New pregnane glycosides from the roots of Cynanchum otophyllum

Article abstract of DOI:10.1016/j.steroids.2007.06.001

Six new pregnane glycosides with an acyl at C-12 and a straight sugar chain at C-3, namely otophyllosides H-M (1-6), were isolated from the roots of Cynanchum otophyllum (Asclepiadaceae) collected from Eryuan County in Yunnan province of China. Their stru

Full text of DOI:10.1016/j.steroids.2007.06.001

steroids 7 2 ( 2 0 0 7 ) 778–786
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
New pregnane glycosides from the
roots of Cynanchum otophyllum
a,b
c
a
Xiao-Xia Ma , Fang-Ting Jiang , Qing-Xiong Yang ,
c
a,∗
a,∗∗
Xiu-Hua Liu , Ying-Jun Zhang , Chong-Ren Yang
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650204, PR China
Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China
b
c
College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Six new pregnane glycosides with an acyl at C-12 and a straight sugar chain at C-3,
namely otophyllosides H–M (1–6), were isolated from the roots of Cynanchum otophyllum
(Asclepiadaceae) collected from Eryuan County in Yunnan province of China. Their struc-
tures were characterized to be qingyangshengenin 3-O-␤-d-glucopyranosyl-(1 → 4)-␤-d-
glucopyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-␤-d-oleandropyranosyl-(1 → 4)-␤-d-
Received 23 January 2007
Received in revised form 5 June 2007
Accepted 28 June 2007
Published on line 4 July 2007
digitoxopyranoside
(1),
qingyangshengenin
3-O-␤-d-glucopyranosyl-(1 → 4)-␤-d-
oleandropyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-␤-d-digitoxopyranoside (2), qingy-
angshengenin 3-O-␤-d-glucopyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-␤-d-oleandro-
pyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-␤-d-digitoxopyranoside (3), qingyangshen-
genin 3-O-␤-d-glucopyranosyl-(1 → 4)-␤-d-thevetopyranosyl-(1 → 4)-␤-d-cymaropyranosyl-
(1 → 4)-␤-d-digitoxopyranoside (4), caudatin 3-O-␤-d-glucopyranosyl-(1 → 4)-␤-d-gluco-
pyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-␤-d-oleandropyranosyl-(1 → 4)-␤-d-cyma-
ropyranoside (5), caudatin 3-O-␤-d-glucopyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-␤-
d-oleandropyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-␤-d-cymaropyranoside (6), res-
pectively, on the basis of detailed spectroscopic analysis and chemical method.
Keywords:
Cynanchum otophyllum
Asclepiadaceae
Pregnane glycosides
Otophyllosides H–M
©
2007 Elsevier Inc. All rights reserved.
1
.
Introduction
were determined as effective ingredients and otophyllo-
sides A–G as main constituents were previously reported
[2,8,9]. In a continuation of our phytochemical investiga-
tion of traditional Chinese medicinal plants to search for
novel biologically active compounds, six new pregnane gly-
cosides were isolated from the roots of this plant. Their
structures were determined by detailed spectroscopic anal-
ysis, including 2D NMR techniques, and acidic hydrolysis.
This paper describes the structure elucidation of these new
compounds.
Cynanchum otophyllum Schneid (Chinese name Qingyangshen)
is a folk medicinal plant endemic to Yunnan province of
China, whose root was used for the treatment of epilepsy,
rheumatic pain, kidney weakness, and muscle injures by
the local people of its growing area. Based on the chemi-
cal and pharmacological experiments, it has been developed
as an anti-epilepsy remedy and put into industrial produc-
tion for many years in China [1–7]. The pregnane glycosides
∗
Corresponding author. Tel.: +86 871 5223235; fax: +86 871 5150124.
∗
∗
Corresponding author.
E-mail addresses: zhangyj@mail.kib.ac.cn (Y.-J. Zhang), cryang@mail.kib.ac.cn (C.-R. Yang).
039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2007.06.001
0

steroids 7 2 ( 2 0 0 7 ) 778–786
779
Table 1 – 13C NMR data for the aglycone moieties of 1–6a
ı in ppm, in CD3OD, 100 MHz)
2
.
Experimental
(
Position
1
2
3
4a
5
6a
2
.1.
General methods
1
2
3
4
5
6
7
8
9
39.8
30.1
79.3
39.8
30.1
79.3
39.8
30.1
79.3
39.2
29.9
77.6
39.8
30.2
79.3
39.8
140.3
119.6
35.2
75.0
45.2
38.1
25.4
73.3
58.7
89.9
34.2
33.2
93.0
10.4
18.5
211.5
27.5
167.4
114.3
167.4
39.3
21.3
21.2
16.7
39.3
29.9
77.7
39.0
139.4
119.2
34.8
74.4
44.6
37.5
25.1
72.6
57.5
89.5
33.9
33.0
92.4
10.7
18.2
209.5
27.6
166.0
114.2
165.5
38.2
20.9
21.0
16.5
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 spec-
tra were determined on a Bruker Tensor 27 spectrometer in
KBr pellets. NMR spectra were performed in CD3OD unless
otherwise noted and recorded on Bruker DRX-400 and -500
instruments with TMS as internal standard. MS data were
detected on a VG Auto Spec-3000 spectrometer. 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. Inc.
l-Glucose, d-glucose, l-cysteine methyl ester hydrochloride,
and 1-(trimethylsilyl)imidazole were purchased from Sigma
39.8
39.8
39.8
39.1
140.3
119.4
35.2
75.0
45.1
38.1
25.5
73.7
59.1
90.0
34.3
33.4
93.1
10.6
18.6
211.9
27.7
167.3
119.6
132.8
117.6
168.3
117.6
132.8
140.3
119.6
35.2
75.0
45.1
38.1
25.5
73.5
59.1
90.0
34.3
33.4
93.0
10.6
18.6
211.8
27.7
167.5
118.3
132.8
118.1
169.9
118.1
132.8
140.3
119.6
35.2
75.0
45.1
38.1
25.5
74.0
59.1
90.0
34.3
33.5
93.1
10.6
18.5
212.0
27.8
166.9
121.5
132.8
116.6
164.9
116.6
132.8
139.4
119.2
34.8
74.3
44.5
37.4
25.2
73.4
58.4
89.6
33.9
33.2
92.5
10.9
18.2
209.9
27.9
165.4
122.0
132.5
116.2
163.6
116.2
132.5
10
1
1
1
1
1
1
2
3
4
5
16
17
1
1
2
2
8
9
0
1
1
2
3
4
5
6
7
(
USA), Supelco (USA), Aldrich (USA), and Fluka (Switzerland),
respectively.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2
.2.
