Cynanauriculoside C-E, three new antidepressant pregnane glycosides from Cynanchum auriculatum

Article abstract of DOI:10.1016/j.phytol.2011.02.009

Based on the bioactive screening results, three new pregnane glycosides named as cynanauriculoside C-E (1-3), were isolated from the roots of Cynanchum auriculatum Royle ex Wight (Asclepiadaceae), together with two known ones, otophylloside L (4) and cynauricuoside C (5). On the basis of detailed spectroscopic analysis and chemical method, the structures of new compounds were characterized to be qingyangshengenin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-cymaropyranoside (1), qingyangshengenin 3-O-β-d-glucopyranosyl- (1 → 4)-β-d-glucopyranosyl-(1 → 4) -α-l-cymaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-cymaropyranoside (2) and caudatin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 4)-β-d-cymaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (3). In the despair mice models, these pregnane glycosides showed significant antidepressant activity at the dosage of 50 mg/kg (i.g.). The most potent one was cyanauriculatoisde D (2), which was close to the positive control fluoxetine (20 mg/kg).

Full text of DOI:10.1016/j.phytol.2011.02.009

Phytochemistry Letters 4 (2011) 170–175
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Cynanauriculoside C–E, three new antidepressant pregnane glycosides from
Cynanchum auriculatum
a
b
c
Qing-Xiong Yang a, , Yong-Chang Ge , Xiao-Yan Huang , Qian-Yun Sun
*
a School of Chemistry & Materials Science, Guizhou Normal University, 116 Baoshan North Road, Guiyang 550001, Guizhou, PR China
b School of Life Sciences, Guizhou Normal University, 116 Baoshan North Road, Guiyang 550001, PR China
c The Key Laboratory of Chemistry of Natural Products, Guizhou Province and Chinese Academy of Sciences, Guiyang 550002, PR China
A R T I C L E I N F O
A B S T R A C T
Article history:
Based on the bioactive screening results, three new pregnane glycosides named as cynanauriculoside C–
E (1–3), were isolated from the roots of Cynanchum auriculatum Royle ex Wight (Asclepiadaceae),
together with two known ones, otophylloside L (4) and cynauricuoside C (5). On the basis of detailed
spectroscopic analysis and chemical method, the structures of new compounds were characterized to be
Received 13 December 2010
Received in revised form 17 February 2011
Accepted 25 February 2011
Available online 16 March 2011
qingyangshengenin 3-O-
genin 3-O-
-glucopyranosyl-(1 ! 4)-
-oleandropyranosyl-(1 ! 4)- -cymaropyranoside (2) and caudatin 3-O-
(1 ! 4)- -glucopyranosyl-(1 ! 4)- -cymaropyranosyl-(1 ! 4)- -oleandropyranosyl-(1 ! 4)-
b
-
D
-oleandropyranosyl-(1 ! 4)-
b-D-cymaropyranoside (1), qingyangshen-
b
-
D
b
-
D
-glucopyranosyl-(1 ! 4) -
a
-L
-cymaropyranosyl-(1 ! 4)-
b
-
Keywords:
D
b
-
D
b
-D
-glucopyranosyl-
Cynanchum auriculatum
Asclepiadaceae
b
-
D
b
-
D
b-
D
b
-
D
-digitoxopyranoside (3). In the despair mice models, these pregnane glycosides showed significant
Cynanauriculoside C
Cynanauriculoside D
Cynanauriculoside E
Antidepressant
antidepressant activity at the dosage of 50 mg/kg (i.g.). The most potent one was cyanauriculatoisde D
(2), which was close to the positive control fluoxetine (20 mg/kg).
ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
and here we report the pregnane glycosides isolated from the
antidepressant fraction of C. auriculatum.
