Two new flavonol glycosides from the Tibetan medicinal plant Aconitum tanguticum

Article abstract of DOI:10.1080/10286020.2013.799144

Two new flavonol glycosides characterized as quercetin 3-O-α-l- rhamnopyranosyl-(1 → 2)-[β-d-glucopyranosyl-(1 → 3)-α-l-(4-O-trans-p-coumaroylrhamnopyranosyl)-(1 → 6)]-β-d-galactopyranoside-7-O-α-l-rhamnopyranoside (1) and kaempferol 3-O-α-l-rhamnopyranos

Full text of DOI:10.1080/10286020.2013.799144

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Two new flavonol glycosides from the
Tibetan medicinal plant Aconitum
tanguticum
Lu Xu c , Xiao Zhang d , Li-Mei Lin e , Chun Li b , Zhi-Min Wang b
Yong-Ming Luo c
&
a Institute of Chinese Materia Medica, China Academy of Chinese
Medicine Sciences , Beijing , China Phone: 100700
b National Engineering Laboratory for Quality Control Technology
of Chinese Herbal Medicines , Beijing , 100700 , China
c School of Pharmaceutical Science, Jiangxi University of
Traditional Chinese Medicine , Nanchang , 712046 , China
d School of Pharmaceutical Science, Hunan University of
Traditional Chinese Medicine , Changsha , 410208 , China
e School of Pharmaceutical Science, Zhengzhou University ,
Zhengzhou , 450000 , China
Published online: 14 Jun 2013.
To cite this article: Lu Xu , Xiao Zhang , Li-Mei Lin , Chun Li , Zhi-Min Wang & Yong-Ming Luo (2013)
Two new flavonol glycosides from the Tibetan medicinal plant Aconitum tanguticum , Journal of
Asian Natural Products Research, 15:7, 737-742, DOI: 10.1080/10286020.2013.799144
To link to this article: http://dx.doi.org/10.1080/10286020.2013.799144
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Journal of Asian Natural Products Research, 2013
Vol. 15, No. 7, 737–742, http://dx.doi.org/10.1080/10286020.2013.799144
Two new flavonol glycosides from the Tibetan medicinal plant
Aconitum tanguticum
Lu Xua,b,c, Xiao Zhangd, Li-Mei Line, Chun Lia,b*, Zhi-Min Wanga,b and Yong-Ming Luoc
aInstitute of Chinese Materia Medica, China Academy of Chinese Medicine Sciences, Beijing
100700, China; bNational Engineering Laboratory for Quality Control Technology of Chinese
Herbal Medicines, Beijing 100700, China; cSchool of Pharmaceutical Science, Jiangxi University of
d
Traditional Chinese Medicine, Nanchang 712046, China; School of Pharmaceutical Science,
Hunan University of Traditional Chinese Medicine, Changsha 410208, China; School of
Pharmaceutical Science, Zhengzhou University, Zhengzhou 450000, China
e
(Received 29 December 2012; final version received 22 April 2013)
Two new flavonol glycosides characterized as quercetin 3-O-a-L-rhamnopyranosyl-
(1 ! 2)-[b-D-glucopyranosyl-(1 ! 3)-a-L-(4-O-trans-p-coumaroylrhamnopyrano-
syl)-(1 ! 6)]-b-D-galactopyranoside-7-O-a-L-rhamnopyranoside (1) and kaempferol
3-O-a-L-rhamnopyranosyl-(1 ! 2)-[b-D-glucopyranosyl-(1 ! 3)-a-L-(4-O-trans-p-
coumaroyl rhamnopyranosyl)-(1 ! 6)]-b-D-galactopyranoside-7-O-a-L-rhamnopyra-
noside (2), together with two known flavonol glycosides quercetin 3-O-a-L-
rhamnopyranosyl-(1 ! 2)- [a-L-rhamnopyranosyl-(1 ! 6)]-b-D-galactopyranoside-
7-O-a-L-rhamnopyranoside (3) and kaempferol 3-O-a-L-rhamnopyranosyl-(1 ! 2)-
[a-L-rhamnopyranosyl-(1 ! 6)]-b-D-galactopyranoside-7-O-a-L-rhamnopyranoside
(4), were isolated from the whole plant of Aconitum tanguticum (Maxim.) Stapf. The
structures of the new compounds were elucidated by spectroscopic methods, and the
total 1H and 13C NMR chemical shifts were assigned.
