New phenanthrene glycosides from Dioscorea opposita

Article abstract of DOI:10.1080/10286020.2013.837459

Two new phenanthrene glycosides, dioscopposide A and dioscopposide B (1 and 2), were isolated from the rhizomes of Dioscorea opposita. Their structures were determined primarily on the basis of 1D and 2D NMR techniques, MS studies, and chemical methods. All the isolates were evaluated for their inhibitory effects on the lipopolysaccharide-induced nitric oxide production using murine macrophage RAW 264.7 cells. The IC₅₀ values of dioscopposide A and dioscopposide B were 5.8 and 7.2 M, respectively.

Full text of DOI:10.1080/10286020.2013.837459

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New phenanthrene glycosides from
Dioscorea opposita
abc
b
a
Ken Yu-Zhong Zheng , Zhen-Xia Zhang , Wenliang Zhou , Hui
c
cd
Cao & Fei-Jun Xiang
a
The School of Life Science, Sun Yat-Sen University, Guangzhou,
10275, China
5
b
Department of Biology, Hanshan Normal University, Chaozhou,
21041, China
5
c
Research and Development Department, Guangdong Kangmei
Pharmaceutical Research Institute Company Limited, Guangzhou,
5
10006, China
d
Research and Development Department, Kangmei
Pharmaceutical Company Limited, Puning, 515300, China
Published online: 29 Oct 2013.
To cite this article: Ken Yu-Zhong Zheng, Zhen-Xia Zhang, Wenliang Zhou, Hui Cao & Fei-Jun Xiang
(2014) New phenanthrene glycosides from Dioscorea opposita, Journal of Asian Natural Products
Research, 16:2, 148-152, DOI: 10.1080/10286020.2013.837459
To link to this article: http://dx.doi.org/10.1080/10286020.2013.837459
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 2, 148–152, http://dx.doi.org/10.1080/10286020.2013.837459
New phenanthrene glycosides from Dioscorea opposita
a,b,c
Ken Yu-Zhong Zheng *, Zhen-Xia Zhang , Wenliang Zhou , Hui Cao and
b
a
c
c,d
Fei-Jun Xiang
a
b
The School of Life Science, Sun Yat-Sen University, Guangzhou 510275, China; Department of
c
Biology, Hanshan Normal University, Chaozhou 521041, China; Research and Development
Department, Guangdong Kangmei Pharmaceutical Research Institute Company Limited,
d
Guangzhou 510006, China; Research and Development Department, Kangmei Pharmaceutical
Company Limited, Puning 515300, China
(Received 18 June 2013; final version received 20 August 2013)
Two new phenanthrene glycosides, dioscopposide A and dioscopposide B (1 and 2),
were isolated from the rhizomes of Dioscorea opposita. Their structures were
determined primarily on the basis of 1D and 2D NMR techniques, MS studies, and
chemical methods. All the isolates were evaluated for their inhibitory effects on the
lipopolysaccharide-induced nitric oxide production using murine macrophage RAW
264.7 cells. The IC50 values of dioscopposide A and dioscopposide B were 5.8 and
7.2 mM, respectively.
Keywords: Dioscorea opposita; phenanthrene glycosides; dioscopposide A, B
1
.
Introduction
2. Results and discussion
The Dioscorea opposita Thunb. grows
wild in China, and the tuber is used as a
food. This plant is also included in the
Pharmacopoeia of the People’s Republic
of China and is widely used to treat
anorexia, chronic diarrhea, diabetes, semi-
nal emission and excessive leukorrhea
Dioscopposide A (1) was isolated as an
amorphous solid. Its molecular formula
was determined as C H O by negative
2
6 30 13
2
HR-ESI-MS, showing an [M–H] peak at
m/z 549.1603. The IR spectrum exhibited
2
1
absorptions at 3288, 1730, and 1432 cm
1
(aromatic rings). The H NMR spectrum
of 1 (Table 1) showed the presence of
seven aromatic protons of aglycone (H-1,
H-2, H-5, H-7, H-8, H-9, and H-10) and
[1,2]. Previous investigation on the phy-
tochemistry of this plant leads to isolation
of phenanthrene and bibenzyl derivatives,
which showed anti-inflammatory, neuro-
protective, and radical scavenging activi-
ties [3–6]. In the course of a search for
biologically active compounds from Chi-
nese medicinal plants, we have investi-
gated the rhizomes of D. opposita. Two
phenanthrene glycosides were isolated and
characterized (1 and 2) (Figure 1). This
paper describes the isolation and structural
elucidation of these compounds and the
evaluation of their effects against nitric
oxide (NO) production.
protons of sugar in the range of d 3.63–
H
1
5.12. The H NMR and
13
C NMR
(Table 1), combined with distortionless
enhancement by polarization transfer
(DEPT) and HMQC, spectra of 1 revealed
that 1 contained seven aromatic methine
carbons (C-1, C-2, C-5, C-7, C-8, C-9, and
C-10), seven quaternary aromatic carbons
(C-3, C-4, C-4a, C-4b, C-8a, and C-10a),
0
00
two anomeric carbons (C-1 and C-1 ), two
0
00
methylene carbons (C-6 and C-6 ), and
0
0
eight methine carbons (C-2 –C-5 and
*Corresponding author. Email: zhengyuzhong@gmail.com
q 2013 Taylor & Francis