Cynanchum auriculatum Royle ex Wight (Asclepiadaceae) is
widely distributed in China. Its root is a famous tonic herbal drug in
traditional Chinese medicine, and has been widely used in clinics
as a beneficial and tonic agent since ancient times. A recent
ethnobotanical revealed that Baishouwu is an important herbal
drug for the treatment of gastric disorders (e.g., indigestion and
stomach ache) in the ethnomedicine of the Tujia and Miao
nationalities in Southwest of China (Shan et al., 2006). According to
the current knowledge, C. auriculatum contains a variety of
bioactive constituents, including pregnane glycosides and
baishouwubenzophenone (Chen et al., 1990; Gu et al., 2009;
Wang et al., 2002; Zhang et al., 2000a,b, 2007a; Sun et al., 2009). C.
auriculatum possesses widely useful activities including anti-
tumor (Shan et al., 2005; Wang et al., 2003, 2007, 2009; Yao et al.,
2009; Zhang et al., 2000a,b), gastroprotective (Shan et al., 2006)
and anti-aging (Zhang et al., 2007b). However, there was no report
about the antidepressant evaluation of C. auriculatum. In our
research, the ethanol extract and its fractions of C. auriculatum
were evaluated for antidepressant activities in mice (Yang, 2007),
2. Results and discussion
Following the screening results, the active fraction of the CHCl3
extract of the roots of C. auriculatum was repeatedly chromato-
graphed and five pregnane glycosides (1–5) were yielded. All of
them showed positive effects on Libermann–Buchard (Xu and
Chen, 1981) and Keller–Kiliani reactions, indicating the presence of
a steroidal skeleton with 2-deoxysugar moiety. Spectroscopic
analysis demonstrated that all the glycosides had a pregnane
skeleton with an acyl group at C-12 position and a straight sugar
chain connected to C-3 group position of the aglycone. In
comparison of 13C NMR and 1H-NMR spectra with reported data,
compounds 4 and 5 were identified as otophylloside L (4) (Ma et al.,
2007) and cynauricuoside C (5) (Chen et al., 1990) (Fig. 1).
The presence of the monosaccharides in the hydrolysates of
each compound was confirmed by co-TLC comparison with
authentic sugars. Further GC analysis of the corresponding
trimethylsilylated
of glucose. For the deoxysugars, since only
samples could be obtained, their absolute configurations could not
L
-cysteine adducts confirmed
D-configuration
-form authentic
D
be assigned by GC analysis, but determined to be
D-forms by
comparison of their 13C NMR spectroscopic data with those
reported data. The most significant differences in the 13C NMR data
* Corresponding author. Tel.: +86 851 6702134; fax: +86 851 6700050.
E-mail address: yangqx@gznu.edu.cn (Q.-X. Yang).
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2011.02.009

[
Q.-X. Yang et al. / Phytochemistry Letters 4 (2011) 170–175
171
Fig. 1. Pregnane glycosides from Cynanchum auriculatum.
between
D
- and
L
-configuration cymaropyranose involve the
(1710 cmꢀ1) groups and benzene ring (1612 and 1591 cmꢀ1). The
1H NMR spectrum of 1 revealed the presence of three singlet
resonances of C-2. The chemical shift of C-2 in the
anose is less than 35.0 ppm, but that of C-2 in the
L-cymaropyr-
D
-cymaropyr-
methyl groups
d
H 1.14, 1.64, 2.05 (each 3H, s, Me-19, -18, -21)] one
anose appears above 36.0 ppm (Hamed et al., 2004; Li et al., 2006;
Wang et al., 2002; Zhu et al., 1999). As to digitoxopyranosyl and
oleandropyranosyl, the configuration was supposed as D-configu-
ration (Ma et al., 2007), and also due to their similar 13C NMR
chemical shifts with those reported in literatures (Bai et al., 2005;
Mu et al., 1986; Yoshikawa et al., 1996, 1999; Zhang and Zhou,
1983).
Compound 1 was obtained as a white amorphous powder. Its
molecular formula was assigned to be C42H60O14 on the basis of
negative HRESI-MS m/z (787.3978 [M (C42H60O14)ꢀH]ꢀ, calculat-
ed: 787.3849) and 13C NMR (DEPT) data (Table 1). IR spectrum
showed the absorption bands for hydroxyl (3443 cmꢀ1), carbonyl
olefinic proton [
dH 5.36 (br. s, H-6)] and four aromatic protons on a
para-substituted benzene ring [dH 6.81 (2H, d, J = 8.8 Hz, H-40,60)
and dH 7.80 (2H, d, J = 8.8 Hz, H-30,70)] (Tables 1 and 2) in its
aglycone moiety. The above observation suggested qingyangshen-
genin seemed to be the aglycone of compound 1. Proton signals
were also assigned to two secondary methyl groups [dH 1.20 (d,
J = 6.4 Hz) and 1.27 (d, J = 6.4 Hz)], two methoxyl groups [dH 3.45
and 3.42] and two anomeric protons [dH 4.84 (dd, J = 1.6, 9.6 Hz),
4.61 (d, J = 8.4 Hz)] whose multiplicities suggested the presence of
three 2,6-dideoxy-sugar in a disaccharides chain and the
configuration of the two hexose units. Acid hydrolysis of 1 gave
qingyangshengenin (1a) as the aglycone, and oleandrose as well as
b-

172
Q.-X. Yang et al. / Phytochemistry Letters 4 (2011) 170–175
Table 1
13C NMR spectroscopic data of compounds 1–3 (C5D5N,
d in ppm).
