A new furostanol saponin from Asparagus cochinchinensis

Article abstract of DOI:10.1007/s12272-011-1001-7

A new furostanol saponin, (25S)-26-O-β-d-glucopyranosyl-5β- furost-20(22)-en-3β, 15β,26-triol-3-O-[α-l-rhamnopyranosyl-(1-4) ]-β-d-glucopyranoside, namely, aspacochioside D (1) were isolated from Asparagus cochinchinensis (Lour.) Merr, along with three known saponins, aspacochioside C (2), (25S)-5β-spirostan-3β-yl-O-[O-α-L- rhamnopyranosyl-(1-4)]-β-d-glucopyranoside (3), and pseudoprotoneodioscin (4). The structure of 1 was elucidated on the basis of chemical reactions and spectral analysis (IR, GC, ESI-MS, ¹H-NMR, ¹³C-NMR, DEPT, HMBC, HMQC and NOESY). The antiproliferative effects of 1-4 were evaluated in a cytotoxicity assay against the human tumor cell line, A549. Compound 2 (Aspacochioside C) exhibited moderate cytotoxicity against A-549, with an IC₅₀ value of 3.87 μg/mL.

Full text of DOI:10.1007/s12272-011-1001-7

Arch Pharm Res Vol 34, No 10, 1587-1591, 2011
DOI 10.1007/s12272-011-1001-7
A New Furostanol Saponin from Asparagus cochinchinensis
Yang Shen, Cong-Li Xu, Wei-Dong Xuan, Hui-Liang Li, Run-Hui Liu, Xi-Ke Xu, and Hai-Sheng Chen
Department of Natural Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433,
China
(Received April 6, 2010/Revised December 1, 2010/Accepted December 26, 2010)
A new furostanol saponin, (25S)-26-O-
3-O-[ -rhamnopyranosyl-(1-4)]- -glucopyranoside, namely, aspacochioside D (
ed from Asparagus cochinchinensis (Lour.) Merr, along with three known saponins, aspacochi-
oside C ( ), (25S)-5 -spirostan-3 -yl-O-[O- -rhamnopyranosyl-(1-4)]- -glucopyranoside ( ),
and pseudoprotoneodioscin ( ). The structure of was elucidated on the basis of chemical
reactions and spectral analysis (IR, GC, ESI-MS, H-NMR, 13C-NMR, DEPT, HMBC, HMQC
and NOESY). The antiproliferative effects of were evaluated in a cytotoxicity assay
β-D-glucopyranosyl-5β-furost-20(22)-en-3
β
, 15
β,26-triol-
α-L
β-D
1) were isolat-
2
β
β
α-L
β-D
3
4
1
1
1-4
against the human tumor cell line, A549. Compound
cytotoxicity against A-549, with an IC50 value of 3.87
2
µ
(Aspacochioside C) exhibited moderate
g/mL.
Key words: Asparagus cochinchinensis, Furostanol glycosides, Cytotoxicity
and cytotoxicity of a new furostanol glycoside are dis-
cussed in this paper.
INTRODUCTION
Asparagus cochinchinensis (Chinese name: Tiandong),
a Traditional Chinese Medicine, is the tuberous root of
A. cochinchinensis (Lour.) Merr (Liliaceae) which is dis-
tributed in many provinces of China. A. cochinchinen-
sis was often used for treatments of fever, cough, hemo-
MATERIALS AND METHODS
General procedures
Melting points were determined on an RY-2 melting
ptysis, diabetes, constipation, swelling, throat pain and point apparatus and were uncorrected. IR spectra were
lung cancer (Jiangsu Medical College, 1985). A few recorded on a Bruker Vector-22 spectrometer with
substances such as β-sitosterol, some amino acids, some KBr pellets. 1D and 2D NMR spectra were recorded
monosaccharides, oligosaccharides, polysaccharides on a Bruker DRX500 NMR spectrometer in pyridine-
and several furostanol saponins have been isolated d5. GC was performed with a HP-5892 II gas chroma-
from the plant (Aquino et al., 1986; Ju et al., 1994; tograph. An Agilent mass spectrometer (for ESI) and
Meng et al., 1998a; Shi et al., 2004). In recent years, Q-Tof micro mass spectrometer (for HR-ESI) were
some steroidal saponins which possess cytotoxic used (in m/z). Column chromatography (CC) was perf-
activities in a human lung carcinoma (A549) tumor ormed with silica gel (200-300 mesh) and Sephadex
cell line have been isolated from other Asparagus LH-20 (40-70
µm, GE Healthcare Biosciences AB).
species, such as A. filicinus (Cong et al., 2000). In search Macroporous resin was from Zhen Tiancheng Tech-
for the bioactive constituents from Chinese herbs, nology Co. Ltd. TLC and preparative TLC silica gel
compounds from A. cochinchinensis were isolated in plates were from Huiyou Silical Gel Development Co.
the present study from the extract of the tuberous Ltd. and visualized by spraying with 5% H2SO4 in
roots. Extraction, separation, structural identification ethanol followed by heating.
Plant material
Correspondence to: Hai-Sheng Chen, Department of Phy-
Roots of A. cochinchinensis were collected in Huang-
tochemistry, School of Pharmacy, Second Military Medical Uni-
shi city (Hubei province) in June 2006 and authenti-
cated by Prof. Han-Chen Zheng, Department of Pharma-
cognosy, Second Military Medical University. A voucher
versity, Shanghai 200433, China
Tel: 86-21-81871250, Fax: 86-21-81871250
E-mail: haishengc@hotmail.com
1587