Keywords: Aconitum tanguticum; Ranunculaceae; flavonol glycosides
1. Introduction
components from Chinese medicinal
plants, we obtained from the ethanol
extract of the whole plant of A. tanguti-
cum, two new flavonol glycosides 1 and 2
as well as two known flavonol glycosides 3
and 4 (Figure 1). The structures of the new
compounds were established mainly on the
basis of HR-ESI-MS, 1H, 13C and 2D
NMR spectroscopic methods.
Aconitum tanguticum (Maxim.) Stapf.
(Ranunculaceae) is a perennial herb
distributed around an altitude of 3200–
4800 m in the alpine meadows of Tibet
Autonomous Region, Qinghai Province,
Gansu Province, Sichuan Province, and
Yunnan Province [1]. The whole plant,
commonly called Ponka, has been used in
traditional Tibetan medicine for the treat-
ment of gastricism, hepatitis, nephritis,
and other diseases for thousands of years
[2]. Although the chemical constituents of
plants of the genus Aconitum have been
extensively studied [3], there are few
reports on the chemical constituents and
biological activities of A. tanguticum [4–
8]. In order to find biologically active
2. Results and discussion
The 30% ethanol fraction was subjected to
repeated column chromatography (CC)
over silica gel and octadecylsilane (ODS)
to obtain two new compounds 1 and 2
(Figure 1), along with two known com-
pounds 3 and 4. Compounds 3 and 4 were
isolated from A. tanguticum for the first
*Corresponding author. Email: wenyeli@yahoo.com.cn
q 2013 Taylor & Francis

738
L. Xu et al.
and 1515 cm21). The UV spectrum exhib-
R
'
3
OH
'
4
ited absorption maxima characteristic for
flavonols at 256 and 315 nm. The 1H NMR
spectrum showed the typical signal pattern
for a quercetin derivative (Table 1). A pair
of doublets (dH 7.50, 6.33) with a coupling
constant of 15.6 Hz revealed the presence
of a trans olefinic double bond. Two ortho-
coupled doublets at dH 7.53 (J ¼ 8.4 Hz)
and 6.82 (J ¼ 8.4 Hz), each integrating
for two proton signals, showed a further o,
p-disubstituted aromatic ring, indicating
the presence of a coumaric acid residue.
The anomeric signals of five sugar units
were observed at dH 4.25 (J ¼ 7.8 Hz),
4.53 (brs), 5.06 (brs), 5.54 (brs), and 5.63
(J ¼ 7.8 Hz). The HMQC and TOCSY
data, in combination with the literature
data for similar compounds [9,10], showed
that the sugars appeared as one b-D-
glucopyranose unit, one b-D-galactopyr-
anose, and three a-L-rhamnopyranose
units. This suggestion was further sup-
ported by chemical means. Acid hydroly-
sis of 1 liberated D-galactopyranose, D-
glucopyranose, and L-rhamnopyranose,
which were identified by gas chromatog-
raphy (GC) analysis of their thiazolidine
derivatives. An HMBC correlation was
OH
α–Rha(I)
O
O
O
1
7
CH3
3
H
5
OH OH
O
OH
O
O
α–Rha(III)
2
6
6
8
3
O
C
9
O
O
1
4
5
OH
HO
O
1
7
CH3
1
β–Gal
OH
4
5
HO
H
O
H
2
OH
O
3
H
1
O
OH
OH
O
1
OH
β–Glc
CH3
α–Rha(II)
H
OH
H
OH OH
1 R=OH
2 R=H
Figure 1. The structures of compounds 1 and 2.
time. Their structures were identified by
means of spectroscopic methods and
compared with the reported data.