Position
1
2
3
Position
1
2
3
1
2
39.8 (CH2)
30.2 (CH2)
79.3 (CH)
39.8 (CH2)
140.2 (C)
119.7 (CH)
35.2 (CH2)
75.0 (C)
39.7 (CH2)
30.2 (CH2)
79.3 (CH)
39.7 (CH2)
140.2 (C)
119.7 (CH)
35.2 (CH2)
75.0 (C)
39.8 (CH2)
30.2 (CH2)
79.3 (CH)
39.8 (CH2)
140.2 (C)
119.7 (CH)
35.2 (CH2)
75.0 (C)
100
b
-
D
-ole 97.3 (CH)
b
-
D
-ole 97.2 (CH)
b
-D-digit 97.2 (CH)
200
36.7 (CH2)
78.6 (CH)
83.9 (CH)
70 .0 (CH)
18.5 (CH3)
58.5 (CH3)
36.7 (CH2)
78.5 (CH)
83.8 (CH)
69.9 (CH)
18.6 (CH3)
58.5 (CH3)
39.0 (CH2)
67.7 (CH)
83.9 (CH)
70.7 (CH)
18.3 (CH3)
3
300
4
400
5
500
6
600
7
OMe
1000
8
b
-D
-cym 102.8 (CH)
b-
D
-cym 102.7 (CH)
b-D-ole 101.6 (CH)
9
45.1 (CH)
38.1 (C)
45.0 (CH)
38.1 (C)
45.2 (CH)
38.1 (C)
2000
37.4 (CH2)
81.6 (CH)
77.0 (CH)
73.3 (CH)
18.4 (CH3)
57.4 (CH3)
37.3 (CH2)
80.1 (CH)
83.2 (CH)
72.8 (CH)
18.7 (CH3)
57.0 (CH3)
36.7 (CH2)
80.9 (CH)
83.6 (CH)
71.7 (CH)
18.6 (CH3)
58.6 (CH3)
10
11
12
13
14
15
16
17
18
19
20
21
10
3000
25.5 (CH2)
74.1 (CH)
59.1 (C)
25.5 (CH2)
74.1 (CH)
59.1 (C)
25.4 (CH2)
73.3 (CH)
58.8 (C)
4000
5000
6000
90.0 (C)
90.0 (C)
89.9 (C)
OMe
10000
20000
30000
40000
50000
60000
OMe
100000
200000
300000
400000
500000
600000
1000000
2000000
3000000
4000000
5000000
6000000
34.3 (CH2)
33.5 (CH2)
93.1 (C)
34.3 (CH2)
33.5 (CH2)
93.1 (C)
34.2 (CH2)
33.3 (CH2)
93.0 (C)
a-
L
-cym 98.5 (CH)
b-D-cym 100.6 (CH)
32.4 (CH2)
74.2 (CH)
79.3 (CH)
65.1 (CH)
18.1 (CH3)
56.9 (CH3)
36.3 (CH2)
80.9 (CH)
83.3
10.6 (CH3)
18.5 (CH3)
212.1 (C)
27.9 (CH3)
166.8 (C)
120.4 (C)
132.8 (CH)
116.2 (CH)
163.6 (C)
116.2 (CH)
132.8 (CH)
10.6 (CH3)
18.6 (CH3)
212.1 (C)
27.8 (CH3)
166.7 (C)
122.3 (C)
132.8 (CH)
116.1 (CH)
163.5 (C)
116.1 (CH)
132.8 (CH)
10.4 (CH3)
18.2 (CH3)
211.6 (C)
27.6 (CH3)
167.5 (C)
114.3 (CH)
167.4 (C)
39.4 (CH)
21.4 (CH3)
21.3 (CH3)
16.7 (CH3)
72.5 (CH)
18.5 (CH3)
57.7 (CH3)
b-
D
-glc 101.5 (CH)
b-D-glc 104.6 (CH)
20
75.0 (CH)
76.6 (CH)
80.9 (CH)
76.2 (CH)
62.0 (CH2)
75.3 (CH)
70.73 (CH)
81.5 (CH)
77.1 (CH)
62.8 (CH2)
30
40
50
60
70
b-
D
-glc 104.6 (CH)
b-D-glc 106.1 (CH)
74.9 (CH)
78.1 (CH)
71.3 (CH)
77.8 (CH)
62.4 CH2)
74.9 (CH)
78.4 (CH)
71.4 (CH)
77.6 (CH)
62.5 (CH2)
cymarose as sugar residues. The 13C NMR shifts of each sugar unit
were assigned unambiguously by HSQC, HMBC and HSQC-TOCSY
Based on the HRESI-MS data (m/z 1255.5938 [M
(C61H92O27)ꢀH]ꢀ, calculated: 1255.5640) and 13C NMR (DEPT) data
(Table 1), compound 2 was shown to have a molecular formula
