Bull. Natl. Mus. Nat. Sci., Ser. B, 46(4), pp. 195–214, November 20, 2020

Further Identiﬁcation of Flavonoids Deposited in the National Museumof Nature and Science in Japan — Flavonoids Isolated from
Cirsium Taxa and Carduus nutans (Asteraceae) —Tsukasa Iwashina1,*, Takayuki Mizuno1 and Hari Prasad Devkota2
1

Departmentof Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan
2
Graduate School of Pharmaceutical Sciences, Kumamoto University,
5–1 Oe-honmachi, Kumamoto 862–0973, Japan*E-mail: iwashina@kahaku.go.jp(Received 6 July 2020; accepted 23 September 2020)

Abstract Chemical structures of 59 ﬂavonoid powder and crystal samples which are deposited in
Department of Botany, National Museum of Nature and Science, Japan, were determined or conﬁrmed by UV spectral survey, LC-MS, NMR and/or HPLC comparisons with authentic samples.
They were isolated from 37 Cirsium taxa and Carduus nutans (Asteraceae) in 1980–2001 anddonated to National Museum of Nature and Science. Many crystal samples were identiﬁed as pectolinarigenin 7-O-rutinoside with minor acacetin 7-O-rutinoside. They are popular ﬂavonoids inthe genus Cirsium. As other ﬂavonoids, hispidulin 7-O-neohesperidoside, cirsimaritin 4′-O-glucoside, and luteolin 7-O-glucoside, 5-O-glucoside, 3′-O-glucoside, 4′-O-glucoside and 7-O-vicianoside were identiﬁed. Of their ﬂavonoids, luteolin 7-O-vicianoside which was obtained from C.
buergeri was reported from the genus Cirsium for the ﬁrst time. Hispidulin 7-O-neohesperidoside
was newly found in Japanese Cirsium species. In this survey, ﬂavonoids of 20 Cirsium taxa, e.g. C.
boninense, C. hanamakiense, C. alpicola, C. aidzuense and C. ishizuchiense, were reported for theﬁrst time.
Key words : Carduus nutans, Cirsium species, crystal samples, luteolin 7-O-vicianoside, pectolinaigenin 7-O-rutinoside.

Introduction
In Department of Botany, National Museum of
Nature and Science, Tsukuba, many ﬂavonoid
and related compounds such as aromatic acids,
xanthones and tannins are deposited as crystals
and powders. Many samples were isolated andcharacterized by one of the authors (T. Iwashina)
and Dr. Kozo Hayashi (former Professor ofTokyo Kyoiku University). The compounds
which were recently obtained were identiﬁed byacid hydrolysis, UV spectral survey according to
Mabry et al. (1970), LC-MS and NMR spectra.
© 2020 National Museum of Nature and Science

However, some compounds which were isolated
in the early times are insuﬃciently characterized
without LC-MS and NMR. In this survey, we
performed complete characterization of the ﬂavonoids which are deposited in National
Museum of Nature and Science. As a series offurther identiﬁcation of insuﬃciently characterized ﬂavonoids, we performed the identiﬁcationof the ﬂavonoids which were obtained from Cirsium taxa and related Carduus nutans (Asteraceae) in 1980–2001, by LC-MS and NMR, and/or direct HPLC comparisons with authentic samples.

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Materials and Methods
Materials
Of their ﬂavonoids which were characterized
in this survey, some compounds have been published as some papers (Iwashina et al., 1988,
1989, 1995, 1999; Iwashina and Ootani, 1998).
Other samples were isolated from Cirsium
species which were collected by Dr. Yuichi
Kadota (Curator Emeritus, National Museum of
Nature and Science, Japan). Their ﬂavonoids
were not published as the papers until now.
Moreover, some powders and crystals were isolated from Cirsium species by Mr. Tsutomu Ito,published as graduation thesis of Faculty of Agriculture, Tokyo University of Agriculture, andpresented to National Museum of Nature andScience in 1986 and 1989. However, they wereincompletely characterized. We examined 59
ﬂavonoid powder and crystal samples from 37
Cirsium taxa and Carduus nutans in this study.
GeneralAnalytical high performance liquid chromatography (HPLC) was performed with Shimadzu
HPLC systems using Inertsil ODS-4 (I.D.
6.0×150 mm, Chemicals Evaluation and ResearchInstitute, Tokyo) at a ﬂow-rate of 1.0 ml min−1.
Detection wavelength was 350 nm. Eluent was
MeCN/H2O/H3PO4 (20 : 80 : 0.2). Liquid chromatograph-mass spectra (LC-MS) was performed with Shimadzu LC-MS systems using
Inertsil ODS-4 (I.D. 2.1×100 mm) at ﬂow-rate
of 0.2 ml min−1, ESI＋ 4.5 kV and ESI− 3.5 kV,
250°C. Eluents were MeCN/H2O/HCOOH(20 : 75 : 5 or 40 : 55 : 5). Acid hydrolysis was
performed in 12%HCl, 100°C, 30 min. Aftershaking with diethyl ether, aglycones were
migrated to the organic layer, and sugars wereleft in aqueous layer. Aglycones were identiﬁedby HPLC comparisons with authentic samples.
On the other hand, sugars were identiﬁed bypaper chromatographic comparisons with authentic sugars, glucose, galactose, glucuronic acid,rhamnose, xylose and arabinose, using solvent
systems, BBPW (n-BuOH/benzene/pyridine/H2O＝5 : 1 : 3 : 3) and BTPW (n-BuOH/toluene/pyridine/H2O＝5 : 1 : 3 : 3). Sugar spots were visualized by spraying 1% methanolic aniline hydrochloride on the chromatograms and heating. 1H
and 13C NMR were recorded on a Brucker
AV-600 in pyridine-d5 at 600 MHz (1H NMR)
and 150 MHz (13C NMR).
Results and Discussion
1. Flavonoids from Cirsium aidzuense Nakai ex
Kitam.
Pectolinarigenin 7-O-rutinoside (Pectolinarin,136-13). UV: λmax (nm) MeOH 275, 328;
＋NaOMe 296, 377 (dec.); ＋AlCl3 286sh, 299,
353, 387sh; ＋AlCl3/HCl 286, 299, 346, 386sh;＋NaOAc 280, 324; ＋NaOAc/H3BO3 276, 331.
LC-MS: m/z 623 [M＋H]＋, 621 [M-H]− (molecular ion peaks, pectolinarigenin＋each 1 mol of
glucose and rhamnose), m/z 477 [M-146＋H]＋,
475 [M-146-H]− (fragment ion peaks, pectolinarigenin＋1 mol glucose), and m/z 315 [M-308＋H]＋ (fragment ion peak, pectolinarigenin).
The sample was obtained as pale yellow needle(ca. 20 mg) from C. aidzuense leaves which werecollected by Dr. Yuichi Kadota (collection site is
unknown). Flavonoid was identiﬁed as pectolinarigenin 7-O-rutinoside (Fig. 1) by UV, LC-MS
and HPLC comparison with 136-10. Acacetin

Fig. 1.

Pectolinarigenin 7-O-rutinoside.

Fig. 2. Acacetin 7-O-rutinoside.

Further identiﬁcation of ﬂavonoids from Cirsium spp.

7-O-rutinoside (Linarin, Fig. 2) was also contained as minor compound (11.7%). Flavonoids
of C. aidzuense were reported for the ﬁrst time.
2. Flavonoids from Cirsium alpicola Nakai
Pectolinarigenin 7-O-rutinoside (136-29). UV:
λmax (nm) MeOH 273, 329; ＋NaOMe 296, 380(dec.); ＋AlCl3 283sh, 300, 352, 386sh;＋AlCl3/HCl 284, 298, 347, 386sh; ＋NaOAc
279, 325; ＋NaOAc/H3BO3 274, 332. LC-MS:
m/z 623 [M＋H]＋ (molecular ion peak,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The sample was obtained as pale yellow needle from C. alpicola leaves which was collectedby Dr. Yuichi Kadota in 2000 (collection site is
unknown). Flavonoid was identiﬁed as pectolinarigenin 7-O-rutinoside by UV, LC-MS and
HPLC comparison with authentic sample (13610) from C. brevicaule. Acacetin 7-O-rutinoside
was contained in this sample as minor compound(19.8%). Flavonoids of C. alpicola were found
for the ﬁrst time.
3. Flavonoids from Cirsium bitchuense Nakai
Pectolinarigenin 7-O-rutinoside (136-15). UV:
λmax (nm) MeOH 275, 328; ＋NaOMe 297, 377(dec.); ＋AlCl3 287sh, 299, 353, 387sh; ＋AlCl3/
HCl 283, 298, 347, 386sh; ＋NaOAc 278, 324;
＋NaOAc/H3BO3 274, 332. LC-MS: m/z 623[M＋H]＋, 621 [M-H]− (molecular ion peaks,
pectolinarigenin＋each 1 mol of glucose andrhamnose), m/z 313 [M-308-H]− (fragment ion
peak, pectolinarigenin).
The sample was obtained as pale yellow needle (ca. 8 mg) from the leaves of C. bitchuense
which was collected in 1993 by Dr. Yuichi
Kadota (collection site is unknown). Flavonoid
was identiﬁed as pectolinarigenin 7-O-rutinoside
by UV, LC-MS and HPLC comparison with 13610. Although pectolinarin has been reported fromthis species (Iwashina et al., 1995), it was provedby this survey that acacetin 7-O-rutinoside is also
contained as minor ﬂavonoid (14.2%).

