Cardenolides from Saussurea stella with cytotoxicity toward cancer cells

Article abstract of DOI:10.1021/np070150o

Three new cardenolides, 3-O-β-D-fucopyranosylstrophanthidin (1), 3-O-β-D-quinovopyranosylperiplogenin (2), and 3-O-β-D-glucopyranosyl- (1→4)-α-L-rhamnopyranosylcannogenin (3), together with seven known cardenolides (4-10), were isolated from a cytotoxic ethanol extract of the whole dried plants of Saussurea Stella. The structures of these compounds were established by spectroscopic and chemical methods. When the cytotoxicity of compounds 2-10 toward Bel-7402 human hepatoma cells and BGC-823 human gastric cancer cells was evaluated, all compounds showed IC₅₀ values of <1 μM for both cell lines. This is the first report of cardenolides occurring in a species of the family Asteraceae.

Full text of DOI:10.1021/np070150o

J. Nat. Prod. 2007, 70, 1429–1433
1429
Cardenolides from Saussurea stella with Cytotoxicity toward Cancer Cells
Tian-Min Wang,† Toshinari Hojo,‡ Fu-Xiang Ran,† Ru-Feng Wang,† Rui-Qing Wang,† Hu-Biao Chen,*,†,§ Jing-Rong Cui,†
Ming-Ying Shang,† and Shao-Qing Cai*,†
The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking UniVersity Health Science Center,
Beijing 100083, People’s Republic of China, ARSOA Honsha Corporation ARSOA R&D Center, Yamanashi, Japan, and School of Chinese
Medicine, Hong Kong Baptist UniVersity, Hong Kong, People’s Republic of China
ReceiVed April 3, 2007
Three new cardenolides, 3-O-ꢀ-D-fucopyranosylstrophanthidin (1), 3-O-ꢀ-D-quinovopyranosylperiplogenin (2), and 3-O-
ꢀ-D-glucopyranosyl-(1f4)-R-L-rhamnopyranosylcannogenin (3), together with seven known cardenolides (4–10), were
isolated from a cytotoxic ethanol extract of the whole dried plants of Saussurea stella. The structures of these compounds
were established by spectroscopic and chemical methods. When the cytotoxicity of compounds 2–10 toward Bel-7402
human hepatoma cells and BGC-823 human gastric cancer cells was evaluated, all compounds showed IC50 values of
<1 µM for both cell lines. This is the first report of cardenolides occurring in a species of the family Asteraceae.
Saussurea stella Maxim. of the family Asteraceae is distributed in
the southwest of mainland China; about 30 species of this genus are
used as folk medicines.1 As recorded in the “Zhonghua Bencao”, S.
stella has “heat-evil clearing”, “wind-evil dispelling”, and “dampness
eliminating” effects and can be used for the treatment of rheumatism.2
In the present study, an EtOH extract of S. stella strongly inhibited
the growth of several human cancer cell lines including Bel-7402
hepatoma cells and BGC-823 gastric cancer cells. There are only two
published reports on the chemistry of S. stella;3,4 while these studies
mainly concerned flavonoid constituents, some of these compounds
were shown to have antioxidant properties.5 The lack of chemical
studies together with the potent biological activity stimulated us to
investigate the chemical constituents of S. stella further.
Cardenolides, also known as cardiotonic steroids, occur in members
of the families Apocynaceae, Asclepiadaceae, Cruciferae, Liliaceae,
and Scrophulariaceae. Asteraceae, one of the biggest families of
flowering plants, comprises many species distributed all over the world,
and research on the chemical constituents of its members is extensive.
However, there have been no reports of cardenolides having been
isolated from Asteraceae. In this study, 10 cardenolides (1–10),
including three new compounds (1–3), were isolated from S. stella
and evaluated against two cancer cell lines for their cytotoxic effects.
