One new pregnane glycoside from the seeds of cultivated Brucea javanica

Article abstract of DOI:10.1007/s12272-011-0809-5

A new pregnane glycoside, named (20R)-O-(3)-β-D-glucopyranosyl- (1→2)-α-L-arabinopyranosyl- pregn-5-en-3β,20-diol (1), and seven known compounds, brusatol (2), bruceine B (3), bruceine D (4), yadanziolide A (5), bruceine E (6), yadanzioside G (7), and yadanzioside B (8), were isolated from the cultivated dry seeds of Brucea javanica. The structure of 1 was elucidated on the basis of 1D- and 2D-NMR spectroscopic analyses. Their inhibitory effects on tumor cells were also tested. Compound 1 was slightly active against HL-60, SMMC-7721, A-549, and MCF-7 tumor cells. Compounds 2 and 3 demonstrated significant inhibitory activities against all tested cells. These results indicate that cultivated B. javanica could replace the wild plant as an antitumor plant resource.

Full text of DOI:10.1007/s12272-011-0809-5

Arch Pharm Res Vol 34, No 8, 1297-1300, 2011
DOI 10.1007/s12272-011-0809-5
One New Pregnane Glycoside from the Seeds of Cultivated Brucea
javanica
Jie-Qing Liu, Cui-Fang Wang, Xing-Yao Li, Jian-Chao Chen, Yan Li, and Ming-Hua Qiu
State Key Laboratory of Photochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Acad-
emy of Sciences, Kunming 650204, Yunnan, China
(Received September 22, 2010/Revised March 29, 2011/Accepted May 3, 2011)
A new pregnane glycoside, named (20
syl-pregn-5-en-3 ,20-diol ( ), and seven known compounds, brusatol (
ine D ( ), yadanziolide A ( ), bruceine E ( ), yadanzioside G ( ), and yadanzioside B (
isolated from the cultivated dry seeds of Brucea javanica. The structure of was elucidated on
the basis of 1D- and 2D-NMR spectroscopic analyses. Their inhibitory effects on tumor cells
were also tested. Compound was slightly active against HL-60, SMMC-7721, A-549, and
MCF-7 tumor cells. Compounds and demonstrated significant inhibitory activities against
R
)-
O
-(3)-β-
D
-glucopyranosyl-(1
→
2)-
α
-
L
-arabinopyrano-
), bruce-
), were
β
1
5
2
), bruceine B (
3
4
6
7
8
1
1
2
3
all tested cells. These results indicate that cultivated B. javanica could replace the wild plant
as an antitumor plant resource.
Key words: Brucea javanica, Pregnane glycoside, Antitumor activity, Brusatol
INTRODUCTION
1979), yadanziolide A (
bruceine E ( ) (Li and Tso, 1980), yadanzioside G (
Brucea Javanica (L.) Merr. is distributed mainly in and yadanzioside B ( ) (Saka et al., 1984). In addition,
5) (Yoshimura et al., 1984),
6
7)
8
south China and exhibits bio-activities including anti- their inhibitory effects on tumor cells were also inves-
tumor and anti-malaria effects (Sharma and Agarwal, tigated. This study showed that cultivated B. javanica
1993). In previous phytochemical research, the plant could replace the wild plant as an antitumor plant
material of B. javanica used was mainly the wild plant. resource.
As a potential crude drug with anti-pancreatic cancer
agents (Lau et al., 2008), the plant of B. javanica was MATERIALS AND METHODS
widely cultivated in south of China. However, the
differences in the phytochemistries of cultivated and General procedures
wild material are not clear.
In order to study the differences in the chemical con- Column chromatography (CC): silica gel (SiO
Glucose and arabinose were purchased from Sigma.
; 200-
2
stituents of wild and cultivated B. javanica, the con- 300 mesh; Qingdao Marine Chemical Products Industry
stituents of cultivated B. javanica were investigated. Factory); Sephadex LH-20 (Pharmacia). TLC: silica
In this paper, we describe the isolation and structure gel G precoated plates (Qingdao Haiyang Chemical
elucidation of a new pregnane glycoside (
cultivated plant of B. javanica, together with 7 known
compounds, brusatol ( ) (Harigaya et al., 1989), bruceine 17A gas chromatograph equipped with an H
B ( ) (Anderson et al., 1987), bruceine D ( ) (Lee et al., ionization detector; column: TC-1 capacity column (30
0.25 mm); detector, FID. Optical rotations: Horiba
SEAP-300 spectropolarimeter. IR Spectra: Shimadzu
1
) from the Co.); spots were visualized by spraying with 10% aq.
SO solvent, followed by heating. GC: Shimadzu GC-
flame
H
2
4
2
2
3
4
m
×
Correspondence to: Ming-Hua Qiu, State Key Laboratory of
Photochemistry and Plant Resources in West China, Kunming
Institute of Botany, Chinese Academy of Sciences, Kunming
−
1
IR-450 instrument, with KBr pellets; in cm . NMR
Spectra: Bruker AV-400 or DRX-500 instruments;
chemical shifts
6
50204, Yunnan, China
Tel: 86-871-522-3257, Fax: 86-871-522-3255
E-mail: mhchiu@mail.kib.ac.cn
4
δ in ppm relative to Me Si, coupling
constants in Hz. FAB-MS (negative-ion mode;
J
1297

