Two new steroidal glycosides from Cynanchum otophyllum Schneid

Article abstract of DOI:10.1080/10286020.2014.956308

Two new C21 steroidal glycosides were isolated from Cynanchum otophyllum Schneid. Their structures were elucidated as qinyangshengenin-3-O-β-d-digitoxopyranoside (1) and rostratamine-3-O-β-d-cymaropyranosyl-(1 → 4)-β-d-digitoxopyranoside (2) on the basis of chemical and spectroscopic methods, including 1D and 2D NMR experiments.

Full text of DOI:10.1080/10286020.2014.956308

This article was downloaded by: [University of Connecticut]
On: 01 April 2015, At: 05:18
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 steroidal glycosides from
Cynanchum otophyllum Schneid
Xin-Xin Yanga, Yong-Rui Baoa, Li-Fen Wangb, Shuai Wanga, Li-Na
Baoa, Yu-Peng Guana & Xian-Sheng Menga
a College of Pharmacy, Liaoning University of Traditional Chinese
Medicine, Dalian 116600, China
b Department of Pathology, The Second Affiliated Hospital
University, Dalian 116027, China
Published online: 07 Oct 2014.
Click for updates
To cite this article: Xin-Xin Yang, Yong-Rui Bao, Li-Fen Wang, Shuai Wang, Li-Na Bao, Yu-Peng Guan
& Xian-Sheng Meng (2015) Two new steroidal glycosides from Cynanchum otophyllum Schneid,
Journal of Asian Natural Products Research, 17:3, 285-288, DOI: 10.1080/10286020.2014.956308
To link to this article: http://dx.doi.org/10.1080/10286020.2014.956308
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, 2015
Vol. 17, No. 3, 285–288, http://dx.doi.org/10.1080/10286020.2014.956308
Two new steroidal glycosides from Cynanchum otophyllum Schneid
Xin-Xin Yanga, Yong-Rui Baoa*, Li-Fen Wangb*, Shuai Wanga, Li-Na Baoa,
Yu-Peng Guana and Xian-Sheng Menga
aCollege of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600,
China; bDepartment of Pathology, The Second Affiliated Hospital University, Dalian 116027,
China
(Received 14 April 2014; final version received 17 August 2014)
Two new C21 steroidal glycosides were isolated from Cynanchum otophyllum Schneid.
Their structures were elucidated as qinyangshengenin-3-O-b-D-digitoxopyranoside (1)
and rostratamine-3-O-b-D-cymaropyranosyl-(1 ! 4)-b-D-digitoxopyranoside (2) on
the basis of chemical and spectroscopic methods, including 1D and 2D NMR
experiments.
Keywords: Asclepiadaceae; Cynanchum otophyllum; C21 steroidal glycosides; 2,6-
dideoxypyranose
1. Introduction
glycosides from the roots of C. otophyllum
(Figure 1).
Cynanchum otophyllum Schneid, Qin-
gyangshen, is a plant of the genus
Cynanchum L. (Asclepiadaceae), and a
traditional Chinese medicine distributed
extensively over Southwest China, whose
root was used for the treatment of epilepsy,
rheumatic pain, kidney weakness, and
muscle injures. The main constituents in
this folk medicine were proved to be
steroidal glycosides with deoxysugars.
In order to have its constituents further
revealed, a systematic research was carried
out to illustrate the possible chemical
foundation of the clinical effects it has
been exhibiting.
2. Results and discussion
Compound 1 was obtained as a white
20
amorphous powder, with ½aꢀD þ 10:5 (c
0.57, MeOH). The molecular formula was
determined to be C34H46O11 by HR-FAB-
MS at m/z 653.2935 [M þ Na]þ. 1H NMR
spectrum gave three methyl signals at dH
1.28 (3H, s, 19-Me), 2.07 (3H, s, 18-Me),
and 2.40 (3H, s, 21-Me) and one group of
AA0BB0 proton signals at dH 7.23 (2H, d,
J ¼ 8.6 Hz, 40,60-H) and 8.28 (2H, d,
J ¼ 8.6 Hz, 30,70-H). The 13C NMR spec-
trum of 1 (Table 1) showed one anomeric
carbon signal at dC 96.5 and two aromatic
carbon signals at dC 116.2 and 132.4
whose intensities are twice as much as the
ordinary carbon signals, which indicated
the presence of a p-substituted benzyl
system and a monosaccharide. All the
carbon signals assignable to the aglycone
moiety were similar to those of qinyang-
shengenin moiety of the literature [1].
In our study, two new steroidal
glycosides with deoxysugars were isolated
from the ethyl acetate extract of the roots of
C. otophyllum Schneid. Their structures
were identified as qinyangshengenin-3-O-
b-D-digitoxopyranoside (1) and rostrata-
mine-3-O-b-D-cymaropyranosyl-(1 ! 4)-
b-D-digitoxopyranoside (2). In this paper,
we report the isolation and structural
elucidation of the two new steroidal
*Corresponding authors. Email: baorongrui1026@163.com; lifen_w@163.com
q 2014 Taylor & Francis