analyses (Table 1). The existence of one
D-oleandropyranosyl and
one -cymaropyranosyl units were confirmed by their comparison
D
C61H92O27. Acidic hydrolysis 2 afforded 1a as the aglycone and
with the spectroscopic data in the literatures (Mu et al., 1986;
Yoshikawa et al., 1996, 1999). Compared with spectral data of
qingyangshengenin (Ma et al., 2007), the glycosylation shifts
effects of C-2 (ꢀ0.2 ppm), C-3 (+7.9 ppm), and C-4 (ꢀ1.7 ppm)
showed the linkage position of the sugar moiety was at the C-3
hydroxyl group of the aglycone, confirmed by the HMBC
b-D-cymaropyranosyl)
to dC 79.3 (C-3). The sequence of the two sugar units was
demonstrated by HMBC spectrum, in which distinct correlations
glucose, cymarose as well as oleandropyranose as sugar residues.
The 13C NMR shifts of each sugar unit were assigned unambiguously
by HSQC, HMBC and HSQC-TOCSY analyses (Table 1). The existence
of two
D-glucopyranosyl, one
L-cymaropyranosyl, one D-oleandro-
pyranosyl and one
D-cymaropyranosyl units were confirmed by
their comparison with the spectroscopic data in the literatures (Ma
et al., 2007; Gu et al., 2009; Xiang et al., 2009). In the HMBC
experiment, the linkage of the sugar moiety to C-3 of the aglycone
was proved by the correlation observed from dH 4.89 (H-100 of
correlation from dH 4.84 (H-100 of terminal
from
(C-400 of inner
3.28 (H-400 of inner
oleandropyranosyl) were observed, respectively. Thus, the struc-
ture of compound 1 was established as qingyangshengenin-3-O-
-cymaropyranoside, and named
d
H 4.61 (H-1000 of terminal
b
-
D
-oleandropyranosyl) to
-cymaropyranosyl) and correlation from H dH
-cymaropyranosyl) to
C 102.8 (C-1000 of
d
C 83.9
terminal
the five sugar units was elucidated by significant correlations
observed from -oleandropyranosyl) to
H 4.61 (H-1000 of terminal
C 83.8 (C-400 of inner H 3.23 (H-4000 of
-cymaropyranosyl), from
inner -cymaropyr-
-oleandropyranosyl) to dC 98.5 (C-1000 of
anosyl), from
H 4.88 (H-10000 of terminal
83.2 (C-4000 of inner
-oleandropyranosyl), from
b-D-cymaropyranosyl)todC 79.3 (C-3), and the sequence of
b
-D
b
-D
d
b
-D
-
d
b-D
d
b-D
d
b
-
b-D
a-L
D
-oleandropyranosyl- (1 ! 4)-
b
-
D
d
a-L-cymaropyranosyl) to dC
cynanauriculoside C.
b-D
d
H 4.43 (H-100000 of
Table 2
Effect of compounds 1–5 on the forced swimming test and tail suspension test in mice (means ꢁ S.E.M.).