1298
J.-Q. Liu et al.
Fig. 1. Structures of 1-8
glycerol matrix) and HR-ESI-MS: VG Auto-Spec 3000 and then over Sephadex LH-20 column eluted with
and Thermo-Finnigan LCQ Advantage spectrometer; MeOH to yield compounds (30 mg), (25 mg), and
7
8
1
in m/z (rel. int. in % of the base peak).
(10 mg).
Plant material
(20R)- -(3)-
O
β
-
D
-glucopyranosyl-(1
→2)-α-L-arabino-
The seeds of Brucea javanica were purchased from pyranosyl-pregn-5-en-3
XiShuangBanNa, Yunnan Province, China, in August White amorphous powder; [
009, and identified by Prof. Shukun Chen, Kunming UV (MeOH) λmax (log
β
, 20-diol (1)
25
α
]
D
+ 82.0 (
c 0.10, MeOH);
2
ε
) 208 (3.08) nm; IR (KBr) υmax:
−1
1
13
Institute of Botany, Chinese Academy of Sciences.
3411, 2927, 1464, 1358, 1076 cm ; H- and C-NMR
see Table I; negative FABMS m/z 611 [M - H] ;
−
Extraction and isolation
HRFABMS (
m/z
calcd. 612.3510, found at 612.3501
−
The dry fruits of B. javanica (5 kg) were extracted [M] ).
three times with MeOH under reflux. After removal of
solvent, the extract (300 g) was suspended in water and Sugar analysis
then successively partitioned with petroleum ether Compound
1
(2 mg) in 2 N HCl/1,4-dioxane 1 : 1 (2
o
(PE) and ethyl acetate (EtOAc). After evaporation of mL) was heated at 90 C for 4 h in a water bath. The
the solvent, the EtOAc extract (80 g) was subjected to mixtures were neutralized with AgCO , filtered, and
3
column chromatography over silica gel with gradient then extracted with CHCl
elution with CH Cl-MeOH (60:1-1:1) to obtain fractions tration, the H O layer (monosaccharide portion) was
I-VIII. Fraction II (10 g) was separated over silica gel dissolved in 0.6 mL of pyridine, then 0.4 mL of hexa-
with CH Cl-MeOH (30:1) to yield (520 mg) and (83 methyl disilazane and 0.2 mL trimethyl chlorosilane
3
(3 × 1 mL). After concen-
3
2
3
2
3
mg). Fraction IV (12 g) was separated over silica gel were added successively. The mixture was kept at
o
with CH Cl-MeOH (25:1) and then over Sephadex LH- 60 C for 10 min in a water-bath. Next, the mixture
3
4
2
0 column eluted with MeOH to obtain compounds
20 mg), (18 mg), and
was separated over silica gel with CH Cl-MeOH (18:1) lowing conditions: Shimadzu GC-17A gas chromatog-
4
was centrifuged for 20 min at 1.0 × 10 rpm. The su-
(
5
6 (30 mg). Fraction VI (13 g) pernatant was subjected to GC analysis under the fol-
3