Results and Discussion
A cytotoxic EtOH extract of the entire herb of S. stella was
suspended in H2O and partitioned with petroleum ether, EtOAc,
and n-BuOH, successively. The EtOAc and n-BuOH extracts
Compound 1 was isolated as an amorphous powder. It was
assigned the molecular formula C29H42O10, which was deduced from
HRESIMS (m/z 573.2650, [M + Na]+). This substance was
determined to be a cardenolide possessing one sugar moiety,
were separated and purified by chromatographic methods. Three
new cardenolides, 1–3, together with seven known cardenolides,
3-O-ꢀ-D-xylopyranosylperiplogenin (4), 3-O-ꢀ-D-quinovopyra-
nosylstrophanthidin (5), 3-O-ꢀ-D-xylopyranosylstrophanthidin (6),
3-O-ꢀ-D-fucopyranosylperiplogenin (7), 3-O-R-L-rhamnopyranosylc-
annogenol (8), 3-O-R-L-rhamnopyranosylstrophanthidin (convallatoxin,
9), and 3-O-R-L-rhamnopyranosylacovenosigenin A (10), were isolated
from the EtOAc and n-BuOH extracts. The structures of compounds
1–3 were elucidated through detailed 1D and 2D NMR analysis, and
the NMR data for the known compounds 4–7 are being reported for
the first time. Compounds 8–10 were identified by comparing their
spectroscopic data with those reported in the literature.6,7
1
according to the UV, IR, and 1D NMR spectra. The H (Table 1)
and 13C NMR data (Table 2) for the aglycon were similar to those
reported for 3-O-substituted strophanthidins.6,8 Thus, the aglycon
of 1 was identified as strophanthidin. An anomeric proton (δ 4.32,
m, H-1′), a methyl group as a doublet (δ 1.25, J ) 6.5 Hz, H-6′),
and four additional protons between δ 3.44 and 3.63 in the 1H NMR
spectrum suggested that 1 bears a 6-deoxyhexose unit. The
correlation of H-1′ (δ 4.32) with C-3 (δ 75.4) in the HMBC
spectrum demonstrated that the sugar moiety is substituted at C-3.
The presence of a D-fucosyl unit was established by acid hydrolysis
and by comparing the 13C NMR data for the sugar moiety of 1
with those reported for the D-fucosyl group.9 A 1H NMR spectrum
measured in D2O was performed to determine the configuration of
the glycosidic bond in 1. A doublet for the anomeric proton (δ
* Corresponding authors. S.-Q.C., Tel/Fax: 86 10 82801693. E-mail:
sqcai@hsc.pku.edu.cn. H.-B.C., Tel: 852 34112060. Fax: 852 34112461.
E-mail: hbchen@hkbu.edu.hk.
† Peking University Health Science Center.
‡ ARSOA Honsha Corporation.
§ Hong Kong Baptist University.
4.48, J ) 8.0 Hz) in the H NMR spectrum of 1 showed that the
1
10.1021/np070150o CCC: $37.00
2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/11/2007

1430 Journal of Natural Products, 2007, Vol. 70, No. 9
Wang et al.
Table 1. 1H NMR Spectroscopic Data (500 MHz, CD3OD) for 1–7 (δ values, J in Hz)