Compound 1 was isolated as a yellow
amorphous powder. The HR-ESI-MS
spectrum showed a positive ion at m/z
1211.3663 [M þ H]þ, indicating a mol-
ecular formula of C54H66O31. The IR
spectrum exhibited the absorptions
of hydroxyl (3402 cm21), carbonyl
(1653.8 cm21), and aromatic rings (1603
OH
OH
OH
O
O
O
O
CH3
H
OH OH
O
OH
O
O
H
O
C
O
OH
O
O
HO
CH3
O
OH
HO
H
H
OH
O
OH
OH
O
OH
CH3
H
OH
H
OH OH
Figure 2. Key HMBC correlations (H ! C) in compound 1.

Journal of Asian Natural Products Research
739
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of compounds 1 and 2
(DMSO-d6).
1
2
Position
dH
dC
dH
dC
2
3
4
5
6
7
8
157.2
133.6
177.8
161.4
99.8
157.2
133.4
177.9
161.4
99.8
6.43 (1H, d, 1.8 Hz)
6.74 (1H, d, 1.8 Hz)
6.44 (1H, d, 1.8 Hz)
6.77 (1H, d, 1.8 Hz)
162.1
94.8
162.1
95.1
9
156.3
106
121.4
116.3
145
149.1
115.6
122.7
156.3
106
100
10
121.1
131.4
115.6
160.6
115.6
131.4
20
7.53 (1H, d, 1.2 Hz)
8.10 (1H, d, 9.0 Hz)
6.87 (1H, d, 9.0 Hz)
30
40
50
6.82 (1H, d, 8.4 Hz)
7.72 (1H,dd, 8.4,1.8 Hz)
6.87 (1H, d, 9.0 Hz)
8.10 (1H, d, 9.0 Hz)
6
7-O-Rha (I)
1
2
3
4
5.54 (1H, brs)
3.85 (1H, m)
3.64 (1H, m)
98.9
70.1
70.5
72.1
70.3
18.4
5.54 (1H, brs)
3.85 (1H, m)
3.64 (1H, m)
98.9
70.1
70.5
72.1
70.3
18.4
3.30 (1H, m)
3.42 (1H, m)
1.13 (3H, d, 6.0 Hz)
3.30 (1H, m)
3.42 (1H, m)
1.12 (3H, d, 6.0 Hz)
5
6
3-O-Gal
1
2
3
4
5
6
5.63 (1H, d, 7.8 Hz)
3.83 (1H, m)
3.65 (1H, m)
99.5
75.4
74.3
71.1
73.9
5.62 (1H, d, 7.8 Hz)
3.84 (1H, m)
3.64 (1H, m)
99.4
75.4
74.2
71.1
73.8
68.7
3.76 (1H, m)
3.65 (1H, m)
3.65 (1H, m), 3.75 (1H, m)
3.75 (1H, m)
3.64 (1H, m)
68.7 3.64 (1H, m), 3.75(1H, m)
2Gal-O-Rha (II)
1
2
3
4
5
6
5.06 (1H, brs)
3.73 (1H, m)
3.49 (1H, m)
101.1
70.7
71.1
72.4
68.8
17.7
5.05 (1H, brs)
3.75 (1H, m)
3.43 (1H, m)
101.1
70.7
71.1
72.3
68.8
17.7
3.13 (1H, m)
3.77 (1H, m)
0.79 (3H, d, 6.6 Hz)
3.30 (1H, m)
3.79 (1H, m)
0.76 (3H, d, 6.0 Hz)
6Gal-O-Rha(III)
1
2
3
4
5
6
4.53 (1H, brs)
3.13 (1H, m)
3.74 (1H, m)
100.2
72.4
76.8
72.6
66.6
18
4.51 (1H, brs)
3.13 (1H, m)
3.75 (1H, m)
100.2
72.4
76.7
72.5
66.6
18
4.99 (1H, m)
3.68 (1H, m)
0.96 (3H, d, 6.0 Hz)
4.98 (1H, m)
3.64 (1H, m)
0.96 (3H, d, 6.6 Hz)
3Rha (III) –O-Glc
1
2
3
4
5
6
4.25 (1H, d, 7.8 Hz)
2.88 (1H, m)
3.04 (1H, m)
103.8
73.4
76.9
70
4.25 (1H, d, 7.8 Hz)
2.88 (1H, m)
3.04 (1H, m)
103.8
73.4
76.9
69.9
77
3.63 (1H, m)
3.80 (1H, m)
3.43 (1H, m), 3.58 (1H, m)
3.64 (1H, m)
3.80 (1H, m)
77
61.2 3.44 (1H, m), 3.57 (1H, m)
61.2