Plant material
The roots of C. otophyllum Schneid were collected at Eryuan
County, in the northwest of Yunnan province, China, and iden-
tified by Prof. C.R. Yang (Kunming Institute of Botany, Chinese
Academy of Sciences). A voucher specimen is deposited in the
herbarium of Kunming Institute of Botany, Chinese Academy
of Sciences.
a
The spectral data were obtained in C5D5N.
2
.3.
Extraction and isolation
2
.3.1. Otophylloside H (1)
◦
25
◦
The air-dried roots of C. otophyllum (10 kg) were extracted
with 90% EtOH at room temperature for three times (72,
White amorphous powder, mp 213–216 C, [˛]D + 8.0 (c = 0.83,
MeOH), IR (KBr) ꢀmax 3442, 2934, 1709, 1610, 1591, 1504,
1452, 1384, 1309, 1276, 1163, 1072, 911, 854, 773 cm 1. FAB-
−
7
2, and 48 h, 15 L × 3). After removal of the organic sol-
−
−
vent in vacuo, the residue was suspended in water (2 L)
and partitioned with CHCl3 for three times (1.5 L × 3) to
give a CHCl3 extract (155 g), part of which (150 g) was sub-
jected to a silica gel CC (11 cm × 120 cm) and eluted with
CHCl3–MeOH (10:1.5, 7.6 L) to give five fractions (Frs. 1–5).
Fr. 3 (16.1 g) was repeatedly chromatographed over silica gel
MS (negative ion mode): m/z 1242 [M] , 1105 [M − 137] ,
−
943 [M − 137 − 162] . HRFAB-MS (negative ion mode): m/z
−
1
13
1241.5628 [M(C
6
0
90 27
H O )–H] (calcd. 1241.5591). H and
C
NMR: see Tables 1–3 .
2.3.2. Otophylloside I (2)
[
CHCl3–MeOH (10:0.8) and EtOAc–EtOH–H2O (12:1:0.5)] and Rp-
◦
27
◦
White amorphous powder, mp 201–203 C, [˛]D + 1.5 (c = 0.55,
MeOH), IR (KBr) ꢀmax 3447, 2934, 1708, 1609, 1590, 1504,
8
4
(MeOH–H2O, 68% → 85%) to afford 1 (370 mg) and 4 (46 mg). Fr.
(6.4 g) was chromatographed on silica gel [CHCl3–MeOH (9:1)
1
7
452, 1383, 1369, 1309, 1276, 1163, 1093, 1004, 912, 854,
and EtOAc–EtOH–H2O (8:1:0.5)], Rp-8 and Rp-18 (MeOH–H2O,
−
1
−
73 cm . FAB-MS (negative ion mode): m/z 1080 [M] , 942
6
5% → 100%) columns to give 2 (87 mg), 3 (92 mg), and 5
−
[
[
M − 1 − 137] , HRFAB-MS (negative ion mode): m/z 1079.5030
(
(
59 mg). Fr. 5 (20.0 g) was subjected repeatedly to silica gel
CHCl3–MeOH, 9:1.5) and Rp-18 (MeOH–H2O, 60% → 100%) CC
−
1
13
M(C54H80O22)–H] (calcd. 1079.5063). H and C NMR: see
Tables 1–3.
to yield 6 (210 mg).
Another part of the CHCl3 extract (5 g) was dissolved in
◦
MeOH (50 ml) and treated with 5% HCl (10 ml) at 90 C for
2.3.3. Otophylloside J (3)
◦
27
◦
3
0 min. After cooled down to room temperature and neutral-
White amorphous powder, mp 179–182 C, [˛] + 12.1
D
ized with NaOH (4 mol/L) to pH 6–7, the reaction mixture was
evaporated under reduced pressure to remove of MeOH. The
condensation was diluted with water and extracted by CHCl3
for three times (50 ml × 3). The CHCl3 phase (1.8 g) was chro-
matographed repeatedly on silica gel column to give 7 (76 mg)
and 8 (93 mg).
(c = 0.46, MeOH), IR (KBr) ꢀmax 3449, 2934, 1711, 1610, 1594,
1515, 1452, 1382, 1276, 1165, 1094, 1005, 912, 866, 853,
772 cm . ESI-MS (negative ion mode): m/z 1224 [M] , 1062
−
1
−
−
[M − 162] . HRESI-MS (negative ion mode): m/z 1223.5840
−
1
13
[M(C61H92O25)–H] (calcd. 1223.5849). H and C NMR: see
Tables 1–3.

