Identification of new qingyangshengenin and caudatin glycosides from the roots of Cynanchum otophyllum

Article abstract of DOI:10.1016/j.steroids.2011.03.019

HPLC analysis of the roots of Cynanchum otophyllum Scheind (Asclepiadaceae) led to the isolation of six new pregnane glycosides, specifically otophyllosides N-P (2-4) and otophyllosides Q-S (7-9), in addition to the identification of three known C-21 steroidal glycosides, otophylloside A (1), otophylloside B (5) and caudatin 3-O-β-d-glucopyranosyl-(1→4)-β- d-oleandropyranosyl-(1→4)-β-d-cymaropyranosyl-(1→4) -β-d-cymaropyranoside (6). The structure of each glycoside was determined by detailed spectroscopic analysis and chemical methods. All compounds contain qingyangshengenin or caudatin aglycones and a straight sugar chain consisting of 4-7 hexosyl moieties with the mode of 1→4 linkage. The optically isomeric monosaccharides, d- and l-cymarose, coexisted in both otophyllosides R (8) and S (9).

Full text of DOI:10.1016/j.steroids.2011.03.019

Steroids 76 (2011) 1003–1009
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Identification of new qingyangshengenin and caudatin glycosides from the roots
of Cynanchum otophyllum
Xiao-Xia Maa,b, Dong Wanga, Ying-Jun Zhanga,∗, Chong-Ren Yanga,∗∗
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, PR China
b Yunnan University of Traditional Chinese Medicine, Kunming 650500, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
HPLC analysis of the roots of Cynanchum otophyllum Scheind (Asclepiadaceae) led to the isola-
tion of six new pregnane glycosides, specifically otophyllosides N-P (2–4) and otophyllosides Q-S
(7–9), in addition to the identification of three known C-21 steroidal glycosides, otophylloside A (1),
otophylloside B (5) and caudatin 3-O-␤-d-glucopyranosyl-(1→4)-␤-d-oleandropyranosyl-(1→4)-␤-d-
cymaropyranosyl-(1→4)-␤-d-cymaropyranoside (6). The structure of each glycoside was determined by
detailed spectroscopic analysis and chemical methods. All compounds contain qingyangshengenin or
caudatin aglycones and a straight sugar chain consisting of 4-7 hexosyl moieties with the mode of 1→4
linkage. The optically isomeric monosaccharides, d- and l-cymarose, coexisted in both otophyllosides R
(8) and S (9).
Received 8 December 2010
Received in revised form 26 March 2011
Accepted 29 March 2011
Available online 7 April 2011
Keywords:
Cynanchum otophyllum
Asclepiadaceae
Pregnane glycosides
Otophyllosides
© 2011 Elsevier Inc. All rights reserved.
1. Introduction
on a Bruker DRX-500 instrument with TMS as the internal stan-
dard. MS data were detected on a VG Auto Spec-3000 spectrometer.
Cynanchum otophyllum Schneid (Chinese name Qingyangshen)
has been used for the treatment of some nervous system and men-
tal disorders, such as epilepsy, depression and Menier’s syndrome.
The C-21 steroidal glycosides are thought to be the bioactive prin-
ciples [1–3]. Previously, we reported the isolation and structure
elucidation of six pregnane glycosides from the roots [4]. In fur-
ther studies, Cynanchum otophyllum was subjected to preparative
HPLC and nine C-21 steroidal glycosides were purified, including
six new otophyllosides N-S (2–4 and 7–9). Their structures were
determined by detailed spectroscopic analysis and chemical meth-
ods. This paper describes the isolation and structural elucidation of
the new compounds.
Preparative HPLC was performed on Waters Delta 600-2487 Dual
␭ Absorbance Detector (Manual-injection, Analytical 7725i, Semi-
prep 3725i-119). Silica gel HF254 prepared for TLC and silica gel
(200–300 mesh) for column chromatography (CC) were obtained
from Qingdao Marine Chemical Company (Qingdao, China). A
preparative reverse phase C18 column (Agilent ZORBAX StableBond
C-18 ∅ 21.2 mm × 250 mm, 7 ␮m) and an analytical reversed phase
C
18 column (Agilent ZORBAX StableBond C-18 ∅ 4.6 mm × 250 mm,
5 ␮m) were employed.
2.2. Plant material
The roots of C. otophyllum Schneid were collected in Eryuan
County, in the northwest of Yunnan province of China, and iden-
tified by Prof. C.R. Yang (Kunming Institute of Botany, Chinese
Academy of Sciences). An example specimen is available at the
herbarium of Kunming Institute of Botany.
2. Experimental
2.1. General methods
Melting points were measured on an XRC-I micromelting point
apparatus and were uncorrected. Optical rotations were obtained
using a JASCO P-1020 automatic digital polarimeter. IR spectra were
determined using a Bruker Tensor 27 spectrometer in KBr pel-
lets. NMR spectra were performed in C5D5N and were recorded
2.3. Extraction and isolation
The components of the air-dried roots of C. otophyllum (10 kg)
were extracted by three consecutive treatments of 90% EtOH at
room temperature. After removal of the organic solvent by vacuum,
the residue was suspended in water and partitioned with CHCl3 to
yield a CHCl3 fraction (155 g). As described in our previous paper
[4], part of the CHCl3 fraction (150 g) was subjected to a silica gel
CC and eluted with CHCl3–MeOH (10:1.5) to give five fractions (Frs.
∗
Corresponding author. Tel.: +86 871 5223235; fax: +86 871 5150124.
Corresponding author.
E-mail addresses: zhangyj@mail.kib.ac.cn (Y.-J. Zhang),
∗∗
cryang@mail.kib.ac.cn (C.-R. Yang).
0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.03.019

1004
X.-X. Ma et al. / Steroids 76 (2011) 1003–1009
Table 1
13C NMR data for the aglycone moieties of compounds 2–4 and 7–9 (ı in ppm, in C5D5N, 100 MHz).
No.
2
3
4
7
8
9
No.
