Four new immunomodulating steroidal glycosides from the stems of Stephanotis mucronata

Article abstract of DOI:10.1016/j.steroids.2006.04.007

Four new C-21 steroidal glycosides, mucronatosides E (1), F (2), G (3), and H (4), were isolated from the stems of Stephanotis mucronata. Two of them had the rare aglycone with a double bond between C-6 and C-7. Their structures were elucidated on the bas

Full text of DOI:10.1016/j.steroids.2006.04.007

steroids 7 1 ( 2 0 0 6 ) 683–690
available at www.sciencedirect.com
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Four new immunomodulating steroidal glycosides from the
stems of Stephanotis mucronata
Xiaoyu Lia,b, Hongxiang Sunc, Yiping Yeb, Fengyang Chenb, Jue Tuc, Yuanjiang Pana,∗
a
Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China
Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou 310013, PR China
College of Animal Sciences, Zhejiang University, Hangzhou 310029, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Four new C-21 steroidal glycosides, mucronatosides E (1), F (2), G (3), and H (4), were isolated
from the stems of Stephanotis mucronata. Two of them had the rare aglycone with a double
bond between C-6 and C-7. Their structures were elucidated on the basis of spectroscopic
data and chemical evidence. These isolated compounds were assayed for their immunolog-
ical activities in vitro against concanavalin A (Con A)- and lipopolysaccharide (LPS)-induced
proliferation of mice splenocytes. Compounds 2 and 4 showed immunosuppressive activi-
ties in a dose-dependent manner, while compounds 1 and 3 possessed immunoenhancing
activities.
Received 15 September 2005
Received in revised form 29 March
2006
Accepted 17 April 2006
Published on line 21 June 2006
Keywords:
© 2006 Elsevier Inc. All rights reserved.
Stephanotis mucronata
Asclepiadaceae
Immunomodulating activities
1.
Introduction
adaceae) are used for the treatment of rheumatoid arthritis
and rheumatic aches in Chinese folk medicine. We have ear-
Immunosuppressive agents are the mainstay treatment for
patients that have received organ grafts and are becoming
increasingly important in the treatment of autoimmune dis-
eases. There are, however, many problems with both the con-
cept and reality of long-term immunosuppression as a ther-
apeutic modality, both in terms of the nonspecific toxicity of
the drugs that are currently available and the increased risk
of infections and tumors arising from global suppression of
the immune system. The immunosuppressant with less side
effects is still a challenge to the medical system.
C-21 steroidal glycosides have attracted much attention
for their antitumor, antiepilepsy, and antifertility activities,
and many such compounds have been isolated from plants,
especially from those of the Asclepiadaceae family [1–4]. The
dried roots of Stephanotis mucronata (Blanco) Merr. (Asclepi-
lier reported the isolation and structural elucidation of 12
pregnane glycosides: mucronatosides A–D, and stephanoside
E from the stems of S. mucronata [5,6], and stemucronatosides
A–G from the roots of S. mucronata [7,8]. We have also reported
the immunomodulating activity of stemucronatosides A–G
[7,8]. Among those compounds, stemucronatosides A–C, and
F showed immunomodulating activities, while stemucronato-
sides D, E, and G showed immunosuppressive activities in a
dose-dependent manner.
In this paper, the chemical studies on the CHCl3 soluble
extract from the stems of this plant were further undertaken,
leading to four new immunomodulating C-21 steroidal glyco-
sides: mucronatosides E–H (1–4) (Fig. 1). These results might be
responsible, at least in part, for the treatment of rheumatoid
arthritis and rheumatic aches in folk.
∗
Corresponding author. Tel.: +86 571 879 51264; fax: +86 571 879 51264.
E-mail address: panyuanjiang@css.zju.edu.cn (Y. Pan).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2006.04.007

684
steroids 7 1 ( 2 0 0 6 ) 683–690
2.2.
Plant material
The stems of S. mucronata were obtained from Yueqing, Zhe-
jiang province, China. A voucher specimen (No. 200208) was
identified by Prof. Zhang Zhi-Guo and deposited in the Lab-
oratory of Natural Products Chemistry, Institute of Materia
Medica, Zhejiang Academy of Medical Sciences, Hangzhou,
China.
2.3.
Extraction and isolation
The dried stems of S. mucronata (6.0 kg) were ground and
extracted three times with 95% EtOH under reflux for 2 h. The
extracts were evaporated in vacuo to give the EtOH extract
(570 g). The ethanolic extract was partitioned with CHCl3
under reflux. Concentration of the CHCl3 extract yielded a
yellow residue (223.5 g). The CHCl3 extract was partitioned
with hexane under reflux. The hexane insoluble portion corre-
sponded to a crude glycoside (193.5 g). The crude glycoside was
subjected to CC (silica gel, gradient CHCl3/MeOH 40:1 → 5:1,
v/v) to give 8 main fractions. Fr. 3 (6.2 g) was subjected to CC
(Rp-18, MeOH/H2O 1:1 → 2:1, v/v; then HPLC, MeOH/H2O 70:30,
v/v) to give 4 (135 mg). Fr. 4 (9.4 g) was subjected to CC (Rp-18,
MeOH/H2O 1:1 → 2:1, v/v; then HPLC, MeOH/H2O 67:33, v/v) to
give 1 (155 mg), 2 (142 mg), and 3 (86 mg).
2.3.1. Mucronatoside E (1)
C48
H
78O21
:
white amorphous powder; mp 155–158◦;
[˛]2D0 + 34.1◦ (MeOH; ca. 0.1); UV (MeOH) ꢀmax
: 207.0 nm;
IR (KBr) ꢁmax: 3510 (OH), 1701 (C O), 1640 (C C), 1165 (C O),
1080 (C
O
C) cm−1. HRESI-MS: 1013.4923 ([C48H78O21 + Na]+,
calcd. 1013.4928). ESI-MS (positive) m/z: 1013.4 [M + Na]+,
851.4 [M + Na − 162]+, 691.3 [M + Na − 162-160]+, 547.2
[M + Na − 162-160-144]+, 403.1 [M + Na − 162 − 160-144-144]+,
Fig. 1 – The chemical structures of compounds 1–4.