A New Pregnane Glycoside from Brucea javanica
1299
raph equipped with an H flame ionization detector. its H- and 13C-NMR spectra. The two anomeric H-
1
2
Column: TC-1 capacity column (30 m × 0.25 mm). atom signals of H-1' at 5.10 (d,
J
= 5.6 Hz) and of H-1''
= 7.6 Hz), respectively, suggested the pre-
-arabinopyranoside and a -glucopyranosyl
injection volume: 1 mL; split ratio: 1/50; glucose and moiety, respectively.
o
Column temp. 200-260 C, programmed increase: 38/ at 5.16 (d,
J
α
o
min; injection temp. 250 C; carrier gas: N (1 mL/min); sence of an
β
2
1
arabinose 11.99 min and 9.13 min, respectively.
The H-NMR spectrum of
methyl singlets at 0.97 (s) and
methyl doublet at 1.42 (d, = 6.1 Hz), suggesting a
1
(Table I) contained two
δ
δ
0.70 (s) and one
Cytotoxicity assay
J
13
A panel of human tumor cell lines was used: breast pregnane skeleton. This was supported by the C-
cancer MCF-7, hepatocellular carcinoma SMMC7721, NMR (3 × CH , 8 × CH , 7 × CH and 3 × C for the agly-
3
2
human myeloid leukemia HL-60, colon cancer SW480 cone). The chemical shift of the two olefinic carbons (a
and lung cancer A549. The cells lines were obtained quaternary carbon at 141.0 and a tertiary one at 121.9)
from Shanghai Cell Bank in China. All the cells were were in good agreement with the pregn-5-en-3-
O
-gly-
cultured in RPMI-1640 or DMEM medium (Hyclone), cosides (Srivastar et al., 1994). The C-NMR spectral
supplemented with 10% fetal bovine serum (Hyclone), features of and (20 )- -(3)- -L-arabinopyranosyl-
,20-diol (Christine et al., 1995) were nearly
cytotoxicity assay was performed by the MTT (2-(4,5- identical to each other, except for one additional glu-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cosyl unit in
method in 96-well microplates (Mosmann, 1983). After The linkage sites and sequences of the saccharides
compound treatment, cell viability was assessed and and the aglycone were determined by HMBC experi-
the cell growth curve was graphed. IC50 values were ments. Long-range correlations between H-3 ( 3.77)
calculated by Reed and Muench’s method (Reed and and C-1' ( 103.2), together with correlations between
4.56-4.60) and C-1', C-3' ( 74.7), and C-1'' (
05.8), were observed (Fig. 2). Meanwhile, some of the
signals were observed in the H-H COSY experiments
Fig. 2), and these data further illustrated the struc-
. The configuration of at C-20 was deter-
was obtained as white powder. Its mole- mined by comparison with the methyl proton shifts of
cular formula was C H O on the basis of HRFABMS the 20-epimers of pregn-5-en-3 ,20-diol (Cooley et al.,
13
1
β
R
O
α
o
at 37 C in a humidified atmosphere with 5% CO . The pregn-5-en-3
2
1.
δ
δ
Muench, 1938).
H-2' (
1
δ
δ
δ
RESULTS AND DISCUSSION
(
Chemical structure elucidation of compound 1 ture of
1
1
Compound
1
β
3
2
52 11
−
(
m
/
z
calcd. 612.3510, found at 612.3501 [M] ). The 1977), whereas good correspondence was found for the
FABMS spectrum of
611 and the [M - glu - H] peak at
−
1
showed the [M - H] peak at
m/
values of the (20
R
)-configuration. Thus, the structure
was determined as (20 )- -(3)- -glucopyranosyl-
2)- -arabinopyranosyl-pregn-5-en-3 ,20-diol.
−
z
of
m/z
449. Hydrolysis of
1
R
O
β-D
1
yielded D-glucose and L-arabinose on GC and in (1
→
α-L
β
Table I. 1H, 13C-NMR spectral data of
1
(in pyridine-d
5
, at 500 MHz and 125 MHz, respectively)
Position
δ
C
δ
H
(ppm) ( in Hz)
J
Position
δ
C
H
δ (ppm) (J in Hz)
1
2
3
4
5
6
7
8
9
137.6 t
130.3 t
178.6 d
139.1 t
141.0 s
121.9 d
132.2 t
131.8 d
150.4 d
137.0 s
121.1 t
139.1 t
141.6 s
156.8 d
124.5 t
126.8 t
1.74-1.78, 1.00-1.03 2m
2.16-2.20, 1.78-1.82 2m
3.77 (d, J = 10.5 )
17
18
19
20
21
1'
2'
3'
4'
5'
1''
2''
3''
4''
5''
6''
159.4 d
112.6 q
119.5 q
169.0 d
124.8 q
100.9 d
181.7 d
173.1 d
168.1 d
165.0 t
106.2 d
176.4 d
178.7 d
171.5 d
178.2 d
162.7 t
1.50-1.54 m
0.70 s
0.97 s
2.73 (dd, J = 12.6, 2.5); 2.55 (t,
J
= 12.1)
3.93 m
1.42 (d, J = 6.1)
5.10 (d, J = 5.6)
4.56-4.60 m
4.38-4.42 m
4.37-4.41 m
4.28-4.31, 3.70-3.75 2m
5.16 (d, J = 7.6)
4.11 (t, J = 8.8)
3.88-3.91 m
4.22-4.28 m
4.21 (t, J = 8.8)
4.50-4.53, 4.40-4.45 2m
5.34 br s
1.82-1.86, 1.44-1.49 2m
1.32-1.36 m
0.85-0.89 m
10
11
12
13
14
15
16
1.36-1.41 m
1.87-1.90, 1.03-1.13 2m
0.92-0.96 m
1.58-1.61, 1.12-1.10 m
2.21-2.16, 1.91-1.89 2m

1300
J.-Q. Liu et al.
tion of State Key Laboratory of Phytochemistry and
Plant Resources in West China (P2008-ZZ05 and
P2010-ZZ14), and the Knowledge Innovation Program
of the CAS (Grant No. KSCX2-YW-G-038, 027, and
KSCX2-YW-R-194, 29, as well as KZCX2-XB2-15-03).
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Fig. 2. Selected HMBC and H-H COSY correlations of
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, and SW480 cell lines using the MTT assay. Com-
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1
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7
and
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In our research, we found that the main compounds,
including brusatol, bruceine B, bruceine D, and yan-
danziolide A, from cultivated B. javanica were similar
to those from wild B. javanica. Furthermore, these
main compounds had significant antitumor effects in
the cytotoxicity assay. Therefore, in our opinion, this
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ACKNOWLEDGEMENTS
The project was financially supported by Founda-
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