7
80
steroids 7 2 ( 2 0 0 7 ) 778–786
Table 2 – 13C NMR data for the sugar moieties of 1–6a (ı in ppm, in CD3OD, 100 MHz)
Position
1
2
3
4a
5
6a
␤
-d-digit
␤-d-digit
␤-d-digit
␤-d-digit
␤-d-cym
␤-d-cym
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1
2
3
4
5
6
97.0
38.8
68.3
83.7
69.5
18.5
97.0
38.8
68.3
83.6
69.4
18.5
97.0
38.8
68.3
83.6
69.5
18.5
96.4
38.9
67.6
83.2
69.4
18.5
97.2
36.7
78.4
83.9
69.8
18.6
58.4
96.5
37.3
78.1
83.4
69.1
18.7
58.7
OMe
Position
1
2
3
4a
5
6a
␤
-d-ole
␤-d-cym
␤-d-cym
␤-d-cym
␤-d-ole
␤-d-cym
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
1
2
3
4
5
6
102.6
37.7
80.1
83.6
72.6
18.8
57.9
100.7
36.3
78.4
83.7
69.9
18.4
58.6
100.6
36.3
78.4
83.8
70.0
18.6
58.6
99.8
36.7
78.2
83.4
68.6
18.7
58.9
102.6
37.8
80.2
83.8
72.7
18.8
57.6
100.5
37.1
77.8
83.2
69.0
18.6
59.0
OMe
Position
1
2
3
4a
5
6a
␤
-d-cym
␤-d-ole
␤-d-ole
␤-d-the
␤-d-cym
␤-d-ole
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
1
2
3
4
5
6
100.6
36.3
78.4
83.6
70.0
18.6
58.6
102.6
37.9
80.1
83.5
72.7
18.8
58.2
102.7
37.7
80.0
84.0
72.4
18.8
57.7
106.0
74.7
85.9
83.0
72.0
18.7
60.6
101.2
36.4
78.5
83.8
70.0
18.6
58.6
102.0
37.8
78.8
82.7
71.8
18.7
58.0
OMe
Position
1
2
3
4a
5
6a
␤
-d-glc
␤-d-glc
␤-d-cym
␤-d-glc
␤-d-glc
␤-d-cym
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
1
2
3
4
5
6
104.0
75.3
76.7
81.0
76.4
62.1
104.1
75.5
78.2
71.7
78.0
63.0
99.6
36.5
78.8
83.8
70.4
18.6
58.5
104.9
75.9
78.7
72.0
78.0
63.1
104.1
75.3
76.7
81.1
76.5
62.2
98.4
36.8
78.2
83.2
69.7
18.7
58.9
OMe
Position
1
3
5
6a
␤
-d-glc
␤-d-glc
␤-d-glc
␤-d-glc
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
1
2
3
4
5
6
104.6
74.9
78.1
71.4
77.8
62.4
106.2
75.3
78.0
71.8
77.9
63.0
104.6
74.9
78.1
71.4
77.9
62.5
106.6
75.4
78.5
71.9
78.4
63.1
digit: digitoxopyranosyl; cym: cymaropyranosyl; ole: oleandropyranosyl; the: thevetopyranosyl; glc: glucopyranosyl.
a
The spectral data were obtained in C5D5N.
2
.3.4. Otophylloside K (4)
2.3.5. Otophylloside L (5)
◦
28
D
◦
◦
29
◦
White amorphous powder, mp 169–172 C, [˛] + 4.0
White amorphous powder, mp 191–193 C, [˛]D + 1.3 (c = 0.74,
(c = 0.21, MeOH), IR (KBr) ꢀmax 3442, 2934, 1710, 1610,
MeOH), IR (KBr) ꢀmax 3443, 2968, 2934, 1713, 1642, 1452,
1
7
516, 1453, 1383, 1369, 1277, 1165, 1076, 911, 865, 853,
72 cm . FAB-MS (negative ion mode): m/z 1096 [M] , 933
1383, 1369, 1317, 1224, 1164, 1060, 1004, 912, 864 cm 1. FAB-
−
−
1
−
−
−
MS (negative ion mode): m/z 1246 [M] , 1118 [M − 128] ,
−
−
[
M − 1 − 162] . HRFAB-MS (negative ion mode): m/z 1095.5004
956 [M − 128 − 162] . HRFAB-MS (negative ion mode): m/z
−
1
13
−
1
13
[M(C54H80O23)–H] (calcd. 1095.5012). H and C NMR: see
1245.6261 [M(C61H98O26)–H] (calcd. 1245.6268). H and
C
Tables 1–3.
NMR: see Tables 1–3.

steroids 7 2 ( 2 0 0 7 ) 778–786
781
Table 3 – 1H NMR spectral data of compounds 1–6a (ı in ppm, J in Hz, in CD3OD, 500 MHz)
Position
1
2
3
4a
5
6a
3
6
3.54 m
3.57 m
3.57 m
3.86 m
3.57 m
3.83 m
5.28 brs
5.04b
1.97 s
1.31 s
2.50 s
5.85 s
5.38 d, 4.7
4.72 dd, 11.6, 4.1
1.63 s
1.14 s
2.08 s
5.38 d, 4.8
4.74 dd, 11.6, 4.1
1.66 s
1.18 s
2.11 s
5.38 d, 4.4
4.78 dd, 11.6, 4.1
1.67 s
1.18 s
2.10 s
5.25 d, 3.2
5.33 dd, 11.4, 4.1
2.09 s
1.29 s
2.41 s
5.34 d, 4.1
4.48 dd, 11.6, 4.1
1.50 s
1.14 s
2.17 s
1
1
1
2
2
3
4
5
6
7
2
8
9
1
ꢀ
5.54 s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
7.73 d, 8.8
6.71 d, 8.8
7.73 d, 8.8
6.69 d, 8.8
7.81 d, 8.8
6.82 d, 8.8
8.30 d, 8.6
7.23 d, 8.6
2.40 m
2.44 m
1.07 d, 6.8
1.07 d, 6.8
2.11 s
0.94 d, 7.3
0.95 d, 7.4
2.26 s
6.71 d, 8.8
7.73 d, 8.8
6.69 d, 8.8
7.73 d, 8.8
6.82 d, 8.8
7.81 d, 8.8
7.23 d, 8.6
8.30 d, 8.6
Position
1
2
3
4a
5
6a
␤
-D-digit
␤-D-digit
␤-D-digit
␤-D-digit
␤-D-cym
␤-D-cym
ꢀ
ꢀ
ꢀ
ꢀ
1
2
4.94 dd, 9.7, 1.7
1.67 m
4.98 dd, 9.7, 1.7
1.65 m
4.97 dd, 9.7,1.7
1.68 m
5.47 brd, 8.9
1.77 m
4.86 dd, 9.6, 1.6
