A new diterpenoid and other constituents from Acanthopanax brachypus harms

Article abstract of DOI:10.1002/jccs.200900171

Phytochemical investigation of the stem bark of Acanthopanax brachypus afforded a new labdanetype diterpene glycoside, 3α-trans-sinapoyloxy- jhanol 18-O-β-D-glucopyranoside (1), together with four known compounds, including one diterpene acid, acanthoic a

Full text of DOI:10.1002/jccs.200900171

1
186
Journal of the Chinese Chemical Society, 2009, 56, 1186-1190
A New Diterpenoid and Other Constituents from Acanthopanax brachypus
Harms
a,b
b
) and Jun Fan * (
Hao-Bin Hu
College of Chemical Engineering, Northwest University, Xi’an 710069, P. R. China
(
)
a
b
College of Chemistry and Chemical Engineering, Longdong University, Qingyang 745000, P. R. China
Phytochemical investigation of the stem bark of Acanthopanax brachypus afforded a new labdane-
type diterpene glycoside, 3a-trans-sinapoyloxy-jhanol 18-O-b-D-glucopyranoside (1), together with
four known compounds, including one diterpene acid, acanthoic acid (2), one coumarin, isofraxidin (3),
one phenolic glycoside, sasanquin (4), as well as one chalcone glycoside, okanin 4-methyl ether-3¢-O-b-
D-glucopyranoside (5). All of the structures were characterized by means of spectroscopic methods, in-
1
13
cluding H, C, 2D-NMR and HR-MS, as well as chemical methods and comparison with the literature
data.
Keywords: Acanthopanax brachypus; Araliaceae; Diterpene glycoside; Phenolic glycoside.
INTRODUCTION
The genus Acanthopanax belonging to the Araliaceae
chemical composition and biological activity, only triter-
penoid saponins, stilbene and phenolic glycosides have
1
5-17
family includes 37 species around the world, which are
widely distributed in Korea, Japan, China and the fareast-
ern region of Russia. Of these, 26 species and 18 varieties
been previously isolated from this plant.
Further sys-
tematic phytochemical investigation led to the isolation of
one new diterpene glycoside, viz. 3a-trans-sinapoyloxy-
jhanol 18-O-b-D-glucopyranoside (1), together with four
known compounds (2-5) from the stem bark of A. brachypus.
In the present communication, we describe the isolation
and structural elucidation of these compounds, their chemi-
cal structures are shown in Fig. 1.
1
,2
grow in mainland China, especially in the North. The
root and stem bark of these plants have been used as a tonic
and sedative, as well as for the treatment of rheumatism, di-
abetes, chronic bronchitis, hypertension, antistress and
3
-6
ischemic heart disease, and gastric ulcer.
As a endangered shrub in the wild due to overhar-
vesting and loss of habitat through deforestation, Acan-
thopanax brachypus Harms is distributed in a narrow geo-
graphical area, most in the loess plateau of the Northwest of
RESULTS AND DISCUSSION
The ethanolic extract of the dried stem bark of A.
2
brachypus was suspended in H O and successively ex-
7
,8
China. The seeds of A. brachypus have been reported to
contain a variety of microelement indispensable to human
body, alleviate women menopause syndrome, and exhibit
tracted with n-hexane, EtOAc and n-BuOH. The EtOAc
fraction was subjected to repeated column chromatography
to yield compounds 1-5. The four known compounds were
9
,10
18
immunostimulating and anticancer activities. Rhizome
identified as (-)-pimara-9(11),15-dien-19-oic acid (2),
1
9
20
isofraxidin (3), sasanquin (4) and okanin 4-methyl
extracts have been used to inhibit the allergic response in
1
1
21
ether-3¢-O-b-D-glucopyranoside (5), respectively, by
China and Korea. The other parts of this plant such as the
roots, leaves and flowers are also employed for various
1
spectral analysis results compared with those already re-
ported in the literature. Of these compounds, 2 and 3 were
first isolated from this plant whereas 4 and 5 were reported
from the family Araliaceae for the first time. The structure
of the new compound was elucidated as follows.
2-14
therapeutic purposes.
For effective utilization and
quality control of the natural resources, analysis of the ac-
tive compounds in A. brachypus could be very important.
However, to the best of our knowledge, previous studies of
A. brachypus was mainly focused on the reproductive biol-
ogy and ecology, there has been a few reports to date on its
Compound 1 was obtained as pale yellow amorphous
powder from MeOH, and assigned the molecular formula
*
Corresponding author. Tel: +86-29-88303085; Fax: +86-29-88302348; E-mail: fanjun1959@126.com

