and C-glycosyl derivatives based on apigenin type). type). On the other hand ，UV spec 町a of 12 and luteolin. We have observed the flavonoids exhibited exhibited the presence of free 5，7 ，3' ，4' ーtetraOH in the leaves of thirty-three Cornus taxa uteolin (l uteolin type). Hot acid treatment of 14 gave (l washina and Hatta 1990 ，1992 ，1993 ，1994). another another spot (an isomer of 14) on the chroma- Their major flavonoids were various flavonol togram togram in addition to original one ，which was 3-0- monoglycosides based on kaempferol and caused caused by Wessely-Moser rearrangement quercetin ，i.e. ，glucoside ，galactoside ，glucuro- (Markham 1982). On the other hand ，no rear- nide ，rhamnoside ，xyloside and arabinoside ， in rangement rangement occurred in 12 and 13. Thus ，it was company with minor myricetin 3-0-rhamno- shown that 12 and 13 are 6，8-di-C- side and flavonol 3-0-diglycosides such as glycosylflavones glycosylflavones and 14 is 6- or 8-mono-C- quercetin 3-0-rutinoside ， kaempferol 3-0- glycosylflavone. glycosylflavone. rutinoside ，quercetin 3-0-xylosylgalactoside From the results described above ，flavonoids etc. (Table 1). Flavonoid characters of Cornus 12 and 13 were characterized as 6，8-di-C- species were similar to those of Aucuba spe- glycosylluteolin glycosylluteolin and 6，8-di-C-glycosylapi- cies with regard to occu 町 ence of flavonols

genin ，respectively. However ，retention times alone. However ，major flavonol 3-0 同 of of 12 and 13 did not agree with those of 6，8-di- monoglycosides which were found from all C-glucosylluteolin C-glucosylluteolin (lucenin-2) and 6，8-di-C- Co rnus species examined (1 washina and Hatta

glucosylapigenin glucosylapigenin (vicenin 白 2). 1994) were completely absent from Aucuba Flavonoid 14 was identified as 8-C- japonica. Instead ，flavono13 ，7 -di-O-glycosides glucosylapigenin glucosylapigenin (vitexin) by PC and HPLC such as kaempferol and quercetin 3-0- comparison comparison with authentic specimen. xyloside-7 -O-glucosides were found from In In addition to these flavonoid glycosides ， Aucuba japonica together with 3-0-xylosyl- some phenolic compounds were isolated from glucosides as major components. In addition ， the the leaves of Helwingiajaponica. One ofthem two anthocyanins ，3-0-xylosylglucosides of was identified as chlorogenic acid (3- both pelargonidin and cyanidin have been iso- 344 植物研究雑誌第72 巻第6号 平成9年12 月

Table Table 1. The flavonoid glycosides isolated from Cornus ，Aucuba and Helwingia species

Genera Glycosides

Cornus kaempferol 3-g1ucoside (1 2) ，3 ・・・ galactoside(13) ，3-g1ucuronide(5) ， 3-diglucoside( 3-diglucoside( 1) ，3-rutinoside(2) ，3- xylogalactoside(2) ， 3， 7 -dirhamnoside( 1) ，3-g1ucoside-7 - rhamnoside( 1) ，

3-galactoside-7 3-galactoside-7 -I ・hamnoside( 1) quercetin quercetin 3-g1ucoside(30) ，3-galactoside (1 4) ，3-g1ucuronide (1 1) ， 3-rhamnoside(9) ，3-arabinoside(5) ，3-xyloside(5) ， 3-diglucoside(2) ，3-rutinoside( 15) ，3- xyloglucoside (1)， 3-xylogalactoside(4) ，3-g1ucoside-7-rhamnoside(1) ， 3-galactoside-7 3-galactoside-7 -rhamnoside(l) myricetin myricetin 3-rhamnoside(5)

Aucub α kaempferol 3- xyloglucoside ，3- xyloside-7 -glucoside

quercetin quercetin 7 同 glucoside ，3- xy loglucoside ，3 ，7 -diglucoside ， 3- xyloside-7 -glucoside ，3- xyloside-7 -xyloglucoside

Helwingia Helwingia apigenin 7 -glucoside ，7 -diglucoside ，7 -neohesperidoside ，

7-xyloglucoside ，8-C-glucosyl and 6，8-di-C 国 glycosyl derivatives derivatives luteolin luteolin 7-g1ucoside ，7-diglucoside ，7-neohesperidoside ， 7-xyloglucoside ， 6 ，8-di-C-glycosyl derivative

)= Numbers of species which the glycoside was found from examined 33 Cornus species species (l washina and Hatta 1990 ，1992 ，1993 ，1994).

