Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum

Article abstract of DOI:10.1016/j.phytochem.2013.03.010

In the search for plant alternatives to Hoodia gordonii containing P57, a pregnane glycoside with potential appetite suppressant effect, the roots of Cynanchum auriculatum were investigated. As a result, 15 pregnane glycosides including nine never previously reported were isolated. Their structures were elucidated on the basis of extensive spectroscopic analyses and chemical methods. Appetite suppressant effect and body weight loss were observed when tested with the most abundant pregnane glycoside, wilfoside K1N, in an in vivo test with rats.

Full text of DOI:10.1016/j.phytochem.2013.03.010

Phytochemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum
⇑
Shuangzhu Liu, Zhenhua Chen, Jian Wu, Luoyi Wang, Hongmin Wang, Weimin Zhao
Department of Natural Products Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 March 2012
Received in revised form 17 September
In the search for plant alternatives to Hoodia gordonii containing P57, a pregnane glycoside with potential
appetite suppressant effect, the roots of Cynanchum auriculatum were investigated. As a result, 15 preg-
nane glycosides including nine never previously reported were isolated. Their structures were elucidated
on the basis of extensive spectroscopic analyses and chemical methods. Appetite suppressant effect and
body weight loss were observed when tested with the most abundant pregnane glycoside, wilfoside K1N,
in an in vivo test with rats.
2
012
Available online xxxx
Keywords:
Cynanchum auriculatum
Asclepiadaceae
Ó 2013 Elsevier Ltd. All rights reserved.
Pregnane glycoside
Appetite suppressant
1
. Introduction
The WHO estimates that at least one tenth of adults worldwide
Gu et al., 2009; Wang et al., 2002; Zhang et al., 2000a,b, 2007a;
Sun et al., 2009; Yang et al., 2011; Lu et al., 2011a,b; Teng et al.,
2011), and anti-tumor (Shan et al., 2005; Zhang et al., 2000a,b),
gastroprotective (Shan et al., 2006), anti-aging (Zhang et al.,
2007b), and anti-depressant (Yang et al., 2011) activities were also
reported in the literature.
The structures of pregnane glycosides identified from C. auricul-
atum resembled that of P57 from H. gordonii. In order to find new
plant sources as an alternative of H. gordonii for development of
anti-obesity food supplements, chemical constituents of the roots
of C. auriculatum collected in Binhai County, Jiangsu Province were
investigated. Reported herein are the isolation and identification of
nine new pregnane glycosides (1–9) (Fig. 1) and six known compo-
nents, and appetite suppressant effects of the two major pregnane
glycosides wilfoside K1N (10) and wilfoside C1N (11).
are obese. Obesity has become a serious concern because it increases
the risk of cardiovascular disease, type 2 diabetes, and some cancers,
among other health problems (Wisse et al., 2007). To reduce food in-
take is an efficient way to control both obesity and overweight, and
searching for a safe appetite suppressant has been attracting consid-
erable interest. Hoodia gordonii (Asclepiadaceae), a succulent plant
indigenous to the African continent, was used among the San tribe
of South Africa to suppress appetite when away on long hunting
trips (Pereira et al., 2010). P57, a natural pregnane glycoside isolated
from H. gordonii (Van Heerden et al., 2007; Van Heerden, 2008) was
reported to up-regulate hypothalamic ATP level, and affect the sense
of satiety to stop eating (MacLean and Luo, 2004). Nowadays, various
Hoodia supplements are popular in the weight management market,
and counterfeit products are common due to the scarcity of Hoodia
plant sources. The efficacy and safety of these products still needs
further investigation.
2
. Results and discussion
Cynanchum auriculatum Royle ex Wight (Asclepiadaceae) is a
plant species native to China, and the roots of C. auriculatum have
been used as local food in Binhai County, Jiangsu Province of China
for more than 100 years, and in Chinese folk medicine for the treat-
ment of geriatric diseases, and for promoting immunity and lon-
gevity (Jiangsu College of New Medicine, 2004). Several thousand
tons of the roots of C. auriculatum, accounting for 95% of the na-
tional output, are harvested in Binhai County each year. Previous
investigations of C. auriculatum established the existence of preg-
nane glycosides and baishouwubenzophenone (Chen et al., 1990;
Powdered air-dried roots of C. auriculatum (3.5 kg) were ex-
tracted with 95% EtOH three times, with the combined filtrates con-
centrated in vacuo to obtain a crude extract which was subjected to
column chromatography (CC) over silica gel and divided into five
fractions. Those fractions were further purified by repeated normal
and/or reversed phase CC and preparative HPLC to afford nine new
pregnane glycosides (1–9) and six known compounds which were
identified as wilfoside K1N (10, Tsukamoto et al., 1985b), wilfoside
C1N (11, Tsukamoto et al., 1985a), wilfoside M1N (Tsukamoto
et al., 1985b), wilfoside C1G (Tsukamoto et al., 1985a), cynauricu-
side A (Chen et al., 1990) and cynandione A (Huang et al., 1995),
respectively.
⇑
Corresponding author. Tel./fax: +86 21 50806052.
E-mail address: wmzhao@mail.shcnc.ac.cn (W. Zhao).
0
031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.03.010
Please cite this article in press as: Liu, S., et al. Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum. Phytochemistry (2013),
http://dx.doi.org/10.1016/j.phytochem.2013.03.010

2
S. Liu et al. / Phytochemistry xxx (2013) xxx–xxx
1
R = R , R = Cin
1 1a 2
O
O
O
1#
2 R = R , R = Cin
1 1e 2
OR2
= Ac
2#
1
8
21
3 R = R , R = Cin
1 1f 2
2
0
1
2
1
7
3#
#
4 R = R , R = Ac
9#
1#
1 1e 2
OH
8#
5 R = R , R = Ac
1
9
= Cin
1
1f
2
OH
4
5
2#
1
4
6 R = R , R = H
1 1e 2
#
7#
7 R = R , R = H
6#
1
1f
2
8
#
7
OH
O
8 R = R , R = H
1 1c 2
3#
1#
= Ikem
5#
9
R = R , R = H
1 1d 2
1 1b 2
R1O
3
4#
6
2#
10 R = R , R = Cin
6#
OCH3
1
1 R = R , R = Ikem
1
1b
2
4
'''
1''
4'
O
O
O
HO
O
O
O
O
= R1a
1
'''
4''
OCH3
1'
1'
OCH3
'''
OCH3
4'
1
''
4
H CO
3
O
O
O
1
''''
=
R1b
O
O
HO
1'''
4''
OCH3
4
''''
OCH3
4
'
4
''
O
= R1c (R = H)
3
O
4
''''
H CO
3
O
O
O
HO
1'''
O
=
R1d (R = Ac)
3
O
1
'
1
''
1
''''
OH
OCH3
4
'''
OR3
4
'
4
''
O
O
= R1e (R = H)
4
= R1f (R4 = Ac)
O
4
''''
H CO
3
O
O
O
1
'''
H3CO O
O
O
1
'
1''
1
'''''
OH
1
''''
OR4
4
'''
HO
O
OCH3
4
'''''
O
O
OH
O
O
O
HO
H CO
O
O
O
3
HO
OCH3
OCH3
P57
Fig. 1. Structures of pregnane glycosides (1–11) isolated from C. auriculatum and P57.
Compounds 1–9 were obtained as white amorphous powders,
Three anomeric carbon resonances at d
responding to three anomeric proton signals at d
J = 3.3 Hz), 5.10 (dd, J = 9.8, 1.8 Hz), and 5.24 (dd, J = 9.4, 1.8 Hz),
C
101.0, 99.5, and 96.1, cor-
5.15 (d,
and exhibited a blue color on TLC by spraying with 5% H
heating.
