Eight new C-21 steroidal glycosides from Dregea sinensis var. corrugata

Article abstract of DOI:10.1016/j.steroids.2007.03.002

Eight new steroidal glycosides, dresiosides were isolated from the stems of Dregea sinensis var. corrugate and their structures were deduced on the basis of chemical and spectral evidence. The sugar units of dresiosides were determined as deoxysugars afte

Full text of DOI:10.1016/j.steroids.2007.03.002

steroids 7 2 ( 2 0 0 7 ) 514–523
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Eight new C-21 steroidal glycosides from
Dregea sinensis var. corrugata
Yunbao Liu, Wenzhao Tang, Shishan Yu∗, Jing Qu, Jing Liu, Yue Liu
Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education & Institute of Materia
Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, No. 1 Xian Nong Tan Street, Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight new steroidal glycosides, dresiosides were isolated from the stems of Dregea sinensis
var. corrugate and their structures were deduced on the basis of chemical and spectral evi-
dence. The sugar units of dresiosides were determined as deoxysugars after hydrolysis and
isolation from natural glycosides. The absolute configurations of deoxysugars were deter-
mined as D on the basis of their optical rotation values in comparison with appropriate
standards.
Received 19 July 2006
Received in revised form
11 February 2007
Accepted 6 March 2007
Published on line 18 March 2007
© 2007 Elsevier Inc. All rights reserved.
Keywords:
Dregea sinensis var. corrugata
Asclepiadaceae
Steroidal glycosides
1.
Introduction
2.
Experimental
Dregea sinensis var. corrugata (Asclepiadaceae) has been used
as an antiepileptic and diuretic in China. A pharmacological
study showed that the crude glycosides from D. sinensis var.
corrugata exhibited antiepileptic activity. Shen et al. [1] iso-
lated one steroidal glycoside from D. sinensis var. corrugate;
Jin et al. [2–6] also isolated six steroidal glycosides from this
plant. In this paper we describe the isolation and character-
ization of eight new polyhydroxy C-21 steroidal glycosides
(1–8). The structures of these compounds are based on the
skeleton of dihydrosarcostin, as well as acetyl, methylbutyryl,
benzoyl, cinnamoyl, and nicotinoyl ester moieties linked at
C-12 or C-20 of the aglycon, respectively. In addition, all com-
pounds possess an oligosaccharide chain consisting of three
or four sugar units at C-3 of the aglycon. The structures were
elucidated mainly through the use of NMR techniques and
chemical methods.
2.1.
General methods
Melting points were measured on an XT-4 micromelting point
apparatus and were uncorrected. Optical rotations were deter-
mined on a Perkin-Elmer 241 automatic digital polarimeter. IR
spectra were recorded on a Nicolet-Impact 400 IR spectrom-
eter with KBr disks. UV spectra were obtained on a Shimadzu
UV-260 spectrometer. 1D NMR and 2D NMR experiments were
performed on an Inova 400 FT-NMR spectrometer. TMS was
used as internal standard. ESIMS were measured on Agilent
1100 Series LC/MSD Trap mass spectrometer. HRESIMS were
measured on Bruker FTMS APEXIII 7.0T mass spectrometer.
Silica gel GF254 for TLC and silica gel (200–300 mesh) for
column chromatography were obtained from Qingdao Marine
Chemical Company, Qingdao, People’s Republic of China.
Rp-18 (40–75 ␮m) silica gel was purchased from Fuji Silysica
∗
Corresponding author. Tel.: +86 10 63165324; fax: +86 10 63017757.
E-mail address: yushishan@imm.ac.cn (S. Yu).
0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2007.03.002

steroids 7 2 ( 2 0 0 7 ) 514–523
515
100 MHz): see Tables 1 and 2; ESIMS m/z: 1090 [M + Na]+, HRES-
IMS m/z: 1068.5504 [M + H]+ (calcd. for C57H82O18N: 1068.5532).
Chemical Ltd. Amersham Pharmacia Biotech AB, Sweden,
produced Sephadex LH-20. HPLC was carried on Shimadazu
LC-6AD and the detector was SPD-6A. Solvents were analyt-
ical grade and purchased from Beijing Chemical Company,
Beijing, People’s Republic of China. A preparative reversed
phase C18 column (YMC-Pack ODS-A 20 mm × 250 mm, 10 ␮m)
and an analytical reversed phase C18 column (Diamonsil C18
4.6 mm × 250 mm, 5 ␮m) were employed.
2.3.1. Mild acid hydrolysis of 1
A solution of 1 (20 mg) in CH3OH (5 ml) was added to 0.05 M
H2SO4 (5 ml), and the solution was kept at 50 ◦C for 1 h. Then
the solution was neutralized with Ba(OH)2 saturated with H2O
and the precipitate was filtered off. The filtrate was partitioned
with CHCl3 to give compound 1a (12 mg).
2.2.
Plant material
Compound 1a colorless needles (CHCl3); ESIMS m/z: 642
[M + Na]+; 1H NMR (pyridine-d5, 400 MHz): ı 1.12 (3H, s, H-19),
1.53 (3H, d, J = 6.0 Hz, H-21), 2.05 (3H, s, H-18), 3.55 (1H, m, H-3),
5.21 (1H, dd, J = 6.8, 11.2 Hz, H-12), 5.24 (1H, q, J = 7.2 Hz, H-20),
6.51 (1H, d, J = 16.0 Hz, H-2 of cin), 7.72 (1H, d, J = 16.0 Hz, H-3 of
cin), 7.33 (3H, m, H-5/7/9 of cin), 7.39 (2H, m, H-6/8 of cin), 8.75
(1H, dd, J = 5.6, 1.6 Hz, H-3 of nic), 8.23 (1H, dd, J = 6.0, 2.0 Hz, H-5
of nic), 7.15 (1H, J = 4.8, 8.0 Hz, H-6 of nic), 9.47 (1H, J = 1.6 Hz,
H-7 of nic).
The stems of D. sinensis var. corrugata were collected in
November 2004 from Jiangxi Province, China. It was iden-
tified by associate Prof. Lin Ma, Department of Chemistry
of Natural Products, Institute of Materia Medica, Chinese
Academy of Medical Sciences & Peking Union Medical College.
The stems were harvested and air-dried at room temper-
ature in shade. A voucher specimen (No. 20040618) was
deposited in the herbarium of Institute of Materia Medica, Chi-
nese Academy of Medical Sciences & Peking Union Medical
College.
2.3.2. Alkaline hydrolysis of 1a
1a (7 mg) was dissolved in 5% methanolic KOH (20 ml) and
refluxed for 5 h. After adding H2O (20 ml), CH3OH was removed
under reduced pressure. The aqueous concentrated was
extracted with CHCl3–CH3OH (9:1), dried over Na2SO4, filtered
and evaporated to dryness yielding 1b (4 mg).
2.3.