286
X.-X. Yang et al.
O
O
3'
1
3'
6'
21
18
21
18
5'
1'
4'
5'
O
O
6'
7'
1'
O
O
2'
7'
4'
3
2'
20
20
OH
OH
HO
19
17
17
19
OH
OH
1
H
OH
H
OH
3
5
O
5
O
O
O
1''
1''
O
1
O
OH
OH
1'''
2
OH
OH
OCH3
Figure 1. Structures of compounds 1 and 2.
Thus, compound 1 was considered a
qinyangshengenin-3-O-glycoside.
the presence of one 2,6-dideoxy-sugar with
b configuration at the anomeric carbon.
Finally, one digitoxopyranosyl was eluci-
dated by comparison of its NMR data with
those in the literature [1,2] and in the
hydrolysis experiment. The NMR data for
the sugar moiety were assigned by HMBC
and HSQC spectra, and are shown in
Table 1.
Besides signals assignable to the
aglycon, signals assignable to sugars were
observed. Proton signals of the sugar
moiety were assigned to one secondary
methyl at dH 1.60 (3H, d, J ¼ 6.0 Hz): one
anomeric proton at dH 5.51 (1H, br d,
J ¼ 9.0 Hz), whose multiplicities showed
Table 1. 13C (150 Hz) NMR spectral data of compouds 1 and 2 (d in ppm).
Compound 1
Compound 2
No.
13C
No.
13C
No.
13C
No.
13C
1
2
3
4
5
6
7
8
39.2
29.9
77.6
39.1
139.4
119.1
34.8
74.3
44.5
37.4
25.2
73.4
58.4
89.6
33.9
33.2
92.5
10.9
18.2
209.8
27.8
10
20
30
40
50
60
70
165.4
122.0
132.4
116.2
163.6
116.2
132.4
Digitoxose
96.5
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
39.9
29.8
77.6
38.9
139.4
119.2
34.7
74.2
44.5
37.4
25.0
73.6
58.1
89.5
34.7
33.8
92.4
10.7
18.1
209.9
27.7
10
165.8
119.2
144.9
135.0
128.6
129.3
130.6
20
30
40
50(90)
60(80)
70
Digitoxose
9
100
200
300
400
500
600
100
96.4
39.1
67.5
83.5
68.6
18.6
10
11
12
13
14
15
16
17
18
19
20
21
39.1
68.8
74.3
70.4
200
300
400
500
18.8
600
Cymarose
1000
2000
99.7
35.6
78.8
74.1
70.9
18.9
58.0
3000
4000
5000
6000
3000-OMe