740
L. Xu et al.
Table 1 – continued
1
2
Position
dH
dC
dH
dC
4Rha (III) -O-
trans–coumaroyl
1
125.7
130.7
116.2
160.2
145.3
114.9
166.9
125.7
130.7
116.2
160.2
145
2,6
3,5
4
7
8
7.53 (2H, d, 8.4 Hz)
6.82 (2H, d, 8.4 Hz)
7.53 (2H, d, 9.0 Hz)
6.81 (2H, d, 9.0 Hz)
7.50 (1H, d, 15.6 Hz)
6.33 (1H, d, 15.6 Hz)
7.50 (1H, d, 15.6 Hz)
6.33 (1H, d, 16.2 Hz)
114.9
166.6
9
observed between the anomeric proton
signal of Rha I (brs, 5.54) and the carbon
resonance at dC 162.1 (C-7). These data
revealed the linkage between the Rha I and
the C-7 of the quercetin unit. The fact that
a long-range correlation was observed
between the proton signal at dH 5.63
(J ¼ 7.8 Hz, H-1 of Gal) and the carbon
resonance at dC 133.6 (C-3) suggested that
the second position of glycosidation was
C-3 of quercetin unit. The HMBC
correlations between the H-1 of Rha II
(dH 5.06) and the C-2 of Gal (dC 75.4),
between the H-1 of Rha III (dH 4.53) and
the C-6 of Gal (dC 68.7), and between the
H-1 of Glc (dH 4.25) and the C-3 of Rha III
(dC 76.8) established that the residue was
linked to position C-3 as a b-D-glucopyr-
anosyl-(1 ! 3)-a-L-rhamnopyranosyl-
(1 ! 6)-[a-L-rhamnopyranosyl-(1 ! 2)]-
b-D-galactopyranosyl unit. The HMBC
correlation between the H-4 of Rha III (dH
4.99) and the carbon resonance at dC 166.9
(C-900) revealed esterification of the
coumaric acid at position 4 of this third
a-L-rhamnopyranose unit. The main cor-
relations between H and C in the HMBC
spectrum are shown in Figure 2.
Thus, compound 1 was characterized as
quercetin 3-O-a-L-rhamnopyranosyl-
(1 ! 2)-[b-D-glucopyranosyl-(1 ! 3)-a-
L-(4-O-trans-p-coumaroylrhamnopyrano-
syl)-(1 ! 6)]-b-D-galactopyranoside-7-O-
a-L-rhamnopyranoside.
Compound 2 was also isolated as a
yellow amorphous powder. Its molecular
formula was deduced to be C54H66O30 by a
positive
HR-ESI-MS
at
m/z
1195.37293[M þ H]þ. The UV spectrum
showed absorption maxima characteristic
for flavonols at 266 and 320 nm. The IR
spectrum exhibited the absorptions of
hydroxyl
(3417 cm21),
carbonyl
(1654.6 cm21), and aromatic rings (1603
and 1514cm21). Comparison of the NMR
spectroscopic data of 2 with those of 1
indicated that the structure of 2 was very
close to that of 1, except that the aglycone
quercetinin1wassubstitutedbykaempferol
in 2 (Table 1). Moreover, 2D experiments
resulted in the assignments of all signals and
the linkage of structural units of 2.
Consequently, compound 2 was established
as kaempferol 3-O-a-L-rhamnopyranosyl-
(1 ! 2)-[b-D-glucopyranosyl-(1 ! 3)-a-
L-(4-O-trans-p-coumaroylrhamnopyrano-
syl)-(1 ! 6)]-b-D-galactopyranoside-7-O-
a-L-rhamnopyranoside.