633.2 [M + Na − 380]+, 489.2 [M + Na − 380-144]+. For 1H and 13
C
NMR data, see Tables 1 and 2.
2.3.2. Mucronatoside F (2)
80O21: white amorphous powder; mp 165–167◦; [˛]D20
+
2.
Experimental
C48
H
18.0◦ (MeOH; ca. 0.1); UV (MeOH) ꢀmax: 207.0 nm; IR (KBr)
2.1.
General methods
ꢁmax: 3480 (OH), 1640 (C C), 1165 (C O), 1080 (C O .
C) cm−1
HRESI-MS: 1015.5103 ([C48H80O21 + Na]+, calcd. 1015.5084). ESI-
MS (positive) m/z: 1015.4 [M + Na]+, 853.4 [M + Na − 162]+,
693.3 [M + Na − 162-160]+, 549.3 [M + Na − 162-160-144]+, 633.2
[M + Na − 382]+, 489.1 [M + Na − 382-144]+, 345.1 [M + Na − 382-
144-144]+. For 1H and 13C NMR data, see Tables 1 and 2.
Melting points were determined on an MRK Prism Scope
instrument and are uncorrected. Optical rotations were mea-
sured in MeOH with a Rudolph Research Autopol IV polarime-
ter. UV spectra were performed on a TU-1800PC spectrometer.
IR spectra were recorded in a KBr pellet on a Perkin-Elmer 577
spectrometer. 1H NMR, 13C NMR, DEPT, 1H, 1H COSY, HMQC,
and HMBC spectra were recorded at 500 MHz for 1H, and at
125 MHz for 13C, with a Bruker DRX 500 instrument in pyridine-
d5 solution. HRESI-MS spectra were recorded on a Bruker Dal-
tonics FTMS APEX III mass spectrometer. ESI-MS spectra were
recorded on a Bruker Esquire 3000plus mass spectrometer. TLC
was performed on precoated Kieselgel 60 F254 plates and Rp C18
(Merck), and the detection was achieved by spraying with 10%
H2SO4 followed by heating. Separation and purification were
performed by column chromatography on silica gel (200–300
mesh, Qingdao), Rp C18 (Merck), and on semi-prep HPLC using
an Agilent 1100 instrument (Zorbax column 9 mm × 250 mm,
DAD).
2.3.3. Mucronatoside G (3)
C62H92O24: white amorphous powder; mp 158–161◦; [˛]D20
+
80.1◦ (MeOH; ca. 0.1); UV (MeOH) ꢀmax: 222.5, 279.0 nm; IR (KBr)
ꢁmax: 3480 (OH), 1710 (C O), 1640 (C C), 1170 (C O),
1080 (C
C) cm−1. HRESI-MS: 1243.5894 ([C62H92O24 + Na]+,
C
C
O
calcd. 1243.5871). ESI-MS (positive) m/z: 1243.4 [M + Na]+, 1095.4
[M + Na − 148]+, 1143.4 [M + Na − 100]+, 995.4 [M + Na − 148-
100]+, 633.3 [M + Na − 610]+, 489.2 [M + Na − 610-144]+. For 1H
and 13C NMR data, see Tables 1 and 2.
2.3.4. Mucronatoside H (4)
C
53H78O19
:
white amorphous powder; mp 153–156◦;
[˛]2D0 + 72.1◦ (MeOH; ca. 0.1); UV (MeOH) ꢀmax: 222.0, 278.5 nm;

steroids 7 1 ( 2 0 0 6 ) 683–690
685
Table 1 – 13C NMR data of compounds 1–4 (ı in ppm,
Table 1 (Continued)
C5D5N)a
1
2
3
4
1
2
3
4
5ꢀꢀꢀ
6ꢀꢀꢀ
72.0 (d)
18.7 (q)
72.1 (d)
18.8 (q)
71.9 (d)
18.5 (q)
72.7 (d)
18.5 (q)
1
2
3
4
5
6
7
8
39.0 (t)
29.4 (t)
78.0 (d)
39.3 (t)
139.4 (s)
119.5 (d)
35.1 (t)
74.3 (s)
45.0 (d)
37.4 (s)
29.9 (t)
68.9 (d)
60.4 (s)
89.3 (s)
34.2 (t)
32.8 (t)
92.6 (s)
9.4 (q)
39.2 (t)
29.2 (t)
77.9 (d)
39.5 (t)
139.3 (s)
119.9 (d)
35.5 (t)
74.2 (s)
44.7 (d)
37.5 (s)
30.1 (t)
70.9 (d)
58.8 (s)
89.0 (s)
34.7 (t)
34.3 (t)
89.1 (s)
11.5 (q)
18.5 (q)
73.2 (d)
17.9 (q)
27.6 (t)
26.6 (t)
74.9 (d)
39.1 (t)
74.8 (s)
136.6 (d)
127.4 (d)
74.0 (s)
36.6 (d)
39.6 (s)
23.7 (t)
75.6 (d)
58.0 (s)
88.2 (s)
33.2 (t)
34.3 (t)
87.8 (s)
12.4 (q)
21.6 (q)
74.5 (d)
15.6 (q)
27.6 (t)
26.6 (t)
74.9 (d)
39.0 (t)
74.8 (s)
136.6 (d)
127.4 (d)
74.0 (s)
36.7 (d)
39.6 (s)
23.7 (t)
75.8 (d)
58.0 (s)
88.2 (s)
33.1 (t)
33.9 (t)
87.5 (s)
12.4 (q)
21.6 (q)
74.5 (d)
15.4 (q)
OCH3
60.6 (q)
60.7 (q)
60.6 (q)
61.0 (q)
d-Glc
1ꢀꢀꢀꢀ
2ꢀꢀꢀꢀ
3ꢀꢀꢀꢀ
4ꢀꢀꢀꢀ
5ꢀꢀꢀꢀ
6ꢀꢀꢀꢀ
106.0 (d)
75.8 (d)
78.7 (d)
72.0 (d)
78.1 (d)
63.1 (t)
106.1 (d)
75.9 (d)
78.8 (d)
72.1 (d)
78.2 (d)
63.2 (t)
105.9 (d)
75.8 (d)
78.6 (d)
71.9 (d)
78.2 (d)
63.1 (t)
9
10
11
12
13
14
15
16
17
18
19
20
21
Multiplicities by DEPT experiments in parentheses; s: quaternary,
d: CH, t: CH2, and q: CH3 C-atoms. allme: 6-deoxy-3-O-methyl-
␤-allopyranosyl; cym: cymaropyranosyl; glc: glucopyranosyl; thv:
thevetopyranosyl.