n
Forced swimming test
Immobility duration (s)
Tail suspension test
Reduction (%)
Immobility duration (s)
Reduction (%)
Control
8
8
8
8
8
8
8
89.7 ꢁ 35.5
31.3 ꢁ 11.5**
52.5 ꢁ 13.5**
42.7 ꢁ 15.2**
55.3 ꢁ 12.6**
62.5 ꢁ 16.5*
69.3 ꢁ 15.1*
112.7 ꢁ 21.5
46.1 ꢁ 18.5**
73.2 ꢁ 28.1**
55.5 ꢁ 21.9**
91.3 ꢁ 27.6*
86.1 ꢁ 25.7*
75.3 ꢁ 24.5**
Fluoxetine
65.1
41.5
52.4
38.4
30.3
22.7
59.1
35.0
50.8
19.0
23.6
33.2
1
2
3
4
5
*
p< 0.05
**
p < 0.01.

Q.-X. Yang et al. / Phytochemistry Letters 4 (2011) 170–175
173
terminal
cymaropyranosyl), and from dH 4.41 (H-1000000 of terminal
glucopyranosyl) to dC 80.9 (C-400000 of inner
Thus, the structure of 2 was established as qingyangshengenin 3-
b
-
D
-glucopyranosyl) to dC 79.3 (C-40000 of inner
a
-
L
-
noted and recorded on Bruker DRX-400 at 400 MHz (1H) and
100 MHz (13C), with TMS as internal standard. HRESI-MS data
were detected on a Bruker Daltonics micrO-TOF-II MS system.
Silica gel HF254 prepared for TLC and silica gel (200–300 mesh)
for column chromatography (CC) were obtained from Qingdao
Marine Chemical Company, Qingdao, China. Reversed phase
silica gel Rp-18 and Rp-8 for CC were purchased from Merck & Co.
b-D
-
b-D-glucopyranosyl).
O-
cymaropyranosyl-(1 ! 4)-
cymaropyranoside, and named cynanauriculoside D.
b-D
-glucopyranosyl-(1 ! 4)-
b
-
D
-glucopyranosyl-(1 ! 4)-
a
-L-
-
b-D
-oleandropyranosyl-(1 ! 4)-
b-D
The negative HRESI-MS of 3 showed the quasi-molecular ion
peak at m/z 1231.6278 ([MꢀH]ꢀ, calculated: 1231.6008), in
agreement with the molecular formula C60H96O26, which was
supported by the 13C NMR and DEPT experiments (Table 1). IR
Inc. L-Glucose, L-glucose, L-cysteine methyl ester hydrochloride,
and 1-(trimethylsilyl)imidazole were purchased from Sigma
(USA), Supelco (USA), Aldrich (USA), and Fluka (Switzerland),
respectively.
spectrum showed the absorption bands for hydroxyl (3445 cmꢀ1
and carbonyl (1713 cmꢀ1
groups as well as C55C band
)
)
3.2. Plant material
(1644 cmꢀ1). Acidic hydrolysis of 3 afforded a sugar mixture of
cymarose, oleandrose, digitoxose and glucose, and the aglycone,
which was identical to caudatin (3a), by co-TLC comparison with
authentic samples. Inspection of the NMR spectral data of
compound 3 (Table 1) showed that besides the signals arising
from the aglycone (1a), it contained five anomeric carbons at dC
97.2 (digit C-1), 100.6 (cym C-1), 101.6 (ole C-1),104.6 (glu C-1),
and 106.1 (glu C-1), corresponding to five anomeric proton signals
at dH 4.88 (d, J = 9.6, 1.6 Hz), 4.79 (d, J = 8.4 Hz), 4.62 (dd, J = 9.6,
1.6 Hz), 4.43 (d, J = 8.8 Hz), and 4.39 (d, J = 7.6 Hz), respectively.
The 13C signals of each sugar unit (Table 1) were assigned by HSQC
C. auriculatum was collected in Duyun, Guizhou province, China,
in September 2007, and identified by Qing-Fu Chen (School of Life
Sciences, Guizhou Normal University, Guiyang, China). A voucher
specimen (No. 0709-08) is deposited at Herbarium of School of Life
Sciences, Guizhou Normal University, Guiyang, China.