1588
Y. Shen et al.
Table I. 13C-NMR data of compound
1
in C5D5N (150 M)
specimen (No. 20060764) was kept in the Herbarium
of Second Military Medical University.
NO
δ
(C)
NO
δ (C)
1
2
3
4
5
6
7
8
30.6
26.6
74.9
29.9
36.6
25.5
26.6
34.8
39.8
34.9
21.0
39.7
43.6
54.4
84.4
90.1
64.6
14.2
23.5
105.0
11.4
153.8
34.1
39.4
30.7
75.4
17.7
3-Glu
1’
2’
3’
4’
Extraction and isolation
102.7
75.2
78.2
78.1
76.8
61.3
Air-dried and ground roots of A. cochinchinensis (9.5
kg) were extracted twice with 75% ethanol for 3 days
each time at room temperature. The solvent was re-
moved under reduced pressure to yield a dark residue
(1516 g). The residue was suspended in water and
then partitioned with CHCl3. The aqueous layer was
subjected to column chromatography over macropor-
ous resin, eluting first with H2O then successively
with 30%, 50%, 80%, and 90% EtOH. Combining 50%
partition with 80% partition, the new partition was
evaporated under reduced pressure to yield a residue
(612 g). The residue (600 g) was subjected to silica gel
column chromatography eluting with a CHCl3-MeOH-
H2O gradient (40:10:1-65:35:10) to afford 5 fractions
(Fr.1-Fr.5). Fraction 3 (18 g) was rechromatographed
on a silica gel column with CHCl3-MeOH-H2O (40:10:1
5’
6’
9
4’-Rha
1’’
2’’
3’’
4’’
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
102.4
72.3
72.5
73.7
70.0
18.2
5’’
6’’
26-Glu
1’’’
2’’’
3’’’
4’’’
104.9
74.9
78.1
71.4
76.5
62.5
-20:10:1) as the eluent to give the known compounds
2
(24 mg) and (17 mg). Fraction 4 (14) was purified by
3
column chromatography over silica gel using a CHCl3-
MeOH-H2O gradient (90:35:6-65:35:10) and further
purified with a C-18 silica gel column by using MeOH-
5’’’
6’’’
H2O (65:35) as the eluent to give compound
1 (53 mg).
Compound (24 mg) was obtained from Fraction 5 (21
4
g) by using a C-18 silica gel column with acetonitrile-
H2O (28:72) as the eluent.
(25S)-26-
, 15 , 26-triol-3-
-D-glucopyranoside (1)
O-β-D-glucopyranosyl-5β-furost-20(22)-en-
3
β
β
β
O-[
α-L
-rhamnopyranosyl-(1-4)]- 747[M+Na]+ (Jadhav and Bhutani, 2006).
1
White amorphous powder, m.p. 210-212oC; H-NMR Pseudoprotoneodioscin (4)
(600 MHz, in C5D5N):
4.12 (1H, dd J = 10.0 Hz, 7.3, H-26), 4.05 (1H,
15), 3.48 (1H, dd J = 10.0 Hz, 7.3, H-26), 2.50 (1H,
J = 10.1 Hz, H-17), 1.72 (3H, , H-21), 1.18 (3H, d, J =
6.0 Hz, H-27), 1.16 (1H, , H-14), 0.85 (3H, , H-19),
, H-18); sugar portions: 5.90 (1H, brs, H-1''),
J = 7.0 Hz, H-1'), 4.81 (1H,
J = 7.0 Hz, anol solution (3 mL) was heated for 4 h at 70oC. After
J = 6.2 Hz, H-6''); 13C-NMR (150 the reaction was completed and checked by HPTLC, 1
δ
4.94 (1H, H-16), 4.27 (1H, H-3), White amorphous powder. C51H82O21. ESI-MS m/z
,
m
, H- 1053 [M+Na]+ (Yoshikawa et al., 2007).
,
d,
s
Acidic hydrolysis of compound 1 and prepara-
tion of the aglycon
m
s
0.70 (3H,
4.82 (1H,
s
d
A solution of compound 1 (5 mg) in 2 M HCl meth-
,
d,
H-1'''), 1.70 (3H, d,
MHz, in C5D5N) see Table I; ESI-MS m/z 925 [M+ M NaOH was added dropwise until neutral pH (7)
Na]+, HR-ESI-MS m/z 925.9913 [M+Na]+ (Calcd. for was achieved. Removal of methanol was done under
C45H74O17Na, 926.0615).
reduced pressure, the water solution was extracted
with CHCl3 (2 mL 3), the organic layers were com-
bined and solvent was removed under reduced pres-
×
Aspacochioside C (2)
White amorphous powder. C45H75O17. ESI-MS m/z sure to give the aglycon (Meng et al., 1998b).
909 [M+Na]+ (Shi and Bhutani, 2004).
Hydrolysis of compound 1, preparation of nitrile
acetate derivatives and GC analysis
(25S)-5
syl-(1-4)]-
β
-spirostan-3
β-yl-O-[O-α-L-rhamnopyrano-
β
-
D-glucopyranoside
A solution of each compound (5 mg) in 2 M HCl
White amorphous powder. C39H64O12. ESI-MS m/z methanol solution (3 mL) was heated for 4 h at 70oC.