2
3
4
7
8
9
1
2
3
4
5
6
7
8
39.3
29.9
77.6
38.9
39.3
29.9
77.6
38.9
39.3
29.9
77.7
38.9
39.3
29.9
77.7
39.0
39.2
29.9
77.6
39.0
39.2
29.9
77.7
39.0
15
16
17
18
19
20
21
1ꢀ
33.9
33.2
92.5
10.9
18.2
33.9
33.2
92.5
10.9
18.2
33.9
33.2
92.5
10.9
18.2
33.9
33.0
92.4
10.8
18.2
209.5
27.6
166.0
114.2
165.5
38.2
33.8
33.0
92.4
10.8
18.2
209.5
27.6
166.0
114.2
165.5
38.2
33.8
33.0
92.4
10.8
18.2
209.5
27.6
166.0
114.2
165.5
38.2
139.4
119.2
34.8
74.3
44.5
37.4
25.2
73.4
58.4
89.6
139.4
119.2
34.8
74.3
44.5
37.4
25.2
73.4
58.4
89.6
139.4
119.2
34.8
74.4
44.5
37.4
25.2
73.4
58.4
89.6
139.3
119.3
34.8
74.3
44.6
37.4
25.1
72.6
58.0
89.5
139.3
119.2
34.8
74.3
44.6
37.4
25.1
72.6
58.0
89.5
139.3
119.2
34.8
74.3
44.6
37.4
25.1
72.6
58.0
89.5
209.9
27.9
209.9
27.9
209.8
27.8
165.4
122.0
132.5
116.2
163.7
116.2
132.5
165.4
122.0
132.5
116.3
163.7
116.3
132.5
165.4
122.0
132.4
116.2
163.6
116.2
132.5
9
2ꢀ
10
11
12
13
14
3ꢀ
4ꢀ
5ꢀ
20.9
21.0
16.5
20.9
20.9
16.5
20.9
20.9
16.5
6ꢀ
7ꢀ
1–5). Further purification was focused on Fr. 1, 2 and 5, mainly by
preparative HPLC. Fr. 1 (18.8 g) was separated by silica gel CC with
CHCl3–MeOH mixture (95:5, 9:1) to give three fractions (Frs. A–C).
Fr. A was chromatographed on silica gel (petrol ether:acetone 2:1,
3:2) to yield 1 (23 mg) and 2 (10 mg). Fr. B was purified by prepar-
ative HPLC with MeOH:H2O (82:18, v/v) to give 5 (8 mg). Fr. C was
performed on preparative HPLC and was eluted with MeOH:H2O
(78:22, v/v) to yield 3 (29 mg). Fr. 2 (21.7 g) was subjected to silica
gel (CHCl3:MeOH 93:7, 9:1, 85:15) to give two fractions (Frs. I and
II). Fr. I was subjected to silica gel (petrol ether:acetone 2:3, 1:2)
to yield 8 (12 mg) and 9 (9 mg). Fr. II was purified by preparative
HPLC with MeOH:H2O (77:23, v/v) to give 6 (19 mg). Compounds
4 (18 mg) and 7 (14 mg) were obtained from the portions of Fr.
5 (20.0 g) by preparative HPLC purification through elution with
MeOH:H2O (82:18, v/v) and (65:35, v/v), respectively.
2.3.5. Otophylloside R (8)
White amorphous powder, mp 158–160 ◦C, [˛]D19 − 27.4◦
(c = 0.20, MeOH), IR (KBr) ꢀmax 3457, 2971, 2934, 1714, 1645, 1549,
1452, 1381, 1369, 1317, 1275, 1223, 1195, 1164, 1102, 1059, 1003,
913, 863, 823 cm−1. FAB-MS (negative ion mode) m/z 1515 [M−1]−,
1389 [M−127]−. HRESI-MS (negative ion mode) m/z 1551.7863
[M(C76H124O30)+Cl]− (calcd. 1551.7865). 1H and 13C NMR: see
Tables 1, 2 and 4.
2.3.6. Otophylloside S (9)
White amorphous powder, mp 155–158 ◦C, [˛]D19 − 41.7◦
(c = 0.16, MeOH), IR (KBr) ꢀmax 3453, 2971, 2934, 1715, 1644, 1549,
1452, 1382, 1369, 1317, 1276, 1224, 1195, 1165, 1102, 1058, 1003,
935, 913, 863, 821 cm−1. FAB-MS (negative ion mode) m/z 1515
[M−1]−, 1388 [M−1−127]−. HRESI-MS (negative ion mode) m/z
1551.7868 [M(C76H124O30)+Cl]− (calcd. 1551.7865). 1H and 13C
NMR: see Tables 1, 2 and 4.
2.3.1. Otophylloside N (2)
White amorphous powder, mp 163–165 ◦C, [˛]D18 + 7.7◦
(c = 0.20, MeOH), IR (KBr) ꢀmax 3446, 2971, 2932, 1713, 1610, 1594,
1516, 1452, 1382, 1369, 1308, 1275, 1164, 1093, 1004, 912, 866,
852, 772 cm−1. FAB-MS (negative ion mode) m/z 1061 [M−1]−,
HRESI-MS (negative ion mode) m/z 1061.5284 [M(C55H82O20)−H]−
(calcd. 1061.5321). 1H and 13C NMR: see Tables 1–3.
2.3.7. Acid hydrolysis of 2–4 and 7–9
Compounds 2–4 and 7–9 (each 5 mg) were subjected to acid
hydrolysis as described in a previous study [4]. The aglycones, as
well as monosaccharides, were detected by TLC analysis combined
with comparison to known samples. Qingyangshengenin was iden-
tified as the aglycone of compounds 2–4, and caudatin was revealed
to be the aglycones of compounds 7–9. Regarding the monosac-
charide component of each compound, cymarose and oleandrose
were detected in all of the six new glycosides (2–4 and 7–9);
glucose was detected in 4 and 7–9; digitoxose was detected only
in 2.
