Three new steroidal glycosides from the roots of Cynanchum auriculatum

Article abstract of DOI:10.1002/hlca.201000424

Three new C₂₁ steroidal glycosides with a cinnamoyl group at C(12) and a 2-methylbutanoyl group at C(20), and a straight sugar chain at C(3), namely cyanoauriculosides C-E (1-3, resp.), together with three known steroidal derivatives, were isolated from the roots of Cynanchum auriculatum (Asclepiadaceae). Their structures were determined by spectroscopic analyses and chemical methods. The known constituents were identified as wilfoside K1N (4), cynanauriculoside II (5), and auriculoside IV (6). Copyright

Full text of DOI:10.1002/hlca.201000424

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Helvetica Chimica Acta – Vol. 94 (2011)
Three New Steroidal Glycosides from the Roots of Cynanchum auriculatum
by Yu Lu a), Hui Xionga), Hong Li Tengb), Guang Zhong Yanga), and Zhi Nan Mei*a)
a) College of Pharmacy, South Central University for Nationalities, Wuhan 430074, P. R. China
(phone: þ 86-27-67843220; fax: þ 86-27-67843220; e-mail: meizhinan@163.com)
b) Guangxi Institute of Minority Medicine, Nanning 530001, P. R. China
Three new C21 steroidal glycosides with a cinnamoyl group at C(12) and a 2-methylbutanoyl group at
C(20), and a straight sugar chain at C(3), namely cyanoauriculosides C – E (1 – 3, resp.), together with
three known steroidal derivatives, were isolated from the roots of Cynanchum auriculatum (Asclepia-
daceae). Their structures were determined by spectroscopic analyses and chemical methods. The known
constituents were identified as wilfoside K1N (4), cynanauriculoside II (5), and auriculoside IV (6).
Introduction. – C21 Steroidal glycosides have been extensively studied as an
important class of biological active compounds. They have been reported to possess
cytotoxic [1], acetylchlolinesterase inhibitory [2], and immunosuppressive activities
[3]. Several of these compounds have been isolated from plants, especially from those
of the Asclepiadaceae family [4 – 8]. The roots of Cynanchum auriculatum Royle ex
Wight (Asclepiadaceae), serve as a famous folk medicine in China, and has widely
been used for the treatment of geriatric diseases, enhancing immunity, anti-aging effect,
and prolonging life [9]. Previous phytochemical investigations on the genus Cynan-
chum revealed steroidal glycosides [10], acetophenones [11], and polysaccharides [12].
Steroidal glycosides were considered as the major biologically active constituents of the
genus Cynanchum [10]. In our search for biologically active compounds from
ethnomedicinal plants, three new C21 steroidal glycosides, 1 – 3, together with three
known compounds, 4 – 6, were isolated from the roots of this plant (Fig.). Their
structures were determined by detailed spectroscopic analyses, including 2D-NMR
techniques, and acidic hydrolysis. Here, we describe the structure elucidation of these
new compounds.
Results and Discussion. – Compound 1, isolated as a colorless amorphous powder,
showed positive reaction on LibermannꢀBuchard and KellerꢀKiliani reactions,
indicating the presence of a steroid skeleton with a 2-deoxysugar moiety. The
molecular formula was established as C62H94O22 according to HR-ESI-MS (m/z
1189.6166 ([M ꢀ H]ꢀ)). The 1H- and 13C-NMR data (Tables 1 and 2) suggested that 1
was a C21 steroidal glycoside. The 1H-NMR spectrum of 1 showed signals of three Me
groups (d(H) 1.56 (s), 1.18 (s), and 1.14 (d, J ¼ 6.3)), three O-bearing CH groups (d(H)
4.81 – 4.83 (m), 3.86 – 3.88 (m), and 3.52 – 3.54 (m)), one olefinic H-atom (d(H) 5.36
(br. s)), one cinnamoyl group (d(H) 6.38 (d, J ¼ 16.0, 1 H), 7.64 (d, J ¼ 16.5, 1 H),
7.65 – 7.67 (m, 2 H), and 7.42 – 7.44 (m, 3 H)), which was supported by the ion fragment
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 94 (2011)
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Figure. Structures of compounds 1 – 6