2
SO
4
and
H
The molecular formula of 1 was established as C51
HRESIMS ion peak at m/z 965.4843 [M+Na] . Its IR spectrum
H
74
O
16 by its
respectively, indicated the existence of three sugar units in the
+
structure of 1, with two b glycosidic linkages, and one
a glycosidic
ꢀ1
showed absorption bands due to OH groups (3465 cm ), a car-
linkage according to the coupling constants of the anomeric proton
ꢀ
1
ꢀ1
1
1
bonyl group (1712 cm ), an olefinic bond (1637 cm ), and a phe-
resonances. Further analysis of its H– H COSY, HSQC, and HMBC
spectra of 1 enabled identification of the three sugar units as 2,6-
dideoxy-3-O-methylated saccharide and assignment of their pro-
ton and carbon signals. The coupling constants at the key reso-
ꢀ
1
nyl function (1450, 1167 cm ). Compound
1
possessed
1
13
characteristic H and C NMR signals of pregnane glycosides iso-
lated from Cynanchum plants. The aglycone of 1 was deduced to
0
000
be kidjoranin based on three methyl resonances at d
and 2.49 (each 3H, s), cinnamoyl resonances at d
H
1.35, 2.03,
8.00 (d, 1H,
nances of H-4 (d
H H
3.48, dd, J = 9.3, 2.8 Hz) and H-4 (d 3.56, dd,
H
J = 9.6, 3.0 Hz) indicated that the two sugar units were cymaropyr-
0
0
J = 16.0 Hz), 7.33 (3H, m), 7.66 (2H, m), and 6.82 (d, 1H, J = 16.0)
anose, while the H-4 (d
H
4.06, t, J = 1.8 Hz) signal suggested the su-
1
in its H NMR spectrum (Tables 1 and 2) along with carbon signals
gar to be diginopyranose (Tsukamoto et al., 1985a). In the HMBC
1
13
of the aglycone, including three oxygenated quaternary carbons at
spectrum of 1 (Fig. 2), H– C long-range correlation signals were
0
00
0
d
C
74.2, 89.5, and 92.4, one ketone carbonyl at d
C
209.9, and one es-
observed between H-1 and C-3 (d
C
77.6), H-1 and C-4 (d
C
82.5),
165.8 in its 13C NMR spectrum (Tables 3 and 4).
and H-1 and C-4 (d 73.8). The C NMR spectroscopic data of
000
00
13
ter carbonyl at d
C
C
Please cite this article in press as: Liu, S., et al. Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum. Phytochemistry (2013),
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S. Liu et al. / Phytochemistry xxx (2013) xxx–xxx
3
Table 1
1
5
H NMR spectroscopic data of the aglycone moieties of compounds 1–9 (pyridine-d , 400 MHz).
Proton
1
2
3
4
5
6
7
8
9
1
1
2
2
3
4
4
6
7
7
9
1
1
1
1
1
1
1
1
2
a
b
a
b
1.11, m
1.83, m
1.80, m
2.08, m
3.85, m
2.42, m
2.55, m
5.30, brs
2.32, m
2.48, m
1.75, m
2.18, m
2.30, m
5.18, m
2.12, m
2.02, m
3.26, m
2.03, s
1.10, m
1.78, m
1.78, m
2.06, m
3.85, m
2.41, m
2.51, m
5.26, brs
2.33, m
2.43, m
1.72, m
2.10, m
2.32, m
5.16, m
2.10, m
2.00, m
3.25, m
2.01, s
1.11, m
1.80, m
1.78, m
2.06, m
3.87, m
2.42, m
2.52, m
5.27, brs
2.32, m
2.46, m
1.74, m
2.18, m
2.34, m
5.15, m
2.12, m
2.02, m
3.26, m
2.02, s
1.06, m
1.78, m
1.78, m
2.04, m
3.86, m
2.40, m
2.50, m
5.25, brs
2.34, m
2.44, m
1.67, m
2.08, m
2.20, m
4.94, m
2.10, m
2.08, m
3.32, m
1.90, s
1.06, m
1.88, m
1.82, m
2.08, m
3.84, m
2.42, m
2.52, m
5.24, brs
2.32, m
2.44, m
1.68, m
2.12, m
2.20, m
4.92, m
2.10, m
2.08, m
3.32, m
1.90, s
1.08, m
1.86, m
1.82, m
2.10, m
3.86, m
2.42, m
2.52, m
5.31, brs
2.34, m
2.42, m
1.60, m
1.82, m
2.38, m
3.90, m
2.10, m
2.08, m
3.32, m
1.97, s
1.10, m
1.86, m
1.84, m
2.09, m
3.86, m
2.44, m
2.56, m
5.33, brs
2.32, m
2.48, m
1.61, m
1.86, m
2.42, m
3.92, m
2.10, m
2.10, m
3.32, m
1.97, s
1.10, m
1.90, m
1.84, m
2.04, m
3.86, m
2.44, m
2.56, m
5.32, brs
2.32, m
2.50, m
1.60, m
1.78, m
2.44, m
3.93, m
2.09, m
2.09, m
3.35, m
1.98, s
1.07, m
1.84, m
1.80, m
2.07, m
3.87, m
2.42, m
2.52, m
5.30, brs
2.32, m
2.44, m
1.58, m
1.83, m
2.38, m
3.90, m
2.09, m
2.09, m
3.34, m
1.96, s
a
b
a
b
1a
1b
2
5
6a
6b
8
9
1
1.35, s
2.49, s
1.33, s
2.48, s
1.33, s
2.49, s
1.31, s
2.46, s
1.31, s
2.45, s
1.36, s
2.61, s
1.38, s
2.62, s
1.36, s
2.62, s
1.36, s
2.61, s
1
2-OCin
12-OAc
2.05, s
2#
6.81, d (16.0)
8.00, d (16.0)
7.63, m
7.34, m
7.33, m
6.79, d (16.0)
7.98, d (16.0)
7.62, m
7.35, m
7.34, m
6.81, d (16.0)
8.00, d (16.0)
7.63, m
7.34, m
7.33, m
2.04, s
#
3
#
#
5
(9 )
#
#
6
(8 )
#
7
0
00
00000
the sugar moiety of 1 matched well with those of wilfoside C3N
Tsukamoto et al., 1985a), which was isolated from the genus
the L configuration [C-2 (d
C
32.4) and C-2 (d
C
32.1)], and one the
0
000
(
D configuration [C-2 (d 36.7)]. Acidic hydrolysis of 2 afforded
C
Cynanchum. Acidic hydrolysis of 1 afforded cymarose and diginose,
cymarose and digitoxose, which were identified by analyses of
the H NMR spectra (Bai et al., 2005). The purified cymarose from
1
1
which were identified by H NMR spectra (Bai et al., 2005). The
2
D
2
22
D
optical rotation value ½
a
ꢁ
þ 39:3 (c 0.14, H
2
O) of purified cyma-
2 gave the optical rotation value ½
established it to the mixture of
1:2, and the purified digitoxose was in the D configuration accord-
a
ꢁ
ꢀ 14:7 (c 0.14, H
2
O), which
rose established it in the D configuration, and meanwhile, the ob-
D- and L-cymarose in the ratio
tained diginose was confirmed in the L configuration according to
2
D
2
22
its optical rotation value ½
et al., 1985a). The structure of 1 was thus characterized as kidjor-
anin 3-O-b- -cymaropyranosyl-(1?4)- -diginopyranosyl-
1?4)-b- -cymaropyranoside, and named cynauricoside A.
The molecular formula of 2 was established as C63 22 by its
HRESIMS ion peak at m/z 1225.6139 [M+Na] , taken together with
a
ꢁ
ꢀ 40:8 (c 0.13, H
2
O) (Tsukamoto
ing to ½
a
ꢁ
þ 51:5 (c 0.13, H
2
O) (Tsukamoto et al., 1985a). In the
HMBC spectrum (Fig. 2) of 2, H– C long-range correlation signals
D
1
13
0
00
D
a-
L
were found between H-1 (d
H
5.44) and C-3 (d
5.02) and C-4 (d
C
77.7), H-1 (d
H
5.29)
5.20)
0
000
00
0000
(
D
and C-4 (d
C
83.4), H-1 (d
77.1), H-1
H
00000
C
81.2), H-1 (d
H
0
00
0000
H
94
O
and C-4 (d
structure 2 was proposed as kidjoranin 3-O-
syl-(1?4)-b- -cymaropyranosyl-(1?4)- -cymaropyranosyl-(1?
4)-b- -digitoxopyranosyl-(1?4)-b- -digitoxopyranoside, named
cynauricoside B.