2.3.2. Otophylloside O (3)
White amorphous powder, mp 157–160 ◦C, [˛]D18 + 20.3◦
(c = 0.19, MeOH), IR (KBr) ꢀmax 3455, 2972, 2934, 1713, 1610,
1594, 1516, 1451, 1382, 1368, 1309, 1275, 1164, 1096, 1059,
1004, 987, 913, 865, 853, 772 cm−1. FAB-MS (negative ion mode)
m/z 1076 [M]−, 931 [M−1−144]−. HRESI-MS (negative ion mode)
2.3.8. Determination of glucose moiety configuration
Neutralized hydrolysates of 4 and 7–9 were treated as described
in a previous study [4], and the GC analysis of the corresponding
trimethylsilylated l-cysteine adducts confirmed a d-configuration
of the glucose molecules.
m/z 1111.5239 [M(C56H84O20)+Cl]− (calcd. 1111.5244). 1H and 13
C
NMR: see Tables 1–3.
2.3.3. Otophylloside P (4)
White amorphous powder, mp 200–202 ◦C, [˛]D17 + 13.2◦
(c = 0.19, MeOH), IR (KBr) ꢀmax 3439, 2932, 1711, 1634, 1610, 1551,
1515, 1449, 1383, 1368, 1308, 1275, 1164, 1097, 1060, 1004, 912,
854, 773 cm−1. FAB-MS (negative ion mode) m/z 1255 [M−1]−,
3. Results and discussion
Nine pregnane glycosides (1–9) were further isolated from
the CHCl3 extracts of the roots of C. otophyllum through open
column chromatography and preparative HPLC. Six of these were
otophyllosides N-S (2–4 and 7–9) and had not been previously
isolated. Acid hydrolysis indicated that compounds 2–4 and
7–9 were qingyangshengenin and caudatin glycosides, respec-
tively, which were confirmed by the comparison of their spectra
data with qingyangshengenin and caudatin [4]. Their structural
characteristics were determined by 1D and 2D spectroscopic
analysis as well as chemical methods. Concerning the three
known compounds, 1 and 5 were identified as otophyllosides
A and B, respectively, which are the active ingredients for the
HRESI-MS (negative ion mode) m/z 1255.5745 [M(C61
H
92O27)−H]−
(calcd. 1255.5747). 1H and 13C NMR: see Tables 1–3.
2.3.4. Otophylloside Q (7)
White amorphous powder, mp 202–204 ◦C, [˛]D18 − 0.0◦
(c = 0.22, MeOH), IR (KBr) ꢀmax 3442, 2969, 2933, 1714, 1643,
1550, 1452, 1381, 1369, 1317, 1276, 1224, 1195, 1164, 1098, 1059,
1004, 912, 865, 824 cm−1. FAB-MS (negative ion mode) m/z 1390
[M]−, 1262 [M−1−127]−, 1228 [M−162]−. HRESI-MS (negative ion
mode) m/z 1425.6831 [M(C68H110O29)+Cl]− (calcd. 1425.6821). 1
H
and 13C NMR: see Tables 1, 2 and 4 .

X.-X. Ma et al. / Steroids 76 (2011) 1003–1009
1005
Table 2
Table 3
13C NMR data for the sugar moieties of compounds 2–4 and 7–9 (ı in ppm, in C5D5N,
1H NMR spectral data of compounds 2–4 (ı in ppm, J in Hz, in C5D5N, 500 MHz).
100 MHz).
No.
2
3a
4
No.
2
3
4
7
8
9
3
6
3.87 m
3.85 m
3.85 m
d-digit
d-cym
d-cym
d-cym
d-cym
d-cym
5.26 br s
5.33 dd, 11.5, 4.0
2.09 s
1.30 s
2.41 s
8.30 d, 8.6
7.23 d, 8.6
7.23 d, 8.6
8.30 d, 8.6
5.29 br sb
5.33 dd, 11.9, 3.8
2.09 s
5.29 br sb
1ꢀꢀ
96.4
39.1
67.6
83.4
68.6
18.7
96.4
37.2
78.1
83.4
69.1
18.7
58.9
96.4
37.3
78.1
83.5
69.1
18.5
58.9
96.4
37.2
78.0
83.4
69.1
18.5
58.7
95.6
36.8
77.8
83.1
68.9
18.5
58.8
95.5
36.7
77.6
83.1
68.9
18.6
58.9
12
18
19
21
3ꢀ
5.33 dd, 11.8, 3.5
2.09 s
1.30 s
2ꢀꢀ
3ꢀꢀ
1.30 s
2.42 s
8.30 d, 8.4
7.23 d, 8.4
7.23 d, 8.4
8.30 d, 8.4
4ꢀꢀ
2.41 s
5ꢀꢀ
8.30 d, 8.6
7.22 d, 8.6
7.22 d, 8.6
8.30 d, 8.6
6ꢀꢀ
4ꢀ
OMe
6ꢀ
7ꢀ
No.
2
3
4
7
8
9
d-cym
d-cym
d-cym
d-cym
d-ole
d-ole
No.
2
3a
4
d-digit
d-cym
d-cym
1ꢀꢀꢀ
99.8
36.7
77.7
83.2
69.1
18.5
58.9
100.5
37.0
77.8
83.2
69.0
18.6
59.0
100.5
37.0
77.8
83.2
68.9
18.6
59.0
100.5
37.0
77.8
83.2
68.9
18.7
59.0
101.8
37.2
79.0
81.6
72.1
18.5
56.4
101.9
37.2
79.0
81.6
72.1
18.6
56.4
2ꢀꢀꢀ
1ꢀꢀ
5.48 br d, 9.2
2.07 m, 2.40 m
4.64 m
3.52 m
4.30 m
5.28 br db
1.90 m, 2.32 m
4.08 m
3.52 m
4.21 m
1.39 d, 6.1
3.62 s
5.12 br d, 9.9
1.81 m, 2.30 m
4.01 m
5.28 br db
1.91 m, 2.32 m
4.08 m
3.66 m
4.22 m
3ꢀꢀꢀ
2ꢀꢀ
4ꢀꢀꢀ
3ꢀꢀ
5ꢀꢀꢀ
4ꢀꢀ
6ꢀꢀꢀ
5ꢀꢀ
OMe
6ꢀꢀ
1.43 d, 6.4
1.35 d, 6.2
3.60 s
OMe
d-cym-1ꢀꢀꢀ
2ꢀꢀꢀ
5.17 br d, 9.8
1.78 m, 2.29 m
4.01 m
3.41 m
4.18 m
1.32 d, 6.1
3.56 s
4.68 br d, 9.7
1.73 m, 2.45 m
3.55 m
5.10 br dc
1.79 m, 2.31 m
4.23 m
No.