New Furostanol Saponin with Cytotoxicity
1589
After the reaction was completed checked by HPTLC, DMSO and diluted with Phosphate Buffered Saline
methanol was removed under reduced pressure. Pyri- (PBS) to a final concentration 1000 g/mL. The solu-
dine (0.5 mL) and hydroxylamine hydrochloride (5 tions were serially diluted with PBS to obtain the lower
mg) was added, the mixture was shaken, for 0.5 h at concentrations from 100-1.5625 g/mL. The cells were
90oC and then cooled to RT. Acetic anhydride (0.5 mL) incubated in 96-well microtitre plates (Nunc and
was added in, heated for 0.5 h at 90oC and the mixture Tarson) at a density of 4 104 - 6 104 cell/mL. Sam-
was then analyzed by GC (Zhang, 1999). Glucose, ples of different concentrations (10 L/well) were
µ
µ
×
×
µ
xylose, galactose and rhamnose standards were pre- added 24 h after cell seeding. The microtitre plates
pared by the same method. GC condition: HP-5892 II were incubated for 72 h in a humidified atmosphere
gas chromatograph, FID, HP-20M (Carbowx 20 M), 25 with 5% CO2 at 37oC. The cellular viability was deter-
m
× 0.32 mm × 0.3 µ
m, sample temperature: 220oC, mined using the MTT assay (Li et al., 2002). The optical
detector temperature: 280oC, column temperature: densities (OD) were read on an ELISA plate reader
210oC (constant temperature), column pressure: 60 psi. (Denley MK-2) at a wavelength of 570 nm.
Bioassay for cytotoxic activity
RESULTS AND DISCUSSION
An MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
terazolium bromide] colorimetric method to determine
the cytotoxic activity against human cell cultures (A-
Chemical structure elucidation of compound 1
Compound
1, white amorphous powder, m.p. 210-
549 cells) in vitro was performed; doxorubicin hydro- 212oC. Liebermann-Burchard reaction was positive.
chloride (DOX) was used as a positive control. The Ehrlich reagent reaction showing a red color indicated
samples (purity > 95%) were individually dissolved in that the compound was a furostanol saponin. ESI-MS
Fig. 1. Chemical structures of compounds 1-4

