Five new pregnane glycosides from the stems of Marsdenia tenacissima

Article abstract of DOI:10.1080/10286020.2011.570263

Activity-guided fractionation of the stems of Marsdenia tenacissima led to the isolation of five new pregnane glycosides, namely marstenacissides E (1), F (2), G (3), H (4), and I (5). Their structures were determined on the basis of ¹H and ¹³C NMR, COSY, TOCSY, ROESY, and FABMS experiments.

Full text of DOI:10.1080/10286020.2011.570263

Journal of Asian Natural Products Research
Vol. 13, No. 6, June 2011, 477–485
Five new pregnane glycosides from the stems of Marsdenia tenacissima
Zeng-Hua Xiaa, Shi-Long Maob*, Ai-Na Laoa, Jun Uzawac, Shigeo Yoshidac and
Yasuo Fujimotod*
aInstitute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 201203, China; Department of Pharmacy, Shanghai Xuhui Central Hospital,
b
c
Shanghai 200031, China; The Institute of Physical and Chemical Research (RIKEN), Wako,
d
Saitama 351-0198, Japan; College of Pharmacy, Nihon University, 7-7-1, Narashinodai,
Funabashi, Chiba 274-8555, Japan
(Received 10 November 2010; final version received 6 March 2011)
Activity-guided fractionation of the stems of Marsdenia tenacissima led to the isolation
of five new pregnane glycosides, namely marstenacissides E (1), F (2), G (3), H (4), and
I (5). Their structures were determined on the basis of 1H and 13C NMR, COSY,
TOCSY, ROESY, and FABMS experiments.
Keywords: Marsdenia tenacissima; Asclepiadaceae; pregnane glycoside; Marstena-
cissides E, F, G, H, I
1. Introduction
determined from its negative ion FAB-MS
(m/z 1227 [M 2 H]2) as well as 13C NMR
and DEPT spectral data. The IR spectrum
of 1 showed that the absorption bands are
due to hydroxyl group (3425 cm21) and
glycoside linkages (1060 cm21). Com-
pound 1 exhibited positive Liebermann–
Burchard and Keller–Kiliani reactions. Its
spectral features and physicochemical
properties suggested that 1 should be a
steroidal glycoside with 2-deoxy sugar
units. Of the 61 carbons, 28 were assigned
to the aglycon part and 33 to the
Marsdenia tenacissima (Roxb.) Wight et
Arn. is distributed in the southwest of
China. Its stems are used for the treatment
of cancer and asthma in Chinese folk
medicine [1]. In previous studies on the
stems of this plant, the isolation of
tenacissosides A-E [2], F-I [3], marsdeno-
sides A-H [4], two C21 steroids [5],
tenacissosides L, M [6], and five C21
steroidal glycosides [7] has been reported.
In the course of our search for bioactive
compounds from this plant, we reported
the isolation of marstenacissides A–D
recently [8]. In this paper, we describe the
isolation and structural elucidation of five
new pregnane glycosides, namely marste-
nacissides E (1), F (2), G (3), H (4), and I
(5) from M. tenacissima (Figure 1).
1
oligosaccharide moiety. The H and 13C
NMR spectra of the aglycon moiety of 1
indicated the presence of three methyl
groups [dH 1.19, 2.20 (each 3H, s) and 1.27
(3H, d, J ¼ 6.1 Hz), dC 12.2, 13.0, and
19.5], three oxygenated methine groups [dH
3.91 (1H, m), 4.09 (1H, q, J ¼ 6.0 Hz) and
5.32 (1H, dd); dC 70.7, 75.9, and 76.7], one
benzoyl group, and five quaternary carbons
(dC 36.6, 57.5, 76.0, 88.4, and 88.9)
2. Results and discussion
Marstenacisside E (1), an amorphous solid,
has a molecular formula C61H96O25,
*Corresponding authors. Email: maoshilong@hotmail.com; fujim-y@chs.nihon-u.ac.jp
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2011.570263
http://www.informaworld.com

478
Z.-H. Xia et al.
O
C
O
OH
OH
OH
H
OH
RO
H
Me
Me
Me
O
O
OH
OH
O
OH
HO
HO
MeO
HO
OH
Dig=D-digitoxose
OMe
Cym=D-cymarose
Ole=D-oleandrose
Me
HOH2C
HO
O
OH
O
OH
HO
MeO
HO
OH
OH
Thv=D-thevetose
Glc=D-glucose
1
2
3
4
5
R=Glc(1→4)Thv(1→4)Ole(1→4)Cym(1→4)Dig
R=Glc(1→4)Thv(1→4)Ole(1→4)Cym
R=Glc′(1→4)Glc(1→4)Thv(1→4)Ole(1→4)Cym
R=Glc′(1→4)Glc(1→4)Thv(1→4)Ole(1→4)Cym(1→4)Dig
R=Glc′(1→4)Glc(1→4)Ole(1→4)Cym(1→4)Cym′
Figure 1. Structure of compounds 1–5.