A New Diterpenoid from Acanthopanax brachypus Harms
J. Chin. Chem. Soc., Vol. 56, No. 6, 2009 1187
Fig. 1. Chemical structures of compounds 1-5.
C
37
H
54
O
12 accounting for 11 degrees of unsaturation, based
and 111.8], a trans-sinapoyl group [two methoxyl proton
+
on HR-FAB-MS (m/z 691.3701 [M+H] ; calc. 691.3693).
signals at d
H
3.91 (6H, s), two aromatic proton signals at d
H
H
The UV spectrum exhibited absorptions at 225, 238 and
6.78 (2H, s), and two trans-olefinic proton signals at d
3
26 nm, suggesting the presence of a conjugated aromatic
6.41 and 7.57 (each H, d, J = 15.9 Hz), corresponding to
56.4, 105.8, 116.4, 126.1, 137.0, 145.6 and 147.2, car-
bonyl carbon at d 167.1] and one glucopyranosyl group
[anomeric proton signal at d 4.87 (d, J = 7.8 Hz), anomeric
carbon (d 104.5), four methines (d 74.8, 78.1, 71.1 and
77.2) and one methylene (d 62.8)]. According to these
spectral data, 1 was considered to be a trans-sinapoyloxy-
2
system. The IR spectrum apart from hydroxyl (3408-
2
d
C
-
1
030 cm ), monosubstituted double bond (3081, 1642, 982
3
C
-
and 918 cm ), aromatic ring (1600 and 1512 cm ) and
1
-1
H
-
glycosidic bond (1075 cm ) characteristic absorption
1
C
C
bands, showed an additional a,b-unsaturated ester (1701,
C
-
628 and 970 cm ), indicating the presence of an ester
1
1
2
3,24
25-27
group conjugated with a trans-double bond.
The frag-
8,13-epoxylabdane diterpene glucoside.
In the FAB-
+
ment ion peaks at m/z 485 [M+H-206] , 323 [M+H-206-
MS spectrum of 1, the significant ion peaks at m/z 485
+
+
[M+H-sinapoyl] , 323 [M+H-sinapoyl-glucosyl] and 207
+
1
62] and 207 indicated that 1 contains a hexose and a
sinapoyl moiety.
The gross structure of 1 was deduced from detailed
+
[sinapoyl] also indicated the presence of sinapoyl and
glucosyl groups in 1. The coupling constant (J = 7.8 Hz) of
1
13
analysis of the H and C-NMR spectroscopic data aided
by DEPT, HMBC and NOESY experiments. All of these
spectra of 1 revealed the presence of four tertiary methyl
the signals due to the anomeric and methine protons indi-
cated that the glucopyranose (Glc) unit is b-configuration
4
in a C
1
conformation when the glucopyranose is assumed
groups [d
sponding to d
methylene [d
one oxygenated methine [d
genated quanternary carbons [d
nal mono-substituted vinyl [ABX system, d
H
1.26 (s), 1.31 (s), 0.89 (s) and 0.86 (s), corre-
29.6, 24.3, 17.2 and 15.8], one oxygenated
2.98, 3.64 (each H, d, J = 9.3 Hz)/d 78.1],
3.57 (brs)/d 79.5], two oxy-
78.6 and 75.2], one termi-
5.98 (dd, J =
7.6, 10.8 Hz), 5.02 (dd, J = 10.8, 1.0 Hz, H-15-cis) and
.22 (dd, J = 17.6, 1.0 Hz, H-15-trans), along with d 147.3
to be in the D-form. These aforementioned conclusions
were further evidenced by the detection of trans-sinapic
acid and D-glucose (in the ratio of 1:1, determined by co-
C
H
C
2
8
H
C
TLC with authentic sample and the optical rotation ) after
9,30
acid and alkaline hydrolysis.
2
C
H
The ester substituent was placed at C-3 as a result of
1
the downfield shifts observed for H-3 and C-3 in the H and
1
5
1
3
C
C-NMR spectra, respectively, and the correlations ob-

