Two new steroidal glycosides from Cynanchum wallichii

Article abstract of DOI:10.1080/10286020.2014.915816

Two new C21 steroidal glycosides were isolated from Cynanchum wallichii Wight. Their structures were elucidated as caudatin-3-O-β-d-glucopyranosyl- (1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-cymaropyranosyl-(1 → 4)-β-d-digitoxopyranoside (1) and caudatin-3

Full text of DOI:10.1080/10286020.2014.915816

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Two new steroidal glycosides from
Cynanchum wallichii
Ding Wanga, Wen-Hao Pana, Yong-Rui Baob, Xin-Xin Yangb & Xian-
Sheng Mengb
a Department of Pharmacy, Affiliated Hospital of Liaoning
University of Traditional Chinese Medicine, Shenyang 110032,
China
b College of Pharmacy, Liaoning University of Traditional Chinese
Medicine, Dalian 116600, China
Published online: 02 May 2014.
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To cite this article: Ding Wang, Wen-Hao Pan, Yong-Rui Bao, Xin-Xin Yang & Xian-Sheng Meng
(2014) Two new steroidal glycosides from Cynanchum wallichii, Journal of Asian Natural Products
Research, 16:7, 759-763, DOI: 10.1080/10286020.2014.915816
To link to this article: http://dx.doi.org/10.1080/10286020.2014.915816
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 7, 759–763, http://dx.doi.org/10.1080/10286020.2014.915816
Two new steroidal glycosides from Cynanchum wallichii
Ding Wanga, Wen-Hao Pana, Yong-Rui Baob*, Xin-Xin Yangb and Xian-Sheng Mengb
aDepartment of Pharmacy, Affiliated Hospital of Liaoning University of Traditional Chinese
Medicine, Shenyang 110032, China; College of Pharmacy, Liaoning University of Traditional
Chinese Medicine, Dalian 116600, China
b
(Received 20 November 2013; final version received 14 April 2014)
Two new C21 steroidal glycosides were isolated from Cynanchum wallichii Wight. Their
structures were elucidated as caudatin-3-O-b-D-glucopyranosyl-(1 ! 4)-b-D-oleandro-
pyranosyl-(1 ! 4)-b-D-cymaropyranosyl-(1 ! 4)-b-D-digitoxopyranoside (1) and
caudatin-3-O-b-D-glucopyranosyl-(1 ! 4)-b-D-cymaropyranosyl-(1 ! 4)-b-D-olean-
dropyranosyl-(1 ! 4)-b-D-cymaropyranosyl-(1 ! 4)-b-D-digitoxopyranoside (2) by
spectroscopic methods including 1D and 2D NMR experiments.
Keywords: Cynanchum wallichii; C21 steroidal glycosides; 2,6-dideoxy-pyranose
1. Introduction
The 13C NMR spectrum of 1 (Table 1)
showed four anomeric carbon signals at
dC 96.4, 99.7, 101.9, and 104.5. The
carbon signals assignable to the aglycone
moiety were similar to those of caudatin
moiety of otophylloside B [4,5]. Thus,
compound 1 was considered to be a
caudatin-3-O-glycoside.
Cynanchum wallichii Wight, also named
Duanjieshen, is a traditional Chinese
medicine distributed extensively over
southwest China. In the famous Chinese
prescription “hulisan”, C. wallichii has
been used as the primary drug to treat
arthrophlogosis and injury from fall or
fracture. Several C21 steroidal glyco-
sides with 2,6-dideoxysugars were iso-
lated from the title plant [1–3]. In order
to find out more C21 steroidal glyco-
sides in this folk medicine, systematic
research on the roots of C. wallichii was
carried out and two new steroidal
glycosides were found. In this paper,
we report isolation and structural eluci-
dation of two new glycosides 1 and 2
(Figure 1).
Besides signals assignable to the
aglycon, signals assignable to sugars
were also observed. Proton signals of the
sugar moiety were assigned to three
secondary methyl groups at dH 1.30 (3H,
d, J ¼ 6.0 Hz), 1.42 (3H, d, J ¼ 6.0 Hz),
and 1.69 (3H, d, J ¼ 6.0 Hz); two meth-
oxyl groups at dH 3.52 (s) and 3.56(s); four
anomeric protons at dH 4.65 (1H, br d,
J ¼ 9.0 Hz), 5.12 (1H, br d, J ¼ 8.0 Hz),
5.16 (1H, br d, J ¼ 9.0 Hz), and 5.45 (1H,
br d, J ¼ 9.0 Hz), whose multiplicities
showed the presence of three 2,6-dideoxy-
sugars with b configuration at the anome-
ric carbon. Finally, one glucopyranosyl,
one oleandropyranosyl, one cymaropyra-
nosyl, and one digitoxopyranosyl were
elucidated by comparing NMR data with
those in the literatures [6,7] and the
2. Results and discussion
Compound 1 was obtained as white
20
amorphous powder, with ½aꢀD þ 15:5
(c ¼ 0.61, MeOH). The molecular formula
was determined to be C54H86O21 by HR-
FAB-MS at m/z 1093.5552 [M þ Na]þ.
*Corresponding author. Email: baorongrui1026@163.com
q 2014 Taylor & Francis

