A new phenylpropanoid glucoside from the aerial parts of Lygodium japonicum

Article abstract of DOI:10.1080/10286020.2011.650690

A new compound, 4-O-caffeoyl-D-glucopyranose (1), and a new natural product, 3-Ocaffeoyl- D-glucopyranose (2), together with six known compounds (3-8) were isolated from the aerial parts of Lygodium japonicum. Their structures were elucidated on the basis of extensive spectroscopic data analyses. The oxygen radical absorbance capacity assay was applied to evaluate their antioxidative capacities in vitro, which revealed that 1-8 showed strong antioxidative properties.

Full text of DOI:10.1080/10286020.2011.650690

Journal of Asian Natural Products Research
Vol. 14, No. 3, March 2012, 286–292
NOTE
A new phenylpropanoid glucoside from the aerial parts
of Lygodium japonicum
ab
Ying-Hui Duan , Yi Dai , Rong-Rong He , Hiroshi Kurihara , Yao-Lan Li and
ac
ac
ac
ac
ad
Xin-Sheng Yao *
a
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy,
Jinan University, Guangzhou 510632, China; Integrated Traditional Chinese and Western
b
Medicine Postdoctoral Station, School of Medicine, Jinan University, Guangzhou 510632, China;
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs
c
d
Research, Jinan University, Guangzhou 510632, China; School of Traditional Materia Medica,
Shenyang Pharmaceutical University, Shenyang 110016, China
(Received 20 July 2011; final version received 14 December 2011)
A new compound, 4-O-caffeoyl-D-glucopyranose (1), and a new natural product, 3-O-
caffeoyl-D-glucopyranose (2), together with six known compounds (3–8) were isolated
from the aerial parts of Lygodium japonicum. Their structures were elucidated on the
basis of extensive spectroscopic data analyses. The oxygen radical absorbance capacity
assay was applied to evaluate their antioxidative capacities in vitro, which revealed that
1
–8 showed strong antioxidative properties.
Keywords: Lygodium japonicum; phenylpropanoid glucoside; antioxidative activity
1
.
Introduction
phenylpropanoid glucoside, 4-O-caffeoyl-
D-glucopyranose (1), and a new natural
product, 3-O-caffeoyl-D-glucopyranose (2)
Lygodium japonicum (Thunb.) Sw, local
name ‘Hai-Jin-Sha’, is a herbaceous
perennial plant mainly distributed in the
south area of China. It has been widely used
for the treatment of pneumonia, dysentery,
hepatitis, etc. [1,2]. The previous studies on
the chemical constitutes of this plant led to
the isolation of several flavones [3], stero-
sides [4], phenolic acids [5,6], and phenolic
glycosides [7]. In our preliminary study,
the water extract of the aerial parts of L.
japonicum was found to exhibit significant
antioxidative activity in the oxygen radical
absorbance capacity (ORAC) assay. In
order to find new and bioactive com-
ponents, a further chemical investigation
on the aerial parts of this plant was carried
out, which resulted in the isolation of a new
[
8], together with six known compounds, 2-
O-caffeoyl-D-glucopyranose (3) [9], 6-O-
caffeoyl-D-glucopyranose (4) [10], 4-O-p-
coumaroyl-D-glucopyranose (5) [11], 6-O-
p-coumaroyl-D-glucopyranose (6) [10],
caffeic acid (7) [12], and p-coumaric acid
(
8) [13] (Figure 1). Here, we report the
isolation and structural elucidation of the
new compounds and their antioxidative
activities.
2.
Results and discussion
The dried aerial parts of L. japonicum
were extracted with water to give a
residue, which was chromatographed on
Diaion HP-20, ODS, Sephadex LH-20,
*Corresponding author. Email: tyaoxs@jnu.edu.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.650690
http://www.tandfonline.com