The molecular formula of 3 was determined as C65
C
(d
H
4.96) and C-4 (d
C
82.3). Thus,
+
a-
L-cymaropyrano-
13
the 63 carbon resonances in its C NMR spectrum. Analyses of its
D
a-L
1
13
H and C NMR spectra (Tables 1–4) indicated that 2 possessed
the same aglycone as 1. Five anomeric carbon signals were ob-
served at d 95.9, 96.3, 98.5, 99.0, and 99.8, corresponding to five
anomeric proton resonances at d 5.20 (dd, J = 9.5, 1.4 Hz), 5.46
dd, J = 9.4, 1.2 Hz), 5.02 (t, J = 3.4 Hz), 4.96 (d, J = 3.2 Hz), and
D
D
C
96
H O23 by its
+
1
13
H
HRESIMS ion peak at m/z 1267.6235 [M+Na] . The H and C NMR
spectroscopic data (Tables 1–4) of 3 resembled those of 2, except
(
5
.29 (dd, J = 9.8, 1.2 Hz), respectively, according to the HSQC spec-
C
for the presence of an additional acetyl group (d 170.3, 21.0 and
trum of 2. Among the five sugar units, three were in b glycosidic
linkages, while the other two were in glycosidic linkages based
on the coupling constants of the anomeric proton signals. Two
d
H
2.03). Hydrolysis of 3 using K CO –MeOH yielded 2. Analyses
2
3
1
1
a
of H– H COSY, HSQC, and HMBC spectra (Fig. 2) of 3 established
the connecting sequence of the five sugar units according to key
correlations between H-1 (d
77.7), H-1 (d
0
2
,6-dideoxy sugars were determined to be b-digitoxopyranose
H
5.46, dd, J = 8.4, 1.0 Hz) and C-3 (d
C
000
0
00
0
(Tsukamoto et al., 1985a) as deduced from the H-4 (d
H
3.48, dd,
4.13, dq,
H
5.11, dd, J = 8.4, 1.2 Hz) and C-4 (d
C
83.5), H-1
00
00
00
0000
J = 9.5, 2.5 Hz), H-3 (d
J = 9.4, 6.3 Hz), though H-4 was overlapped with methoxy reso-
nances at d 3.36. The absolute configuration of the digitoxopyr-
H
4.43, q, J = 2.7 Hz), and H-5 (d
H
(d
H
4.96, d, J = 2.5 Hz) and C-4 (d
C
79.9), H-1 (d
H
5.20, dd,
00
000
00000
J = 9.4, 1.2 Hz) and C-4 (d
J = 2.5 Hz) and C-4 (d 82.2). An upfield shift of C-2 and C-4
C
and a downfield shift of C-3 and H-3 suggested an acetyl substi-
tution at position C-3 . A H– C long-range correlation signal be-
tween H-3 (d
in its HMBC spectrum confirmed this location. Therefore, com-
pound 3 was established as kidjoranin 3-O- -cymaropyranosyl-
(1?4)-b- -cymaropyranosyl-(1?4)- -cymaropyranosyl-(1?4)-
b- -3-O-acetyldigitoxopyranosyl-(1?4)-b- -digitoxopyranoside,
named cynauricoside C.
The molecular formula of 4 was established as C56
C
77.5), and H-1
(d
H
00
4.95, d,
0
000
00
H
0
0
00
anosyl unit was assigned as D according to the configuration
found so far for this residue in the family Asclepiadaceae (Gu
et al., 2009). Three 3-O-methylated sugars were identified to be
0
0
1
13
0
0
H C
5.65) and the carbonyl carbon (d 170.3) observed
0
00
cymaropyranose by the J values at H-4 (d
H
3.81, dd, J = 8.0,
0000
00000
3
.0 Hz), H-4 (d
H
3.44, dd, J = 9.5, 2.5 Hz), and H-4 (d
H
3.58, dd,
a-L
J = 9.2, 3.2 Hz). According to the literature, the absolute configura-
tion of the cymaropyranosyl unit could be identified based on the
chemical shift of C-2 of cymaropyranose (with L configuration, dC-
D
a-L
D
D
2
< 33.0 ppm; with D configuration, dC-2 > 35.0 ppm) (Gan et al.,
90
H O
22 by its
+
2
008). Therefore, two of the cymaropyranosyl units were assigned
HRESIMS ion peak at m/z 1137.5864 [M+Na] . Five anomeric
Please cite this article in press as: Liu, S., et al. Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum. Phytochemistry (2013),
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Table 2
1
H NMR spectroscopic data of the sugar moieties of compounds 1–9 (pyridine-d
5
, 400 MHz).
Proton
1
2
3
4
5
6
7
8
9
D-Cym
D-Digit
D-Digit
D-Digit
D
-Digit
D-Digit
D-Digit
D-Digit
D
-Digit
0
1
5.24, dd (9.4, 1.8)
1.80, m
2.34, m
3.93, q (2.8)
3.48, dd (9.3, 2.8)
4.23, dq (9.3, 6.3)
1.37, d (6.3)
5.46, dd (9.4 1.2)
2.02, m
2.40, m
4.62, q (2.5)
3.48, dd (9.4, 2.5)
4.26, dq (9.4, 6.3)
1.39, d (6.3)
5.46, dd (8.4, 1.0)
2.04, m
2.42, m
4.64, q (2.7)
3.49 overlap
4.27, dq (9.3, 6.2)
1.37, d (6.2)
5.45, dd (8.2, 1.0)
2.04, m
2.42, m
4.62, q (2.6)
3.48, dd (9.4, 2.6)
4.27, dq (9.4, 6.2)
1.39, d (6.2)
5.44, dd (8.4, 1.0)
2.04, m
2.40, m
4.62, q (2.7)
3.46 overlap
4.24, dq (9.3, 6.0)
1.36, d (6.0)
5.46, dd (8.8, 1.2)
2.02, m
2.38, m
4.62, q (2.6)
3.48, dd (9.3, 2.6)
4.26, dq (9.3, 6.2)
1.38, d (6.2)
5.47, dd (9.2, 1.0)
2.04, m
2.42, m
4.63, q (2.5)
3.49 overlap
4.26, dq (9.4, 6.0)
1.35, d (6.0)
5.47, dd (8.4, 1.0)
2.02, m
2.44, m
4.62, q (2.6)
3.47, dd (9.4, 2.6)
4.27, dq (9.4, 6.2)
1.37, d (6.2)
5.46, dd (9.2, 1.0)
2.04, m
2.40, m
4.62, q (2.7)
3.48 overlap
4.24, dq (9.3, 6.3)
1.36, d (6.3)
0
2
2
3
4
5
6
a
b
0
0
0
0
0
OMe
3.52, s
L-Digin
D-Digit
D-Digit
D-Digit
D
-Digit
D-Digit
D-Digit
D-Digit
D-Digit
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
2
3
4
5
6
5.15, d (3.3)
2.02, m
2.34, m
3.81overlap
4.06, t (1.8,)
4.26, dq (1.8, 6.0)
1.45, d (6.0)
5.29, dd (9.8, 1.2)
1.86, m
2.36, m
4.43, q (2.7)
3.36 overlap
4.13, dq (9.4, 6.3)
1.29, d (6.3)
5.11, dd (8.4, 1.2)
1.88, m
2.36, m
5.65, q (2.8)
3.37 overlap
4.10, dq (9.5, 6.2)
1.31, d (6.2)
5.30, dd (8.2, 1.0)
1.90, m
2.36, m
4.45, q (2.7)
3.38 overlap
4.15, dq (9.2, 6.1)
1.31, d (6.1)
5.10, dd (9.2, 1.2)
1.90, m
2.38, m
5.64, q (2.8)
3.39 overlap
4.10, dq (9.3, 6.2)
1.31, d (6.2)
5.30, dd (9.0, 1.0)
1.88, m
2.36, m
4.44, q (2.7)
