A new drimane sesquiterpenoid glycoside from the seeds of Antiaris toxicaria

Article abstract of DOI:10.1080/10286020.2011.573479

A new drimane sesquiterpenoid glycoside, named 7-drimen-3β,11-diol 3-O-β-d-glucopyranoside, was isolated from the 95% EtOH extract of the seeds of Antiaris toxicaria (Pers.) Lesch. The chemical structure was completely elucidated using a combination of 1D and 2D NMR techniques (COSY, HMQC, HMBC, and ROESY) and HR-ESI-MS analysis. The compound showed inhibitory activities toward methicillin-resistant Staphylococcus aureus (MRSA), chronic myelogenous leukemia (K562), and human hepatoma (SMMC-7721) cell lines.

Full text of DOI:10.1080/10286020.2011.573479

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A new drimane sesquiterpenoid
glycoside from the seeds of Antiaris
toxicaria
a b
a b
a b
a b
Wen-Hua Dong
, Wen-Li Mei
, You-Xing Zhao
, Yan-Bo Zeng
a b
a b
,
Hui Wang
& Hao-Fu Dai
a
Key Laboratory of Tropical Crop Biotechnology, Ministry of
Agriculture, Institute of Tropical Bioscience and Biotechnology,
Chinese Academy of Tropical Agricultural Sciences , Haikou,
5
71101, China
b
Hainan Key Laboratory for Research and Development of Natural
Product from Li Folk Medicine , Haikou, 571101, China
Published online: 25 May 2011.
To cite this article: Wen-Hua Dong , Wen-Li Mei , You-Xing Zhao , Yan-Bo Zeng , Hui Wang & Hao-Fu
Dai (2011) A new drimane sesquiterpenoid glycoside from the seeds of Antiaris toxicaria , Journal of
Asian Natural Products Research, 13:06, 561-565, DOI: 10.1080/10286020.2011.573479
To link to this article: http://dx.doi.org/10.1080/10286020.2011.573479
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Journal of Asian Natural Products Research
Vol. 13, No. 6, June 2011, 561–565
A new drimane sesquiterpenoid glycoside from the seeds of
Antiaris toxicaria
ab
Wen-Hua Dong , Wen-Li Mei , You-Xing Zhao , Yan-Bo Zeng , Hui Wang and
ab
ab
ab
ab
ab
Hao-Fu Dai *
a
Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical
Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101,
b
China; Hainan Key Laboratory for Research and Development of Natural Product from Li Folk
Medicine, Haikou 571101, China
(
Received 26 December 2010; final version received 16 March 2011)
A new drimane sesquiterpenoid glycoside, named 7-drimen-3b,11-diol 3-O-b-D-
glucopyranoside, was isolated from the 95% EtOH extract of the seeds of Antiaris
toxicaria (Pers.) Lesch. The chemical structure was completely elucidated using a
combination of 1D and 2D NMR techniques (COSY, HMQC, HMBC, and ROESY)
and HR-ESI-MS analysis. The compound showed inhibitory activities toward
methicillin-resistant Staphylococcus aureus (MRSA), chronic myelogenous leukemia
(
K562), and human hepatoma (SMMC-7721) cell lines.
Keywords: Antiaris toxicaria; drimane sesquiterpenoid glycoside; 7-drimen-3b,11-
diol 3-O-b-D-glucopyranoside; anti-MRSA; cytotoxicity
1
.
Introduction
this plant. In our study of the seeds of
A. toxicaria collected from Lingshui
County of Hainan Province for the first
time, a new drimane sesquiterpenoid
glycoside (1) was isolated from the 95%
EtOH extract of the seeds. Drimane
sesquiterpenes showed attractive bioactive
properties including antibacterial, antifun-
gal, antifeedant, plant growth regulatory,
cytotoxic, phytotoxic, piscicidal, and
molluscicidal effects [16]. In this paper,
we described the structural elucidation of
compound 1 and its anti-methicillin-
resistant Staphylococcus aureus (MRSA)
and cytotoxic activities (Figure 1).
Antiaris toxicaria (Pers.) Lesch. (Mora-
ceae) is well known as ‘upas tree’ because
it contains a complex mixture of cardeno-
lide glycosides used as arrow and dart
poisons prepared throughout southeastern
Asia, from Burma to Indonesia [1–3]. The
genus Antiaris (Moraceae) comprises four
species, of which only A. toxicaria is
distributed in China, mainly in Guangxi,
Guangdong, Yunnan, and Hainan pro-
vinces [4]. Earlier studies of the toxicity of
this plant in Indonesia, Malaysian, or
China led to the isolation of cardenolides
[
[
5–11], flavonoids [12–14], and lignans
15]. Phytochemistry study of the seeds of
2
.
Results and discussion
A. toxicaria (Java, Indonesia) was only
reported by Muhlradt et al. [9], and the
chemical constituents isolated from the
seeds are quite different from the latex of
Compound 1 was obtained as a colorless
oil and had a molecular formula C H O
1 36 7
2
based on its HR-ESI-MS at m/z 400.2459,
*Corresponding author. Email: hfdai2001@yahoo.com.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2011.573479
http://www.informaworld.com

