Cytotoxicity of pregnane glycosides of Cynanchum otophyllum

Article abstract of DOI:10.1016/j.steroids.2015.08.010

Fourteen new pregnane glycosides, including nine caudatin glycosides (1-9), three qinyangshengenin glycosides (10-12), one kidjoranin glycosides (13) and one gagaminin glycosides (14), along with twelve known analogs (15-26) were isolated from roots of Cy

Full text of DOI:10.1016/j.steroids.2015.08.010

Steroids xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Cytotoxicity of pregnane glycosides of Cynanchum otophyllum
b,
Mi Zhang a,1, Xiang Li a,1, Cheng Xiang a, Yi Qin a, Jing He a, Bao-Cai Li a, , Peng Li
⇑
⇑
a Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China
b State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau 999078, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Fourteen new pregnane glycosides, including nine caudatin glycosides (1–9), three qinyangshengenin
glycosides (10–12), one kidjoranin glycosides (13) and one gagaminin glycosides (14), along with twelve
known analogs (15–26) were isolated from roots of Cynanchum otophyllum Schneid. Their structures were
deduced by detailed analysis of 1D and 2D NMR spectra, as well as HRESIMS. In this study, all pregnane
glycosides obtained (1–26) were evaluated for their cytotoxic activities using three cancer cell lines
(HepG2, Hela, U251). As results, except 6 and 10, other twenty-four pregnane glycosides showed cytotox-
icities at different degrees against three cell lines.
Received 27 January 2015
Received in revised form 2 April 2015
Accepted 15 August 2015
Available online xxxx
Keywords:
Cynanchum otophyllum
Pregnane glycosides
Cytotoxicity
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
(HepG2, Hela, U251). The results showed all compounds, except
6 and 10, displayed cytotoxicities against several cell lines at differ-
Pregnane glycosides, also known as C21 steroidal glycosides, are
the characteristically chemical and bioactive constituents in the
genus Cynanchum (subfamily Asclepiadoideae within Apocy-
naceae) [1,2]. Their basic skeletons are the pregnane derivative,
whose C-8, C-13, C-15 and C-17 are easy to be substituted by oxy-
gen-containing groups, and C- or D-rings of aglycones are opening
occasionally. The sugar moieties in pregnane glycosides consist of
2,6-dideoxysugars mainly, which are usually connected to C-3 of
aglycones as a chain [3]. Previous pharmacological experiments
have displayed that pregnanes and their glycosides from Cynan-
ent degrees.
2. Experimental
2.1. General methods
Optical rotations were obtained on a JASCO P-1020 polarimeter.
IR spectra were measured on Bruker Tensor 27 spectrometer in KBr
pellets. NMR spectra were performed in CDCl3 or C5D5N, recorded
on Bruker DRX-500 instrument with TMS as internal standard. UV
spectra were recorded on Shimadzu UV-2450 spectropolarimeter.
HRESIMS were measured on Agilent LC-MSD TOF spectrometer.
Silica gel GF254 prepared for TLC and silica gel (100–200 mesh)
for column chromatography (CC) were obtained from Qingdao
Marine Chemical Company, Qingdao, China. Compounds were
purified by semi-preparative HPLC (Beijing Tong Heng Innovation
Technology Co., Ltd. LC3000), equipped with a column Zorbax
chum have
a variety of bioactivities, such as cytotoxicity,
immunoregulation, multidrug-resistance modulation, and anti-
fungus, etc. [4–7].
The perennial herbaceous plant Cynanchum otophyllum
Schneid., is mainly distributed in southwest of China. Its roots have
been used as folk medicine for treatment of rheumatism, lumbago,
abdominal pain and distension, etc. [8–12]. In previously chemical
studies, our group and other researchers have found this plant is
rich in pregnane glycosides [13–23]. As a part of our ongoing
research program to isolate novel pregnane glycosides from
Cynanchum plants, fourteen new pregnane glycosides (1–14) and
twelve known analogs (15–26) were isolated from roots of
C. otophyllum (Fig. 1). All steroidal glycosides obtained (1–26) were
evaluated for their cytotoxicities using three cancer cell lines
SB-C18 (9.4 mm ꢀ 250 mm, 5
lM).
2.2. Plant material
The roots of C. otophyllum were collected from Traditional
Chinese Medicine market in Kunming, China, and identified by
Prof. Shiming Guo (Yunan Institute of Traditional Chinese
Medicine and Material Medica, China).
A voucher specimen
⇑
Corresponding authors at: 727# Jingming Road, Chenggong, Kunming, Yunnan
650500, China (B.-C. Li).
(No. Co-2012-05) has been deposited in the laboratory of Faculty
of Life Science and Technology, Kunming University of Science
and Technology.
E-mail addresses: baocaili@hotmail.com (B.-C. Li), pli1978@hotmail.com (P. Li).
Those authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.steroids.2015.08.010
0039-128X/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: M. Zhang et al., Cytotoxicity of pregnane glycosides of Cynanchum otophyllum, Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.08.010

2
M. Zhang et al. / Steroids xxx (2015) xxx–xxx
O
R2
H3C
H3C
H3C
R2O
O
O
O
a
b
c
HO
O
A
B
C
Ikem
O
OH
OH
H3CO
1''
OH
OH
OCH3
cym
OCH3
cym
p-OH-Bz
Cin
ole
H3C
H
R1O
H3C
H3C
O
O
O
HO
H3CO
O
H3CO
O
OH
1''
1''
1''
R3O
OCH3
cym
R2O
ole
the
R2
R3
Nic
H3C
H3C
OH
OH
H3C
O
Cin
D
O
O
HO
O
O
H
H3CO
OH
OCH3
cym
R1O
OCH3
cym
the
the
O
1'
O
1'
H3C
H3C
H3C
O
O
O
d
O
HO
O
H3CO
OH
OCH3
cym
OH
digit
OH
p-OH-Bz=p-Hydroxybenzoyl
Ikem=Ikemaoyl
H3C
H3C
H3C
O
O
O
O
e
HO
H3CO
O
O
O
O
O
1'
O
1'''
OCH3
cym
OH
digit
1''
ole
ole
N
H3C
H3C
H3C
H3C
O
O
f
O
Cin=Cinnamoyl
Nic=Nicotinoyl
HO
O
H3CO
1''
OCH3
cym
OCH3
OH
digit
cym
R1
OH
H3C
a
b
c
1
2
3
A
A
A
O
O
O
O
CH3
H3C
H3CO
g
O
O
O
OCH3
H3CO
O
CH3
1''
1''
d
f
4
5
A
A
OH
digit
L-cym
O
cym
L-cym
H3C
g
h
l
H3C
H3C
6
7
8
A
A
A
H3C
O
O
h
HO
H3CO
O
O
O
OH
H3CO
OCH3
cym
OCH3
cym
ole
the
m
i
9
10
11
A
B
B
H3C
H3C
H3C
H3C
O
O
i
O
O
HO
O
O
O
O
j
OH
H3CO
1''
OCH3
cym
OCH3
cym
OH
digit
m
k
12
13
14
B
C
D
the
OH
H3C
e
H3C
H3C
O
O
O
O
j
O
O
CH3
H3CO
H3CO
1''
OCH3
cym
OCH3
cym
L-cym
ole
H3C
H3C
H3C
H3C
O
O
O
k
O
HO
O
O
O
H3CO
H3CO
1''
OCH3
cym
OCH3
cym
ole
ole
OH
H3C
O
O
O
O
H3C
CH3
H3C
H3CO
O
O
O
l
OCH3
O
O
CH3
H3CO
1''
1''
OCH3
cym
OH
digit
L-cym
O
cym
H3C
L-cym
OH
H3C
O
O
O
O
O
H3C
H3C
CH3
O
H3CO
O
O
m
OCH3
cym
OCH3
H3CO
O
H3CO
O
CH3
OCH3
cym
L-cym
cym
L-cym
ole
Fig. 1. Pregnane glycosides (1–14) isolated from the roots of C. otophyllum.
Please cite this article in press as: M. Zhang et al., Cytotoxicity of pregnane glycosides of Cynanchum otophyllum, Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.08.010

