Two new glycosides from Carduus acanthoides

Article abstract of DOI:10.1080/10286020.2013.837452

Two new glycosides, syringic acid-4-O-β-l-arabinopyranoside (1) and kaempferol-3-O-α-l-rhamnopyranosyl-7-O-β-d-glucuronopyranoside (2), were isolated from whole plants of Carduus acanthoides (Asteraceae), and their structures were elucidated on the basis

Full text of DOI:10.1080/10286020.2013.837452

This article was downloaded by: [Ryerson University]
On: 02 December 2014, At: 11:17
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Asian Natural Products
Research
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/ganp20
Two new glycosides from Carduus
acanthoides
Sui-Ku Liua, Sheng Queb, Wei Chenga, Qing-Ying Zhanga, Min Yea &
Hong Lianga
a State Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University Health Science Center,
Beijing, 100191, China
b Department of Chemistry, Teachers College for Nationalities,
Qinghai Normal University, Xining, 810008, China
Published online: 01 Nov 2013.
To cite this article: Sui-Ku Liu, Sheng Que, Wei Cheng, Qing-Ying Zhang, Min Ye & Hong Liang (2013)
Two new glycosides from Carduus acanthoides, Journal of Asian Natural Products Research, 15:12,
1243-1248, DOI: 10.1080/10286020.2013.837452
To link to this article: http://dx.doi.org/10.1080/10286020.2013.837452
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Journal of Asian Natural Products Research, 2013
Vol. 15, No. 12, 1243–1248, http://dx.doi.org/10.1080/10286020.2013.837452
Two new glycosides from Carduus acanthoides
Sui-Ku Liua, Sheng Queb, Wei Chenga, Qing-Ying Zhanga, Min Yea and Hong Lianga*
aState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University Health Science Center, Beijing 100191, China; Department of Chemistry,
Teachers College for Nationalities, Qinghai Normal University, Xining 810008, China
b
(Received 6 March 2013; final version received 7 August 2013)
Two new glycosides, syringic acid-4-O-b-L-arabinopyranoside (1) and kaempferol-3-
O-a-L-rhamnopyranosyl-7-O-b-D-glucuronopyranoside (2), were isolated from whole
plants of Carduus acanthoides (Asteraceae), and their structures were elucidated on the
basis of spectroscopic analysis.
Keywords: Carduus acanthoides; flavonol glycoside; phenolic glycoside; structural
elucidation
1. Introduction
formula of 1 was determined to be
C14H18O9 on the basis of quasi-molecular
ion peaks at m/z 329.0869 [M 2 H]2,
353.0844 [M þ Na]þ in the negative and
The genus Carduus, which belongs to the
Asteraceae family, comprises about 95
species all over the world, among which
three species are widely distributed in
China [1]. The whole plants or roots of
Carduus acanthoides were used in China
for the treatment of rheumatism, urethritis,
chyluria, hematemesis, epistaxis, hema-
turia, menorrhagia, furuncle, scald, and
edema [2,3]. Recent pharmacological
studies demonstrated that ethanol extracts
of Carduus plants had various activities
such as anticancer [4], hepatoprotection
[5–7], antioxidation [8], bacteriostasis [9],
etc. Chemical studies on flavonoids
[10,11], lignans [10], coumarins [12], and
alkaloids [13,14] have been reported from
the plants of the genus Carduus. This
paper deals with the isolation and struc-
tural elucidation of two new glycosides,
syringic acid-4-O-b-L-arabinopyranoside
(1) and kaempferol-3-O-a-L-rhamnopyra-
nosyl-7-O-b-D-glucuronopyranoside (2).