197

4. Flavonoids from Cirsium boninense Koidz.
Pectolinarigenin 7-O-rutinoside (136-23). UV:
λmax (nm) MeOH 275, 329; ＋NaOMe 296, 376(dec.); ＋AlCl3 286sh, 299, 354, 387sh; ＋AlCl3/
HCl 285sh, 297, 345, 386sh; ＋NaOAc 280, 320;
＋NaOAc/H3BO3 275, 331. LC-MS: m/z 623[M＋H]＋, 645 [M＋H＋Na]＋, 621 [M-H]−(molecular ion peaks, pectolinarigenin＋each 1 molof glucose and rhamnose), m/z 477 [M-146＋H]＋(fragment ion peak, pectolinarigenin＋1 mol glucose).
The sample was obtained as pale yellow needle (ca. 2 mg) from the leaves of C. boninense
which was collected in 1999 in Ogasawara
Islands, Japan by Dr. Yuichi Kadota. Molecularweight and UV absorption properties wereagreed with those of pectolinarin. Finally, it was
identiﬁed as pectolinarigenin 7-O-rutinoside by
HPLC comparison with 136-10. The ﬂavonoids
of C. boninense were reported for the ﬁrst time.
Acacetin 7-O-rutinoside was mixed in the sample
as minor compound (8.9%).
5. Flavonoids from Cirsium borealinipponense
Kitam.
Pectolinarigenin 7-O-rutinoside (136-18). UV:
λmax (nm) MeOH 275, 328; ＋NaOMe 295, 378(dec.); ＋AlCl3 283sh, 300, 350, 386sh; ＋AlCl3/
HCl 283, 299, 346, 385sh; ＋NaOAc 281, 321;＋NaOAc/H3BO3 273, 333. LC-MS: m/z 623[M＋H]＋, 621 [M-H]− (molecular ion peaks,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The sample was isolated as pale yellow needle(ca. 40 mg) from the leaves of C. borealinipponense which was collected by Dr. Yuichi Kadota
in 2001 (collection site is unknown), and identiﬁed as pectolinarigenin 7-O-rutinoside by
LC-MS, UV spectra, and HPLC comparison with
136-10 from C. brevicaule. Acacetin 7-O-rutinoside was contained in the sample as minor ﬂavonoid (25.5%). Flavonoids of C. borealinipponense were reported for the ﬁrst time.
6. Flavonoids from Cirsium brevicaule A. Gray
Pectolinarigenin 7-O-rutinoside (136-10, 136-

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32). UV: λmax (nm) MeOH 276, 329; ＋NaOMe
295, 377 (dec.); ＋AlCl3 283sh, 299, 355, 385sh;＋AlCl3/HCl 284, 298, 346, 386sh; ＋NaOAc
278, 323; ＋NaOAc/H3BO3 274, 331. LC-MS:
m/z 623 [M＋H]＋ (molecular ion peak,
pectolinarigenin＋each 1 mol of glucose andrhamnose), m/z 477 [M-146＋H]＋ (fragment ion
peak, pectolinarigenin＋1 mol glucose), and m/z
313 [M-308-H]− (fragment ion peak, pectolinarigenin). 1H NMR (600 MHz, pyridine-d5): δH 8.16
(2H, d, J＝8.9 Hz, H-2′,6′), 7.36 (2H, d,
J＝8.9 Hz, H-3′,5′), 7.31 (1H, s, H-8), 6.97 (1H,s, H-3), 5.78 (1H, d, J＝7.7 Hz, glucosyl H-1),
5.45 (1H, d, J＝1.0 Hz, rhamnosyl H-1), 4.70
(1H, brd, J＝10.2 Hz, glucosyl H-6a), 4.64 (1H,
dd, J＝1.5 and 13.4 Hz, rhamnosyl H-2), 4.55
(1H, dd, J＝3.4 and 9.2 Hz, rhamnosyl H-3),
4.48 (1H, t, J＝9.1 Hz, glucosyl H-5), 4.40 (1H,
dd, J＝7.8 and 9.1 Hz, glucosyl H-2), 4.35 (1H,m, glucosyl H-3), 4.29 (1H, dd, J＝6.1 and9.4 Hz, rhamnosyl H-5), 4.24 (1H, t, J＝9.3 Hz,
rhamnosyl H-4), 4.15 (1H, brd, J＝9.5 Hz, glucosyl H-6b), 4.12 (1H, brd, J＝9.3 Hz, glucosylH-4), 4.12 (3H, s, 6-OCH3), 3.88 (3H, s,
4′-OCH3), 1.57 (3H, d, J＝6.1 Hz, rhamnosyl
CH3). 13C NMR (150 MHz, pyridine-d5): (pectolinarigenin) δC 153.5 (C-2), 104.2 (C-3), 183.2(C-4), 165.1 (C-5), 133.7 (C-6), 157.5 (C-7),
95.2 (C-8), 153.2 (C-9), 107.0 (C-10), 123.7(C-1′), 129.0 (C-2′,6′), 115.4 (C-3′,5′), 163.2(C-4′), 61.1 (6-OCH3), 55.8 (4′-OCH3); (glucose)δC 102.1 (C-1), 74.4 (C-2), 77.3 (C-3), 71.0(C-4), 77.7 (C-5), 67.4 (C-6); (rhamnose) δC
102.0 (C-1), 71.8 (C-2), 72.4 (C-3), 73.7 (C-4),
69.7 (C-5), 18.3 (C-6).
The ﬂavonoid was obtained as pale yellow
needle (ca. 60 mg 136-10, small amount 136-32).
Molecular ion peak, m/z 623 [M＋H]＋, and two
fragment ion peaks, m/z 477 [M-146＋H]＋ and313 [M-308-H]−, appeared on LC-MS, showing
the attachment of each 1 mol of hexose andrhamnose to dihydroxy-dimethoxyﬂavone. The
presence of free 5-hydroxyl and substituted 7and 4′-hydroxyl groups was shown by UV spectral survey according to Mabry et al. (1970).
Moreover, the additional presence of substitutedhydroxyl group to 6- or 8-position was presumed
by Band II value of absorption maxima in
MeOH solution. Practically, pectolinarigenin(5,7-dihydroxy-6,4′-dimethoxyﬂavone), glucose
and rhamnose were liberated by acid hydrolysis.
In NMR, proton and carbon signals wereassigned by HSQC, HMBC, COSY and NOESY.
Four aromatic proton signals, δH 8.16 (2H), 7.36
(2H), 6.97 (1H) and 7.31 (1H) according toH-2′,6′, H-3′,5′, H-3 and H-8, appeared, together
with glucosyl and rhamnosyl anomeric protons,
δH 5.78 (d, J＝7.7 Hz) and 5.45 (d, J＝1.0 Hz),and two methoxyl proton signals, δH 4.12 and3.88. Of their proton signals, two methoxyl proton signals correlated with the carbon signals at
δC 133.7 and 163.2 corresponding to C-6 andC-4′ by HMBC, respectively. On the other hand,sugar anomeric proton signals, δH 5.78 and 5.45
were correlated with the carbon signals at δC
157.5 and 67.4 corresponding to C-7 of pectolinarigenin and C-6 of glucose. From their results,
the ﬂavonoid was identiﬁed as pectolinarigenin
7-O-α-L-rhamnopyranosyl-(1→6)-β-Dglucopyranoside (pectolinarin, Fig. 1). Pectolinarin has already been reported from some Cirsium species (e.g. Iwashina et al., 1988, 1995). In
C. brevicaule, it has been isolated from theleaves (Morita et al., 1964). However, it wasproved by HPLC comparison with authentic
sample that acacetin 7-O-rutinoside (linarin, Fig.
2) is also contained in their samples as minor
compound (10.3% in 136-10 and 8.4% in 13632). Linarin was found in C. brevicaule for theﬁrst time.
7. Flavonoids from Cirsium buergeri Miq.
Luteolin 7-O-glucoside (304-1, 304-11, 306-1,
306-2). UV: λmax (nm) MeOH 255, 266sh, 349;
＋NaOMe 270, 397 (inc.); ＋AlCl3 273, 425;
＋AlCl3/HCl 270, 294sh, 358, 384sh; ＋NaOAc
259, 404; ＋NaOAc/H3BO3 260, 371. LC-MS:
m/z 449 [M＋H]＋, 447 [M-H]− (molecular ion
peaks, luteolin＋1 mol glucose), m/z 287 [M-162＋H]＋, 285 [M-162-H]− (fragment ion peaks,
luteolin).
The samples were isolated by Mr. Tsutomu ItoFurther identiﬁcation of ﬂavonoids from Cirsium spp.

(the student of Tokyo University of Agricultureof the day) from the leaves of C. buergeri(ca.10 mg, 304-1, small amount, 304-11, ca.
40 mg, 306-1 and ca. 20 mg, 306-2) and conﬁrmed as luteolin 7-O-glucoside (Fig. 3) by UV,
LC-MS and HPLC comparison with authentic
sample from Carl Roth (Germany) in this survey.
Luteolin 7-O-vicianoside (304-2, 304-5). UV:
λmax (nm) MeOH 256, 266sh, 347; ＋NaOMe
270, 394 (inc.); ＋AlCl3 273, 425; ＋AlCl3/HCl
271, 294sh, 357, 383; ＋NaOAc 262, 400;
＋NaOAc/H3BO3 260, 371. LC-MS: m/z 581[M＋H]＋, 579 [M-H]− (molecular ion peaks,
luteolin＋each 1 mol of glucose and arabinose),m/z 287 [M-294＋H]＋ (fragment ion peak, luteolin). 1H NMR (600 MHz, pyridine-d5): δH 13.66(1H, s, 5-OH), 8.04 (1H, d, J＝2.3 Hz, H-2′),
7.59 (1H, dd, J＝2.3 and 8.3 Hz, H-6′), 7.37 (1H,d, J＝8.3 Hz, H-5′), 7.03 (1H, d, J＝2.2 Hz, H-8),
6.90 (1H, s, H-3), 6.83 (1H, d, J＝2.1 Hz, H-6),
5.68 (1H, d, J＝7.7 Hz, glucosyl H-1), 4.95 (1H,d, J＝6.5 Hz, arabinosyl H-1), 4.84 (1H, brd,
J＝9.3 Hz, glucosyl H-6a), 4.55 (1H, dd, J＝6.5
and 8.3 Hz, arabinosyl H-2), 4.37 (1H, m, glucosyl H-6b), 4.36 (1H, m, glucosyl H-5), 4.33 (1H,m, glucosyl H-3), 4.32 (1H, m, arabinosyl H-4),
4.31 (1H, m, arabinosyl H-5a), 4.27 (1H, m, glucosyl H-2), 4.25 (1H, m, glucosyl H-4), 4.22
(1H, dd, J＝3.5 and 8.4 Hz, arabinosyl H-3), 3.74
(1H, dd, J＝1.8 and 11.9 Hz, arabinosyl H-5b).
13
C NMR (150 MHz, pyridine-d5): δC 165.5(C-2), 104.1 (C-3), 182.8 (C-4), 162.5 (C-5),
95.4 (C-6), 164.1 (C-7), 100.9 (C-8), 158.0(C-9), 106.8 (C-10), 122.7 (C-1′), 114.8 (C-2′),
147.7 (C-3′), 151.9 (C-4′), 117.0 (C-5′), 120.0(C-6′); (glucose) δC 102.1 (C-1), 74.7 (C-2), 77.6
(C-3), 71.2 (C-4), 79.2 (C-5), 69.4 (C-6); (arabinose) δC 105.3 (C-1), 71.3 (C-2), 74.3 (C-3), 69.0(C-4), 66.2 (C-5).
Luteolin (hydrolysate of 304-2). UV: λmax(nm) MeOH 256, 266, 348; ＋NaOMe 273, 320,404 (inc.); ＋AlCl3 274, 422; ＋AlCl3/HCl
263sh, 275, 294sh, 356, 385sh; ＋NaOAc 270,
394; ＋NaOAc/H3BO3 263, 372, 427sh.
Their samples were isolated from the leaves of
C. buergeri (ca. 10 mg 304-2 and ca. 20 mg

Fig. 3.

Luteolin 7-O-glucoside.

Fig. 4.

Luteolin 7-O-vicianoside.