M. Zhang et al. / Steroids xxx (2015) xxx–xxx
3
2.3. Extraction and isolation
2.3.4. Compound 4
22
White amorphous powder; [
a]
+3.2 (c 2.01, MeOH); UV
D
The air-dried roots of C. otophyllum (9.0 kg) were extracted with
EtOH (75%) for three times (3 h, 10 L ꢀ3). The organic solvent was
removed at vacuum, and the residue suspended in water (10 L).
Then, it was extracted with CHCl3 (5 L ꢀ4) to afford CHCl3 soluble
fraction (240 g). CHCl3 extract (240 g) was subjected to a silica gel
(15 cm ꢀ 100 cm) column and eluted with CHCl3–MeOH (40:1,
20:1, 10:1, 5:1, v/v) to get six fractions (Frs. 1–6). Purification
was focused on Frs. 2–5 by mainly methods of semi-preparative
HPLC. Fr. 2 (30.5 g) was separated by MCI CC with MeOH–H2O
(85:15 ? 75:25, v/v) to give five fractions (Frs. 2A–2F). Fr. 2A
was subjected to ODS CC with MeOH–H2O (85:15 ? 80:20, v/v)
to give three fractions (Frs. 2A1–2A3). Frs. 2A3 was performed on
preparative HPLC and eluted with MeOH–H2O (85:15 ? 82:18 ?
80:20, v/v) to yield 15 (9.4 mg, Rt = 33.5 min), 10 (60.2 mg,
Rt = 35.3 min), 2 (7.5 mg, Rt = 38.4 min), 18 (6.5 mg, Rt = 42.0 min),
1 (10.1 mg, Rt = 45.2 min) and 9 (66.3 mg, Rt = 47.8 min). Fr. 2B
was separated by ODS CC with MeOH–H2O (83:17 ? 78:22, v/v)
to give two fractions (Frs. 2B1–2B2). Frs. 2B1–2B2 were purified
by preparative HPLC, eluted with MeOH–H2O (80:20 ? 78:22, v/
v) to yield 26 (10.1 mg, Rt = 45.9 min), 3 (110 mg, Rt = 50.4 min),
5 (48.3 mg, Rt = 55.6 min) and 16 (30.2 mg, Rt = 60.5 min). Fr. 2D
was purified by preparative HPLC with MeOH–H2O (77:23, v/v)
to yield 25 (5.6 mg, Rt = 36.9 min), 24 (22.3 mg, Rt = 40.3 min), 23
(13.2 mg, Rt = 45 min) and 13 (5.4 mg, Rt = 50.3 min). Fr.3 was sub-
jected to ODS CC with MeOH–H2O (80:20 ? 76:24, v/v) to give
three fractions (Frs. 3A1–3A3). Frs. 3A1 was performed on prepar-
ative HPLC, eluted with MeOH–H2O (76:24, v/v) to yield 17
(40.1 mg, Rt = 43.9 min), 7 (23.3 mg, Rt = 46.1 min), 8 (12.5 mg,
Rt = 50.9 min) and 19 (20.1 mg, Rt = 55.8 min). Fr. 4 was subjected
to ODS CC with MeOH–H2O (78:22 ? 74:26, v/v) to give four frac-
tions (Frs. 4A1–4A4). Fr. 4A2 was performed on preparative HPLC,
eluted with MeOH–H2O (77:23 ? 75:25 ? 74:26, v/v) to obtain 14
(18.1 mg, Rt = 40.5 min), 6 (13.4 mg, Rt = 45.9 min), 20 (8.5 mg,
Rt = 48.9 min) and 12 (40.9 mg, Rt = 57.9 min). Fr. 5 was subjected
to ODS CC with MeOH–H2O (75:25 ? 70:30, v/v) to give two frac-
tions (Frs. 5A1–5A2). Fr. 5A1 was performed on preparative HPLC,
eluted with MeOH–H2O (73:27, v/v) to yield 22 (4.1 mg,
(MeOH) kmax (loge) 220 (3.58) nm; IR (KBr) õmax 3461, 2969, 2934,
1713, 1643, 1451, 1384, 1369, 1317, 1224, 1166, 1084, 1007, 912,
864; HRESIMS m/z 947.4976 ([M+Na]+, C48H76O17Na, calcd.
947.4974). 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N,
125 MHz) see Tables 1–3.
2.3.5. Compound 5
21
White amorphous powder; [
a
]
+2.6 (c 1.16, MeOH); UV
D
(MeOH) kmax (log
e
) 224 (3.92) nm; IR (KBr) õmax 3442, 2968, 2934,
1713, 1641, 1450, 1385, 1370, 1317, 1224, 1165, 1063, 1004, 912,
865; HRESIMS m/z 1075.5810 ([M+Na]+, C55H88O19Na, calcd.
1075.5812). 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3,
125 MHz) see Tables 1–3.
2.3.6. Compound 6
22
White amorphous powder; [
(MeOH) kmax (log
a]
ꢁ67.2 (c 1.05, MeOH); UV
D
e
) 219 (3.74) nm; IR (KBr) õmax 3445, 2969, 2932,
1714, 1641, 1452, 1384, 1367, 1316, 1223, 1164, 1055, 1005, 913,
865; HRESIMS m/z 1075.5815 ([M+Na]+, C55H88O19Na, calcd.
1075.5812). 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N,
125 MHz) see Tables 1–3.
2.3.7. Compound 7
22
White amorphous powder; [
(MeOH) kmax (log
a]
+7.8 (c 1.60, MeOH); UV
D
e
) 219 (3.75) nm; IR (KBr) õmax 3452, 2969, 2933,
1714, 1643, 1451, 1383, 1368, 1317, 1223, 1164, 1059, 1004, 912,
864; HRESIMS m/z 1105.5912 ([M+Na]+, C56H90O20Na, calcd.
1105.5917). 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N,
125 MHz) see Tables 1–3.
2.3.8. Compound 8
White amorphous powder; [
(MeOH) kmax (log
22
a]
ꢁ2.2 (c 1.63, MeOH); UV
D
e
) 230 (3.98) nm, 225 3.95)nm; IR (KBr) õmax
3447, 2968, 2934, 1712, 1641, 1450, 1384, 1369, 1317, 1224, 1166,
1061, 1004, 910, 866; HRESIMS m/z 1219.6604 ([M+Na]+,
C
62H100O22Na, calcd. 1219.6598). 1H NMR (C5D5N, 500 MHz) and
Rt = 33.9 min), 21 (13.2 mg, Rt = 36.3 min),
Rt = 42.9 min) and 11 (20.3 mg, Rt = 46.9 min).
4
(60.2 mg,
13C NMR (C5D5N, 125 MHz) see Tables 1–3.
2.3.9. Compound 9
2.3.1. Compound 1
21
21
White amorphous powder; [
(MeOH) kmax (log
a]
ꢁ9.9 (c 1.00, MeOH); UV
D
White amorphous powder; [
(MeOH) kmax (log
a]
ꢁ4.7 (c 1.06, MeOH); UV
D
e
) 220 (3.93) nm; IR (KBr) õmax 3443, 2969, 2932,
e
) 226 (3.86) nm; IR (KBr) õmax 3442, 2965, 2933,
1714, 1642, 1452, 1382, 1368, 1317, 1223, 1165, 1065, 1004, 912,
864; HRESIMS m/z 1377.7556 ([M+Na]+, C70H114O25Na, calcd.
1377.7552). 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3,
125 MHz) see Tables 1–3.
1713, 1641, 1450, 1384, 1368, 1316, 1224, 1166, 1063, 1004, 911,
863; HRESIMS m/z 945.5180 ([M+Na]+, C49H78O16Na, calcd.
945.5182). 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3,
125 MHz) see Tables 1–3.
2.3.10. Compound 10
2.3.2. Compound 2
White amorphous powder; [
(MeOH) kmax (log
21
22
White amorphous powder; [
(MeOH) kmax (log
a]
+9.9 (c 1.08, MeOH); UV
a
]
D
ꢁ5.9 (c 3.46, MeOH); UV
D
e
) 259 (3.57) nm; IR (KBr) õmax 3447, 2972, 2934,
e
) 228 (3.83) nm, 225 (3.81) nm; IR (KBr) õmax
1711, 1610, 1596, 1515, 1451, 1382, 1310, 1276, 1167, 1087, 1059,
911, 856, 772; HRESIMS m/z 1101.5242 ([M+Na]+, C55H82O21Na,
calcd. 1101.5240). 1H NMR (C5D5N, 500 MHz) and 13C NMR
(C5D5N, 125 MHz) see Tables 4–6.
3443, 2965, 2932, 1713, 1641, 1450, 1384, 1369, 1315, 1224, 1165,
1065, 1005, 913, 862; HRESIMS m/z 961.5140 ([M+Na]+, C49H78O17Na,
calcd. 961.5141). 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N,
125 MHz) see Tables 1–3.
2.3.3. Compound 3
2.3.11. Compound 11
22
22
White amorphous powder; [
a
]
+2.8 (c 1.04, MeOH); UV
White amorphous powder; [
a
]
ꢁ25.5 (c 1.32, MeOH); UV
D
D
(MeOH) kmax (log
e
) 224 (3.87) nm, 226 (3.86) nm; IR (KBr) õmax
(MeOH) kmax (loge) 214 (4.00) nm, 259 (3.51) nm; IR (KBr) õmax
3453, 2968, 2933, 1713, 1642, 1451, 1384, 1368, 1317, 1224, 1167,
1059, 1005, 912, 864; HRESIMS m/z 961.5134 ([M+Na]+, C49H78O16Na,
calcd. 961.5141). 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N,
125 MHz) see Tables 1–3.
3451, 2972, 2933, 1711, 1611, 1515, 1452, 1383, 1311, 1276, 1164,
1099, 1057, 912, 853, 773; HRESIMS m/z 1099.5448 ([M+Na]+,
C
56H84O20Na, calcd. 1099.5446). 1H NMR (C5D5N, 500 MHz) and
13C NMR (C5D5N, 125 MHz) see Tables 4–6.
Please cite this article in press as: M. Zhang et al., Cytotoxicity of pregnane glycosides of Cynanchum otophyllum, Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.08.010

4
M. Zhang et al. / Steroids xxx (2015) xxx–xxx
Table 1
13C NMR data for the aglycone moieties of compounds 1–9 (d in ppm, 125 MHz).
No.
1a
2b
3b
4b
5a
38.7
6b
39.2
7b
39.0
8b
39.0
9a
38.7
1
38.9
39.0
39.0
39.0
2
3
4
5
6
7
8
9
29.0
79.0
38.8
140.8
117.6
34.2
74.4
43.9
37.2
24.3
72.5
58.0
88.1
33.9
32.0
91.5
9.4
18.6
208.7
27.1
166.7
113.1
165.9
38.2
21.0
20.9
16.5
29.7
77.7
39.3
139.4
118.8
34.7
74.3
44.4
37.4
25.1
72.6
57.9
89.4
33.3
32.5
92.4
10.7
18.2
209.1
27.7
165.6
114.0
164.9
38.1
20.8
20.9
16.7
29.9
77.7
39.3
139.5
118.9
34.9
74.4
44.6
37.5
25.1
72.6
58.0
89.5
33.9
33.0
92.5
10.8
18.2
209.4
27.6
166.0
114.2
165.5
38.2
20.9
21.0
16.5
29.9
77.7
39.3
139.4
119.1
34.8
74.3
44.6
37.4
25.1
72.6
58.0
89.5
33.8
33.0
92.4
10.7
18.2
209.3
27.5
166.0
114.2
165.3
38.1
20.8
20.9
16.5
28.8
77.8
38.7
140.7
117.6
34.2
74.3
43.7
37.1
24.2
72.2
57.9
87.9
33.0
31.8
91.4
9.4
18.6
208.9
27.2
166.9
112.9
165.9
38.2
21.0
20.8
16.5
29.8
77.7
39.0
139.4
119.1
34.8
74.3
44.6
37.4
25.0
73.3
58.0
89.4
33.8
33.0
92.4
10.7
18.1
209.3
27.5
165.9
114.2
165.3
38.1
20.8
20.9
16.7
29.9
77.7
39.3
139.4
119.9
34.8
74.3
44.6
37.4
25.1
72.6
58.0
89.5
33.8
33.0
92.4
10.7
18.2
209.4
27.5
166.0
114.2
165.4
38.2
20.9
21.0
16.5
29.8
77.7
39.3
139.4
119.1
34.8
74.3
44.6
37.4
25.0
72.6
58.0
89.4
33.8
33.0
92.4
10.7
18.1
209.3
27.5
165.9
114.2
165.3
38.1
20.8
20.9
16.7
28.8
77.8
38.7
140.7
117.6
34.2
74.8
43.6
37.1
24.2
72.7
57.9
88.0
33.0
31.8
91.5
9.4
18.6
208.9
27.2
166.9
112.9
165.9
38.1
20.9
20.8
16.5
10
11
12
13
14
15
16
17
18
19
20
21
10
20
30
40
50
60
70
a
Measured in CDCl3.
Measured in C5D5N.
b
2.3.12. Compound 12
White amorphous powder; [
(MeOH) kmax (log ) 260 (3.89) nm; IR (KBr) õmax 3446, 2972, 2933,
1713, 1611, 1610, 1516, 1452, 1381, 1313, 1274, 1164, 1100, 1059,
913, 853, 772; HRESIMS m/z 1387.7018 ([M+Na]+, C70H108O26Na,
calcd. 1387.7021). 1H NMR (C5D5N, 500 MHz) and 13C NMR
(C5D5N, 125 MHz) see Tables 4–6.
was confirmed by TLC, comparing with its Rf value of authentic
sugar, respectively. As results, cymaroses were detected from 1–
14, digitoxoses were detected from 3–6, 8 and 10, oleandroses
were detected from 1, 2, 5, 6, 8 and 11–14, and thevetoses were
detected from 3–4, 7 and 10. The Rf values of thevetose, digitoxose,
cymarose, and oleandrose were 0.10, 0.18, 0.38, and 0.40 with sol-
vent CHCl3–MeOH (10:1); 0.05, 0.09, 0.15, and 0.17 with solvent
petroleum ether–Me2CO (7:3), respectively. All authentic com-
pounds were purchased from National Institutes for Food and Drug
Control, China.
22
a]
ꢁ52.8 (c 0.75, MeOH); UV
D
e
2.3.13. Compound 13
21
White amorphous powder; [
(MeOH) kmax (log
a]
+15.8 (c 1.90, MeOH); UV
D
e
) 216 (4.30) nm, 278 (3.80) nm; IR (KBr) õmax
3442, 2992, 1713, 1636, 1496, 1450, 1384, 1368, 1311, 1165, 1088,
911, 864, 769; HRESIMS m/z 1109.5649 ([M+Na]+, C58H86O19Na,
calcd. 1109.5655). 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3,
125 MHz) see Tables 4–6.
2.5. Determination of the absolute configuration of the sugars
Compound 10 (20.0 mg) was hydrolyzed by the above method
to get digitoxose, cymarose, thevetose, respectively, which abso-
lute configurations were confirmed by comparison of their optical
21
rotations with those reported in literatures (digitoxose [
a
]
+42.5
2.3.14. Compound 14
D
20
21 +53.5 (c
+36.5 (c
22
(c 0.16, H2O), [15]: D-digitoxose [
a
D
]
+43.0; cymarose [a]
White amorphous powder; [
a
]
+111.3 (c 1.49, MeOH); UV
D D
D
27
21
0.20, H2O), [24]: D-cymarose [
a
]
+56.0; thevetose [
a]
D
(MeOH) kmax (log
e
) 217 (3.89) nm, 282 (3.81) nm; IR (KBr) õmax
27
0.10, H2O), [24]: D-thevetose [
a
]
+35.0). By the same method, D-
oleandrose and L-cymarose were detected from 6 (5.0 mg), and their
3447, 2972, 2934, 2309, 1717, 1637, 1593, 1450, 1379, 1286, 1165,
D
1074, 989, 952, 911, 866, 743; HRESIMS m/z 1036.5267 ([M+H]+,
C
56H78NO17, calcd. 1036.5264). 1H NMR (C5D5N, 500 MHz) and 13C
optical rotations were determined and compared with reported data
21
27
(oleandrose [
a
]
ꢁ7.5 (c 0.10, H2O), [24]: D-oleandrose [
a
]
D
ꢁ12.0;
NMR (C5D5N, 125 MHz) see Tables 4–6.
D
21
20
cymarose [
a
]
ꢁ44.9 (c 0.10, H2O); [23]: L-cymarose [
a
]
D
ꢁ47.3).
D
2.4. Acidic hydrolysis
2.6. Cytotoxic bioassay
A solution of 1–14 (each 2.0 mg) in MeOH (2 mL) was added
0.025 M H2SO4 (1 mL). The solution was kept at 60 °C for 2 h, and
diluted with H2O (5 mL). The hydrolyzed mixture was neutralized
with saturated aqueous Ba(OH)2 solution. The precipitate was fil-
tered and the filtrate evaporated to dryness. The residue was iso-
lated to CC (Toyopearl-HW-40C gel, MeOH) to afford sugars and
aglycone parts successively. The total yields of digitoxoses, cym-
aroses, oleandroses and thevetoses from the hydrolysates of 1–14
were 0.5 mg, 1.1 mg, 0.6 mg and 0.3 mg, and each monosaccharide
The protocol of the cytotoxic bioassay was provided in a previ-
ously published paper [25]. Briefly, each cell line (HepG2, HeLa,
U251 human cancer cell lines) was cultured in DMEM medium in
5% CO2 at 37 °C. Then 100 lL of the cell suspension was seeded onto
96-well cell culture plates at an initial density of 1 ꢀ 104 cells/mL
and allowed to adhere for 12 h. Each tumor cell line was exposed
to each test compound at various concentrations for 48 h, with
5-FU and cisplatin (Sigma, St. Louis, MO, USA) used as positive
Please cite this article in press as: M. Zhang et al., Cytotoxicity of pregnane glycosides of Cynanchum otophyllum, Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.08.010