a
1H NMR, 13C NMR, DEPT, 1H, 1H COSY, HMQC and HMBC spectra
were obtained at 500 and 125 MHz at room temperature, respec-
tively.
18.5 (q)
209.6 (s)
27.9 (q)
12-O-Acyl
cin
cin
1
2
3
4
5, 9
6, 8
7
166.7 (s)
120.4 (d)
143.8 (d)
135.2 (s)
128.5 (d)
129.3 (d)
130.5 (d)
166.8 (s)
120.5 (d)
144.1 (d)
135.1 (s)
128.6 (d)
129.4 (d)
130.5 (d)
IR (KBr) ꢁmax: 3485 (OH), 1735 (C O), 1711 (C
C C O), 1638
(C C), 1165 (C O), 1080 (C
O
C) cm−1. HRESI-MS: 1041.4990
([C53H78O19 + Na]+, calcd. 1041.5030). ESI-MS (positive) m/z:
1041.4 [M + Na]+, 981.4 [M + Na − 60]+, 893.4 [M + Na − 148]+,
875.4 [M + Na − 148-18]+, 833.4 [M + Na − 148-60]+, 715.4
[M + Na − 148-18-160]+, 571.3 [M + Na − 148-18-160-144]+, 471.2
[M + Na − 570]+, 327.1 [M + Na − 570-144]+. For 1H and 13C NMR
data, see Tables 1 and 2.
20-O-Acyl
tig
ac
169.8 (s)
21.4 (q)
1
2
3
4
5
166.7 (s)
129.4 (s)
137.7 (d)
14.1 (q)
12.2 (q)
2.3.5. Acid hydrolysis of 1, 2, 3 and 4
Each solution of 40 mg of 1, 2, 3, and 4 in 10 ml MeOH was
treated with 10 ml of 0.1N H2SO4, and each mixture was kept
at 60 ◦C for 2 h, then diluted with H2O (20 ml) and concen-
trated to 30 ml. The solution was kept for 60 ◦C for another
hour, then neutralized with aq. Satd Ba(OH)2. The precipita-
tion was then filtered off. The filtrate was concentrated to
dryness and chromatographed on a column of silica gel with
CHCl3/MeOH(100:1 → 50:1, v/v) to afford 5 (12.1 mg) from 1,
6 (10.3 mg) from 2, 7 (9.4 mg) from 3, and 8 (11.4 mg) from
4, respectively. The sugar components in each hydrolysate
were identified by TLC comparison with authentic samples.
Cymarose was identified with the solvent A: CHCl3/MeOH
(9:1); solvent B: Me2CO/petrol ether (2:3); and solvent C:
CH2Cl2/EtOH (9:1). Glucose was identified with the solvent
CHCl3/MeOH/H2O (4:3:1).
d-Cym
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
96.4 (d)
37.0 (t)
78.1 (d)
83.1 (d)
69.3 (d)
18.4 (q)
96.5 (d)
37.1 (t)
78.2 (d)
83.5 (d)
69.2 (d)
18.7 (q)
97.7 (d)
36.9 (t)
78.0 (d)
82.9 (d)
69.3 (d)
18.4 (q)
97.7 (d)
36.8 (t)
77.8 (d)
82.9 (d)
69.1 (d)
18.4 (q)
OCH3
58.9 (q)
59.0 (q)
58.9 (q)
58.8 (q)
d-Cym
1ꢀꢀ
2ꢀꢀ
3ꢀꢀ
4ꢀꢀ
5ꢀꢀ
6ꢀꢀ
100.4 (d)
37.3 (t)
77.7 (d)
83.0 (d)
69.0 (d)
18.6 (q)
100.5 (d)
37.4 (t)
78.1 (d)
83.1 (d)
69.4 (d)
18.6 (q)
100.3 (d)
36.8 (t)
77.9 (d)
82.9 (d)
69.1 (d)
18.6 (q)
100.3 (d)
36.8 (t)
78.1 (d)
83.0 (d)
69.3 (d)
18.5 (q)
2.3.6. 12-O-deacetylmetaplexigenin (5)
C
21H32O6: colorless needles (MeOH); ESI-MS m/z 403.1
OCH3
59.0 (q)
59.1 (q)
58.8 (q)
58.8 (q)
[M + Na]+. 13C NMR (C5D5N, 125 MHz): 39.0 (C-1), 31.9 (C-2), 71.4
(C-3), 43.2 (C-4), 140.1 (C-5), 118.6 (C-6), 34.0 (C-7), 74.2 (C-8),
44.8 (C-9), 37.2 (C-10), 29.3 (C-11), 68.8 (C-12), 60.2 (C-13), 89.2
(C-14), 34.9 (C-15), 32.6 (C-16), 92.4 (C-17), 9.2 (C-18), 18.3 (C-19),
209.4 (C-20), 27.7 (C-21). 1H NMR (C5D5N, 500 MHz): ı 3.93 (1H,
m, H-3), 5.42 (1H, br s, H-6), 3.98 (1H, dd, J = 11.5, 4.0 Hz, H-12),
2.04 (3H, s, H-18), 1.49 (3H, s, H-19), 2.68 (3H, s, H-21).