3.3. Extraction and isolation
The air-dried roots of C. auriculatum (4.5 kg) were extracted
with 90% EtOH at room temperature for three times (72, 72, and
48 h, 10 L ꢂ 3). After removal of the organic solvent in vacuo, the
residue was suspended in water (2 L) and partitioned with CHCl3
for three times (1.5 L ꢂ 3) to give a CHCl3 extract (125.0 g), part of
which (80.0 g) was subjected to a silica gel CC (11 cm ꢂ 120 cm)
and eluted with CHCl3-MeOH (10:1.5) to give five fractions (Frs. 1-
5). (The Fr. 5 was found to be the most active fraction in our
previous screening for antidepressant activity). Fr. 5 (6.2 g) was
repeatedly subjected to silica gel [CHCl3–MeOH (10:1) and EtOAc–
EtOH–H2O (10:1:0.5)], and gave two fractions (Frs. 5–1 to 5–3). Fr.
5–1 (200 mg) was subjected to repeatedly Rp-18 (MeOH–H2O,
60% ! 100%) to afford 1 (80 mg). Fr 5–2 (2.1 g) was subjected
repeatedly to silica gel (CHCl3–MeOH, 9:1) and Rp-18 (MeOH–H2O,
60% ! 100%) CC to yield 3 (120 mg) and 4 (70 mg), Fr 5–3 (1.8 g)
was subjected repeatedly to silica gel (CHCl3–MeOH, 7:1) and Rp-
18 (MeOH–H2O, 60% ! 90%) CC to yield 2 (150 mg) and 5
(100 mg).
and HMBC analyses, suggesting the existence of one
D-digitoxose,
one -cymaropyranosyl, one -oleandropyranosyl, and two D-
D
D
glucopyranosyl units compared with the spectroscopic data in the
literatures (Li et al., 2006; Bai et al., 2005, 2008; Warashina and
Noro, 1997; Xiang et al., 2009). Compared to the 13C chemical shifts
of 3a (Ma et al., 2007), glycosylation shifts were observed at the C-3
(+8.0 ppm), and C-4 (ꢀ1.7 ppm) positions, thus proving the
attachment of the sugar chain at the C-3 hydroxyl group of the
aglycone in 3. The sugar sequence of 3 was demonstrated by HMBC
correlations from dH 4.39 (H-1000000of terminal
to -glucopyranosyl); from
C 81.5 (C-400000 of inner
of outer
-glucopyranosyl) to dC 83.3 (C-40000 of inner
cymaropyranosyl); from dH 4.79 (H-10000 of outer
-cymaropyr-
anosyl) to -oleandropyranosyl); from dH
C 83.6 (C-4000 of inner
4.62 (H-1000 of C 83.9 (C-400 of inner
-oleandropyranosyl) to
digitoxoyranosyl); from dH 4.88 (H-100 of inner
-digitoxopyr-
anosyl) to dC 79.3 (C-3). Consequently, the structure of 3 was
deduced to be caudatin 3-O-
-glucopyranosyl-(1 ! 4)-
-cymaropyranosyl-(1 ! 4)-
-digitoxopyranoside, and named
b-
D
-glucopyranosyl)
d
d
b
-D
H 4.43 (H-100000
b
-D
b-D-
b
-D
d
b-D
b
-
D
d
b-D-
b
-D
b
-
D
b
-D
-
3.4. Cynanauriculoside C (1)
glucopyranosyl-(1 ! 4)-
b
-
D
b-D-
oleandropyranosyl-(1 ! 4)-
b
-D
White amorphous powder, mp 215–218 8C,
½
a 2D6
ꢃ
þ 2:8ꢄ
cynanauriculoside E.
(c = 0.60, MeOH), IR (KBr) nmax 3443, 2938, 2934, 1710, 1612,
1591, 1512, 1383, 1317, 1254, 1066, 911, 860, 781 cmꢀ1. HRESI-
MS: m/z 787.3978 [M (C42H60O14)ꢀH]ꢀ (calculated: 787.3849). 1H
and 13C NMR: see Table 1 and Supplementary Material II.
Compounds (1–5) were evaluated for their antidepressant
activities on a forced swimming test and a tail suspension test in
mice (Porsolt et al., 1977; Steru et al., 1985). Treating compounds
(1–5) in mice by gavage for five consecutive days respectively, the
immobility duration of forced swimming test and tail suspension
test was significantly reduced (Table 2), while the open field test
showed that compounds (1–5) did not affect the spontaneous
activity of mice (Supplementary Material III). These results
confirmed the antidepressant activity of the compounds (1–5).