In addition, the known compounds
were identified as quercetin 3-O-a-L-
rhamnopyranosyl-(1 ! 2)- [a-L-rhamno-
pyranosyl-(1 ! 6)]-b-D-galactopyrano-
side-7-O-a-L-rhamnopyranoside (3) and
kaempferol 3-O-a-L-rhamnopyranosyl-
(1 ! 2)-[a-L-rhamnopyranosyl-(1 ! 6)]-
b-D-galactopyranoside-7-O-a-L-rhamno-
pyranoside (4) by comparison of their
NMR spectroscopic data with those
reported in literatures [9,10].

Journal of Asian Natural Products Research
741
3. Experimental
3.2 Plant material
The whole plant of A. tanguticum (collected
from Qinghai Province, China, in August
2010) was boughtfrom XiningSan Jiangbao
Trade Co., Ltd and identified by Researcher
Xue-feng Feng of the Institute of Chinese
Materia Medica, China Academy of Chi-
nese Medical Sciences. The specimen (No.
20100813) was deposited in the Institute of
Chinese Materia Medica, China Academy
of Chinese Medical Sciences.
3.1 General experimental procedures
Optical rotation was taken on a RUD-
OLPH Autopol Vplus polarimeter
(Rudolph, Research Flanders, NJ, USA;
Part No of Polarization tube (10 mm):
40T-5.0-10-0.2). UV spectra were
measured on T6 New Century UV–vis
spectrophotometer (Pgeneral, Beijing,
China). IR spectra were recorded on a
Nicolet 5700 FT-IR spectrometer (Thermo
Nicolet Corporation, Madison, WI, USA).
HR-ESI-MS data were recorded on an
Agilent Technologies 6250 Accurate-
Mass Q-TOF LC/MS spectrometer (Agi-
lent, Santa Clara, CA, USA). ESI-MS were
measured on an Agilent 6130SQ-MSD
(Agilent). NMR spectra were recorded on
a Bruker Advance 600 spectrometer
(Bruker, Faellanden, Switzerland). GC
data were recorded on an Agilent 7890A
instrument with a Flame Ionization Detec-
tor (FID) (Agilent 6130SQ-MSD). Pre-
parative HPLC was performed on an
LC3000 instrument (Beijing Chuangxin-
tongheng Science and Technology Co.,
Ltd, Beijing, China) connected to a UV
3000 detector, using an Intersil-ODS
column (250 £ 20 mm, 10 mm; GL
Sciences, Inc., Tokyo, Japan). A TLC
plate precoated with silica gel GF254
(20 £ 20 cm) was produced by Merck
(Darmstadt, Germany). Sephadex LH-20
(TOSOH Corporation, Tokyo, Japan),
ODS (Octadecylsilyl, 50 mm, YMC Com-
pany, Kyoto, Japan), silica gel (200–300
mesh, Qingdao Marine Chemical Industry,
Qingdao, China), and AB-8 macroporous
adsorption resin (The Chemical Plant of
Nankai University, Tianjin, China) were
used for CC. Spots were visualized under
UV light or by spraying with 10% H2SO4
in EtOH–H2O (95:5, v/v) followed by
heating. Solvents [petroleum ether (60–
908C), CHCl3, EtOAc, MeOH, CH2Cl2,
and EtOH] were of analytical grade and
purchased from Beijing Chemical Com-
pany, Beijing, China.
3.3 Extraction and isolation
Dried and powdered plants of A. tanguti-
cum (10 kg) were extracted under reflux
with 95% EtOH under reflux, and the EtOH
extract was concentrated to dryness. The
residue (1.8kg) was suspended in H2O and
then extracted with petroleum ether and
EtOAc. The residual aqueous solution was
chromatographed over macroporous resin
AB-8 with H2O containing increasing
amounts of EtOH as eluent to afford H2O
eluate 605 g (A), 30% EtOH eluate 407.9 g
(B), 60% EtOH eluate 107.0 g (C), and 95%
EtOH eluate 2.3 g (D). The fraction D was
chromatographed on silica gel and eluted
with gradient CHCl3 –MeOH–H2O
(8:2:0.125/7:3:0.5/6:4:1/5:5:1) to give frac-
tions 1 , X. Fr. X (31.5 g) was repeatedly
purified on silica gel CC using gradient
CHCl3–MeOH–H2O (7:3:0.5/65:35:10/6:
4:1/5:5:1) followed by ODS CC with
gradient MeOH–H2O (2:8/3:7/10:0) to
afford compounds 1 (878mg), 2 (114mg),
3 (522mg) and 4 (50 mg).