(Table 1). All of these spectral data
suggested that the aglycon of 1 was a
pregnane highly oxidized at C-3, C-8, C-
12, C-14, C-17, and C-20. The presence of
one benzoyl group at C-12 was confirmed
by observation of the correlation between
H-12 at dH 5.32 and CvO of the benzoyl
group at dc166 .6in the HMBC spectrum of
1. Comparison of the 13C NMR spectrum of
the aglycon moiety of 1 with that of 12-O-
benzoyl-dihydrosarcostin (A) [8] (Table 1)
showed that the aglycon moiety of 1 was
12-O-benzoyl-dihydrosarcostin. The car-
bon signals at dC 25.2 and 34.9 of 12-O-
benzoyl-dihydrosarcostin (A) have been
assigned to C-7 and C-11, respectively, by
Yuan et al. [9]. However, the correlation
between the H-12 at dH 5.32 and the carbon
at dC 24.6 in the HMBC spectrum of 1
suggested that the signal at dC 24.6 should
be assigned to C-11. Therefore, the assign-
ments for C-7 and C-11 of 12-O-benzoyl-
dihydrosarcostin(A)reportedbyYuanetal.
should be revised to C-11 and C-7,
respectively. The glycosylation shifts of 1
[C-3 (þ5.8 ppm), C-2 (22.4 ppm), and C-
4 (24.4 ppm)] indicated that the sugars of 1
were bound to the C-3 position of the
aglycon.
1
In the H NMR and 13C NMR spectra
of the sugar moiety of 1 (Tables 2 and 3),
the signals due to five anomeric protons
and carbons [dH 4.67 (1H, dd, J ¼ 9.9,
2.0 Hz), 4.89 (1H, d, J ¼ 8.6 Hz), 5.13

Journal of Asian Natural Products Research
479
Table 1. 13C NMR spectral data for the aglycon moieties of compounds 1, 2, 3, 4, 5, and 12-
O-benzoyl-dihydrosarcostin (A) (Pyridine-d5, d in ppm, 150 MHz).
1
2
3
4
5
Aa
1
2
3
4
5
6
7
8
9
38.1
29.6
76.7
34.5
45.3
25.2
34.3
76.0
46.9
36.6
24.6
75.9
57.5
88.4
32.8
33.8
88.9
12.2
13.0
70.7
19.5
38.0
29.6
76.7
34.7
45.3
25.2
34.7
76.0
46.9
36.6
24.6
75.9
57.5
88.4
32.8
33.8
88.9
12.2
13.0
70.7
19.5
38.1
29.6
76.7
34.6
45.3
25.2
34.1
76.0
46.8
36.5
24.6
75.8
57.5
88.9
32.8
33.8
88.8
12.2
13.0
70.7
19.5
38.3
29.5
76.9
34.4
45.4
24.5
34.6
76.0
47.0
36.7
25.1
75.9
58.0
88.6
34.0
34.6
89.0
12.1
13.0
70.8
19.5
38.0
29.6
76.6
34.5
45.3
25.2
34.5
75.9
46.8
36.5
24.8
75.8
57.5
88.4
32.8
33.8
88.8
12.2
12.9
70.8
19.6
38.6
32.0
70.9
38.9
46.1
25.5
25.2
76.1
47.6
36.6
34.9
75.9
59.2
89.0
34.1
34.1
88.8
11.7
13.3
71.7
17.7
10
11
12
13
14
15
16
17
18
19
20
21
Bez
CvO
10
166.6
131.6
130.4
129.0
133.0
166.6
131.8
130.4
128.8
133.2
166.6
131.7
130.4
128.8
133.3
166.7
131.6
130.4
128.7
133.3
166.6
131.8
130.4
128.8
133.2
167.0
131.6
130.4
129.0
133.0
20, 60
30, 50
40
Note: Bez: benzoyl group.
a Reported data for 12-O-benzoyl-dihydrosarcostin by Yuan et al.
(1H, d, J ¼ 8.0 Hz), 5.19 (1H, dd, J ¼ 9.8,
2.1 Hz), and 5.53 (1H, dd, J ¼ 9.2,
2.1 Hz); dC 101.9, 104.0, 104.0, 99.7, and
95.8], four methyl groups [dH 1.33, 1.49,
1.67, and 1.77 (each 3H, d)], and three
methoxyl groups [dH 3.52, 3.58, and 3.96
(each 3H, s)] suggested that one hexose
and four deoxy sugars were present in 1.