199

304-5) and characterized as luteolin 7-O-arabinosylglucoside by Mr. Tsutomu Ito (graduation thesis of Tokyo University of Agriculture in 1986).
In the present survey, luteolin, glucose and arabinose were liberated from the original glycosideby acid hydrolysis. Attachment of each 1 mol of
glucose and arabinose to 7-position of luteolin
was conﬁrmed by UV spectral survey according
to Mabry et al. (1970) and LC-MS. Final identiﬁcation of the original glycoside was performed
by NMR. Six aromatic proton signals, δH 8.04,
7.59, 7.37, 7.03, 6.90 and 6.83 corresponding toH-2′, H-6′, H-5′, H-8, H-3 and H-6, which were
determined by COSY, NOESY, HMQC and
HMBC, appeared on 1H NMR. Moreover, twosugar anomeric proton signals, δH 5.68 (glucosylH-1) and 4.95 (arabinosyl H-1) also appeared.
Attachment of glucose to 7-position of luteolin
was conﬁrmed by HMBC correlation of glucosyl
anomeric proton signal at δH 5.68 with δC 164.1
corresponding to C-7. On the other hand, attachment of arabinose to 6-position of glucose wasshown by HMBC correlation of arabinosyl anomeric proton signal at δH 4.95 with δC 69.4 corresponding to C-6 of glucose. Since coupling constants of glucosyl and arabinosyl anomeric
proton signals were J＝7.7 and 6.5 Hz, respec-

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Tsukasa Iwashina et al.

tively, they were determined as β-glucopyranoseand α-arabinopyranose. Thus, the ﬂavonoid was
identiﬁed as luteolin 7-O-α-arabinopyranosyl(1→6)-β-glucopyranoside (luteolin 7-O-vicianoside, Fig. 4). The plants which their ﬂavonoids
were isolated were collected in Mt. Ibuki, Shiga
Pref., Japan by Mr. Tatsuya Ueno in 1986. Flavonoids were reported from C. buergeri for the ﬁrst
time. Although luteolin 7-O-vicianoside has beenfound in Dacrydium spp. (Podocarpaceae)(Markham et al., 1987), it was newly reported
from Cirsium species in this survey.
8. Flavonoids from Cirsium confertissimum
Nakai
Luteolin 7-O-glucoside (138-10, 138-11, 2782, 304-7, 304-9, 304-10). UV: λmax (nm) MeOH
255, 265sh, 348; ＋NaOMe 269, 392 (inc.);＋AlCl3 274, 427; ＋AlCl3/HCl 273, 294sh, 357,
386; ＋NaOAc 260, 402; ＋NaOAc/H3BO3 259,
372. LC-MS: m/z 449 [M＋H]＋, 447 [M-H]−(molecular ion peaks, luteolin＋1 mol glucose),m/z 287 [M-162＋H]＋, 285 [M-162-H]− (fragment ion peaks, luteolin).
The ﬂavonoid was isolated from the leaves of
C. confertissimum which was collected in Mt.
Ibuki, Shiga Prefecture by Mr. Tatsuya Ueno in1986 and crystalized by Mr. Tsutomu Ito as paleyellow needle (ca. 3 mg 138-10, 7 mg 138-11,50 mg 278-2, 10 mg 304-7, 40 mg 304-9 and
15 mg 304-10). Luteolin and glucose were liberated by acid hydrolysis. Attachment of 1 mol glucose to 7-position of luteolin was determined by
LC-MS and UV spectral survey according to
Mabry et al. (1970). Finally, the ﬂavonoid was
identiﬁed as luteolin 7-O-glucoside by HPLC
comparison with authentic sample from Carl
Roth. Although luteolin 7-O-glucoside has beenreported from some Cirsium species, e.g. C. suffultum, C. yakushimense and C. chikushiense(Iwashina et al., 1995), it was found in C. confertissimum for the ﬁrst time.
Luteolin 4′-O-glucoside (141-1). UV: λmax(nm) MeOH 246, 269, 335; ＋NaOMe 267,
295sh, 374 (dec.); ＋AlCl3 259sh, 278, 292sh,
350, 380; ＋AlCl3/HCl 258, 279, 292sh, 342,

Fig. 5.

Luteolin 4′-O-glucoside.

Fig. 6.

Luteolin 3′-O-glucoside.

379sh; ＋NaOAc 270, 376; ＋NaOAc/H3BO3
270, 337. LC-MS: m/z 449 [M＋H]＋, 447[M-H]− (molecular ion peaks, luteolin＋1 mol
glucose).
The sample was obtained from C. confertissimum leaves as pale yellow powder (ca. 3 mg).
Luteolin and glucose were produced by acidhydrolysis. Attachment of 1 mol glucose to luteolin was determined by LC-MS. Since bathochromic shift, and decrease of absorbance of Band I
occurred in addition of NaOMe to MeOH solution in UV spectral survey, the presence of substituted 4′-hydroxyl group was shown. Finally,
the compound was identiﬁed as luteolin
4′-O-glucoside (Fig. 5) by HPLC comparison
with authentic sample from Extrasynthese(France). Luteolin 4′-O-glucoside was found in
Cirsium species for the ﬁrst time.
Luteolin 3′-O-glucoside (148). UV: λmax (nm)
MeOH 239sh, 269, 338; ＋NaOMe 275, 329,
397 (inc.); ＋AlCl3 258, 276, 294sh, 353, 380;
＋AlCl3/HCl 254, 277, 293sh, 346, 377sh;＋NaOAc 274, 331, 396; ＋NaOAc/H3BO3 268,
348. LC-MS: m/z 449 [M＋H]＋, 447 [M-H]−(molecular ion peaks, luteolin＋1 mol glucose).
The sample was obtained by Mr. Tsutomu ItoFurther identiﬁcation of ﬂavonoids from Cirsium spp.

201

as pale yellow powder (ca. 1 mg) in 1986. The
original glycoside produced luteolin and glucose
by acid hydrolysis. In UV spectral survey, thepresence of free 5-, 7- and 4′-hydroxyl groups
was proved by addition of NaOMe, AlCl3, AlCl3/
HCl or NaOAc, showing the attachment of glucose to 3′-position of luteolin. Thus, the ﬂavonoid was characterized as luteolin 3′-O-glucoside(Fig. 6). Luteolin 3′-O-glucoside was newly
reported from Cirsium species. Flavonoids of C.
confertissimum were reported for the ﬁrst time.

nose), m/z 315 [M-308＋H]＋, 313 [M-308-H]−(fragment ion peaks, pectolinarigenin).
The sample was isolated as pale yellow needle
from C. grayanum leaves, which were collected
in 2000 by Dr. Yuichi Kadota (collection site is
unknown), and identiﬁed as pectolinarigenin
7-O-rutinoside by UV, LC-MS and HPLC comparison with 136-10. Flavonoids of C. grayanum
were reported for the ﬁrst time. Minor acacetin
7-O-rutinoside (19.9%) was contained in thesample.

9. Flavonoids from Cirsium esculentum (Sieb.)
C.A.Mey
Pectolinarigenin 7-O-rutinoside (136-22). UV:
λmax (nm) MeOH 273, 329; ＋NaOMe 296, 375(dec.); ＋AlCl3 282sh, 299, 352, 386sh; ＋AlCl3/
HCl 284, 299, 346, 387sh; ＋NaOAc 284, 316sh;＋NaOAc/H3BO3 274, 331. LC-MS: m/z 623[M＋H]＋, 621 [M-H]− (molecular ion peaks,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The sample was obtained from the leaves of C.
esculentum (TNS9522069) which was collected
between Semipalatinsk and Ayaguz, Kazakhstan
in 18 July 1995 by T. Iwashina, as pale yellow
needle (ca. 40 mg). The ﬂavonoid was identiﬁed
as pectolinarigenin 7-O-rutinoside by UV,
LC-MS and HPLC comparison with 136-10.
Although two ﬂavonol glycosides, kaempferol
3-O-rhamnosylglucosideandquercetin
3-O-rutinoside, have been reported (Kozyra etal., 2010; Kasterova et al., 2019), pectolinarigenin 7-O-rutinoside newly found in this survey.
Acacetin 7-O-rutinoside (24.9%) was accompanied with pectolinarin as minor ﬂavonoid.

11. Flavonoids from Cirsium hanamakiense
Koidz.
Pectolinarigenin 7-O-rutinoside (136-17). UV:
λmax (nm) MeOH 273, 328; ＋NaOMe 296, 375(dec.); ＋AlCl3 281sh, 299, 351, 387sh; ＋AlCl3/
HCl 282sh, 299, 343, 386sh; ＋NaOAc 278, 325;
＋NaOAc/H3BO3 273, 331. LC-MS: m/z 623[M＋H]＋, 645 [M＋H＋Na]＋, 621 [M-H]−(molecular ion peaks, pectolinarigenin＋each
1 mol of glucose and rhamnose), m/z 477[M-146＋H]＋ (fragment ion peak, pectolinarigenin＋1 mol glucose), and m/z 315 [M-308＋H]＋, 313 [M-308-H]− (fragment ion peaks,
pectolinarigenin).
The sample was isolated as pale yellow needle(ca. 30 mg) from the leaves of C. hanamakiense
which was collected by Dr. Yuichi Kadota in
1996 (collection site is unknown). It was identiﬁed as pectolinarigenin 7-O-rutinoside by UVproperties, LC-MS and HPLC comparison withthe sample 136-10 from C. brevicaule. The ﬂavonoids of C. hanamakiense were reported for theﬁrst time. It was shown by HPLC that linarin is
contained in the sample as minor ﬂavonoid(27.6%).

10. Flavonoids from Cirsium grayanum (Maxim.)
Nakai
Pectolinarigenin 7-O-rutinoside (136-28). UV:
λmax (nm) MeOH 274, 328; ＋NaOMe 296, 377(dec.); ＋AlCl3 284sh, 300, 353, 388sh; ＋AlCl3/
HCl 284sh, 298, 345, 386sh; ＋NaOAc 280, 322;
＋NaOAc/H3BO3 274, 331. LC-MS: m/z 623[M＋H]＋, 621 [M-H]− (molecular ion peaks,
pectolinarigenin＋each 1 mol of glucose and rham-

12. Flavonoids from Cirsium happoense Kadota
Acacetin 7-O-rutinoside (280-1). UV: λmax(nm) MeOH 269, 327; ＋NaOMe 293, 371(dec.); ＋AlCl3 277, 300, 345, 377sh; ＋AlCl3/
HCl 278, 298, 339, 380sh; ＋NaOAc 272, 316;
＋NaOAc/H3BO3 270, 330. LC-MS: m/z 593[M＋H]＋ (molecular ion peak, acacetin＋each
1 mol of glucose and rhamnose), m/z 447 [M-146

202

Tsukasa Iwashina et al.