Extraction and isolation
The air-dried stems (11 kg) of D. sinensis var. corrugate were
extracted with 95% EtOH under reflux. The 95% EtOH solu-
tion was combined and evaporated in vacuum to yield 980 g
residue. The residue was suspended in water and partitioned
with petroleum, EtOAc, and n-BuOH, successively. The par-
tial EtOAc extract (100 g) was chromatographed on the silica
gel (3000 g) column (12 cm × 150 cm) eluted with CHCl3/CH3OH
(100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, 0:100, v/v) to yield 8 frac-
tions (Frs. 1–8). Fraction 3 (8 g) was subjected to Sephadex
LH-20 eluted with CH3OH to obtain three fractions (A–C). Frac-
tion A was submitted to Rp-18 silica gel column eluted with
CH3OH/H2O (60:40 → 100:1, v/v) and then submitted to prepar-
ative HPLC on a Rp-18 column (250 mm × 20 mm, wavelength
208 nm, flow rate 5 ml/min) with CH3OH/H2O (90:10, v/v) to
give compound 6 (25 mg, tR = 31.7 min). Fraction 4 (12 g) was
subjected to Sephadex LH-20 eluted with CH3OH to obtain
two fractions (I and II). Fraction I was submitted to Rp-18 sil-
ica gel column eluted with CH3OH/H2O (50:50 → 90:10, v/v)
and then purified with preparative HPLC on a Rp-18 column
(250 mm × 20 mm, wavelength 208 nm, flow rate 5 ml/min)
with CH3OH/H2O (80:20, v/v) to give compounds 1 (52 mg,
tR = 39.1 min), 2 (22 mg, tR = 40.2 min), 3 (25 mg, tR = 41.2 min),
Compound 1b C21H36O6; colorless needles (CHCl3); mp
244–247 ◦C; [˛]D25 : +49.8◦ (c 0.1, CH3OH); 1H NMR (pyridine-d5,
400 MHz): ı 1.11 (3H, s, H-19), 1.54 (3H, d, J = 6.0 Hz, H-21), 2.07
(3H, s, H-18), 3.57 (1H, m, H-3), 4.02 (1H, dd, J = 6.8, 11.2 Hz,
H-12␣), 4.26 (1H, q, J = 7.2 Hz, H-20). The spectral data were
identical to those of dihydrosarcostin in literature [7].
Compound 2 C55H86O20; white amorphous powder; mp
220–222 ◦C; [˛]D25 : +17.9◦ (c 0.1, CH3OH); UV (CH3OH): ꢀmax
230.4 nm; IR (KBr): ꢁmax 3452, 2935, 1711, 1601, 1452, 1383,
1279, 1068, 1001, 964, 864, 717, 669 cm−1 1H NMR (pyridine-
;
d5, 400 MHz): ı 1.15 (3H, s, H-19), 1.58 (3H, d, J = 6.0 Hz, H-21),
2.17 (3H, s, H-18), 3.88 (1H, m, H-3), 4.34 (1H, q, J = 7.2 Hz, H-
20), 5.26 (1H, dd, J = 6.8, 11.2 Hz, H-12), 7.36 (2H, t, J = 7.6 Hz,
H-4/6 of Bz), 7.45 (1H, t, J = 7.6 Hz, H-5 of Bz), 8.51 (2H, d,
J = 7.6 Hz, H-3/7 of Bz); Other 1H NMR data see Table 4. 13C
NMR (pyridine-d5, 100 MHz): see Tables 1 and 2; ESIMS m/z:
1089 [M + Na]+, HRESIMS m/z: 1089.5649 [M + Na]+ (calcd. for
C55H86O20Na: 1089.5610).
Compound 3 C57H81O18N; white amorphous powder; mp
215–217 ◦C; [˛]D25 : +34.2◦ (c 0.1, CH3OH); UV (CH3OH): ꢀmax
218.6 nm; IR (KBr): ꢁmax 3437, 2935, 1709, 1682, 1641, 1452, 1383,
7
(45 mg, tR = 42.5 min),
4
(32 mg, tR = 43.8 min),
8
(55 mg,
1281, 1205, 1138, 1070, 1003, 839, 800, 769, 723, 669 cm−1 1H
;
tR = 52.2 min), and 5 (53 mg, tR = 55.6 min).
NMR (pyridine-d5, 400 MHz): ı 1.10 (3H, s, H-19), 1.54 (3H, d,
J = 6.0 Hz, H-21), 2.09 (3H, s, H-18), 3.89 (1H, m, H-3), 5.24 (1H,
dd, J = 6.8, 11.2 Hz, H-12), 5.26 (1H, q, J = 7.2 Hz, H-20), 6.53 (1H,
d, J = 16.0 Hz, H-2 of cin), 7.79 (1H, d, J = 16.0 Hz, H-3 of cin), 7.32
(3H, m, H-5/9 and 7 of cin), 7.36 (2H, m, H-6/8 of cin), 8.79 (1H,
dd, J = 5.6, 1.6 Hz, H-3 of nic), 8.24 (1H, dd, J = 6.0, 2.0 Hz, H-5 of
nic), 7.12 (1H, J = 4.8, 8.0 Hz, H-6 of nic), 9.48 (1H, J = 1.6 Hz, H-7
of nic); Other 1H NMR data see Table 3. 13C NMR (pyridine-d5,
100 MHz): see Tables 1 and 2; ESIMS m/z: 1090 [M + Na]+, HRES-
IMS m/z: 1068.5581 [M + H]+ (calcd. for C57H82O18N: 1068.5532).
Compound 4 C57H88O20; white amorphous powder; mp
234–236 ◦C; [˛]D25 : +26.0◦ (c 0.1, CH3OH); UV (CH3OH): ꢀmax
245.6 nm; IR (KBr): ꢁmax 3436, 2935, 1709, 1639, 1595, 1452, 1383,
Compound 1 C57H81O18N; white amorphous powder; mp
211–213 ◦C; [˛]D25 : +49.4◦ (c 0.1, CH3OH); UV (CH3OH): ꢀmax
242.8 nm; IR (KBr): ꢁmax 3438, 2935, 1716, 1714, 1639, 1595,
1452, 1381, 1284, 1203, 1167, 1066, 1003, 945, 916, 864, 769, 744,
719 cm−1 1H NMR (pyridine-d5, 400 MHz): ı 1.11 (3H, s, H-19),
;
1.54 (3H, d, J = 6.0 Hz, H-21), 2.07 (3H, s, H-18), 3.87 (1H, m, H-3),
5.22 (1H, dd, J = 6.8, 11.2 Hz, H-12), 5.28 (1H, q, J = 7.2 Hz, H-20),
6.52 (1H, d, J = 16.0 Hz, H-2 of cin), 7.73 (1H, d, J = 16.0 Hz, H-3 of
cin), 7.32 (3H, m, H-5/9/7 of cin), 7.39 (2H, m, H-6/8 of cin), 8.79
(1H, dd, J = 5.6, 1.6 Hz, H-3 of nic), 8.24 (1H, dd, J = 6.0, 2.0 Hz, H-5
of nic), 7.14 (1H, J = 4.8, 8 Hz, H-6 of nic), 9.47 (1H, J = 1.6 Hz, H-7
of nic); Other 1H NMR data see Table 3. 13C NMR (pyridine-d5,

516
steroids 7 2 ( 2 0 0 7 ) 514–523
Table 1 – 13C NMR chemical shifts of compounds 1–8 in pyridine-d5 (100 MHz)
Aglycon moiety
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
37.8
29.6
76.6
34.4
45.2
24.6
34.4
76.0
46.7
36.4
25.1
75.0
57.4
87.6
33.5
34.0
88.7
12.0
12.9
76.3
15.4
38.0
29.6
76.6
33.8
45.3
24.6
34.5
75.8
46.8
36.5
25.2
76.0
57.3
88.2
33.8
32.8
88.8
12.2
12.9
70.7
19.5
37.8
29.5
76.6
34.4
45.2
24.6
34.4
75.8
46.7
36.4
25.1
76.0
57.3
88.2
33.5
34.0
88.7
12.0
12.9
76.3
15.4
38.0
29.5
76.5
34.4
45.2
24.6
34.4
75.9
46.9
36.4
25.2
76.3
57.2
88.1
33.8
33.8
88.8
12.1
12.9
70.7
19.3
37.8
29.5
76.5
34.4
45.2
24.5
34.4
76.0
46.7
36.4
25.1
75.0
57.4
87.6
33.5
34.0
88.7
12.0
12.8
76.3
15.4
37.6
29.5
76.6
34.4
45.2
24.6
34.4
76.0
46.8
36.4
25.1
75.9
57.3
87.6
33.5
34.3
88.4
11.5
13.0
76.3
15.4
38.0
29.5
76.6
34.4
45.2
23.9
34.4
76.0
47.1
36.5
25.1
74.5
58.7
89.3
33.6
33.5
92.3
11.3
13.0
210.5
27.8
38.1
29.5
76.6
34.4
45.2
23.9
34.5
76.0
47.2
36.5
25.1
74.1
58.5
89.3
33.6
34.5
92.3
11.2
13.0
210.2
27.7
9
10
11
12
13
14
15
16
17
18
19
20
21
Aglycon moiety
1
2
3
4
5
6
7
8
Cin at C-12 Bz at C-12 Nic at C-12 Cin at C-12 Cin at C-12 Ace at C-12 Bz at C-12 Cin at C-12
1
2
3
4
5/9
6/8
7
166.6
120.2
143.9
134.8
129.2
128.5
130.5
166.6
131.7
130.4
128.8
133.2
128.8
130.4
164.6
166.9
119.7
145.1
135.0
129.1
128.6
130.5
166.6
120.2
143.9
134.8
129.2
128.5
130.5
171.5
21.4
165.2
131.2
129.9
128.9
133.2
128.9
129.9
165.8
119.2
144.9
135.0
129.3
128.6
130.6
153.8
126.8
137.3
123.5
151.4
Aglycon moiety
1
2
3
4
5
6
7
8
Nic at C-20
Cin at C-20
Mebu at C-20
1
2
3
4
5
6
7
164.6
166.7
120.3
144.0
134.8
129.2
128.5
130.5
175.7
41.7
26.5
11.9
16.5
153.8
126.8
137.3
123.6
151.3
1277, 1205, 1136, 1068, 1003, 866, 800, 769, 721 cm−1
;
1H NMR
dd, J = 5.6, 1.6 Hz, H-3 of nic), 8.24 (1H, dd, J = 6.0, 2.0 Hz, H-5 of
nic), 7.14 (1H, J = 4.8, 8.0 Hz, H-6 of nic), 9.47 (1H, J = 1.6 Hz, H-7
of nic); Other 1H NMR data see Table 4. 13C NMR (pyridine-d5,
100 MHz): see Tables 1 and 2; ESIMS m/z: 1234 [M + Na]+, HRES-
IMS m/z: 1212.6294 [M + H]+ (calcd. for C64H94O21N: 1212.6318).