position
1
1
2
3
4
5
6
7
1.32, m
2.15, m
1.65, m
1.89, m
4.17, brs
1.72, m
2.10, m
1.36, m
1.81, m
1.70, m
1.70, m,
4.15, brs
1.69, m
2.12, m
1.41, m
1.59, m
1.64, m
1.70, m
3.98, brs
1.79, m
1.89, m
2.18, m
1.61, m
1.65, m
1.27, m
1.85, m
1.84, m
1.88, m
1.51, m
1.51, m
1.49, m
1.53, m
1.68, m
2.18, m
1.90, m
2.16, m
2.83, dd
(8.5, 6.0)
0.94, s
1.39, m
1.81, m
1.71, m
1.71, m
4.17, brs
1.73, m
2.15, m
1.32, m
2.15, m
1.65, m
1.89, m
4.16, brs
1.72, m
2.10, m
1.32, m
2.14, m
1.66, m
1.82, m
4.14, brs
1.72, m
2.12, m
1.35, m
1.83, m
1.71, m
1.71, m
4.16, brs
1.69, m
2.12, m
2
3
4
5
6
1.67, m
2.09, m
1.69, m
2.20, m
1.95, m
1.71, m
1.21, m
1.53, m
1.44, m
1.48, m
1.68, m
2.13, m
1.85, m
2.16, m
2.82, dd
(9.0, 6.0)
0.85, s
10.03, s
4.90, dd
(18.5, 1.5)
5.02, dd
(18.5, 1.5)
5.89, brs
Fuc
1.38, m
1.73, m
1.20, m
1.92, m
1.64, m
1.61, m
1.33, m
1.45, m
1.48, m
1.51, m
1.70, m
2.14, m
1.87, m
2.18, m
2.83, dd
(8.0, 5.5)
0.88, s
1.41, m
1.75, m
1.25, m
1.94, m
1.67, m
1.65, m
1.36, m
1.49, m
1.51, m
1.54, m
1.74, m
2.15, m
1.88, m
2.19, m
2.86, dd
(8.5, 5.5)
0.91, s
1.67, m
2.09, m
1.69, m
2.20, m
1.95, m
1.71, m
1.21, m
1.53, m
1.44, m
1.48, m
1.68, m
2.13, m
1.85, m
2.16, m
2.82, dd
(9.0, 5.0)
0.85, s
10.04, s
4.90, dd
(18.5, 2.0)
5.02, dd
(18.5, 1.5)
5.89, brs
Qui
1.65, m
2.10, m
1.68, m
2.16, m
1.95, m
1.70, m
1.20, m
1.51, m
1.44, m
1.48, m
1.68, m
2.13, m
1.87, m
2.18, m
2.82, dd
(9.0, 5.5)
0.84, s
10.04, s
4.90, dd
(18.5, 1.5)
5.07, dd
(18.5, 1.5)
5.89, brs
Xyl
1.37, m
1.73, m
1.19, m
1.92, m
1.64, m
1.61, m
1.32, m
1.45, m
1.48, m
1.52, m
1.70, m
2.14, m
1.88, m
2.18, m
2.83, dd
(8.0, 6.0)
0.88, s
7
8
9
11
12
15
16
17
18
19
21
0.93, s
9.57, s
0.96, s
0.93, s
4.91, s
4.91, dd
(18.5, 1.5)
5.02, dd
(18.5, 1.5)
5.89, t (1.5)
Qui
4.91, dd
(18.5, 1.0)
5.03, dd
(18.5, 1.0)
5.90, brs
Rha
4.94, dd
(18.5, 1.5)
5.06, dd
(18.5, 1.5)
5.92, brs
Xyl
5.02, s
22
5.89, s
Fuc
sugar moieties
1′
2′
4.32, m
4.36, d (7.5)
3.16, dd
(7.5, 9.0)
3.31a
4.79, d (1.5)
3.81, m
4.36, d (7.5)
3.18, dd
(7.5, 9.0)
3.35a
4.37, d (7.5)
3.15, dd
(7.5, 9.0)
3.31a
4.33, d (7.5)
3.14, dd
(7.5, 9.0)
3.31a
4.32, m
3.46a
3.46a
3′
4′
5′
3.46a
3.91, dd
(9.5, 3.0)
3.61, t (9.5)
3.46a
3.58, brs
3.63, q (6.5)
2.98, t (9.0)
3.31a
3.50, ddd
(5.5, 10.5, 9.0)
3.86, dd
2.99, t (9.0)
3.31a
3.47, ddd
(5.5, 9.0, 10.5)
3.84, dd
3.58, d (1.0)
3.69a
3.62, dq
(5.5, 11.5)
3.22, dd
(5.5, 11.5)
3.19, dd
(6.5, 1.0)
(10.5, 11.5)
(10.5, 11.5)
6′
1.25, d (6.5)
1.25, d (6.0)
1.3, d (6.0)
Glc
1.26, d (6.0)
1.25, d (6.5)
1′′
2′′
4.58, d (7.5)
3.20, dd
(7.5, 8.5)
3.36, t
3′′
(8.5, 8.5)
4′′
5′′
6′′
3.28a
3.28a
3.84, dd
(12.0, 2.0)
3.69a
a Overlapped signals.