＋H]＋ (fragment ion peak, acacetin＋1 mol glucose), m/z 285 [M-308＋H]＋ (fragment ion
peak, acacetin).
The sample (ca. 20 mg) was isolated from theleaves of C. happoense which was collected in
Nagano Pref., Japan by Dr. Yuichi Kadota in
1994. The ﬂavonoid was identiﬁed as acacetin
7-O-rutinoside by UV, LC-MS, acid hydrolysis
and HPLC comparison with authentic sample(Carl Roth). Pectolinarigenin 7-O-rutinoside wasaccompanied as minor ﬂavonoid (38.4%).
Although the plant was collected as C. happoense, the species name is not recorded in“Flora of Japan” (Kadota, 1995).
13. Flavonoids from Cirsium hidakamontanum
KadotaPectolinarigenin 7-O-rutinoside (282-6). UV:
λmax (nm) MeOH 275, 327; ＋NaOMe 297, 376(dec.); ＋AlCl3 285sh, 300, 355, 387sh; ＋AlCl3/
HCl 284sh, 298, 344, 386sh; ＋NaOAc
280, 323; ＋NaOAc/H3BO3 273, 330. LC-MS:
m/z 623 [M＋H]＋ (molecular ion peak,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The sample was isolated as pale yellow needle
from the leaves of C. hidakamontanum which
was collected in Mt. Kamui-Ekuuchikaushi,
Hokkaido, Japan by Dr. Yuichi Kadota in 1998.
C. hidakamontanum was recorded as a new Cirsium species by Kadota (1999). The ﬂavonoid
was identiﬁed as pectolinarigenin 7-O-rutinoside
by UV, LC-MS and HPLC comparison with 13610. Acacetin 7-O-rutinoside was accompanied asminor compound (30.5%). Flavonoids werereported from C. hidakamontanum for the ﬁrst
time.
14. Flavonoids from Cirsium inundatum Makino
Pectolinarigenin 7-O-rutinoside (136-12). UV:
λmax (nm) MeOH 274, 328; ＋NaOMe 296, 375(dec.); ＋AlCl3 282sh, 299, 352, 386sh; ＋AlCl3/
HCl 283, 299, 344, 385sh; ＋NaOAc 282, 321;＋NaOAc/H3BO3 273, 333. LC-MS: m/z 623[M＋H]＋, 621 [M-H]− (molecular ion peaks,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The sample was obtained as pale yellow needle (ca. 20 mg) from C. inundatum leaves whichwere collected in 1995 by Dr. Yuichi Kadota(collection site is unknown). Flavonoid was characterized as pectolinarigenin 7-O-rutinoside by
UV, LC-MS and HPLC comparison with 136-10.
Pectolinarigenin 7-O-rutinoside has already beenreported from the species (Nakaoki and Morita,1960). In this survey, acacetin 7-O-rutinoside(31.8%) was newly found as minor compound in
the sample.
15. Flavonoids from Cirsium irumtiense Kitam.
Pectolinarigenin 7-O-rutinoside (136-4). UV:
λmax (nm) MeOH 276, 329; ＋NaOMe 297, 378(dec.); ＋AlCl3 287sh, 300, 354, 387sh; ＋AlCl3/
HCl 284sh, 299, 347, 386sh; ＋NaOAc 283, 319;
＋NaOAc/H3BO3 275, 331. LC-MS: m/z 623[M＋H]＋, 645 [M＋H＋Na]＋ (molecular ion
peaks, pectolinarigenin＋each 1 mol of glucose
and rhamnose), m/z 477 [M-146＋H]＋ (fragment
ion peak, pectolinarigenin＋1 mol glucose), andm/z 315 [M-308＋H]＋, 313 [M-308-H]− (fragment ion peaks, pectolinarigenin).
The sample was isolated as pale yellow needle(ca. 30 mg) from C. irumtiense leaves whichwere collected by Dr. Yuichi Kadota in 1994
(collection site is unknown). Flavonoid was identiﬁed as pectolinarigenin 7-O-rutinoside by UV,
LC-MS, and HPLC comparison with 136-10.
Acacetin 7-O-rutinoside (12.4%) was accompanied as minor ﬂavonoid. Flavonoids werereported from C. irumtiense for the ﬁrst time.
Kadota (1995) described that the species is thesame with C. brevicaule. It was shown by thissurvey that ﬂavonoid composition of the both
species was the same with each other.
16. Flavonoids from Cirsium ishizuchiense(Kitam.) Kadota
Pectolinarigenin 7-O-rutinoside (136-1). UV:
λmax (nm) MeOH 275, 328; ＋NaOMe 296, 378(dec.); ＋AlCl3 286sh, 300, 353, 387sh; ＋AlCl3/
HCl 284sh, 298, 346, 386sh; ＋NaOAc 280, 325;
＋NaOAc/H3BO3 275, 332. LC-MS: m/z 623

Further identiﬁcation of ﬂavonoids from Cirsium spp.

[M＋H]＋, 621 [M-H]− (molecular ion peaks,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The sample (ca. 40 mg) was obtained from C.
ishizuchiense leaves which were collected by Dr.
Yuichi Kadota in 1996 (collection site is
unknown). Flavonoid was identiﬁed as pectolinarigenin 7-O-rutinoside by UV, LC-MS and
HPLC comparison with 136-10. Acacetin
7-O-rutinoside was contained in the sample asminor compound (13.3%). Flavonoids werereported from C. ishizuchiense for the ﬁrst time.
C. ishizuchiense was independent as a species
from a variety, C. tenue Kitam. var. ishizuchiense
Kitam. by Kadota (2000).
17. Flavonoids from Cirsium japonicum Ledeb.
ex DC.
Pectolinarigenin 7-O-rutinoside (136-30, 2903). UV: λmax (nm) MeOH 273, 330; ＋NaOMe
296, 379 (dec.); ＋AlCl3 281, 299, 352, 387sh;＋AlCl3/HCl 280sh, 298, 345, 386sh; ＋NaOAc
284, 314; ＋NaOAc/H3BO3 272, 331. LC-MS:
m/z 623 [M＋H]＋, 645 [M＋H＋Na]＋ (molecular ion peaks, pectolinarigenin＋each 1 mol of
glucose and rhamnose).
Their samples were obtained from C. japonicum leaves which were collected by Mr. Tatsuya
Ueno (136-30) (collection site is unknown) and
Mr. Tsutomu Ito (collection site, Atsugi City,
Kanagawa Prefecture, Japan, 290-3) in 1985 and1987 as pale yellow needle. The ﬂavonoid was
identiﬁed as pectolinarigenin 7-O-rutinoside by
UV, LC-MS and HPLC comparison with 136-10.
Acacetin 7-O-rutinoside (linarin) was accompanied with pectolinarin as minor compound(24.8% in 136-30 and 6.0% in 290-3). Pectolinarin and linarin have already been reported
from the species (Nakaoki and Morita, 1959;
Iwashina et al., 1988, 1995).
Cirsimaritin 4′-O-glucoside (290-2). UV: λmax(nm) MeOH 276, 328; ＋NaOMe 294, 359(dec.); ＋AlCl3 262sh, 288sh, 299, 356; ＋AlCl3/
HCl 259sh, 287sh, 298, 346; ＋NaOAc 277, 326,
385; ＋NaOAc/H3BO3 276, 330. LC-MS: m/z
477 [M＋H]＋ (molecular ion peak, cirsimari-203

tin＋1 mol glucose), m/z 315 [M-162＋H]＋, 313[M-162-H]− (fragment ion peaks, cirsimaritin).
1
H NMR (600 MHz, pyridine-d5): δH 13.55 (1H,s, 5-OH), 7.92 (2H, d, J＝8.9 Hz, H-2′,6′), 7.49
(2H, d, J＝8.9 Hz, H-3′,5′), 6.95 (1H, s, H-3),
6.83 (1H, s, H-8), 5.80 (1H, d, J＝7.2 Hz, glucosyl H-1), 4.65 (1H, dd, J＝2.1 and 12.1 Hz, glucosyl H-6a), 4.43 (1H, m, glucosyl H-3), 4.42
(1H, m, glucosyl H-2), 4.40 (1H, m, glucosylH-6b), 4.35 (1H, t, J＝8.6 Hz, glucosyl H-4),
4.24 (1H, m, glucosyl H-5), 4.02 (3H, s,
6-OCH3), 3.94 (3H, s, 7-OCH3). 13C NMR(150 MHz, pyridine-d5): (cirsimaritin) δC 153.5(C-2), 104.7 (C-3), 183.2 (C-4), 164.1 (C-5),
133.2 (C-6), 159.5 (C-7), 91.7 (C-8), 153.7(C-9), 106.4 (C-10), 125.0 (C-1′), 129.0 (C-2′,6′),
117.0 (C-3′,5′), 161.4 (C-4′), 60.6 (7-OCH3),
56.5 (6-OCH3); (glucose) δC 101.8 (C-1), 74.9
(C-2), 78.6 (C-3), 71.4 (C-4), 79.3 (C-5), 62.5
(C-6).
Cirsimaritin (hydrolysate of 290-2). UV: λmax(nm) MeOH 275, 333; ＋NaOMe 278, 302, 357(inc.); ＋AlCl3 285sh, 301, 358, 387sh; ＋AlCl3/
HCl 286sh, 300, 351, 386sh; ＋NaOAc 272, 389;
＋NaOAc/H3BO3 275, 336.
The sample was obtained as pale yellow powder (ca. 40 mg) from the leaves of C. japonicum
which was collected in Atsugi City by Mr. Tsutomu Ito in 1986. The presence of free
5-hydroxyl, and substituted 7- and 4′-hydroxyl
groups was shown by UV spectral properties. An
aglycone and glucose were liberated by acidhydrolysis of the original glycoside. Attachmentof 1 mol glucose to dihydroxy-dimethoxyﬂavone
was proved by appearance of the molecular ion
peak, m/z 477 [M＋H]＋ and fragment ion peaks,m/z 315 [M-162＋H]＋ and 313 [M-162-H]−. In
1
H NMR, four aromatic proton signals, δH 7.92
(H-2′,6′), 7.49 (H-3′,5′), 6.95 (H-3) and 6.83
(H-8) appeared, together with glucosyl anomeric
proton signal, δH 5.80 (d, J＝7.2 Hz) and two
methoxyl proton signals, δH 4.02 and 3.94.
Attachment of glucose to 7-position of ﬂavone
was shown by HMBC correlation of anomeric
proton signal δH 5.80 with δC 161.4 corresponding to C-4′. On the other hand, two methoxyl

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Tsukasa Iwashina et al.

Fig. 7.