M. Zhang et al. / Steroids xxx (2015) xxx–xxx
5
Table 2
13C NMR data for the sugar moieties of compounds 1–9 (d in ppm, 125 MHz).
No.
1a
-cym
2b
-cym
3b
-cym
4b
-digit
5a
-digit
6b
7b
-cym
8b
9a
-cym
D
D
D
D
D
D
D
D
D
D-digit
D
D
-digit
D
100
96.1
35.8
77.0
82.3
68.5
18.2
58.3
96.3
37.2
77.9
83.1
68.9
18.5
58.8
96.5
37.3
78.1
83.2
69.1
18.6
58.9
96.4
39.0
67.5
83.5
68.6
18.7
95.9
37.1
66.7
82.5
68.7
18.3
96.4
38.8
67.8
82.3
68.8
18.6
96.4
37.4
78.1
81.6
69.0
18.7
58.9
95.6
39.0
67.5
83.4
68.7
18.6
94.6
35.1
77.1
82.4
68.8
18.2
57.7
200
300
400
500
600
OMe
D
-ole
D
-the
D
-cym
-cym
-cym
L-cym
D
-cym
D
-cym
D-ole
1000
101.4
36.4
79.0
82.7
71.2
18.3
56.6
104.1
75.2
88.2
83.4
72.0
18.8
60.9
100.5
37.1
78.2
83.4
69.4
18.6
58.9
99.7
36.7
78.1
83.2
69.5
18.5
58.8
98.4
35.3
76.9
82.4
68.2
18.2
58.3
99.0
32.5
73.6
77.7
66.3
18.2
57.1
100.5
37.3
78.0
82.4
69.3
18.6
58.9
99.7
36.7
77.7
82.3
69.3
18.6
58.5
101.3
35.9
78.5
81.8
71.5
17.7
56.8
2000
3000
4000
5000
6000
OMe
D
-cym
D
-ole
D
-the
-the
-ole
D-cym
D
-the
L
-cym
L
-cym
10000
20000
30000
40000
50000
60000
OMe
98.2
33.9
77.6
71.7
71.1
18.4
57.1
101.6
36.8
79.2
76.2
72.9
18.5
57.3
106.3
75.1
87.9
75.9
72.8
18.6
61.0
106.3
75.1
87.9
75.9
72.8
18.5
60.9
101.4
36.4
78.9
82.2
71.1
18.2
56.7
96.0
36.7
77.3
81.1
69.4
18.6
58.3
106.0
74.8
85.6
83.2
71.6
18.6
60.4
99.0
32.2
73.2
77.7
65.3
18.5
56.6
98.4
31.6
72.0
76.9
63.8
18.1
56.3
D
-cym
L-cym
D-ole
D
-cym
D-cym
100000
200000
300000
400000
500000
600000
OMe
98.2
33.7
77.5
71.5
71.2
18.3
57.1
98.4
32.2
76.4
73.3
66.6
18.5
56.6
100.4
37.0
83.4
76.3
73.1
18.5
57.1
96.4
36.8
77.7
82.1
69.3
18.5
58.2
96.6
35.4
76.9
82.4
68.5
18.2
57.9
L
-cym
D-cym
1000000
2000000
3000000
4000000
5000000
6000000
OMe
98.9
32.3
76.4
73.3
66.4
18.4
56.9
98.3
35.5
76.9
81.9
68.3
18.2
58.3
L-cym
10000000
20000000
30000000
40000000
50000000
60000000
OMe
97.4
31.8
74.5
71.6
65.5
18.2
56.2
D
-digit: b-
D
-digitoxopyranosyl.
D-cym: b-
D-cymaropyranosyl.
L
-cym:
a
-
L
-cymaropyranosyl.
D-ole: b-
D-oleandropyranosyl.
D-the: b-
D-thevetopyranosyl.
a
Measured in CDCl3.
Measured in C5D5N.
b
controls. At the end of the treatment period, 20
l
L (5 mg/mL) of the
preliminarily determine the existence of pregnane glycosides.
The TLC spots, showing characteristically green color (Lieber-
mann–Burchard Reaction) of C21 steroids, were rich in CHCl3 frac-
tion from 75% ethanol extract of its roots. Thus, the CHCl3 fraction
were successively and repeatedly subjected to silica gel and ODS
column chromatography, and purified by semi-preparative HPLC
to afford 26 pregnane glycosides.
MTT was added to each well and the plates were incubated for 4 h
at 37 °C. The medium was removed and MTT reduction product
(formazan crystals) was dissolved in dimethylsulfoxide (DMSO)
and measured with an ELISA reader (Bio-Rad, USA) at a wavelength
of 492 nm. IC50 value of each test compound was calculated, given
in Table 7. Each assay was done in triplicate.
Compound 1 was obtained as a white powder. Its molecular for-
mula C49H78O16 was defined by HRESIMS. The 13C NMR spectrum
(Tables 1 and 2) showed 49 carbon signals, seven of which at dC
166.7, 113.1, 165.9, 38.2, 21.0, 20.9 and 16.5 were assigned to an
ikemaoyl group by study of HSQC and HMBC spectra. The aglycone
part of 1 was identified as caudatin considering the NMR data [21].
3. Results and discussion
This study aims to systematically elucidate the structure and
cytotoxicities of C21 steroids in C. otophyllum. TLC was used to
Please cite this article in press as: M. Zhang et al., Cytotoxicity of pregnane glycosides of Cynanchum otophyllum, Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.08.010