1
positive HR-ESI-MS. The H NMR spec-
trum showed the presence of two aromatic
proton signals at dH 7.35 (2H, s) as an A2
spin system and two methoxyl signals at dH
3.89 (6H, s). In addition, six proton signals
were found in the range of dH 3.30–5.40,
which suggested the existence of a sugar
moiety. The signal at dH 5.40 (1H, d,
J ¼ 4 Hz) was assigned as the anomeric
proton. The 13C NMR and DEPT spectra
clearly exhibited 14 carbon signals, includ-
ing a carboxyl group (dC 169.4), four sp2
quaternary carbons (dC 154 £ 2, 138.6,
128), two sp2 methines (dC 108.2 £ 2), two
methoxyls (dC 56.8 £ 2), and a pentosyl
residue (dC 102.7, 73.0, 71.7, 66.6, 63.5).
Except for the pentosyl residue, the signals
observed above were quite similar to those
of the aglycone moiety of syringic acid-4-
O-a-L-rhamnopyranoside [15], which
suggested that the aglycone moiety of 1
was syringicacid (4-hydroxy-3,5-dimethox-
ybenzoic acid). The sugar was identified as
L-arabinose by gas chromatography (GC)
2. Results and discussion
Compound 1 was isolated as a white
needle crystal (MeOH). The molecular
*Corresponding author. Email: lianghong@bjmu.edu.cn
q 2013 Taylor & Francis

1244
S.-K. Liu et al.
analysis, in which the retention times of
trimethylsilyl ether derivatives of the sugar
residue and authentic sugars were com-
pared, as described in Section 3. According
to the coupling constant of the anomeric
proton at dH 5.40 (1H, d, J ¼ 4 Hz), the
arabinosyl unit was determined to be
b configuration. In HMBC spectrum,
the correlation of the anomeric proton
signal at dH 5.40 (H-10) with the carbon
signal at dC 138.6 (C-4) revealed that the
arabinopyranose was attached to C-4. The
1H and 13C NMR data were completely
assigned on the basis of the analysis of
1H–1H COSY, HSQC, and HMBC spectra.
Thus, the structure of 1 was characterized
as syringic acid-4-O-b-L-arabinopyrano-
side (Figure 1).
belong to the meta-coupled aromatic
protons H-6 and H-8, suggesting that 2
was a 5,7,40-trisubstituted flavone. In
addition, a methyl proton signal at dH
0.81 (3H, d, J ¼ 5.4 Hz) and 10 complex
proton signals in the range of dH 3.00–
5.50 suggested the existence of two sugar
moieties, among which the two proton
signals at dH 5.32 (1H, br s) and 5.22 (1H,
d, J ¼ 7.3 Hz) were assigned as the
anomeric protons of two sugar moieties.
The 13C NMR spectrum exhibited 27
carbon signals, among which 15 carbons
were attributed to the skeleton of a 5,7,40-
trisubstituted flavone, and 12 carbons
were attributed to two sugar moieties. b-
Glucuronidase hydrolysis yielded D-glu-
curonic acid by co-thin layer chromatog-
raphy (co-TLC) analysis comparing with
the authentic sugar (D-glucuronic acid).
And further acid hydrolysis of the
glucuronidase hydrolysate afforded L-
rhamnose, determined by preparation of
trimethylsilyl ether derivatives and com-
paring with the authentic sugar in GC
analysis. According to the coupling
constant of the anomeric proton at dH
5.22 (1H, d, J ¼ 7.3 Hz), the glucurono-
pyranosyl unit was determined to be b
configuration. Comparing 13C NMR data
of the rhamnopyranosyl to those of a- and
b-rhamnopyranose, the rhamnopyranosyl
unit was determined to be a configuration
[16]. In HMBC spectrum, the correlation
of the anomeric proton signal at dH 5.32
(H-1000) with the carbon signal at dC 134.5
(C-3) showed that the rhamnopyranosyl
unit was linked to C-3, and the corre-
lation of the anomeric proton signal at dH
5.22 (H-100) with the carbon signal at dC
162.6 (C-7) showed that the glucurono-
pyranosyl unit was linked to C-7. The 1H
and 13C NMR data were completely
assigned on the basis of the analysis of
1H–1H COSY, TOCSY, HSQC, and
HMBC spectra. Thus, the structure of 2
was characterized as kaempferol-3-O-a-
L-rhamnopyranosyl-7-O-b-D-glucurono-
pyranoside (Figure 2).