Cirsimaritin 4′-O-glucoside.

proton signals, δH 4.02 and 3.94 correlated with
δC 133.2 and 159.5 corresponding to C-6 andC-7. Thus, this was identiﬁed as 5,4′-dihydroxy6,7-dimethoxyﬂavone 4′-O-β-D-glucopyranoside(cirsimaritin 4′-O-glucoside, Fig. 7). Cirsimaritin
4′-O-glucoside has been reported in Cirsium
maritimum (Morita and Shimizu, 1963) and C.
kamtschaticum (Iwashina et al., 1988). It was
found in C. japonicum for the ﬁrst time.
18. Flavonoids from Cirsium japonicum Fisch.
ex DC. var. diabolicum (Kitam.) Kitam.
Pectolinarigenin 7-O-rutinoside (136-31). UV:
λmax (nm) MeOH 274, 329; ＋NaOMe 296,375 (dec.); ＋AlCl3 282, 300, 352, 386sh;＋AlCl3/HCl 282, 298, 346, 386sh; ＋NaOAc
280, 319; ＋NaOAc/H3BO3 272, 331. LC-MS:
m/z 623 [M＋H]＋ (molecular ion peak,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The sample was obtained as pale yellow needle from C. japonicum var. diabolicum which
was collected in Toyama Prefecture, Japan by Dr.
Yuichi Kadota in 1994. The ﬂavonoid was identiﬁed as pectolinarigenin 7-O-rutinoside by UV,
LC-MS and HPLC comparison with the sample
136-10. Acacetin 7-O-rutinoside (27.8%) was
accompanied in the sample as minor compound.
Flavonoids of C. japonicum var. diabolicum werereported for the ﬁrst time.
19. Flavonoids from Cirsium kagamontanum
Nakai
Pectolinarigenin 7-O-rutinoside (279-5). UV:
λmax (nm) MeOH 276, 329; ＋NaOMe 296, 377(dec.); ＋AlCl3 286sh, 300, 353, 387sh; ＋AlCl3/
HCl 285sh, 298, 345, 386sh; ＋NaOAc 286, 320;
＋NaOAc/H3BO3 275, 331. LC-MS: m/z 623[M＋H]＋, 621 [M-H]−, 645 [M＋H＋Na]＋,

Fig. 8.

Luteolin 5-O-glucoside.

(molecular ion peaks, pectolinarigenin＋each
1 mol of glucose and rhamnose), m/z 315 [M-308＋H]＋ (fragment ion peak, pectolinarigenin).
The ﬂavonoid was obtained as pale yellow
needle (ca. 80 mg) from the leaves of C. kagamontanum which was collected by Dr. Yuichi
Kadota (collection site is unknown). It was identiﬁed as pectolinarigenin 7-O-rutinoside by UV,
LC-MS and HPLC comparison with 136-10.
Acacetin 7-O-rutinoside was contained as minor
ﬂavonoid (18.0%).
Luteolin 5-O-glucoside (279-8, 279-9). UV:
λmax (nm) MeOH 240, 247sh, 258sh, 342;
＋NaOMe 260, 312, 402 (inc.); ＋AlCl3 265,
390; ＋AlCl3/HCl 261, 350; ＋NaOAc 260,276sh, 319, 388; ＋NaOAc/H3BO3 252, 260sh,
305, 363. LC-MS: m/z 447 [M-H]−, 471[M＋H＋Na]＋ (molecular ion peaks, luteolin＋1 mol glucose).
Their samples were isolated by Mr. Tsutomu
Ito from C. kagamontanum leaves which werecollected in Mts. Hira, Shiga Pref., Japan by Mr.
Tatsuya Ueno, and identiﬁed as luteolin 5-O-glucoside (galuteolin, Fig. 8) by UV, LC-MS, acidhydrolysis and HPLC comparison with 308-2.
Pectolinarigenin 7-O-rutinoside has beenreported from the species (Nakaoki and Morita,1960). Acacetin 7-O-rutinoside was found in thespecies for the ﬁrst time. In this survey, sinceluteolin 5-O-glucoside, and pectolinarigenin andacacetin 7-O-rutinosides were obtained from two
individuals, respectively, the occurrence of geographic ﬂavonoid variation was presumed in thespecies.

Further identiﬁcation of ﬂavonoids from Cirsium spp.

20. Flavonoids from Cirsium kamtschaticum
Ledeb. ex DC.
Pectolinarigenin 7-O-rutinoside (317-2). UV:
λmax (nm) MeOH 273, 329; ＋NaOMe 296, 376(dec.); ＋AlCl3 280sh, 300, 351, 387sh; ＋AlCl3/
HCl 282sh, 298, 344, 385sh; ＋NaOAc 282, 321;＋NaOAc/H3BO3 273, 331. LC-MS: m/z 623[M＋H]＋, 645 [M＋H＋Na]＋ (molecular ion
peaks, pectolinarigenin＋each 1 mol of glucose
and rhamnose), m/z 313 [M-308-H]− (fragment
ion peak, pectolinarigenin).
The ﬂavonoid was isolated as pale yellow needle (ca. 20 mg) from the leaves of C. kamtschaticum and identiﬁed as pectolinarigenin 7-O-rutinoside by UV, LC-MS and HPLC comparison
with 136-10. Acacetin 7-O-rutinoside wasaccompanied as minor ﬂavonoid (31.3%). Pectolinaigenin and acacetin 7-O-rutinosides havebeen reported from the species (Morita et al.,1973). Additionally, cirsimaritin 4′-O-glucosidehas also been found in C. kamtschaticum (Iwashina et al., 1988).
21. Flavonoid from Cirsium longe-pedunculatum
Kitam.
Luteolin 5-O-glucoside (308-2). UV: λmax(nm) MeOH 240, 248sh, 258sh, 343; ＋NaOMe
260, 311, 406 (inc.); ＋AlCl3 264, 396; ＋AlCl3/
HCl 261, 297, 351; ＋NaOAc 260, 317, 387;
＋NaOAc/H3BO3 251sh, 259sh, 305, 364.
LC-MS: m/z 449 [M＋H]＋, 447 [M-H]− (molecular ion peaks, luteolin＋1 mol glucose), m/z 287[M-162＋H]＋ (fragment ion peak, luteolin). 1H
NMR (600 MHz, pyridine-d5): δH 7.90 (1H, d,
J＝1.7 Hz, H-2′), 7.52 (1H, dd, J＝2.2 and8.3 Hz, H-6′), 7.49 (1H, d, J＝2.2 Hz, H-6), 7.30
(1H, d, J＝8.3 Hz, H-5′), 6.96 (1H, d, J＝2.0 Hz,
H-8), 6.90 (1H, s, H-3), 5.38 (1H, d, J＝7.7 Hz,
glucosyl H-1), 4.44 (1H, m, glucosyl H-2), 4.41
(1H, m, glucosyl H-6a), 4.38 (1H, m, glucosylH-3), 4.36 (1H, m, glucosyl H-6b), 4.35 (1H, m,
glucosyl H-4), 4.04 (1H, m, glucosyl H-5). 13C
NMR (150 MHz, pyridine-d5): (luteolin) δC 162.5(C-2), 107.0 (C-3), 182.8 (C-4), 160.2 (C-5),
104.0 (C-6), 164.3 (C-7), 99.5 (C-8), 158.5(C-9), 109.6 (C-10), 123.0 (C-1′), 114.4 (C-2′),

205

147.7 (C-3′), 151.3 (C-4′), 116.9 (C-5′), 119.6(C-6′); (glucose) δC 106.7 (C-1), 75.4 (C-2), 77.6
(C-3), 71.2 (C-4), 79.3 (C-5), 62.5 (C-6).
Luteolin and glucose were liberated by acidhydrolysis of the original glycoside. Attachmentof 1 mol glucose to luteolin was shown by
LC-MS (appearance of molecular ion peaks, m/z
449 [M＋H]＋ and 447 [M-H]−, and fragment
ion peak, m/z 287 [M-162＋H]＋). In 1H NMR,
six aromatic proton signals, δH 7.90 (H-2′), 7.52
(H-6′), 7.49 (H-6), 7.30 (H-5′), 6.96 (H-8) and6.90 (H-3), appeared together with glucosyl anomeric proton signal δH 5.38 (d, J＝7.7 Hz).
Attachment of glucose to 5-position of luteolin
was shown by HMBC correlation of anomeric
proton signal δH 5.38 with C-5 of luteolin at δC
160.2. Thus, the glycoside was identiﬁed as luteolin 5-O-β-D-glucopyranoside, and has beenreported from C. longe-pedunculatum (Iwashina
et al., 1989).
22. Flavonoid from Cirsium nambuense Nakai
Pectolinarigenin 7-O-rutinoside (287). UV:
λmax (nm) MeOH 276, 328; ＋NaOMe 296, 376(dec.); ＋AlCl3 284sh, 300, 353, 387sh; ＋AlCl3/
HCl 283sh, 298, 346, 387sh; ＋NaOAc 282,
320; ＋NaOAc/H3BO3 274, 331. LC-MS:
m/z 623 [M＋H]＋ (molecular ion peak,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The sample was obtained as pale yellow needle (ca. 3 mg) from the leaves of C. nambuense.
The plants were collected by Dr. Yuichi Kadota(collection site is unknown). The ﬂavonoid wascharacterized as pectolinarigenin 7-O-rutinoside
by UV, LC-MS and comparison with 136-10 by
HPLC. Minor ﬂavonoid was accompanied with
pectolinarin and identiﬁed as acacetin 7-O-rutinoside (30.2%) by HPLC comparison withauthentic sample. Flavonoids of C. nambuense
were reported in this survey for the ﬁrst time.
23. Flavonoid from Cirsium norikurense Nakai
Acacetin 7-O-rutinoside (310-1, 310-2). UV:
λmax (nm) MeOH 268, 327; ＋NaOMe 288, 371(dec.); ＋AlCl3 276, 300, 343, 382; ＋AlCl3/HCl

206

Tsukasa Iwashina et al.

277, 299, 337, 381; ＋NaOAc 269, 318;
＋NaOAc/H3BO3 268, 333. LC-MS: m/z 593[M＋H]＋ (molecular ion peak, acacetin＋each
1 mol of glucose and rhamnose), m/z 447 [M-146＋H]＋ (fragment ion peak, acacetin＋1 mol glucose), m/z 285 [M-308＋H]＋, 283 [M-308-H]−(fragment ion peak, acacetin).
The ﬂavonoids (ca. 40 mg 310-1 and ca. 1 mg
310-2) were isolated from C. norikurense by Mr.
Tsutomu Ito. The leaves of the species were collected in Izumi Village, Fukui Pref., Japan by Mr.
Tatsuya Ueno. The ﬂavonoid was identiﬁed asacacetin 7-O-rutinoside by UV, LC-MS and
HPLC comparison with authentic sample (CarlRoth). Flavonoid of C. norikurense was found
for the ﬁrst time in this survey.
24. Flavonoid from Cirsium occidentalinipponense Kadota
Luteolin 7-O-glucoside (303). UV: λmax (nm)
MeOH 255, 266sh, 346; ＋NaOMe 270, 394(inc.); ＋AlCl3 274, 425; ＋AlCl3/HCl 272,
295sh, 355, 383; ＋NaOAc 260, 402; ＋NaOAc/
H3BO3 256, 372. LC-MS: m/z 449 [M＋H]＋,
447 [M-H]− (molecular ion peaks, luteolin＋1 mol glucose).
The sample was obtained from the leaves of C.
occidentalinipponense which was collected in
Mt. Sanʼnomine, Fukui Pref., Japan by Dr. Yuichi
Kadota in 1996. The species was recorded as a
new Cirsium species by Kadota (1997). The ﬂavonoid was identiﬁed as luteolin 7-O-glucoside
by UV, LC-MS and comparison with authentic
sample (Carl Roth) by HPLC. Flavonoid wasreported from the species for the ﬁrst time.
25. Flavonoids from Cirsium oligophyllum(Franch. et Sav.) Matsum.
Pectolinarigenin 7-O-rutinoside (136-3). UV:
λmax (nm) MeOH 273, 329; ＋NaOMe 296,375 (dec.); ＋AlCl3 281sh, 299, 353, 387sh;＋AlCl3/HCl 285, 299, 343, 386sh; ＋NaOAc
277, 324; ＋NaOAc/H3BO3 275, 332. LC-MS:
m/z 623 [M＋H]＋ (molecular ion peak,
pectolinarigenin＋each 1 mol of glucose andrhamnose).