6
M. Zhang et al. / Steroids xxx (2015) xxx–xxx
Table 3
1H NMR spectral data of compounds 1–9 (d in ppm, J in Hz, 500 MHz).
No.
1
1a
2b
3b
4b
5a
6b
7b
8b
9a
1.08 m; 1.87 m 1.11 m;
1.81 m
1.60 m; 1.91 m 1.80 m;
2.08 m
1.08 m; 1.78 m
1.10 m; 1.80 m
1.08 m; 1.87 m 1.08 m; 1.79 m 1.09 m;
1.08 m; 1.79 m 1.08 m;
1.85 m
1.78 m; 2.07 m 1.59 m;
1.90 m
1.78 m
1.61 m; 1.89 m 1.78 m; 2.06 m 1.78 m;
2.05 m
2
1.77 m; 2.04 m
1.78 m; 2.08 m
3
4
3.57 m
2.29 m; 2.36 m 2.40 m;
2.55 m
3.84 m
3.84 m
2.39 m; 2.55 m
3.86 m
2.40 m; 2.51 m
3.56 m
3.84 m
3.84 m
3.84 m
3.55 m
2.28 m; 2.36 m 2.39 m; 2.50 m 2.41 m;
2.52 m
5.35 s
2.18 m
1.51 m
1.81 m
2.39 m; 2.50 m 2.28 m;
2.36 m
6
7
9
11
5.36 s
5.28c
5.27c
2.44 m
1.70 m
2.13 m; 2.23 m
5.25 s
2.45 m
1.72 m
2.15 m; 2.25 m
5.28 s
2.44 m
1.70 m
5.27c
2.44 m
1.72 m
5.26c
2.43 m
1.70 m
5.36 s
2.17 m
1.49 m
2.18 m
1.51 m
1.84 m
2.47 m
1.73 m
2.16 m;
2.26 m
5.04d
2.13 m; 2.24 m 2.14 m;
2.24 m
2.14 m; 2.23 m 1.80 m
12
4.56 t (7.5)
5.02 dd (4.0,
11.5)
2.08 m
5.02d
4.55 t (7.5)
1.93 m
1.83 m; 2.84 m 2.02 m; 3.26 m 2.03 m;
3.26 m
5.05d
5.0 5d
5.04d
4.54 t (7.5)
1.93 m
15
16
1.95 m
1.84 m; 2.84 m 2.02 m;
3.27 m
2.11 m
2.10 m
2.02 m; 3.27 m
2.10 m
2.09 m
2.09 m
2.01 m; 3.26 m 1.84 m;
2.84 m
2.01 m; 3.27 m
18
19
21
20
1.40 s
1.12 s
2.12 s
5.51 s
2.35 m
1.05 d (7.0)
1.06 d (7.0)
2.16 s
1.98 s
1.32 s
2.51 s
5.86 s
2.25 m
0.92 d (7.5)
0.94 d (7.5)
2.27 s
1.97 s
1.31 s
2.50 s
5.85 s
2.26 m
0.92 d (7.5)
0.99 d (7.5)
2.26 s
1.98 s
1.30 s
2.50 s
5.86 s
2.25 m
0.92 d (6.5)
0.94 d (6.5)
2.25 s
1.40 s
1.12 s
2.16 s
5.51 s
2.36 m
1.05 d (7.0)
1.06 d (7.0)
2.12 s
1.99 s
1.31 s
2.50 s
5.86 s
2.25 m
0.92 d (7.5)
0.94 d (7.5)
2.27 s
1.98 s
1.31 s
2.50 s
5.86 s
2.25 m
0.92 d (6.5)
0.93 d (6.5)
2.26 s
1.99 s
1.31 s
2.50 s
5.86 s
2.25 m
0.92 d (7.5)
0.94 d (7.5)
2.27 s
1.39 s
1.11 s
2.14 s
5.50 s
2.34 m
1.04 d (7.0)
1.05 d (7.0)
2.13 s
40
50
60
70
D-cym
D
-cym
D
-cym
D-digit
D-digit
D-digit
D-cym
D-digit
D-cym
100
200
4.82 dd (9.5,
1.5)
1.58 m; 2.10 m 1.88 m;
2.33 m
5.26 d (9.5)
5.25 d (10.0)
5.46 dd (10.0,
1.0)
2.06 m; 2.38 m
4.91 dd (9.5,
1.5)
1.68 m; 2.10 m 2.03 m; 2.38 m 1.85 m;
2.33 m
5.41 dd (9.0,
1.0)
5.26c
5.47 dd (9.5,
1.0)
2.07 m; 2.39 m 1.61 m;
2.09 m
4.84c
1.85 m; 2.29 m
300
400
500
600
3.81 m
3.17 m
3.86 m
1.29 d (5.5)
4.05 m
3.49 m
4.21 m
1.41 d (6.0)
3.56 s
4.05 m
3.48 m
4.20 m
1.37 d (6.0)
3.60 s
4.62 m
3.48 m
4.30 m
1.42 d (6.0)
4.23 m
3.18 m
3.76 m
1.28 d (6.0)
4.48 m
3.48 m
4.19 m
1.40 d (6.5)
4.06 m
3.49 m
4.21 m
1.36 d (6.0)
3.60 s
4.62 m
3.45 m
4.28 m
1.42 d (6.0)
3.73 m
3.22 m
3.81 m
1.19 d (6.0)
3.42 s
OMe 3.41 s
D-ole
D
-the
D
-cym
D-cym
D-cym
L-cym
D-cym
D-cym
D-ole
1000
2000
4.44 dd (9.5,
1.5)
1.52 m; 2.32 m 3.92 m
4.94 d (8.0)
5.10 d (9.5)
5.16 dd (9.5, 1.0) 4.87 dd (9.5,
1.5)
4.97 d (3.0)
5.11d
5.12 dd (9.5,
1.0)
1.78 m; 2.33 m 1.53 m;
2.32 m
4.44 d (9.5)
1.81 m; 2.31 m
1.78 m; 2.32 m
1.61 m; 2.33 m 1.69 m; 2.26 m 1.81 m;
2.30 m
3000
4000
5000
6000
3.55 m
3.23 m
3.58 m
1.22 d (6.5)
3.60 m
3.67 m
3.57 m
1.70 d (6.0)
3.88 s
4.04 m
3.58 m
4.19 m
1.57 d (6.0)
3.55 s
4.05 m
3.52 m
4.21 m
1.53 d (6.0)
3.55 s
3.80 m
3.21 m
3.90 m
1.22 d (6.0)
3.42 s
3.73 m
3.89 m
4.55 m
1.40 d (6.0)
3.36 s
4.03 m
3.55 m
4.22 m
1.47 d (5.5)
3.61 s
3.85 m
3.45 m
4.18 m
1.36 d (6.0)
3.52 s
3.24 m
3.19 m
3.26 m
1.17 d (6.0)
3.36 s
OMe 3.40 s
D-cym
D
-ole
D
-the
D-the
D-ole
D-cym
D-the
L-cym
L-cym
10000
20000
4.84 dd (9.5,
1.5)
1.94 m; 2.20 m 1.78 m;
2.47 m
4.69 d (9.5)
4.75 d (7.5)
3.91 m
4.75 d (6.0)
3.90 m
4.44 dd (9.5,
1.5)
1.71 m; 2.10 m 1.77 m; 2.30 m 3.88 m
5.23 dd (9.5,
1.5)
4.71 d (8.0)
4.96 d (3.5)
4.83c
1.82 m; 2.32 m 1.83 m;
2.24 m
30000
40000
50000
60000
3.62 m
3.29 m
3.64 m
1.29 d (5.5)
3.58 m
3.62 m
3.73 m
1.58 d (6.0)
3.52 s
3.61 m
3.60 m
3.72 m
1.58 d (7.0)
3.89 s
3.61 m
3.60 m
3.70 m
1.56 d (6.0)
3.89 s
3.21 m
3.16 m
3.29 m
1.30 d (6.0)
3.40 s
3.60 m
3.45 m
4.17 m
1.35 d (6.0)
3.52 s
3.69 m
3.68 m
3.67 m
1.58 d (6.0)
3.88 s
3.60 m
3.85 m
4.62 m
1.50 d (6.5)
3.35 s
3.56 m
3.88 m
4.27 m
1.24 d (6.0)
3.33 s
OMe 3.45 s
D-cym
L-cym
D-ole
D-cym
D-cym
100000
200000
4.80 dd (9.5,
1.5)
1.56 m; 2.19 m 1.78 m; 2.27 m 1.74 m;
2.47 m
5.05 s
4.99 d (9.5)
4.54 dd (9.5,
1.0)
1.78 m; 2.33 m 1.61 m;
2.09 m
4.82c
300000
400000
500000
600000
OMe
3.60 m
3.21 m
3.59 m
1.22 d (6.0)
3.45 s
3.73 m
3.60 m
4.56 m
1.51 d (6.0)
3.37 s
3.48 m
3.50 m
3.61 m
1.58 d (6.0)
3.48 s
3.85 m
3.45 m
4.18 m
1.36 d (6.0)
3.52 s
3.78 m
3.24 m
3.83 m
1.19 d (6.0)
3.45 s
L-cym
D-cym
1000000
2000000
4.93 d (2.5)
1.80 m; 2.35 m 1.53 m;
2.11 m
4.73 d (9.5)
Please cite this article in press as: M. Zhang et al., Cytotoxicity of pregnane glycosides of Cynanchum otophyllum, Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.08.010

M. Zhang et al. / Steroids xxx (2015) xxx–xxx
7
Table 3 (continued)
No.
1a
2b
3b
4b
5a
6b
7b
8b
9a
3000000
4000000
5000000
6000000
OMe
3.70 m
3.73 m
4.53 m
1.50 d (6.0)
3.51 s
3.80 m
3.06 m
3.84 m
1.19 d (6.0)
3.46 s
L
-cym
10000000
20000000
4.79 d (3.5)
1.83 m;
2.21 m
30000000
40000000
50000000
60000000
OMe
3.68 m
3.27 m
4.04 m
1.24 d (6.0)
3.33 s
D
-digit: b-
D
-digitoxopyranosyl.
D
-cym: b-
D-cymaropyranosyl.
L
-cym:
a
-
L
-cymaropyranosyl.
D-ole: b-
D-oleandropyranosyl.
D-the: b-
D-thevetopyranosyl.
a
Measured in CDCl3.
Measured in C5D5N.
Partial overlapped with other signals.
Overlapped with H2O signals.
b
c
d
Table 4
13C NMR data for the aglycone moieties of compounds 10–14 (d in ppm, 125 MHz).
(d, J = 5.5 Hz)] in 1H NMR (Table 3) and of the anomeric protons,
as well as other signals in 1D NMR spectra (Tables 1–3) indicated
No.
10b
11b
12b
13a
14b
a b-linkage for the sugar chain, which was formed by two
D-cy-
marose and one -oleandrose units. This fact was confirmed by
D
1
39.0
39.3
39.0
38.7
39.3
comparison of the TLC and optical rotation (OR) analysis of acid
hydrolysate of 1 with those of authentic samples. The sequence
of these three sugar units linked C-3 of the caudatin was illustrated
2
3
4
5
6
7
8
9
29.9
77.7
39.3
139.4
119.1
34.8
74.3
44.6
37.4
25.1
72.6
58.0
89.5
33.8
33.0
92.4
10.7
18.2
209.3
27.5
166.0
114.2
165.3
38.1
20.8
20.9
16.5
29.9
77.7
38.9
139.5
119.1
34.8
74.4
44.5
37.4
25.2
73.4
58.4
89.5
33.9
33.1
92.5
10.8
18.2
29.8
77.7
39.2
139.4
119.1
34.8
74.3
44.5
37.4
25.2
73.4
58.4
89.5
33.9
33.2
92.5
10.8
18.1
29.1
77.8
38.7
140.7
117.5
34.5
75.4
43.6
37.1
24.5
72.7
58.7
87.7
33.1
31.9
91.4
9.4
18.6
29.9
77.7
39.1
139.4
119.3
34.9
74.3
44.1
37.3
25.7
74.6
57.2
89.0
34.1
33.7
87.5
11.5
18.0
by distinct HMBC correlations from dH 4.82 (b-
H-100) to dC 78.0 (C-3), from dH 4.44 (inner b-
-oleandropyranosyl
H-1000) to dC 82.3 (b- -cymaropyranosyl C-400), from dH 4.86 (termi-
nal b- -oleandropyranosyl
-cymaropyranosly H-10000) to dC 82.7 (b-
C-4000). Thus, the structure of 1 was determined to be caudatin-3-O-
b- -cymaropyranosyl-(1 ? 4)-b- -oleandropyranosyl-(1 ? 4)-b-
cymaropyranoside.
D-cymaropyranosyl
D
D
D
D
10
11
12
13
14
15
16
17
18
19
20
21
10
D
D
D-
Comparison of 1D NMR data (Tables 1 and 3) and the key corre-
lations in 2D NMR with those reported in the literatures [21,22]
suggested that aglycone parts of 2–9 were caudatin-3-O-multio-
sides, as the same as 1. The differences observed among them were
orders of linkage and types of sugar units, which were identified by
NMR analysis, HRESIMS, and TLC and optical rotation comparison
with authentic samples, respectively.
209.7
27.8
209.8
27.9
209.1
27.6
76.4
15.4
165.4
122.0
132.4
116.2
163.6
116.2
132.4
165.3
122.0
132.4
116.2
163.6
116.2
132.4
165.7
117.7
145.4
134.3
128.1
128.9
130.4
128.9
128.1
166.8
120.3
144.0
134.9
128.5
129.3
128.5
129.3
128.5
164.7
153.8
127.0
137.3
123.0
151.4
Compounds
2 and 3 had the same molecular formula
20
C
49H78O17 based on HRESIMS. The 13C NMR data of sugar parts
30
40
in 2 (Tables 1 and 2) exhibited three anomeric carbon signals
at dC 96.3, 104.1, 101.6, correlating with anomeric protons at
dH 5.26 (brd, 9.5 Hz), 4.94 (brd, J = 8.0 Hz), and 4.69 (brd,
J = 9.5 Hz) confirmed by HSQC experiment, respectively, along
with three groups of well recognized methoxyl proton [dH
3.60, 3.55, 3.98 (each 3H, s)] and three methyl signals [dH 1.41
(d, J = 6.0 Hz), 1.70 (d, J = 6.0 Hz), 1.58 (d, J = 6.0 Hz)] in 1H
NMR (Table 3), suggesting that the sugar chain for 2 consisted
50
60
70
80
90
100
200
300
400
500
600
of one b-
[22]. The sequence of those sugar units of 2 was inferred by
the correlations in HMBC analysis between dH 4.69 (b- -olean-
dropyranosyl H-10000) and dC 83.4 (middle b-
-thevetopyranosyl
C-4000), and dH 4.94 (middle b- -thevetopyranosyl H-1000) and dC
83.1 (b-
-cymaropyranosyl C-400). Similarly, three anomeric car-
D-cymarose, one b-D-oleandrose and one b-D-thevetose
D
a
Measured in CDCl3.
Measured in C5D5N.
b
D
D
D
bons at dC 96.5, 100.5, 106.3, and the corresponding protons at
dH 5.25 (d, J = 10.0 Hz), 5.10 (d, J = 9.5 Hz) and 4.75 (d,
J = 7.5 Hz), were shown in HSQC experiment of 3. The coupling
constants of three anomeric protons and of three secondary
methyl groups [dH 1.37 (d, J = 6.0 Hz), 1.57 (d, J = 6.0 Hz) and
1.58 (d, J = 7.0 Hz)] in 1H NMR (Table 3), as well as other signals
The anomeric carbons at dC 96.1 (cym C-1), 101.4 (ole C-1), and
98.2 (cym C-1), and the corresponding proton signals at dH 4.82
(dd, J = 9.5, 1.5 Hz), 4.44 (dd, J = 9.5, 1.5 Hz), and 4.86 (dd, J = 9.5,
1.5 Hz), respectively, showed correlations in the HSQC experiment.
Analysis of the coupling constants of three secondary methyl
groups [dH 1.29 (d, J = 5.5 Hz), 1.22 (d, J = 6.5 Hz) and 1.29
Please cite this article in press as: M. Zhang et al., Cytotoxicity of pregnane glycosides of Cynanchum otophyllum, Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.08.010