glycosidic bond is in a ꢀ-configuration. Furthermore, the coupling
constants of H-1′ with H-2′ (J ) 8.0 Hz), H-2′ with H-3′ (J )
10.0 Hz), and H-3′ with H-4′ (J ) 3.5 Hz) in the 1H NMR spectrum
measured in D2O gave support for the identification of the ꢀ-D-
fucosyl unit in compound 1. Thus, the structure of 1 was elucidated
as 3-O-ꢀ-D-fucopyranosylstrophanthidin.
the correlations of H-1′ (δ 4.36) with C-3 (δ 76.2) and H-3 (δ
4.15) with C-1′ (δ 101.9) in the HMBC spectrum of 2. Acid
hydrolysis of 2 yielded D-quinovose. The coupling constants of H-1′
with H-2′ (J ) 7.5 Hz), H-2′ with H-3′ (J ) 9.0 Hz), H-3′ with
H-4′ (J ) 9.0 Hz), and H-4′ with H-5′ (J ) 9.0 Hz) supported the
conclusions that the glycosyl of 2 is a D-quinovosyl and that the
glycosidic bond is in a ꢀ-configuration. Consequently, the structure
of 2 was determined as 3-O-ꢀ-D-quinovopyranosylperiplogenin.
Compound 3 was obtained as an amorphous powder. As deduced
from its HRESIMS (m/z 719.3247, [M + Na]+), its molecular
formula was determined to be C35H52O14. The similarities of the
1H NMR (Table 1) and 13C NMR (Table 2) spectra of compound
3 to those of compounds 1 and 2 revealed that 3 is also a cardenolide
glycoside. One main difference between the 13C NMR data for the
aglycons of 3 and 1 was the absence of the quaternary carbon signal
(δ 75.3) associated with the presence of a methine signal (δ 31.7,
The molecular formula of compound 2 was determined to be
C29H44O9 from its HRESIMS (m/z 559.2901, [M + Na]+). The 1H
NMR (Table 1) and 13C NMR (Table 2) spectra indicated that
compound 2 is also a cardenolide bearing a hexose unit. The 13C
NMR data for the aglycon of 2 were identical with those for 3-O-
substituted periplogenins reported in the literature.10 Accordingly,
the aglycon of 2 was assigned as periplogenin. The sugar moiety
of 2 is also a 6-deoxyhexose, as deduced from a methyl doublet (δ
1
1.25, J ) 6.0 Hz), which was assigned to H-6′, in the H NMR
spectrum. The 3-O-linkage of the sugar moiety was ascertained by

Cytotoxic Cardenolides from Saussurea stella
Journal of Natural Products, 2007, Vol. 70, No. 9 1431
Table 2. 13C NMR Spectroscopic Data (CD3OD, 125 MHz) for 1–7 (δ values)
position
1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
25.9
26.5
75.4
36.1
75.3
38.3
19.5
43.1
40.8
56.7
23.7
41.0
51.2
86.5
33.0
28.4
52.3
16.7
210.6
178.7
75.8
118.4
177.7
102.9
72.8
75.8
73.6
72.6
17.3
26.5
27.0
76.2
34.8
75.1
36.2
25.0
41.6
40.1
41.8
22.6
40.9
50.9
86.3
33.4
28.0
51.9
16.3
17.2
178.3
75.3
117.8
177.2
101.9
75.4
78.0
77.0
73.4
18.1
29.8
26.5
73.1
30.4
31.7
23.6
23.0
43.6
36.4
52.7
22.5
41.3
51.5
86.5
33.3
28.5
52.5
16.7
208.6
178.8
75.8
118.4
177.7
100.3
73.2
73.1
84.0
69.3
18.6
106.2
76.6
78.7
72.0
78.6
63.3
26.5
26.9
76.4
34.9
75.1
36.2
25.0
41.6
40.1
41.8
22.6
40.9
50.8
86.3
33.4
28.0
51.9
16.3
17.2
178.4
75.3
117.8
177.2
102.9
75.0
78.0
71.2
67.1
25.8
26.6
75.4
36.1
75.3
38.3
19.5
43.1
40.8
56.7
23.7
41.0
51.2
86.4
33.0
28.4
52.3
16.7
210.5
178.7
75.8
118.4
177.7
102.3
75.8
78.5
77.5
73.9
18.6
25.4
26.1
75.2
35.7
74.8
37.8
19.0
42.6
40.3
56.2
23.2
40.5
50.7
85.9
32.5
27.9
51.7
16.2
210.0
178.2
75.3
117.9
177.2
102.8
74.9
78.0
71.2
67.1
26.5
26.9
76.1
34.8
75.1
36.2
25.0
41.6
40.1
41.8
22.6
40.9
50.9
86.3
33.4
28.0
52.0
16.3
17.2
178.4
75.3
117.8
177.2
102.6
72.3
75.3
73.1
72.1
16.8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1′
2′
3′
4′
5′
6′
1′′
2′′
3′′
4′′
5′′
6′′
Table 3. IC50 Values of Cardenolides 2–10 toward Two
Cancer Cell Lines (µM)
cell linesa
compound
BGC-823
Bel-7402
2
3
4
5
6
7
8
9
0.070
0.29
0.23
0.10
0.016
0.12
0.077
0.016
0.056
0.31
0.87
0.48
0.020
0.13
0.40
0.028
0.050
0.24
Figure 1. Key HMBC correlations for compound 3.