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Helvetica Chimica Acta – Vol. 94 (2011)
1
at m/z 147 ([C9H7O2]þ) arising from the aglycone moiety. From H,1H-COSY and
HSQC, one 2-methylbutyanoyl group (d(H) 2.28 – 2.30 (m, 1 H), 1.17 – 1.19 (m, 3 H),
1.36 – 1.38, 1.68 – 1.70 (2m, 2 H), and 0.91 (t, J ¼ 7.5, 3 H); d(C) 42.6 (d), 16.7 (q), 27.2
(t), 12.1 (q), and 176.9 (s)) was also identified in aglycone moiety. When comparing the
13C-NMR data of the aglycone portion of 1 with the data of penupogenin [13][14], it
was found that the major difference was the presence of an additional 2-methylbu-
tanoyl group in 1, and the chemical shift of C(20) of 1 was deshielded by ca. 5 ppm. This
suggested that a 2-methylbutanoyl group was located at C(20). Thus, the aglycone of
compound 1 was proposed to be 20-O-(2-methylbutanoyl)penupogenin. The glycosidic
portion contained four anomeric C-atoms with signals at d(C) 96.9 (C(1I)), 101.7
(C(1II)), 100.3 (C(1III)), and 106.3 (C(1IV)), corresponding to anomeric H-atom signals
at d(H) 4.89 (d, J ¼ 9.9), 4.99 (d, J ¼ 3.6), 4.82 (d, J ¼ 9.6), and 4.36 (d, J ¼ 7.8 ) ,
respectively, which indicated four sugar units in 1 with three b-linkages and one a-
linkage. Upon acidic hydrolysis, this glycoside liberated cymarose, diginose, and
glucose as sugar components, by TLC comparison with authentic samples. Compared
with penupogenin [14], the glycosidation shifts were observed at C(2) (ꢀ1.9 ppm),
C(3) (þ 7.7 ppm), and C(4) (ꢀ 3.6 ppm) in aglycone moiety, indicating the attachment
of the sugar chain at C(3)ꢀO of the aglycone. This was corroborated by the HMBC
between HꢀC(1I) at d(H) 4.89 and C(3) at d(C) 79.2, and ROESY correlation was
observed between HꢀC(1I) at d(H) 4.89 and HꢀC(3) at d(H) 3.52 – 3.54. On the basis
of the 1H,1H-COSY, HSQC, HMBC, and ROESY spectra, the data of these four sugars
were assigned as two b-cymaropyranosyls, one a-diginopyranosyl and one b-glucopyr-
anosyl. By comparing the 1H- and 13C-NMR data of the deoxysugar moieties of 1 with
those of the corresponding deoxysugar moieties of cynanoside K [15] and cynanaur-
iculoside II [16], as well as with those which have been reported in contrast for d-
diginopyranosyl [17] and l-cymaropyranosyl [13][18] residues, the configuration of the
diginopyranose and cymaropyranose units in 1 were assigned to l and d, respectively.
The sugar sequence and the interglycosidic linkages were established from the HMBC
spectrum, in which distinct correlations between HꢀC(1I) at d(H) 4.89 and C(3) at
d(C) 79.2, between HꢀC(1II) at d(H) 4.99 and C(4I) at d(C) 82.1, between HꢀC(1III) at
d(H) 4.82 and C(4II) at d(C) 75.3, and between HꢀC(1IV) at d(H) 4.36 and C(4III) at
d(C) 83.7 were detected. Thus, compound 1 was determined to be 20-O-(2-
methylbutanoyl)penupogenin-3-O-b-glucopyranosyl-(1 ! 4)-b-d-cymaropyranosyl-
(1 ! 4)-a-l-diginopyranosyl-(1 ! 4)-b-d-cymaropyranoside, named cyanoauriculoside
C.
Compound 2 was obtained as a colorless amorphous powder. The molecular
formula was established as C69H106O25 according to HR-ESI-MS (m/z 1369.6713 ([M þ
Cl]ꢀ)). Acidic hydrolysis of 2 afforded a mixture of sugars, which were identified as
1
cymarose, diginose, and glucose by TLC comparison with authentic samples. The H-
and 13C-NMR data indicated that 2 possessed the same aglycone as 1. Comparison of
the spectroscopic data of the sugar moieties with those of 1 indicated the presence of
one additional cymaropyranosyl moiety in 2. The sugar sequence and the interglyco-
sidic linkages were determined by HMBC spectrum, in which correlations between
HꢀC(1II) at d(H) 4.96 and C(4I) at d(C) 82.7, between HꢀC(1III) at d(H) 4.79 and
C(4II) at d(C) 75.3, between HꢀC(1IV) at d(H) 4.83 and C(4III) at d(C) 83.1, and
between HꢀC(1V) at d(H) 4.40 and C(4IV) at d(C) 78.6 were observed. Thus,

Helvetica Chimica Acta – Vol. 94 (2011)
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Table 1. NMR Data (CD3OD) of the Aglycone Moiety of Compounds 1 – 3. d in ppm, J in Hz.