Compound 2 was isolated as a yellow
amorphous powder (MeOH). The mol-
ecular formula of 2 was determined to be
C27H28O16 on the basis of quasi-molecu-
lar ion peaks at m/z 607.1296 [M 2 H]2
and 609.1461 [M þ H]þ in the negative
and positive HR-ESI-MS. The UV
spectrum with absorption maxima at
261 and 340 nm indicated the presence
of flavone. The 1H NMR spectrum
displayed six aromatic proton signals, of
which four aromatic proton signals at dH
7.77 (2H, d, J ¼ 8.5 Hz) and 6.93 (2H, d,
J ¼ 8.5 Hz) belong to H-20, 60 and H-30, 50
as an AA0BB0 spin system, and the other
two proton signals at dH 6.78 (1H, d,
J ¼ 1.5 Hz) and 6.47 (1H, d, J ¼ 1.5 Hz)
Figure 1. Structure and key HMBC
correlations of 1.

Journal of Asian Natural Products Research
1245
HO
Mitsubishi Chemical Corp., Kyoto, Japan),
HP20 (75–150 mm, Mitsubishi Chemical
Corp., Kyoto, Japan), and MDS-5 (75 um,
Beijing Medicine Technology Center,
Beijing, China) were used for normal
pressure column chromatography. Frac-
tions were monitored and analyzed by TLC
(Qingdao Marine Chemical, Inc.). L-
Arabinose, D-arabinose, D-glucuronic
acid, and L-rhamnose were obtained from
Sigma-Aldrich Co., Ltd (St Louis, MO,
USA). L-Cysteine methyl ester hydrochlo-
ride was obtained from TCI Co., Ltd
(Shanghai, China). HMDS–TMCS–Pyri-
dine (3:1:9) and b-glucuronidase (Type
HP-2 from Helix pomatia) were obtained
from Sigma-Aldrich Co., Ltd (St Louis,
MO, USA). High purity water was provided
by Peking University Health Science
Center, while reagents used in HPLC
were HPLC grade (J&K Scientific Ltd,
Beijing, China). Other solvents used were
of analytical grade (Beijing Chemical
Works, Beijing, China).
HOOC
HO
HO
O
1''
O
O
O
OH
O
1'''
OH
Me
O
OH
OH
OH
Figure 2. Structure and key HMBC
correlations of 2.
3. Experimental
3.1 General experimental procedures
Melting points (uncorrected) were deter-
mined on a X-5 micromelting point
apparatus (Cany Precision Instruments
Co., Ltd, Shanghai, China). Optical
rotations were recorded on a AUTOPOLw
automatic polarimeter (Rudolph Research
Analytical, Hackettstown, NJ, USA). UV
spectra were obtained on a UV-2201
spectrophotometer (Shimadzu, Kyoto,
Japan). HR-ESI-MS was obtained on a
Xevo G2 Q-TOF mass spectrometer
(Waters, Milford, MA, USA). NMR spectra
were recorded on AVANCE III-400 and
INOVA-500 spectrometers (Bruker, Fael-
landen, Switzerland) with TMS (tetra-
methylsilane) as the internal standard.
High-performance liquid chromatography
(HPLC) preparation was performed on a
Preparative HPLC system (Gilson, Mid-
dleton, WI, USA) equipped with two 306
pumps, a 811C dynamic mixer, a 806
manometric module, a 118 UV/Vis detec-
tor, and a Grace ODS column (Allsphere
ODS-2, 5 mm, 22 mm £ 250 mm) with a
flow rate of 10 ml/min. HPLC analysis was
performed on an analysis HPLC system
(Shimadzu, Kyoto, Japan) consisting of a
LC-10AVP pump, a DGU-14A degasser, a
SCL-10AVP system controller, a SPD-
M10AVP diode-array detector, and a Grace
ODS column (Allsphere ODS-2, 5 mm,
4.6 mm £ 250 mm) with a flow rate of 1 ml/
min. Silica gel (200–300 mesh, Qingdao
Marine Chemical, Inc., Qingdao, China),
Sephadex LH-20 (GE Healthcare, Uppsala,
Sweden), MCI gel CHP 20P (75–150 mm,
3.2 Plant material
The whole plants of C. acanthoides were
collected in September 2010 in Guide
County, Qinghai Province, China. Species
identification was confirmed by Associate
Prof. Ming-Ying Shang and Associate
Prof. Ying-Tao Zhang, School of Pharma-
ceutical Sciences, Peking University.