Journal of Asian Natural Products Research
149
9
10
8
8a
4b
10a
4a
4
1
7
2
6
5
3
HO
HO
HO
O
O
H3CO
HO
HO
O
6'
O
OH
OH
O
OH
1'
OH
HO
O
OH
Glc1
4
'' OH
1''
OH
OH
Glc2
1
2
Figure 1. The structures of compounds 1 and 2.
1
13
Table 1. H and C NMR spectral data of compounds 1 and 2 (500 MHz, in pyridine-d5).
1
2
Position
d
H
d
C
d
H
d
C
1
2
3
4
7.02 d (8.0)
7.22 d (8.0)
115.8
118.3
151.1
152.7
109.3
136.7
123.9
143.7
118.8
118.6
127.6
121.3
111.5
118.6
7.01 d (8.0)
7.23 d (8.0)
115.7
118.2
151.2
152.6
109.2
136.6
121.5
144.9
118.2
117.8
127.5
121.2
111.4
118.5
4
4
5
6
7
8
8
9
1
1
a
b
7.56 d (1.5)
7.29 d (1.5)
7.01 dd (7.5, 1.5)
7.95 dd (7.5, 1.0)
6.82 dd (7.0, 1.5)
7.94 dd (7.0, 1.0)
a
7.04 dd (7.5, 1.0)
7.41 d (7.5)
7.04 dd (7.5, 1.0)
7.41 d (7.5)
0
0a
1
Glc
0
1
5.04 d (8.0)
3.84 dd (9.0, 8.0)
4.12 dd (9.0, 8.0)
4.21 m
3.63 m
4.25 dd (12.0, 6.0)
4.11 dd (12.0, 2.0)
102.8
74.2
76.6
81.2
76.7
61.9
5.14 d (8.0)
4.12 dd (9.5, 8.0)
4.26 dd (9.5, 8.0)
4.14 m
3.96 m
4.31 dd (12.0, 6.0)
4.22 dd (12.0, 2.0)
102.6
74.3
77.5
70.8
77.6
61.5
0
2
0
3
0
4
0
5
0
6
6
a
b
0
2
Glc
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
6
5.12 d (8.0)
3.98 dd (9.5, 8.0)
4.20 dd (9.5, 8.0)
4.15 m
3.92 m
4.50 dd (13.0, 7.0)
4.20 dd (13.0, 2.0)
104.6
74.9
78.3
71.5
78.4
62.3
a
b
OMe
3H, 3.70 s
55.2