(pyridine-d5, 400 MHz): ı 1.20 (3H, s, H-19), 1.52 (3H, d, J = 6.0 Hz,
H-21), 2.11 (3H, s, H-18), 3.87 (1H, m, H-3), 4.24 (1H, q, J = 7.2 Hz,
H-20), 5.15 (1H, dd, J = 6.4, 11.2 Hz, H-12), 6.89 (1H, d, J = 16.0 Hz,
H-2 of cin), 8.11 (1H, d, J = 16.0 Hz, H-3 of cin), 7.27 (3H, m, H-5/9
and 7 of cin), 7.47 (2H, m, H-6/8 of cin); Other 1H NMR data see
Table 4. 13C NMR (pyridine-d5, 100 MHz): see Tables 1 and 2;
ESIMS m/z: 1115 [M + Na]+, HRESIMS m/z: 1115.5791 [M + Na]+
(calcd. for C57H88O20Na: 1115.5767).
Compound 6 C49H82O18; white amorphous powder; mp
203–205 ◦C; [˛]D25 : +19.4◦ (c 0.1, CH3OH); UV (CH3OH): ꢀmax
242.6 nm; 1H NMR (pyridine-d5, 400 MHz): ı 0.88 (3H, t, H-4 of
mebu), 1.08 (3H, d, H-5 of mebu), 1.12 (3H, s, H-19), 1.56 (3H, d,
J = 6.0 Hz, H-21), 1.92 (3H, s, H-18), 2.24 (3H, s, CH3CO), 3.93 (1H,
m, H-3), 5.04 (1H, q, J = 7.2 Hz, H-20), 5.29 (1H, dd, J = 6.8, 11.2 Hz,
H-12); Other 1H NMR data see Table 3. 13C NMR (pyridine-d5,
100 MHz): see Tables 1 and 2; ESIMS m/z: 981 [M + Na]+, HRES-
IMS m/z: 981.5391 [M + Na]+ (calcd. for C49H82O18Na: 981.5399).
Compound 7 C55H84O20; white amorphous powder; mp
243–245 ◦C; [˛]D25 : +123.1◦ (c 0.2, CH3OH); UV (CH3OH): ꢀmax
244.8 nm; IR (KBr): ꢁmax 3437, 2935, 1720, 1714, 1639, 1593, 1450,
Compound 5 C64H93O21N; white amorphous powder; mp
227–229 ◦C; [˛]D25 : +11.4◦ (c 0.1, CH3OH); UV (CH3OH): ꢀmax
268.4 nm; IR (KBr): ꢁmax 3446, 2935, 1714, 1712, 1641, 1590,
1452, 1383, 1275, 1165, 1070, 1003, 866, 800, 715, 669 cm−1 1H
;
NMR (pyridine-d5, 400 MHz): ı 1.11 (3H, s, H-19), 1.54 (3H, d,
J = 6.0 Hz, H-21), 2.08 (3H, s, H-18), 3.87 (1H, m, H-3), 5.22 (1H,
dd, J = 6.8, 11.2 Hz, H-12), 5.26 (1H, q, J = 7.2 Hz, H-20), 6.52 (1H,
d, J = 16.0 Hz, H-2 of cin), 7.73 (1H, d, J = 16.0 Hz, H-3 of cin), 7.32
(3H, m, H-5/9 and 7 of cin), 7.39 (2H, m, H-6/8 of cin), 8.79 (1H,
1381, 1282, 1167, 1065, 1003, 866, 744 cm−1 1H NMR (pyridine-
;

steroids 7 2 ( 2 0 0 7 ) 514–523
517
Table 2 – 13C NMR chemical shifts of compounds 1–8 in pyridine-d5 (100 MHz)
Sugar moiety
1
2
3
4
5
6
7
8
␤-d-cym
␤-d-cym
␤-d-cym
␤-d-cym
␤-d-cym
␤-d-cym
␤-d-cym
␤-d-cym
1
2
3
4
5
6
95.9
37.2
77.9
83.6
68.9
18.5
58.8
95.9
39.1
77.7
83.1
68.6
18.7
58.9
95.9
37.3
77.9
83.6
68.9
18.5
58.8
95.8
36.7
77.7
83.4
68.9
18.5
58.9
95.9
37.0
78.0
83.4
68.9
18.5
58.9
95.9
37.3
77.9
83.6
68.9
18.5
58.8
95.9
39.1
77.7
83.4
68.8
18.7
58.9
95.9
37.0
78.0
83.4
68.9
18.5
58.9
3-OMe
Sugar moiety
1
2
3
4
5
6
7
8
␤-d-ole
␤-d-dig
␤-d-ole
␤-d-dig
␤-d-cym
␤-d-ole
␤-d-dig
␤-d-ole
1
2
3
4
5
6
101.9
37.6
79.2
83.1
72.0
18.8
57.3
99.7
37.6
67.5
83.0
69.0
18.4
101.9
37.7
79.2
83.1
72.0
18.8
57.3
99.7
37.5
67.5
83.1
69.0
18.4
100.4
37.2
77.8
83.2
69.0
18.7
58.8
101.9
37.7
79.2
83.1
72.0
18.8
57.3
99.7
37.6
67.5
83.1
69.0
18.4
100.4
37.2
77.8
83.2
69.0
18.7
58.8
3-OMe
Sugar moiety
1
2
3
4
5
6
7
8
␤-d-the
␤-d-ole
␤-d-the
␤-d-ole
␤-d-ole
␤-d-the
␤-d-ole
␤-d-ole
1
2
3
4
5
6
104.1
75.2
88.2
76.0
72.8
18.7
60.9
101.9
36.7
79.2
83.4
72.0
18.8
57.4
104.1
75.2
88.2
76.0
72.9
18.7
60.9
101.9
37.5
79.1
83.1
72.0
18.8
57.2
101.9
37.6
79.2
83.0
72.0
18.8
57.3
104.1
75.2
88.2
76.0
72.9
18.7
60.9
101.9
36.7
79.2
83.0
72.0
18.8
57.3
101.9
37.6
79.2
83.0
72.0
18.8
57.3
3-OMe
Sugar moiety
1
2
3
4
5
6
7
8
␤-d-the
␤-d-the
␤-d-the
␤-d-the
␤-d-the
1
2
3
4
5
6
104.1
75.2
88.4
76.0
72.8
18.5
60.9
104.1
75.2
88.3
75.9
72.8
18.7
60.9
104.1
75.2
88.2
76.0
72.8
18.5
60.9
104.1
75.2
88.2
76.0
72.8
18.5
60.9
104.1
75.2
88.2
76.0
72.8
18.5
60.9
3-OMe
cym: Cymaropyranosyl; dig: digitoxopyranosyl; ole: oleandropyranosyl; the: thevetopyranosyl.