A voucher specimen (FL2010090901) is
maintained in the Department of Natural
Medicines, School of Pharmaceutical
Sciences, Peking University.
3.3 Extraction and isolation
The air-driedwholeplantsofC. acanthoides
(7.5kg) were crushed into raw powders and
successively refluxed with 95% EtOH
(3 £ 60 liters, 2 h each) and 50% EtOH
(3 £ 60 liters, 2 h each). The EtOH extract
(870 g) was suspended in water and
successively partitioned with petroleum
ether, EtOAc, and n-BuOH, respectively,

1246
S.-K. Liu et al.
to obtain petroleum ether extract (120 g),
EtOAc extract (95 g), n-BuOH extract
(220 g), and water extract (430 g). The
dried n-BuOH extract was subjected to
HP-20 CC (column chromatography)
(10 cm £ 70 cm, EtOH–H2O 0:100–95:5,
v/v) to give five fractions (Fr. 1–Fr. 5).
Fraction 2 (50 g) was further subjected to
MCI CC (6 cm £ 70 cm, EtOH–H2O
10:90–70:30, v/v) to yield five subfrac-
tions (Fr. 2A–Fr. 2E). Fraction 2A (13 g)
was separated on silica gel CC (3 cm
£ 55 cm, CHCl3–MeOH–H2O 100:0:0–
60:40:10, v/v/v) to obtain five fractions
(Fr. 2A-1–Fr. 2A-5). Fraction 2A-4 (1.9 g)
was further separated by MCI CC (2 cm
£ 40 cm, MeOH–H2O 0:100–40:60, v/v),
3.3.1 Syringic acid-4-O-b-L-
arabinopyranoside (1)
White needle crystal (MeOH); melting
13
point (mp) 196–1988C; ½aꢀD 264.68
(c ¼ 0.46, MeOH); UV (MeOH) lmax
(log 1): 229 (2.47), 254 (4.13), and 298
1
(1.84) nm; for H NMR and 13C NMR
spectral data see Table 1; HR-ESI-MS m/z:
329.0869 [M 2 H]2 (calcd for C14H17O9,
329.0873), 353.0844 [M þ Na]þ (calcd
for C14H18O9Na, 353.0849).
3.3.2 Kaempferol-3-O-a-L-
rhamnopyranosyl-7-O-b-D-
glucuronopyranoside (2)
Yellow amorphous powder (MeOH); mp
14
216–2188C; ½aꢀD 21438 (c ¼ 0.44,
MeOH); UV (MeOH) lmax (log 1): 236
(2.56), 261 (4.20), 316 (3.87), and 340
preparative
HPLC
(MeOH–H2O–
1
(4.09) nm; for H NMR and 13C NMR
HCOOH 15:85:0.5, flow rate 10 ml/min,
wavelength 260 nm), and then purified by
Sephadex LH-20 CC (1.8cm £ 100 cm,
MeOH–H2O 20:80, v/v) to afford 1
(30 mg). Fraction 2B (9 g) was subjected
to MDS-5 reversed phase CC (4 cm
£ 60cm, MeOH–H2O 30:70–70:30, v/v),
preparative HPLC (MeOH–H2O–HCOOH
35:65:0.5, flow rate 10ml/min, wavelength
300 nm), and further purified by Sephadex
LH-20 CC (2 cm £ 100 cm, MeOH–H2O
50:50, v/v) to afford compound 2 (20 mg).
spectral data see Table 2; HR-ESI-MS m/z:
607.1296 [M 2 H]2 (calcd for C27H27O16,
607.1299), 609.1461 [M þ H]þ (calcd for
C27H29O16, 609.1456).