760
D. Wang et al.
O
7'
O
R1
1''
5'
O
O
OH
1'''
O
3'
4'
6'
1'
O
2'
O
O
OCH3
1''''
OCH3
OH
OH
17
OH
O
O
OH
1
1'''''
OH
O
OH
OH
H
1''
O
OH
1'''
O
5
RO
3
R2
O
O
OCH3
OCH3
1''''
O
O
1
R = R1
R = R2
1'''''
O
OH
OCH3
O
2
OH
1''''''
H
OH
Figure 1. Structures and key HMBC correlations of compounds 1 and 2.
Table 1. 1H and 13C NMR spectral data of compound 1 in C5D5N (600 MHz).
No.
dC
dH (J in Hz)
No.
dH (J in Hz)
dC
1
2
3
4
5
6
7
8
39.0
29.9
77.7
39.3
139.4
119.2
34.8
74.3
44.6
37.4
25.1
72.6
58.0
89.5
33.9
32.9
92.4
10.7
18.2
209.4
27.6
166.0
114.2
165.4
38.2
20.8
20.9
16.5
2.43 (m) and 2.52 (m)
1.78 (m) and 2.06 (m)
3.86 (m)
b-D-Dgt
100
200
5.45 (br d, 9.0)
96.4
38.9
67.6
83.3
68.6
18.5
99.7
36.7
77.6
83.1
69.0
18.5
58.9
101.9
37.6
79.3
83.2
72.0
18.7
57.2
104.5
75.7
78.7
71.9
78.0
63.1
a
300
4.60 (m)
3.51 (br d, 9.0)
4.27 (m)
a
400
–
5.30 (br t)
2.30 (m) and 2.41 (m)
–
1.70 (m)
–
2.12 (m) and 2.25 (m)
500
600
1.42 (d, 6.0)
5.16 (br d, 9.0)
b-D-Cym
1000
a
2000
9
3000
3.87 (m)
3.38 (br d, 9.0)
4.17 (m)
10
11
12
13
14
15
16
17
18
19
20
21
10
4000
500
5.03 (dd, 11.6, 3.9)
–
6000
1.30 (d, 6.0)
3.56 (s)
4.65 (br d, 9.0)
3000-OMe
10000
20000
30000
40000
50000
60000
30000-0O0000Me
100000
200000
300000
4
–
2.07 (m, 2H)
2.48 (m, 2H)
–
2.02 (s, 3H)
1.30 (s, 3H)
b-D-Ole
a
3.68b
3.72 (m)
3.60 (m)
1.69 (d, 6.0)
3.52 (s)
5.12 (br d, 8.0)
4.01b
4.24 (m)
3.63
3.97 (m)
4.35 (dd, 11.4, 5.4)
4.53 (br d 11.4)
–
2.50 (s, 3H)
b-D-Glu
–
–
–
20
30
40
1.10 (m)
0.93 (d, 6.0, 3H)
0.95 (d, 6.0, 3H)
2.26 (s, 3H)
500000
600000
50
60
70
a Overlapped with other sugar signals.
b Overlapped with other signals.