d5, 400 MHz): ı 1.15 (3H, s, H-19), 2.02 (3H, s, H-18), 2.30 (3H,
s, H-21), 3.87 (1H, m, H-3), 5.27 (1H, dd, J = 4, 11.2 Hz, H-12),
7.44 (3H, t, J = 7.6 Hz, H-4/6/5 of Bz), 8.25 (2H, d, J = 7.6 Hz, H-3/7
of Bz); Other 1H NMR data see Table 4. 13C NMR (pyridine-
d5, 100 MHz): see Tables 1 and 2; ESIMS m/z: 1087 [M + Na]+,
HRESIMS m/z: 1087.5434 [M + Na]+ (calcd. for C55H84O20Na:
1087.5454).
Compound 7a colorless needles (CHCl3); ESIMS m/z: 509
[M + Na]+; 1H NMR (pyridine-d5, 400 MHz): ı 1.14 (3H, s, H-19),
2.01 (3H, s, H-18), 2.33 (3H, s, H-21), 5.24 (1H, dd, J = 4.0, 11.2 Hz,
H-12), 3.53 (1H, m, H-3), 7.44 (3H, t, J = 7.6 Hz, H-4/6/5 of Bz), 8.26
(2H, d, J = 7.6 Hz, H-3/7 of Bz).
Compound 7b colorless needles (CHCl3); mp 193–196 ◦C;
[˛]2D5 + 39.6◦ (c 0.1, CH3OH); ESIMS m/z: 407 [M + Na]+; 1H NMR
(pyridine-d5, 400 MHz): ı 1.14 (3H, s, H-19), 2.01 (3H, s, H-18),
2.33 (3H, s, H-21), 3.24 (1H, dd, J = 4.0, 11.2 Hz, H-12), 3.53 (1H,
m, H-3). The spectral data were identical to those of tayloron
in literature [8].
2.3.3. Mild acid and alkaline hydrolysis of 7
A solution of 7 (20 mg) in CH3OH (5 ml) was added to 0.05 M
H2SO4 (5 ml), and the solution was kept at 50 ◦C for 1 h. Then
the solution was neutralized with Ba(OH)2 saturated with H2O
and the precipitate was filtered off. The filtrate was partitioned
with CHCl3 to give compound 7a (12 mg).
7a (7 mg) was dissolved in 5% methanolic KOH (10 ml)
and refluxed for 5 h. After adding H2O (10 ml), CH3OH was
removed under reduced pressure. The aqueous concentrated
was extracted with CHCl3, dried over Na2SO4, filtered and
evaporated to dryness yielding 7b (4.5 mg).
2.3.4. NaBH4 reduction of 7b
7b (4 mg) was dissolved in CH3OH (5 ml). NaBH4 was added,
and the mixture was kept for 2 h at room temperature, then
treated with cold 2N HCl, and extracted with ether. The
extracts were washed with 5% NaHCO3 and saturated NaCl
successively, dried over anhydrous sodium sulfate (Na2SO4),
and concentrated. Dihydrosarcostin was identified in the

518
steroids 7 2 ( 2 0 0 7 ) 514–523
tallized, [˛]2D5 + 38.6◦ (c 0.5, H2O) and was found to be identical
Table 3 – 1H NMR data of sugar moieties of compounds
1, 3 and 6 in pyridine-d5 (400 MHz)
to free d-digitose (50 mg) [9]. Methyl-␤-d-digitose was isolated
in the course of methanolic hydrolysis of glycosides. 1H NMR
(500 MHz, CDCl3) for methyl-␤-d-digitose: 4.71 (1H, dd, J = 10,
2 Hz, H-1), 4.11 (1H, d, J = 3 Hz, H-3), 3.74 (1H, m, H-5), 3.48
(3H, s, Me-1), 3.32 (1H, dd, J = 9, 2.5 Hz, H-4), 2.11 (1H, ddd,
J = 14, 5.5, 3 Hz, H-2), 1.71 (1H, ddd, J = 14, 10, 3 Hz, H-2ꢀ), 1.31
(3H, J = 6 Hz, 6-Me). 13C NMR (125 MHz, CDCl3) for methyl-␤-d-
digitose: 98.8 (C-1), 73.1 (C-4), 69.4 (C-5), 68.0 (C-3), 56.5 (OMe of
1), 37.6 (C-2), 18.1 (C-6). The remained mixture was separated
by column chromatography on silica gel [eluent: CHCl3–MeOH
(9:1)] to afford three free sugars, which were determined as d-
cymarose [9], d-oleandrose [10], and d-thevetose [11] according
to their [˛]D and NMR data.
Sugar moiety
1
3
6
␤-d-cym
␤-d-cym
␤-d-cym
1
2
3
4
5.29 (brd, 7.2)
1.82, 2.21 (m)
4.11 (brd, 2.4)
3.50 (m)
5.29 (brd, 8)
1.80, 2.23 (m)
4.11 (brd, 2.4)
3.52 (m)
5.29 (brd, 8.4)
1.90, 2.23(m)
4.05 (brd, 2.4)
3.53 (m)
5
6
4.17 (dd, 6,2)
1.36 (d, 6.4)
3.51 (s)
4.19 (dd, 6, 2.4)
1.42 (d, 6)
3.54 (s)
4.19 (dd, 6,2.4)
1.46 (d, 6.4)
3.53 (s)
3-OMe
Sugar moiety
1
3
6
␤-d-ole
␤-d-ole
␤-d-ole
1
2
3
4.69 (brd, 9.6)
1.74, 2.45 (m)
3.63 (m)
4.69 (brd, 9.6)
1.74, 2.45 (m)
3.65 (m)
4.70 (brd, 9.2)
1.70, 2.46 (m)
3.65 (m)
d-Cymarose: [˛]D25 + 47.6◦ (c 0.1, H2O) [Ref. [9] [˛]17 + 49.8◦,
D
H2O]. The ratio of ␣,␤-d-cymarose is considered to be ca.