2
3
4
7
8
9
d-ole
d-ole
d-ole
d-ole
l-cym
l-cym
3ꢀꢀꢀ
1ꢀꢀꢀꢀ
2ꢀꢀꢀꢀ
3ꢀꢀꢀꢀ
4ꢀꢀꢀꢀ
5ꢀꢀꢀꢀ
6ꢀꢀꢀꢀ
OMe
102.0
37.7
78.9
82.7
71.8
18.7
57.4
102.0
37.7
78.9
82.8
71.8
18.7
57.4
101.9
37.5
79.5
83.4
72.0
18.8
57.3
102.0
37.8
78.8
82.6
71.8
18.7
57.5
97.4
32.4
73.5
78.1
64.7
18.4
57.2
97.4
32.3
73.6
78.0
64.8
18.4
57.3
4ꢀꢀꢀ
3.45 m
4.17 m
3.41 m
4.16 m
5ꢀꢀꢀ
6ꢀꢀꢀ
1.39 d, 6.1
3.57 s
4.69 br d, 9.5
1.73 m, 2.48 m
3.56 m
3.50 m
3.54 m
1.44 d, 5.2
3.52 s
1.37 d, 6.2
3.55 s
4.66 br d, 9.3
1.73 m, 2.48 m
3.58 m
3.51 m
3.63 m
1.69 d, 5.6
3.47 s
OMe
d-ole-1ꢀꢀꢀꢀ
2ꢀꢀꢀꢀ
3ꢀꢀꢀꢀ
4ꢀꢀꢀꢀ
3.51 m
3.52 m
1.43 d, 6.4
3.51 s
5ꢀꢀꢀꢀ
No.
2
3
4
7
8
9
6ꢀꢀꢀꢀ
d-cym
d-cym
d-glc
d-cym
d-cym
d-cym
OMe
1ꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀ
OMe
98.6
35.9
79.0
74.2
71.3
19.1
58.1
98.6
35.9
79.0
74.2
71.2
19.1
58.1
104.3
75.3
77.0
81.7
76.4
62.5
98.3
36.8
78.1
83.3
69.6
18.7
58.9
96.4
37.2
78.1
83.4
69.0
18.6
58.9
96.4
37.2
78.0
83.4
69.1
18.6
59.0
No.
2
3a
d-cym
4
d-cym
d-glc
1ꢀꢀꢀꢀꢀ
5.25 br d, 9.3
1.77 m, 2.38 m
3.77 m
3.54 m
4.14 m
5.26 br db
1.78 m, 2.39 m
3.78 m
3.54 m
4.13 m
5.06 dc
4.00 m
4.26 m
4.30 m
3.91 m
4.48 m, 4.52 m
2ꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀ
No.
2
2
2
3
3
3
4
7
8
9
6ꢀꢀꢀꢀꢀ
1.54 d, 6.3
3.46 s
1.54 d, 6.0
3.46 s
d-glc
d-glc
d-cym
d-cym
OMe
d-glc-1ꢀꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀꢀ
1ꢀꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀꢀ
OMe
105.1
74.9
78.5
71.6
78.3
62.5
106.3
74.9
76.6
81.4
76.5
62.4
100.5
37.0
77.8
83.3
69.0
18.6
59.0
100.5
37.0
77.8
82.5
69.1
18.6
58.4
5.18 d, 7.4
4.10 m
4.21 m
4.20 m
4.02 m
4.30 m, 4.54 m
d-digit: ␤-d-digitoxopyranosyl; d-cym: ␤-d-cymaropyranosyl; l-cym: ␣-l-
cymaropyranosyl; d-ole: ␤-d-oleandropyranosyl; d-glc: ␤-d-glucopyranosyl.
No.
4
7
8
9
a
d-glc
d-ole
l-cym
Obtained in 400 MHz.
Partial overlap with each other.
Overlap with H2O signal.
b
1ꢀꢀꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀꢀꢀ
OMe
105.0
74.8
78.6
71.5
78.3
62.4
101.9
37.5
79.3
83.3
72.1
18.9
57.3
99.0
32.3
73.3
78.9
65.1
18.5
56.9
c
treatment of epilepsy [1]. Compound 6 was identified as caudatin
3-O-␤-d-glucopyranosyl-(1→4)-␤-d-oleandropyranosyl-(1→4)-
␤-d-cymaropyranosyl-(1→4)-␤-d-cymaropyranoside, which was
first reported as the product of wallicoside hydrolyzed with
␤-glucosidase [5], and was then later isolated as a natural product
from Cynanchum caudatum [6] (Scheme 1).
No.
4
7
8
9
d-glc
d-glc
1ꢀꢀꢀꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀꢀꢀꢀ
104.5
75.8
78.7
71.9
78.2
63.1
102.3
75.3
78.8
71.8
78.5
63.0
3.1. Otophylloside N (2)
Compound 2 was obtained as a white amorphous powder with
[˛]1D8 + 7.7◦ (c = 0.20, MeOH). Its molecular formula, C55H82O20
,
was determined by negative HRESI-MS (m/z 1061.5284 [M−H]−,
calcd. 1061.5321) and 13C NMR (DEPT) data (Tables 1 and 2).