1
188 J. Chin. Chem. Soc., Vol. 56, No. 6, 2009
Hu and Fan
served between H-3/C-1, C-2, C-4, C-18, C-19 and C-9¢ of
the trans-sinapoyloxy unit in the HMBC spectrum. Like-
wise, the glucosyl group was connected to C-18 of the
aglycon through an O-glycosidic bond because of the pres-
ence of HMBC correlations between H-1²/C-18 and H-
termined on a Bruker DRX-400 spectrometer using tetra-
methylsilane as the internal standard. Chemical shifts (d)
are given in ppm and coupling constants (J) in Hz. Mass
spectra were obtained on VG Auto-Spec-3000 mass spec-
trometer using glycerol as matrix. Silica-gel (100-200,
200-300 mesh, Qingdao Marine Chemical Plant, Qingdao)
and Sephadex LH-20 gel (20-100 mm, Pharmacia) were
used for column chromatography (CC). Thin-layer chro-
matography (TLC) was performed on precoated sillica-gel
plates (GF254), and spots were visualized under ultraviolet
(UV) light (254 and 365 nm) by spraying proper colored re-
agent. All solvents used were of analytical grade.
1
8/C-1², as well as the downfield chemical shift of H
2
-18
and C-18, respectively.
The relative stereochemistry of the chairl centres at
C-3, C-4, C-5, C-8, C-9, C-10 and C-13 was deduced from
the NOESY spectrum and the J-values. In the NOESY of 1
(
Fig. 2) couplings were seen between H-3/H
3 2
-19, H -18,
H
a
-2 and H -2, this indicated that H-3 must be equatorial
b
and on the b-side of the molecule. This was also supported
by the small J-values for the equatorial-axial and equato-
Plant Material
The stem bark of A. brachypus were collected from
Qingyang of Gansu Province, P. R. China, in August 2007,
and identified by Prof. Xiao-Qiang Guo (Department of
Life Sciences, Longdong University, P. R. China). A
voucher specimen (No. 2007-18) was deposited in the Her-
barium of the Department of Life Sciences, Longdong
University.
rial-equatorial couplings between H-3/H
presence of NOESY couplings between H-5/H
cated that H-5 had to be a-oriented like the C-18 methy-
lene. The NOESY correlations between H -17/H -20/H
1, H -19/H -20 and H -16/H -17/H -7 indicated the close
a
-2 and H
b
-2. The
2
-18 indi-
3
3
2
-
1
3
3
3
3
a
proximity of these groups and their b-dispositions.
On the basis of the foregoing studies, the structure of
Extraction and Isolation
1
was established as 3a-trans-sinapoyloxy-jhanol 18-O-
The air-dried and pulverized stem bark of A. brachypus
(5.0 kg) were extracted three times with 95% aqueous
EtOH (15 L ´ 7 d, each time) at room temperature, and then
the extracts were combined and concentrated under re-
duced pressure at 60 °C to yield 242 g of a brown viscous
residue. The EtOH extract was suspended in distilled water
(1500 mL) and partitioned successively with n-hexane
(500 mL), EtOAc (500 mL) and n-BuOH (saturated with
water, 500 mL). The concentrated EtOAc-soluble extract
(188 g) was subjected to column chromatography [silica
gel (100-200 mesh, 480 g), polarity gradient with n-hex-
b-D-glucopyranoside.
EXPERIMENTAL PART
General Experimental Procedures
Melting points were obtained on an X-4 digital mi-
cro-melting point apparatus and uncorrected. Optical rota-
tions were measured on a Perkin-Elmer 341 digital polari-
meter. UV spectra were recorded on a Shimadzu UV-300
spectrophotometer, lmax (log e) in nm. IR spectra were re-
KBr
corded on Perkin-Elmer 577 spectrometer, n max in cm . H
-1 1
1
400 MHz), C (100 MHz) and 2D NMR spectra were de-
3
(
3 2
ane, CHCl , Me CO and MeOH, pure or in mixtures] to af-
Fig. 2. The key HMBC (H®C) and NOESY (H«H) correlations of compound 1.