2.06 m
5.26 brd, 9.4
1.89 m
1
.93 m
1.93 m
4.25 m
3.20 m
3.79 m
1.96 m
4.24 m
3.19 m
3.82 m
2.03 m
4.61 m
3.47 m
4.20 m
2.17 m
3.86 m
3.26 m
3.82 m
1.18 d, 6.3
3.42 s
2.30 m
4.07 m
3.66 dd, 8.7, 2.4
4.21 m
1.38 d, 6.0
3.51 s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3
4
5
6
4.21 m
3.27 m
3.80 m
1.21 d, 5.8
1.23 d, 5.8
1.23 d, 6.3
1.49 d, 6.2
OMe
Position
1
2
3
4a
5
6a
␤
-D-ole
␤-D-cym
␤-D-cym
␤-D-cym
␤-D-ole
␤-D-cym
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
1
2
4.60 dd, 9.6, 1.6
1.41 m
4.86 dd, 9.6, 1.6
1.59 m
4.86 dd, 9.6, 1.6
1.63 m
5.15 brd, 8.7
1.75 m
4.61 dd, 9.6, 1.6
1.94 m
5.10 brd, 9.5
1.77 m
2
.34 m
2.11 m
3.87 m
3.27 m
3.86 m
1.24 d, 5.8
3.46 s
2.16 m
3.87 m
3.27 m
3.86 m
1.23 d, 6.2
3.46 s
2.31 m
3.97 m
3.48 m
4.29 m
1.42 d, 6.2
3.55 s
2.33 m
3.42 m
3.23 m
3.41 m
1.38 d, 6.1
3.45 s
2.28 m
4.00 m
3.50 m
4.16 m
1.37 d, 5.9
3.55 s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
3
4
5
6
3.41 m
3.18 m
3.40 m
1.38 d, 6.2
3.48 s
OMe
Position
1
2
3
4a
5
6a
␤
-D-cym
␤-D-ole
␤-D-ole
␤-D-the
␤-D-cym
␤-D-ole
ꢀ
ꢀ
ꢀꢀꢀ
ꢀꢀꢀ
1
2
4.82 dd, 9.6, 1.6
1.64 m
4.64 dd, 9.6, 1.6
1.44 m
4.60 dd, 9.6, 1.6
1.44 m
4.68 d, 7.7
3.88 m
4.78 dd, 9.6, 1.6
1.58 m
4.67 brd, 9.6
1.81 m
2
.13 m
2.33 m
3.43 m
3.41 m
3.44 m
1.41 d, 6.1
3.50 s
2.43 m
3.38 m
3.11 m
3.32 m
1.30 d, 6.2
3.45 s
2.14 m
3.87 m
3.20 m
3.85 m
1.21 d, 6.3
3.42 s
2.29 m
3.51 m
3.46 m
3.49 m
1.40 d, 5.8
3.50 s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
3
4
5
6
3.84 m
3.24 m
3.85 m
1.20 d, 5.9
3.46 s
3.70 t, 9.0
3.85 m
4.00 m
1.73 d, 6.1
3.93 s
OMe
Position
1
2
3
4a
5
6a
␤
-D-glc
␤-D-glc
␤-D-cym
␤-D-glc
␤-D-glc
␤-D-cym
ꢀ
ꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
1
2
4.48 d, 7.9
3.23 m
4.48 d, 7.8
3.22 m
4.93 dd, 9.8, 1.8
1.60 m
5.13 d, 7.6
4.03 m
4.47 d, 8.0
3.22 m
5.24 brd, 8.2
2.32 m
2
.13 m
2.45 m
4.14 m
3.40 m
4.26 m
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
3
4
5
6
3.29 m
3.52 m
3.51 m
3.26 m
3.25 m
3.37 m
3.67 dd, 5.8, 12.0
3.88 dd, 2.2, 12.0
3.94 m
3.33 m
3.90 m
1.34 d, 6.2
4.23 m
3.74 m
4.00 m
4.35 dd, 5.4, 11.4
4.53 dd, 2.2, 11.4
3.40 m
3.55 m
3.54 m
3.85 m
3.94 dd, 2.2, 12.0
c
c
3.82 m
1.61 d, 6.2
3
.92 dd, 2.2, 12.0
OMe
3.48 s
3.60 s

7
82
steroids 7 2 ( 2 0 0 7 ) 778–786
Table 3 (Continued )
Position
1
2
3
4a
5
6a
␤
-D-glc
␤-D-glc
␤-D-glc
␤-D-glc
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
1
2
3
4
5
6
4.38 d, 7.8
3.21 m
3.34 m
3.28 m
3.32 m
4.36 d, 7.7
3.18 m
3.36 m
3.26 m
3.27 m
3.67 dd, 5.8, 12.0
3.91 mc
4.38 d, 7.8
3.19 m
3.36 m
3.31 m
3.34 m
3.69 dd, 5.8, 12.0
3.90 dd, 2.2, 12.0
4.92 d, 7.7
3.98 m
3.96 m
4.17 m
4.23 m
4.37 dd, 5.3, 11.5
4.57 brd, 9.6
3.64 dd, 5.8, 12.0
3
.87 dd, 2.2, 12.0
digit: digitoxopyranosyl; cym: cymaropyranosyl; ole: oleandropyranosyl; the: thevetopyranosyl; glc: glucopyranosyl.
a
The spectral data were obtained in C5D5N.
Overlapped by H2O signal.
Overlapped by deoxy sugar signals.
b
c
2
.3.6. Otophylloside M (6)
monosaccharides in the hydrolysates of each compound was
confirmed by TLC comparison with authentic sugars, digitox-
ose was detected from 1–4; oleandrose was detected from 1–3,
5 and 6; glucose and cymarose were detected from 1–6. The
Rf values of qingyangshengenin, caudatin, digitoxose, olean-
drose, and cymarose were 0.35, 0.47, 0.16, 0.37, and 0.10 with
solvent CHCl3–MeOH (9:1); 0.10, 0.38, 0.09, 0.19, and 0.02 with
solvent petroleum ether–Me2CO (3:2); 0.23, 0.53, 0.19, 0.30, and
◦
29
◦
White amorphous powder, mp 162–164 C, [˛] + 2.4
D
(c = 0.91, MeOH), IR (KBr) ꢀmax 3456, 2970, 2934, 1713, 1643,
1
8
452, 1382, 1369, 1317, 1280, 1224, 1165, 1088, 1004, 913,
65 cm . FAB-MS (negative ion mode): m/z 1228 [M] , 1099
−
1
−
−
[
M − 1 − 128] . HRFAB-MS (negative ion mode): m/z 1227.6513
−
1
13
[M(C62H100O24)–H] (calcd. 1227.6526). H and C NMR: see
Tables 1–3.