9:10 based on integration of the anomeric signals. 1H NMR
(400 MHz, CD3OD) for ␣-d-cymarose: ı 5.49 (brs, H-1), 4.01 (brs,
4
3.63 (m)
3.62 (m)
3.64 (m)
5
3.61 (m)
3.61 (m)
3.60 (m)
H-4), 3.30 (3H, s, 3-OMe), 2.28–1.90 (m, H-2), 1.17 (s, 6-Me); 13
NMR (100 MHz, CD3OD) for ␣-d-cymarose: ı 99.5 (C-1), 89.3 (C-
3), 78.8 (C-4), 69.0 (C-5), 57.2 (3-OMe), 41.0 (C-2). 19.4 (6-Me). 1
C
6
1.69 (d, 6)
3.56 (s)
1.69 (d, 6)
3.58 (s)
1.68 (d, 6)
3.59 (s)
3-OMe
H
Sugar moiety
1
3
6
NMR (400 MHz, CD3OD) for ␤-d-cymarose: ı 4.93 (brd, J = 9.6 Hz,
H-1), 3.92 (1H, brs, H-4), 3.26 (3H, s, 3-OMe), 2.28–1.90 (m, H-2),
1.17 (s, 6-Me); 13C NMR (100 MHz, CD3OD) for ␤-d-cymarose:
ı 92.2 (C-1), 82.1 (C-3), 73.6 (C-4), 68.4 (C-5), 56.9 (3-OMe), 36.6
(C-2), 18.3 (6-Me).
␤-d-the
␤-d-the
␤-d-the
1
2
3
4
4.94 (d, 8)
3.85 (m)
3.63 (m)
3.59 (m)
4.94 (d, 7.6)
3.86 (m)
3.61 (m)
4.95 (brd, 7.8)
3.83 (m)
3.62 (m)
d-Oleandrose: [˛]D25 − 11.3◦ (c 0.1, H2O) [Ref. [10] [˛]25 − 12.5◦,
3.59 (m)
3.58 (m)
D
5
3.71 (dd, 8,2) 3.70 (dd, 8.4, 2.4) 3.70 (dd, 8.4, 2.4)
H2O]. The ratio of ␣, ␤-d-oleandrose is considered to be ca.
10:8 based on integration of the anomeric signals. 1H NMR
(400 MHz, CD3OD) for ␣-d-oleandrose: ı 5.16 (brd, J = 2.8 Hz, H-
1), 3.78 (m, H-3), 3.43 (m, H-3), 3.13 (m, H-5), 2.94 (m, H-4), 2.26
(ddd, J = 6, 4.8, 1.2 Hz, H-2), 1.42–1.24 (m, H-2), 1.19 (d, J = 6 Hz,
6-Me); 13C NMR (100 MHz, CD3OD) for ␣-d-oleandrose: ı 92.6
(C-1), 79.2 (C-3), 76.9 (C-4), 68.8 (C-5), 57.3 (OMe-3), 36.4 (C-2),
18.3 (6-Me). 1H NMR (400 MHz, CD3OD) for ␤-d-oleandrose: ı
4.68 (dd, J = 2, 9.6 Hz, H-1), 3.43 (m, H-3), 3.13 (m, H-5), 2.94
(m, H-4), 2.16 (dd, J = 4.8, 12.8 Hz, H-2), 1.42–1.24 (m, H-2ꢀ),
1.17 (d, J = 6.8 Hz, 6-Me); 13C NMR (100 MHz, CD3OD) for ␤-d-
oleandrose: ı 94.9 (C-1), 81.7 (C-3), 77.8 (C-4), 73.2 (C-5), 57.4
(OMe-3), 38.6 (C-2), 18.4 (6-Me).
6
1.58 (d, 6)
3.87 (s)
1.58 (d, 6)
3.86 (s)
1.58 (d, 6)
3.88 (s)
3-OMe
cym: Cymaropyranosyl; dig: digitoxopyranosyl; ole: oleandropyra-
nosyl; the: thevetopyranosyl.
concentrated solution by TLC comparison with authentic
sample.
Compound 8 C57H86O20; white amorphous powder; mp
231–233 ◦C; [˛]D25 : +22.4◦ (c 0.1, CH3OH); UV (CH3OH): ꢀmax
247.0 nm; IR (KBr): ꢁmax 3452, 2933, 1712, 1639, 1580, 1450,
1383, 1311, 1275, 1203, 1167, 1068, 1003, 866, 800, 769, 721, 669,
602, 561 cm−1 1H NMR (pyridine-d5, 400 MHz): ı 1.22 (3H, s, H-
;
d-Thevetose: [˛]D25 + 3.5◦ (c 0.5, H2O) [Ref. [11] [˛]25 + 3.9◦,
D
19), 1.99 (3H, s, H-18), 2.49 (3H, s, H-21), 5.12 (1H, dd, J = 4.4,
10.8 Hz, H-12), 3.91 (1H, m, H-3), 6.78 (1H, d, J = 16.0 Hz, H-2 of
cin), 7.77 (1H, d, J = 16.0 Hz, H-3 of cin), 7.33 (2H, m, H-5/9 of
cin), 7.34 (1H, m, H-7 of cin), 7.61 (2H, m, H-6/8 of cin); Other
1H NMR data see Table 4. 13C NMR (pyridine-d5, 100 MHz):
see Tables 1 and 2; ESIMS m/z: 1113 [M + Na]+, HRESIMS m/z:
1113.5593 [M + Na]+(calcd. for C57H86O20 Na: 1113.5610).
H2O]. The ratio of ␣, ␤-d-thevetose is considered to be ca.
1:2 based on integration of the anomeric signals. 1H NMR
(400 MHz, CD3OD) for ␣-d-thevetose: ı 4.70 (brd, J = 1.6 Hz, H-
1), 3.89 (m, H-4), 3.61 (s, 3-OMe), 1.22 (d, 5.6 Hz, Me-6); 13C
NMR (100 MHz, CD3OD) for ␣-d-thevetose: ı 102.6 (C-1), 86.3
(C-3), 79.5 (C-4), 75.7 (C-2), 72.5 (C-5), 63.2 (OMe-3), 18.6 (Me-
6). 1H NMR (400 MHz, CD3OD) for ␤-d-thevetose: ı 4.43 (1H, dd,
5.6, 9.6 Hz, H-1), 3.89 (m, H-4), 3.39 (s, 3-OMe), 1.22 (d, 5.6 Hz,
Me-6); 13C NMR (100 MHz, CD3OD) for ␤-d-thevetose: ı 99.4
(C-1), 83.9 (C-3), 78.4 (C-4), 75.2 (C-2), 71.9 (C-5), 61.2 (OMe-3),
18.0 (Me-6).
2.3.5. Mild acid hydrolysis of crude glycosides and
isolation of sugars which contained in compounds 1–8
A solution of crude glycosides (1 g) in MeOH (100 ml) was mixed
with 0.05 M H2SO4 (100 ml) and warmed for 30 min at 50◦.
H2O (100 ml) was added and the whole mixture was concen-
trated and again warmed at 50◦ for a further 30 min, then the
solution was extracted with CHCl3. The aqueous layer was
neutralized with Ba(OH)2 saturated with H2O. The precipitates
were filtered off and the solution was evaporated to dryness.