A New Diterpenoid from Acanthopanax brachypus Harms
J. Chin. Chem. Soc., Vol. 56, No. 6, 2009 1189
1 13
Table 1. H and C-NMR (DEPT) data of compound 1 (400/100 MHz, CD OD)
3
Position
dH
dC (DEPT) Position
dH
dC (DEPT)
1
2
3
4
5
6
7
8
9
1.81, 1.06 m
1.86, 1.51 m
3.57 brs
036.1 (CH2)
025.8 (CH2)
079.5 (CH)
043.8 (C)
20
1¢
2¢
3¢
4¢
5¢
6¢
7¢
8¢
9¢
0.86 s
6.78 s
015.8 (CH3)
126.1 (C)
105.8 (CH)
147.2 (C)
1.69 m
039.8 (CH)
020.6 (CH2)
041.8 (CH2)
078.6 (C)
137.0 (C)
1.54, 1.28 m
1.47, 1.84 m
147.2 (C)
6.78 s
105.8 (CH)
145.6 (CH)
116.4 (CH)
167.1 (C)
7.57 (d, J = 15.9 Hz)
6.41 (d, J = 15.9 Hz)
1.34 m
055.8 (CH)
037.8 (C)
1
0
1
1
1.59, 1.45 m
1.63, 1.89 m
015.3 (CH2) CH O-3¢
3
3.91 s
056.4 (CH3)
056.4 (CH3)
104.5 (CH)
074.8 (CH)
078.1 (CH)
071.1 (CH)
077.2 (CH)
1
1
1
1
2
3
4
5
035.7 (CH2) CH O-5¢
3
3.91 s
075.2 (C)
5.98 (dd, J = 17.6, 10.8 Hz) 147.3 (CH)
1²
2²
3²
4²
5²
4.87 (d, J = 7.8 Hz)
3.82 m
5.02 (dd, J = 10.8, 1.0 Hz)
.22 (dd, J = 17.6, 1.0 Hz)
111.8 (CH2)
3.77 m
5
3.94 m
1
1
1
1
6
7
8
9
1.26 s
029.6 (CH3)
024.3 (CH3)
078.1 (CH2)
017.2 (CH3)
3.86 m
1.31 s
4.05 (dd, J = 11.3, 1.6 Hz)
3.96 (dd, J = 11.3, 6.0 Hz)
6
²
062.8 (CH2)
2.98, 3.64 (d, J = 9.3 Hz)
0.89 s
J values are in parentheses and reported in Hz; chemical shifts are given in ppm.
ford Fr. A
1
-A18 according to TLC analysis. Fr. A
3
(5.6 g)
Acid hydrolysis of compound 1 and the determina-
tion of the absolute configuration of glucose
was applied to a silica gel column (200-300 mesh, 75 g)
with elution of petroleum ether/acetone (10:1®1:5) to di-
vide into Subfr. A301-A307 on the basis of TLC analysis.
Compound 2 (15 mg) and 3 (21 g) were obtained from
Subfr. A302 and A305, respectively. Compound 1 (16 mg)
A solution of 1 (3 mg) in 2N HCl-MeOH (3 mL) was
refluxed with magnetic stirring in a water bath at 90 °C for
2 h. After cooling, the reaction mixture was diluted with
2
H O (5 mL), then extracted twice with EtOAc. The aque-
was isolated from Fr. A
umn (200-300 mesh, 90 g) with CHCl
and preparative TLC with MeOH/CHCl
and compound 4 (18 mg) and 5 (9 mg) were isolated from
Fr. A (10.6 g) by subjected silica gel column (200-300
mesh, 130 g) with a successive gradient mixture of CHCl
4
(3.2 g) by repeated silica gel col-
-MeOH (2:1®1:4)
/n-hexane (1:6:3),
ous layer were neutralized with 5% NaOH, then concen-
trated and dried to furnish a monosaccharide residue. The
residue was purified through Sephadex LH-20 column
3
3
3
eluting with CHCl /MeOH (1:1) to afford sugar, which was
6
identified as glucose by co-TLC with authentic sample.
3
-
The glucose was determined as D-form by optical rotation
2
5
MeOH (3:1®1:10) and Sephadex LH-20 eluting with
D
2
[[a] +4.8° (c 0.092, H O)].
MeOH, respectively.
Alkaline hydrolysis of compound 1 and the determi-
nation of trans-sinapic acid
3
a-trans-sinapoyloxy-jhanol 18-O-b-D-glucopyranoside
(
1)
Asolution of 1 (3 mg) in 5% KOH-MeOH (4 mL) was
Pale yellow amorphous powder (MeOH), molecular
refluxed (5 h), diluted with H
with EtOAc. The aqueous layer were neutralized with 5%
HCl, then extracted with CH Cl . The CH Cl layer con-
2
O (5 mL), then extracted
25
formula: C37
H O12; mp 212-215 °C; [a] -18.3° (c 0.07,
54
MeOH
MeOH); UV lmax nm (log e): 225 (4.21), 238 (3.56) and
D
2
2
2
2
KBr
-1
26 (4.06); IR n max cm : 3408-3030, 3081, 1701, 1642,
3
1
centrated and repeated crystallized to furnish trans-sinapic
1
acid (0.4 mg), which was identified by comparison of H-
30
NMR spectral data with those reported in the literature.
628, 1601, 1512, 1456, 1280, 1140, 1075, 982, 970, 918;
+
HR-FAB-MS m/z: 691.3701 [M+H] (calculated for
+
37 55
C H O12, 691.3693). FAB-MS m/z: 691 [M+H] , 485
+
M+H-206] , 323 [M+H-206-162] and 207 [sinapoyl] ;
13
H and C-NMR see Table 1.
+
+
[
1
ACKNOWLEDGEMENTS
We gratefully acknowledge the financial support of

1
190 J. Chin. Chem. Soc., Vol. 56, No. 6, 2009
Hu and Fan
1
4. Deyama, T.; Nishibe, S.; Nakazawa, Y. Acta Pharmacol.
the “Postgraduates’ Innovative Education Program” (NWU
Graduate Cross-discipline Funds, No. 08YJC20) of North-
west University, P. R. China.
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Received May 11, 2009.
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