IR spectrum showed the absorption bands for hydroxyl groups
d-Digit: ␤-d-digitoxopyranosyl; d-cym: ␤-d-cymaropyranosyl; l-cym: ␣-l-
cymaropyranosyl; d-ole: ␤-d-oleandropyranosyl; d-glc: ␤-d-glucopyranosyl.

1006
X.-X. Ma et al. / Steroids 76 (2011) 1003–1009
Table 4 (Continued )
Table 4
1H NMR spectral data of compounds 7–9 (ı in ppm, J in Hz, in C5D5N, 500 MHz).
No.
7
8a
9a
No.
7
8a
9a
d-glc
d-ole
l-cym
3
6
12
18
3.84 m
3.84 m
3.85 m
5.28 br sb
5.04c
1.98 s
1.30 s
2.49 s
5.86 s
2.23 m
0.92 d, 7.3
0.93 d, 7.3
2.25 s
5ꢀꢀꢀꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀꢀꢀꢀ
3.95 m
4.35 dd, 11.5, 5.3
4.52 br d, 11.5
4.25 m
4.38 dd, 11.2, 4.9
4.57 br d, 11.2
5.28 br sb
5.04 dd, 12.1, 4.2
1.98 s
5.27 br sb
5.04c
1.98 s
1.31 s
2.50 s
5.85 s
d-digit: ␤-d-digitoxopyranosyl; d-cym: ␤-d-cymaropyranosyl; l-cym: ␣-l-
cymaropyranosyl; d-ole: ␤-d-oleandropyranosyl; d-glc: ␤-d-glucopyranosyl.
19
1.31 s
2.50 s
5.85 s
2.25 m
0.92 d, 7.0
0.94 d, 7.0
2.26 s
21
a
Obtained in 400 MHz.
Partial overlap with each other.
Overlap with H2O signal.
2ꢀ
b
4ꢀ
2.26 m
c
5ꢀ
0.92 d, 6.7
0.93 d, 6.7
2.26 s
6ꢀ
7ꢀ
d-cym-1ꢀꢀ
5.26 br db
1.90 m, 2.31 m
4.07 m
3.52 m
4.21 m
1.37 d, 6.0
3.48 s
5.26 br db
1.83 m, 2.30 m
4.00 m
5.24 br db
1.80 m, 2.29 m
4.00 m
3.42 m
4.15 m
1.35 db
3.53 s
(3446 cm−1), carbonyl groups (1713 cm−1), and benzene rings
(1610 and 1594 cm−1). The 1H NMR spectrum of 2 revealed the
presence of three singlet methyl groups [ıH 1.30, 2.09, 2.41 (each
3H, s, Me-19, 18, 21)], one olefinic proton [ıH 5.26 (brs, H-6)]
and four aromatic protons on a para-substituted benzene ring
[ıH 7.23 (2H, d, J = 8.6 Hz, H-4ꢀ,6ꢀ), and 8.30 (2H, d, J = 8.6 Hz,
H-3ꢀ,7ꢀ)] in its aglycone moiety. Its negative FAB-MS exhibited
a base peak at m/z 137, which could represent the fragmented
ion peak of a hydroxybenzoyl ester group. The above features,
together with the TLC comparison with known samples after acid
hydrolysis, suggested that the aglycone was qingyangshengenin
[4]. Proton signals of the sugar moiety could be assigned to four
secondary methyl groups [ıH 1.32 (3H, d, J = 6.1 Hz), 1.43 (2 × 3H,
d, J = 6.4 Hz), and 1.54 (3H, d, J = 6.3 Hz)], three methoxyl groups
[ıH 3.46, 3.51, and 3.56] and four anomeric protons [ıH 4.68 (brd,
J = 9.7 Hz), 5.17 (brd, J = 9.8 Hz), 5.25 (brd, J = 9.3 Hz), and 5.48 (brd,
J = 9.2 Hz)]. The multiples suggested that the sugar chain consisted
of four 2,6-dideoxy-sugar units with ␤-linkage. The 13C NMR
shifts of each sugar unit were assigned unambiguously by HSQC,
HMBC and HSQC-TOCSY analyses (Table 2), and we identified
the presence of one digitoxopyranosyl, one oleandropyranosyl
and two cymaropyranosyl units. Comparison with published
spectroscopic data, the absolute configuration of the deoxysugars
was consistent with d-series [1,7,8]. The sequence of the four
sugar units located at C-3 of the aglycone was elucidated by
HMBC spectrum, in which distinct correlations from ıH 5.48
(␤-d-digitoxopyranosyl H-1ꢀꢀ) to ıC 77.6 (C-3); from ıH 5.17 (inner
␤-d-cymaropyranosyl H-1ꢀꢀꢀ) to ıC 83.4 (␤-d-digitoxo-pyranosyl
C-4ꢀꢀ); from ıH 4.68 (␤-d-oleandropyranosyl H-1ꢀꢀꢀꢀ) to ıC 83.2
(inner ␤-d-cymaropyranosyl C-4ꢀꢀꢀ); from ıH 5.25 (terminal ␤-d-
2ꢀꢀ
3ꢀꢀ
4ꢀꢀ
3.43 m
4.16 m
1.36 d, 6.2
3.52 s
5ꢀꢀ
6ꢀꢀ
OMe
No.
7
8a
d-ole
9a
d-ole
d-cym
1ꢀꢀꢀ
5.11 br dc
1.80 m, 2.30 m
4.00 m
3.49 m
4.15 m
4.64 br d, 9.8
1.62 m, 2.48 m
3.39 m
4.64 br d, 9.8
1.62 m, 2.48 m
3.40 m
2ꢀꢀꢀ
3ꢀꢀꢀ
4ꢀꢀꢀ
3.37 m
3.36 m
5ꢀꢀꢀ
3.45 m
3.45 m
6ꢀꢀꢀ
1.38 d, 6.0
3.55 s
1.37 db
1.36 db
OMe
3.29 s
3.27 s
No.