The sample was isolated as pale yellow needle(ca. 10 mg) from C. oligophyllum which was collected in 1993 by Dr. Yuichi Kadota (collection
site is unknown). Flavonoid was identiﬁed as
pectolinarigenin 7-O-rutinoside by UV, LC-MS
and HPLC comparison with 136-10. Acacetin
7-O-rutinoside was contained in the sample asminor compound (26.5%). Nepetin and hispidulin 4′-O-glucosides have been reported from thespecies (Iwashina et al., 1999). However, pectolinaigenin 7-O-rutinoside was newly isolated.
26. Flavonoids from Cirsium otayae Kitam.
Pectolinarigenin 7-O-rutinoside (281-4). UV:
λmax (nm) MeOH 275, 328; ＋NaOMe 296, 377(dec.); ＋AlCl3 285sh, 300, 353, 390sh; ＋AlCl3/
HCl 284sh, 298, 346, 387sh; ＋NaOAc 277, 325;
＋NaOAc/H3BO3 275, 331. LC-MS: m/z 623[M＋H]＋, 645 [M＋H＋Na]＋ (molecular ion
peaks, pectolinarigenin＋each 1 mol of glucose
and rhamnose).
The sample was isolated by Mr. Tsutomu Itofrom the leaves of C. otayae which was collectedin Mt. Norikura, Nagano Pref., Japan by Mr. Tatsuya Ueno. The ﬂavonoid was identiﬁed as pectolinarigenin 7-O-rutinoside by UV, LC-MS and
HPLC comparison with 136-10. Acacetin
7-O-rutinoside was contained as minor ﬂavonoid(11.8%). The ﬂavonoids of C. otayae werereported in this survey for the ﬁrst time.
27. Flavonoid from Cirsium pectinellum A. Gray
Cirsimaritin 4′-O-glucoside (134). UV: λmax(nm) MeOH 277, 325; ＋NaOMe 294, 374(dec.); ＋AlCl3 262, 291sh, 297, 351, 389sh;＋AlCl3/HCl 261, 288sh, 295, 343, 387sh;＋NaOAc 278, 324; ＋NaOAc/H3BO3 276, 327.
LC-MS: m/z 477 [M＋H]＋ (molecular ion peak,cirsimaritin＋1 mol glucose), m/z 315 [M-162＋H]＋, 313 [M-162-H]− (fragment ion peaks,
cirsimaritin).
The ﬂavonoid was isolated by Mr. Tsutomu Itofrom C. pectinellum leaves which were collectedin Rikubetsu Town, Hokkaido, Japan by one of
the authors (T. Iwashina) in 1985. The compound
was identiﬁed as cirsimaritin 4′-O-glucoside byFurther identiﬁcation of ﬂavonoids from Cirsium spp.

UV, LC-MS and HPLC comparison with 290-2.
Although pectolinarigenin 7-O-rutinoside andacacetin 7-O-rutinoside have been reported fromC. pectinellum (Morita et al., 1973; Iwashina etal., 1988), cirsimaritin 4′-O-glucoside was found
for the ﬁrst time in this survey.
28. Flavonoids from Cirsium pendulum Fisch. ex
DC.
Pectolinarigenin 7-O-rutinoside (288, 298-1).
UV: λmax (nm) MeOH 275, 327; ＋NaOMe 297,378 (dec.); ＋AlCl3 286sh, 300, 354, 392sh;＋AlCl3/HCl 285sh, 298, 344, 387sh; ＋NaOAc
280, 324; ＋NaOAc/H3BO3 275, 330. LC-MS:
m/z 623 [M＋H]＋ (molecular ion peak,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
Of two samples which are deposited in the
National Museum of Nature and Science, 288
was isolated as pale yellow needle (ca. 5 mg)from the leaves of C. pendulum. The plants werecollected by Dr. Yuichi Kadota in 1998 (collection site is unknown). On the other hand, originof 298-1was unknown (may be from Dr. Kozo
Hayashi). Both samples were identiﬁed as pectolinarigenin 7-O-rutinoside by UV, LC-MS andcomparison with 136-10 by HPLC. Acacetin
7-O-rutinoside was accompanied in their samples(31.7% in 288 and 23.7% in 298-1). Flavonoids
were reported from C. pendulum for the ﬁrst
time.
29. Flavonoid from Cirsium purpuratum (Maxim.)
Matsum.
Acacetin 7-O-rutinoside (305). UV: λmax(nm) MeOH 268, 326; ＋NaOMe 286, 375(dec.); ＋AlCl3 275, 299, 345, 375sh; ＋AlCl3/
HCl 278, 299, 336, 378sh; ＋NaOAc 269, 322;
＋NaOAc/H3BO3 266, 341. LC-MS: m/z 593[M＋H]＋ (molecular ion peak, acacetin＋each
1 mol of glucose and rhamnose), m/z 285 [M-308＋H]＋ (fragment ion peak, acacetin).
The sample was isolated from the leaves of C.
purpuratum as powder (ca. 30 mg) by Mr. Tsutomu Ito. The plants were collected in Izumi Village, Fukui Pref. by Mr. Tatsuya Ueno in 1986.

207

The ﬂavonoid was identiﬁed as acacetin
7-O-rutinoside by UV, LC-MS and HPLC comparison with authentic sample (Carl Roth). Acacetin 7-O-rutinoside has already been reported fromthe species (Nakaoki and Morita, 1959).
30. Flavonoids from Cirsium spinosum Kitam.
Pectolinarigenin 7-O-rutinoside (136-24). UV:
λmax (nm) MeOH 275, 329; ＋NaOMe 297, 380(dec.); ＋AlCl3 284sh, 300, 353, 387sh;＋AlCl3/HCl 285, 299, 346, 386sh; ＋NaOAc
280, 326; ＋NaOAc/H3BO3 274, 332. LC-MS:
m/z 623 [M＋H]＋ (molecular ion peak,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The sample was isolated from C. spinosum
leaves which were collected in 1999 by Dr.
Yuichi Kadota (collection site is unknown). Flavonoid was identiﬁed as pectolinarigenin
7-O-rutinoside by UV, LC-MS and HPLC comparison with 136-10. Flavonoid of acacetin
7-O-rutinoside type has been reported from C.
spinosum (Morita et al., 1964). However, acacetin 7-O-rutinoside was shown to be minor ﬂavonoid (17.0%) by HPLC survey. Instead, pectolinarigenin 7-O-rutinoside was found as major
compound in this survey.
31. Flavonoids from Cirsium suﬀultum Nakai
Pectolinarigenin 7-O-rutinoside (136-21). UV:
λmax (nm) MeOH 276, 328; ＋NaOMe 297, 378(dec.); ＋AlCl3 287sh, 299, 355, 387sh; ＋AlCl3/
HCl 286sh, 298, 346, 386sh; ＋NaOAc
285, 317sh; ＋NaOAc/H3BO3 275, 331. LC-MS:
m/z 623 [M＋H]＋ (molecular ion peak,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The sample was obtained as pale yellow needle (ca. 40 mg) from the leaves of C. suﬀultum
which was collected in 1996 by Dr. Yuichi
Kadota (collection site is unknown). Flavonoid
was identiﬁed as pectolinarigenin 7-O-rutinoside
by UV, LC-MS and HPLC comparison with 13610. Acacetin 7-O-rutinoside (15.4%) was accompanied with pectolinarin as minor ﬂavonoid.
Although luteolin 7-O-glucoside has been

208

Tsukasa Iwashina et al.

reported from the species (Iwashina et al., 1995),
pectolinarigenin 7-O-rutinoside was found in thissurvey for the ﬁrst time.
32. Flavonoid from Cirsium suzukaense Kitam.
Luteolin 7-O-glucoside (309-3, 309-4, 309-5).
UV: λmax (nm) MeOH 255, 267sh, 350;
＋NaOMe 271, 397 (inc.); ＋AlCl3 273, 424;
＋AlCl3/HCl 270, 294sh, 358, 382; ＋NaOAc
260, 405; ＋NaOAc/H3BO3 259, 372. LC-MS:
m/z 449 [M＋H]＋, 447 [M-H]− (molecular ion
peaks, luteolin＋1 mol glucose), m/z 287 [M-162＋H]＋ (fragment ion peak, luteolin).
Luteolin (hydrolysate of 309-3, 309-5) (309-1,
309-2). UV: λmax (nm) MeOH 254, 266, 349;
＋NaOMe 272, 409 (inc.); ＋AlCl3 273, 422;
＋AlCl3/HCl 261sh, 273, 294sh, 355, 384sh;＋NaOAc 267, 396; ＋NaOAc/H3BO3 260, 373,
427sh. LC-MS: m/z 287 [M＋H]＋, 285 [M-H]−(molecular ion peaks, tetrahydroxyﬂavone).
Their samples (309-3, 309-4, 309-5) wereobtained as pale yellow needles from the leaves ofthree individuals of C. suzukaense which was collected in Mt. Ryozen, Shiga Pref., Japan by Mr.
Tatsuya Ueno in 1986. Luteolin (309-1, 309-2)
and glucose were liberated by acid hydrolysis.
The original glycoside was identiﬁed as luteolin
7-O-glucoside by acid hydrolysis, UV, LC-MS
and comparison with authentic sample (CarlRoth) by HPLC. Flavonoid was reported from C.
suzukaense for the ﬁrst time.
33. Flavonoids from Cirsium tashiroi Kitam.
Pectolinarigenin 7-O-rutinoside (136-2). UV:
λmax (nm) MeOH 275, 328; ＋NaOMe 297,377 (dec.); ＋AlCl3 287sh, 299, 354, 387sh;＋AlCl3/HCl 284, 297, 345, 386sh; ＋NaOAc
280, 319; ＋NaOAc/H3BO3 274, 332. LC-MS:
m/z 623 [M＋H]＋ (molecular ion peak,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The sample was isolated as pale yellow needle(ca. 8 mg) from C. tashiroi leaves which werecollected in Mt. Ibuki, Shiga Prefecture by Mr.
Tatsuya Ueno in 1991. The compound was identiﬁed as pectolinarigenin 7-O-rutinoside by UV,