Table Table 2. Comparison of t1 avonoid characters among the genera Cornus ， Aucuba and Helwingia

Flavonoids Flavonoids Cornus Aucuba Helwingia

Flavonols Flavonols myncetm + 3- monogl ycosides + 3-diglycosides 3-diglycosides + + 7-g1ycosides 3，7-g1ycosides + + 7-g1ycoside 7-g1ycoside + Flavones Flavones 7-g1ycosides 7-g1ycosides + -diglycosides 7 -diglycosides + C-glycosyl C-glycosyl derivatives + December 1997 Joumal of Japanese Botany Vo l. 72 No. 6 345

Giannasi Giannasi D. E. 1988. Fl avonoids and evolution in the dicotyle- dons. dons. In: Harbome J. B. (ed.) The Flavonoids: Advances in 1971) showing that the flavonoid 3，7- Research Research Since 1980. Chapman and Hall ，London ， pp. 479- diglycosides diglycosides and 3-0-xylosylglycosides are 504. principal principal chemical character of the species or HaraH. 1989. Comaceae. In: Satake Y. ，HaraH. ， Watari S. and Tominari Tominari T. (eds.) Wild Flowers of Japan. Woody Plants. genus. genus. It is noteworthy that both flavonoid Heibonsha ，Tokyo ， pp. 109-112 (in Japanese). characters characters were reported from Chinese Cornus Harbome J. B. 1975. The biochemical systematics of flavonoids. species ，C. multinervosa (Pojar k.) Q. Y. Xiang In: Harbome J. B. ，Mabry T. J. and Mabry H. (eds.) The Fl avonoids. Chapman and Hall ，London ， pp. 1056-1095. belonging belonging to subgenus Benthamia (I washina 一一一一一 and Tumer B. L. 1984. Plant Chemosystematics. and Hatta 1994). Academic Press ，London ， pp. 49-386. On the other hand ，major flavonoids of 一一一一一 and Williams C. A. 1988. Flavone and flavonol glycosides. glycosides. In: Harbome J. B. (ed.) The Fl avonoids: Aι Helwingia Helwingia were flavone 7-0-g1ycosides to- vances in Research Since 1980. Chapman and Hall; London ， gether gether with minor C-glycosides ，and their pp. 303-328. chemical chemical characters were clearly distinguish- Ishikura N. 1971. Pelargonidin glycosides in fruits. Experientia 27: 27: 1006. able able from those of Cornus ，Aucuba and also 一一一一一 1982. Fl avonol glycosides in the flowers of Hibiscus Mastixi α(1 washina and Hatta unpublished mutabilis f. versicolor. Agric. Bio l. Chem. 46: 1705 ー1706. data) data) containing flavonol glycosides as major Itokawa H. ，Oshida Y.， Ikuta A ， Inatomi H. and Ikegami S. 198 1. Flavonol glycosides from the.flowers of Cucurbita compounds (Table 2). Bate-Smith et al. (1 975) pepo. Phytochemistry 20: 2421-2422. have also proposed to raise the genUS Iwashina T. and Hatta H. 1990. The flavonoid glycosides in the Helwingia Helwingia to family rank within Cornales due leaves of Cornus species 1. The flavonoids of C. controversa ， C. C. brachypoda ， C. darvasica and C. drummondii. Ann. to to the absence of any tannins and iridoids (the Tsukuba Bo t. Gard. 9: 41 -4 7 (in Japanese with English presence presence in Cornus ，Aucuba and Mastixia). summary). The facts described above show thatHelwingia 一一一一一 and 一一一一一 1992. The flavonoid glycosides in the leaves leaves of Cornus species 11. The flavonoids of C. canadensis phytochemically phytochemically keeps aloof from Cornus ， and C. suecica. Ann. Tsukuba Bo t. Gard. 11: 23-34. Aucuba and Mastixia. Moreover ， they were 一一一一-and 一一一一一 1993. The flavonoid glycosides in the also also supported by rbcL sequence data (Xiang leaves of Cornus species III. The flavonoids of three Himalayan Cornus species. Ann. Tsukuba Bo t. Gard. 12: et et al. 1993). 49-56. In In delineating the position of Cornus ，char- 一一一一 -and 一一一一一 1994. The flavonoid glycosides in the leaves leaves of Cornus species IV. The distribution of flavonoids acterization acterization of flavonoids in other Cornaceae in genus Cornus. Ann. Tsukuba Bo t. Gard. 13: 29 -4 0. genera genera and related families ，Alangiaceae ， Jensen S. R. ， Kjaer A. and Nielsen B. J. 1975. The genus Nyssaceae and Davidiaceae are needed and it Cornus. Non-flavonoid glucosides as taxonomic markers. Biochem. Sys t. Eco l. 3: 75-78. is is nOW in progress by us. Larsen ， L. M. ， Nielsen J. K. and Sφrensen H. 1982. Identifica-