0.05 with solvent n-hexane–Me2CO (1:1), respectively. The Rf
2
.3.7. Qingyangshengenin (7)
C28H36O8, white amorphous powder. 1H NMR (CDCl3,
00 MHz): ı 1.14 (3H, s, H-19), 1.56 (3H, s, H-18), 2.08 (3H, s,
H-21), 3.53 (1H, m, H-3), 4.79 (1H, dd, J = 4.4, 11.4 Hz, H-12), 5.37
values of glucose was 0.30 with solvent CHCl3–MeOH–H2O
(7:3:0.5).
5
2.3.10. Determination of absolute configuration of glucose
ꢀ ꢀ
(
1H, brs, H-6), 6.83 (2H, d, J = 8.7 Hz, H-4 ,6 ), 7.82 (2H, d, J = 8.7 Hz,
moieties
H-3 ,7 ). 13C NMR (CDCl3, 125 MHz): ı 38.5 (C-1), 30.4 (C-2), 71.4
C-3), 41.5 (C-4), 140.0 (C-5), 117.7 (C-6), 34.0 (C-7), 73.9 (C-8),
3.4 (C-9), 36.7 (C-10), 24.1 (C-11), 72.5 (C-12), 58.0 (C-13), 88.2
C-14), 33.0 (C-15), 31.9 (C-16), 91.5 (C-17), 9.5 (C-18), 18.2 (C-19),
ꢀ
ꢀ
Each neutralized hydrolysates of 1–6 were condensed to dry-
ness after TLC analyses and then suspended into water
(
4
(
(
2 ml) and extracted with CHCl3 (2 ml × 3). The aqueous layer
was concentrated to dryness and then dissolved in pyridine
2 ml) to give a monosaccharide mixture, which was added
ꢀ ꢀ ꢀ ꢀ
2
1
10.2 (C-20), 27.2 (C-21), 165.4 (C-1 ), 120.9 (C-2 ), 131.6 (C-3 ,7 ),
15.1 (C-4 ,6 ), 161.5 (C-5 ).
(
ꢀ
ꢀ
ꢀ
to l-cysteine methyl ester hydrochloride (1.5 mg) and kept
◦
at 60 C for 1 h. After 1-(trimethylsilyl)imidazole was added
2
.3.8. Caudatin (8)
to the reaction mixture slowly under ice bath condition,
it was then kept at 60 C for 30 min. The supernatant was
subjected to GC analysis under the following conditions: Shi-
madzu GC-14C gas chromatograph equipped with an H2 flame
ionization detector. Column: 30QC2/AC-5 quartz capillary col-
C28H42O7, white amorphous powder. 1H NMR (CDCl3,
◦
ꢀ
ꢀ
5
1
00 MHz): ı 1.07 (6H, d, J = 6.8 Hz, H-5 , H-6 ), 1.15 (3H, s, H-19),
.47 (3H, s, H-18), 2.13 (3H, s, H-7 ), 2.20 (3H, s, H-21), 3.52 (1H,
ꢀ
m, H-3), 4.54 (1H, dd, J = 4.2, 11.5 Hz, H-12), 5.36 (1H, brs, H-6),
.53 (1H, s, H-2 ). 13C NMR (CDCl3, 125 MHz): ı 38.5 (C-1), 30.3 (C-
), 71.3 (C-3), 41.5 (C-4), 140.1 (C-5), 117.6 (C-6), 33.9 (C-7), 73.9
ꢀ
5
2
◦
◦
umn (30 m × 0.32 mm). Column temperature: 180 C/280 C,
◦
programmed increase: 3 C/min, carrier gas: N2 (1.5 ml/min).
Injector and detector temperature: 250 C, injection volume:
(
8
C-8), 43.5 (C-9), 36.7 (C-10), 24.1 (C-11), 71.4 (C-12), 57.4 (C-13),
8.2 (C-14), 32.8 (C-15), 31.7 (C-16), 91.5 (C-17), 9.4 (C-18), 18.2
◦
4
␮l, split ratio: 1/50. The absolute configurations of the glu-
ꢀ
ꢀ
(
(
C-19), 209.6 (C-20), 27.0 (C-21), 166.7 (C-1 ), 112.7 (C-2 ), 166.0
C-3 ), 38.0 (C-4 ), 20.7 (C-5 ), 20.8 (C-6 ), 16.4 (C-7 ).
coses were confirmed to be d-series by comparison of the
retention times of glucose derivatives with those of standard
samples: d-glucose (23.373 min) and l-glucose (23.832 min).
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2
.3.9. Acid hydrolysis of 1–6
A solution of 1–6 (each 6 mg) in MeOH (5 ml) was treated sep-
◦
arately with 5% HCl (5 ml) at 50 C for 15 min. After added H2O
3.
Results and discussion
(
5 ml), the reaction mixture was evaporated to 10 ml under vac-
◦
uum for removing of MeOH, and then kept in 60 C for another
Six new pregnane glycosides (1–6) were isolated from the
CHCl3 extracts of the roots of C. otophyllum through repeated
column chromatography. All of them showed positive effects
on Libermann–Buchard and Keller–Kiliani reactions, indicat-
ing the presence of a steroidal skeleton with 2-deoxysugar
moiety. Spectroscopic analysis demonstrated that all the gly-
cosides had a pregnane skeleton with an acyl group at C-12
1
5 min. The hydrolyzed mixture was neutralized to pH 7 with
NaOH (4 mol/L) and condensed to dryness under reduced pres-
sure. The residue was dissolved in MeOH, and compared by
TLC analysis with authentic samples of qingyangshengenin
(7) and caudatin (8) which thus revealed to be the aglycones
of compounds 1–4 and 5–6, respectively. The presence of the

steroids 7 2 ( 2 0 0 7 ) 778–786
783
Scheme 1 – Pregnane glycosides (1–6) isolated from Cynanchum otophyllum.
position and a straight sugar chain consisting of four or five
sugar units connected to C-3 group position of the aglycone.