C-5) in the 13C NMR spectrum of 3. This difference suggested that
the hydroxyl group located at C-5 in 1 is missing from 3. The
ꢀ-orientation of H-5 was confirmed by comparing the chemical shift
of C-5 with that reported for 5R-H and 5ꢀ-H cardenolides.11–13
Accordingly, the aglycon of 3 was characterized as cannogenin.11
Acid hydrolysis of 3 produced D-glucose and L-rhamnose. The
glycosyl moiety of 3 was found to be the same as that of
5R-oleandrigenin 3-[O-ꢀ-D-glucopyranosyl-(1f4)-R-L-rhamnopy-
ranoside],13 as deduced from the similarities in the 13C NMR data
for their sugar moieties. Therefore, the structure of 3 was assigned
as 3-O-ꢀ-D-glucopyranosyl-(1f4)-R-L-rhamnopyranosylcannoge-
nin. This identification was also supported by the COSY, HMQC,
and HMBC (Figure 1) spectra.
10
a Cell lines: BGC-823, human gastric cancer; Bel-7402, human hep-
otama.
The known compounds 4–7, on which there are only a few
published reports, are all rare natural products. Compound 4 has
been isolated only from the secretions of a chrysomelid beetle,
Chrysolina coerulans L. G. Scriba.14 With the exception of
compound 3, which has a two-sugar chain including a terminal
glucopyranosyl moiety, compounds 1–10 are linked to a single
6-deoxyhexose moiety. While the aglycons of these cardenolides
isolated from S. stella are common, the common 6-deoxyhexoses
including quinovose,19,20 fucose,21–23 and xylose14,24–27 are not
frequently encountered among cardenolides. Moreover, the carde-
nolides reported herein have been isolated from a plant in the
Asteraceae family for the first time.
Compounds 2–10, isolated from the cytotoxic EtOH extract of
S. stella, showed potent inhibitory effects on the growth of two
cell lines tested: BGC-823 human gastric cancer cells and Bel-
7402 human hepatoma cells (Table 3). The IC50 values of
compounds 2–10 were all <1 µM. Previously, compound 9 has
been shown to inhibit the growth of the Eagle KB strain of human
Compounds 4–7 have been reported earlier.14–18 Their 1H (Table
1) and 13C NMR (Table 2) data are presented herein because they
have not appeared in the literature previously. The aglycons of
compounds 4 and 7 are the same as that of 2, while the aglycons
of compounds 5 and 6 are identical with that of 1. Acid hydrolysis
of 4–7 furnished D-xylose, D-quinovose, D-xylose, and D-fucose,
respectively. Thus, the structures of compounds 4–7 were ascer-
tained, in turn, as 3-O-ꢀ-D-xylopyranosylperiplogenin, 3-O-ꢀ-D-
quinovopyranosylstrophanthidin, 3-O-ꢀ-D-xylopyranosylstrophanthi-
din, and 3-O-ꢀ-D-fucopyranosylperiplogenin.