1
50
K.Y.-Z. Zheng et al.
0
0
00
1
13
H, C, H– H COSY, 1D TOCSY,
1
1
C-2 –C-5 ). A detailed analysis of the
1
1
H– H correlated spectroscopy (COSY)
HSQC, HMBC, and NOESY NMR exper-
iments. From these results, the structure of
1 was determined as shown in Figure 1 and
named as dioscopposide A.
spectrum of 1 allowed to elucidate the
following three pairs of ortho-coupled
aromatic protons at d 7.02 (H-1) and 7.22
H
(
(
H-2), 7.01 (H-7) and 7.95 (H-8), and 7.04
H-9) and 7.41 (H-10). In the HMBC
Dioscopposide B (2) was isolated as an
amorphous solid. Its molecular formula
spectrum, H-1 showed correlations with
two aromatic carbons C-3 and C-10, and
H-2 correlated with C-3 and C-4, respect-
ively. H-5, an isolated proton, showed
HMBC correlations with three quaternary
carbons C-4a, C-4b, and C-8a. Cross-
peaks were equally observed between H-7
and C-6 and C-8a. Additional HMBC
correlations were observed between H-8
and C-4b and C-9, between H-9 and C-4b
and C-10a, and between H-10 and C-8a
and C-10a (Figure 2). All these data
suggested the presence of a 3,4,6-trisub-
was determined as C21H O by negative
22 8
2
HR-ESI-MS, showing an [M–H] peak at
m/z 401.1233. Comparison of the NMR
data for 1 and 2 revealed that the
compounds are similar except for the
2
absence of signals for Glc connected to
1
Glc –C-4 in 1. This was confirmed by the
0
0
C-4 appeared comparatively upfield at d
70.8. In addition, the connection of a
methoxyl group (d
C-6 was confirmed by HMBC correlation
of d OCH -3.70/C-6. Thus, compound 2
3.70 and d 55.2) and
H
C
H
3
was determined as shown in Figure 1 and
named as dioscopposide B.
1
13
stitued phenanthrene [4]. The H and
C
NMR data, as well as acid hydrolysis and
GC comparison with an authentic sample,
indicated the presence of two D-glucose
moieties. The b-configuration of the
glycosidic linkage was determined from
the coupling constant of the anomeric
Compounds 1 and 2 were evaluated for
their inhibitory activity against NO pro-
duction. Aminoguanidine was used as a
positive control. The IC50 values of 1, 2,
and aminoguanidine were 5.8, 7.2 and
15.3 mM, respectively.
1
0
proton. The connections of the Glc -C-1 /
0
2
aglycone-C-3 and Glc -C-1 /Glc -C-4
00
1
3.
Experimental
were revealed by HMBC correlations H-
0
0
00
3.1 General experimental procedures
1 /C-3 and H-1 /C-4 . The proton and
carbon signals of 1 were assigned using
Optical rotations were measured on a
Jasco DIP-360 digital polarimeter (Jasco
Corporation, Tokyo, Japan). UV spectra
were obtained on a TU-1901 spectrometer
H
H
H
H
(Beijing Purkinje General Instrument Co.,
Ltd, Beijing, China). IR spectra were
detected by a Nexus-470 FT-IR (Nicolet)
spectrometer (Thermo Electron, Madison,
WI, USA). NMR spectra were recorded on
a Bruker Inova 500 spectrometer (Varian,
Palo Alto, CA, USA) with tetramethylsi-
lane as an internal standard. HR-ESI-MS
data were measured using a Q-TOF micro
mass spectrometer (Waters, Millford, MA,
USA). GC was obtained on an Agilent
H
H
HO
H
HO
HO
O
O
OH
H
HO
O
O
OH
OH
H
OH
OH
6
890N gas chromatography (Agilent
Technologies, CA, USA). Column chro-
matography was carried out on D101
Figure 2. Selected HMBC correlations of
compound 1.