3.4 Hydrolysis of compounds 1 and 2
Compound 2 (5 mg) was dissolved in
0.5 ml of KH2PO4/KOH buffer solution
(pH 4.90) before 0.2 ml of b-glucuroni-
dase solution was added, and incubated at
378C for 5 h. The reaction mixture (about
0.7 ml) was mixed with 3 ml of methanol,
Table 1. 1H (400 MHz) and 13C (100 MHz) NMR spectral data of 1 in CD3OD.
Number
dH (mult, J in Hz)
dC
HMBC
1
2, 6
3, 5
4
128.0
108.2
154.0
138.6
169.4
56.8
102.7
71.7
73.0
7.35, s
C-1, 3, 4, 6/2, 7
7
3,5-OCH3
3.89, s
5.40, d (4.0)
4.02, dd (5.6, 4.0)
3.78, dd (5.6, 2.4)
3.90, m
C-3/5
C-4, 30
C-10, 30
10
20
30
C-10, 20, 40, 50
C-20, 50
40
66.6
63.5
50a
50b
3.45, dd (14.8, 7.2)
3.93, m
C-10, 30, 40
C-10, 30, 40

Journal of Asian Natural Products Research
1247
Table 2. 1H (500 MHz) and 13C (125 MHz) NMR spectral data of 2 in DMSO-d6.
Number
dH (mult, J in Hz)
dC
Number
dH (mult, J in Hz)
dC
2
3
4
5
6
7
8
9
10
10
20, 60
30, 50
40
157.8
134.5
178.0
161.0
99.3
100
200
5.22, d (7.3)
3.29, m
3.33, m
3.36, m
3.94, d (9.2)
99.3
72.8
75.8
71.4
75.0
170.7
101.8
70.1
70.3
70.6
71.1
17.4
300
400
6.47, d (1.5)
6.78, d (1.5)
500
162.6
94.5
600
1000
2000
3000
4000
5000
6000
5.32, brs
3.99, m
3.48, dd (8.4, 2.9)
3.13, m
3.15, m
156.1
105.9
120.3
130.6
115.4
160.2
7.77, d (8.5)
6.93, d (8.5)
0.81, d (5.4)
vortexed at 1500 rpm for 5 min, and
centrifuged at 10,000 rpm for 10 min. The
supernatant was transferred to a clean test
tube and dried under a gentle flow of
nitrogen gas at 40 8C. The residue was
partitioned between EtOAc and H2O
(4 £ 1 ml). The water-soluble part was
reduced to dryness and then analyzed by
co-TLC over silica gel (EtOAc–EtOH–
H2O–HCOOH 6:4:1:1), by comparison
with authentic sugar (D-glucuronose), and
the EtOAc-soluble part was analyzed by
LC-MSn compared with compound 2
[Agilent 1100 series HPLC system
coupled with Finnigan LCQ Advantage
ion trap mass spectrometer via ESI inter-
face, Grace Allsphere ODS-2 column
(5 mm, 4.6 mm £ 250 mm), mobile phase
consisted of acetonitrile (A) and water
containing 0.1% (v/v) formic acid (B) in
the following gradient program: 0 min,
10% A; 60 min, 60% A; flow rate 1 ml/
min], which confirmed the presence of
D-glucuronic acid.
hydrochloride were dissolved in 0.3 ml of
anhydrous pyridine, stirred at 60 8C for 2 h.