Among the four deoxy sugars, three were
2,6-dideoxy sugars and the other one
should be 6-deoxy sugar. It was disclosed
that the sugars of 1 should be composed of
one 2,6-dideoxypyranose (S1), two 2,6-
dideoxy-3-O-methylpyranoses (S2 and
S3), one 6-deoxy-3-O-methylpyranose
(S4), and one glucose (S5) by detailed
analyses of TOCSY, FOCSY, HMQC,
SELTOCSY, HMBC, and SELROESY
spectra of 1. The comparison of the
chemical shift values of the anomeric
protons [d 5.19 (1H, dd, J ¼ 9.8, 2.1 Hz)
and 4.67 (1H, dd, J ¼ 9.9, 2.0 Hz)] of S2
and S3 with those of cymarose (Cym: d
5.08–5.29) and oleandrose (Ole: d 4.66–
4.90, in a higher field than Cym) [10]
suggested that S2 and S3 should be Cym
and Ole, respectively. In the light of the 1H
and 13C NMR spectra of the sugar moiety
of 1 (Tables 2 and 3), S1, S4, and S5 were
assigned to be digitoxose (Dig), thevetose
(Thv), and glucose (Glc), respectively.
Finally, the result of acid hydrolysis of 1
(see Section 3) further confirmed that the
sugars of 1 were composed of digitoxose
(S1), cymarose (S2), oleandrose (S3),
thevetose (S4), and glucose (S5). The
connectivity of these sugars was

480
Z.-H. Xia et al.
Table 2. 1H NMR spectral data for the sugar moieties of compounds 1, 2, 3, 4, and 5 (Pyridine-d5, d
in ppm, J in Hz, 600 MHz).
1
2
3
4
5
dig
5.53, dd, (9.2,
2.1)
Cym
5.31, dd, (9.6,
2.0)
cym
5.31, dd, (9.2,
2.0)
Dig
5.54, dd, (9.8,
2.1)
Cym0
5.33, dd, (9.8,
2.0)
1
2
3
4
2.43, 2.07
4.65, q, (2.5)
3.52, dd, (9.0,
2.5)
2.32, 1.89
4.09, q, (3.1)
3.49, dd, (9.1,
3.0)
2.34, 1.89
4.07, q, (3.1)
3.50, dd, (9.0,
3.1)
2.45, 2.06
4.65, q, (2.5)
3.51, dd, (9.0,
2.5)
2.35, 1.82
4.11, q, (3.1)
3.53, dd, (9.0,
3.1)
5
4.35
1.49, d, (6.1)
4.30
1.49, d, (6.1)
4.27
1.48, d, (6.1)
4.35
1.33, d, (6.1)
4.28
*
1.42, d, (6.0)
3.65
Cym
5.13, dd (9.8,
2.1)
6
OCH3
3.61
Ole
3.59
ole
*
*
cym
5.19, dd, (9.8,
2.1)
Cym
5.18,dd, (9.8,
2.0)
1
4.71, dd, (9.8,
2.1)
4.72, dd, (9.7,
2.0)
2
3
4
2.32, 1.78,
4.05, q, (3.0)
3.40, dd, (9.1,
3.0)
2.50, 1.75
3.55
3.60, t, (9.1)
2.50,1.76
3.58
3.60, t, (9.1)
2.34, 1.80,
3.93, q, (3.0)
3.40, dd, (9.1,
3.0)
2.33, 1.88
4.02, q, (3.0)
3.44, dd, (9.1,
3.0)
5
4.19
3.57
1.67, d, (6.1)
3.59
4.17
4.18
*
6
OCH3
1.33, d, (5.5)
3.58
ole
1.67, d, (6.0)
3.51
Thv
1.45, d, (5.5)
3.52
Ole
1.38, d, (6.0)
3.58
Ole
3.61
Thv
*
1
4.67, dd, (9.9,
2.0)
4.89, d, (8.8)
4.87, d, (8.6)
4.79, dd, (9.8,
2.0)
4.70, dd, (9.9,
2.0)
2
3
4
5
2.46, 1.74
3.57
3.60, t, (9.1)
3.75
3.90
3.91
2.46, 1.75
3.57
3.62, t, (9.1)
3.54
2.46, 1.79
3.60
3.68, t, (9.1)
3.65
3.90, t, (9.2)
3.88, t, (9.2)
3.56
3.67, t, (9.1)
3.82, t, (9.1)
3.74
6
OCH3
1.67, d, (6.0)
3.52
thv
1.78, d, (6.0)
3.91
Glc
1.75, d, (6.1)
3.93
Glc
1.67, d, (6.0)
3.60
Thv
1.72, d, (6.0)
3.50
Glc
1
2
3
4
4.89, d, (8.6)
3.91
3.70, t, (9.2)
3.88, t, (9.2)
3.74
5.13, d, (8.0)
4.05
4.24
4.21
3.98
5.08, d, (8.0)
4.03
4.28
4.30
3.92
4.89, d, (8.8)
3.88
3.70, t, (9.2)
3.87, t, (9.2)
3.75
5.08, d, (8.0)
4.03
4.28
4.30
3.92
*
5
6
OCH3
1.77, d, (5.8)
3.96
glc
4.55,4.38
4.49, 4.30
1.76, q, (5.8)
3.91
Glc
4.55, 4.50
glc0
glc0
1
2
3
4
5
6
5.13, d, (8.0)
4.03
4.24
4.21
3.98
5.18, d, (7.8)
4.10
5.12, d, (7.8)
4.03
4.25
5.18, d, (7.8)
4.10
4.29
4.20
4.04
4.22§
4.20
4.20
3.97
*
4.22§
4.54, 4.28
4.53, 4.36
4.55, 4.35
glc0
4.54, 4.28
5.21, d, (7.8)
4.12
4.22
4.20
4.10
*
4.56, 4.31
Note: ,§, overlapped signals.