8
M. Zhang et al. / Steroids xxx (2015) xxx–xxx
in 1D NMR spectra (Tables 1–3) indicated that the sugar chain in
3 was formed by two b- -cymaropyranosides and one b- -theve-
to dC 77.3 (b-
maropyranosyl H-1000) to dC 82.3 (b-
Therefore, the structure of and
caudatin-3-O-b- -cymaropyranosyl-(1 ? 4)-b-
(1 ? 4)-b- -cymaropyranosyl-(1 ? 4)-b- -digitoxopyranoside and
caudatin-3-O- -cymaropyranosyl-(1 ? 4)-â- -cymaropyranosyl-
(1 ? 4)- -cymaropyranosyl-(1 ? 4)-b- -digitoxopyranoside,
respectively.
L
-cymaropyranosyl C-4000), and from dH 4.97 (
-digitoxopyranosyl C-400).
were established as
-oleandropyranosyl-
a-L-cy-
D
D
D
topyranoside. The sequence of sugar units of 3 was elucidated
by HMBC experiment in which the correlations between dH
5
6
D
D
4.75 (b-
maropyranosyl C-4000), and dH 5.10 (middle b-
H-1000) and dC 83.2 (b- -cymaropyranosyl C-400) were observed.
D
-thevetopyranosyl H-10000) and dC 83.4 (middle b-
D
-cy-
D
D
D
-cymaropyranosyl
a
-
L
D
D
a
-
L
D
Comparing with reported spectral data, sugar moiety of 3 bore
a resemblance to that of stemucronatoside J [5]. Further, all
Compound 7 was suggested to have a molecular formula
the monosaccharides in
2
and
3
were also confirmed by
C56H90O20 on the basis of the HRESIMS. The analysis of acid hydro-
comparison of the TLC and OR analysis of acid hydrolysates of
lysate of 7 suggested that the sugar units consisted of cymarose,
2 and 3 with those of authentic samples. Hence, the structures
thevetose and oleandrose. Based on the 1D NMR data of sugar part,
of 2 and 3 was determined to be caudatin-3-O-b-
nosyl-(1 ? 4)-b- -thevetopyranosyl(1 ? 4)-b- -cymaropyranoside
and caudatin-3-O-b- -thevetopyranosyl-(1 ? 4)-b- -cymaropy-
ranosyl-(1 ? 4)-b- -cymaropyranoside, respectively.
D
-oleandropyra-
it suggested two b-
D-cymaropyranosyl, one b-D-oleandropyranosyl
D
D
and one b- -thevetopyranosyl existed in 7. These four sugar units
D
D
D
were formed as one chain connected to C-3 of the aglycone, which
was confirmed by long-range correlations in HMBC analysis from
D
Compound 4 had a molecular formula C48H76O17. Its 1H NMR
spectral data of sugar part existed three anomeric protons at dH
5.46 (dd, J = 10.0, 1.5 Hz), 5.16 (dd, J = 9.5, 1.0 Hz), and 4.75 (brd,
J = 7.5 Hz), whose coupling constants revealed the existence of
two 2, 6-dideoxy sugars and one 6-dideoxy sugar with all b-link-
ages. According to 2D NMR analysis and comparison of the spectral
data, TLC and OR data of acid hydrolysate of 4 with those of analogs
dH 4.99 (outer b-
topyranosyl C-40000), from dH 4.71 (b-
82.4 (b-
-cymaropyranosyl C-4000), from dH 4.98 (b-
nosyl H-1000) to dC 81.6 (b- -cymaropyranosyl C-400), and from dH
5.26 (b-
-cymaropyranosyl H-100) to dC 77.7 (C-3). Thus, 7 was
determined to be caudatin-3-O-b- -oleandropyranosyl-(1 ? 4)-b-
-thevetopyranosyl-(1 ? 4)-b- -cymaropyranosyl-(1 ? 4)-b-
cymaropyranoside.
Compound 8 was isolated as colorless powder. According to the
D
-oleandropyranosyl H-100000) to dC 83.3 (b-
D
-theve-
-thevetopyranosyl C-10000) to dC
-cymaropyra-
D
D
D
D
D
D
D
D
D-
[22], it indicated the existence of one b-
-cymaropyranosyl and one b- -thevetopyranosyl in 4. The
sequence of those sugar units of 4 was inferred by the long-range
correlations in HMBC analysis from dH 4.75 (b- -thevetopyranosyl
H-10000) to dC 83.2 (b- -cymaropyranosyl C-4000), and from dH 5.15
(b- -digitoxopyranosyl
-cymaropyranosyl H-1000) to dC 83.5 (b-
C-400). Consequently, 4 was established to be caudatin-3-O-b-
D-digitoxopyranosyl, one b-
D
D
HRESIMS, its molecular formula was determined to be C62H100O22
.
D
Its 1H NMR showed five anomeric proton signals [dH 5.47 (H, dd,
J = 9.5, 1.0 Hz), 5.12 (H, dd, J = 9.5, 1.0 Hz), 4.96 (H, d, J = 3.5 Hz),
5.24(H, dd, J = 9.5, 1.0 Hz)and 4.93 (H, d, J = 2.5 Hz)], whosecoupling
constants observed suggested that 8 had three sugar units with b-
D
D
D
D
-
thevetopyranosyl-(1 ? 4)-b-
D
-cymaropyranosyl-(1 ? 4)-b-
D-digi-
linkages and two sugar units with a-linkages (Table 3), respectively.
toxopyranoside.
Acid hydrolysis of 8 gave digitoxose and cymarose as residues. The
The molecular formulas of 5 and 6 were both determined as
NMR data of sugar part of 8 bore a resemblance to that of 6, except
C
55H88O19 by their HRESIMS. TLC analysis of the acidic hydrolysate
for the presence of an additional b-
sequenceof the sugarpart was assignedby HMBC spectrumin which
distinct correlations from dH 4.93 (outer -cymaropyranosyl H-
1000000) to dC 82.1 (b- -cymaropyranosyl C-400000), from dH 5.24 (b-
-cy-
maropyranosyl H-100000) to dC 77.7 ( -cymaropyranosyl C-40000), from
dH 4.96 ( -cymaropyra-
-cymaropyranosyl H-10000) to dC 82.3 (b-
nosyl C-4000), and from dH 5.15 (b- -cymaropyranosyl H-1000) to dC
83.4 (b-
-digitoxopyranosyl C-400) were observed. Hence, 8 was
deduced to be caudatin-3-O- -cymaropyranosyl-(1 ? 4)-b- -cy-
maropyranosyl-(1 ? 4)- -cymaropyranosyl-(1 ? 4)-b- -cy-
maropyranosyl-(1 ? 4)-b- -digitoxopyranoside.
The molecular formula of 9 was inferred to be C70H114O25
according to HRESIMS. In 1H NMR spectrum, six methoxyl groups
and six secondary methyl groups were shown, suggesting that
the sugar chain consisted of six 2,6-deoxysugar units. The coupling
constants of six anomeric proton signals, which were shown at dH
4.84 (brd, partially overlapped), 4.44 (brd, J = 9.5 Hz), 4.82 (d, par-
tially overlapped), 4.83 (brd, partially overlapped), 4.74 (brd,
J = 9.5 Hz), 4.79 (d, J = 3.5 Hz) in 1H NMR spectrum, indicated 9
D-cymaropyranosyl in 8. The
displayed there were cymarose, oleandrose and digitoxose in 5,
and cymarose and digitoxose in 6, respectively. The 1H NMR spec-
trum of 5 showed four secondary methyl signals [dH 1.28 (d,
J = 6.0 Hz), 1.22 (d, J = 6.0 Hz), 1.30 (d, J = 6.0 Hz), 1.22 (d,
J = 6.0 Hz)], three methoxyl signals [dH 3.42, 3.40, 3.45 (each 3H,
s) and four anomeric proton signals [dH 4.91 (1H, dd, J = 9.5,
1.5 Hz), 4.80 (1H, dd, J = 9.5, 1.5 Hz), 4.44 (1H, dd, J = 9.5, 1.5 Hz),
4.87 (1H, dd, J = 9.5, 1.5 Hz)] (Table 3). Those information proved
that four 2,6-dideoxysugars were composed as sugar residue with
a b-linkage in 5. The carbon shifts of each sugar unit were assigned
by HMBC and HSQC experiments (Table 2). Compared with the
spectral data of analogs [26–29], it suggested the sugar chain of
a-L
D
D
a
-L
a
-L
D
D
D
a
-L
D
a
D
-
L
D
5 was formed from two
nosyl and one -oleandropyranosyl units. The sequence of those
four sugar units of 5 was elaborated by HMBC long correlations
from dH 4.87 (terminal b-
-cymaropyranosyl H-100000) to dC 82.2
(inner b- -olean-
-oleandropyranosyl C-40000), from dH 4.44 (b-
dropyranosyl H-10000) to dC 82.4 (inner b- -cymaropyranosyl C-4000),
and from dH 4.81 (b-
-cymaropyranosyl H-1000) to dC 82.5 (inner
b-
-digitoxopyranosyl C-400). Similarly, based on the reporting data
[21] that the chemical shift of C-2 is <dC 35.0 ppm in an -cy-
-cymaropyranosyl, respec-
D-cymaropyranosyl, one D-digitoxopyra-
D
D
D
D
D
D
possessed six sugar units with two
(Table 3), respectively. Each sugar unit signal was assigned to three
-cymaropyranosyl units, two -cymaropyranosyl units and one D-
a-linkages and four b-linkages
D
L
D
L
maropyranosyl and >dC 36.0 ppm in a
D
oleandropyranosyl unit by 1D and 2D NMR analysis (Tables 2 and
3) and comparison of their NMR information with those of analogs
[26–29]. The sequence of four sugar units in 9 was elaborated by
tively, as well as the 13C NMR signals of the five sugar unit
assigning unambiguously by HSQC and HMBC analysis (Table 2),
and the resonance of C-2 at dC 32.5, 36.7 and 32.2 in cymaropyra-
nosyl, respectively, it suggested the existence of two
HMBC correlations from dH 4.79 (terminal
H-10000000) to dC 81.9 (inner b- -cymaropyranosyl C-4000000), from dH
4.73 (b- -cymaropy-
-cymaropyranosyl H-1000000) to dC 82.4 (inner b-
ranosyl C-400000), from dH 4.83 (b- -cymaropyranosly H-100000) to dC
76.9 ( -cymaropyra-
-cymaropyranosyl C-40000), from äH 4.82 (
nosyl H-10000) to dC 81.8 (b- -oleandropyranosyl C-4000), and from
äH 4.44 (b- -cymaropyra-
-oleandropyranosyl H-1000) to dC 82.4 (b-
a-L-cymaropyranosyl
D
a-L-cymaropyranosyl, one b-D-cymaropyranosyl and one b-D-
digitoxopyranosyl in sugar moieties in 6. The sequence of sugar
units was determined by the analysis of HMBC correlations from
D
D
D
a-
L
a-L
dH 5.05 (outer
a-
L
-cymaropyranosyl H-100000) to dC 81.1 (b-
D
-cy-
D
maropyranosyl C-40000), from dH 5.23 (b-
D
-cymaropyranosyl H-10000
)
D
D
Please cite this article in press as: M. Zhang et al., Cytotoxicity of pregnane glycosides of Cynanchum otophyllum, Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.08.010