d-Allme
d-Thv
1ꢀꢀꢀ
2ꢀꢀꢀ
3ꢀꢀꢀ
4ꢀꢀꢀ
104.8 (d)
74.8 (d)
85.9 (d)
83.4 (d)
104.9 (d)
74.9 (d)
86.0 (d)
83.3 (d)
104.8 (d)
74.7 (d)
85.8 (d)
83.1 (d)
106.2 (d)
75.0 (d)
87.8 (d)
75.8 (d)

686
steroids 7 1 ( 2 0 0 6 ) 683–690
Table 2 – 1H NMR data of compounds 1–4 (ı in ppm, J in Hz, C5D5N)a
1
2
3
4
3
3.96, m
3.86, m
4.19, m
4.19, m
6
7
5.36, br s
5.37, br s
5.96, d (10.0)
6.27, d (10.0)
5.38, dd (10.5, 4.0)
2.17, s
1.56, s
5.13, q (6.0)
1.49, d (6.0)
5.96, d (10.5)
6.27, d (10.5)
5.38, dd (10.0, 3.5)
2.12, s
1.58, s
5.02, q (6.0)
1.43, d (6.0)
12
18
19
20
21
3.94, m
2.00, s
1.42, s
3.89, m
1.93, s
1.41, s
4.44, q (6.0)
1.51, d (6.0)
2.65, s
12-O-Acyl
cin
cin
2
3
5, 9
6, 8
7
6.76, d (16.0)
7.95, d (16.0)
7.67, d (6.5)
7.43, m
6.88, d (15.5)
8.04, d (15.5)
7.75, d (7.0)
7.41, m
7.40, m
7.38, m
20-O-Acyl
tig
ac
2
3
4
5
2.01, s
7.01, q (7.0)
1.51, d (7.0)
1.79, s
d-Cym
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
5.30, d (9.5)
1.73, m; 2.16, m
3.98, m
3.54, dd (9.5, 2.5)
4.24, m
5.29, d (9.5)
1.69, m; 2.16, m
3.95, m
3.47, dd (9.5, 2.5)
4.22, m
5.19, d (9.5)
1.76, m; 2.22, m
4.02, m
3.45, dd (9.5, 3.0)
4.23, m
5.19, d (9.0)
1.72, m; 2.23, m
4.03, m
3.46, dd (9.5, 2.5)
4.17, dq (9.5, 6.5)
1.37, d (6.0)
1.38, d (6.0)
1.36, d (6.0)
1.40, d (6.0)
OCH3
3.62, s
3.60, s
3.63, s
3.63, s
d-Cym
1ꢀꢀ
2ꢀꢀ
3ꢀꢀ
4ꢀꢀ
5ꢀꢀ
6ꢀꢀ
5.12, d (9.5)
1.78, m; 2.17, m
4.03, m
3.88, dd (10.5, 5.0)
4.22, m
5.08, d (10.0)
1.75, m; 2.18, m
4.05, m
3.84, m
4.19, m
5.12, d (10.0)
1.78, m; 2.24, m
4.08, m
3.91, m
4.21, m
5.13, d (10.0)
1.80, m; 2.26, m
4.08, m
3.61, dd (10.0, 2.0)
4.23, dq (9.5, 6.0)
1.62, d (6.0)
1.58, d (6.5)
1.55, d (6.0)
1.58, d (6.0)
OCH3
3.58, s
3.56, s
3.59, s
3.58, s
d-Allme
d-Thv
1ꢀꢀꢀ
2ꢀꢀꢀ
3ꢀꢀꢀ
4ꢀꢀꢀ
5ꢀꢀꢀ
6ꢀꢀꢀ
5.15, d (10.0)
3.92, t (9.0)
3.72, t (8.0)
3.54, dd (9.5, 2.5)
3.81, m
5.12, d (10.0)
3.88, t (9.0)
3.69, t (8.0)
3.49, dd (10.0, 2.5)
3.74, dq (8.5, 6.0)
1.73, d (6.0)
5.16, d (10.5)
3.93, m
3.75, m
3.53, dd (9.5, 2.0)
3.79, dq (8.5, 6.0)
1.79, d (6.0)
4.80, d (7.5)
3.95, m
3.66, m
3.64, m
3.76, dq (8.5, 6.0)
1.61, d (6.0)
1.76, d (6.0)
OCH3
3.96, s
3.92, s
3.97, s
3.93, s
d-Glc
1ꢀꢀꢀꢀ
2ꢀꢀꢀꢀ
3ꢀꢀꢀꢀ
4ꢀꢀꢀꢀ
5ꢀꢀꢀꢀ
6””
4.72, d (7.5)
4.04, m
4.25, m
4.21, m
4.03, m
4.69, d (7.5)
4.05, m
4.22, m
4.20, m
4.01, m
4.73, d (7.5)
4.07, m
4.28, m
4.22, m
4.09, m
4.37, dd (11.5, 5.5)
4.55, dd (11.5, 2.5)
4.35, dd (11.0, 5.0)
4.52, d (10.5)
4.40, dd (11.0, 5.5)
4.58, dd (11.0, 2.5)
cym: cymaropyranosyl; glc: glucopyranosyl; thv: thevetopyranosyl.
a
Allme: 6-deoxy-3-O-methyl-␤-allopyranosyl.