Position
1
2
3
d(H)
d(C)
d(H)
d(C)
d(H)
d(C)
1
2
1.10 – 1.12 (m),
1.79 – 1.82 (m)
1.56 – 1.58 (m),
1.85 – 1.87 (m)
3.52 – 3.54 (m)
2.20 – 2.22 (m),
2.34 – 2.36 (m)
39.7
1.10 – 1.12 (m),
1.78 – 1.80 (m)
1.57 – 1.59 (m),
1.84 – 1.86 (m)
3.51 – 3.53 (m)
2.20 – 2.22 (m),
2.34 – 2.36 (m)
39.7
1.14 – 1.16 (m),
1.78 – 1.81 (m)
1.57 – 1.59 (m),
1.85 – 1.87 (m)
3.53 – 3.55 (m)
2.20 – 2.22 (m),
2.35 – 2.37 (m)
39.8
30.1
30.1
30.1
3
4
79.2
39.7
79.1
39.7
79.2
39.8
5
6
7
140.0
119.7
35.1
140.0
119.9
35.1
140.1
119.8
35.2
5.36 (br. s)
2.14 – 2.16 (m),
2.22 – 2.24 (m)
5.34 (br. s)
2.15 – 2.17 (m),
2.23 – 2.25 (m)
5.33 (br. s)
2.15 – 2.17 (m),
2.21 – 2.23 (m)
8
9
10
11
75.0
44.7
38.0
26.2
75.0
44.7
38.0
26.2
74.9
44.7
38.0
26.2
1.52 – 1.54 (m)
1.52 – 1.54 (m)
1.52 – 1.54 (m)
1.62 – 1.64 (m),
2.03 – 2.05 (m)
4.81 – 4.83 (m)
1.62 – 1.64 (m),
2.01 – 2.03 (m)
4.82 – 4.84 (m)
1.64 – 1.66 (m),
2.02 – 2.04 (m)
4.82 – 4.84 (m)
12
13
14
15
16
17
18
19
20
21
1’
2’
3’
4’
5’
6’
7’
8’
9’
75.4
57.4
88.3
34.0
34.1
89.5
11.0
18.5
75.3
75.0
57.5
88.4
34.0
34.0
89.4
11.0
18.3
75.2
75.4
57.6
88.4
34.1
34.1
89.6
11.0
18.6
74.1
1.98 – 2.00 (m)
1.87 – 1.89 (m)
1.94 – 1.96 (m)
1.87 – 1.89 (m)
1.96 – 1.98 (m)
1.88 – 1.90 (m)
1.56 (s)
1.18 (s)
3.86 – 3.88 (m)
1.14 (d, J ¼ 6.3)
1.54 (s)
1.15 (s)
3.82 – 3.84 (m)
1.14 (d, J ¼ 6.4)
1.56 (s)
1.15 (s)
3.80 – 3.82 (m)
1.14 (d, J ¼ 6.3)
15.2
15.2
15.2
168.4
120.3
145.5
135.9
129.2
130.0
131.5
130.0
129.2
176.9
42.6
168.4
120.3
145.5
135.8
129.2
130.0
131.5
130.0
129.2
176.9
42.6
168.4
120.3
145.5
135.8
129.2
130.0
131.5
130.0
129.2
177.0
42.6
6.38 (d, J ¼ 16.0)
7.64 (d, J ¼ 16.5)
6.35 (d, J ¼ 16.0)
7.60 (d, J ¼ 16.0)
6.35 (d, J ¼ 15.8)
7.60 (d, J ¼ 15.5)
7.65 – 7.67 (m)
7.42 – 7.44 (m)
7.42 – 7.44 (m)
7.42 – 7.44 (m)
7.65 – 7.67 (m)
7.56 – 7.58 (m)
7.39 – 7.41 (m)
7.39 – 7.41 (m)
7.39 – 7.41 (m)
7.56 – 7.58 (m)
7.56 – 7.58 (m)
7.39 – 7.41 (m)
7.39 – 7.41 (m)
7.39 – 7.41 (m)
7.56 – 7.58 (m)
10’
11’
12’
13’
2.28 – 2.30 (m)
1.17 – 1.19 (m)
1.36 – 1.38 (m),
1.68 – 1.70 (m)
0.91 (t, J ¼ 7.5)
2.29 – 2.31 (m)
1.15 – 1.17 (m)
1.36 – 1.38 (m),
1.67 – 1.69 (m)
0.89 (t, J ¼ 7.5)
2.29 – 2.31 (m)
1.13 – 1.15 (m)
1.36 – 1.38 (m),
1.67 – 1.69 (m)
0.89 (t, J ¼ 7.5)
16.7
27.2
16.8
27.2
16.7
27.2
14’
12.1
12.2
12.2
compound 2 was determined to be 20-O-(2-methylbutanoyl)penupogenin-3-O-b-
glucopyranosyl-(1 ! 4)-a-l-cymaropyranosyl-(1 ! 4)-b-d-cymaropyranosyl-(1 ! 4)-a-l-
diginopyranosyl-(1 ! 4)-b-d-cymaropyranoside, named cyanoauriculoside D.