The mixture of sugars which were obtained from the aqueous
layer was dissolved in MeOH, from which a sugar was crys-
2.3.6. Determination of the absolute configurations of
sugars in compounds 1–8 [12]
A solution of l-(−)-MBA (␣-methylbenzylamine) (20 mg) and
NaBH3CN (sodium cyanoborohydride) (4 mg) in 0.5 mL of
MeOH was added to a solution of an authentic sugar sample
(20 mg) in 0.5 mL of H2O. The mixture was allowed to stand
overnight, acidified to pH 3–4 by glacial acetic acid, and evap-

steroids 7 2 ( 2 0 0 7 ) 514–523
519
Table 4 – 1H NMR data of sugar moieties of compounds 2, 4, 5, 7 and 8 in pyridine-d5 (400 MHz)
Sugar moiety
2
4
7
8
5
␤-d-cym
␤-d-cym
␤-d-cym
␤-d-cym
␤-d-cym
1
2
3
4
5.50 (brd, 8Hz)
2.46 (m), 2.06 (m)
3.97 (brd, 2)
3.63 (m)
5.48 (brd, 9.6)
2.45 (m), 2.08 (m)
3.96 (brd, 2)
3.61 (m)
5.48 (brd, 9.2)
2.45 (m), 2.05 (m)
3.99 (brd, 2)
3.61 (m)
5.49 (brd, 9.2)
2.46 (m), 2.05 (m)
3.99 (brd, 2)
3.62 (m)
5.29 (brd, 7.2)
1.81, 2.25(m)
4.09 (brd, 2.4)
3.56 (m)
5
6
4.32 (m)
1.46 (d, 6)
3.56 (s)
4.31 (m)
1.45 (d, 6)
3.56 (s)
4.32 (m)
1.45 (d, 6.4)
3.56 (s)
4.31 (m)
1.46 (d, 6)
3.57 (s)
4.21 (dd, 6, 2.4)
1.36 (d, 6.8)
3.51 (s)
3-OMe
Sugar moiety
2
4
7
8
5
␤-d-dig
␤-d-dig
␤-d-dig
␤-d-dig
␤-d-cym
1
2
3
4
5
6
5.16 (brd, 8.4)
2.47 (m)
4.65 (m)
3.38 (m)
3.82 (m)
5.15 (brd, 8.4)
2.49 (m)
4.65 (m)
3.38 (dd, 2, 9.2)
3.83 (m)
1.30 (d, 6)
5.15 (brd, 9.6)
2.46 (m)
4.63 (m)
3.38 (dd, 2, 9.2)
3.83 (m)
1.30 (d, 6)
5.14 (brd, 8.8)
2.45 (m)
4.63 (m)
3.38 (dd, 2, 9.2)
3.85 (m)
1.31 (d, 6)
5.12 (dd, 8.4, 1.2)
1.79, 1.92(m)
4.01 (brd, 2.8)
3.42 (dd, 9.6, 2.8)
4.15 (brd, 7.2)
1.41 (d, 6)
1.24 (d, 6)
3-OMe
3.63 (s)
Sugar moiety
2
4
7
8
5
␤-d-ole
␤-d-ole
␤-d-ole
␤-d-ole
␤-d-ole
1
2
3
4.65 (dd, 7.8, 2.8)
1.75, 2.45 (m)
3.65 (m)
4.65 (brd, 9.6)
1.75, 2.46 (m)
3.63 (m)
4.65 (brd, 10)
1.75, 2.45 (m)
3.65 (m)
4.65 (brd, 10)
1.75, 2.46 (m)
3.65 (m)
4.69 (dd, 9.6, 1.6)
1.78, 2.47 (m)
3.66 (m)
4
3.63 (m)
3.63 (m)
3.65 (m)
3.65 (m)
3.62 (m)
5
3.55 (m)
3.58 (m)
3.57 (m)
3.57 (m)
3.60 (m)
6
1.68 (d, 6)
3.52 (s)
1.67 (d, 6)
3.52 (s)
1.65 (d, 6)
3.51 (s)
1.68 (d, 6)
3.52 (s)
1.69 (d, 6)
3.56 (s)
3-OMe
Sugar moiety
2
4
7
8
5
␤-d-the
␤-d-the
␤-d-the
␤-d-the
␤-d-the
1
2
3
4
5
6
4.94 (d, 7.6)
3.87 (m)
3.60 (m)
3.57 (m)
3.71 (m)
1.57 (d, 6)
3.88 (s)
4.92 (d, 8)
3.83 (m)
3.61 (m)
3.54 (m)
3.70 (m)
1.58 (d, 6)
3.85 (s)
4.93 (d, 8)
3.86 (m)
3.61 (m)
3.55 (m)
3.71 (m)
1.57 (d, 6)
3.83 (s)
4.94 (d, 8)
3.86 (m)
3.60 (m)
3.55 (m)
3.71 (m)
1.58 (d, 6)
3.87 (s)
4.94 (d, 8)
3.88 (m)
3.61 (m)
3.60 (m)
3, 71 (dd, 8.4, 2.4)
1.58 (d, 6)
3.87 (s)
cym: Cymaropyranosyl; dig: digitoxopyranosyl; ole: oleandropyranosyl; the: thevetopyranosyl.
orated to dryness. The resultant oily material was acetylated
by acetic anhydride-pyridine (1:1) (2 mL) at 100 ◦C for 1 h in a
sealed tube. After codistillation of the acetic anhydride with
toluene, H2O (2 mL) was added to the residue and the mixture
was extracted with CHCl3 (1 mL). The CHCl3 layer was evapo-
rated to give an oily residue, and subjected to HPLC (Inertsil sil-
100A column, 250 mm × 4.6 mm, 5 ␮m; eluant, CH2Cl2/MeOH
(65:1); flow rate, 1.0 mL/min; detection at 230 nm). The
retention times of the derivatives of authentic sugar
samples were determined: d-cymaropyranose 5.627 min, d-
digitoxopyranose 11.012 min, d-oleandropyranose 6.388 min
and D-thevetopyranose 7.588 min. To each solution of 1–8
in CH3OH (5 ml) was added 0.05 M H2SO4 (5 ml), and the
solution was kept at 50 ◦C for 1 h. Then the solution was neu-
tralized with aq. saturated Ba(OH)2 and the precipitate was
filtered off. The filtrate was partitioned with CHCl3 to give
aglycone and the aqueous phase was retained to study the
sugar components by HPLC. The aqueous phase obtained from
the acid hydrolysis of compounds 1–8 were treated as the
authentic samples in the same method, and the retention
times were compared with those of the authentic samples.
According to this reported procedure, the absolute config-
urations of the sugars were determined by HPLC on the
different behaviors of acyclic diastereoisomeric 1-deoxy-1-(N-
acetyl-␣-methylbenzylamino)-alditol acetates derived from
monosaccharides (Fig. 1).
3.
Results and discussion
Compound
1 showed positive Libermann–Buchard and
Keller–Kiliani reactions, suggesting it to be a steroidal gly-
coside with 2-deoxysugar moieties. Acid hydrolysis of 1 also
confirmed the existence of deoxysugars by TLC analysis. Mild
acid hydrolysis of 1 afforded crystalline 1a. Complete alkaline
hydrolysis of 1a with 5% KOH/CH3OH afforded a crystalline

520
steroids 7 2 ( 2 0 0 7 ) 514–523
Fig. 1 – The structures of compounds 1–9.
product 1b which was identified as dihydrosarcostin [11].
The positive HRESIMS gave a [M + H]+ peak at m/z 1068.5504
(calcd., 1068.5532), corresponding to the molecular formula
J = 6.0 Hz, 21-Me) and 2.07 (3H, s, 18-Me), and three signals at ı
3.87 (1H, m, 3␣-H), 5.22 (1H, dd, J = 6.8, 11.2 Hz, H-12), 5.28 (1H,
q, J = 7.2 Hz, H-20) corresponding to secondary oxygenated
carbons. Through the proton signals at ı 6.52 (1H, d, J = 16.0 Hz,
cin-2), 7.73 (1H, J = 16.0 Hz, cin-3), 7.32 (3H, m, cin-5/9 and 7),
7.39 (2H, m, cin-6/8), and the 13C signals at ı 166.6 (cin-1), 120.2
(cin-2), 143.9 (cin-3), 134.8 (cin-4), 129.2 (cin-5/9), 128.5 (cin-6/8)
and 130.5 (cin-7), the cinnamoyl group was identified. The
C
57H81O18N. IR spectrum showed the absorption bands for
hydroxyl (3438 cm−1), conjugated carbonyl (1714, 1716 cm−1),
olefin (1639 cm−1) and phenyl (1595, 1452 cm−1) groups. The
1H NMR spectrum of the aglycon portion showed signals
for three methyl groups at ı 1.11 (3H, s, 19-Me), 1.53 (3H, d,

steroids 7 2 ( 2 0 0 7 ) 514–523
521
position of the cinnamoyl group was identified as at C-12
based on the long-range correlation between its carbonyl
group and H-12 (ı 5.22) in the HMBC spectrum. The identi-
fication of nicotinoyl group was deduced from the proton
signals at ı 8.79 (1H, dd, J = 5.6 Hz, 1.6 Hz, nic-3), 8.24 (1H, dd,
J = 6.0, 2.0 Hz, nic-5), 7.14 (1H, dd, J = 4.8, 8.0 Hz, nic-6), 9.47 (1H,
J = 1.6 Hz, nic-7), and the 13C signals at ı 164.6 (nic-1), 153.8
(nic-3), 126.8 (nic-4), 137.3 (nic-5), 123.6 (nic-6), 151.3 (nic-7).