*

Journal of Asian Natural Products Research
481
Table 3. 13C NMR spectral data for the sugar moieties of compounds 1, 2, 3, 4, and 5 (Pyridine-d5,
d in ppm, 150 MHz).
1
2
3
4
5
dig
cym
cym
Dig
Cym0
1
2
3
4
95.8
39.1
67.6
83.5
68.6
18.7
95.9
95.9
95.8
95.9
37.2
77.9
83.6
68.9
18.8
58.9
ole
101.9
37.6
79.2
83.3
71.9
18.6
57.4
thv
37.5
77.8
83.6
68.8
18.6
58.9
ole
101.9
37.5
79.1
83.4
71.9
18.7
57.4
thv
39.1
67.6
83.1
68.6
18.5
37.3
78.1
83.5
69.0
18.7
58.9
5
6
OCH3
cym
99.7
36.7
77.7
83.2
69.0
18.7
58.9
ole
Cym
99.8
36.7
77.9
83.4
69.0
18.7
58.9
ole
cym
1
2
3
4
100.5
37.0
77.8
83.2
68.9
18.5
59.0
ole
5
6
OCH3
1
2
3
4
101.9
37.6
79.3
83.3
72.0
18.7
57.4
thv
104.0
74.8
86.3
83.4
71.9
18.5
60.7
glc
104.0
74.8
86.4
83.5
72.0
18.7
60.7
glc
101.9
38.3
79.2
83.1
72.0
18.8
57.4
thv
101.9
37.6
79.3
83.3
72.0
18.8
57.4
glc
5
6
OCH3
1
2
3
4
104.0
75.0
86.3
83.3
72.1
18.7
60.6
glc
104.8
75.8
78.4
72.1
78.1
63.1
105.0
75.9
78.6
72.0
78.2
62.4
104.7
75.4
78.2
81.7
76.3
62.5
103.9
74.9
86.3
83.2
72.0
18.7
60.7
glc
104.8
75.8
78.4
82.1
78.1
63.1
glc0
104.3
75.4
76.8
81.5
76.5
62.5
5
6
OCH3
glc0
105.0
75.0
78.3
71.6
78.5
62.5
glc0
1
2
3
4
5
6
105.0
74.8
78.3
71.6
78.5
62.5
1
2
3
4
5
6
105.0
75.0
78.3
71.6
78.5
62.5
investigated by analysis of HMBC and
ROESY spectra of 1.
In the HMBC spectrum of 1 (Figure 2),
the cross peaks between the following
proton and carbon signals were observed:
H-3 of aglycon/C-1 of Dig, H-4 of Dig/C-1
of Cym, H-4 of Cym/C-1 of Ole, H-4 of
Ole/C-1 of Thv, and H-4 of Thv/C-1 of
Glc. Furthermore, the ROESY spectrum of
1 (Figure 2) exhibited the cross peaks

482
Z.-H. Xia et al.
H
H
H
OH
H
HO
OH
H
Me
O
76.7
Me
O
MeO
O
HO
HO
O
O
O
O
Aglycone
O
O
Me
O
MeO
Me
H
H
HO
OMe
H
H
HMBC
Figure 2. HMBC and NOESY correlations of the sugar moiety of 1.