Compound 3 was obtained as a colorless amorphous powder. The molecular
formula was established as C75H116O30 according to HR-ESI-MS (m/z 1531.7179

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Helvetica Chimica Acta – Vol. 94 (2011)
Table 2. NMR Data (CD3OD) of the Sugar Moieties of Compounds 1 – 3. d in ppm, J in Hza)
Position
1
2
3
d(H)
b-d-cym
d(C)
d(H)
b-d-cym
d(C)
d(H)
b-d-cym
d(C)
1I
2I
4.89 (d, J ¼ 9.9)
1.50 – 1.52 (m),
2.15 – 2.17 (m)
3.65 – 3.67 (m)
3.23 – 3.25 (m)
3.86 – 3.88 (m)
1.22 (d, J ¼ 6.0)
3.44 (s)
96.9
35.3
4.84 (d, J ¼ 10.0)
1.52 – 1.54 (m),
2.14 – 2.16 (m)
3.67 – 3.69 (m)
3.22 – 3.24 (m)
3.83 – 3.85 (m)
1.19 (d, J ¼ 6.0)
3.47 (s)
96.9
35.3
4.86 (overlap)
1.53 – 1.55 (m),
2.15 – 2.17 (m)
3.63 – 3.65 (m)
3.22 – 3.24 (m)
3.84 – 3.86 (m)
1.20 (d, J ¼ 6.5)
3.42 (s)
97.0
35.3
3I
78.2
82.1
70.3
18.5
57.4
78.2
82.7
70.2
18.5
57.4
78.2
82.7
70.2
18.5
57.5
4I
5I
6I
MeO
a-l-dig
a-l-dig
a-l-dig
1II
2II
4.99 (d, J ¼ 3.6)
1.73 – 1.75 (m),
2.19 – 2.21 (m)
3.92 – 3.94 (m)
3.59 – 3.61 (m)
3.96 – 3.98 (m)
1.26 (d, J ¼ 6.4)
3.43 (s)
101.7
32.6
4.96 (d, J ¼ 3.1)
1.74 – 1.76 (m),
2.24 – 2.26 (m)
3.92 – 3.94 (m)
3.59 – 3.61 (m)
3.95 – 3.97 (m)
1.24 (d, J ¼ 6.5)
3.39 (s)
101.7
32.6
4.96 (br. s)
101.7
32.6
1.78 – 1.80 (m),
2.26 – 2.28 (m)
3.92 – 3.94 (m)
3.60 – 3.62 (m)
3.95 – 3.97 (m)
1.23 (d, J ¼ 6.5)
3.39 (s)
3II
75.1
75.3
68.2
17.8
56.1
75.2
75.3
68.2
17.8
56.1
74.9
75.0
68.3
17.8
56.1
4II
5II
6II
MeO
b-d-cym
b-d-cym
b-d-cym
1III
2III
4.82 (d, J ¼ 9.6)
1.62 – 1.64 (m),
2.23 – 2.25 (m)
3.70 – 3.72 (m)
3.24 – 3.26 (m)
3.86 – 3.88 (m)
1.23 (d, J ¼ 6.0)
3.48 (s)
100.3
35.6
4.79 (d, J ¼ 9.5)
1.61 – 1.63 (m),
2.24 – 2.26 (m)
3.68 – 3.70 (m)
3.22 – 3.24 (m)
3.83 – 3.85 (m)
1.22 (d, J ¼ 6.0)
3.48 (s)
100.3
35.7
4.79 (d, J ¼ 9.5)
1.61 – 1.63 (m),
2.26 – 2.28 (m)
3.63 – 3.65 (m)
3.22 – 3.24 (m)
3.84 – 3.86 (m)
1.22 (d, J ¼ 6.0)
3.48 (s)
100.3
35.7
3III
78.6
83.7
70.2
18.7
58.4
78.7
83.1
70.1
18.7
58.3
78.7
83.2
70.2
18.7
58.3
4III
5III
6III
MeO
b-glu
a-l-cym
a-l-cym
1IV
2IV
4.36 (d, J ¼ 7.8)
3.18 – 3.20 (m)
106.3
75.0
4.83 (d, J ¼ 3.0)
1.78 – 1.80 (m),
2.25 – 2.27 (m)