7
8a
l-cym
9a
l-cym
d-ole
1ꢀꢀꢀꢀ
4.66 br d, 9.6
1.71 m, 2.43 m
3.51 m
3.47 m
3.49 m
5.07 br s
1.87 m, 2.32 m
3.77 m
3.88 dd, 8.5, 2.7
4.74 m
5.07 br s
1.84 m, 2.33 m
3.77 m
3.88 m
4.71 m
2ꢀꢀꢀꢀ
3ꢀꢀꢀꢀ
4ꢀꢀꢀꢀ
5ꢀꢀꢀꢀ
6ꢀꢀꢀꢀ
1.39 d, 5.7
3.49 s
1.56 d, 6.2
3.38 s
1.54 d, 6.4
3.40 s
OMe
d-cym-1ꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀ
5.25 br db
1.76 m, 2.29 m
4.05 m
5.27 br db
1.89 m, 2.31 m
4.07 m
5.27 br db
1.89 m, 2.30 m
4.08 m
3ꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀ
3.61 m
4.25 m
1.61 d, 6.2
3.60 s
3.50 m
3.51 m
5ꢀꢀꢀꢀꢀ
4.19 m
4.20 m
6ꢀꢀꢀꢀꢀ
1.37 db
1.36 db
OMe
3.58 s
3.59 s
cymaropyranosyl H-1ꢀꢀꢀꢀꢀ) to ıC 82.7 (␤-d-oleandropyranosyl C-4ꢀꢀꢀꢀ
)
were observed. Thus, the structure of otophylloside N (2) was
established as qingyangshengenin-3-O-␤-d-cymaropyranosyl-
(1→4)-␤-d-oleandropyranosyl-(1→4)-␤-d-cymaropyranosyl-
(1→4)-␤-d-digitoxopyra-noside.
No.
7
8a
d-cym
9a
d-cym
d-glc
1ꢀꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀꢀ
OMe
4.88 d, 7.7
3.98 m
4.27 m
4.28 m
3.93 m
5.10 br d, 9.5
1.78 m, 2.30 m
3.99 m
5.11 br d, 9.8
1.78 m, 2.30 m
3.99 m
3.63 m
3.45 m
3.2. Otophylloside O (3)
4.19 m
4.19 m
4.50 m, 4.52 m
1.38 db
1.37 db
3.54 s
3.52 s
The molecular formula of compound 3 was determined to be
C
56H84O20 on the basis of negative HRESI-MS (m/z 1111.5239
[M+Cl]−, calcd. 1111.5244). Similar IR absorption frequency, NMR
characteristics and acidic hydrolysis indicated that 3 possessed the
same aglycone, qingyangshengenin, as compound 2. The fragment
ion peak m/z 931 [M−1−144]− in negative FAB-MS confirmed a
terminal deoxysugar in 3. TLC results from acid hydrolysis revealed
the presence of cymarose and oleandrose. The sugar moiety was
elucidated to be a single chain connected to C-3 of the aglycone,
and the sequence of the sugar units was identical to those of the 3-
O-tetraglycosides isolated from Cynanchum caudatum identifying
cynanchogenin or caudatin as the aglycone [9,10]. The structure of
otophylloside O (3) was determined to be qingyangshengenin-3-
O-␤-d-cymaropyranosyl-(1→4)-␤-d-oleandropyranosyl-(1→4)-
␤-d-cymaropyranosyl-(1→4)-␤-d-cymaropyranoside.
No.
7
8a
d-ole
9a
l-cym
d-glc
1ꢀꢀꢀꢀꢀꢀꢀ
5.19 d, 7.7
4.10 m
4.20 m
4.20 m
4.01 m
4.67 br d, 10.4
1.71 m, 2.47 m
3.61 m
3.70 m
3.63 m
1.69 d, 5.9
3.50 s
5.11 d, 7.7
4.00 m
4.22 m
4.20 m
4.94 br s
1.77 m, 2.32 m
3.93 m
3.97 m
4.68 m
1.46 d, 6.4
3.42 s
5.01 d, 7.8
3.99 m
3.97 m
4.22 m
2ꢀꢀꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀꢀꢀ
5ꢀꢀꢀꢀꢀꢀꢀ
6ꢀꢀꢀꢀꢀꢀꢀ
4.29 m, 4.52 m
OMe
d-glc-1ꢀꢀꢀꢀꢀꢀꢀꢀ
2ꢀꢀꢀꢀꢀꢀꢀꢀ
3ꢀꢀꢀꢀꢀꢀꢀꢀ
4ꢀꢀꢀꢀꢀꢀꢀꢀ

X.-X. Ma et al. / Steroids 76 (2011) 1003–1009
1007
4'
O
21
O
O
1'
2'
H3C
3' 2'
4'
O
HO
C O
18
20
CH C CH C O
OH
12
1'
H3C
6'
OH
17
S
CH3
19
5'
7'
OH
1
14 16
5
6
7
8
9
Ra
Re
Rf
Rg
Rh
OH
OH
S
10
8
1
2
3
4
Ra
Rb
Rc
Rd
OH
3
5
SO
SO
6
R
H
H3C
H3C
H3C
Ra
O
O
RO
O
O
O
Rc cym
Re glc
O
OCH3
cym
CH3
OCH3
cym
ole
H3C
H3C
H3C
O
O
H3C
O
HO
O
O
O
O
Rb
H3CO
OCH3
cym
OCH3
cym
OH
digit
ole
H3C
O
O
H3C
H3C
HO
HO
HO
O
HO
O
O
O
Rd
O
O
O
HO
O
CH3
OCH3
cym
OCH3
cym
OH
OH
glc
glc
ole
H3C
H3C
H3C
HO
HO
HO
H3C
HO
O
O
O
O
O
O
O
O
O
O
O
Rf
O
CH3
HO
OCH3
cym
OCH3
cym
OH
OH
OCH3
cym
glc
glc
O
ole
H3C
H3C
O
O
O
O
Rg
H3CO
O
CH3
H3C
H3C
H3C
O
H3C
HO
HO
HO
OCH3
cym
O
O
O
O
ole
O
O
O
O
CH3
L-cym
OCH3
OCH3
cym
OH
glc
ole
cym
H3C
H3C
O
O
Rh
O
O
H3CO
H3C
O
H3C
H3C
O
O
CH3
OCH3
cym
O
O
O
O
H3CO
ole
HO
HO
HO
L-cym
H3C
OCH3
cym
O
OCH3
cym
O
O
L-cym
OH
glc
Scheme 1. Qingyangshengenin and caudatin glycosides (1–9) isolated from Cynanchum otophyllum.