HMQC
between the following proton signals: H-3
of aglycon/H-1 of Dig, H-4 of Dig/H-1 of
Cym, H-4 of Cym/H-1 of Ole, H-4 of
Ole/H-1 of Thv, and H-4 of Thv/H-1 of
Glc. The b configurations for all of the
sugars were determined from their large
3JH1,H2 coupling constants (7.8–9.8 Hz).
Thus, the sequence and the linkage sites of
the pentasaccharide of 1 was represented
as shown in Figure 2. Therefore, 1 was
established as 3-O-b-D-glucopyranosyl-
(1 ! 4)-b-D-thevetopyranosyl-(1 ! 4)-b-
D-oleandropyranosyl-(1 ! 4)-b-D-cymar-
opyranosyl-(1 ! 4)-b-D-digitoxopyrano-
syl-12-O-benzoyl-dihydrosarcostin.
absence of a digitoxose moiety. In the
HMBC spectrum of 2, the correlations
were observed between the following
proton and carbon signals: H-3 of agly-
con/C-1 of Cym, H-4 of Cym/C-1 of Ole,
H-4 of Ole/C-1 of Thv, and H-4 of Thv/C-1
of Glc. The ROESY spectrum showed the
cross peaks between the following proton–
proton signals: H-3 of aglycon/H-1 of
Cym, H-4 of Cym/H-1 of Ole, H-4 of
Ole/H-1 of Thv, and H-4 of Thv/H-1 of
Glc. Therefore, the structure of 2 was
established to be 3-O-b-D-glucopyranosyl-
(1 ! 4)-b-D-thevetopyranosyl-(1 ! 4)-b-
D-oleandropyranosyl-(1 ! 4)-b-D-cymar-
opyranosyl-12-O-benzoyl-dihydrosarcos-
tin.
Marstenacisside F (2), an amorphous
solid, had a molecular formula C55H86O22
determined from its negative ion FAB-MS
(m/z: 1097 [M 2 H]2) as well as 13C NMR
and DEPT spectral data. The spectral
evidence indicated that compound 2 had
the same aglycon as that of 1 (Table 1) and
the structural difference should be in the
sugar part. In the 1H and 13C NMR spectra
of the sugar moiety of 2 (Tables 2 and 3),
the signals due to four anomeric protons
and carbons [dH 4.71 (1H, dd, J ¼ 9.8,
2.1 Hz), 4.89 (1H, d, J ¼ 8.8 Hz), 5.13
(1H, d, J ¼ 8.0 Hz), and 5.31 (1H, dd,
J ¼ 9.6, 2.0 Hz); dC 101.9, 104.0, 105.0,
and 95.9] indicated the presence of four
sugars in 2. Comparison of the 13C NMR,
1H–1H COSY, TOCSY, ROESY, HMQC,
and HMBC spectra of 2 with those of 1
suggested that the four sugars of 2 were
identical to those of 1 except for the
Marstenacisside G (3), an amorphous
solid, had a molecular formula C61H96O27
determined from its negative ion FAB-MS
(m/z: 1259 [M 2 H]2) as well as 13C NMR
and DEPT spectral data. The spectral
evidence indicated that 3 had the same
aglycon as that of 2 (Table 1), but differed
1
in the sugar part. The H and 13C NMR
spectra of the sugar moiety of 3 (Tables 2
and 3) [dH 4.72 (1H, dd, J ¼ 9.7, 2.0 Hz),
4.87 (1H, d, J ¼ 8.6 Hz), 5.08 (1H, d,
J ¼ 8.0 Hz), 5.18 (1H, d, J ¼ 7.8 Hz), and
5.31 (1H, dd, J ¼ 9.2, 2.0 Hz); dC 101.9,
104.0, 104.7, 105.0, and 95.9 assignable to
the anomeric protons and carbons] mani-
fested the pentasaccharide feature of sugar
moiety of 3. Comparison of the 13C NMR,
1H–1HCOSY, TOCSY, ROESY, HMQC,
and HMBC spectra of 3 with those of 2

Journal of Asian Natural Products Research
483
indicated that four sugars were identical
with those of 2, except that an additional
glucose existed in the sugar chain of 3. The
glycosylation shift (þ9.6 ppm) of the C4
signal of the glucose (Glc) in 3 suggested
that the additional glucose (Glc0) in 3
should be bound to C4 of Glc. Finally, the
sequence and the linkage sites of the
pentasaccharide of 3 were further con-
firmed by HMBC and ROESY exper-
iments. Thus, 3 was established to be 3-O-
b-D-glucopyranosyl-(1 ! 4)-b-D-gluco-
pyranosyl-(1 ! 4)-b-D-thevetopyranosyl-
(1 ! 4)-b-D-oleandropyranosyl-(1 ! 4)-
b-D-cymaropyranosyl-12-O-benzoyl-
dihydrosarcostin.