M. Zhang et al. / Steroids xxx (2015) xxx–xxx
9
nosyl C-400). Thus, the structure of 9 was established to be
caudatin-3-O- -cymaropyranosyl-(1 ? 4)-b- -cymaropyranosyl-
(1 ? 4)-b- -cymaropyranosyl-(1 ? 4)- -cymaropyranosyl-(1 ? 4)-
b- -oleandropyranosyl-(1 ? 4)-b- -cymaropyranoside.
Compound 10, isolated as white powder, had a molecular for-
Table 5
13C NMR data for the sugar moieties of compounds 10–14 (d in ppm, 125 MHz).
a-
L
D
10b
-digit
11b
-cym
12b
-cym
13a
-cym
14b
D
a-L
No.
D
D
D
D
D
D
D
D
D
D
100
96.4
39.0
67.6
83.5
69.5
18.6
96.4
37.2
78.1
83.5
69.1
18.7
59.0
95.6
36.7
77.6
83.1
68.9
18.6
58.9
96.1
35.7
77.0
82.4
68.3
18.2
58.5
mula C55H82O21. For aglycone moiety, the 1H NMR signals of 10
suggested the presence of four aromatic protons attributed to a
benzene ring [dH 8.30 (2H, m, H-30, 70), 7.26 (2H, m, H-40, 60)], three
singlet methyl groups [dH 1.29, 2.08, 2.41 (each 3H, s, H-19, 18,
20)], and an olefinic proton (dH 5.27, partially overlapped, H-6),
which features were in good agreement with qingyangshengenin
by comparing with spectral data (Table 6) of analogs in the litera-
tures [21,22]. Furthermore, four anomeric protons [dH 5.47 (H, brd,
J = 9.5 Hz), 5.15 (H, brd, partially overlapped), 4.69 (H, brd,
J = 8.0 Hz), 5.26 (H, brd, partially overlapped)], four secondary
methyl groups [dH 1.44 (H, d, J = 6.5 Hz), 1.42 (H, d, J = 6.0 Hz),
1.54 (H, d, J = 6.0 Hz), 1.53 (H, d, J = 5.5 Hz)], and three methoxyl
groups [dH 3.56, 3.87, 3.48, s, each 3H] were exhibited in 1H
NMR, indicating that three 2,6-dideoxy sugar units and one 6-
deoxy sugar unit with b-linkage formed the sugar moiety in 10.
The carbon signals of each sugar unit were assigned unambigu-
ously by HSQC and HMBC analysis (Tables 5 and 6), suggesting
the presence of one digitoxopyranosyl, one thevetopyranosyl, and
two cymaropyranosyl in 10. Comparing spectral data of 10 with
those of analogs [21,22], the absolute configurations of sugar units
were all elucidated as D-series. According to HMBC spectrum, the
sequence of the deoxysugars located at C-3 of the aglycone as a
chain was confirmed by the HMBC long range correlations
200
300
400
500
600
OMe
-cym
D-cym
D-ole
-cym
D
D
D
-digit
1000
99.8
36.8
78.1
83.2
68.6
18.7
58.9
100.5
37.1
77.8
83.2
69.0
18.6
58.8
101.9
37.2
79.1
81.6
72.1
18.5
56.4
99.7
35.7
78.0
82.2
68.5
18.4
58.3
96.4
38.8
67.5
83.4
68.6
18.7
2000
3000
4000
5000
6000
OMe
-the
D-cym
L-cym
-ole
-cym
10000
20000
30000
40000
50000
60000
OMe
106.1
74.7
85.5
82.4
71.7
18.6
60.7
101.9
37.3
79.1
81.5
72.2
18.5
56.9
97.4
32.3
73.6
78.0
64.9
18.3
57.2
101.3
36.6
79.1
82.5
71.6
18.2
56.9
99.7
36.7
77.8
83.2
69.1
18.7
58.9
D-cym
L-cym
D-cym
D-ole
-ole
100000
200000
300000
400000
500000
600000
OMe
98.9
36.0
79.0
74.2
71.3
19.1
58.1
97.3
32.2
76.7
73.4
66.1
18.5
56.4
96.4
37.2
77.9
83.1
69.1
18.6
58.8
100.2
35.8
80.7
75.4
70.9
17.9
56.6
102.2
37.2
81.4
76.2
73.0
18.5
57.1
observed between dH 4.69 (outer b-
and dC 82.4 (b-
-thevetopyranosyl C-40000), from dH 4.69 (b-
topyranosyl H-10000) and dC 83.2 (b- -cymaropyranosyl C-4000), from
dH 5.15 (b- -digitoxopyra-
-cymaropyranosyl H-1000) and dC 83.5 (b-
nosyl C-400), from dH 5.47 (b- -digitoxopyranosyl H-100) and dC 77.7
(C-3). Thus, 10 was determined to be qingyangshengenin-3-O-b-
cymaropyranosyl-(1 ? 4)-b- -thevetopyranosyl-(1 ? 4)-b- -cy-
maropyranosyl-(1 ? 4)-b- -digitoxopyranoside.
D
-cymaropyranosyl H-100000
-theve-
)
D
D
D
D
D
D
D-cym
D
-
1000000
2000000
3000000
4000000
5000000
6000000
OMe
100.5
37.0
77.7
82.3
69.4
18.6
58.3
D
D
D
Compound 11 was obtained as colorless powder and its molec-
ular formula was established as C56H84O20. The NMR characteris-
tics implied that 11 had the same aglycone of qingyangshengenin
as 10. Observation of the NMR spectral data showed that sugar
moiety of 11 contained four anomeric carbons at dC 96.4, 100.5,
101.9 and 97.3, and four corresponding anomeric proton signals
at dH 5.28 (dd, partially overlapped), 5.10 (dd, J = 9.5, 1.5 Hz),
4.66 (dd, J = 9.5, 2.0 Hz), and 5.08 (dd, J = 3.5, 1.0 Hz), confirmed
by HSQC experiment. On the basis of further analysis of HSQC
and HMBC spectra and comparison of the spectral data with those
of analogs [22] (Tables 5 and 6), the signals of the sugar units were
L-cym
10000000
20000000
30000000
40000000
50000000
60000000
OMe
99.0
32.3
76.4
73.3
66.4
18.5
56.9
exactly assigned, implying the existence of two b-
nosyl, one b- -oleandropyranosyl, and one -cymaropyranosyl
in 11. The sugar chain was attached at the C-3 of aglycone by
HMBC analysis in which correlations from dH 5.08 (outer -cy-
maropyranosyl H-100000) to dC 81.5 (b- -oleandropyranosyl C-40000),
from dH 4.66 (b-
-oleandropyranosyl C-10000) to dC 83.2 (b-
maropyranosyl C-4000), from dH 5.10 (b-
to dC 83.5 (b-
-cymaropyranosyl C-400), and from dH 5.28 (b-
D-cymaropyra-
a
Measured in CDCl3.
Measured in C5D5N.
D
a-L
b
a
-L
D
spectroscopic data of sugar part of 12 (Tables 2, 3, 5 and 6) to those
of 9 indicated that 12 possesses the same substituted sugar moiety
as 9, which was confirmed by analysis of the key HMBC
correlations in 12. Thus, 12 was confirmed to be qingyangshen-
genin-3-O-
(1 ? 4)-b-
(1 ? 4)-b-
D
D
-cy-
-cy-
D
-cymaropyranosyl H-1000)
D
D
maropyranosyl H-100) to dC 77.7 (C-3) were observed. Therefore,
a
D
-
L
-cymaropyranosyl-(1 ? 4)-b-
-cymaropyranosyl-(1 ? 4)-
-oleandropyranosyl-(1 ? 4)-b-
D
-cymaropyranosyl-
-cymaropyranosyl-
D-cymaropyranoside.
11 was established to be qingyangshengenin-3-O-
nosyl-(1 ? 4)-b- -oleandropyranosyl-(1 ? 4)-b- -cymaropyranosyl-
(1 ? 4)-b- -cymaropyranoside.
a-L-cymaropyra-
a-L
D
D
D
D
Compound 13, obtained as a colorless powder, had a molecular
formula C58H86O19, established by HRESIMS and 13C NMR spec-
trum. Its 1H NMR spectrum (Table 6) showed a series of character-
istic proton signals at dH 6.30 (d, H-20, J = 16.0 Hz), 7.62 (d, H-30,
J = 16.0 Hz), 7.51 (m, H-50 and H-90), 7.38 (m, H-60 and H-80), and
7.39 (m, H-70), corresponding to the carbon signals at dC 165.7
(C-10), 117.6 (C-20), 145.4 (C-30), 134.3 (C-40), 128.1 (C-50, C-90),
Compound 12 had a molecular formula C70H108O26. The NMR
features of aglycone demonstrated that 12 was a qingyangshen-
genin-type steroid (Table 4). Its sugar moiety consisted of three
b-D-cymaropyranosyl, two a-L-cymaropyranosyl and one b-D-ole-
andropyranosyl based on analysis of 1D and 2D NMR data of 12
and comparison of its NMR information with reported data of ana-
logs [22] (Tables 5 and 6). Meantime, the similarity of the 1D NMR
Please cite this article in press as: M. Zhang et al., Cytotoxicity of pregnane glycosides of Cynanchum otophyllum, Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.08.010