steroids 7 1 ( 2 0 0 6 ) 683–690
687
J = 7.0 Hz, H-5ꢀ,9ꢀ), 7.38 (2H, m, H-6ꢀ,8ꢀ), 7.35 (1H, m, H-7ꢀ), 2.02
(1H, s, H-2ꢀꢀ).
2.3.7. Sarcostin (6)
C21 34O6: colorless needles (MeOH). ESI-MS m/z 405.1
H
[M + Na]+. 13C NMR (C5D5N, 125 MHz): 38.9 (C-1), 31.2 (C-2), 70.7
(C-3), 42.2 (C-4), 139.4 (C-5), 118.5 (C-6), 34.8 (C-7), 73.6 (C-8),
44.1 (C-9), 36.9 (C-10), 29.4 (C-11), 70.3 (C-12), 58.4 (C-13), 88.4
(C-14), 34.1 (C-15), 33.6 (C-16), 88.9 (C-17), 10.6 (C-18), 18.3 (C-
19), 72.5 (C-20), 17.8 (C-21). 1H NMR (C5D5N, 500 MHz): ı 3.93
(1H, m, H-3), 5.40 (1H, br s, H-6), 3.97 (1H, m, H-12), 1.96 (3H, s,
H-18), 1.39 (3H, s, H-19), 4.40 (1H, q, J = 6.0 Hz, H-20), 1.51 (3H,
d, J = 6.0 Hz, H-21).
2.4.
Splenocyte proliferation assay
Single cell suspensions were prepared as previously described
[9]. Splenocytes were seeded into four wells of a 96-well flat-
bottom microtiter plate (Nunc) at a cell density of 1 × 107 per
ml in 100 ␮l of complete medium, into which 100 ␮l of 1–4
and CsA (0.01–10 ␮g/ml) were added, followed by Con A (final
concentration 5 mg l−1), LPS (final concentration 10 mg l−1), or
medium. The plate was incubated at 37 ◦C under a humidified
2.3.8. 12-O-cinnamoyl-20-O-tigloyl(20S)-pregn-6-ene-
3ˇ,5˛,8ˇ,12ˇ,14ˇ,17ˇ,20-heptanol (7)
atmosphere of 5% CO2. After 44 h, 50 ␮l of MTT solution (2 g l−1
)
C35
H
46O9: colorless needles (MeOH). ESI-MS m/z 633.1
was added to each of the wells, which were incubated for 4 h.
The microtiter plates were centrifuged (1400 × g, 5 min), and
the untransformed MTT was removed carefully by pipetting.
Next, 200 ␮l of a Me2SO working solution (192 ␮l Me2SO with
8 ␮l HCl 1 mol l−1) was added to each well, and the absorbance
(A) was evaluated after 15 min in an ELISA reader at 570 nm
with a 630 nm reference.
[M + Na]+. 13C NMR (C5D5N, 125 MHz): 27.3 (C-1), 28.7 (C-2),
66.7 (C-3), 41.3 (C-4), 75.3 (C-5), 136.8 (C-6), 127.1 (C-7), 74.1
(C-8), 36.4 (C-9), 39.8 (C-10), 23.8 (C-11), 75.7 (C-12), 58.0 (C-
13), 88.3 (C-14), 33.2 (C-15), 34.5 (C-16), 87.8 (C-17), 12.4 (C-18),
21.8 (C-19), 74.6 (C-20), 15.6 (C-21), 166.9 (C-1ꢀ), 120.5 (C-2ꢀ),
143.7 (C-3ꢀ), 135.1 (C-4ꢀ), 128.7 (C-5ꢀ,9ꢀ), 129.3 (C-6ꢀ,8ꢀ), 130.5
(C-7ꢀ), 166.9 (C-1ꢀꢀ), 129.4 (C-2ꢀꢀ), 138.1 (C-3ꢀꢀ), 14.2 (C-4ꢀꢀ), 12.3
(C-5ꢀꢀ). 1H NMR (C5D5N, 500 MHz): ı 4.32 (1H, m, H-3), 5.98
(1H, d, J = 10.0 Hz, H-6), 6.23 (1H, d, J = 10.0 Hz, H-7), 5.39 (1H,
dd, J = 10.0, 4.0 Hz, H-12), 2.12 (3H, s, H-18), 1.60 (3H, s, H-
19), 5.12 (1H, q, J = 6.0 Hz, H-20), 1.49 (3H, d, J = 6.0 Hz, H-21),
6.75 (1H, d, J = 16.0 Hz, H-2ꢀ), 7.92 (1H, d, J = 16.0 Hz, H-3ꢀ), 7.64
(2H, dd, J = 7.0, 1.5 Hz, H-5ꢀ, 9ꢀ), 7.39 (2H, dd, J = 7.0, 6.5 Hz,
H-6ꢀ, 8ꢀ), 7.36 (1H, ddd, J = 7.0, 6.5, 1.5 Hz, H-7ꢀ), 6.96 (1H, q,
J = 6.5 Hz, H-3ꢀꢀ), 1.46 (3H, d, J = 6.5 Hz, H-4ꢀꢀ), 1.77 (3H, s, H-
5ꢀꢀ).
3.
Results and discussion
The EtOH extract of the stems of S. mucronata was extracted
with CHCl3. The CHCl3 soluble portion was subsequently sep-
arated using silica, reversed-phase silica gel column chro-
matography and semi-prep HPLC to yielded four compounds
(1–4). These compounds showed positive Libermann–Buchard
and Keller–Kiliani reactions, indicating the presence of
a
13C NMR (DMSO-d6, 125 MHz): 26.3 (C-1), 27.4 (C-2), 66.0
(C-3), 40.1(C-4), 74.3 (C-5), 135.7 (C-6), 126.1 (C-7), 73.0 (C-8),
34.8 (C-9), 38.4 (C-10), 22.6 (C-11), 74.8 (C-12), 57.1 (C-13), 87.2
(C-14), 32.2 (C-15), 33.4 (C-16), 87.1 (C-17), 11.7 (C-18), 21.3
(C-19), 73.6 (C-20), 15.2 (C-21), 165.7 (C-1ꢀ), 119.3 (C-2ꢀ), 143.5
(C-3ꢀ), 134.1 (C-4ꢀ), 128.4 (C-5ꢀ,9ꢀ), 129.0 (C-6ꢀ,8ꢀ), 130.1 (C-7ꢀ),
166.1 (C-1ꢀꢀ), 128.4 (C-2ꢀꢀ), 137.7 (C-3ꢀꢀ), 14.3 (C-4ꢀꢀ), 12.1 (C-5ꢀꢀ).