TOCSY, ROESY, HMQC, and HMBC
spectra of 4 with those of 3 suggested that
the sugar moiety of 4 was similar to that of 3
except for the presence of an additional
digitoxopyranose (Dig). By analysis of the
HMBC and ROESY spectra, 4 was estab-
lished as 3-O-b-D-glucopyranosyl-(1 ! 4)-
b-D-glucopyranosyl-(1 ! 4)-b-D-theveto-
pyranosyl-(1 ! 4)-b-D-oleandropyranosyl-
(1 ! 4)-b-D-cymaropyranosyl-(1 ! 4)-b-
D-digitoxopyranosyl-12-O-benzoyl-dihy-
drosarcostin.
Marstenacisside F (5), an amorphous
solid, had the molecular formula C61H96O26
determined from its negative ion FAB-MS
(m/z: 1243 [M 2 H]2) as well as 13C NMR
and DEPT spectral data. The spectral
evidence indicated that 5 had the same
aglycon as that of 4 (Table 1), but differed in
the sugar moiety. The pentasaccharide
The structure of compound 3 reported
by Liu et al. [11] is the same as that of
marstenacisside G (3). However, com-
pound 3 exists in a fraction of extract from
Dregea sinensis var. corrugate instead of
being isolated. Its structure was deduced
from HPLC-HR-ESI-MS and HPLC-
DADESIMSLC-ESI. Also, the reported
data (Table 2 in Liu’s article: ESI-MS
[M þ Na]þ 1267 and the molecular
formula C61H96O25Na) of compound 3
are in agreement neither with its structure
nor with Marstenacisside G (3) (FAB-MS
1259 [M 2 H]2 and the molecular for-
mula is C61H96O27). No further data of
compound 3 were reported in Liu’s article.
Marstenacisside F (4), an amorphous
solid, had the molecular formula
C67H106O30 determined from its negative
ion FAB-MS (m/z: 1389 [M 2 H]2) as well
as 13C NMR and DEPT spectral data. The
spectral evidence indicated that 4 had the
same aglycon as that of 3 (Table 1), but
1
feature of 5 was proved by its H and 13C
NMR spectra [dH 4.70 (1H, dd, J ¼ 9.9,
2.0 Hz), 5.08 (1H, d, J ¼ 8.0 Hz), 5.13 (1H,
dd, J ¼ 9.8, 2.1 Hz), 5.18 (1H, d,
J ¼ 7.8 Hz), and 5.33 (1H, dd, J ¼ 9.8,
2.0 Hz); dC 101.9, 104.3, 95.9, 100.5, and
105.0] (Tables 2 and 3). Detailed analysis of
the 13C NMR, 1H–1H COSY, TOCSY,
ROESY, HMQC, and HMBC spectra of 5
indicated that the sugar moiety of 5 was
composed of one oleandropyranose, two
cymaropyranoses, and two glucopyranoses.
The connectivity of the sugar units of 5 was
deduced from the results of the HMBC and
ROESY experiments. Therefore, 5 was
established as 3-O-b-D-glucopyranosyl-
(1 ! 4)-b-D-glucopyranosyl-(1 ! 4)-b-D-
oleandropyranosyl-(1 ! 4)-b-D-cymaro-
pyranosyl-(1 ! 4)-b-D-cymaropyranosyl-
12-O-benzoyl-dihydrosarcostin.
differed in the sugar moiety. The 1H and 13
C
NMR spectra of the sugar moiety of 4
(Tables 2 and 3) [dH 4. 79 (1H, dd, J ¼ 9.8,
2.0 Hz), 4.89 (1H, d, J ¼ 8.8 Hz), 5.12(1H,
d, J ¼ 7.8 Hz), 5.18 (1H, dd, J ¼ 9.8,
2.0 Hz), 5.21 (1H, d, J ¼ 7.8 Hz), and 5.54
(1H, dd, J ¼ 9.8, 2.1 Hz); dC 101.9, 103.9,
104.8, 99.8, 105.0, and 95.8] indicated that
the sugar moiety of 4 was a hexasaccharide.
Finally, these above results showed the
presence of the b configuration at 1 ! 4
linkage in all of the sugar moieties of five
new pregnane glycosides 1, 2, 3, 4, and 5.
In their HMBC and ROESY spectra,
correlations of H-1/C-4 and H-1/H-4
signals were observed without an excep-
tion, respectively. On the other hand, all of
the C-4 signals at the connected position in
1
Comparison of 13C NMR, H–1H COSY,

484
Z.-H. Xia et al.
the sugar moieties appeared ca. 10 ppm
downfield without regard to 2-hydroxy
(Glc, Thev) or 2-deoxy (Cym, Ole) sugars.