3.36 overlap
4.12, dq (9.4, 6.3)
1.34, d (6.3)
5.10, dd (9.6, 1.0)
1.90, m
2.38, m
5.65, q (2.6)
3.38 overlap
4.10, dq (9.3, 6.1)
1.31, d (6.1)
5.30, dd (8.6, 1.2)
1.86, m
2.42, m
4.45, q (2.8)
3.37 overlap
4.16, dq (9.4, 6.2)
1.31, d (6.2)
5.08, dd (9.8, 1.2)
1.88, m
2.36, m
5.64, q (2.8)
3.42 overlap
4.08, dq (9.3, 6.0)
1.30, d (6.0)
a
b
OMe/Ac
3.42, s
2.03, s
2.05, s
2.04, s
2.02, s
D-Cym
L
-Cym
L-Cym
L-Cym
L
-Cym
L
-Cym
L-Cym
L-Cym
L-Cym
0
0
0
0
0
0
0
00
00
00
00
00
00
00
1
2
2
3
4
5
6
5.10, dd (9.8, 1.8)
1.82, m
2.46, m
3.72, q (3.0)
3.56, dd (9.6, 3.0)
4.11, dq (9.6, 6.2)
1.52, d (6.2)
5.02, t (3.4)
1.82, m
2.26, m
3.72, q (3.0)
3.81, dd (8.0, 3.0)
4.54, dq (8.0 6.1)
1.37, d (6.1)
4.95, d (2.5)
1.81, m
2.36, m
3.69, q (3.2)
3.79, dd (8.7, 3.2)
4.48, dq (8.7, 6.4)
1.51, d (6.4)
5.02, brs
1.86, m
2.34, m
3.73 overlap
3.83, dd (8.6, 2.8)
4.56, dq (8.6, 6.3)
1.38, d (6.3)
4.96, brs
1.82, m
2.32, m
3.72 overlap
3.78, dd (8.8, 2.8)
4.46, dq (8.8, 6.2)
1.51, d (6.2)
5.02, brs
1.84, m
2.28, m
3.72 overlap
3.84, dd (8.6, 3.0)
4.56, dq (8.6, 6.1)
1.60, d (6.1)
4.98, brs
1.82, m
2.32, m
3.73 overlap
3.79, dd (8.7, 2.8)
4.48, dq (8.7, 6.4)
1.51, d (6.4)
5.05, d (3.4)
1.92, m
2.34, m
3.78 overlap
3.88, dd (8.8, 2.8)
4.58, dq (8.8, 6.3)
1.39, d (6.3)
4.96, d (2.8)
1.84, m
2.30, m
3.78 overlap
3.82, dd (8.6, 2.7)
4.48, dq (8.6, 6.3)
1.50, d (6.3)
a
b
OMe
3.45, s
3.35, s
3.36, s
3.36, s
3.36, s
3.32, s
3.36, s
3.38, s
3.36, s
D-Cym
D-Cym
D-Cym
D
-Cym
D-Cym
D-Cym
D-Cym
D-Cym
0
0
0
0
0
0
0
000
000
000
000
000
000
000
1
2
2
3
4
5
6
5.20, dd (9.4, 1.4)
1.78, m
2.31, m
3.88 overlap
3.44, dd (9.5, 2.5)
4.16, dq (9.5, 6.2)
1.35, d (6.2)
3.52, s
5.20, dd (9.4, 1.2)
1.74, m
2.25, m
3.85 overlap
3.43, dd (9.5, 3.2)
4.16, dq (9.5, 6.2)
1.34, d (6.2)
3.50, s
5.22, dd (9.6, 1.6)
1.82, m
2.36, m
3.90 overlap
3.46, dd (9.4, 2.8)
4.17, dq (9.4, 6.2)
1.35, d (6.2)
3.53, s
5.18, dd (9.6, 1.4)
1.76, m
2.23, m
3.82 overlap
3.42, dd (9.6, 3.2)
4.14, dq (9.6, 6.3)
1.34, d (6.3)
3.49, s
5.22, dd (9.6, 1.2)
1.78, m
2.30, m
3.88 overlap
3.44, dd (9.5, 3.0)
4.16, dq (9.5, 6.3)
1.32, d (6.3)
3.50, s
5.20, dd (9.5, 1.2)
1.78, m
2.28, m
3.84 overlap
3.44, dd (9.4, 2.8)
4.15, dq (9.4, 6.3)
1.34, d (6.3)
3.50, s
5.22, dd (9.5, 1.4)
1.82, m
2.40, m
3.76, d (2.8)
3.56, dd (9.5, 2.8)
4.12, dq (9.5, 6.2)
1.53, d (6.2)
3.45, s
5.18, dd (9.4, 1.2)
1.76, m
2.32, m
3.70, d (3.0)
3.52, dd (9.4, 3.0)
4.10, dq (9.4, 6.2)
1.34, d (6.2)
3.38, s
a
b
OMe
L
-Cym
L-Cym
L-Cym
L
-Cym
L
-Cym
L-Cym
0
0
0
0
0
0
0
0000
0000
0000
0000
0000
0000
0000
1
2
2
3
4
5
6
4.96, d (3.2)
1.80, m
2.36, m
3.70, q (3.2)
3.58, dd (9.2, 3.2)
4.51, dq (9.2, 6.2)
1.50, d (6.2)
4.95, d (2.5)
1.78, m
2.26, m
3.71, q (3.3)
3.58, dd (8.8, 3.3)
4.51, dq (8.8, 6.3)
1.51, d (6.3)
4.96, brs
1.84, m
2.38, m
3.70 overlap
3.60, dd (8.9, 3.3)
4.54, dq (8.9, 6.2)
1.52, d (6.2)
4.96, brs
1.84, m
2.38, m
3.70 overlap
3.58, dd (8.9, 3.2)
4.52, dq (8.9, 6.3)
1.49, d (6.3)
4.96, brs
1.82, m
2.36, m
3.70 overlap
3.58, dd (8.8, 3.3)
4.52, dq (8.8, 6.2)
1.52, d (6.2)
4.95, brs
1.84, m
2.40, m
3.70 overlap
3.58, dd (8.9, 3.2)
4.52, dq (8.9, 6.2)
1.49, d (6.2)
a
b
OMe
3.37, s
3.36, s
3.37, s
3.36, s
3.34, s
3.36, s
Cym = cymaropyranosyl; Digin = diginopyranosyl; Digit = digitoxopyranosyl.

S. Liu et al. / Phytochemistry xxx (2013) xxx–xxx
5
Table 3
1
3C NMR spectroscopic data of the aglycone moieties of compounds 1–11 (pyridine-d
5
, 100 MHz).
Carbon
1
2
3
4
5
6
7
8
9
10
39.2
11
39.2
1
2
3
4
5
6
7
8
9
38.9
29.8
77.6
39.2
38.9
29.8
77.7
39.2
38.9
29.8
77.7
39.2
38.9
29.8
77.7
39.2
38.9
29.8
77.7
39.2
39.0
30.0
77.7
39.3
139.3
119.5
35.0
74.3
44.9
37.4
29.4
68.9
60.4
89.3
34.2
32.7
92.5
9.4
39.1
30.0
77.7
39.3
39.0
30.0
77.7
39.3
39.0
30.0
77.7
39.3
29.9
77.6
38.9
139.3
119.2
34.7
74.5
44.5
37.4
25.0
73.6
58.1
89.5
33.8
33.0
92.4
10.7
18.6
209.9
27.5
29.9
77.6
38.9
139.3
119.2
34.8
74.2
44.5
37.4
25.0
73.5
57.9
89.4
33.8
32.9
92.4
10.7
18.4
209.4
27.5
139.2
119.1
34.7
74.2
44.5
37.4
25.0
73.6
58.1
89.5
33.8
33.0
92.4
10.7
18.1
209.9
27.7
139.3
119.1
34.7
74.3
44.5
37.4
25.0
73.6
58.1
89.5
33.8
33.1
92.4
10.7
18.2
209.8
27.7
139.3
119.2
34.7
74.3
44.5
37.4
25.0
73.6
58.1
89.5
33.8
33.1
92.4
10.7
18.4
209.9
27.7
139.3
119.1
34.7
74.2
44.5
37.3
24.8
73.5
57.9
89.4
33.7
32.8
92.4
10.4
18.5
210.2
27.6
139.3
119.1
34.7
74.2
44.5
37.3
24.8
73.5
58.3
89.4
33.7
32.8
92.4
10.4
18.5
210.2
27.6
139.3
119.5
35.0
74.3
44.9
37.4
29.4
69.0
60.4
89.3
34.2
32.8
92.3
9.4
139.4
119.5
35.0
74.2
44.9
37.4
29.4
68.9
60.4
89.3
34.2
32.8
92.5
9.4
139.3
119.5
35.0
74.2
44.9
37.4
29.4
68.9
60.4
89.3
34.2
32.7
92.5
9.4
10
11
12
13
14
15
16
17
18
19
20
21
18.3
209.5
27.9
18.4
209.6
27.9
18.4
209.6
27.9
18.3
209.5
27.9
1
2-OCin
12-OAc
12-OCin
12-OIkem
1#
165.8
119.2
144.9
135.0
128.6
129.3
130.6
165.8
119.1
144.9
135.0
128.6
129.3
130.6
165.8
119.2
144.9
135.0
128.6
129.3
130.6
169.9
20.8
169.9
20.8
165.8
119.2
145.0
135.0
128.6
129.3
130.6
166.0
114.1
165.5
38.1
20.8
20.9
#
2
#
3
#
4
#
#
5
(9 )
#
#
6
(8 )
#
7
16.4
proton signals at d
.0 Hz), 5.02 (brs), 4.96 (brs), and 5.30 (dd, J = 8.2, 1.0 Hz), and five
anomeric carbon resonances at d 95.9, 96.3, 98.5, 99.0, and 99.8
were observed in the H and C NMR spectra (Tables 1–4) of 4.