Journal of Asian Natural Products Research
761
D-glucopyranosyl-(1 ! 4)-b-D-oleandro-
pyranosyl-(1 ! 4)-b-D-cymaropyranosyl-
(1 ! 4)-b-D-digitoxopyranoside.
hydrolysis experiment. The NMR data for
the sugar moiety were assigned by HMBC
and HSQC spectra and are shown in
Table 1.
Compound 2 was obtained as white
20
amorphous powder, with ½aꢀD þ 9:3
The sequence of the sugar moiety was
determined by the HMBC spectrum, in
which00c00orrelations from H-100000 at dH 5.12
to C-4 at dC 83.2, from H-10000 at dH 4.65
to C-4000 at dC 83.1, from H-1000 at dH 5.16
to C-400 at dC 83.3, and from H-100 at dH
5.45 to C-3 at dC 77.7 of the aglycone
were observed. Thus, compound 1 was
finally determined as caudatin-3-O-b-
(c ¼ 0.32, MeOH). The molecular formula
was determined to be C61H98O24 by HR-
ESI-MS at m/z 1237.6342 [M þ Na]þ.
The 13C NMR spectrum of 2 (Table 2)
assignable to the aglycone moiety was
identical to that of compound 1. Hence,
compound 2 was also considered to be a
caudatin-3-O-glycoside.
Table 2. 1H and 13C NMR spectral data of compound 2 in C5D5N (600 MHz).
No.
dC
dH (J in Hz)
No.
dH (J in Hz)
dC
1
2
3
4
5
6
7
8
39.0
29.9
77.7
39.3
139.4
119.2
34.8
74.3
44.6
37.4
25.1
72.6
58.0
89.5
33.9
32.9
92.4
10.7
18.2
209.4
27.6
166.0
114.2
165.4
38.2
20.8
20.9
16.5
2.43 (m) and 2.52 (m)
1.78 (m) and 2.06 (m)
3.86 (m)
b-D-Dgt
100
200
5.45 (br d, 9.0)
96.4
38.9
67.6
83.2
68.6
18.5
99.7
36.7
77.6
83.4
69.0
18.5
58.9
101.9
37.8
78.7
82.6
71.7
18.7
58.0
98.4
36.8
78.3
83.2
69.7
18.7
58.9
106.5
75.3
78.4
71.8
78.4
63.1
a
300
4.60 (m)
3.51 (br d, 9.0)
4.27 (m)
a
400
–
5.30 (br t)
2.30 (m) and 2.41 (m)
–
1.70 (m)
–
2.12 (m) and 2.25 (m)
500
600
1.42 (d, 6.0)
5.16b
b-D-Cym
1000
a
2000
9
3000
3.87 (m)
3.38 (br d, 9.0)
4.17 (m)
10
11
12
13
14
15
16
17
18
19
20
21
10
4000
500
5.03 (dd, 11.6, 3.9)
–
6000
1.30 (d, 6.0)
3.56 (s)
4.66 (br d, 9.0)
3000-OMe
10000
–
2.07 (m, 2H)
2.48 (m, 2H)
–
2.02 (s, 3H)
1.30 (s, 3H)
b-D-Ole
b-D-Cym
b-D-Glu
a
20000
30000
3.51 (m)
3.46 (m)
3.49b
40000
50000
60000
1.40 (m)
3.50 (s)
–
2.50 (s, 3H)
30000-0O0000Me
100000
2
5.26b
a
–
–
–
20
300000
400000
500000
600000
3000-OMe
1000000
2000000
3000000
4000000
5000000
6000000
4.14 (m)
3.40 (m)
4.26 (m)
1.61 (d, 6.0)
3.52 (s)
4.91 (br d, 8.0)
3.98 (m)
3.96 (m)
4.17 (m)
4.23 (m)
4.37 (dd, 11.4, 5.4)
4.57 (br d, 11.4)
30
40
1.10 (m)
0.93 (d, 6.0, 3H)
0.95 (d, 6.0, 3H)
2.26 (s, 3H)
50
60
70
a Overlapped with other sugar signals.
b Overlapped with other signals.

762
D. Wang et al.
1
Proton signals of the sugar moiety
(600 MHz for H and 150 MHz for 13C)
with TMS as internal standard (Bruker
Co.). HR-FAB-MS data were measured on
Micross Mass Autospec-Ultima-TOF
spectrometer (Bruker Co.). Silica gel
GF254 for TLC and silica gel (200–
300 mesh) for column chromatography
were obtained from Qingdao Marine
Chemical Company, Qingdao, China.
HPLC was carried on Shimadzu LC-8A
(Shimadzu, Kyoto, Japan) and the detector
was Shimadzu SPD-10A. An analytical
reversed phase C18 column (Diamonsil
C18 B 4.6 mm £ 250 mm, Zuanshi Com-
pany, Shanghai, China) and a preparative
reversed phase C18 column (Inertsil Prep-
ODS B 10 mm £ 250 mm, Zuanshi
Company) were employed.
were also assigned to four secondary
methyl groups at d 1.30 (d, J ¼ 6.0 Hz),
1.40 (m), 1.42 (d, J ¼ 6.0 Hz), and 1.61 (d,
J ¼ 6.0 Hz); three methoxyl groups at d
3.50 (s), 3.52 (s), and 3.56 (s); and five
anomeric protons at d 4.66 (br d,
J ¼ 9.0 Hz), 4.91 (br d, J ¼ 8.0 Hz), 5.16,
5.26, and 5.45 (br d, J ¼ 9.0 Hz), whose
multiplicities showed the presence of four
2,6-dideoxy-sugar units. The 13C shifts of
each sugar unit were assigned unambigu-
ously by HMBC and HMQC spectra and
are shown in Table 2. The NMR data for
the sugar moiety of compound 2 were
almost identical to those of otophylloside
M [7] except that the inner cymaropyr-
anosyl unit disappeared and one digitox-
opyranosyl moiety [2] appeared. The
linkage of the sugar units was finally
further confirmed by the HMBC corre-
lations from H-1000000 at dH 4.91 to C-400000 at
dC 83.2, from H-100000 at dH 5.26 to C-40000
at dC 82.6, from H-10000 at dH 4.66 to C-4000
at dC 83.4, from H-1000 at dH 5.16 to C-400 at
dC 83.2, and from H-100 at dH 5.45 to C-3 at
dC 77.7 of the aglycone. Hence, compound
2 was elucidated as caudatin-3-O-b-D-
glucopyranosyl-(1 ! 4)-b-D-cymaropyr-
anosyl-(1 ! 4)-b-D-oleandropyranosyl-
(1 ! 4)-b-D-cymaropyranosyl-(1 ! 4)-
b-D-digitoxopyranoside.
3.2 Plant material
The roots of C. wallichii were collected in
August 2011 at Xinxiang City, Henan
Province, China and was identified by
Prof. Yanjun Zhai. A voucher specimen
was deposited in the Liaoning University
of Traditional Chinese Medicine (No.
20110510).
3.3 Extraction and isolation
The cytotoxic activities of compounds
1 and 2 were bioassayed against HL-60
cell lines with the same procedures
described in the literature [3] and their
IC50 values were 47.2 and 26.4 mM,
respectively
Roots of C. wallichii were extracted three
times by means of reflux with hot 95%
EtOH for 2 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
(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 18 min for 1
and 25 min for 2) to give compounds 1
(20 mg) and 2 (15 mg).
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