Journal of Asian Natural Products Research
287
The location of the sugar moiety was
confirmed by the HMBC spectrum, in
which the correlation from H-100 at dH 5.51
to C-3 at dC 77.6 of the aglycone was
observed. Thus, compound 1 was finally
determined as qinyangshengenin-3-O-b-
D-digitoxopyranoside.
aropyranosyl-(1 ! 4)-b-D-digitoxopyra-
noside.
3. Experimental
3.1 General experimental procedures
Optical rotations were measured on a P-E
241 MC (Perkin-Elmer Co., Jena,
Germany). IR spectra were recorded on a
Bruker IFS-55 infrared spectrophotometer
with KBr disks (Bruker Co., Zurich,
Switzerland). The NMR spectral data
were recorded on Bruker AV-600
Compound 2 was obtained as a white
20
amorphous powder, with ½aꢀD þ 9:9 (c
0.42, MeOH). The molecular formula was
determined to be C43H60O13 by HR-FAB-
MS at m/z 807.3934 [M þ Na]þ. The
NMR signals of 2 were quite similar to
those of 1 except for the obvious existence
of two more proton signals assigned to one
double bond at d 6.83 (1H, d, J ¼ 16.0 Hz,
20-H) and 8.00 (1H, d, J ¼ 16.0 Hz, 30-H)
and one more anomeric carbon signal at d
99.7. The 13C NMR spectrum of 2
(Table 1) assignable to the aglycone
moiety was identical to that of rostrata-
mine moiety in the literature [2]. Hence,
compound 2 was also considered a
rostratamine-3-O-glycoside.
1
(600 MHz for H NMR and 150 MHz for
13C NMR; Bruker, Fallanden, Switzer-
land) in dimethyl sulfoxide-d6 with TMS
as the internal standard. The HR-FAB-MS
data were obtained on the Micross Mass
Autospec-UltimaE TOF mass spectropho-
tometer (Bruker Co., Zurich, Switzerland).
Chromatography was performed on silica
gel (200–300 mesh; Qingdao Haiyang
Chemical Factory, Qingdao, China),
Sephadex LH-20 (Pharmacia, Piscataway,
NJ, USA), and reversed-phase HPLC
(Shimadzu LC-10 AVP, Kyoto, Japan).
Proton signals of the sugar moiety
were also assigned to two secondary
methyl groups at d 1.44 (d, J ¼ 6.1 Hz)
and 1.45 (d, J ¼ 6.1 Hz): one methoxyl
group at d 3.45 (s) and two anomeric
protons at d 5.11 (dd, J ¼ 9.5, 1.3 Hz) and
5.46 (dd, J ¼ 9.5, 1.3 Hz), whose multi-
plicities showed the presence of two 2,6-
dideoxy-sugar units. The 13C NMR
spectrum gave two anomeric carbon
signals at d 96.4 and 99.7, and the 13C
shifts of each sugar unit were assigned
unambiguously by HMBC and HMQC
spectra, and are shown in Table 1. Finally,
one digitoxopyranosyl and one cymaro-
pyranosyl were elucidated by comparison
of NMR data with those in the literature
[1,2] and in the hydrolysis experiment.
The linkage of the sugar moiety was
determined by the HMBC correlations
from H-1000 at d 5.11 to C-400 at d 83.5 and
from H-100 at d 5.46 to C-3 at dC 77.6 of the
aglycone. Thus, compound 2 was finally
determined as rostratamine-3-O-b-D-cym-
3.2 Plant material
The roots of C. otophyllum Schneid were
obtained in October 2008 in Kunming,
Yunnan Province, China. A voucher speci-
men (No. 6050) has been identified and
deposited in the Liaoning University of
Traditional Chinese Medicine.
3.3 Extraction and isolation
Roots of C. otophyllum were extracted
three times by means of reflux with hot
95% EtOH for 3 h each, and the combined
solution was concentrated in vacuo to give
a syrup (1100 g), followed by suspension
in water. The suspension was then
extracted with petroleum ether, chloro-
form, acetic ether, and n-butanol, succes-
sively. The acetic ether (90 g) was further
fractionated by silica gel column chroma-
tography eluted with chloroform/CH3OH