LC-MS and HPLC comparison with 136-10.
Acacetin 7-O-rutinoside was accompanied asminor ﬂavonoid (17.9%).
Hispidulin 7-O-neohesperidoside (122). UV:
λmax (nm) MeOH 275, 332; ＋NaOMe 278, 357(inc.); ＋AlCl3 284, 300, 359, 386sh; ＋AlCl3/
HCl 286, 298, 351, 384sh; ＋NaOAc 272, 390;
＋NaOAc/H3BO3 275, 336. LC-MS: m/z 631[M＋H＋Na]＋, 607 [M-H]− (molecular ion
peaks, hispidulin＋each 1 mol of glucose andrhamnose), m/z 301 [M-308＋H]＋ (fragment ion
peak, hispidulin). 1H NMR (600 MHz, pyridined5): δH 7.89 (2H, d, J＝8.8 Hz, H-2′,6′), 7.26 (1H,s, H-8), 7.21 (2H, d, J＝8.8 Hz, H-3′,5′), 6.90
(1H, s, H-3), 6.57 (1H, d, J＝1.1 Hz, rhamnosylH-1), 5.91 (1H, d, J＝7.7 Hz, glucosyl H-1), 4.83
(1H, dd, J＝5.7 and 9.4 Hz, rhamnosyl H-5),
4.83 (1H, dd, J＝6.5 and 9.4 Hz, rhamnosyl H-3),
4.64 (1H, dd, J＝7.8 and 8.9 Hz, glucosyl H-2),
4.60 (1H, dd, J＝3.4 and 9.4 Hz, rhamnosyl H-2),
4.55 (1H, dd, J＝2.0 and 12.2 Hz, glucosylH-6a), 4.44 (1H, t, J＝8.9 Hz, glucosyl H-3),
4.34 (1H, t, J＝9.4 Hz, rhamnosyl H-4), 4.31
(1H, dd, J＝5.6 and 11.7 Hz, glucosyl H-6b),4.23 (1H, t, J＝8.9 Hz, glucosyl H-4), 4.20 (3H,s, 6-OCH3), 4.18 (1H, m, glucosyl H-5), 1.78
(3H, d, J＝6.1 Hz, rhamnosyl CH3). 13C NMR(150 MHz, pyridine-d5): (hispidulin) δC 154.0(C-2), 103.5 (C-3), 183.1 (C-4), 165.0 (C-5),
134.1 (C-6), 156.7 (C-7), 95.2 (C-8), 152.8(C-9), 107.0 (C-10), 122.1 (C-1′), 128.9 (C-2′,6′),
116.9 (C-3′,5′), 162.8 (C-4′), 61.0 (6-OCH3);(glucose) δC 100.0 (C-1), 77.1 (C-2), 79.5 (C-3),
71.3 (C-4), 79.2 (C-5), 62.3 (C-6); (rhamnose) δC
101.8 (C-1), 72.5 (C-2), 72.6 (C-3), 74.1 (C-4),
70.1 (C-5), 19.0 (C-6).
Hispidulin (hydrolysate of 122) (128-1). UV:
λmax (nm) MeOH 274, 333; ＋NaOMe 276,
324, 394 (inc.); ＋AlCl3 283sh, 302, 358, 387sh;＋AlCl3/HCl 287sh, 299, 350, 387sh; ＋NaOAc
275, 311, 331, 389; ＋NaOAc/H3BO3 276, 339.
LC-MS: m/z 301 [M＋H]＋, 299 [M-H]− (molecular ion peaks, trihydroxy-monomethoxyﬂavone).
The sample was obtained as pale yellow powder (ca. 5 mg) from C. tashiroi leaves which wereFurther identiﬁcation of ﬂavonoids from Cirsium spp.

209

hesperidoside were isolated, respectively, werecollected from diﬀerent populations, the occurrence of chemical geographic variation was presumed.

Fig. 9.

Hispidulin 7-O-neohesperidoside.

collected in another population of Mt. Ibuki by
Mr. Tatsuya Ueno in 1992. Attachment of each
1 mol of hexose and rhamnose to trihydroxymonomethoxyﬂavone was shown by LC-MS. An
aglycone, glucose and rhamnose were liberated
by acid hydrolysis of the original glycoside. The
presence of free 5- and 4′-hydroxyl groups wasproved by UV spectral survey according to
Mabry et al. (1970). In 1H NMR, four aromatic
proton signals, δH 7.89 (H-2′,6′), 7.21 (H-3′,5′),
7.26 (H-8) and 6.90 (H-3) appeared, together
with two anomeric proton signals, δH 6.57 (d,J＝1.1 Hz) and 5.91 (d, J＝7.7 Hz) corresponding to rhamnosyl and glucosyl H-1, and amethoxyl proton signal δH 4.20. Attachment ofmethoxyl group to 6-position was shown by
HMBC correlation of δH 4.20 with the carbon
signal at δC 134.1 corresponding to C-6. On theother hand, attachment of glucose to 7-position of
hispidulin was proved by HMBC correlation ofanomeric proton signal (δH 5.91) with the carbon
signal at δC 156.7 corresponding to C-7. Moreover,rhamnosyl anomeric proton signal (δH 6.57) correlated with carbon signal at δC 77.1 corresponding
to C-2 of glucose. Thus, the ﬂavonoid was identiﬁed as 5,7,4′-trihydroxy-6-methoxyﬂavone 7-O-αL-rhamnopyranosyl-(1→2)-β-D-glucopyranoside(hispidulin 7-O-neohesperidoside, Fig. 9). Hispidulin 7-O-neohesperidoside has been reported
from the ﬂowers of Ipomoea purpurea Roth (Convolvulaceae) for the ﬁrst time (Ragunathan and
Sulochana, 1994). Although the glycoside has
been reported from the whole plants of Cirsium
japonicum var. ussuriense (Park et al., 1995), it
was newly found in Japanese Cirsium species.
Flavonoids of C. tashiroi were reported for theﬁrst time. Since two plant materials, which pectolinarigenin 7-O-rutinoside and hispidulin 7-O-neo-

34. Flavonoids from Cirsium magofukui×C.
microspicatum var. kiotoense
Pectolinarigenin 7-O-rutinoside (136-5). UV:
λmax (nm) MeOH 276, 329; ＋NaOMe 297, 376(dec.); ＋AlCl3 285sh, 299, 353, 387sh; ＋AlCl3/
HCl 284sh, 298, 347, 387sh; ＋NaOAc 281, 324;
＋NaOAc/H3BO3 274, 331. LC-MS: m/z 623[M＋H]＋, 645 [M＋H＋Na]＋ (molecular ion
peaks, pectolinarigenin＋each 1 mol of glucose
and rhamnose).
The sample was isolated from the leaves of C.
magofukui×C. microspicatum var. kiotoense as
pale yellow needle (ca. 90 mg). The plants werecollected in 1992 and identiﬁed by Dr. Yuichi
Kadota (collection site is unknown). The ﬂavonoid was characterized as pectolinarigenin
7-O-rutinoside by UV, LC-MS and HPLC comparison with 136-10. Trace of acacetin 7-O-rutinoside was accompanied as minor compound(3.3%).
35. Flavonoid from Cirsium microspicatum×C.
norikurense
Pectolinarigenin 7-O-rutinoside (284-12). UV:
λmax (nm) MeOH 276, 328; ＋NaOMe 296,377 (dec.); ＋AlCl3 286sh, 299, 354, 390sh;＋AlCl3/HCl 286sh, 299, 345, 387sh; ＋NaOAc
280, 324; ＋NaOAc/H3BO3 276, 331. LC-MS:
m/z 623 [M＋H]＋ (molecular ion peak,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The ﬂavonoid was isolated from the leaves of
C. microspicatum×C. norikurense as pale yellow needle (ca. 15 mg). The plants were collected
and identiﬁed by Dr. Yuichi Kadota. The ﬂavonoid was identiﬁed as pectolinarigenin 7-O-rutinoside by UV, LC-MS and HPLC comparison
with 136-10.

210

Tsukasa Iwashina et al.

36. Flavonoids from Cirsium yezoense×C.
magofukui
Pectolinarigenin 7-O-rutinoside (285-2). UV:
λmax (nm) MeOH 276, 328; ＋NaOMe 296, 376(dec.); ＋AlCl3 286sh, 300, 354, 389sh; ＋AlCl3/
HCl 284sh, 298, 345, 387sh; ＋NaOAc
281, 323; ＋NaOAc/H3BO3 275, 331. LC-MS:
m/z 623 [M＋H]＋ (molecular ion peak,
pectolinarigenin＋each 1 mol of glucose andrhamnose).
The ﬂavonoid was isolated from C.
yezoense×C. magofukui leaves. The species wascollected and identiﬁed by Dr. Yuichi Kadota(collection site is unknown). The sample was
identiﬁed as pectolinarigenin 7-O-rutinoside by
UV, LC-MS and comparison with 136-10 by
HPLC. Acacetin 7-O-rutinoside was contained asminor ﬂavonoid (12.9%).
37. Flavonoids from Cirsium sp.
Pectolinarigenin 7-O-rutinoside and acacetin
7-O-rutinoside (278-1). UV: λmax (nm) MeOH
272, 327; ＋NaOMe 294, 373 (dec.); ＋AlCl3
279, 300, 352, 387sh; ＋AlCl3/HCl 279sh, 298,
342, 384sh; ＋NaOAc 274, 324; ＋NaOAc/
H3BO3 272, 330. LC-MS: (major ﬂavonoid) m/z
623 [M＋H]＋, 621 [M-H]− (molecular ion
peaks, pectolinarigenin＋each 1 mol of glucose
and rhamnose), m/z 313 [M-308-H]− (fragment
ion peak, pectolinarigenin). LC-MS: (minor ﬂavonoid) m/z 593 [M＋H]＋ (molecular ion peak,acacetin＋each 1 mol of glucose and rhamnose),m/z 285 [M-308-H]− (fragment ion peak, acacetin)
The ﬂavonoids were isolated as pale yellow
needle from the leaves of Cirsium sp. which wascollected in Rikubetsu Town, Hokkaido, Japanby one of the authors (T. Iwashina). The crystal
was the mixture of two ﬂavonoids and identiﬁed
as pectolinarigenin 7-O-rutinoside (62.8%) andacacetin 7-O-rutinoside (37.2%), by UV, LC-MS
and HPLC comparisons with authentic samples.
38. Flavonoids from Carduus nutans L.
Acacetin 7-O-rutinoside and pectolinarigenin
7-O-rutinoside (300). UV: λmax (nm) MeOH