1432 Journal of Natural Products, 2007, Vol. 70, No. 9
Wang et al.
epidermoid carcinoma,28 and compound 10 was found to be
cytotoxic to mouse L1210 leukemia cells, human HeLa cervical
cancer cells, human A549 lung cancer cells, and human HL-60
leukemia cells.7 Of the nine compounds tested here, compound 3,
which has a two-sugar side chain, showed the least potent inhibitory
effects toward the cancer cell lines tested. Compounds with an
aldehyde group at C-10 (compounds 5, 6, and 9) were more potent
than those with a methyl group at C-10 (compounds 2, 4, and 7) in
inhibiting the growth of these cancer cell lines. The BGC-823 cell
line seems to be a little more sensitive than the Bel-7402 cell line
to the compounds tested.
with gradient mixtures of CHCl3–MeOH (1:0 f 0:1) to produce 14
subfractions. Further separations of the subfractions were conducted
on semipreparative HPLC using different conditions to furnish com-
pounds 2 (60 mg), 4 (10 mg), 6 (12 mg), and 7 (14 mg). The final
solvents used to isolate compounds 2, 4, and 6 were mixtures of
MeOH–H2O (60:40), while the final solvents used for purification of
compound 7 were mixtures of 50% MeOH and 50% H2O.
3-O-ꢀ-D-Fucopyranosylstrophanthidin (1): white, amorphous
powder; mp 178–180 °C; [R]20 +12.5 (c 0.4, MeOH); UV (MeOH)
D
λmax (log ε) 216 (4.42) nm; IR (KBr) νmax 3456, 2933, 1741, 1621,
1071 cm-1; 1H NMR (CD3OD, 500 MHz), see Table 1; 1H NMR (D2O,
500 MHz) δ 10.14 (1H, s, H-19), 5.97 (1H, s, H-22), 5.05 (1H, dd, J
) 1.0, 19.0 Hz, H-21a), 5.00 (1H, dd, J ) 1.0, 19.0 Hz, H-21b), 4.22
(1H, brs, H-3), 4.48 (1H, d, J ) 8.0 Hz, H-1′), 3.78 (1H, q, J ) 6.5
Hz, H-5′), 3.74 (1H, d, J ) 3.5 Hz, H-4′), 3.64 (1H, dd, J ) 10.0, 3.5
Hz, H-3′), 3.46 (1H, dd, J ) 8.0, 10.0 Hz, H-2′), 2.88 (1H, m, H-17),
1.24 (3H, d, J ) 6.5 Hz, H-6′), 0.83 (3H, s, H-18); 13C NMR (CD3OD,
125 MHz), see Table 2; HRESIMS [M + Na]+ m/z 573.2650 (calcd
for C29H42O10Na, 573.2670).
Experimental Section
General Experimental Procedures. Melting points were measured
on an XT-4A micromelting point apparatus without correction. Optical
rotations were determined on a Perkin-Elmer 243 B polarimeter. UV
spectra were measured with a Cary 300 UV–vis spectrophotometer,
while IR spectra were collected from a Nicolet NEXUS-470 FTIR
spectrophotometer. Varian INOVA-500 and Bruker DRX-500 spec-
trometers were used to obtain the NMR spectra. The chemical shifts
are expressed as δ values using solvent as internal standard. HRESIMS
and ESIMS were detected with Bruker APEX IV FT and ABI Q-STAR
mass spectrometers, respectively. Column chromatography was per-
formed with silica gel (200–300 mesh, Qingdao Marine Chemical Co.,
Ltd.) and macroporous resin D101 (Tianjin Resin Co., Ltd.). Semi-
preparative HPLC was conducted on an Alltima C18 column (10 mm
i.d. × 250 mm, 10 µm) equipped with an Alltech 426 HPLC pump
and an Alltech single-wavelength UV detector. Analytical HPLC was
carried out on an Agilent 1100 liquid chromatography system with a
DAD UV detector using a Zobax SB-C18 column (4.6 mm i.d. × 250
mm, 5 µm). D-Fucose, D-quinovose, D-xylose, L-(–)-R-methylbenzyl-
amine, and NaBH3CN were purchased from Sigma (St. Louis, MO).
D-glucose and L-rhamnose were obtained from Beijing Chemical
Reagent Company. All other chemical solvents used for isolation were
of analytical grade (Beijing Beihua Fine Chemicals Co., Ltd.).