1H NMR (DMSO-d6, 500 MHz): ı 3.97 (1H, m, H-3), 5.38 (1H,
d, J = 10.0 Hz, H-6), 5.65 (1H, d, J = 10.0 Hz, H-7), 4.72 (1H, dd,
J = 10.0, 4.5 Hz, H-12), 1.46 (3H, s, H-18), 0.92 (3H, s, H-19), 4.45
(1H, q, J = 6.0 Hz, H-20), 1.09 (3H, d, J = 6.0 Hz, H-21), 6.21 (1H, d,
J = 16.0 Hz, H-2ꢀ), 7.42 (1H, d, J = 16.0 Hz, H-3ꢀ), 7.60 (2H, dd, J = 7.5,
1.5 Hz, H-5ꢀ,9ꢀ), 7.39 (2H, m, H-6ꢀ,8ꢀ), 7.38 (1H, m, H-7ꢀ), 6.75
(1H, q, J = 7.0 Hz, H-3ꢀꢀ), 1.61 (3H, d, J = 7.0 Hz, H-4ꢀꢀ), 1.53 (3H, s,
H-5ꢀꢀ).
steroidal glycoside with a 2-deoxysugar moiety. Each of the
isolates was subjected to detailed spectroscopic analysis to
establish their chemical structures.
Mucronatoside E (1) was isolated as an amorphous pow-
der. Based on the HRESI-MS data, the molecular formula of 1
was determined to be C48H78O21 (1013.4923 [M + Na]+, calcd.
1013.4928). Mild acid hydrolysis of 1 afforded an aglycone (5),
cymarose, d-glucose, and an unidentified sugar (on TLC). The
aglycone was identified as 12-O-deacetylmetaplexigenin, by
comparison of its spectroscopic data to those in the literature
[7]. The NMR (1H NMR, 13C NMR, DEPT, HMQC, and HMBC)
spectral data of compound 1 showed that it contained four
anomeric carbon signals at ı 96.4, 100.4, 104.8, and 106.0,
correlating with anomeric protons at ı 5.30 (d, J = 9.5 Hz),
5.12 (d, J = 9.5 Hz), 5.15 (d, J = 10.0 Hz), and 4.72 (d, J = 7.5 Hz),
respectively, which indicated that there were four sugar units
in the compound. The sugar moieties of 1 were identical
to those of stemucronatoside C, by comparison of their 1H
NMR and 13C NMR data and TLC evidence with those in
the literature [7]. The sugar sequence of compound 1 was
confirmed by the HMBC spectrum, which showed long-range
correlations between (i) H-1ꢀ of the ␤-d-cymaropyranose (ıH
5.30, 1H, d, J = 9.5 Hz) and C-3 of the aglycone (ıC 78.0); (ii)
H-1ꢀꢀ of the ␤-d-cymaropyranose (ıH 5.12, 1H, d, J = 9.5 Hz)
and C-4ꢀ of the ␤-d-cymaropyranose (ıC 83.1); (iii) H-1ꢀꢀꢀ of the
6-deoxy-3-O-methyl-␤-d-allopyranose (abbreviated as allme)
(ıH 5.15, 1H, d, J = 10.0 Hz) and C-4ꢀꢀ of the ␤-d-cymaropyranose
(ıC 83.0); and (iv) H-1ꢀꢀꢀꢀ of the ˇ-d-glucopyranose (ıH 4.72,
1H, d, J = 7.5 Hz) and C-4ꢀꢀꢀ of the 6-deoxy-3-O-methyl-␤-d-
allopyranose (ıC 83.4). Thus, the sequence and linkage sites
2.3.9. 12-O-cinnamoyl-20-O-acetyl(20S)-pregn-6-ene-
3ˇ,5˛,8ˇ,12ˇ,14ˇ,17ˇ,20-heptanol (8)
C32H42O9: colorless needles (MeOH). ESI-MS m/z 593.1
[M + Na]+. 13C NMR (C5D5N, 125 MHz): 27.5 (C-1), 28.7 (C-2), 66.6
(C-3), 41.3 (C-4), 75.1 (C-5), 136.5 (C-6), 127.3 (C-7), 74.2 (C-8),
36.5 (C-9), 39.7 (C-10), 23.7 (C-11), 75.7 (C-12), 58.1 (C-13), 88.3
(C-14), 33.1 (C-15), 34.1 (C-16), 87.7 (C-17), 12.5 (C-18), 21.7 (C-
19), 74.6 (C-20), 15.6 (C-21), 166.9 (C-1ꢀ), 120.5 (C-2ꢀ), 143.8 (C-3ꢀ),
135.1 (C-4ꢀ), 128.6 (C-5ꢀ,9ꢀ), 129.1 (C-6ꢀ,8ꢀ), 130.5 (C-7ꢀ), 169.7 (C-
1ꢀꢀ), 21.5 (C-2ꢀꢀ). 1H NMR (C5D5N, 500 MHz): ı 4.26 (1H, m, H-3),
5.94 (1H, d, J = 10.5 Hz, H-6), 6.22 (1H, d, J = 10.5 Hz, H-7), 5.36
(1H, dd, J = 10.0, 3.5 Hz, H-12), 2.12 (3H, s, H-18), 1.59 (3H, s, H-
19), 5.06 (1H, q, J = 6.0 Hz, H-20), 1.47 (3H, d, J = 6.0 Hz, H-21), 6.85
(1H, d, J = 16.0 Hz, H-2ꢀ), 7.97 (1H, d, J = 16.0 Hz, H-3ꢀ), 7.70 (2H, d,

688
steroids 7 1 ( 2 0 0 6 ) 683–690
of the sugar units were established as glc-(1 → 4)-allme-
(1 → 4)-cym-(1 → 4)-cym-(1 → 3)-aglycone. This assignment
was consistent with the ESI-MS data, which showed the
quasi-molecular ion at m/z 1013.4 [M + Na]+, and other
prominent fragments at m/z 851.4 [M + Na − glc]+, 691.3
[M + Na − glc-allme]+, 547.2 [M + Na − glc-allme-cym]+, and
403.1, which corresponded to the aglycone moiety. In the
13C NMR spectrum of 1, glycosidation shifts were observed
at C-2, C-3, and C-4 by −2.5, +6.6 and −3.9, respectively,
when compared with 13C chemical shifts of 5, indicating
that the sugar chain was linked to the C-3 hydroxyl group
of the aglycone. Thus, the structure of 1 was established
as 12-O-deacetylmetaplexigenin 3-O-␤-d-glucopyranosyl-
(1 → 4)-6-deoxy-3-O-methyl-␤-d-allopyranosyl-(1 → 4)-␤-d-
cymaropyranosyl-(1 → 4)-␤-d-cymaropyranoside.