5
62
W.-H. Dong et al.
OH
was further confirmed by its acid hydroly-
sis. The other 15 carbon signals at d 135.0
11
1
3
C
12
1
9
6
(s), 123.8 (d), 90.8 (d), 61.3 (t), 58.3 (d),
2
10
8
OH
'
51.4 (d), 40.1 (s), 38.9 (t), 36.6 (s), 28.6
(q), 27.3 (t), 24.3 (t), 22.2 (q), 16.7 (q),
and 15.0 (q) in 1 indicated an aglycone
6
3
5
4'
7
5'
2'O
HO
O
3'
HO
1'
H
14
15
OH
13
of sesquiterpene, and these C NMR
spectral data were similar to a known
drimane sesquiterpene, 3b-hydroxydrime-
nol [18,19]. The obvious differences were
the significant down-fielded shift of C-3
from dC 79.6 to 90.8, as well as the
presence of six sugar carbons in compound
Figure 1. The structure of compound 1.
1
3
which was supported by C NMR and
distortionless enhancement by polarization
transfer (DEPT) spectral data (Table 1).
The IR spectrum showed characteristic
1
anomeric proton signal at d 4.30 (H-1 ) to
. The key HMBC correlation from the
0
absorption
2
for
hydroxyl
3511 cm ) and for double bonds (1652
groups
1
(
H
2
1
13
C-3 confirmed that the sugar moiety was
linked to the drimane sesquiterpenoid
aglycone at C-3 (Figure 2). The anomeric
configuration of the glucose was deter-
mined as b on the basis of the coupling
constant J ¼ 7.8 Hz. The relative configur-
ation of aglycone was same to the 3b-
hydroxydrimenol and confirmed by the
cm ). The C NMR and DEPT spectra
Table 1) showed 21 carbon resonances,
(
including four methyl, five methylene (two
oxygenated), nine methine, and three
quaternary carbons. The presence of a
glucose moiety was determined by six
characteristic carbon signals at d 106.7,
C
7
8.3, 77.7, 75.7, 71.8, and 62.9 [17], which
1 13
Table 1. H NMR (400 MHz) and C NMR (100 MHz) spectral data of compound 1 (in CD OD,
d).
3
No.
1
dH
dC
No.
12
dH
dC
2.02 (1H, m),
38.9 (t)
1.72 (3H, s)
22.2 (q)
1
.22 (1H, m)
1.99 (1H, m),
.98 (1H, m)
3.18 (1H, dd,
J ¼ 9.0, 2.8 Hz)
2
3
27.3 (t)
90.8 (d)
13
14
15
1.02 (3H, s)
0.90 (3H, s)
28.6 (q)
16.7 (q)
1
4
5
40.1 (s)
51.4 (d)
0.79 (3H, s)
4.30 (1H, d,
J ¼ 7.8 Hz)
3.17 (1H,
overlapped)
3.32 (1H,
15.0 (q)
106.7 (d)
0
1.19 (1H, m)
1
0
6
7
8
9
1.95 (1H, m),
1.94 (1H, m)
5.42 (1H, m)
24.3 (t)
123.8 (d)
135.0 (s)
2
75.7 (d)
78.3 (d)
71.8 (d)
0
3
overlapped)
3.27 (1H,
0
4
overlapped)
3.25 (1H, m)
3.69 (1H, dd,
J ¼ 11.9, 4.9 Hz)
0
0
1.78 (1H, m)
58.3 (d)
36.6 (s)
5
6
77.7 (d)
62.9 (t)
1
0
3
.82 (1H, d,
J ¼ 11.9 Hz)
11
3.77 (1H, dd,
J ¼ 11.0, 3.0 Hz)
61.3 (t)
3
.53 (1H, dd,
J ¼ 11.0, 6.4 Hz)