269, 327; ＋NaOMe 292, 371 (dec.); ＋AlCl3
277, 299, 347, 377sh; ＋AlCl3/HCl 278, 299,
339, 380sh; ＋NaOAc 270, 323; ＋NaOAc/
H3BO3 269, 330. LC-MS: (major ﬂavonoid)
m/z 593 [M＋H]＋ (molecular ion peak,acacetin＋each 1 mol of glucose and rhamnose).
LC-MS: (minor ﬂavonoid) m/z 623 [M＋H]＋(molecular ion peak, pectolinarigenin＋each
1 mol of glucose and rhamnose).
The ﬂavonoids were isolated from the leavesof Carduus nutans as pale yellow needle (ca.
90 mg). The plant (TNS9522025) was collected
in near Suusanmyr, Kyrgyz Republic in 1995 by
T. Iwashina. It was shown by HPLC survey thatthe crystal was the mixture of two ﬂavonoids. Of
their ﬂavonoids, major compound (69.9%) was
identiﬁed as acacetin 7-O-rutinoside by UV,
LC-MS and HPLC comparison with authentic
sample (Carl Roth). On the other hand, minor ﬂavonoid (37.1%) was characterized as pectolinarigenin 7-O-rutinoside by LC-MS and comparison
with 136-10 by HPLC. Flavones, apigenin andits 7-O-glucoside and 7-O-neohesperidoside, andluteolin and its 7-O-glucoside, 7-O-galactoside,
7-O-rutinoside, 7-O-diglucoside and 7-O-digalactoside, have been reported from the leaves ofthe species (Bain and Desrochers, 1988; Warwick et al., 1989; Jordon-Thaden and Louda,
2003). Flavonols, kaempferol and its 3-O-rhamnoside, 3-O-glucoside-7-O-rhamnoside, isorhamnetin, and quercetin 3-O-rutinoside, have also
been found in the species (Kaloshina et al., 1975;
Kaloshina and Mazulin, 1988; Jordon-Thaden
and Louda, 2003). Although acacetin 7-O-glucoside has been isolated from C. nutans (Kaloshina
et al., 1975), acacetin 7-O-rutioside and pectolinarigenin 7-O-rutinoside were found for the ﬁrst
time. Since C. nutans is widely distributed in
Eurasia and North America, chemical geographic
variation may be occurred.
In this survey, the chemical structures of 48
ﬂavonoid powders and crystals, which are deposited in Department of Botany, National Museumof Nature and Science, Japan, from 38 Cirsium
taxa and Carduus nutans, were newly determinedor reconﬁrmed (Table 1). Of their ﬂavonoids,

Further identiﬁcation of ﬂavonoids from Cirsium spp.

211

Table 1. List of the ﬂavonoids isolated from Cirsium taxa and related Carduus nutans deposited in the National
Museum of Nature and Science, Japan
Accession number

OriginAcacetin 7-O-rutinoside
113-1
Cirsium senjoense
113-3
C. babanum

References
Iwashina et al. (1995)
Iwashina et al. (1995)

280-1
298-4
305
310-1, 310-2

C. happoense
C. sp.
C. purpuratum
C. norikurense

Present paper
Present paper
Nakaoki and Morita (1959)
Present paper

300

Carduus nutans

Present paper

Remarks

Mixed with trace of pectolinarigenin 7-O-rutinoside
Mixed with pectolinaigein 7-O-rutinoside (38.4%)
Mixed with pectolinaigein 7-O-rutinoside (34.1%)

Mixed with pectolinaigein 7-O-rutinoside (37.1%)

Hispidulin 7-O-neohesperidoside122
C. tashiroi

Present paper

Hispidulin
128-1
128-2

C. tashiroi
C. oligophyllum

Present paper
Iwashina et al. (1999)

Hispidulin 4′-O-glucoside
129
C. oligophyllum

Iwashina et al. (1999)

Cirsimaritin 4′-O-glucoside134
C. pectinellum
290-1, 290-2, 290-4
C. japonicum307
C. sp.
311-1, 311-2, 311-3
C. maritimum

Present paper
Present paper
Mixed with cirsimaritin (290-1)
Present paper
Morita and Shimizu (1963)

Hydrolysate of hispidulin 7-O-neohesperidoside
Hydrolysate of hispidulin 4′-O-glucoside

Pectolinarigenin 7-O-rutinoside
136-1
C. ishidzuchiense
Present paper
Mixed with acacetin 7-O-rutinoside (13.3%)
136-2
C. tashiroi
Present paper
Mixed with acacetin 7-O-rutinoside (17.9%)
136-3
C. oligophyllum
Present paper
Mixed with acacetin 7-O-rutinoside (26.5%)
136-4
C. irumtiense
Present paper
Mixed with acacetin 7-O-rutinoside (12.4%)
136-5
C. magofukui × C. Present paper
Mixed with acacetin 7-O-rutinoside (3.3%)
microspicatum var.
kiotoense
136-6
C. senjoense
Iwashina et al. (1995)
136-7
C. spicatum
Iwashina et al. (1995)
Mixed with acacetin 7-O-rutinoside (11.2%)
136-8
C. gratiosum
Iwashina et al. (1995)
136-9
C. babanum
Iwashina et al. (1995)
Mixed with acacetin 7-O-rutinoside (28.7%)
136-10C. brevicaule
Present paper
Mixed with acacetin 7-O-rutinoside (10.3%)
136-12
C. inundatum
Nakaoki and Morita (1960) Mixed with acacetin 7-O-rutinoside (31.8%)
136-13
C. aidzuense
Present paper
Mixed with acacetin 7-O-rutinoside (11.7%)
136-15
C. bitchuense
Iwashina et al. (1995)
Mixed with acacetin 7-O-rutinoside (14.2%)
136-17C. hanamakiense
Present paper
Mixed with acacetin 7-O-rutinoside (27.6%)
136-18
C. borealinipponense Present paper
Mixed with acacetin 7-O-rutinoside (25.5%)
136-21
C. suﬀultum
Morita et al. (1973)
Mixed with acacetin 7-O-rutinoside (15.4%)
Iwashina et al. (1995)
136-22
C. esculentum
Present paper
Mixed with acacetin 7-O-rutinoside (24.9%)
136-23
C. boninense
Present paper
Mixed with acacetin 7-O-rutinoside (8.9%)
136-24
C. spinosum
Present paper
Mixed with acacetin 7-O-rutinoside (17.0%)
136-26
C. yezoense
Iwashina et al. (1988)
Mixed with acacetin 7-O-rutinoside (11.3%)
136-28C. grayanum
Present paper
Mixed with acacetin 7-O-rutinoside (19.9%)
136-29136-30, 290-3

C. alpicola
C. japonicum

136-31
136-32

C. diabolicum
C. brevicaule

Present paper
Mixed with acacetin 7-O-rutinoside (19.8%)
Nakaoki and Morita (1959) Mixed with acacetin 7-O-rutinoside (24.8%, 136-30,
and 36.3%, 294-3)
Iwashina et al. (1988)
Ganzera et al. (2005)
Liu et al. (2007)
Peng (2011)
Present paper
Mixed with acacetin 7-O-rutinoside (27.8%)
Present paper
Mixed with acacetin 7-O-rutinoside (8.4%)

212

Tsukasa Iwashina et al.

Table 1. List of the ﬂavonoids isolated from Cirsium taxa and related Carduus nutans deposited in the National
Museum of Nature and Science, Japan (continued)
Accession number
278-1
279-5
281-4
282-6
283-4
284-1

OriginReferences

Remarks

C. sp.
C. kagamontanum
C. otayae
C. hidakamontanum
C. ugoense
C. microspicatum

Present paper
Mixed with acacetin 7-O-rutinoside (37.2%)
Nakaoki and Morita (1960) Mixed with acacetin 7-O-rutinoside (18.0%)
Present paper
Mixed with acacetin 7-O-rutinoside (11.8%)
Present paper
Mixed with acacetin 7-O-rutinoside (30.5%)
Present paper
Mixed with acacetin 7-O-rutinoside (24.4%)
Nakaoki and Morita (1959) Mixed with acacetin 7-O-rutinoside (17.5%)
Iwashina et al. (1988)
284-12
C. microspicatum×C. Present paper
norikurense
285-2
C. yezoense×
Present paper
Mixed with acacetin 7-O-rutinoside (12.9%)
C. magofukui286-1, 286-7, 286-10, C. kiotoense
Iwashina et al. (1988)
289-2
287
C. nambuense
Present paper
Mixed with acacetin 7-O-rutinoside (30.2%)
288, 298-1
C. pendulum
Present paper
Mixed with acacetin 7-O-rutinoside (31.7%, 288
and 23.7%, 298-1)
290-3
C. japonicum
Nakaoki and Moria (1959) Mixed with acacetin 7-O-rutinoside (36.0%)
Iwashima et al. (1988, 1995)
317-2
C. kamtschaticum
Morita et al. (1973)
Mixed with acacetin 7-O-rutinoside (31.3%)
Luteolin 7-O-glucoside
138-1
C. chikushiense
Iwashina et al. (1995)
138-5
C. hachijoense
Iwashina and Ootani (1998)
Present paper
138-10, 138-11, 278-2, C. confertissimum
297, 304-7, 304-9,
304-10303
C. occidentalinippo- Present paper
nense
304-1, 304-11, 306-1, C. buergeri
Present paper
306-2
Luteolin 4′-O-glucoside
141-1
C. confertissimum

Present paper

Luteolin 3′-O-glucoside148
C. confertissimum

Present paper

Nepetin 4′-O-glucoside
149

C. oligophyllum

Iwashina et al. (1999)

Nepetin
157

C. oligophyllum

Iwashina et al. (1999)

Luteolin 7-O-vicianoside
304-2, 304-5
C. buergeri
Luteolin 5-O-glucoside
279-8, 279-9
308-1
308-2
309-3, 309-4, 309-5
312-1, 312-2
Luteolin309-1, 309-2

Hydrolysate of nepetin 4′-O-glucoside

Present paper

C. kagamontanum
Iwashina et al. (1989)
C. sieboldii
Iwashina et al. (1989)
C. longe-peduncula- Iwashina et al. (1989)tum
C. suzukaense
Present paper
C. magofukui
Iwashina et al. (1989)
C. suzukaense

Present paper

pectolinarigenin 7-O-rutinoside was frequently
found (22 samples), and acacetin 7-O-rutinoside
was accompanied in almost Cirsium taxa andCarduus nutans. As other ﬂavonoids, hispidulin

7-O-neohesperidoside, cirsimaritin 4′-O-glucoside, luteolin 7-O-glucoside, luteolin 4′-O-glucoside, luteolin 3′-O-glucoside, luteolin 7-O-vicianoside and luteolin 5-O-glucoside were found inFurther identiﬁcation of ﬂavonoids from Cirsium spp.

some Cirsium species. Of their ﬂavonoids, luteolin 7-O-vicianoside which was isolated from C.
buergeri was reported in Cirsium species for theﬁrst time. Hispidulin 7-O-neohesperidosidewhich was newly isolated from C. tashiroi wasfound in Japanese Cirsium species.
Acknowledgments
Authors thank to Mr. Tsutomu Ito for donationof ﬂavonoid crystals and powders, and also to Dr.
Yuichi Kadota and Mr. Tatsuya Ueno for oﬀer ofplant materials.
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