288
X.-X. Yang et al.
(100:1 ! 1:1, v/v) to obtain 10 fractions.
Fraction six (632 mg) was further separ-
ated by HPLC eluted with MeOH (68% v/
v, 4 ml/min, 210 nm, RT ¼ 30 min for 1
and 45 min for 2) to give compounds 1
(12 mg) and 2 (10 mg).
5.11 (1H, dd, J ¼ 9.5, 1.3 Hz, 1000-H),
5.17–5.19 (1H, m, 6-H), 5.28 (1H, dd,
J ¼ 10.6, 6.0 Hz, 12-H), 5.46 (1H, dd,
J ¼ 9.5, 1.3 Hz, 100-H), 6.83 (1H, d,
J ¼ 16.0 Hz, 20-H), 7.28–7.30 (3H, m,
60,70,80-H), 7.62–7.64 (2H, m, 50,90-H),
8.00 (1H, d, J ¼ 16.0 Hz, 30-H); for 13C
NMR spectral data, see Table 1; HR-ESI-
MS: m/z 807.3934 [M þ Na]þ (calcd for
C43H60O13Na, 807.3932).
3.3.1 Qinyangshengenin-3-O-b-D-
digitoxopyranoside (1)
20
White amorphous powder; ½aꢀD þ 10:5
(c ¼ 0.57, MeOH); IR (KBr) nmax: 3444,
2930, 1731, 1640, 1451, 1164, 1104, 1062,
1
988 cm21; H NMR d: 1.28 (3H, s, 19-
3.4 Acidhydrolysisofcompounds1and2
Me), 1.60 (d, J ¼ 6.0 Hz, 600-H), 2.07 (3H,
s, 18-Me), 2.40 (3H, s, 21-Me), 3.51 (dd,
J ¼ 9.5, 2.9 Hz, 400-H), 3.62 (dd, J ¼ 9.0,
2.4 Hz, 400-H), 3.86–3.88 (1H, m, 3a-H),
4.29–4.31 (1H, m, 300-H), 4.30–4.32 (1H,
m, 500-H), 5.13 (1H, br t, 6-H), 5.32–5.34
(1H, m, 12-H), 5.51 (1H, br d, J ¼ 9.0 Hz,
100-H), 7.23 (2H, d, J ¼ 8.6 Hz, 40,60-H),
8.28 (2H, d, J ¼ 8.6 Hz, 30,70-H); for 13C
NMR spectral data, see Table 1; HR-FAB-
MS: m/z 653.2935 [M þ Na]þ (calcd for
C34H46O11Na, 653.2938).
A solution of 1 or 2 (10 mg) in MeOH
(5 ml) was treated separately with 0.1 N
H2SO4 (5 ml) at 508C for 15 min. After
H2O (5 ml) was added, the mixture was
evaporated to 10 ml under reduced press-
ure to remove MeOH and then kept in
608C for another 30 min. The hydrolyzed
mixture was neutralized to pH 7 with Ba
(OH)2 and condensed to dryness under
reduced pressure. The monosaccharide
was isolated and determined to be
cymarose and digitoxose by means of
preparative TLC developed by CHCl3–
MeOH–H2O (20:3:1, Rf ¼ 0.55 and 0.26
for cymarose and digitoxse, respectively),
compared with an authentic sample. The
absolute configurations of cymarose and
3.3.2 Rostratamine-3-O-b-D-
cymaropyranosyl-(1 ! 4)-b-D-
digitoxopyranoside (2)
20
White amorphous powder; ½aꢀD þ 9:9
digitoxose were considered D-form by
20
(c ¼ 0.42, MeOH); IR (KBr) nmax: 3444,
their optical values: cymarose: ½aꢀD
þ
20
2930, 1731, 1640, 1451, 1164, 1104, 1062,
49:6 (c 0.02, H2O), digitxose: ½aꢀD þ 46:3
(c 0.02, H2O).
1
988 cm21; H NMR d: 1.34 (3H, s, 19-
Me), 1.44 (1H, d, J ¼ 6.1 Hz, 6000-H), 1.45
(1H, d, J ¼ 6.1 Hz, 600-H), 2.04 (3H, d,
J ¼ 6.0 Hz, 18-Me), 2.42 (3H, s, 21-Me),
3.45 (3H, s, 3000-OCH3), 3.46 (1H, dd,
J ¼ 3.0, 9.5 Hz, 4000-H), 3.71 (1H, d,
J ¼ 3.0 Hz, 3000-H), 3.84–3.86 (1H, m, 3-
H), 4.08–4.10 (1H, m, 5000-H), 4.29–4.31
(1H, m, 500-H), 4.62–4.64 (1H, m, 300-H),
References
[1] G. Chen, N. Xu, Z.F. Li, Q.H. Zhang,
H.H. Wu, and Y.H. Pei, J. Asian Nat. Prod.
Res. 12, 453 (2010).
[2] G. Chen, N. Xu, and Y.H. Pei, J. Asian Nat.
Prod. Res. 11, 177 (2009).