Journal of Asian Natural Products Research
763
3.3.1 Caudatin-3-O-b-D-glucopyranosyl-
(1 ! 4)-b-D-oleandropyranosyl-(1 ! 4)-
b-D-cymaropyranosyl-(1 ! 4)-b-D-
digitoxopyranoside (1)
ure to remove MeOH and then kept in
608C for another 30min. 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 0.55, 0.50, 0.26 for cymarose,
oleandrose, digitoxse, respectively), com-
pared with authentic sample. The absolute
configurations of cymarose, digitoxose, and
oleandrose were considered to be D-form by
20
White amorphous powder; ½aꢀD þ 15:5
(c ¼ 0.61, MeOH); IR (KBr) nmax: 3444,
2930, 1731, 1640, 1451, 1164, 1104, 1062,
and 988 cm–1; for 1H and 13C NMR
spectral data, see Table 1; HR-ESI-MS: m/
z
C54H86O21Na, 1093.5554).
1093.5552 [M þ Na]þ (calcd for
20
3.3.2 Caudatin-3-O-b-D-glucopyranosyl-
(1 ! 4)-b-D-cymaropyranosyl-(1 ! 4)-
b-D- oleandropyranosyl-(1 ! 4)-b-D-
cymaropyranosyl-(1 ! 4)-b-D-
digitoxopyranoside (2)
their optical values: cymarose: ½aꢀD þ 49:9
20
(c ¼ 0.01, H2O), oleandrose: ½aꢀ2D0 2 15:2
(c ¼ 0.01, H2O), digitxose: ½aꢀD þ 47:38
(c ¼ 0.02, H2O).
20
White amorphous powder; ½aꢀD þ 9:3
(c ¼ 0.32, MeOH); IR (KBr) nmax: 3444,
2930, 1731, 1640, 1451, 1164, 1104, 1062,
and 988 cm–1; for 1H and 13C NMR
spectral data, see Table 2; HR-ESI-MS:
m/z 1237.6342 [M þ Na]þ (calcd for
C61H98O24Na, 1237.6340).
References
[1] Z.X. Zhang and J. Zhou, Acta Chim. Sin.
41, 1058 (1983).
[2] G. Chen, N. Xu, and Y.H. Pei, J. Aisan Nat.
Prod. Res. 11, 177 (2009).
[3] G. Chen, N. Xu, Z.F. Li, Q.H. Zhang, H.H.
Wu, and Y.H. Pei, J. Aisan Nat. Prod. Res.
12, 453 (2010).
[4] Q.Z. Mu, J.R. Lu, and Q.L. Zhou, Sci. Sin.
[B] 29, 295 (1986).
[5] X.X. Ma, D. Wang, Y.J. Zhang, and C.R.
Yang, Steroids 76, 1003 (2011).
[6] L.Q. Wang, J.H. Wang, Y.M. Shen, and
J. Zhou, Chin. Chem. Lett. 18, 1235 (2007).
[7] X.X. Ma, F.T. Jiang, Q.X. Yang, X.H. Liu,
Y.J. Zhang, and C.R. Yang, Steroids 72,
778 (2007).
3.4 Acid hydrolysis of compounds 1
and 2
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-