Acidic hydrolysis of the CHCl3 extract furnished two aglycone
compounds which were identified to be qingyangshengenin
cymaropyranosyl unit and >36.0 ppm in ␤-d-cymaropyranosyl
unit [13–15]. Therefore, all of the cymaropyranosyl units were
determined to be d-configurations based on their 13C NMR
chemical shifts (>36.0 ppm). To the best of our knowledge, ␤-
linked digitoxopyranosyl and oleandropyranosyl units were so
far only found d-configurations from the Asclepiadaceae fam-
ily, which were supposed for these sugars in this study as well,
also due to their similar 13C NMR chemical shifts with those
reported in literatures [2,12,13,15–17].
(7) and caudatin (8) by comparison of their spectral data with
those reported in the literatures [1,2,10–12] (Scheme 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 corre-
sponding trimethylsilylated L-cysteine adducts confirmed
d-configuration of glucose. For the deoxysugars, since only
d-form authentic samples could be obtained, their absolute
configurations could not be assigned by GC analysis, but
determined to be d-forms by comparison of their 13C NMR
spectroscopic data with those reported data. According to
the literatures, the chemical shift of C-2 is <35.0 ppm in ␤-l-
3.1.
Otophylloside H (1)
Compound 1 was obtained as a white amorphous powder.
Its molecular formula was assigned to be C60H90O27 on the
−
basis of negative HRFAB-MS (m/z 1241.5628 [M–H] , calcd.
1241.5591) and 13C NMR (DEPT) data (Tables 1 and 2). IR spec-

7
84
steroids 7 2 ( 2 0 0 7 ) 778–786
trum showed the absorption bands for hydroxyl (3442 cm 1),
−
ꢀꢀꢀ
from ıH 4.86 (H-1 of ␤-d-cymaropyranosyl) to ıC 83.6
carbonyl (1709 cm 1) groups and benzene ring (1610 and
−
591 cm 1). The 1H NMR spectrum of 1 revealed the presence
of three singlet methyl groups [ıH 1.14, 1.63, 2.08 (each 3H, s,
Me-19, -18, -21)], one olefinic proton [ıH 5.38 (d, J = 4.7 Hz, H-6)]
and four aromatic protons on a para-substituted benzene
−
ꢀꢀ
ꢀꢀ
(C-4 of ␤-d-digitoxopyranosyl); from ıH 4.98 (H-1 of ␤-d-
digitoxopyranosyl) to ıC 79.3 (C-3). Therefore, compound 2 was
deduced to be qingyangshengenin-3-O-␤-d-glucopyranosyl-
(1 → 4)-␤-d-oleandropyranosyl-(1 → 4)-␤-d-cymaropyranosyl-
(1 → 4)-␤-d-digitoxopyranoside, and named otophylloside I.
1
ꢀ
ꢀ
ring [ıH 6.71 (2H, d, J = 8.8 Hz, H-4 ,6 ) and 7.73 (2H, d, J = 8.8 Hz,
ꢀ
ꢀ
H-3 ,7 )] in its aglycone moiety. Its negative FAB-MS exhibited
fragment ion peaks at m/z 1105 [M − 137]− which can be
ascribed to the loss of a hydroxybenzoyl ester group. The
above observation suggested qingyangshengenin to be the
aglycone of compound 1. Proton signals were also assigned
to three secondary methyl groups [ıH 1.20 (d, J = 5.9 Hz), 1.21
3.3.
Otophylloside J (3)
The molecular formula of 3 was determined as C61H92O25 by
−
its HRESI-MS (m/z 1223.5840 [M–H] , calcd. 1223.5849). The
acidic hydrolysis afforded 7 as the aglycone and the same
sugar compositions as compounds 1 and 2. The 1H NMR
spectrum of 3 showed four secondary methyl signals [ıH 1.23
(d, J = 6.3 Hz), 1.23 (d, J = 6.2 Hz), 1.30 (d, J = 6.2 Hz), and 1.34
(d, J = 6.2 Hz)] and three methoxyl methyl signals [ıH 3.45,
3.46, and 3.48] together with five anomeric proton signals
[ıH 4.36 (d, J = 7.7 Hz), 4.60 (dd, J = 9.6, 1.6 Hz), 4.86 (dd, J = 9.6,
1.6 Hz), 4.93 (dd, J = 9.8, 1.8 Hz), and 4.97 (dd, J = 9.7, 1.7 Hz)].
These observations suggested that four 2,6-dideoxysugar
were part of a pentahexose chain with ␤-linkages in 3. The
13C NMR signals of each sugar unit were assigned exactly
by HMQC and HMBC analyses and indicated the existence
of one ␤-d-digitoxopyranosyl, one ␤-d-oleandropyranosyl,
one ␤-d-glucopyranosyl and two ␤-d-cymaropyranosyl units.