H2O gradient system (0 to 95%) to afford
four fractions (Fr. 1–Fr. 4). Fr. 4 (12 g)
eluted with 95% EtOH was subjected to a
silica gel column eluted with CHCl3:-
MeOH:H2O (7:1:0.05; 5:1:0.1; 4:1:0.1) to
give three fractions (Fr. 4-1–Fr. 4-3),
respectively. Fr. 4-2 (2.0 g) was subjected
to an RP-18 column eluted with 65%
MeOH–H2O and further separated by
silica gel column chromatography eluted
with EtOAc:MeOH:H2O (8:1:1), to get
compounds 1 (60 mg), 2 (52 mg), 3
(105 mg), 4 (40 mg), and 5 (18 mg).
3. Experimental
3.1 General experimental procedures
IR was recorded on a Perkin-Elmer 599
infrared spectrometer. The 1H and 13C
NMR and all 2D NMR spectra were
recorded on a JEOL a600 with AFG-type
field gradient unit, TMS as internal
standard, C5D5N as solvent. FAB-MS
was measured on a MAT-95 mass
spectrometer. Lichroprep RP-18 (25–
40 mm, Merck, Darmstadt, Germany),
Diaion HP-20 (Mitsubishi Kasei, Tokyo,
Japan), and silica gel 60H (Qingdao
Haiyang Chemical Group Co. Qingdao,
China) were used for column chromatog-
raphy. TLC was performed on silica gel
HSGF254 (Zhifu Huangwu Co. Ltd. of
Yantai, Yantai, China). Spots were visu-
alized by spraying with 10% H2SO4 in
95% EtOH followed by heating.
3.3.1 Marstenacisside E (1)
An amorphous solid, IR nKmBaxr: 3425,
1
1060 cm21. The H NMR (C5D5N) spec-
tral data of aglycon moiety of 1: d 1.19
(3H, s, H-19), 1.27 (3H, d, J ¼ 6.1 Hz, H-
21), 2.20 (3H, s, H-18), 3.91 (1H, m, H-3),
4.09 (1H, q, J ¼ 6.1 Hz, H-20), 5.32 (1H,
dd, J ¼ 4.3, 11.5 Hz, H-12), 7.40 (2H, t,
J ¼ 8.2 Hz, H-30, 50), 7.49 (1H, t,
J ¼ 8.2 Hz, H-40), and 8.50 (2H, d,
J ¼ 8.2 Hz, H-20, 60). The 13C NMR
spectral data of the aglycon moiety of 1
are given in Table 1. The 1H and 13C NMR
spectral data of the sugar moiety of 1 are
given in Tables 2 and 3. FAB-MS m/z:
1227 [M 2 H]2.
3.2 Plant materials
The stems of M. tanecissima were
purchased in Kunming, Yunnan Province
(China), in 2000. Botanical identification
was made by Dr Wang-Xing Xing and
Prof. He-Ming Mi (The Second Military
Medical University). A voucher specimen
(No. 7) is deposited at the Herbarium of
the Department of Phytochemistry, Shang-
hai Institute of Materia Medica, Chinese
Academy of Sciences.
3.3.2 Marstenacisside F (2)
An amorphous solid, IR nKmBaxr: 3438,
1060 cm21. The 1H NMR spectral data
(C5D5N) of aglycon moiety of 2: d 1.19
(3H, s, H-19), 1.27 (3H, d, J ¼ 6.1 Hz, H-
21), 2.20 (3H, s, H-18), 3.91 (1H, m, H-3),
4.05 (1H, q, J ¼ 6.1 Hz, H-20), 5.31 (1H,
dd, J ¼ 4.5, 11.5 Hz, H-12), 7.40 (2H, t,
J ¼ 8.2 Hz, H-30, 50), 7.50 (1H, t,
J ¼ 8.2 Hz, H-40), and 8.53 (2H, d,
J ¼ 8.2 Hz, H-20, 60). The 13C NMR
spectral data of the aglycon moiety of 2
are given in Table 1. The 1H and 13C NMR
spectral data of the sugar moiety of 2 are
given in Tables 2 and 3. FAB-MS m/z:
1097 [M 2 H]2.
3.3 Extraction and isolation
The powdered stems of M. tenacissima
(15 kg) were extracted with 95% EtOH
under reflux. After evaporation of ethanol
in vacuo, the residue was suspended in
water and then extracted successively with
petroleum ether, EtOAc, and n-BuOH. The
n-BuOH fraction (180 g) was subjected to
a Diaion HP-20 column using an EtOH–

Journal of Asian Natural Products Research
485
3.3.3 Marstenacisside G (3)
spectral data of the sugar moiety of 5 are
given in Tables 2 and 3. FABMS m/z: 1243
[M 2 H]2.