Plant Material. The entire plants of Saussurea stella Maxim. were
collected from suburbs of Shiqu County, Sichuan Province, People’s
Republic of China, in August 2003 and May 2004. The plant material
was identified by Prof. Hu-Biao Chen. Voucher specimens (030803
and 040501) were deposited in the herbarium of the School of
Pharmaceutical Sciences, Peking University Health Science Center.
Extraction and Isolation. The air-dried and powdered S. stella
(2.5 kg, 030803) was extracted with 95% EtOH at room temperature
(25 L, 23 L, 18 L; 3 × 7 days). The resulting EtOH extract was
concentrated under reduced pressure to obtain a crude extract (100 g).
After suspension in water, the crude extract was partitioned with
petroleum ether, EtOAc, and n-BuOH, successively.
The EtOAc extract (20 g) was subjected to silica gel column
chromatography eluted with gradient mixtures of petroleum–EtOAc–
MeOH (1:0:0 f 0:0:1) to yield fractions E1–E55. Fractions E26–28
(1.2 g) were subjected to silica gel column chromatography using
CHCl3–MeOH mixtures (50:1 f 1:1) for elution and gave subfractions
EC1–EC16. Compounds 10 (6 mg) and 9 (8 mg) were obtained by
purifying EC7 (0.09 g) and EC8 (0.1 g) with semipreparative HPLC
eluted with MeOH–H2O (50:50), respectively.
The n-BuOH extract (20 g) was chromatographed over macroporous
resin D101 eluted with EtOH–H2O (0% f 95%) to give fractions
Bu1–Bu8. Fraction Bu4 (2.5 g) was subjected to silica gel column
chromatography using a CHCl3–MeOH mixture (7:1 f 0:1) as eluants
to afford subfractions BuC1–BuC45. Compound 3 (16 mg) was isolated
from subfraction BuC1 (0.1 g) by semipreparative HPLC (MeOH–H2O,
45:55). Subfraction BuC2 (0.3 g) was subjected to semipreparative
HPLC eluted with MeOH–H2O (45:55) and yielded compounds 1 (3
mg), 5 (14 mg), and 8 (7 mg).
3-O-ꢀ-D-Quinovopyranosylperiplogenin (2): white, amorphous
powder; mp 187–188 °C; [R]20 -10.0 (c 0.2, MeOH); UV (MeOH)
D
λmax (log ε) 212 (4.23) nm; IR (KBr) νmax 3414, 2938, 1740, 1623,
1062 cm-1 1H NMR (CD3OD, 500 MHz), see Table 1; 13C NMR
;
(CD3OD, 125 MHz), see Table 2; HRESIMS [M + Na]+ m/z 559.2901
(calcd for C29H44O9Na, 559.2878).
3-O-ꢀ-D-Glucopyranosyl-(1f4)-r-L-rhamnopyranosylcannoge-
nin (3): white, amorphous powder; mp 208–210 °C; [R]20 -35.0 (c
D
0.2, MeOH); UV (MeOH) λmax (log ε) 212 (4.29) nm; IR (KBr) νmax
1
3420, 2932, 1740, 1624, 1067, 1030 cm-1; H NMR (CD3OD, 500
MHz), see Table 1; 13C NMR (CD3OD, 125 MHz), see Table 2;
HRESIMS [M + Na]+ m/z 719.3247 (calcd for C35H52O14Na, 719.3249).
3-O-ꢀ-D-Xylopyranosylperiplogenin (4): white, amorphous pow-
1
der; mp 174–176 °C; H NMR (CD3OD, 500 MHz), see Table 1; 13C
NMR (CD3OD, 125 MHz), see Table 2; ESIMS [M + Na]+ m/z
545.2224.
3-O-ꢀ-D-Quinovopyranosylstrophanthidin (5): white, amorphous
1
powder; mp 169–171 °C; H NMR (CD3OD, 500 MHz), see Table 1;
13C NMR (CD3OD, 125 MHz), see Table 2; ESIMS [M + Na]+ m/z
573.2149.