According to the distinct long-range correlations from ıH
(
[
d, J = 5.8 Hz), and 1.38 (d, J = 6.2 Hz)], two methoxyl groups
ıH 3.46 and 3.48] and five anomeric protons [ıH 4.38 (d,
J = 7.8 Hz), 4.48 (d, J = 7.9 Hz), 4.60 (dd, J = 9.6, 1.6 Hz), 4.82 (dd,
J = 9.6, 1.6 Hz), and 4.94 (dd, J = 9.7, 1.7 Hz)] whose multiplici-
ties suggested the presence of three 2,6-dideoxy-sugar in a
pentasaccharides chain and the ␤-configuration of the five
hexose units. Acid hydrolysis of 1 gave qingyangshengenin
(
7) as the aglycone, and glucose, digitoxose, oleandrose, as
well as cymarose as sugar residues. The 13C NMR shifts of
each sugar unit were assigned unambiguously by HMQC,
HMBC and HMQC-TOCSY analyses (Table 2). The existence
of one d-digitoxopyranosyl, one d-oleandropyranosyl, one
d-cymaropyranosyl and two d-glucopyranosyl units were
confirmed by their comparison with the spectroscopic data
in the literatures [2,12–17]. The glycosylation shifts effects of
C-2 (−0.3 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. The sequence of these five
sugar units was demonstrated by HMBC spectrum, in which
ꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
4.36 (H-1
of ␤-d-glucopyranosyl) to ıC 83.8 (C-4
of outer
of outer ␤-d-
cymaropyranosyl) to ıC 84.0 (C-4 of ␤-d-oleandropyranosyl);
ꢀ
ꢀꢀꢀꢀ
␤-d-cymaropyranosyl); from ıH 4.93 (H-1
ꢀ
ꢀꢀꢀ
ꢀ
ꢀꢀꢀ
ꢀꢀꢀ
from ıH 4.60 (H-1 of ␤-d-oleandropyranosyl) to ıC 83.8 (C-4
of inner ␤-d-cymaropyranosyl); from ıH 4.86 (H-1 of inner ␤-
d-cymaropyranosyl) to ıC 83.6 (C-4 of ␤-d-digitoxopyranosyl);
from ıH 4.97 (H-1 of ␤-d-digitoxopyranosyl) to ıC 79.3 (C-3),
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀ
distinct correlations from ıH 4.38 (H-1
of terminal ␤-d-
glucopyranosyl) to ıC 81.0 (C-4 of inner ␤-d-glucopyranosyl);
of inner ␤-d-glucopyranosyl) to ıC 83.6
C-4 of ␤-d-cymaropyranosyl); from ıH 4.82 (H-1 of ␤-d-
cymaropyranosyl) to ıC 83.6 (C-4 of ␤-d-oleandropyranosyl);
from ıH 4.60 (H-1 of ␤-d-oleandropyranosyl) to ıC 83.7
C-4 of ␤-d-digitoxopyranosyl); from ıH 4.94 (H-1 of ␤-d-
ꢀ
ꢀꢀꢀꢀ
the structure of otophylloside J (3) was confirmed to be
qingyangshengenin-3-O-␤-d-glucopyranosyl-(1 → 4)-␤-d-
cymaropyranosyl-(1 → 4)-␤-d-oleandropyranosyl-(1 → 4)-␤-d-
cymaropyranosyl-(1 → 4)-␤-d-digitoxopyranoside.
ꢀ
ꢀꢀꢀꢀ
from ıH 4.48 (H-1
(
ꢀ
ꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
(
3.4.
Otophylloside K (4)
digitoxopyranosyl) to ıC 79.3 (C-3), were observed, respectively.
Thus, the structure of otophylloside H (1) was established
Compound 4 possessed the molecular formula C54H80O23
on the basis of HRFAB-MS (m/z 1095.5004 [M-H] , calcd.
−
as
qingyangshengenin-3-O-␤-d-glucopyranosyl-(1 → 4)-␤-
d-glucopyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-␤-d-
oleandropyranosyl-(1 → 4)-␤-d-digitoxopyranoside.
1095.5012). The combination of acidic hydrolysis, GC analysis
and spectra studies indicated that 4 possessed qingyang-
shengenin (7) as aglycone, and d-digitoxose, d-cymarose,
d-glucose and another deoxysugar as sugar residue. The
undefined deoxysugar showed one secondary methyl [ıH
3
.2.
Otophylloside I (2)
1
.73 (d, J = 6.1 Hz)], one methoxyl (ıH 3.93), one methine [ıH
−
Based on the HRFAB-MS data (m/z 1079.5030 [M–H] , calcd.
079.5063) and 13C NMR (DEPT) spectrum, compound 2
3.70 (t, J = 9.0 Hz)], and an anomeric proton doublet [ıH 4.68
(d, J = 7.7 Hz)] as well as seven carbons ıC 106.0 (d), 74.7 (d),
85.9 (d), 83.0 (d), 72.0 (d), 18.7 (q), and 60.6 (q) in its NMR
spectra. These NMR features were identical to those of ␤-
thevetopyranosyl unit [13,16–17], and its configuration was
supposed to be d-form because no l-thevetopyranose was
found from the Asclepiadaceae family so far. The sequence
of these four sugar units was determined by the important
correlations from ıH 5.13 (H-1
83.0 (C-4 of ␤-d-thevetopyranosyl); from ıH 4.68 (H-1 of ␤-
d-thevetopyranosyl) to ıC 83.4 (C-4 of ␤-d-cymaropyranosyl);
from ıH 5.15 (H-1 of ␤-d-cymaropyranosyl) to ıC 83.2
1
was shown to have a molecular formula C54H80O22. The
acidic hydrolysis afforded 7 as the aglycone and the same
sugar compositions as compound 1. The 1H and 13C NMR
spectra of 2 were very similar to those of 1 except that
the signals due to one set of d-glucopyranosyl unit van-
ished in 2. In the HMBC experiment, the sequence of the
four sugar units was elucidated by significant correlations
ꢀ
ꢀꢀꢀꢀ
of ␤-d-glucopyranosyl) to ıC
ꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
observed from ıH 4.48 (H-1 of ␤-d-glucopyranosyl) to ıC 83.5
ꢀ
ꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀ
(
C-4 of ␤-d-oleandropyranosyl); from ıH 4.64 (H-1 of ␤-d-
ꢀ
ꢀꢀ
ꢀꢀꢀ
oleandropyranosyl) to ıC 83.7 (C-4 of ␤-d-cymaropyranosyl);

steroids 7 2 ( 2 0 0 7 ) 778–786
785
ꢀ
ꢀ
ꢀꢀ
(
C-4 of ␤-d-digitoxopyranosyl); from ıH 5.47 (H-1 of ␤-d-
(brd, J = 9.5 Hz), 5.24 (brd, J = 8.2 Hz), and 5.26 (brd, J = 9.4 Hz),
and five relevant anomeric carbon signals at ıC 102.0 (ole C-1),
106.6 (glu C-1), 100.5 (cym C-1), 98.4 (cym C-1), and 96.5 (cym
C-1) were observed in the NMR spectra, respectively. Based
on the HMQC, HMBC and TOCSY spectra, the data of these
five sugars were assigned to be three d-cymaropyranosyl,
one d-oleandropyranosyl and one d-glucopyranosyl units
comparing with the spectroscopic data in the literatures
digitoxopyranosyl) to ıC 77.6 (C-3) observed in the HMBC
spectrum. Hence, the structure of otophylloside K (4) was
concluded to be qingyangshengenin-3-O-␤-d-glucopyranosyl-
1 → 4)-␤-d-thevetopyranosyl-(1 → 4)-␤-d-cymaropyranosyl-
1 → 4)-␤-d-digitoxopyranoside.