The reaction mixture was dried under
vacuum and was trimethylsilylated with
0.5 ml of HMDS–TMCS–pyridine (3:1:9)
at 60 8C for 2 h. The mixture was then
concentrated and partitioned between
n-hexane and H2O. The n-hexane extract
was analyzed by GC under the following
conditions: Varian CP-3800 GC, DB-5 GC
column (30 m £ 0.25 mm £ 0.25 mm), col-
umn temperature increased from 50 to
260 8C at 68C/min, carrier gas N2. The tR
values (min) of trimethylsilyl ether deriva-
tives of authentic sugars (L-Ara, D-Ara,
and L-Rha) prepared in a similar way were
24.12, 24.51 and 24.96 min, respectively.
L-Ara and L-Rha were detected from
compounds 1 (tR 24.13 min) and 2 (tR
24.92 min).
Acknowledgments
The authors are grateful to Associate Prof.
Sheng Que (Department of Chemistry, Tea-
chers College for Nationalities, Qinghai
Normal University, Xining, China) for col-
lecting the plant material, and they thank
Associate Prof. Ming-Ying Shang and
Associate Prof. Ying-Tao Zhang (School of
Pharmaceutical Sciences, Peking University,
Beijing, China) for identifying the plant
material.
Compound 1 (2 mg) and compound 2
(the dried EtOAc extract) were hydrolyzed
with 1 N HCl (2 ml) for 6 h at 1008C,
respectively. After lyopilization, the dry
residue was partitioned between Et2O and
H2O (3 £ 2 ml). The dried water extract
and 3 mg of L-cysteine methyl ester

1248
S.-K. Liu et al.
[7] G. Aktay, D. Deliorman, E. Ergun, F.
References
Ergun, E. Yesilada, and C. C¸ evik,
J. Ethnopharmacol. 73, 121 (2000).
[8] D.M. Jeong, H.A. Jung, and J.S. Choi,
Arch. Pharm. Res. 31, 28 (2008).
[1] Delectis Florae Reipublicae Popularis
Sinicae Agendae Academiae Sinicae
Edita, Flora Reipublicae Popularis Sini-
cae (Beijing, Science Press, 1979), Vol.
78, p. 155.
[9] Y.F. Yu, H. Ma, Y.J. Han, and B. Tao,
J. Northeast Agric. Univ. 40, 15 (2009).
[10] I. Fernandez, B. Garcia, J.R. Pedro, and
A. Varea, Phytochemistry 30, 1030 (1991).
[11] A.M. EL-Lakany, M.S. Abdel-Kader,
H.M. Hammoda, N.M. Ghazy, and Z.F.
Mahmoud, Die Pharmazie 52, 78 (1997).
[12] L. Cardona, B. Garcia, J.R. Pedro, and
J. Perez, Phytochemistry 31, 3989 (1992).
[13] B. Frydman and V. Deulofeu, Tetrahe-
dron 18, 1063 (1962).
[14] Q.Y. Zhang, G.Z. Tu, Y.Y. Zhao, and
T.M. Cheng, Tetrahedron 58, 6795 (2002).
[15] X.H. Ran, W. Ni, G. Wei, C.X. Chen, and
H.Y. Liu, Acta Bot. Yunnan. 32, 83 (2010).
[16] D.Q. Yu and J.S. Yang, Handbook of
Analytical Chemistry, 2nd ed., (Chemical
Industry Press, Beijing, 1999), Vol. 7,
p. 902.
[2] Nanjing University of Chinese Medicine,
Dictionary of Chinese traditional medi-
cine, 2nd ed., (Shanghai Scientific &
Technical Publishers, Shanghai, 2006),
p. 379.
[3] D.S. Luo, Tibetan Materia Medica of
China (The Ethnic Publishing House,
Beijing, 1997), p. 248.
[4] I. Ramirez-Erosa, Y.G. Huang, R.A. Hickie,
R.G. Sutherland, and B. Barl, Can.
J. Physiol. Pharmacol. 85, 1160 (2007).
[5] R. Gebhardt, Environ. Toxicol. Pharm.
27, 402 (2009).
[6] P. Lu, Y. Zeng, F.X. Guo, X.Z. Ma, M.
Kang, and J. Qinghai, J. Qinghai Normal
Univ. Nat. Sci. Ed. 26, 42 (2010).