tion tion of 3-0-[2-0-(s-D-xylopyranosyl) ーβD-galacto- The authors thank to M r. Zenshu Miyara flavonoids pyranosyl] in horseradish leaves acting as feed- ing ing stimulants for a flea beetle. Phytochemistry 21: 1029- (Ishigaki (Ishigaki Junior High School) ， Mrs. Kayoko 1033. Inoue Inoue (Hakone Botanical Garden ofWetLands) Markham K. R. ，Temai B. ， Stanley R.， Geiger H. and Mabry T. and The Botanic Garden ， Faculty of Agricul- J. 1978. Carbon-13 N 九1R studies of flavonoids- lI1. Natu- rally rally occu 汀 ing flavonoid glycosides and their acylated ture ，Hokkaido University for collection of derivatives. Tetrahedron 34: 1389-1397. plant plant materials. 一一一一一 1982. Techniques of Flavonoid Identification. Aca- demic Press ，London. 一一一一一 and Geiger H. 1994. lH-nuclear magnetic resonance References References spectroscopy spectroscopy of flavonoids and their glycosides in Agrawal B. K. and Bansal M. C. 1989. Fl avonoid glycosides. In: hexadeuterodimethylsulfoxide. hexadeuterodimethylsulfoxide. In: Harbome J. B. (ed.) Agrawal P. K. (ed.) Carbon-13 NMR ofFlavonoids. Elsevier ， The Fl avonoids: Advances in Research Since 1986. Chapman Amsterdam ， pp. 283-364. & Hall ，London ， pp. 441 -4 97. Bate-Smith Bate-Smith E. c.， Ferguson 1. K.， Hutson K.， Jensen S. R.， Webby R. F. 199 1. A flavonol triglycoside from Actinidia Nielsen Nielsen B. J. and Swain T. 1975. Phytochemical interrela- arguta arguta var. giraldii. Phytochemistry 30: 2443-2444. tionships tionships in the Comaceae. Biochem. Syst. Eco l. 3: 79-89. 346 346 植物研究雑誌第72 巻第6号 平成9年12 月

Xiang Xiang Q.-Y. ，Soltis D. E. ，Morgan D. R. and Soltis P. S. 1993. putative relatives inferred from rbcL sequence data. Ann. Phylogenetic Phylogenetic relationships of Cornus L. sensu lato and Missouri Bo t. Gard. 80: 723-734.

岩科 司，亀之園浩治，八田洋章:アオキおよび ハナイカ夕、(ミズキ科)の葉に含まれるフラボノ イド配糖体:ミズキ属植物との植物化学的関係 キ属植物のもの(岩科・八田 1990 ，Iwashina and アオキ，ヒメアオキ，ハナイカダおよびリュウ Hatta 1992 ， 1993 ， 1994) と比較すると，アオキ キュウハナイカダの葉に含まれるフラボノイド配 とミズキ属植物のフラボノイドはいずれもフラボ 糖体が今回初めて報告された.アオキとその変種 ノール配糖体である点で一致するが，調査された のヒメアオキは同一のフラボノイドを有しており， ミズキ属すべての植物の主要配糖体が一般的なフ それらは quercetin の 3 -O-sarnbubioside ， 7-0- ラボノール 3-0- モノ配キ唐イ本であるのに文すして， ア glucoside ， 3，7-di-0-glucoside ， 3-0-xyloside-7-0- オキではほとんどがキシロースを結合した 3-0- ジ glucoside および3-0-xyloside-7-0-xylosylglucoside ， 配糖体や 3，7-0 崎配糖体であることで区別された. kaernpferol の 3- 0-xy losy 19lucoside および3-0- また，ハナイカダから分離されたフラボノイド xyloside-7 xyloside-7 -O-glucoside と同定された. はすべてがフラボンの 0- およびC- 配糖体であり， 一方，ハナイカダとその亜種のリュウキュウハ ミズキ属植物やアオキとは明らかに異なっており， ナイカダも同一のフラボノイドを有し， apigenin この属がミズキ属やアオキ属とはかなり離れた位 とluteolin の7-0-g1ucoside ，7-0-diglucoside ，7-0- 置にあることを示唆した. neohesperidoside ， 7-0- xy losy 19lucoside および6，8- ("国立科学博物館筑波実験植物園， di-C-glycoside ，および、 vitexin と同定された. b 日本油脂筑波研究所) これらのフラボノイドを，以前に報告したミズ