3.3.1 Quercetin 3-O-a-L-
rhamnopyranosyl-(1 ! 2)-[b-D-
glucopyranosyl-(1 ! 3)-a-L-(400-O-trans-
p-coumaroylrhamnopyranosyl)-(1 ! 6)]-
b-D-galactopyranoside-7-O-a-L-
rhamnopyranoside (1)
Yellow amorphous powder; [a ]D 2 85.0
(c ¼ 0.02, MeOH); UV (MeOH) lmax
256, 315 nm; IR (KBr) nmax: 3402, 2935,
1654, 1603, 1515, 1170, 1072cm21 1H
:
.

742
L. Xu et al.
NMR (600MHz) and 13C NMR (150MHz)
spectral data, see Table 1. (-) ESI-MS: m/z
1209 [M–H]2; LC-ESI-MS(n): m/z 1209
[M–H]2, 1063 [M–H-rha]2, 917 [M–H-
rha-rha]2, 755 [M–H-rha-rha-glc]2, 753
[M–H-rha-rha-p-coumaroyl]2; HR-ESI-
MS: m/z 1211.3663 [M þ H]þ (calcd for
C54H67O31, 1211.3666).
injection temperature: 2508C; oven tem-
perature gradient: 1008C for 2 min,
1008C ! 2808C (108C/min), and 2808C for
5 min. The same procedure was applied
to authentic samples. By comparing
with the retention time of authentic sample
(tR- -galactose 20.034 min, tR- -galactose
D
L
20.037 min, tR- -glucose 19.849 min, tR- -
D
L
19.822min, tR- -rhamnose 18.697min,
L
glucose
tR-D-rhamnose 18.706min), D-galactose, D-
glucose, and L-rhamnose were identified in
the hydrolysate of 1 and 2.
3.3.2 Kaempferol 3-O-a-L-
rhamnopyranosyl-(1 ! 2)-[b-D-
glucopyranosyl-(1 ! 3)-a-L-(400-O-trans-
p-coumaroylrhamnopyranosyl)-(1 ! 6)]-
b-D-galactopyranoside-7-O-a-L-
rhamnopyranoside (2)
Acknowledgements
This project was financially supported by
Foundation
Beijing
Natural
Science
Yellow amorphous powder; [a ]D 2 30.0
(c ¼ 0.1, MeOH); UV (MeOH) lmax: 266,
320 nm; IR (KBr) nmax: 3417, 2933, 1655,
(7132152) and the Fundamental Research
Funds for the Central Public Welfare Research
Institutes (ZZ070828). The authors are grateful
to the Department of Instrumental Analysis of
Beijing University of Chemical Technology for
the NMR measurements.
1
1603, 1514, 1174, 1069 cm21. H NMR
(600 MHz) and 13C NMR (150 MHz)
spectral data, see Table 1. (-) ESI-MS:
m/z 1193 [M–H]2; HR-ESI-MS: m/z
1195.3729 [M þ H]þ (calcd for
C54H67O30, 1195.3717).
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3.4 Determination of the absolute
configuration of the sugar moieties
According to the reported method [11], each
(2 mg) of the compounds 1 and 2 was
hydrolyzed by 2 M HCl–H2O (2 ml) at 908C
for 4 h. After removal of HCl by evaporation
and extraction with CHCl3, the H2O extract
was evaporated and dried in vacuo to give
the monosaccharide residue. The residue
was treated with L-cysteine methyl ester
hydrochloride (2mg) in pyridine (1.0ml) at
608C for 2 h, then evaporated under N2
stream and dried in vacuo. The residue was
dissolved in 0.2 ml of N-trimethylsilylimi-
dazole and heated at 608C for 1 h. The
reaction mixture was partitioned between n-
hexane and H2O (2ml each), and the n-
hexane extract was analyzed byGC (Agilent
7890A) under the following conditions:
capillary column HP-5 (30 m £ 0.32mm £
0.25mm); detector FID; carrier gas N2, flow
rate 1 ml/min; detector temperature 2808C;