3-O-ꢀ-D-Xylopyranosylstrophanthidin (6): white, amorphous
1
powder; mp 182–183 °C; H NMR (CD3OD, 500 MHz), see Table 1;
13C NMR (CD3OD, 125 MHz), see Table 2; ESIMS [M + Na]+ m/z
559.2020.
3-O-ꢀ-D-Fucopyranosylperiplogenin (7): white, amorphous pow-
1
1
der; mp 166–168 °C; H NMR (CD3OD, 500 MHz), see Table 1; H
NMR (D2O, 500 MHz) δ 5.97 (1H, s, H-22), 5.07 (1H, dd, J ) 1.5,
18.5 Hz, H-21a), 5.01 (1H, dd, J ) 1.5, 18.5 Hz, H-21b), 4.21 (1H,
brs, H-3), 4.49 (1H, d, J ) 8.0 Hz, H-1′), 3.78 (1H, q, J ) 6.5 Hz,
H-5′), 3.74 (1H, d, J ) 3.5 Hz, H-4′), 3.65 (1H, dd, J ) 10.0, 3.5 Hz,
H-3′), 3.48 (1H, dd, J ) 8.0, 10.0 Hz, H-2′), 2.89 (1H, dd, J ) 9.0, 5.0
Hz, H-17), 1.24 (3H, d, J ) 6.5 Hz, H-6′), 0.90 (3H, s, H-19), 0.87
(3H, s, H-18); 13C NMR (CD3OD, 125 MHz), see Table 2; ESIMS [M
+ Na]+ m/z 559.2385.
Acid Hydrolysis of 1–7. Compounds 1–7 (each 2 mg) were
dissolved individually in methanol (0.2 mL) and refluxed with 1 N
HCl for 1 h. Each reaction mixture was evaporated to dryness under
vacuum and then partitioned between CHCl3 and H2O. The H2O layer
was concentrated to yield a sugar residue hydrolyzed from the
corresponding compound. The residue was dissolved in H2O (0.1 mL),
to which a solution of L-(–)-R-methylbenzylamine (2 mg) and
NaBH3CN (1 mg) in 0.1 mL of methanol was added. After warming
at 60 °C for 1 h, the mixture was acidified to pH 4 by addition of
glacial acetic acid (0.05 mL) and evaporated to dryness. The resultant
product was treated with acetic anhydride (0.4 mL) and anhydrous
pyridine (0.4 mL) at 100 °C for 1 h and then concentrated to remove
the solvent. The residue was partitioned between CHCl3 and H2O. The
CHCl3 layer, in which the derivates of the hydrolyzed sugars were
present, was concentrated and redissolved in methanol (0.5 mL) for
HPLC analysis: Zobax SB-C18 column (4.6 mm i.d. × 250 mm, 5 µm);
detection, UV 230 nm; mobile phase, CH3CN–H2O (2:3); flow rate,
0.8 mL/min; column temperature, 25 °C. Identification of the sugars
hydrolyzed from compounds 1–7 was carried out by comparing the
retention times of the derivative products of the hydrolyzed sugars with
those of the authentic known sugars: D-fucose (tR ) 19.51 min) was
The air-dried and powdered larger re-collected sample of S. stella
(9 kg, 040501) was extracted with 95% EtOH under reflux (120 L,
100 L; 2 × 2 h). The extract was filtrated, and the residue was extracted
with 50% EtOH under reflux (100 L, 100 L; 2 × 2 h). All resulting
EtOH extracts were concentrated under reduced pressure to obtain a
crude extract (1.2 kg). About 1.1 kg of the crude extract was subjected
to silica gel column chromatography and eluted with petroleum and
gradient CHCl3–MeOH mixtures to yield 80 fractions. Fractions 43–49
(30 g) were repeatedly chromatographed on silica gel columns eluted

Cytotoxic Cardenolides from Saussurea stella
Journal of Natural Products, 2007, Vol. 70, No. 9 1433
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from 2 and 5; D-xylose (tR ) 17.11 min) was detected from 4 and 6;
D-glucose (tR ) 22.87 min) and L-rhamnose (tR ) 26.33 min) were
detected from 3.
Cytotoxicity Assay. The IC50 values of the inhibitory effects of
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Acknowledgment. This work was supported financially by the
Program for Changjiang Scholar and Innovative Team in University
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