Journal of Asian Natural Products Research
porous polymer resin (Tianjin Chemical 341 nm; IR (KBr) n
151
(cm ): 3288,
2
1
max
1
Industry Co., Ltd, Tianjin, China), silica
gel (200–300 mesh, Qingdao Haiyang
Chemical Co., Ltd, Qingdao, China), and
C18 silica gel (150–200 mesh, Merck,
Darmstadt, Germany). HPLC was per-
formed on Waters 600 Controller (Waters,
Milford, MA, USA) by using an Agilent
Eclipse XDB C18 semi-preparative column
1730, and 1432. H NMR (pyridine-d ,
5
1
500 MHz) and C NMR (pyridine-d5,
3
125 MHz) spectral data, see Table 1. HR-
2
ESI-MS: m/z 549.1603 [M–H] (calcd for
C H O , 549.1608).
26 29 13
3.3.2 Dioscopposide B (2)
2
0
(
Technologies, Inc., CA, USA).
250 mm £ 9.4 mm, i.d. 5 mm, Agilent
Yellow amorphous solid; ½aꢀ 216.6 (c
D
0.25, MeOH); UV (MeOH) l
3
222, 239,
max
2
1
41 nm; IR (KBr) n
(cm ): 3328,
max
1
832, 1690, and 1441. H NMR (pyridine-
2
3
.2 Plant material
1
3
d5, 500 MHz) and C NMR (pyridine-d5,
25 MHz) data, see Table 1. HR-ESI-MS:
The rhizomes of D. opposita were collected
from Wenxian, Henan Province, China, in
July 2012, and were identified by Dr Ken
Yuzhong Zheng, Hanshan Normal Univer-
sity. A voucher specimen (No. 201204) has
been deposited in the herbarium of Depart-
ment of Biology of Hanshan Normal
University.
1
m/z 401.1233 [M 2 H]
2
(calcd for
C H O , 401.1236).
21 21 8
3.4 Acid hydrolysis
Compounds 1 and 2 (5 mg, respectively)
were hydrolyzed separately with 2 M HCl
(
0.5ml) for 10h at 958C. After filtration of
3
.3 Extraction and isolation
the reaction mixture, the filtrate was
evaporated under vacuum. After addition
of H O, the acidic solution was evaporated
The air-dried powdered rhizomes of D.
opposita (15 kg) were successively
extracted with 75% EtOH under reflux.
The resultant extract was combined and
dried under reduced pressure to give
concentrated extractives. The latter was
subsequently suspended in water and parti-
tioned successively with petroleum ether,
EtOAc, and n-butanol. The n-butanol part
2
again to remove HCl. This procedure was
repeated until a neutral solution was
obtained, which was finally evaporated and
dried in vacuo to furnish a monosaccharide
residue. The residue was dissolved in
pyridine (1 ml), to which 2 mg of L-cysteine
methyl ester hydrochloride was added. The
mixture was kept at 608C for 2 h and
(322g) was subjected to column chroma-
evaporated under N stream and dried in
2
tography by a combination of D101
macroporous resin, eluted gradiently with
vacuo. The residue was trimethylsilylated
with N-trimethylsilylimidazole (0.2ml) for
1
0% EtOH and 60% EtOH, successively.
2
hexane and H O (2 ml each), and the n-
h. The mixture was partitioned between n-
The fraction eluted with 60% EtOH (120g)
was subjected to repeated ODS column
chromatography [MeOH:H O (5:95–95:5)]
2
hexane extract was analyzed by GC under
the following conditions: capillary column,
DB-5 (30 m £ 0.25mm £ 0.25mm); detec-
tion, FID; detector temperature, 2808C;
injection temperature, 2508C; initial tem-
perature was maintained at 1008C for 2 min
and then raised to 2808C at the rate of 108C/
min, and final temperature was maintained
2
to afford four subfractions. Subfraction 3
(12.5 g) was separated by semi-preparative
HPLC (MeOH:H O 20:80, 2.0 ml/min) to
yield 1 (33 mg) and 2 (36 mg).
2
3
.3.1 Dioscopposide A (1)
20
Yellow amorphous solid; ½aꢀ 222.6 (c
for 5 min; carrier, N gas. The peak of the
2
D
0
.25, MeOH); UV (MeOH) l
222, 239,
hydrolysate of 1 and 2 was detected at
max

1
52
K.Y.-Z. Zheng et al.
24.94 min (D-glucose) by comparison with
authentic sample [7].
Acknowledgments
This research was financially supported by
grants from Natural Science Foundation of
China (No. 81202907) and Natural Science
Foundation of Guangdong Province (No.
S2012040008079).
3
.5 NO production from
lipopolysaccharide-stimulated
macrophages
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evaluated using the method reported pre-
viously [8]. Briefly, thioglycolate-induced
peritoneal exudate cells (5200 cells/well)
were collected from the peritoneal cavities
of male ddY mice and were suspended in
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1
5
00 ml of RPMI 1640 supplemented with
% fetal calf serum (FCS), penicillin G
(
(
100 units/ml), and streptomycin sulfate
100mg/ml), and were precultured in 96-
[
[
[
wellmicroplatesat378Cin5%CO inairfor
2
1
h. Nonadherent cells were removed by
washing with phosphate buffer solution, and
the adherent cells were cultured in 200 ml of
fresh medium containing 10mg/ml lipopo-
lysaccharide (LPS) and various concen-
trations of each test compound for 20h. NO
production in each well was assessed by
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[
measuring the accumulation of nitrite
2
(NO ) in the culture medium using Griess
reagent.
2