The position of the nicotinoyl group was identified as at C-20
based on the long-range correlation between its carbonyl
group and H-20 (ı 5.28) in the HMBC spectrum. The proton
signals due to three secondary methyl groups [ıH 1.36 (3H, d,
J = 6.8 Hz), 1.69 (3H, d, J = 6.0 Hz), and 1.58 (3H, d, J = 6 Hz)] and
three methoxyl groups [ıH 3.51 (3H, s), 3.56 (3H, s) and 3.87
(3H, s)] of deoxysugars and three anomeric protons [ıH 5.29
(1H, d, J = 7.2 Hz), 4.69 (1H, brd, J = 9.6 Hz), 4.94 (1H, d, J = 8.0 Hz)]
were observed in its sugar moiety. The splitting patterns of
anomeric proton signals indicated that 1 possessed three
sugar units with ␤-linkages. On the basis of the 1H-1H COSY,
HMQC, and HMBC spectra, the 1H NMR and 13C NMR spectra
of 1 suggested that the three sugars were ␤-cymaropyranose,
␤-oleandropyranose and ␤-thevetopyranose. The linkage
positions and sequence of the three sugars in compound
1 were ascertained by the HMBC spectrum, which showed
distinct cross-peaks of correlations from ıH 4.94 (H-1 of
␤-thevetopyranose) to ıC 83.1 (C-4 of ␤-oleandropyranose),
from ıH 4.69 (H-1 of ␤-oleandropyranose) to ıC 83.6 (C-4 of ␤-
cymaropyranose), and from ıH 5.29 (H-1 of ␤-cymaropyranose)
to ıC 76.6 (C-3 of aglycon). By comparing 1H NMR and 13C
NMR spectral data of its deoxysugar units with those of cor-
responding deoxysugar unit of dresioside I [1], the absolute
configurations of the deoxysugars were confirmed to be d
types. The absolute configurations of the deoxysugars were
further confirmed to be d types by HPLC method described
above [12]. Therefore, the structure of compound 1 was elu-
cidated as 12-O-cinnamoyl-20-nicotinoyl-dihydrosarcostin
3-O-␤-d-thevetopyranosyl-(1 → 4)-␤-d-oleandropyranosyl-
(1 → 4)-␤-d-cymaropyranoside.
␤-digitoxopyranosyl) to ıC 83.1 (C-4 of ␤-cymaropyranose),
from ıH 5.50 (H-1 of ␤-cymaropyranose) to ıC 76.6 (C-3 of
aglycon). By comparing 1H NMR and 13C NMR spectral data of
its deoxysugar units with those of corresponding deoxysugar
units of dresioside I [1] and cynaforroside B [13]. The absolute
configurations of the deoxysugars were confirmed to be d
types by HPLC method described above further [12]. Thus, the
structure of 2 was defined as 12-O-benzyl-dihydrosarcostin
3-O-␤-d-thevetopyranosyl-(1 → 4)-␤-d-oleandropyranosyl-
(1 → 4)-␤-d-digitoxopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Compooud 3 was obtained as white amorphous powder.
The molecular formula of 3 was determined to be C57H81O18
N
(1068.5581 [M + H]+, calcd., 1068.5532) on the basis of its posi-
tive HRESIMS data. IR spectrum showed the absorption bands
for hydroxyl (3437 cm−1), carbonyl (1709, 1682 cm−1), olefin
(1641 cm−1) and phenyl (1600, 1452 cm−1) groups. The NMR
data and TLC analysis of the hydrolysate of 3 indicated that
it possessed the same aglycon, the same substituted groups
(cinnamoyl and nicotinoyl) and the same sugar moieties as
those of compound 1. The difference between compounds 3
and 1 was that cinnamoyl and nicotinoyl groups were linked
to different positions. The linkage positions of the two groups
were ascertained by the HMBC spectrum, which showed dis-
tinct cross-peaks of correlations from ıH 5.22 (1H, dd, J = 6.8,
11.2 Hz, H-12) to ıC 164.6 (C-1 of nicotinoyl), from H-20 (ı 5.28)
to ıC 166.6 (C-1 of cinnamoyl).
Sugar analysis was assumed to be the same as that of 1.
Thus, the structure of 3 was identified as 12-O-nicotinoyl-20-
cinnamoyl-dihydrosarcostin 3-O-␤-d-thevetopyranosyl-(1 →
4)-␤-d-oleandropyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Compound 4 was obtained as white amorphous powder.
The molecular formula of 4 was determined to be C57H88O20
(1115.5791 [M + Na]+, calcd., 1115.5767) on the basis of its
positive HRESIMS data. IR spectrum showed the absorption
bands for hydroxyl (3436 cm−1), carbonyl (1709 cm−1), olefin
(1639 cm−1) and phenyl (1595, 1452 cm−1) groups. The NMR
data and TLC analysis of the hydrolysate of 4 indicated that
it possessed the same aglycon and same sugars as those of
compound 2. The difference was that there was a cinnamoyl
group not benzyl substituted group at C-12 of 4 by comparing
with 2. The structure of 4 was further confirmed by the HSQC,
HMBC, and 1H-1H COSY experiments. Thus, the structure
Compound 2 was obtained as white amorphous powder.
The molecular formula of 2 was determined to be C55H86O20
(1089.5649 [M + Na]+, calcd., 1089.5610) on the basis of its
positive HRESIMS data. IR spectrum showed the absorption
bands for hydroxyl (3452 cm−1), carbonyl (1711 cm−1), and
phenyl (1501, 1452 cm−1) groups. The NMR data and TLC
analysis of the hydrolysate of 2 indicated that its aglycon
possessed the same structure as the known compound 9. The
1H NMR spectrum of 2 displayed signals for four secondary
methyl groups and three methoxyl groups of deoxysugars and
four anomeric protons [ıH 5.50 (1H, d, J = 8.0 Hz), 5.16 (1H, brd,
J = 8.4 Hz), 4.65 (1H, dd, J = 7.8, 2.8 Hz), 4.94 (1H, d, J = 7.6 Hz)].
The splitting patterns of anomeric proton signals indicated
that 2 possessed four sugar units with ␤-linkages. The 1H NMR
and 13C NMR spectra of 2 suggested that the four sugars were
␤-cymaropyranose, ␤-digitoxopyranose, ␤-oleandropyranose
and ␤-thevetopyranose. The linkage positions and sequence of
the four sugars of compound 2 were ascertained by the HMBC
spectrum, which showed distinct cross-peaks of correlations
from ıH 4.94 (H-1 of ␤-thevetopyranose) to ıC 83.4 (C-4 of ␤-
oleandropyranose), from ıH 4.65 (H-1 of ␤-oleandropyranose)
to ıC 83.0 (C-4 of ␤-digitoxopyranosyl), from ıH 5.16 (H-1 of
of
4 was identified as 12-O-cinnamoyl-dihydrosarcostin
3-O-␤-d-thevetopyranosyl-(1 → 4)-␤-d-oleandropyranosyl-
(1 → 4)-␤-d-digitoxopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Compound 5 was obtained as white amorphous powder.