3.78 – 3.80 (m)
3.56 – 3.58 (m)
4.28 – 4.30 (m)
1.25 (d, J ¼ 6.5)
99.9
32.2
4.83 (d, J ¼ 3.0)
1.78 – 1.80 (m),
2.26 – 2.28 (m)
3.81 – 3.83 (m)
3.53 – 3.55 (m)
4.26 – 4.28 (m)
1.25 (d, J ¼ 6.5)
100.0
32.3
3IV
4IV
5IV
6IV
3.26 – 3.28 (m)
3.32 – 3.34 (m)
3.24 – 3.26 (m)
3.64 – 3.66 (m),
3.87 – 3.89 (m)
78.0
71.7
77.9
62.8
74.0
78.6
65.9
18.4
74.1
79.1
66.0
18.4
MeO
3.42 (s)
b-glu
56.8
3.39 (s)
b-glu
56.8
1V
2V
3V
4V
5V
6V
4.40 (d, J ¼ 7.7)
3.19 – 3.21 (m)
3.26 – 3.28 (m)
3.35 – 3.37 (m)
3.25 – 3.27 (m)
3.63 – 3.65 (m),
3.83 – 3.85 (m)
101.7
75.0
78.1
71.6
77.8
62.8
4.41 (d, J ¼ 8.0)
3.20 – 3.22 (m)
3.26 – 3.28 (m)
3.37 – 3.39 (m)
3.23 – 3.25 (m)
3.65 – 3.67 (m),
3.85 – 3.87 (m)
101.8
75.4
78.5
71.0
77.1
62.1

Helvetica Chimica Acta – Vol. 94 (2011)
1301
Table 2 (cont.)
Position
1
2
3
d(H)
d(C)
d(H)
d(C)
d(H)
b-glu
d(C)
1VI
2VI
3VI
4VI
5VI
6VI
4.37 (d, J ¼ 7.8)
3.21 – 3.23 (m)
3.36 – 3.38 (m)
3.44 – 3.46 (m)
3.30 – 3.32 (m)
104.6
74.9
78.0
71.3
77.8
62.4
3.65 – 3.67 (m), 3.88 – 3.90 (m)
a) cym ¼ Cymaropyranosyl; dig ¼ diginopyranosyl; glu ¼ glucopyranosyl.
([M þ Cl]ꢀ)). Acidic hydrolysis of 3 afforded a mixture of sugars, which were
identified as cymarose, diginose, and glucose by TLC comparison with authentic
samples. The 1H- and 13C-NMR data indicated that 3 possessed the same aglycone as 2.
Comparison of the spectroscopic data of the sugar moieties with those of 2 revealed the
presence of one additional glucopyranosyl moiety in 3. The sugar sequence and the
interglycosidic linkages were determined by HMBC spectrum, in which correlations
between HꢀC(1II) at d(H) 4.96 and C(4I) at d(C) 82.7, between HꢀC(1III) at d(H) 4.79
and C(4II) at d(C) 74.9, between HꢀC(1IV) at d(H) 4.83 and C(4III) at d(C) 83.2,
between HꢀC(1V) at d(H) 4.41 and C(4IV) at d(C) 79.1, and between HꢀC(1VI) at d(H)
4.37 and C(4V) at d(C) 81.0 were observed. Thus, compound 3 was determined to be 20-
O-(2-methylbutanoyl)penupogenin-3-O-b-glucopyranosyl-(1 ! 4)-b-glucopyranosyl-
(1 ! 4)-a-l-cymaropyranosyl-(1 ! 4)-b-d-cymaropyranosyl-(1 ! 4)-a-l-diginopyra-
nosyl-(1 ! 4)-b-d-cymaropyranoside, named cyanoauriculoside E.