Journal of Asian Natural Products Research
287
OH
O
OH
7'
O
O
5
O
HO
O
5'
9'
1'
O
HO
1
OH
OH
OH
3
HO
OH
3'
HO
OH
1
OH
2
Figure 1. Chemical structures of 1 and 2.
and HW-40 columns to afford a new
phenylpropanoid glucoside (1), a new
natural product (2), and six known
compounds (3–8).
moieties by comparing with those of
4-O-p-coumaroyl-D-glucose [11]. The
location of caffeoyl moiety was deter-
mined to be at the C-4 position of the
glucose moiety from the chemical shift
Compound 1, obtained as amorphous
powder, gave a molecular formula of
C H O by the analysis of the HR-ESI-
1
3
values in the C NMR spectrum. The
signals assignable to the C-4a and C-4b
positions of the glucose moities were each
downfield shifted by þ2.2 and þ2.3 ppm,
respectively, as compared with those of
unsubstituted a-D-glucose and b-D-glu-
cose [15]. This is further confirmed by the
HMBC correlations from the proton at dH
4.80 (H-4a and H-4b) to the carbon at dC
1
5 18 9
MS data. The UV spectrum of 1 exhibited
absorption maxima at 217, 243 (sh), 304,
and 330 nm, suggesting the presence of a
caffeoyl moiety [14]. The IR spectrum of 1
showed absorption bands for hydroxyl
2
1
group (3381 cm ), ester carbonyl
2
1692 cm ), and aromatic ring (1602
1
(
and 1524 cm ).
2
1
0
168.7 (C-9 ; Figure 2). Hydrolyzed with
1
In the H NMR spectrum of 1 (Table 1),
5% hydrochloric acid, compound 1
yielded a glucose residue. The absolute
configuration of the glucose was demon-
strated to be D configuration by gas
chromatographic analysis using the
method of Hara et al. with slight
modification [16,17]. Therefore, com-
pound 1 was characterized as 4-O-
caffeoyl-D-glucopyranose.
there were two trans-olefinic proton
signals at dH 7.58 (1H, d, J ¼ 16.0 Hz,
0
0
H-7 ) and 6.29 (1H, d, J ¼ 16.0 Hz, H-8 ),
and three aromatic proton signals at dH
0
7
.05 (1H, br s, H-2 ), 6.77 (1H, d,
0
J ¼ 8.0 Hz, H-5 ), and 6.95 (1H, br d,
0
J ¼ 8.0 Hz, H-6 ). Meanwhile, nine carbon
0
0
0
signals at d 168.7 (C-9 ), 149.6 (C-4 ),
C
0
0
0
0
1
1
47.5 (C-7 ), 146.8 (C-3 ) 127.7 (C-1 ),
Compound 2 was obtained as amor-
phous powder with a molecular formula of
C H O by HR-ESI-MS data. The NMR
0
13
C
23.0 (C-6 ), 116.5 (C-5 ), 115.2 (C-2 ),
0
and 114.8 (C-8 ) were observed in the
1
5 18 9
NMR spectrum of 1 (Table 1). The pieces
of evidence mentioned above indicated the
presence of a caffeoyl moiety. In addition,
there were two anomeric proton signals at
spectral data of 2 were similar to those of 1
(Table 1), showing the presence of a
caffeoyl moiety and a glucose moiety. The
location of the caffeoyl moiety was
concluded to be at the C-3 position of the
glucose moiety from the observations of
the downfield shift of the C-3a (þ3.6) and
dH 5.16 (1/2H, d, J ¼ 3.6 Hz, H-1a) and
1
4
NMR spectrum and two sets of carbon
.55 (1/2H, d, J ¼ 8.0 Hz, H-1b) in the H
1
3
signals at dC 93.8 (C-1a), 73.8 (C-2a),
the C-3b (þ2.7) in the
C NMR
7
6
7
2.9 (C-3a), 72.6 (C-4a), 71.2 (C-5a), and
2.5 (C-6a); and 98.3 (C-1b), 76.4 (C-2b),
6.2 (C-5b), 75.8 (C-3b), 72.6 (C-4b),
spectrum, as compared with those of
unsubstituted a/b-D-glucose [15], which
was further confirmed by the HMBC
correlations from the proton at dH 5.32
(H-3a), 5.03 (H-3b) to the carbon at dC
1
3
and 62.6 (C-6b) in the C NMR spectrum,
which suggested the presence of glucose

2
88
Y.-H. Duan et al.
d
d
d
d

Journal of Asian Natural Products Research
289
OH
H
OH
O
H
O
O
O
HO
O
OH
O
OH
OH
HO
OH
HO
HO
OH
OH
2
1
Figure 2. Key HMBC ( ! ) and COSY (—) correlations of 1 and 2.
A
1
Blank (+AAPH)
Blank (–AAPH)
LJE 12.5 µg/mL
LJE 6.25 µg/mL
LJE 3.125 µg/mL
Trolox 10 µM/L
0
.75
0
.5
0.25
0
0
20
40
60
80
100
120
Time (min)
B 1600
1
5
2
0 µM
µM
1200
.5 µM
8
00
00
0
4
1
2
3
4
5
6
7
8
Vc
Figure 3. Curves of fluorescence decay induced by AAPH in the presence of L. japonicum extract
A) and compounds isolated (B) at different concentrations. Trolox, a water-soluble vitamin E
analog, was used as a control standard. The antioxidative activity of a sample was expressed as the
(
net area under the curve. V
calculated as U ¼ K(S
c
was used as a positive control in Figure 3(B). One ORAC unit is
2 S )/(STrolox 2 S ), K: samples multiple of dilution and
Sample
þ AAPH
þ AAPH
S: area below the fluorescence decay curve. Data are expressed as means of three experiments.