H
5.22 (dd, J = 9.6, 1.6 Hz), 5.45 (dd, J = 8.2,
of 6 as deacylmetaplexigenin 3-O-
-cymaropyranosyl-(1?4)- -cymaropyranosyl-(1?4)-b-
-digitoxopyranoside, named cynaurico-
a
-
L
-cymaropyranosyl-(1?4)-b-
1
D
a
-
L
D
-dig-
C
itoxopyranosyl-(1?4)-b-D
1
13
side F.
1
13
The H and C NMR spectroscopic data of the sugar part of 4 were
almost identical to those of 2, suggesting a common saccharide
90
Compound 7 shared the same molecular formula C56H O22 as
1
13
that of 4. Comparison of H and C NMR spectra (Tables 1–4) of
7 with those of 3 and 6 suggested that 7 and 6 possess identical
aglycones, and that 7 and 3 contain the same saccharide moiety.
1
13
chain in their structures. Comparison of H and C NMR spectro-
scopic data of the aglycones of 4 and 2 established the only struc-
tural difference to be the substitution of the cinnamoyl group in 2
1
1
Detailed analyses of H– H COSY, HSQC, and HMBC spectra of 7
supported the above assumption, and compound 7 was character-
1
13
with an acetyl group in 4, which was confirmed by the H–
long-range correlation signals between the carbonyl signal (d
69.9) and H-12 (d 4.94) and methyl (d 2.05) in the HMBC spec-
C
C
ized as deacylmetaplexigenin 3-O-
a
-
L
-cymaropyranosyl-(1?4)-b-
-3-O-
acetyldigitoxopyranosyl-(1?4)-b-D-digitoxopyranoside, named
1
H
H
D
-cymaropyranosyl-(1?4)- -cymaropyranosyl-(1?4)-b-D
a
-
L
1
1
trum (Fig. 2). Further analyses of H– H COSY, HSQC, and HMBC
spectra of 4 enabled the characterization of its structure as meta-
cynauricoside G.
plexigenin 3-O-
syl-(1?4)- -cymaropyranosyl-(1?4)-b-
1?4)-b- -digitoxopyranoside, named cynauricoside D.
The molecular formula of 5 was established as C58
a
-
L
-cymaropyranosyl-(1?4)-b-
D
-cymaropyrano-
The HRESIMS of 8 displayed a quasimolecular ion peak at m/z
+
a
-
L
D
-digitoxopyranosyl-
951.4965 [M+Na] , corresponding to the molecular formula
1
(
D
C
47
76
H O
18. The H and 13C NMR spectroscopic data (Tables 1–4)
H
92
O23 by
of the aglycone part were almost identical to those of 6 and 7, indi-
+
HRESIMS ion peak at m/z 1179.5944 [M+Na] . By comparison of
its NMR spectroscopic data (Tables 1–4) with those of 3 and 4,
compound 5 was suggested to share the same saccharide moiety
as 3, and an identical aglycone part to that of 4. Further analysis
cating the aglycone was deacylmetaplexigenin. Four anomeric pro-
1
13
ton and carbon signals were observed in its H and C NMR
spectra, and chemical shifts were similar to those of 2 except for
the absence of signals for the terminal cymaropyranose. Detailed
1
1
1
1
of H– H COSY, HSQC, and HMBC spectra (Fig. 2) of 5 confirmed
its structure as metaplexigenin 3-O- -cymaropyranosyl-(1?4)-
b- -cymaropyranosyl-(1?4)- -cymaropyranosyl-(1?4)-b- -3-
-digitoxopyranoside, named
analyses of H– H COSY, HSQC, and HMBC spectra (Fig. 2) of 8 indi-
cated that its sugar chain was the same as the inner four sugar
units in 2. Therefore, compound 8 was proposed as deacylmeta-
a-L
D
a
-
L
D
O-acetyldigitoxopyranosyl-(1?4)-b-
D
plexigenin 3-O-b-
syl-(1?4)-b- -digitoxopyranosyl-(1?4)-b-
named cynauricoside H.
D
-cymaropyranosyl-(1?4)-
a-L-cymaropyrano-
cynauricoside E.
D
D
-digitoxopyranoside,
88
The molecular formula of 6 was established as C54H O21 by its
+
HRESIMS with an ion peak at m/z 1095.5623 [M+Na] . By compar-
ison of its NMR spectroscopic data (Tables 1–4) with those of 4, the
aglycone of 6 was determined to be deacylmetaplexigenin by the
absence of an acetyl group at 12-OH, while the sugar chain was
indicated to be the same as in 2 and 4 by the good match of
NMR spectroscopic data. Further analysis of 1H– H COSY, HSQC,
and HMBC spectra (Fig. 2) led to the confirmation of the structure
The HRESIMS of 9 displayed a quasimolecular ion peak at m/z
+
993.5065 [M+Na] , corresponding to the molecular formula
C
49
78
H O
19 with one more acetyl group than 8. The NMR spectro-
scopic data (Tables 1–4) of 9 were quite similar to those of 8, ex-
0
0
00
00
cept for the differences of chemical shifts of C-2 , C-3 , C-4 , and
1
00
00
H-3 , suggesting that the additional acetyl was attached to C-3 .
Detailed analyses of H– H COSY, HSQC, and HMBC spectra
1
1
Please cite this article in press as: Liu, S., et al. Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum. Phytochemistry (2013),
http://dx.doi.org/10.1016/j.phytochem.2013.03.010

6
S. Liu et al. / Phytochemistry xxx (2013) xxx–xxx
(Fig. 2) of 9 supported the above assumption, and the structure of 9
appetite-reducing effect of pregnane glycosides from Cynanchum
was proposed as deacylmetaplexigenin 3-O-b-
D
-cymaropyranosyl-
and Hoodia plants is warranted concerning their molecular weight
and the seemingly impossibility of passing through the blood–
brain barrier. Over-consumption of the roots of C. auriculatum
was said to result in abortion of pregnant sows in Binhai County,
Jiangsu Province of China, and the extract of H. gordonii was found
to delay fetal development of mice, which might be a consequence
of reduced maternal feed consumption (Dent et al., 2012). Further
toxicity evaluation of pregnane glycosides from C. auriculatum and
H. gordonii are thus warranted. Nevertheless, to reduce food intake
is an efficient way to control both obesity and overweight,
although appetite suppressants like fenfluramine and sibutramine
have been withdrawn from market, several other appetite suppres-
sants are still waiting for FDA approval marketing as anti-obesity
drugs.
(
1?4)-
a
-
L
-cymaropyranosyl-(1?4)-b-
D-3-O-acetyldigitoxopyr-
anosyl-(1?4)-b-
D
-digitoxopyranoside, named cynauricoside I.