The three known compounds were identified as wilfoside K1N (4) [16],
cynanauriculoside II (5) [17], and auriculoside IV (6) [10] by comparison of their
spectroscopic data with those reported in the literature. They have been already
reported from Cynanchum auriculatum.
Experimental Part
General. HPLC: Ultimate 3000 HPLC system; Ultimate 3000 Variable Wavelength; Ultimate 3000
pump; column, Waters 5C18-MS-II (10 ꢁ 250 mm, 5 mm). Column chromatography (CC): silica gel (SiO2;
200 – 300 mesh; Qingdao Marine Chemical Co., Ltd., P. R. China) and C18 reversed-phase (RP) silica gel
(YMC Co., Ltd., Japan). TLC: Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd.,
P. R. China). 1H- and 13C-NMR spectra: Bruker DRX-500 instrument; d in ppm rel. to Me4Si as internal
standard (¼0 ppm), J in Hz. HR-ESI-MS: Finnigan LCQ-Deca and Waters/Micromass Q-Tof-Ultima
mass spectrometer in m/z (rel. int.).
Plant Material. The roots of the C. auriculatum were collected from Jishou, Hunan Province, P. R.
China, in September 2007, and were identified by Prof. Ding-Rong Wan (College of Pharmacy, South
Central University for Nationalities). The voucher specimen was deposited with the Herbarium of
College of Pharmacy, South Central University for Nationalities.
Extraction and Isolation. The air-dried roots of C. auriculatum (4 kg) were powdered and extracted
with 95% EtOH at r.t. for three times (3 ꢁ 6 l; 48, 48, and 24 h). The EtOH extract (0.6 kg) was
suspended in H2O (1.6 l) and then successively partitioned with petroleum ether (PE; 1.5 l ꢁ 3), CHCl3

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Helvetica Chimica Acta – Vol. 94 (2011)
(1.5 l ꢁ 3), AcOEt (1.5 l ꢁ 3), and BuOH (1.5 l ꢁ 3). The CHCl3 extract (195 g) was chromatographed on
SiO2 column using gradient solvents of cyclohexane/AcOEt (100 :0 ! 0 :100) and AcOEt/MeOH
(100 :0 ! 0 :100) to yield seven fractions, Frs. 1 – 7. Fr. 5 (7.9 g) was subjected to CC (SiO2; CHCl3/MeOH
9 :1 ! 0 :100) to afford three subfractions, Frs. 5.1 – 5.3. Fr. 5.3 (2.5 g) was subjected to CC (ODS; H2O/
MeOH 9 :1 ! 1:9) to afford five subfractions, Frs. 5.3.1 – 5.3.5. Fr. 5.3.1 was purified by semi-prep. HPLC
(MeOH/H2O 82 :18; 3 ml/min) to yield 1 (25 mg, tR 24.6 min). Fr. 5.3.4 was purified by semi-prep. HPLC
(MeOH/H2O 85 :15; 3 ml/min) to yield 2 (37 mg, tR 16.5 min). Fr. 5.3.5 was purified by semi-prep. HPLC
(MeOH/H2O 85 :15; 3 ml/min) to yield 3 (27 mg, tR 14.9 min). Fr. 2 (4.3 g) was repeatedly chromato-
graphed on SiO2, then purified on a RP-C18 SiO2 column to afford 4 (28 mg) and 6 (32 mg). Fr. 7 (18.0 g)
was subjected to CC (SiO2; AcOEt/MeOH 100 :0 ! 0 :100) to afford five subfractions, Frs. 7.1 – 7.5.
Fr. 7.1 (2.1 g) was subjected to CC (ODS; H2O/MeOH 9 :1 ! 1:9) to afford five subfractions, Frs. 7.1.1 –
7.1.5. Fr. 7.1.1 was purified by semi-prep. HPLC (MeOH/H2O 80 :20; 3 ml/min) to yield 5 (38 mg, tR
16.9 min).