An amorphous solid, IR nKmBaxr: 3423,
1060 cm21. The 1H NMR spectral data
(C5D5N) of the aglycon moiety of 3: d 1.18
(3H, s, H-19), 1.27 (3H, d, J ¼ 6.1 Hz, H-
21), 2.19 (3H, s, H-18), 3.91 (1H, m, H-3),
4.08 (1H, q, J ¼ 6.1 Hz, H-20), 5.31 (1H,
dd, J ¼ 4.3, 11.5 Hz, H-12), 7.39 (2H, t,
J ¼ 8.2 Hz, H-30, 50), 7.50 (1H, t,
J ¼ 8.2 Hz, H-40), and 8.52 (2H, d,
J ¼ 8.2 Hz, H-20, 60). The 13C NMR
spectral data of the aglycon moiety of 3
are given in Table 1. The 1H and 13C NMR
spectral data of the sugar moiety of 3 are
given in Tables 2 and 3. FAB-MS m/z:
1259 [M 2 H]2.
3.4 Acid hydrolysis
Compound 1 (5 mg) was heated in 5 ml of
0.1 N HCl:dioxane (1:1) at 958C for 2 h.
The reaction mixture was concentrated in
vacuo and 3 ml of H2O was added. The
mixture was neutralized with 10% KOH
and then partitioned with CHCl3–H2O to
get the aglycon and the sugar fractions.
The sugars were identified by comparison
of Rf values with those of authentic
samples on TLC [(silica gel, developed
with CHCl3:MeOH:H2O (3:1:0.1) and
EtOAc:MeOH:H2O (6:1.1:1)], detected
by spraying with aniline–phthalic acid
reagent [aniline–phthalic acid–n-BuOH
(4:5:0.5)] and then heating to 1108C.
3.3.4 Marstenacisside H (4)
An amorphous solid, IR nKmBaxr: 3448,
1060 cm21. The 1H NMR spectral data
(C5D5N) of the aglycon moiety of 4: d 1.19
(3H, s, H-19), 1.30 (3H, d, J ¼ 6.1 Hz, H-
21), 2.19 (3H, s, H-18), 3.90 (1H, m, H-3),
4.05 (1H, q, J ¼ 6.1 Hz, H-20), 5.31 (1H,
dd, J ¼ 4.3, 11.3 Hz, H-12), 7.40 (2H, t,
J ¼ 8.2 Hz, H-3’, 5’), 7.50 (1H, t,
J ¼ 8.2 Hz, H-40), and 8.51 (2H, d,
J ¼ 8.2 Hz, H-20, 60). The 13C NMR
spectral data of the aglycon moiety of 4
are given in Table 1. The 1H and 13C NMR
spectral data of the sugar moiety of 4 are
given in Tables 2 and 3. FAB-MS m/z:
138M 2 H]2.
References
[1] Jiangsu New College of Medicine, Zhong-
Yao-Da-Ci-Dian (Shanghai Science and
Technology Publishing Co., Shanghai,
1977), p. 1976.
[2] S. Miyakawa, K. Yamaura, and K.
Hayashi, Phytochemistry 25, 2861 (1986).
[3] J.J. Chen, Z.X. Zhang, and J. Zhou, Acta
Bot. Yunnan. 21, 369 (1999).
[4] J. Deng, Z.X. Liao, and D.F. Chen,
Phytochemistry 66, 1040 (2005).
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3.3.5 Marstenacisside I (5)
An amorphous solid, IR nKmBaxr: 3448,
1060 cm21. The 1H NMR spectral data
(C5D5N) of the aglycon moiety of 5: d 1.17
(3H, s, H-19), 1.31 (3H, d, J ¼ 6.1 Hz, H-
21), 2.19 (3H, s, H-18), 3.88 (1H, m, H-3),
4.07 (1H, q, J ¼ 6.1 Hz, H-20), 5.31 (1H,
dd, J ¼ 4.3, 11.5 Hz, H-12), 7.42 (2H, t,
J ¼ 8.2 Hz, H-30, 50), 7.50 (1H, t,
J ¼ 8.2 Hz, H-4’), and 8.53 (2H, d,
J ¼ 8.2 Hz, H-20, 60). The 13C NMR
spectral data of the aglycon moiety of 5
are given in Table 1. The 1H and 13C NMR
[11] Y.B. Liu, E.N. Su, J.B. Li, J.L. Zhang,
S.S. Yu, J. Qu, J. Liu, and Y. Li, J. Nat.
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