(
(
3
.5.
Otophylloside L (5)
[
18–21]. According to the glycosylation shifts, the sugar
The negative HRFAB-MS showed the quasi-molecular ion peak
at m/z 1245.6261 ([M–H] , calcd. 1245.6268), in agreement with
chain was determined to be attached to C-3 position of the
aglycone. Following the same methodology described above,
the sequence of the sugar moieties was assigned from HMQC
and HMBC analysis using the well definite anomeric protons
as starting signals, which was identical to that of caudatoside
P, a glycoside of cynanchogenin isolated from Cynanchum
caudatum [21]. Accordingly, the structure of otophylloside M
(6) was elucidated to be caudatin-3-O-␤-d-glucopyranosyl-
(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-␤-d-oleandropyranosyl-
(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Pregnane glycosides obtained from C. otophllum not only
has prominent performance on epilepsy [2,7], but also can be
used to treat Menier’ syndrome and chronic hepatitis [8,9].
Our recent researches found that the total glycosides of C.
otophllum showed potent influence on hypochondrium [22]
and Alzheimer’s disease (AD). All the isolated glycosides have
a normal four-ring skeleton with an ester group at C-12 posi-
tion and a single sugar chain mainly consisting of deoxy sugar
units connected to C-3 group position of the aglycone. Initial
studies on the structure–activity relationship revealed that the
ester group and deoxy sugars would be important to phar-
macological effects. Whether the various sugars constitute
will affect the medical function remains to be investigated in
future.
−
the molecular formula C61H98O26, which was supported by
the 13C NMR and DEPT experiments. IR spectrum showed
the absorption bands for hydroxyl (3443 cm 1) and carbonyl
−
(
1713 cm 1) groups as well as C C band (1642 cm ). Acidic
− −1
hydrolysis of 5 afforded a sugar mixture of cymarose, olean-
drose and glucose, and the aglycone, which was identical to
caudatin (8), by co-TLC comparison with authentic samples.
Its negative FAB-MS exhibited fragment ion peaks at m/z 1118
−
−
[
M − 128] and 956 [M − 128 − 162] , revealing the existence
of an ikemaoyl ester group in the aglycone and a terminal
hexosyl unit in the sugar moiety. Inspection of the NMR
spectral data of compound 5 showed that besides the signals
arising from the aglycone (8), it contained five anomeric
carbons at ıC 97.2 (cym C-1), 101.2 (cym C-1), 102.6 (ole C-1),
1
04.1 (glu C-1), and 104.6 (glu C-1), corresponding to five
anomeric proton signals at ıH 4.86 (dd, J = 9.6, 1.6 Hz), 4.78
dd, J = 9.6, 1.6 Hz), 4.61 (dd, J = 9.6, 1.6 Hz), 4.47 (d, J = 8.0 Hz),
(
and 4.38 (d, J = 7.8 Hz), respectively. The signals of each sugar
unit (Table 2) were assigned by HMQC and HMBC analyses,
suggesting the existence of two d-cymaropyranosyl, one
d-oleandropyranosyl, and two d-glucopyranosyl units com-
pared with the spectroscopic data in the literatures [12,18–21].
Compared to the 13C chemical shifts of 8, 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 5. The sugar
sequence of 5 was demonstrated by HMBC correlations
Acknowledgements
The authors are grateful to Professor Chen Jijun for supplying
the deoxy sugar samples of d-digitoxose, d-cymarose, and d-
oleandrose, and appreciate the measurements of all spectra
performed by the colleagues of the Analytical Group in State
Key Laboratory of Phytochemistry and Plant Resources in West
China, Kunming Institute of Botany.
ꢀ
ꢀꢀꢀꢀꢀ
from ıH 4.38 (H-1
of terminal ␤-d-glucopyranosyl) to ıC
ꢀꢀꢀꢀ
of inner ␤-d-glucopyranosyl); from ıH 4.47 (H-
ꢀ
8
1
1.1 (C-4
ꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
of inner ␤-d-glucopyranosyl) to ıC 83.8 (C-4 of outer
-d-cymaropyranosyl); from ıH 4.78 (H-1
cymaropyranosyl) to ıC 83.8 (C-4 of ␤-d-oleandropyranosyl);
from ıH 4.61 (H-1
ꢀ
ꢀꢀꢀ
␤
of outer ␤-d-
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
of ␤-d-oleandropyranosyl) to ıC
ꢀ
ꢀ
8
3.9 (C-4 of inner ␤-d-cymaropyranosyl); from ıH 4.86
r e f e r e n c e s
ꢀ
ꢀ
(
H-1
of inner ␤-d-cymaropyranosyl) to ıC 79.3 (C-
). Consequently, the structure of was established
be
3
to
5
caudatin-3-O-␤-d-glucopyranosyl-(1 → 4)-␤-d-
[
[
[
1] Mu QZ, Zhou QL. The constituents of qingyangshen
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named otophylloside L.
(
1
and
3
.6.
Otophylloside M (6)
Compound 6 possessed a molecular formula C62H100O24
−
based on its HRFAB-MS spectrum (m/z 1227.6513 [M–H] ,
calcd. 1227.6526). In the acid hydrolysis experiment, the same
aglycone (8) and sugar components as those of 5 were obtained
by TLC comparison with standard samples. Five anomeric
proton signals at ıH 4.67 (brd, J = 9.6 Hz), 4.92 (d, J = 7.7 Hz), 5.10
[
4] Hu YM, Zhang MQ, Kuang PG, Lang SY, Zhong YL, Wang CL,
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