2
90
Y.-H. Duan et al.
0
1
69.5 (C-9 ; Figure 2). Thus, compound 2
RP C18 column (XB-C18, 5 mm,
21.2 £ 250 mm; Welch Materials, Inc.,
Shanghai, China). Column chromatog-
raphy (CC) was carried out using Diaion
HP-20 (Mitsubishi Chemical, Tokyo,
Japan), ODS (50 mm, YMC, Kyoto,
Japan), Sephadex LH-20 (Pharmacia,
Uppsala, Sweden), and Toyopearl HW-
40C (Tosoh, Tokyo, Japan). Thin-layer
chromatography was carried out using
precoated silica gel plates (silica gel
GF254, Yantai Chemical Industry Research
Institute, Yantai, China).
was identified as 3-O-caffeoyl-D-gluco-
pyranose [8].
The antioxidative properties of
L. japonicum extract and compounds 1–8
were determined by the ORAC assay as
described previously [18]. The antioxida-
tive activity of a sample was expressed as
the net area under the curve. Trolox was
used as the control standard (10 mM).
Figure 3(A),(B) shows the working curves
of fluorescein oxidation used as an index of
resistance time for the oxidative reaction,
which clearly illustrated that L. japonicum
extract and compounds 1–8 displayed
potent ORACs in a dose-dependent
manner (as shown in Figure 3(B)).
3.2 Plant material
The plant material was generously
provided by Guangzhou Wanglaoji Phar-
maceutical Co. (Guangzhou, China),
which was then identified as L. japonicum
3
.
Experimental
3
.1 General experimental procedures
(Thunb.) Sw. by Prof. Guang-Xiong Zhou,
Optical rotations were measured using a
JASCO P-1030 automatic digital polari-
meter (JASCO, Tokyo, Japan). UV spectra
were measured on a JASCO V-550 UV–
vis spectrophotometer. IR spectra were
recorded on a JASCO FT-IR-400 spec-
trometer. 1D and 2D NMR spectra were
recorded on a Bruker AV-400 spec-
College of Pharmacy, Jinan University. A
voucher specimen (2006WZY0044) is
deposited in the Institute of Traditional
Chinese Medicine & Natural Products,
Jinan University, Guangzhou, China.
3.3 Extraction and isolation
1
trometer (400 MHz for H; Bruker BioS-
The dried aerial parts of L. japonicum
(6.5kg) were refluxed twice with water for
2 h eachtime. After filtration, the filtrate was
evaporated to dryness under vacuum. The
dried extract (758.3 g) was chromato-
graphed over Diaion HP-20 column and
eluted with water and 30%, 50%, and 95%
pin, Fallanden, Switzerland). ESI-MS data
were recorded on a Finnigan LCQ
Advantage MAX mass spectrometer
(Thermo Electron, Lowell, MA, USA).
HR-ESI-MS data were determined by an
Agilent 6210 LC/MSD TOF mass spec-
trometer (Agilent, Pala Alto, CA, USA).
HPLC analysis was carried out on a
Dionex HPLC system equipped with a
Dionex P-680 quaternary pump, a PDA-
(V/V) EtOH–H O, successively, to afford
2
four fractions (Fractions A–D). Fraction B
(30% EtOH–H O eluent, 88.2 g) was
2
chromatographed over ODS column and
eluted with CH OH–H O (1:9 to 1:0) to
1
00 diodearray detector, a TCC-100 oven,
and an ASP-100 autosampling system
Dionex, Sunnyvale, CA, USA) using a
3
2
afford four subfractions (B1–B4). Subfrac-
tion B1 (10% CH OH–H O eluent, 16.8g)
(
3
2
reversed-phase (RP) C18 column (XB-
C18, 5 mm, 4.6 £ 250 mm; Welch
Materials, Inc., Shanghai, China). Prepara-
tive HPLC was carried out on a Varian
instrument equipped with UV detectors
was further subjected to Toyopearl HW-40
column using 30% CH OH–H O as elu-
3
2
ents, and then purified by preparative HPLC
with 10% CH OH–H O to afford com-
3
2
pounds 1 (tR 42.3min, 52.2mg), 2 (tR
28.5min, 28.6mg), and 3 (tR 33.0min,
(Varian, Salt Lake City, UT, USA) and a