The molecular formula of 5 was determined to be C64H93O21
N
(1212.6294 [M + H]+, calcd., 1212.6318) on the basis of its posi-
tive HRESIMS data. IR spectrum showed the absorption bands
for hydroxyl (3446 cm−1), carbonyl (1712, 1714 cm−1), olefin
(1641 cm−1) and phenyl (1590, 1452 cm−1) groups. The NMR
data and TLC analysis of the hydrolysate of 5 indicated that it
had the same aglycon and substituted groups as those of com-
pound 1. NMR data indicated that compound 5 had different
sugar moiety from 1. The 1H NMR spectrum for 5 displayed
signals for four secondary methyl groups and three methoxyl
groups of deoxysugars and four anomeric protons [ıH 5.29 (1H,
brd, J = 7.2 Hz), 5.12 (1H, dd, J = 8.4, 1.2 Hz), 4.69 (1H, dd, J = 9.6,
1.6 Hz), 4.94 (1H, d, J = 8 Hz)]. The splitting patterns of anomeric
proton signals indicated that 5 possessed three sugar units

522
steroids 7 2 ( 2 0 0 7 ) 514–523
with ␤-linkages. The 1H NMR and 13C NMR spectra of 5 sug-
gested that the four sugars were two ␤-cymaropyranoses, one
␤-oleandropyranose and one ␤-thevetopyranose. The linkage
positions and sequence of the four sugars in compound 5
were ascertained by the HMBC spectrum, which showed
distinct cross-peaks of correlations from ıH 4.94 (H-1 of
␤-thevetopyranose) to ıC 83.0 (C-4 of ␤-oleandropyranose),
from ıH 4.69 (H-1 of ␤-oleandropyranose) to ıC 83.2 (C-4
of outer ␤-cymaropyranose), from ıH 5.12 (H-1 of outer ␤-
cymaropyranose) to ıC 83.4 (C-4 of inner ␤-cymaropyranose),
and from ıH 5.29 (H-1 of inner ␤-cymaropyranose) to ıC
76.5 (C-3 of aglycon). By comparing 1H NMR and 13C NMR
spectral data of its deoxysugar units with those of corre-
sponding deoxysugar units of dregeoside A [4], the absolute
configurations of the deoxysugars were confirmed to be
d types, which were further confirmed to be d types by
HPLC method described above [12]. Thus, the structure of 5
was identified as 12-O-cinnamoyl-dihydrosarcostin 3-O-␤-d-
thevetopyranosyl-(1 → 4)-␤-d-oleandropyranosyl-(1 → 4)-␤-d-
cymaropyranosyl-(1 → 4)-␤-d-cymaropyranoside.
The NMR data and TLC analysis of the hydrolysate of 8
indicated that it had the same sugar moieties and same
aglycon as compound 7. In addition, the signals of cin-
namoyl group in 8 were observed in the 1H and 13C NMR
spectra. The structure of 8 was further confirmed by the
HMQC, HMBC, and 1H–1H COSY experiments. Thus, the struc-
ture of 8 was identified as 12-cinnamoyl-tayloron 3-O-␤-d-
thevetopyranosyl-(1 → 4)-␤-d-oleandropyranosyl-(1 → 4)-␤-d-
digitoxopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
The cytotoxicity of the eight compounds were tested with
MTT assay against HCT-8, Bel-7402, BGC-823, A549, and A2780,
but they showed no activity. The antiepileptic acitivity of these
compounds to animals was not tested because of the small
amounts of the glycosides obtained.
An experiment was performed to prove that the iso-
lated glycosides were not obtained by transesterication during
extraction. Ten milligrams of benzic acid was dissolved to
10 ml methanol to prepare the 1 mg/ml standard solution.
Ten milligrams of benzic acid and 40 mg dihydrosarcostin
were added to methanol to prepare the test solution 1. Ten
milligrams of benzic acid and 40 mg dihydrosarcostin were
added to air-dried stems (0.1 g) of D. sinensis var. corrugate,
then extracted with 100 ml 95% EtOH under reflux. The extrac-
tion was evaporated in vacuum and then dissolved in 10 ml
methanol to prepare the test solution 2. The test solutions
1 and 2, and standard solution were injected to HPLC to
determine the content of benzic aicd. According to the deter-
mined results, the content of benzic acid was not changed
obviously by comparision with the content of benzic acid of
test solutions 1 and 2. The pH of extracted solution was 6–7
and the extraction temperature was 60–70 ◦C, which were too
mild to catalize the transesterication reaction. So the isolated
glycosides were not transestericated during extraction and
isolation, and might be conformed in the plant metabolities
on the effects of enzyme.
Compound 6 was obtained as white amorphous powder.
The molecular formula of 6 was determined to be C49H82O18
(981.5391 [M + Na]+, calcd., 981.5399) on the basis of its
positive HRESIMS data. The NMR data and TLC analysis of
the hydrolysate of 6 indicated that it possessed the same
aglycon and sugar moieties as those of compound 1. The 1H
NMR and 13C NMR spectra showed a doublet methyl signal
[ı 1.08 (3H, d, J = 7.2 Hz)] and a triplet methyl signal [ı 0.88
(3H, t, J = 7.2 Hz)], and carbon signals at ı 175.7, 41.7, 26.5,
11.9 and 16.5, which were attributed to a 2-methylbutanoyl
group. In addition, the NMR signals of acetyl group were
observed. The substituted positions of 2-methylbutanoyl and
acetyl were identified as at 20-C and 12-C, respectively, by
HMBC experiment. Thus, the structure of 6 was identified
as 12-acetyl-20-methylbutanoyl-dihydrosarcostin 3-O-␤-d-
thevetopyranosyl-(1 → 4)-␤-d-oleandro-pyranosyl-(1 → 4)-␤-
d-cymaropyranoside.
Acknowledgements
Compound 7 was obtained as white amorphous pow-
der. The molecular formula was established as C55H84O20
from HRESIMS at m/z [M + Na]+ 1087.5434 (calcd., 1087.5454).
IR spectrum showed the absorption bands for hydroxyl
Financial support was provided by grants from the National
Science Fund for Distinguished Young Scholars, Project No.
30625040, National Natural Science Foundation of China,
Project No. 20432030, and National Key Basic R&D (973)
Project, No. 2004CB518906. We are grateful to the Depart-
ment of Instrumental Analysis, Institute of Materia Medica,
(3437 cm−1), carbonyl (1720, 1714 cm−1), olefin (1639 cm−1
)
and phenyl (1593, 1450 cm−1) groups. The NMR data and
TLC analysis of the hydrolysate of 7 indicated that it had
the same sugar moieties as those of compound 4. Mild acid
hydrolysis of 7 afforded crystalline 7a. Complete alkaline
hydrolysis of 7a with 5% KOH/CH3OH afforded a crystalline
product 7b which was identified as tayloron [8]. From the
proton signals and the 13C NMR signals, the benzyl group
was identified. The structure of 7 was further confirmed by
the HMQC, HMBC, and 1H–1H COSY experiments. There-
fore, the structure of 7 was identified as 12-benzyl-tayloron
3-O-␤-d-thevetopyranosyl-(1 → 4)-␤-d-oleandropyranosyl-
(1 → 4)-␤-d-digitoxopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Compound 8 was obtained as white amorphous powder.
The molecular formula of 8 was determined to be C57H86O20
(1113.5593 [M + Na]+, calcd., 1113.5610) on the basis of its
positive HRESIMS data. IR spectrum showed the absorp-
tion bands for hydroxyl (3452 cm−1), carbonyl (1712 cm−1),
Chinese Academy of Medical Sciences
& Peking Union
Medical College, for measuring the IR, UV, NMR and MS
spectra.
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