The structures of the wilfoside K1N (10) and wilfoside C1N (11),
the most abundant constituents of C. auriculatum, resembled that
of P57 (Fig. 1), an appetite suppressant from Hoodia species (Van
Heerden et al., 2007). Both 10 and 11 were selected for the testing
of appetite-reducing effects according to the standard bioassay
method. The results showed that 10 could suppress the appetite
of rats at a dose of 50 mg/kg body weight significantly, and the
influences of 10 and 11 on food and water consumption and body
weight are given in Table 5 and Figs. 3–5. The decrease of body
weight and food consumption suggested that the two pregnane
glycosides from C. auriculatum possess similar appetite-reducing
effects to P57, which was reported to up-regulate hypothalamic
ATP level and affect the sense of satiety to reduce the desire for
food (MacLean and Luo, 2004). It is noted that the extract of
Cynanchum otophyllum Schneid rich in pregnane glycosides
has been developed as a formulated Chinese medicine for the
treatment of epilepsy. Further study on the mechanism of
3
. Conclusion
A phytochemical study on the roots of C. auriculatum led to
identification of nine new pregnane glycosides along with six
known compounds. The most abundant known pregnane
Table 4
1
3C NMR spectroscopic data of the sugar moieties of compounds 1–11 (pyridine-d
5
, 100 MHz).
Carbon
1
2
3
4
5
6
7
8
9
10
-cym
11
D-cym
D
-Cym
D-Digit
D-Digit
D
-Digit
D-Digit
D-Digit
D-Digit
D
-Digit
D-Digit
D
0
1
96.1
35.3
77.5
82.5
69.2
18.8
96.3
39.0
67.4
83.4
68.5
18.6
96.5
39.0
67.5
83.5
68.5
18.6
96.3
39.1
67.5
83.4
68.6
18.6
96.3
39.0
67.4
83.4
68.4
18.6
96.3
39.0
67.5
83.5
68.5
18.5
96.3
39.1
67.5
83.5
68.4
18.6
96.3
39.1
67.5
83.5
68.4
18.6
96.3
39.1
67.5
83.5
68.4
18.6
96.1
35.3
77.5
82.5
69.2
18.7
96.1
35.3
77.6
82.4
69.2
18.7
0
2
0
3
0
0
4
5
0
6
OMe
57.2
57.3
57.3
L
-Digin
D-Digit
D-Digit
D
-Digit
D-Digit
D-Digit
D-Digit
D
-Digit
D-Digit
L
-Digin
L-Digin
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
101.0
32.5
74.5
73.8
67.5
17.9
99.8
38.4
67.5
81.3
68.8
18.5
99.5
36.5
70.5
79.9
69.7
18.5
99.8
38.4
67.6
81.3
68.9
18.5
99.5
36.5
70.5
79.9
69.7
18.5
99.8
38.4
67.6
81.3
68.9
18.6
99.5
36.5
70.5
79.9
69.7
18.3
99.8
38.4
67.6
81.3
68.9
18.4
99.5
36.5
70.5
79.9
71.0
18.4
100.9
32.5
74.6
73.8
67.4
17.9
100.9
32.5
74.6
73.8
67.4
17.9
OMe/Ac
55.3
170.3
1.0
170.3
21.0
170.3
21.0
170.3
21.0
55.3
55.3
2
D
-Cym
L-Cym
L-Cym
L
-Cym
L
-Cym
L-Cym
L-Cym
L
-Cym
L
-Cym
D-Cym
D-Cym
0
0
0
0
0
0
00
00
00
00
00
00
1
2
3
4
5
6
99.5
35.3
79.9
74.1
71.0
18.7
98.5
32.4
73.3
77.1
66.3
18.3
99.3
32.1
73.0
77.5
65.0
18.7
98.5
32.4
73.5
77.1
66.4
18.2
99.3
32.2
73.0
77.7
65.0
18.6
98.5
32.2
73.5
77.1
66.4
18.2
99.3
32.2
73.0
77.7
65.1
18.5
98.5
32.4
73.5
77.2
66.5
18.6
99.3
32.2
73.0
77.8
65.2
18.9
99.4
36.3
77.8
82.3
69.4
18.7
99.4
36.3
77.8
82.3
69.4
18.6
OMe
58.0
57.1
56.9
57.7
56.6
56.6
56.6
57.1
56.9
58.3
58.2
D-Cym
D-Cym
D
-Cym
D-Cym
D-Cym
D-Cym
D
-Cym
D-Cym
L
-Cym
L-Cym
0
0
0
0
0
0
000
000
000
000
000
000
1
2
3
4
5
6
95.9
36.7
77.6
82.3
69.4
18.6
95.4
36.7
77.7
82.2
69.4
18.6
95.9
36.7
77.6
82.3
69.4
18.6
95.4
36.6
77.6
82.2
69.3
18.5
95.9
36.7
77.7
82.3
69.4
18.6
95.5
36.7
77.7
82.2
69.4
18.4
96.0
35.6
78.8
74.3
71.1
19.0
95.5
35.6
78.8
74.2
69.7
18.5
99.0
32.1
76.4
73.2
66.4
18.2
99.0
32.1
76.4
73.2
66.4
18.1
OMe
58.3
58.3
58.3
58.3
58.5
58.3
57.9
57.9
56.6
56.6
L-Cym
L-Cym
L
-Cym
L
-Cym
L-Cym
L-Cym
0
0
0
0
0
0
0000
0000
0000
0000
0000
0000
1
2
3
4
5
6
99.0
32.1
76.4
73.5
66.3
18.4
56.6
98.9
32.2
76.4
73.3
66.4
18.3
56.6
99.0
32.1
76.4
73.3
66.4
18.4
56.6
98.9
32.1
76.4
73.3
66.4
18.1
56.9
99.0
32.1
76.4
73.3
66.4
18.2
57.2
98.9
32.1
76.4
73.3
66.4
18.5
56.9
OMe
Please cite this article in press as: Liu, S., et al. Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum. Phytochemistry (2013),
http://dx.doi.org/10.1016/j.phytochem.2013.03.010

S. Liu et al. / Phytochemistry xxx (2013) xxx–xxx
7
O
O
O
OH
OH
OCH3
OH
O
O
HO
O
O
O
O
O
O
OCH3
OCH3
1
OH
OH
OH
OH
H CO
O
3
O
O
O
O
H CO
O
O
3
O
O
O
O
OH
OR4
HO
2
- 7
OCH3
OH
OH
H3CO
O
O
O
O
O
HO
O
O
OH
OR3
OCH3
8, 9
Fig. 2. Key HMBC correlations of new glycosides from C. auriculatum.
glycoside, wilfoside K1N (10) with similar structures to that of P57
isolated from H. gordonii showed significant appetite suppressing
effect, and led to loss of body weight in rats. Further investigation
of the efficacy and safety of pregnane glycosides isolated from the
abundant plant source C. auriculatum is needed, although more
evaluation work should also be undertaken on the popular appetite
suppressing extracts of H. gordonii, a plant that might be listed as
endangered species due to overexploitation.
4.2. Plant material
Roots of C. auriculatum Royle ex Wight were collected from Bin-
hai County, Jiangsu Province, China, in May, 2010, and authenti-
cated by Associate Professor Jin-Gui Shen of Shanghai Institute of
Materia Medica (SIMM), Chinese Academy of Sciences (CAS). A
voucher specimen (SIMM20100501) has been deposited in the her-
barium of SIMM, CAS.
4
. Experimental
4.3. Extraction and isolation
4.1. General experimental procedures
Powdered air-dried roots of C. auriculatum (3.5 kg) were ex-
2
tracted with EtOH–H O (95:5, v/v) under reflux condition (for 5 L
Optical rotations were measured with a Perkin–Elmer 341
polarimeter; IR spectra were recorded using a Perkin–Elmer 577
spectrometer; ESIMS and HRESIMS were measured on a Esquire
000plus and a Bruker Daltonics microTOF QII mass spectrometer,
respectively; NMR spectra were acquired on a Bruker AM 400 with
each, 2 h ꢂ 3). The combined filtrates were concentrated in vacuo
to get the crude extract (110 g) which was subjected to silica gel
CC (petroleum ether–acetone, 20:1–3:2, then CHCl
2:1) to obtain five fractions (A–E). Fraction C (2.4 g) was further ap-
plied to silica gel CC (CHCl –acetone, 10:1–1:1) to afford four sub-
3
–MeOH, 10:1–
3
3
TMS as internal standard, and chemical shifts are expressed in d
fractions (C1–C4). Subfraction C1 (50 mg) was purified on silica gel
CC (petroleum ether–acetone, 5:1–3:1) to yield 1 (20 mg). Subfrac-
tions C2 (2.1 g), C3 (3.2 g) and C4 (2.5 g) were subjected to silica gel
CC (petroleum ether–acetone, 4:1–2:1), respectively, to give wilfo-
side C1N (11, 1.5 g), wilfoside K1N (10, 2.5 g), and cynandione A
(2.0 g), respectively. An aliquot of Fraction E (25 g) was purified
(
ppm); Preparative HPLC was carried out using a Unimicro Tech-
nologies instrument equipped with a Waters XBridge C18 column
m, 19 mm ꢂ 100 mm). Column chromatographic (CC) separa-
tions were carried out by using MCI gel CHP-20P (75–150 m;
(5 l
l
Mitsubishi Chemical Industry Co., Ltd., Tokyo, Japan), silica gel
H60 (300–400 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao,
China), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Swe-
den) as packing materials. HSGF254 silica gel plates (Yantai Chem-
ical Industrial Institute, Yantai, China) were used for TLC analytical
purpose. All solvents used were redistilled from chemical grade be-
fore use (Shanghai Chemical Reagent Company, Ltd., Shanghai,
China).