Acid Hydrolysis. A soln. of 1, 2, and 3 (each 5 mg) in MeOH was treated with 0.05m HCl/4-dioxane
1:1 (1 ml) at 608 for 1.5 h, resp. [13][19]. After removing dioxane, the soln. was extracted with AcOEt
(3 ꢁ 2 ml). The aq. layer was neutralized by NaOH and concentrated under reduced pressure to give the
sugar fraction. The presence of the monosaccharides in the hydrolysates of each compound was
confirmed by TLC comparison with authentic sugars. Diginose, cymarose, and glucose were detected
from the hydrolysates of compounds 1 – 3. The Rf values of diginose and cymarose were 0.66 and 0.76 with
CHCl3/MeOH 95 :5, 0.18 and 0.23 with PE/Me2CO 3 :1, resp.
Cyanoauriculoside C (¼20-O-(2-Methylbutanoyl)penupogenin-3-O-b-glucopyranosyl-(1 ! 4)-b-d-
cymaropyranosyl-(1 ! 4)-a-l-diginopyranosyl-(1 ! 4)-b-d-cymaropyranoside ¼ (3b,12b,14b,17a,20S)-3-
{[b-Glucopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-b-d-ribo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-
methyl-a-l-lyxo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-b-d-ribo-hexopyranosyl]oxy}-8,14,17-
trihydroxy-12-{[(2E)-3-phenylprop-2-enoyl]oxy}pregn-5-en-20-yl 2-Methylbutanoate; 1). Colorless
amorphous powder. [a]2D0 ¼ þ11.4 (c ¼ 0.30, MeOH). UV (MeOH): 278 (4.40), 216 (4.26), 203 (4.33).
1H- and 13C-NMR: see Tables 1 and 2, resp. HR-ESI-MS: 1189.6166 ([M ꢀ H]ꢀ, C62H93O2ꢀ2 ; calc.
1189.6158).
Cyanoauriculoside D (¼20-O-(2-Methylbutanoyl)penupogenin-3-O-b-glucopyranosyl-(1 ! 4)-a-l-
cymaropyranosyl-(1 ! 4)-b-d-cymaropyranosyl-(1 ! 4)-a-l-diginopyranosyl-(1 ! 4)-b-d-cymaro-
pyranoside ¼ (3b,12b,14b,17a,20S)-3-{[b-glucopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-a-l-arabino-
hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-b-d-ribo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-meth-
yl-a-l-lyxo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-b-d-ribo-hexopyranosyl]oxy}-8,14,17-trihy-
droxy-12-{[(2E)-3-phenylprop-2-enoyl]oxy}pregn-5-en-20-yl 2-Methylbutanoate; 2). Colorless amor-
1
phous powder. [a]2D0 ¼ ꢀ18.3 (c ¼ 0.14, MeOH). UV (MeOH): 278 (4.38), 218 (4.22), 203 (4.30). H-
and 13C- NMR: see Tables 1 and 2, resp. HR-ESI-MS: 1369.6713 ([M þ Cl]ꢀ, C69H106ClO2ꢀ5 ; calc.
1369.6711).
Cyanoauriculoside E (¼20-O-(2-Methylbutanoyl)penupogenin-3-O-b-glucopyranosyl-(1 ! 4)-b-glu-
copyranosyl-(1 ! 4)-a-l-cymaropyranosyl-(1 ! 4)-b-d-cymaropyranosyl-(1 ! 4)-a-l-diginopyranosyl-
(1 ! 4)-b-d-cymaropyranoside ¼ (3b,12b,14b,17a,20S)-3-{[b-Glucopyranosyl-(1 ! 4)-b-glucopyranosyl-
(1 ! 4)-2,6-dideoxy-3-O-methyl-a-l-arabino-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-b-d-ribo-
hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-a-l-lyxo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-meth-
yl-b-d-ribo-hexopyranosyl]oxy}-8,14,17-trihydroxy-12-{[(2E)-3-phenylprop-2-enoyl]oxy}pregn-5-en-20-
yl 2-Methylbutanoate; 3). Colorless amorphous powder. [a]2D0 ¼ þ7.8 (c ¼ 0.20, MeOH). UV (MeOH):
1
278 (4.37), 216 (4.24), 203 (4.30). H- and 13C-NMR: see Tables 1 and 2, resp. HR-ESI-MS: 1531.7179
([M þ Cl]ꢀ, C75H116ClO3ꢀ0 ; calc. 1531.7239).
This work was financially supported by the National Key Technology R&D Program in the 11th Five
Year Plan of China (No. 2007BAI48B08).

Helvetica Chimica Acta – Vol. 94 (2011)
1303
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Received November 17, 2010