3.3. Otophylloside P (4)
1425.6821), in agreement with the molecular formula C68H110O29,
which was further supported by 13C NMR and DEPT experiments. IR
spectrum showed absorption bands for hydroxyl (3442 cm−1) and
carbonyl (1714 cm−1) groups as well as a C C band (1643 cm−1).
The 1H NMR spectrum of 7 exhibited four singlet methyl groups
[ıH 1.31, 1.98, 2.26, 2.50 (each 3H, s, Me-19,18,7ꢀ,21)], two doublet
methyl groups [ıH 0.92 and 0.94 (each 3H, d, J = 7.0 Hz, Me-5ꢀ,6ꢀ)],
and two olefinic protons [ıH 5.28 (brs, H-6) and 5.85 (s, H-2ꢀ)].
These spectral characteristics suggested that the aglycone was
caudatin [4], which could be consistent with the fragment ion
peaks at m/z 1262 [M−1−127]− resulting from the loss of ikemaoyl
ester group in negative FAB-MS spectrum. Acid hydrolysis of 7
led to the detection of caudatin and a sugar mixture of cymarose,
oleandrose, and glucose by TLC analysis and comparison to known
samples. Its negative FAB-MS exhibited fragment ion peaks at m/z
1228 [M−162]−, revealing a terminal glucosyl unit in the sugar
moiety. The 1H NMR spectral data of compound 7 contained six
anomeric proton signals [ıH 4.66 (brd, J = 9.6 Hz), 4.88 (d, J = 7.7 Hz),
5.11 (brd, partially overlapped), 5.19 (d, J = 7.7 Hz), 5.25 (brd, par-
The molecular formula of compound 4, C61H92O27, was deter-
mined by the negative HRESI-MS data (m/z 1255.5745 [M−H]−,
calcd. 1255.5747). It was identified as qingyangshengenin 3-O-
pentoside based on its 1H and 13C NMR spectra (Tables 1–3). Acid
hydrolysis showed that its sugar moiety consisted of three types of
sugar units: cymarose, oleandrose, and glucose. Detailed 2D NMR
revealed that the sugar sequence was consistent with that of walli-
coside [5]. Thus, the structure of otophylloside P (4) was elucidated
to be qingyangshengenin-3-O-␤-d-glucopyranosyl-(1→4)-␤-
d-glucopyranosyl-(1→4)-␤-d-oleandropyranosyl-(1→4)-␤-d-
cymaropyranosyl-(1→4)-␤-d-cymaropyranoside.
3.4. Otophylloside Q (7)
Compound 7 was obtained as white amorphous powder with
[˛]1D8 − 0.0◦ (c = 0.22, MeOH). The negative HRESI-MS showed
a quasi-molecular ion peak at m/z (1425.6831 [M+Cl]−, calcd.

1008
X.-X. Ma et al. / Steroids 76 (2011) 1003–1009
the 1H and 13C NMR spectra of 8 and 9 indicated that a set of the
characteristic signals of ␤-d-oleandropyranosyl unit in 8 [ıH 4.67
(brd, J = 10.4 Hz), 1.69 (d, J = 5.9 Hz), 3.50 (s), and ıC 101.9, 37.5,
79.3, 83.3, 72.1, 18.9, 57.3] was absent in 9. Instead, a set of signals
arising from an ␣-l-cymaropyranosyl unit [ıH 4.94 (brs), 1.46 (d,
J = 6.4 Hz), 3.42 (s), and ıC 99.0, 32.3, 73.3, 78.9, 65.1, 18.5, 56.9]
existed in 9. The sequence of the sugars and the location of the
sugar chain were confirmed by HMBC, and the sixth sugar unit was
the only difference. Because of the variation from ␤-d-oleandrosyl
in 8 to ␣-l-cymarosyl in 9, the C-4 signal of the fifth sugar unit
(␤-d-cymaropyranosyl) shifted from ıC 83.3 in 8 to ıC 82.5 in
9, and both the anomeric proton and carbon signals of terminal
glucopyranosyl unit in 9 moved to lower frequency shifts [ıH
5.01 (d, J = 7.8 Hz) and ıC 102.3]. These features were identical
to those of wilfosides C1G and G1G, whose sugar chains implied
tially overlapped), 5.26 (brd, partially overlapped)], four secondary
methyl groups [ıH 1.37 (3H, d, J = 6.0 Hz), 1.38 (3H, d, J = 6.0 Hz), 1.39
(3H, d, J = 5.7 Hz), 1.61 (3H, d, J = 6.2 Hz)], and four methoxyl groups
[ıH 3.48, 3.49, 3.55, and 3.60] in the sugar moiety, indicating the
existence of four deoxy sugars and two glucose units. The signals
of each sugar unit were assigned by HSQC, HMBC and HSQC-
TOCSY analyses (Tables 2 and 4), suggesting that the proportion of
cymarose and oleandrose was 3:1; their absolute configuration was
determined to be d-series by comparison with the spectroscopic
data in the literature [6,9,10]. The sugar sequence of 7 was studied
by the same method described above, and it was found to be
coincident with otophylloside M [4] except for an excess terminal
glucosyl unit. Therefore, the structure of 7 was concluded to be
caudatin-3-O-␤-d-glucopyranosyl-(1→4)-␤-d-glucopyranosyl-
(1→4)-␤-d-cymaropyranosyl-(1→4)-␤-d-oleandropyranosyl-
(1→4)-␤-d-cymaropyranosyl-(1→4)-␤-d-cymaropyranoside and
was named otophylloside Q.
a
␤-d-glucopyranosyl-(1→4)-␣-l-cymaropyranosyl-(1→4)-␤-d-
cymaropyranosyl fragment [11,12]. Therefore, the structure of 9
was confirmed to be caudatin 3-O-␤-d-glucopyranosyl-(1→4)-
␣-l-cymaropyranosyl-(1→4)-␤-d-cymaropyranosyl-(1→4)-
␤-d-cymaropyranosyl-(1→4)-␣-l-cymaropyranosyl-(1→4)-␤-d-
oleandropyranosyl-(1→4)-␤-d-cymaropyranoside and was named
otophylloside S.