H-20 (ıH 4.45) and C-1ꢀꢀ of the tigloyl (ıC 166.1). Thus, 7 was
determined to be 12-O-cinnamoyl-20-O-tigloyl (20S)-pregn-6-
ene-3␤,5␣,8␤,12␤,14␤,17␤,20-heptanol. The 1H and 13C NMR
spectral data for the aglycone moiety of compound 3 were
similar to those of 7 with the major difference being the
absence of signals for an OH group at C-3. The only other
difference in 13C NMR spectral data between 3 and 7 occurred
for C-atoms C-2, C-3, and C-4, by −2.1, +8.2, and −2.2, respec-
tively. The 1H and 13C NMR signals due to the sugar moieties
of 3 were also superimposable onto those of 1. Consequently,
the structure of 3 was established as 12-O-cinnamoyl-20-O-
tigloyl(20S)-pregn-6-ene-3␤,5␣,8␤,12␤,14␤,17␤,20-heptanol 3-
O-␤-d-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-␤-d-allo-
pyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-␤-d-
cymaropyranoside.
Mucronatoside F (2) possessed the molecular formula
Mucronatoside H (4) had the molecular formula C53H78O19
(1041.4990 [M + Na]+, calcd. 1041.5030) on the basis of the
HRESI-MS data. Mild acid hydrolysis of 4 afforded an agly-
cone (8), cymarose, and an unidentified sugar (on TLC). The
molecular formula of 8 was deduced as C32H42O9 by the quasi-
molecular ion peak at m/z 593.1 [M + Na]+ in the ESI-MS spectra
and its 13C NMR data. The spectral analysis of 8 revealed
a distinct similarity to 7. In comparison to 7, 8 showed the
absence of the tigloyl group, but suggested the presence of
an acetyl group. In the HMBC spectrum of 8, the following
long-range correlations were observed: (i) between H-12 (ıH
5.36) and C-1ꢀ of the cinnamoyl (ıC 166.9), and (ii) between
H-20 (ıH 5.06) and C-1ꢀꢀ of the acetyl (ıC 169.7). Thus, 8 was
determined to be 12-O-cinnamoyl-20-O-acetyl(20S)-pregn-6-
C
48H80O21 (1015.5103 [M + Na]+, calcd. 1015.5084) on the basis
of the HRESI-MS. Mild acid hydrolysis of 2 afforded an aglycone
(6) and a sugar mixture. 6 was identical to sarcostin by com-
parison of its spectroscopic data with those in the literature
[10]. In 1975, the absolute configuration of 6 was confirmed
by the X-ray analysis of its 3-O-p-bromobenzoate which
showed S-configuration at C-20 by Hayashi and Mitsuhashi
et al. [11]. Moreover, in the 20-hydroxy-C/d-cis-pregnane type
steroids the free rotation of the side-chain is considered to be
restricted by steric hindrance among the C-18 methyl, C-21
methyl and 20-hydroxyl groups on the basis of a CPK model
[12]. In compound 6, the C-17 OH further restricted the rotate
of the side-chain. Thus, the NOESY evidence of (i) H-12 at ı
3.97 and H-20 at ı 4.40; (ii) H-16␣ at ı 1.90 and H-20 at ı 4.40;
and (iii) H-16␤ at ı 1.85 and H-21 at ı 1.51 can further confirm
the 20-S configuration. The 1H and 13C NMR spectral data for
the aglycone moiety of 2 were similar to those of sarcostin (6)
with the major difference being the absence of signals of an
OH group at C-3. The only other difference in the 13C NMR
data between 2 and 6 occurred for C-2, C-3, and C-4, by −2.0,
+7.2, and −2.7, respectively. Therefore, the sugar moiety was
linked to the C-3 hydroxyl group of the aglycone. The NMR
assignments of 2 were made unambiguously on the basis of
HMQC, HMBC and 1H, and 1H COSY experiments; by compar-
ison to those of 1, 2 was determined to have the same sugar
sequence in the oligosaccharide moiety. Thus, compound
ene -3␤,5␣,8␤,12␤,14␤,17␤,20-heptanol. The NMR (1H NMR, 13
C
NMR, DEPT, HMQC, and HMBC) spectral data of compound 4
showed that it contained three anomeric carbon signals at ı
97.7, 100.3, and 106.2, correlating with anomeric protons at
ı 5.19 (d, J = 9.0 Hz), 5.13 (d, J = 10.0 Hz), and 4.80 (d, J = 7.5 Hz),
respectively, which indicated that there were three sugar units
in compound 1. The sugar moieties of 4 were identical to those
of stemucronatoside B, deduced by comparing of their 1H and
13C NMR data and TLC data to those in the literature [7]. The
sequence of sugars in 4 was confirmed by the HMBC spectrum,
which showed distinct cross-peaks of correlation between (i)
H-1ꢀ of the ␤-d-cymaropyranose (ıH 5.19, 1H, d, J = 9.0 Hz) and
C-3 (ıC 74.9); (ii) H-1ꢀꢀ of the ␤-d-cymaropyranose (ıH 5.13, 1H,