10
M. Zhang et al. / Steroids xxx (2015) xxx–xxx
Table 6
1H NMR spectral data of compounds 10–14 (d in ppm, J in Hz, 500 MHz).
No.
10b
11b
12b
13a
14b
1
2
3
1.09 m; 1.78 m
1.77 m; 2.07 m
3.86 m
1.07 m; 1.77 m
1.77 m; 2.05 m
3.85 m
1.08 m; 1.78 m
1.77 m; 2.05 m
3.86 m
1.09 m; 1.87 m
1.63 m; 1.92 m
3.56 m
1.03 m; 1.71 m
1.77 m; 2.06 m
3.86 m
4
6
2.40 m; 2.51 m
2.40 m; 2.52 m
2.41 m; 2.53 m
2.30 m; 2.34 m
5.37 s
2.33 m; 2.47 m
5.27c
5.28c
5.28c
5.29c
7
2.46 m
2.45 m
2.45 m
2.21 m
2.47 m
9
1.75 m
1.75 m
1.75 m
1.56 m
1.73 m
11
12
15
16
18
19
20
21
20
2.18 m; 2.31 m
5.32 dd (11.5, 4.0)
2.13 m
2.05 m; 3.27 m
2.08 s
2.18 m; 2.31 m
5.29 dd (11.5, 4.0)
2.11 m
2.02 m; 3.27 m
2.09 s
2.18 m; 2.31 m
5.32 dd (11.5, 4.0)
2.12 m
2.03 m; 3.26 m
2.09 s
1.88 m
4.74 t (7.5)
1.99 m
1.90 m; 2.86 m
1.46 s
1.12 s
2.01 m; 2.33 m
5.32d
2.07 m
2.07 m; 2.12 m
2.10 s
1.29 s
1.30 s
1.30 s
1.28 s
5.04d
2.51 s
2.41 s
2.41 s
2.20 s
6.30 d (16.0)
7.62 d (16.0)
1.55c
6.54 d (16.0)
7.85 d (16.0)
30
8.29 m
7.26 m
8.30 m
7.23 m
8.30 m
7.22 m
40
50
7.51 m
7.37 m
7.38 m
7.37 m
7.51 m
7.42 m
7.33 m
7.33 m
7.33 m
7.42 m
60
7.26 m
8.29 m
7.23 m
8.30 m
7.22 m
8.30 m
70
80
90
D-digit
D-cym
D-cym
D-cym
100
5.47 d (9.5)
2.03 m; 2.39 m
4.61 m
3.48 m
4.21 m
5.28c
5.24c
4.84 dd (9.5, 1.5)
1.42 m; 2.07 m
3.80 m
3.16 m
3.85 m
200
1.90 m; 2.35 m
4.08 m
3.52 m
4.22 m
1.38c
1.79 m; 2.30 m
3.99 m
3.49 m
4.16 m
1.36c
9.52 s
300
400
8.29 d (8.0)
7.17c
8.80 d (5.0)
500
600
1.44 d (6.5)
1.22 d (6.5)
3.44 s
OMe
3.60 s
3.54 s
D-cym
D-cym
D-ole
D
-cym
D-digit
1000
5.15d
1.74 m; 2.30 m
4.02 m
3.50 m
4.29 m
5.10d
4.64 d (9.5)
1.62 m; 2.49 m
3.40 m
4.74 dd (9.5, 1.5)
1.54 m; 2.10 m
3.78 m
3.16 m
3.86 m
5.48 dd (9.5, 1.0)
2.01 m; 2.43 m
4.65 m
3.51 m
4.30 m
2000
1.83 m; 2.32 m
4.00 m
3.48 m
4.16 m
1.38c
3000
4000
3.36 m
5000
3.44 m
6000
1.42 d (6.5)
3.56 s
1.37c
1.22 d (6.0)
3.44 s
1.43 d (6.0)
OMe
3.55 s
3.29 s
D-the
D-ole
L
-cym
D
-ole
D-cym
10000
20000
30000
40000
50000
60000
OMe
4.69 d (8.0)
3.86 m
3.65 m
3.64 m
3.63 m
4.66 dd (9.5, 1.5)
1.78 m; 2.46 m
3.41 m
3.40 m
3.47 m
5.06 d (3.0)
1.86 m; 2.32 m
3.77 m
3.88 m
4.71 m
4.44 dd (9.5, 1.5)
1.54 m; 2.30 m
3.37 m
3.22 m
3.31 m
5.17 dd (9.5, 1.0)
1.78 m; 2.32 m
4.05 m
3.46 m
4.20 m
1.54 d (6.0)
3.87 s
1.38 d (6.0)
3.43 s
1.54 d (6.0)
3.40 s
1.30 d (6.0)
3.39 s
1.35 d (6.0)
3.56 s
D-cym
L
-cym
D-cym
D
-ole
D-ole
100000
200000
300000
400000
500000
600000
OMe
5.26c
1.77 m; 2.37 m
3.76 m
3.54 m
4.13 m
5.08 dd (3.5, 1.0)
1.86 m; 2.30 m
3.73 m
3.62 m
4.65 m
5.27c
4.70 dd (9.5, 1.5)
1.55 m; 2.32 m
3.16 m
3.14 m
3.57 m
4.75 dd (9.5, 1.0)
1.74 m; 2.47 m
3.57 m
3.48 m
3.59 m
1.82 m; 2.30 m
4.08 m
3.50 m
4.20 m
1.36c
1.53 d (5.5)
3.48 s
1.54 d (6.0)
3.30 s
1.34 d (6.0)
3.39 s
1.56 d (6.0)
3.45 s
3.60 s
D-cym
1000000
2000000
3000000
4000000
5000000
6000000
OMe
5.13 dd (9.5, 1.0)
1.78 m; 2.30 m
3.88 m
3.44 m
4.20 m
1.38c
3.52 s
L
-cym
10000000
20000000
30000000
4.96 d (3.0)
1.81 m; 2.34 m
3.70 m
Please cite this article in press as: M. Zhang et al., Cytotoxicity of pregnane glycosides of Cynanchum otophyllum, Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.08.010

M. Zhang et al. / Steroids xxx (2015) xxx–xxx
11
Table 6 (continued)
No.
10b
11b
12b
13a
14b
40000000
50000000
60000000
OMe
3.60 m
4.54 m
1.51 d (6.0)
3.36 s
D-digit: b-
D-digitoxopyranosyl.
D-cym: b-
D-cymaropyranosyl. L-cym: a-L-cymaropyranosyl.
D
-ole: b- -oleandropyranosyl.
D
D
-the: b-D-thevetopyranosyl.
a
Measured in CDCl3.
Measured in C5D5N.
Partial overlapped with other signals.
Overlapped with H2O signal.
b
c
d
200), 127.0 (C-300), 137.3 (C-400), 123.0 (C-500), 151.4 (C-600)] were pre-
sent in 14 (Tables 4–6), which were linked in C-12 and C-20 of
aglycone, confirmed by the HMBC correlations from dC 166.8 (C-
10) to dH 5.32 (H-12), and from dC 164.7 (C-10) to dH 5.04 (H-20)
in HMBC, respectively. Comparison of remaining spectral data of
14 with those of analogs [24,30], along with above two character-
istically substituted groups, indicated that aglycone of 14 was
gagaminin. For sugar moiety, the signals of the sugar units were
unambiguously assigned on the basis of 1D and 2D NMR analysis
Table 7
Cytotoxicities of compounds 1–26, 5-FU and Cisplatin against three cancer cell lines.
Compound
IC50
(lM)
HepG2
Hela
U251
1
2
3
4
5
6
41.82 2.91
86.28 2.90
37.87 3.08
34.27 4.35
30.71 4.02
>100
38.69 3.85
86.27 3.97
57.69 5.20
72.35 4.45
27.44 4.88
>100
46.16 4.80
>100
22.52 4.74
>100
40.37 8.92
>100
(Tables 5 and 6), which suggested the existence of one b-
D-cy-
7
8
9
44.44 2.03
25.99 2.66
30.47 5.40
>100
46.92 8.73
44.85 7.82
20.89 6.03
>100
79.47 6.79
96.83 3.87
35.09 5.14
>100
53.94 4.86
34.70 6.28
45.94 4.49
>100
50.39 2.43
83.28 3.87
52.02 7.21
97.18 4.87
69.28 2.51
35.81 4.91
35.37 3.60
23.96 6.06
>100
54.64 3.33
77.98 8.33
64.85 3.95
65.98 5.52
51.71 15.26
>100
69.81 2.85
47.45 0.71
5.38 0.93
maropyranosyl, one b- -oleandropyranosyl, one b- -digitoxopyra-
D
D
nosyl. The linkage position and sequence of three sugar units
were determined by the distinct correlations in HMBC spectrum
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
5-FU
Cisplatin
from dC 102.2 (b-
maropyranosyl H-40000), from dC 99.7 (b-
to dH 3.51 (b-
-digitoxopyranosyl H-4000), and from dC 96.4 (b-
itoxopyranosyl H-1000) to dH 3.86 (H-3). Hence, 14 was confirmed as
gagaminin-3-O-b- -oleandropyranosyl-(1 ? 4)-b- -cymaropyranosyl-
(1 ? 4)-b- -digitoxopyranoside.
Twelve known steroids were otophylloside T (15) [14], cau-
datin-3-O-b- -cymaropyranosyl-(1 ? 4)-b- -oleandropyranosyl-
(1 ? 4)-b- -cymaropyranosyl-(1 ? 4)-b- -cymaropyranoside (16)
[26], caudatin-3-O-b- -oleandropyranosyl-(1 ? 4)-b- -digitox-
opyranosyl-(1 ? 4)-b- -cymaropyranoside (17) [12], caudatin-3-
O-b- -oleandropyranosyl-(1 ? 4)-b- -cymaropyranosyl-(1 ? 4)-
b- -digitoxopyranoside (18) [12], Otophylloside (19) [23],
otophylloside N (20) [23], qinyangshengenin-3-O-b- -oleandropy-
ranosyl-(1 ? 4)-b- -cymaropyranosyl-(1 ? 4)-b- -digitoxopyra-
noside (21) [5], wallicoside J (22) [31], mucronatoside C (23) [32],
gagamine-3-O-b- -oleandropyranosyl-(1 ? 4)-b- -cymaropyra-
nosyl-(1 ? 4)-b- -cymaropyranoside (24) [28], stephanoside H
D
-oleandropyranosyl C-100000) to dH 3.46 (b-
D
-cy-
-cymaropyranosyl C-10000
-dig-
61.91 3.95
72.50 2.28
41.76 1.83
98.19 1.01
96.21 1.85
38.91 3.21
51.84 4.33
16.75 1.42
66.9 1.00
74.75 4.42
95.26 2.89
49.93 1.81
70.69 6.89
43.72 5.99
28.48 3.28
64.15 4.78
20.09 0.79
4.85 0.97
D
)
D
D
>100
D
D
31.24 4.63
45.37 2.05
25.33 3.06
47.48 2.53
89.13 2.48
92.62 3.65
42.71 10.33
>100
D
D
D
D
D
D
D
D
>100
D
D
51.08 4.46
56.88 4.23
7.80 3.85
10.61 1.27
D
O
D
D
D
Data are present as the mean SD of three experiments performed in triplicate.
D
D
D
128.9 (C-60, C-80), and 130.4 (C-70), respectively, confirmed by
HSQC experiment, indicating that a cinnamoyl group was present
in 13 and it was linked in C-12 of aglycone based on the long-range
correlation from dC 165.7 (C-10) to dH 4.74 (m, H-12) in HMBC spec-
trum. Compared to features of NMR data of known aglycones in
Asclepiadaceae subfamily, the aglycone of 13 was determined to
be kidjoranin [26,27]. For the sugar units, detailed analysis of 1D
and 2D NMR of 13 suggested that the sugar sequence and linkage
position to the aglycone were exactly similar to that of gagaminin-
(25) [5], sinomarinoside B (26) [33].
Compounds 1–26 were evaluated for their cytotoxicity using
HepG2, Hela and U251 human cancer cell lines. All of them, except
6
and 10 (IC50 > 100
lM), displayed the cytotoxic activities
(IC50 < 100 M) at different degrees against those cell lines, com-
l
pared with the reference compounds 5-FU and cisplatin against
HepG2, Hela and U251 with IC50 values of 20.09, 7.80 and
47.45 lM, and 4.85, 10.61 and 5.38 lM, respectively (Table 7).
Notably, those steroidal glycosides, whose aglycones were cau-
datin, such as 1, 3, 5, 7–9, 13, 16–18, showed better cytotoxicities
(IC50 < 50 lM) than another analogs against cancer cell lines. It
suggested that the aglycone of caudatin may play an important
role in the cytotoxic activity.
3-O-b-
b- -cymaropyranosyl-(1 ? 4)-b-
13 was elucidated to be kidjoranin-3-O-b-
(1 ? 4)-b- -oleandropyranosyl-(1 ? 4)-b- -cymaropyranosyl-(1 ?
4)-b- -cymaropyranoside.
D
-oleandropyranosyl-(1 ? 4)-b-
-cymaropyranoside [28]. Thus,
-oleandropyranosyl-
D-oleandropyranosyl-(1 ? 4)-
D
D
D
D
D
D
Compound 14 had a molecular formula C56H77NO17. Detailed
analysis of 1D NMR signals assigned to aglycone indicated that a
cinnamoyl group [dH 6.54 (d, H-20, J = 16.0 Hz), 7.85 (d, H-30,
J = 16.0 Hz), 7.42 (m, H-50 and H-90), 7.33 (m, H-60 and H-80), 7.33
(m, H-70); dC 166.8 (C-10), 120.3 (C-20), 144.0 (C-30), 134.9 (C-40),
128.5 (C-50, C-90), 128.5 (C-60, C-80), 130.5 (C-70)] and a nicotinoyl
group [dH 9.52 (s, H-200), 8.27 (d, H-400, J = 8.0 Hz), 7.17 (dd, H-500,
J = 8.0, 5.0 Hz), 8.80 (d, H-600, J = 5.0 Hz); dC 164.7 (C-100), 153.8 (C-
4. Conclusion
Systematic research on the pregnane glycosides from C. otophyl-
lum was carried out in this study. As a result, fourteen new preg-
nane glycosides (1–14) were isolated, along with twelve known
analogs (15–26). Their structures possessed five types of aglycones,
including caudatin, qinyangshengenin, gagaminin, kidjoranin and
Please cite this article in press as: M. Zhang et al., Cytotoxicity of pregnane glycosides of Cynanchum otophyllum, Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.08.010