Journal of Asian Natural Products Research
563
HO
spectrum was obtained using a Nicolet
80 FT-IR instrument from KBr pellets.
H
3
The NMR spectra were recorded on a
Bruker AV-400 spectrometer, using TMS
as an internal standard. The HR-ESI-MS
spectrum was measured with an API
QSTAR Pulsar mass spectrometer.
Column chromatography (CC) was per-
formed with silica gel (Marine Chemical
Industry Factory, Qingdao, China) and
Sephadex LH-20 (Merck, Darmstadt,
Germany). Thin-layer chromatography
OH
3
O
HO
HO
O
H
1'
OH
HMBC
ROESY
Figure 2. Key HMBC and ROESY
correlations of compound 1.
(
TLC) was preformed with silica gel
ROESY correlations (Figure 2). Based on
the above evidence, compound 1 was
determined as 7-drimen-3b,11-diol 3-O-b-
D-glucopyranoside.
GF254 (Marine Chemical Industry Fac-
tory, Qingdao, China).
The antibacterial tests showed that
compound 1 had inhibitory effects on
MRSA, with a diameter of the inhibition
zone of 10.0mm. The diameter of the
inhibition zone of the positive control,
kanamycin sulfate, was 30.0 mm. The
cytotoxicity of compound 1 was evaluated
in vitro by the 3-(4,5-dimethylthiazol-2-yl)-
3.2 Plant material
Seeds of A. toxicaria (Pers.) Lesch were
collected from Lingshui County of Hainan
Province, China, in May 2009, and the plant
wasidentifiedbyProfessorZhu-NianWang.
A voucher specimen (No. AN200905)
has been deposited in the Institute of
Tropical Bioscience and Biotechnology,
Chinese Academy of Tropical Agricultural
Sciences.
2
,5-diphenyltetrazolium bromide (MTT)
method [20]. Compound 1 showed cytotox-
icity against the K562 and SMMC-7721 cell
lines with the IC50 values of 6.60 and
7.20mg/ml, respectively, and mitomycin C
was used as a positive control (Table 2).
3.3 Extraction and isolation
The dried and crushed seeds (25.9 kg) of
A. toxicaria were extracted three times
with 95% of EtOH (50 L £ 3) at room
temperature. The combined extracts
were concentrated under reduced
3
.
Experimental
3
.1 General experimental procedures
pressure and suspended in H O and then
2
Optical rotation was recorded using a
Rudolph Autopol III polarimeter (Rudolph
Research Analytical, New Jersey, USA).
The UV spectrum was measured with a
Shimadzu UV-2550 spectrometer. The IR
partitioned successively with petroleum
ether, EtOAc, and n-BuOH. The EtOAc
fraction (39.5 g), which showed inhibitory
activity against MRSA and cytotoxic
activities, was subjected to CC on silica
gel with a gradient solvent system of
Table 2. In vitro cytotoxicities of compound 1
(
CHCl –MeOH (1:0–0:1, v/v) to afford 10
3
IC50 values, mg/ml).
fractions. Fraction 8 (4.42 g) was sub-
mitted to repeated CC on silica gel CC
eluting with CHCl –MeOH (20:1 v/v)
Compound
K562
SMMC-7721
3
1
Mitomycin C
6.60
7.10
7.20
2.20
and Sephadex LH-20 (CHCl –MeOH 1:1
3
a
v/v) and finally yielded compound 1
(7.6 mg).
a
Note: Positive control.