d, J = 10.0 Hz) and C-4ꢀ of the ␤-d-cymaropyranose (ıC 82.9); and
(iii) H-1ꢀꢀꢀ of the ␤-d-thevetopyranose (ıH 4.80, 1H, d, J = 7.5 Hz)
and C-4ꢀꢀ of the ␤-d-cymaropyranose (ıC 83.0). The glycosida-
tion shifts of the carbon signals of 4 were observed at C-2,
C-3, and C-4, by −2.1, +8.3, and −2.3, respectively, compared
to 8, indicating the sugar chain linked to the C-3 hydroxyl
group of the aglycone. Consequently, the structure of 4 was
established as 12-O-cinnamoyl-20-O-acetyl(20S)-pregn-6-ene-
3␤,5␣,8␤, 12␤,14␤,17␤,20-heptanol 3-O-␤-d-thevetopyranosyl-
(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Comparing the 13C-NMR data of compounds 1–4 (Table 1),
the chemical shifts of the C-1 and C-9 of compounds 3 and 4
are ca. 12 and 9 ppm high-field shifted than that of compounds
1 and 2, respectively. Those phenomena could be relate to that
(i) compounds 3 and 4 had the 5␣-OH group, which make the
␥-position carbons C-1 and C-9 up-field, and (ii) C-1 and C-9 in
compounds 3 and 4 were in the shielding regions of the double
bond between C-6 and C-7.
2
was established as sarcostin 3-O-␤-d-glucopyranosyl-
(1 → 4)-6-deoxy-3-O-methyl-␤-d-allopyranosyl-(1 → 4)-␤-d-
cymaropyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Mucronatoside G (3) possessed the molecular formula
C
62H92O24 (1243.5894 [M + Na]+, calcd. 1243.5871) on the
basis of the HRESI-MS. Mild acid hydrolysis of 3 afforded an
aglycone (7) and a sugar mixture. The molecular formula of 7
was deduced as C35H46O9 by the quasi-molecular ion peak at
m/z 633.1 [M + Na]+ in the ESI-MS spectra and by its 13C NMR
data. 7 was identical to 12-O-cinnamoyl-20-O-tigloyl(20S)-
pregn-6-ene-3␤,5␣,8␤,12␤,14␤,17␤,20-heptanol by comparison
of its spectroscopic data with those in the literature [13],
but the C-1 chemical shifts of the cinnamoyl group and the
tigloyl group were both at 166.9 ppm. In order to locate the
cinnamoyl group and tigloyl group in 7, a HMBC experiment
(in DMSO-d6) had to be carried out. In the HMBC spectrum,
the following long-range correlations were observed between
(i) H-12 (ıH 4.72) and C-1ꢀ of the cinnamoyl (ıC 165.7); and (ii)

steroids 7 1 ( 2 0 0 6 ) 683–690
689
Fig. 2 – Effect of compounds 1–4 on Con A-stimulated splenocyte proliferation in vitro. Splenocytes were cultured with the
various concentrations of compounds 1–4 or CsA and Con A (5 ␮g/ml) in RPMI 1640 medium for 48 h. Cellular proliferation
was measured by the MTT method as described in the text and shown as a stimulation index. The values are presented as
means S.E. (n = 4). Significant differences compared to 0 ␮g/ml are designated as aP < 0.05, bP < 0.01, and cP < 0.001. CsA:
cyclosporin A (positive drug).
Fig. 3 – Effect of compounds 1–4 on LPS-stimulated splenocyte proliferation in vitro. Splenocytes were cultured with the
various concentrations of compounds 1–4 or CsA and LPS (10 ␮g/ml) in RPMI 1640 medium for 48 h. Cellular proliferation
was measured by the MTT method as described in the text and shown as a stimulation index. The values are presented as
means S.E. (n = 4). Significant differences compared to 0 ␮g/ml are designated as aP < 0.05, bP < 0.01, and cP < 0.001. CsA:
cyclosporin A (positive drug).
The effects of 1–4 on mitogen-stimulated mice splenocyte
proliferation in vitro are shown in Figs. 2 and 3. Compounds 2
Acknowledgements
and 4 significantly inhibited Con A- and LPS-stimulated mice
We gratefully acknowledge the Zhejiang Provincial Natu-
splenocyte proliferation in a dose-dependent manner. How-
ral Science Foundation of China (Grant-in Aid No. M303749)
ever, compound 1 and 3 significantly enhanced Con A- and
and the Zhejiang Provincial Science and Technology Council
LPS-induced mice splenocyte proliferation at suitable concen-
(Grant-in Aid No. 2004F13G1360002) for financial support.
trations. There were no significant differences in inhibitory
effect on mitogen-stimulated splenocyte proliferation among
CsA, compounds 2 and 4 at the concentration of 1 ␮g/ml. CsA
r e f e r e n c e s
showed cytotoxic activity mice splenocyte at the concentra-
tion of 10 ␮g/ml, while compounds 2 and 4 had no influences
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