Journal of Asian Natural Products Research
291
þ
6.0 mg). Subfraction B2 (10% CH OH–
H O eluent, 4.4 g) was chromatographed
HR-ESI-MS: m/z 365.0807 [M þ Na]
(calcd for C H O Na, 365.0843).
3
2
15 18 9
over Toyopearl HW-40 column, using
CH OH–H O (2:8 to 1:0) as mobile phase
3
2
3.4 Measurement of antioxidative
capacities
to afford four subfractions (B2A–B2D).
B2B (40% CH OH–H O eluent) was then
purified by preparative HPLC with 23%
3
2
The antioxidative properties of L. japoni-
cum extract and compounds 1–8 were
determined by the ORAC assay as
described previously [18]. Purities of
compounds 1–8 were greater than 98%
as measured by HPLC analyses. The
compounds were dissolved in the potass-
ium phosphate buffer before use. Trolox,
purchased from Wako Pure Chemical
Industries (Osaka, Japan), was used as a
control standard. Peroxyl radicals were
generated at a controlled rate by thermal
decomposition of 2,2-azobis(2-amidino-
propane) dihydrochloride (AAPH). The
180-ml assay solution contained 20 ml of
samples and 20 ml of 75-mM potassium
phosphate buffer (pH 7.4) with 140 ml of
APPH solution (final concentration
12.8 mmol/liter). The reaction was started
by adding 20 ml of fluorescein (final
concentration 63 mmol/liter) to the assay
solution. Decay of the fluorescein signal
was determined at 485/527 nm in a
GENios Lueifcrase microplate reader
CH OH–H O to give compounds 4 (t
R
3
2
2
5
2.3 min, 9.0 mg) and 6 (tR 31.8 min,
0.0 mg). Compound 5 (24.7 mg) was
recrystallized in CH OH–H O (3:7) from
2
3
subfraction B2C (60% CH OH–H O elu-
3
2
ent). Subfraction B3 (30% CH OH–H O
3
2
eluent, 3.4 g) was applied to Toyopearl HW-
0 CC, using 40% CH OH–H O as eluents,
4
3
2
and then subjected to Sephadex LH 20 CC,
eluted with 50% CH OH–H O to yield
compound 7 (320.0mg). Fraction C (50%
3
2
EtOH–H O eluent, 80.0g) was chromato-
2
graphed over ODS CC, eluted with
CH OH–H O (1:9 to 1:0) to afford three
3
2
subfractions (C1–C3). Subfraction C2
50% CH OH–H O eluent, 18.0g) was
(
3
2
further subjected to Toyopearl HW-40
column using 30% CH OH–H O as elu-
3
2
ents, and then purified by Sephadex LH 20
CC with 50% CH OH–H O to afford
compound 8 (135.0mg).
3
2
3
.3.1 4-O-Caffeoyl-D-glucopyranose (1)
(TECAN, Mannedorf, Switzerland) at
378C.
25
Amorphous powder, ½aꢀ þ 14.9 (c ¼ 0.6,
D
CH OH), UV (MeOH) l
3
(loge): 217
3.97), 243 (sh, 3.81), 304 (3.80), and 330
3.99)nm; IR (KBr) v : 3381, 1692, 1602,
max
(
(
Acknowledgements
max
1
21
13
This work was financially supported by the
Fund of National Science and Technology
Development (No. 2006BAIO6A20-09).
The authors are grateful to Dr Hui-nan Zhao,
Dr Ming-yang Liu, and Dr Zhen-qiang Mu,
Institute of Traditional Chinese Medicine and
Natural Products, Jinan University, for measur-
ing the HR-ESI-MS, ESI-MS, and NMR
spectral data.
and 1524cm ; for H and C NMR
spectral data see Table 1; ESI-MS: m/z 365
þ
þ
[
M þ Na] , 707 [2M þ Na] ; HR-ESI-
þ
MS: m/z 365.0816 [M þ Na] (calcd for
C H O Na, 365.0843).
15 18 9
3
.3.2 3-O-Caffeoyl-D-glucopyranose (2)
Amorphous powder, UV (MeOH) lmax
loge): 218 (4.06), 246 (sh, 3.87), 296 (sh,
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