5
64
W.-H. Dong et al.
3.3.1 7-Drimen-3b,11-diol 3-O-b-D-
glucopyranoside (1)
3.6 Cell cultures and in vitro
cytotoxicity assay
2
D
6
Colorless oil; ½aꢀ 22.2 (c ¼ 0.68, MeOH);
21
Compound 1 was dissolved in dimethyl
sulfoxide (DMSO) to make a stock
solution with the concentration of
10 mM, which was then diluted to the
required concentrations with RPMI 1640
medium (Beijing Xin Jing Ke Biotechno-
logy Co. Ltd., Beijing, China). Cytotox-
icity of compound 1 against two human
tumor cell lines, K562 (human myeloid
leukemia cell line) and SMMC-7721
UV (MeOH): l
(
(log1) 192 (5.00), 205
max
KBr
2.82) nm; IR n
044, 2978, 1652 (CvC), 1418, 1080,
13
(cm ): 3512 (OH),
max
3
21
1
28 cm ; for H NMR (400MHz) and
7
NMR (100MHz) spectral data, see Table 1.
C
þ
HR-ESI-MS: m/z 400.2459 [M] (calcd for
C H O , 400.2461).
2
1 36 7
(
human hepatoma cell line), was
3
.4 Acid hydrolysis
measured. Cells were plated in 96-well
plates for 24 h with initial density of
Compound 1 (5 mg) was dissolved in
MeOH (2.0ml) and 5% of H SO solution
2
4
5
000 cells/well and continuously exposed
to different concentrations (0.1, 0.3, 0.9,
.7, 8.1, and 24.3 mg/ml) of compound 1
(2.0ml) and hydrolyzed under reflux at
808C for 3 h. The mixture was diluted
twofold with distilled water and partitioned
2
for 72 h, with mitomycin C (Kyowa Hakko
Kogyo Co. Ltd., Tokyo, Japan) as the
positive control. Inhibition rates of cell
proliferation after treatment were deter-
mined by the MTT method [20]. IC50 was
calculated as the concentration (mg/ml) of
samples causing 50% inhibition of cell
viability. The IC50 values are listed in
Table 2.
between CHCl3 and H O. The aqueous
2
layer was neutralized with aqueous
NaHCO solution (1.0M) and evaporated
3
to afford the residue. The sugar was
determined as D-glucose by TLC (CHCl3–
MeOH–H O 7:3:0.5, R ¼ 0.45) with an
2
f
authentic sample and optical rotation
22
dispersion (½aꢀD þ46.6 (c ¼ 0.2, MeOH)).
3
.5 Antimicrobial activity
Acknowledgements
The compound was tested for in vitro
antimicrobial activity against MRSA
This research was financially supported by
National Basic Research Program of China
(obtained from Hainan Institute of Drug
(
No. 2009CB125900), National Natural
Control, Haikou, China) by the filter-paper
disk agar diffusion method [21]. The strains
were cultured using nutrient agar (NA) in
plates (90mm in diameter). Fifty microliters
of compound 1 (10 mg/ml) were impreg-
nated on sterile filter paper disks (6 mm in
diameter) and then applied aseptically to the
surface of the agar plates. Ten microliters of
kanamycin sulfate (0.08mg/ml) were used
as positive control. As an expression of the
antimicrobial activities, the diameters of the
inhibition zones including the 6 mm disk in
diameter were measured after 24 h of
incubation at room temperature. Experi-
ments were carried out in triplicate and the
results were presented as mean values.
Science Foundation of China (No. 30860345),
and National Nonprofit Institute Research
Grant of CATAS-ITBB (No. ITBBZD0744).
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