The most potent one was compound 2, which show a reduction
degree close to the positive control fluoxetine.
3.5. Cynanauriculoside D (2)
White amorphous powder, mp 203–206 8C,
½
a 2D6
ꢃ
þ 5:6ꢄ
(c = 0.72, MeOH), IR (KBr) nmax 3441, 2936, 1710, 1621, 1503,
1385, 1312, 1162, 1069, 910, 856, 772 cmꢀ1. HRESI-MS: m/z
1255.5938 [M (C61H92O27)ꢀH]ꢀ (calculated: 1255.5640). 1H and
13C NMR: see Table 1 and Supplementary Material II.
3. Experimental
3.6. Cynanauriculoside E (3)
3.1. General experimental procedures
White amorphous powder, mp 187–190 8C,
½
a 2D6
ꢃ
þ 2:8ꢄ
(c = 0.55, MeOH), IR (KBr) nmax 3445, 2971, 2933, 1713, 1644,
Melting points were measured on an XRC-I micromelting
point apparatus and were uncorrected. Optical rotations were
obtained on a SEPA-300 automatic digital polarimeter. IR spectra
were determined on a Bruker Tensor 27 spectrometer in KBr
pellets. NMR spectra were performed in CD3OD unless otherwise
1451, 1382, 1369, 1317, 1224, 1162, 1066, 1003, 914, 865 cmꢀ1
.
HRESI-MS: m/z 1277.6353 [M (C60H96O26) + HCOOH–H]ꢀ,
1231.6278 [M (C60H96O26)ꢀH]ꢀ (calculated: 1231.6008);
917.4585 [M (C60H96O26)–Glc–GlcꢀH]ꢀ. 1H and 13C NMR: see
Table 1 and Appendix B Supplementary Material II.

174
Q.-X. Yang et al. / Phytochemistry Letters 4 (2011) 170–175
3.7. Acid hydrolysis of 1–3
significant difference tests were conducted to identify differences
among means.
A solution of 1 and 2 (each 5 mg) in MeOH (5 mL) was treated
separately with 5% HCl (5 mL) at 50 8C for 15 min. After added H2O
(5 mL), the reaction mixture was evaporated to 10 mL under
vacuum for removing of MeOH, and then kept in 60 8C for another
15 min. The hydrolyzed mixture was neutralized to pH 7 with
NaOH (4 mol/L) and condensed to dryness under reduced pressure.
The residue was dissolved in MeOH, and compared by TLC analysis
with authentic samples of qingyanshengenin (1a) and caudatin
(3a) which thus revealed to be the aglycones of compounds 1–3,
respectively. The presence of the monosaccharides in the hydro-
lysates of each compound was confirmed by TLC comparison with
authentic sugars, TLC Solvent system A: CHCl3–MeOH (10:1) and
Solvent system B: n-hexane–Me2CO (1:1) were used for qingyang-
shengenin, caudatin, digitoxose, oleandrose, and cymarose, while
solvent system C: CHCl3–MeOH–H2O (7:3:0.5) was used for
glucose (Ma et al., 2007). Cymarose was detected from 1–3,
digitoxose was detected from 3; oleandrose was detected from 1–
3; glucose was detected from 2 and 3.
Conflict of interest
The authors report no conflicts of interest.
Acknowledgments
This research work was supported by the NSFC (30760032),
Foundation of Science and Technology Department of Guizhou
Province ([2007] No.2145), Special Foundation for Traditional
Chinese Medicine Modernization of Guizhou Province ([2008]
No.5022), Governor Foundation of Guizhou Province ([2007]
No.34) and National Basic Research Program of China ‘‘973
Program’’ (No2009CB526512). And the authors are grateful to
Professor Chong-Ren Yang (Kunming Institute of Botany, Chinese
Academy of Sciences, Kunming, China) for supplying the 2-deoxy
sugar samples.
Appendix A. Supplementary data
3.8. Determination of absolute configuration of glucose moieties
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytol.2011.02.009.
The procedure was conducted as described (Ma et al., 2007),
and the absolute configurations of the glucoses were confirmed to
be
D-series by comparison of the retention times of glucose
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