by HPLC (MeOH–H
side C1G (20 mg) and cynauricuoside A (30 mg). Fraction D (5.4 g)
was separated on MCI gel CC (MeOH–H O, 45:55–100:0) to give
four subfractions (D1–D4). Subfraction D1 (500 mg) was subjected
to silica gel CC (CHCl –MeOH, 1:0–10:1) to give D1a (35 mg) and
D1b (80 mg), which were further purified by HPLC (MeOH–H O,
50:50–100:0, 4 mL/min, 30 min) and Sephadex LH-20 (EtOH–H
2
O, 75:25–100:0, 4 mL/min, 30 min) to get wilfo-
2
3
2
2
O
Please cite this article in press as: Liu, S., et al. Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum. Phytochemistry (2013),
http://dx.doi.org/10.1016/j.phytochem.2013.03.010

8
S. Liu et al. / Phytochemistry xxx (2013) xxx–xxx
(
95:5, v/v)), respectively, to yield 6 (7 mg) and 9 (8 mg) from D1a,
2
and 7 (10 mg) and 8 (15 mg) from D1b with a MeOH–H O gradient
(
8:2–0:100, v/v). Subfraction D2 (200 mg) was subjected to silica
3
gel CC (CHCl –MeOH, 1:0–10:1) to give wilfoside M1N (80 mg).
Subfraction D3 (180 mg) was separated by silica gel CC (CHCl
MeOH, 1:0–10:1) and then by HPLC (MeOH–H O, 50:50–100:0,
mL/min, 30 min) and Sephadex LH-20 (EtOH–H O (95:5, v/v)) to
obtain 4 (15 mg) and 5 (45 mg). Subfraction D4 (100 mg) was sep-
arated by silica gel CC (CHCl –MeOH, 1:0–10:1) and then by HPLC
MeOH–H O, 50:50–100:0, 4 mL/min, 30 min) and Sephadex LH-
0 (EtOH–H O (95:5, v/v)) to yield 2 (20 mg) and 3 (15 mg).
3
–
2
4
2
3
(
2
2
2
4.4. Cynauricoside A (1)
2
D
2
White amorphous powder; ½
aꢁ
ꢀ 91:4 (c 0.22, MeOH); IR(KBr)
m
1
max 3464, 2974, 2933, 1712, 1637, 1450, 1365, 1279, 1167, 1088,
ꢀ1
003, 943, 769 cm ; Positive mode HRESIMS m/z 965.4843 (calcd
1
13
74
for C51H O16Na, 965.4875); for H and C NMR spectroscopic
data, see Tables 1–4.
4.5. Cynauricoside B (2)
2
D
2
White amorphous powder; ½
aꢁ
ꢀ 41:7 (c 0.24, MeOH); IR(KBr)
m
9
C
max 3454, 2970, 2931, 1714, 1637, 1450, 1383, 1311, 1165, 1055,
95, 868 cm ; Positive mode HRESIMS m/z 1225.6139 (calcd for
H O22Na, 1225.6134); for H and C NMR spectroscopic data,
94
ꢀ
1
1
13
63
see Tables 1–4.
4.6. Cynauricoside C (3)
2
D
2
White amorphous powder; ½
aꢁ
ꢀ 26:7 (c 0.18, MeOH); IR(KBr)
m
1
(
max 3456, 2972, 2931, 1741, 1714, 1637, 1450, 1369, 1244, 1163,
059, 1005, 866 cm ; Positive mode HRESIMS m/z 1267.6235
ꢀ1
1
13
calcd for C65
96
H O23Na, 1267.6240); for H and C NMR spectro-
scopic data, see Tables 1–4.
4.7. Cynauricoside D (4)
2
D
2
White amorphous powder; ½
aꢁ
ꢀ 76:4 (c 0.22, MeOH); IR(KBr)
m
1
C
max 3465, 2970, 2931, 1742, 1712, 1452, 1371, 1238, 1163, 1088,
054, 991 cm ; Positive mode HRESIMS m/z 1137.5864 (calcd for
H O22Na, 1137.5821); for H and C NMR spectroscopic data,
90
ꢀ1
1
13
56
see Tables 1–4.
4.8. Cynauricoside E (5)
2
D
2
White amorphous powder; ½
aꢁ
ꢀ 16:1 (c 0.28, MeOH); IR(KBr)
m
1
C
max 3471, 2972, 2931, 1743, 1712, 1452, 1373, 1240, 1174, 1112,
059, 991 cm ; Positive mode HRESIMS m/z 1179.5944 (calcd for
H O23Na, 1179.5927); for H and C NMR spectroscopic data,
92
ꢀ1
1
13
58
see Tables 1–4.
4.9. Cynauricoside F (6)
2
D
2
White amorphous powder; ½
a
ꢁ
ꢀ 10:6 (c 0.18, MeOH); IR(KBr)
m
1
C
max 3460, 2966, 2927, 2852, 1707, 1637, 1452, 1381, 1163, 1088,
055, 995 cm ; Positive mode HRESIMS m/z 1095.5623 (calcd for
H O21Na, 1095.5716); for H and C NMR spectroscopic data,
88
ꢀ1
1
13
54
see Tables 1–4.
4.10. Cynauricoside G (7)
2
D
2
White amorphous powder; ½
a
ꢁ
ꢀ 20:6 (c 0.16, MeOH); IR(KBr)
m
1
(
max 3452, 2968, 2929, 1741, 1709, 1633, 1452, 1378, 1242, 1161,
113, 1059, 1005 cm ; Positive mode HRESIMS m/z 1137.5786
ꢀ1
1
13
calcd for C56
90
H O22Na, 1137.5821); for H and C NMR spectro-
scopic data, see Tables 1–4.
Please cite this article in press as: Liu, S., et al. Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum. Phytochemistry (2013),
http://dx.doi.org/10.1016/j.phytochem.2013.03.010

S. Liu et al. / Phytochemistry xxx (2013) xxx–xxx
9
Wilfoside K1N
Wilfoside C1N
Sibutramine
Vehicle
Effect of wilfoside K1N and wilfoside C1N
on food consumption in rats
2
2
1
1
5
0
5
0
5
0
Fig. 3. Effect of wilfoside K1N (50 mg/kg body weight) and wilfoside C1N (50 mg/kg body weight) on food consumption in rats.
Wilfoside K1N
Effect of wilfoside K1N and wilfoside C1N
on water consumption in rats
Wilfoside C1N
Sibutramine
Vehicle
2
2
1
1
7.5
2
5
2.5
2
0
7.5
1
5
2.5
1
0
Fig. 4. Effect of wilfoside K1N (50 mg/kg body weight) and wilfoside C1N (50 mg/kg body weight) on water consumption in rats.
Wilfoside K1N
Wilfoside C1N
Sibutramine
Vehicle
Effect of wilfoside K1N and wilfoside C1N
on bodyweight in rats
1
1
1
1
1
1
1
1
1
1
65
60
55
50
45
40
35
30
25
20
Fig. 5. Effect of wilfoside K1N (50 mg/kg body weight) and wilfoside C1N (50 mg/kg body weight) on body weight in rats.
Please cite this article in press as: Liu, S., et al. Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum. Phytochemistry (2013),
http://dx.doi.org/10.1016/j.phytochem.2013.03.010

1
0
S. Liu et al. / Phytochemistry xxx (2013) xxx–xxx
4.11. Cynauricoside H (8)
Manufacturing Program’’, China (No. 2009ZX09301-001) and
Natural Science Foundation of China (No. 81273399), and also to
Prof. Zhou Zhang of SIMM, CAS for guidance in animal experiments.