12
M. Zhang et al. / Steroids xxx (2015) xxx–xxx
[10] Q.Z. Mu, Q.L. Zhou, The constituents of qingyangshen (Cynanchum otophyllum
Schneid.), Acta Pharm. Sin. 18 (1983) 356–362.
[11] Q.Z. Mu, J.R. Lu, Q.L. Zhou, Two new antiepilepsy compounds – otophyllosides
A and B, Sci. Sin. B (Engl. Ed.) 29 (1986) 295–301.
[12] Q.Z. Mu, Y.M. Shen, X.L. Zhou, X.J. Hao, The preparation and application of
otophyllosides C-G, CN 1064048C, Kunming Institute of Botany, Chinese
Academy of Sciences, 2001. April 4.
[13] X. Li, M. Zhang, C. Xiang, B.C. Li, P. Li, Antiepileptic C21 steroids from the roots
of Cynanchum otophyllum, J. Asian Nat. Prod. Res. 2015 (2014), http://dx.doi.
org/10.1080/10286020.2014.1001380.
[14] D.Y. Shen, J.C. Wei, J.B. Wan, X.J. Huang, C. Xiang, B.C. Li, Q.W. Zhang, Y.T.
Wang, P. Li, Four new C21 steroidal glycosides from Cynanchum otophyllum
Schneid, Phytochem. Lett. 9 (2014) 86–91.
[15] W.G. Shan, X. Liu, L.F. Ma, Z.J. Zhan, New polyhydroxypregnane glycosides
from Cynanchum otophyllum, J. Chem. Res. 36 (2012) 38–40.
[16] L.M. Shi, W.H. Liu, Q. Yu, H.T. Wan, Two new C21 steroids from the roots of
Cynanchum otophyllum, J. Chem. Res. 35 (2011) 126–128.
penupogenin, and four types of deoxysugars, including digitoxose,
oleandrose, cymarose, and thevetose, which were the mainly char-
acteristic structures of the pregnane glycosides in the genus Cynan-
chum plants (subfamily Asclepiadoideae). In cytotoxic bioassay, all
steroidal glycosides obtained (1–26), except 6 and 10, showed the
cytotoxicities against HepG2, Hela, U251 cancer cell lines at differ-
ent degrees. And 18 displayed obvious activities against HepG2
with IC50 at 16.75 1.42 lM (IC50 < 20 lM).
Notes
The authors declare no competing financial interest.
[17] L.M. Shi, W.H. Liu, Q. Yu, H.T. Wan, Two new polyhydroxypregnane glycosides
from the roots of Cynanchum otophyllum, J. Chem. Res. 37 (2013) 404–405.
[18] Y.B. Zhao, Y.M. Shen, H.P. He, Q.Z. Mu, X.J. Hao, A new C21 steroidal glycoside
from Cynanchum otophyllum, Acta Bot. Yunn. 27 (2005) 443–446.
[19] Y.B. Zhao, H.P. He, C.H. Lu, Q.Z. Mu, Y.M. Shen, X.J. Hao, C21 steroidal glycosides
of seven sugar residues from Cynanchum otophyllum, Steroids 71 (2007) 935–
941.
Acknowledgment
The research work was financially supported by the Natural
Science Foundation of China (No. 31160065).
[20] Z.M. Zhao, Z.H. Sun, M.H. Chen, Q. Liao, M. Tan, X.W. Zhang, H.D. Zhu, R.B. Pi, S.
Yin, Neuroprotective polyhydroxypregnane glycosides from Cynanchum
otophyllum, Steroids 78 (2013) 1015–1020.
[21] X.X. Ma, F.T. Jiang, Q.X. Yang, X.H. Liu, Y.J. Zhang, C.R. Yang, New pregnane
glycosides from the roots of Cynanchum otophyllum, Steroids 72 (2007) 778–
786.
[22] L.F. Ma, W.G. Shan, Z.J. Zhan, Polyhydroxypregnane glycosides from the roots
of Cynanchum otophyllum, Helv. Chim. Acta 94 (2011) 2272–2282.
[23] X.X. Ma, D. Wang, Y.J. Zhang, C.R. Yang, Identification of new
qingyangshengenin and caudatin glycosides from the roots of Cynanchum
otophyllum, Steroids 76 (2011) 1003–1009.
[24] F. Abe, H. Okeabe, T. Yamauchi, K. Honda, N. Hayashi, Pregnane glycosides
from Marsdenia tomentosa, Chem. Pharm. Bull. 47 (1999) 869–875.
[25] K. Ma, J.S. Wang, J. Luo, M.H. Yang, L.Y. Kong, Tabercarpamines AꢁJ, apoptosis-
inducing indole alkaloids from the leaves of Tabernaemontana corymbosa, J.
Nat. Prod. 77 (2014) 1156–1163.
[26] T. Warashina, T. Noro, Steroidal glycosides from the root of Cynanchum
caudatum M, Chem. Pharm. Bull. 43 (1995) 977–982.
[27] T. Warashina, T. Noro, Steroidal glycosides from roots of Cynanchum caudatum
M. II, Chem. Pharm. Bull. 43 (1995) 1734–1737.
[28] T. Warashina, T. Noro, Steroidal glycosides from roots of Cynanchum caudatum
M. III, Chem. Pharm. Bull. 44 (1996) 358–369.
[29] T. Warashina, T. Noro, Steroidal glycosides from roots of Cynanchum caudatum,
Phytochemistry 44 (1997) 917–923.
Appendix A. Supplementary data
HRESIMS, 1D and 2D NMR spectra of compounds 1–14 are
available in the online version, at http://dx.doi.org/10.1016/j.ster-
oids.2015.08.010.
References
[1] Y. Ni, Y.P. Ye, Distribution of C21 steroidal glycosides in plants of
Asclepiadaceae and their pharmacological activities, Chin. Tradit. Herbal
Drugs 1 (2010) 162–166.
[2] X.F. Tao, J.L. Xu, R.S. Zhang, Research progress on C21 steroidal glycosides from
Asclepiadaceae, Chin. J. Ethnomed. Ethnopharm. 17 (2009) 40–42.
[3] H. Bai, Y.S. Wang, A.Q. Liu, Advances study on the C21 steroid constituents of
Cynanchum plants, Nat. Prod. Res. Dev. 19 (2007) 879–904.
[4] B.Y. Hwang, S.E. Kim, Y.H. Kim, H.S. Kim, Y.S. Hong, J.S. Ro, K.S. Lee, J.J. Lee,
Pregnane glycoside multidrug-resistance modulators from Cynanchum
wilfordii, J. Nat. Prod. 62 (1999) 640–643.
[5] X.Y. Li, H.X. Sun, Y. Ye, F. Chen, J. Tu, Y. Pan, Three new immunomodulating
C
21–steroidal glycosides from the stems of Stephanotis mucronata, Chem.
Biodivers. 2 (2005) 1701–1711.
[6] Y.M. Li, L.H. Wang, S.L. Li, X.Y. Chen, Y.M. Shen, Z.K. Zhang, H.P. He, W.B. Xu, Y.L.
Shu, G.D. Liang, R.X. Fang, X.J. Hao, Seco-pregnane steroids target the
subgenomic RNA of alphavirus-like RNA viruses, Proc. Natl. Acad. Sci. U.S.A.
104 (2007) 8083–8088.
[7] Y.B. Li, J.F. Zhang, X.J. Gu, Y.R. Peng, W.H. Huang, S.H. Qian, Two new Cytotoxic
pregnane glycosides from Cynanchum auriculatum, Planta Med. 74 (2008) 551–
554.
[8] P.G. Kuang, Y.X. Wu, F.J. Meng, P.Z. Kuang, D.S. Shao, Q.Z. Mu, Treatment of
grand mal seizures with ‘‘qingyangshen” (root of Cynanchum otophyllum) and
observations on experimental animals, J. Tradit. Chin. Med. 1 (1981) 19–24.
[9] P.G. Kuang, S.Y. Lang, F.Y. Zhang, Z.L. Yang, Studies on qingyangshen (root of
Cynanchum otophyllum). II. Effect of qingyangshen on metabolism of central
monoamines, J. Tradit. Chin. Med. 5 (1985) 191–194.
[30] K. Yoshikawa, N. Okada, Y. Kann, S. Arihara, Steroidal glycosides from the Fresh
Stem of Stephanotis lutchuensis var. japonica (Asclepiadaceae). Chemical
Structures of Stephanosides A–J, Chem. Pharm. Bull. 44 (1996) 1790–1796.
[31] G. Chen, Studies the constituents of Cynanchum wallichi Wight and Pfaffia
Glometate, PhD Thesis, Shenyang Pharmaceutical University, Shenyang, IN,
2009, p 32.
[32] R.S. Zhang, Y.P. Ye, X.Y. Li, X.Y. Zhang, Oxypregnane-oligoglycosides from the
stems of Stephanotis mucronata (Asclepiadaceae), Chin. Chem. Lett. 16 (2005)
483.
[33] J.J. Chen, Z.X. Zhang, J. Zhou, B.T. Li, New pregnane glycosides from
Sinomarsdenia incise, J. Nat. Prod. 62 (1999) 829–832.
Please cite this article in press as: M. Zhang et al., Cytotoxicity of pregnane glycosides of Cynanchum otophyllum, Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.08.010