3.5. Otophylloside R (8)
The molecular formula of compound 8 was determined to be
C76H124O30 on the basis of negative HRESI-MS (m/z 1551.7863
[M+Cl]−, calcd. 1551.7865) and 13C NMR (DEPT) spectrum. The
aglycone of 8 was inferred to be caudatin based on a comparison
of the NMR features to a known sample [4] and the fragment
ion peak at m/z 1389 [M−127]− in negative FAB-MS. Its 1H
NMR spectrum exhibited seven anomeric proton signals at ıH
4.64 (brd, J = 9.8 Hz), 4.67 (brd, J = 10.4 Hz), 5.07 (brs), 5.10 (brd,
J = 9.5 Hz), 5.11 (d, J = 7.7 Hz), 5.26 (brd, partially overlapped),
and 5.27 (brd, partially overlapped), revealing the existence of
six ␤-linkages and one ␣-linkage. The observation of six sec-
ondary methyl and six methoxyl groups (Table 4) suggested the
sugar moiety contained six deoxysugar units. In HSQC spectrum,
seven anomeric carbon signals at ıC 101.8, 101.9, 97.4, 100.5,
104.5, 95.6, and 96.4 were attributed to the above anomeric
protons, respectively. The other signals were assigned by HSQC,
HMBC and HSQC-TOCSY studies, indicating the existence of
three d-cymaropyranosyl units, two d-oleandro-pyranosyl units,
one l-cymaropyranosyl unit, and one glucopyranosyl unit by
comparison with the data in Refs. [5,6,11,12]. These seven
sugar units were in a single chain connected to C-3 of the agly-
cone, and the sequence of the sugars was illustrated by the
distinct correlations in HMBC spectrum: from ıH 5.11 (␤-d-
glucopyranosyl H-1ꢀꢀꢀꢀꢀꢀꢀꢀ) to ıC 83.3 (outer ␤-d-oleandropyranosyl
C-4ꢀꢀꢀꢀꢀꢀꢀ), from ıH 4.67 (outer ␤-d-oleandro-pyranosyl H-1ꢀꢀꢀꢀꢀꢀꢀ) to
ıC 83.3 (outer ␤-d-cymaropyranosyl C-4ꢀꢀꢀꢀꢀꢀ), from ıH 5.10 (outer
␤-d-cymaropyranosyl H-1ꢀꢀꢀꢀꢀꢀ) to ıC 83.4 (middle ␤-d-cymaro-
pyranosyl C-4ꢀꢀꢀꢀꢀ), from ıH 5.27 (middle ␤-d-cymaropyranosyl
H-1ꢀꢀꢀꢀꢀ) to ıC 78.1 (␣-l-cymaropyranosyl C-4ꢀꢀꢀꢀ), from ıH 5.07 (␣-l-
cymaropyranosyl H-1ꢀꢀꢀꢀ) to ıC 81.6 (inner ␤-d-oleandropyranosyl
C-4ꢀꢀꢀ), from ıH 4.64 (inner ␤-d-oleandropyranosyl H-1ꢀꢀꢀ) to
ıC 83.1 (inner ␤-d-cymaro-pyranosyl C-4ꢀꢀ), and from ıH 5.26
(inner ␤-d-cymaropyranosyl H-1ꢀꢀ) to ıC 77.6 (C-3). There-
fore, the structure of otophylloside R (8) was determined to be
caudatin 3-O-␤-d-glucopyranosyl-(1→4)-␤-d-oleandropyranosyl-
(1→4)-␤-d-cymaropyranosyl-(1→4)-␤-d-cymaropyranosyl-
(1→4)-␣-l-cymaropyranosyl-(1→4)-␤-d-oleandropyranosyl-
(1→4)-␤-d-cymaropyranoside.
It is noted that most pregnane glycosides isolated from C.
otophyllum possessed either qingyangshengenin or caudatin as
aglycones, which contain a straight sugar chain at C-3 of the agly-
cone with different composition and various connecting sequences
[1,4,13]. In addition, otophyllosides R and S (8 and 9) were com-
prised of caudatin as the aglycone and seven sugar units, with the
coexistence of d- and l-cymarosyl optically isomeric monosaccha-
rides. The configurations of the cymarosyl moieties in compounds 8
and 9 were determined by comparing their 13C NMR chemical shifts
with the published configurations [11]. The relationship between
the 13C NMR data and absolute configuration of cymarosyl moi-
ety was confirmed by a detailed analysis of the purified d- and
l-cymarose by acid hydrolysis. It was shown that the orientations
of methyl and oxymethyl at C-3 position affect the carbon signals
strongly in 2,6-dideoxy sugars. Therefore, the configurations of dig-
itoxosyl and oleandrosyl moieties could also be determined by the
13C NMR data.
Acknowledgements
The authors are grateful to Professor Chen Jijun for supplying
the deoxy sugar samples of digitoxose, cymarose, and oleandrose
and appreciate the measurements of all spectra performed by the
colleagues of the Analytical Group in State Key Laboratory of Phyto-
chemistry and Plant Resources in West China, Kunming Institute of
Botany. This work was supported by the Funds 2008ZX09401-004
and 2011CB915503 from the Ministry of Science and Technology
of China.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2011.03.019.
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