2
D
2
White amorphous powder; ½
aꢁ
ꢀ 56:7 (c 0.15, MeOH); IR(KBr)
m
8
O
max 3450, 2968, 2929, 1705, 1635, 1452, 1381, 1165, 1088, 1005,
ꢀ1
68 cm ; Positive mode HRESIMS m/z 951.4965 (calcd for C47
H
76-
Appendix A. Supplementary data
1
13
18Na, 951.4929); for H and C NMR spectroscopic data, see
Tables 1–4.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.
4.12. Cynauricoside I (9)
2
013.03.010.
2
2
White amorphous powder; ½
aꢁ
1:3 (c 0.16, MeOH); IR(KBr)
D
References
m
1
9
max 3465, 2966, 2927, 2852, 1741, 1709, 1452, 1377, 1244,
ꢀ1
165, 1082, 1005, 868 cm
;
Positive mode HRESIMS m/z
Bai, H., Li, W., Koike, K., Satou, T., Chen, Y.J., Nikaido, T., 2005. Cynanoside A–J, ten
19Na, 993.5035); for H and 13C NMR
1
novel pregnane glycosides from Cynanchum atratum. Tetrahedron 61, 5797–
78
93.5065 (calcd for C49H O
5811.
spectroscopic data, see Tables 1–4.
Chen, J.J., Zhang, Z.X., Zhou, J., 1990. Cynauricuosides A, B and C, steroid glycosides
from the roots of Cynanchum auriculatum. Acta Bot. Yunnan 12, 197–210.
Dent, M.P., Wolterbeek, A.P.M., Russell, P.J., Bradford, R., 2012. Safety profile of
Hoodia gordonii extract: Mouse prenatal developmental toxicity study. Food
Chem. Toxicol. 50, S20–S25.
4
.13. Hydrolysis of compounds 1, 2 and 3
A solution of 1 (18 mg) in dioxane (6 mL) was treated with
.2 M H SO (2 mL) at 50 °C for 2 h. After removal of dioxane, the
2 4
solution was neutralized by 1 M NaOH and then extracted with
EtOAc (2 mL ꢂ 3) to remove the aglycone. The aqueous layer was
dried and subjected on silica gel CC to afford cymarose (4.0 mg,
Gan, H., Xiang, W.J., Ma, L., Hu, L.H., 2008. Six new C21 steroidal glycosides from
Cynanchum bungei Dence. Helv. Chim. Acta 91, 2222–2234.
Gu, X.J., Yao, N., Qian, S.H., Li, Y.B., Li, P., 2009. Four new C21 steroidal glycosides
from the roots of Cynanchum auriculatum. Helv. Chim. Acta 92, 88–97.
Huang, P.L., Lu, C.M., Yen, M.H., Wu, R.R., Lin, C.N., 1995. Acetophenones from
Cynanchum taiwanianum. Phytochemistry 22, 537–541.
Jiangsu College of New Medicine, 2004. Dictionary of Traditional Chinese Medicines.
People’s Hygiene Publisher, Shanghai, p. 732.
Lu, Y., Xiong, H., Teng, H.L., Mei, Z.N., 2011a. Three new steroidal glycosides from the
roots of Cynanchum auriculatum. Helv. Chim. Acta 94, 1296–1303.
Lu, Y., Teng, H.L., Yang, G.Z., Mei, Z.N., 2011b. Three new steroidal glycosides from
the roots of Cynanchum auriculatum. Molecules 16, 1901–1909.
MacLean, D.B., Luo, L.G., 2004. Increased ATP content/production in the
hypothalamus may be a signal for energy-sensing of satiety: Studies of the
anorectic mechanism of a plant steroidal glycoside. Brain Res. 1020, 1–11.
Pereira, C.A., Pereira, L.L.S., Corrêa, A.D., 2010. Hoodia gordonii in the treatment of
obesity: A review. J. Med. Plants Res. 4, 2305–2312.
0
2
D
2
22
D
½
a
H
ꢁ
þ 39:3, c 0.14, H
2
O) and diginose (2.8 mg, ½
aꢁ
ꢀ 40:8, c 0.13,
2
O) with petroleum ether–acetone (5:1) as eluent. Using the sim-
2
D
2
ilar procedure, cymarose (4.5 mg, ½
a
ꢁ
ꢀ 14:7, c 0.14, H
2
O) and dig-
O) were isolated from the
hydrolysate of 2 (18 mg) by silica gel CC with Et O–acetone (3:1)
as eluent. A solution of 3 (1 mg) in MeOH (2 mL) was treated with
CO (10 mg) at r.t. for 6 h. Compound 2 was identified from the
solution by co-TLC with authentic sample.
2
D
2
itoxose (4.1 mg, ½
a
ꢁ
þ 51:5, c 0.13, H
2
2
K
2
3
Shan, L., Zhang, W.D., Zhang, C., Su, J., Zhou, Y., 2005. Antitumor activity of crude
extract and fractions from root tuber of Cynanchum auriculatum Royle ex Wight.
Phytother. Res. 19, 259–261.
4.14. Bioassay
Shan, L., Liu, R.H., Shen, Y.H., Zhang, W.D., Zhang, C., Wu, D.Z., Min, L., Su, J., Xu, X.K.,
2006. Gastroprotective effect of a traditional Chinese herbal drug ‘Baishouwu’
on experimental gastric lesions in rats. J. Ethnopharmacol. 107, 389–394.
Sun, Y.S., Liu, Z.B., Wang, J.H., Xiang, L., Zhu, L.X., 2009. Separation and purification
SPF female Wistar rats (body weight 110–150 g), approximately
–7 weeks of age obtained from SLAC laboratory animal, were di-
6
vided into four groups with five animals each. The animals were
housed in polycarbonate material cages with 12 h light and 12 h
dark each day at controlled room temperature 23 ± 1.5 °C and rel-
ative humidity 55 ± 15%, respectively, and had ad libitum access to
standard certified laboratory rodent diet and filter water. The num-
ber of the laboratory animal facility permission was SYXK(Shang-
hai)2008-0049. Food consumption and water consumption were
recorded from Day-7 to the termination of the study. Test samples
and sibutramine were mixed with calculated amount of soluble
of
baishouwubenzophenone,
4-hydroxyacetophenone
and
2,4-
dihydroxyacetophenone from Cynanchum auriculatum Royle ex Wight by
HSCCC. Chromatographia 70, 1–6.
Teng, H.L., Lu, Y., Li, J., Yang, G.Z., Mei, Z.N., 2011. Two new steroidal glycosides from
the root of Cynanchum auriculatum. Chin. Chem. Lett. 22, 77–80.
Tsukamoto, S., Hayashi, K., Mitsuhashi, H., 1985a. Studies on the constituents of
Asclepiadaceae plant-LVII: The structures of six glycosides, wilfoside C1N, C2N,
C3N, C1G, C2G and C3G, with novel sugar chain containing a pair of optically
isomeric sugars. Tetrahedron 41, 927–934.
Tsukamoto, S., Hayashi, K., Mitsuhashi, H., 1985b. Studies on the constituents of
Asclepiadaceae plant-LX: Further studies on glycoside with a novel sugar chain
containing
a pair of optically isomeric sugars, D- and L-cymarose, from
starch (100 mg/mL), suspended in H
by using a constant dose volume of 10 mL/kg body weight. On Days
, 2 and 3 of the procedure, the animals in treatment groups were
administrated wilfoside C1N and wilfoside K1N at the dose of
0 mg/kg body weight once daily. Sibutramine was administrated
2
O, respectively, and gavaged
Cynanchum wilfordii. Chem. Pharm. Bull. 33, 2294–2304.
Van Heerden, F.R., 2008. Hoodia gordonii:
A natural appetite suppressant. J.
1
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.15. Statistical method
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Acknowledgments
D
The authors are grateful to financial support from National
Science & Technology Major Project ‘‘Key New Drug Creation and
Please cite this article in press as: Liu, S., et al. Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum. Phytochemistry (2013),
http://dx.doi.org/10.1016/j.phytochem.2013.03.010