1590
Y. Shen et al.
Table II. GC results of hydrolyzed products of compound
1
Comp.\tR
tR1 (min)
tR2 (min)
tR3 (min)
tR4 (min)
Sugar Standard
4.398 (Rha)
6.489 (Xyl)
12.983 (Glu)
14.840 (Gal)
I
4.438
−
12.473
−
tR: relative retention time
Furthermore, these data indicated that compound
1
possessed a triglycosidic structure. In the HMBC spec-
trum (see Fig. 1), long range correlations from H-1' to
C-3, H-1'' to C-4' and H-1''' to C-26 unequivocally
revealed a disaccharide [
-glucopyra-nosyl moiety and the remaining
glucopyranosyl unit was located at C-3 and C-26 of
the aglycon. The NMR data of were similar to the
known compound aspacochioside C ( ) which isolated
from A. cochinchinensis by Shi et al. (2004) except
that compound had a hydroxy moiety. The OH group
α-L-rhamnopyranosyl-(1-4)]-
β-D
β-D-
1
2
1
was positioned at C-15 (δC 84.4) due to the HMBC
cross-peaks of H-15 (δH 4.05) with C-14 (δC 54.4) and
C-16 (δC 90.1). The relative configuration
15) was deduced by the NOESY correlations of H-15
H 4.05) and H-14 ( H 1.16). Accordingly, the structure
of was confirmed as (25S)-26-O- -glucopyranosyl-
-furost-20(22)-en-3 ,15 ,26-triol-3-O-[ -rhamno-
[M+Na]+ indicating that its moleculer weight was 902. pyranosyl-(1-4)]-
-glucopyranoside. It is a novel com-
δ of OH (C-
Fig. 2. Chemical structure and Key HMBC correlations
(H C) and NOESY correlations (H-H) of compound
→
1
(
δ
δ
1
β-D
showed that the quasi-molecular ion peak was 925
5
β
β
β
α-L
β-D
HR-ESI-MS showed that quasi-molecular ion peak pound and was named aspacochioside D.
was 925.9913 [M+Na]+ (cal. 926.0615) which in-
Compounds 1-4 were tested for their cytotoxicities
dicated that the molecular formula was C45H74O18. against a human lung adenocarcinoma cell line, A-
Aglycon was obtained after acid hydrolysis and its 549. Compounds 1-4 exhibited moderate cytotoxicities
absorption band at 917 cm−1 was higher than 899 cm−1 against A-549 cells with IC50 values of 21.3, 3.87, 17.4,
in the IR spectrum indicating that C25 was in an S and 25.8
µg/mL, respectively.
configuration. 1H-NMR (C5D5N) of Compound
1
at high
field revealed 4 methyl groups [δH 0.70 (3H, s, 18-
CH3), 0.85 (3H, s, 19-CH3), 1.18 (3H, d, J = 6 Hz, 27-
ACKNOWLEDGEMENTS
CH3), 1.72 (3H, s, 21-CH3)], 3 signals for sugar [
(1H, brs), 4.82 (1H, d, J = 7.0 Hz), 4.81 (1H, d, J = 7.0 Foundation of National China (No. 20472113) and
δ
H 5.90
The work was supported by the National Science
Hz)]. Glucose and rhamnose were detected by GC Shanghai Leading Academic Discipline Project (No.
analysis: a
guration of rhamnose were confirmed due to the cou-
pling constant of the end group proton [ H 4.83 (1H, d,
J = 7.0 Hz), H 5.90 (1H, brs), H 4.81 (1H, d, J = 7.0 Hz)].
13C-NMR and DEPT spectrums displayed 3 carbon
signals of the end group [ C 104.9, 102.7 and 102.4], 2
characteristic signals of a double bond (both were
quaternary carbons) at δC 153.8 and 105.0. Further,
the correlations of H-21 with C-22 (δC 153.8) and H-
β-configuration of glucose and an α-confi- B906).
δ
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New Furostanol Saponin with Cytotoxicity
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