A new isoflavone glycoside from the stem bark of Sophora japonica

Article abstract of DOI:10.1007/s12272-010-0805-1

A new isoflavone glycoside, 6-methoxy-7-hydroxy-4′-O-β-d- glucosyl isoflavone, glycitein-4′-O-β-d-glucoside (10), along with nine known flavonoids, were isolated from the stem bark of Sophora japonica. The structures of these compounds were determined by analysis of spectroscopic data (1D -, 2D - NMR and HRMS). The inhibitory effects of all the isolated compounds on aldose reductase were evaluated in vitro. Among these compounds, daidzein (1), puerol A (4), and paratensein-7-O-glucoside (9) exhibited potent inhibitory effects, with IC₅₀ values of 3.2, 6.4, and 1.9 μM, respectively.

Full text of DOI:10.1007/s12272-010-0805-1

Arch Pharm Res Vol 33, No 8, 1165-1168, 2010
DOI 10.1007/s12272-010-0805-1
A New Isoflavone Glycoside from the Stem Bark of Sophora japonica
1
,
1,
1
1
2
3
Hyun Young Park *, Soo Hee Kim *, Gi Beom Kim , Jae Young Sim , Soon Sung Lim , Myong Jo Kim ,
4
1
Wanjoo Chun , and Yong Soo Kwon
College of Pharmacy, Kangwon National University, Chuncheon 200-701, Korea, Department of Food Science and
Nutrition, Division of Life Science, Hallym University, Chuncheon 200-702, Korea, Oriental Bio-Herb Institute, Kang-
won National University, Chuncheon 200-701, Korea, and College of Medicine, Kangwon National University, Chun-
1
2
3
4
cheon 200-701, Korea
(Received November 2, 2009/Revised June 7, 2010/Accepted June 7, 2010)
A new isoflavone glycoside, 6-methoxy-7-hydroxy-4’- -glucosyl isoflavone, glycitein-4’-
O
-
β
-
D
O-β-
D
-glucoside (10), along with nine known flavonoids, were isolated from the stem bark of
Sophora japonica. The structures of these compounds were determined by analysis of spectro-
scopic data (1D -, 2D - NMR and HRMS). The inhibitory effects of all the isolated compounds
on aldose reductase were evaluated in vitro. Among these compounds, daidzein (
1
), puerol A
(4
), and paratensein-7-
O-glucoside (9) exhibited potent inhibitory effects, with IC50 values of
3
.2, 6.4, and 1.9 M, respectively.
µ
Key words: Sophora japonica, Stem bark, Isoflavone glycoside, Glycitein-4’-O-β-D-glucoside,
Aldose reductase
INTRODUCTION
of Sophora japonica showed the best inhibition acti-
vity (data not shown). This paper deals with the
The stem bark of Sophora japonica (Leguminosae) isolation, structure elucidation, and aldose reductase
has been used as an anti-inflammatory and analgesic inhibitory activity of compounds from the barks of
agents in traditional Chinese medicine (Zhong Hua Sophora japonica
.
Ben Cao Editorial Commitee, 1999). The chemical con-
stituents cyclosin, puerol A, trifolirhizin, and genistein MATERIALS AND METHODS
have been isolated from the stem bark of this plant
(Park et al., 2002).
General procedures
Aldose reductase (EC 1.1.1.21; AR) is a key enzyme
Melting point were determined on a Fisher Johns
in the polyol pathway, in which catalyzes glucose to melting point apparatus. UV/Vis spectra were obtain-
sorbitol, and then sorbitol dehydrogenase converts ed using a V-530 spectrophotometer (JASCO). Optical
sorbitol to fructose (Yabe-Nishimura, 1998). The signi- rotation was measured on a DIP 1000 Digital Polari-
ficance of this enzyme has been indicated in the patho- meter (JASCO). IR spectra were obtained using a
genesis of diabetic complications such as neuropathy, re- FTIR-4200 (JASCO). MS spectra were obtained using
tinopathy, and cataract (Santiago, 1993; Feldman et al., a Autospec M363 (Micromass). NMR spectra were re-
1997). Thus, the inhibition of aldose reductase may be corded on a DPX 400 and AVANCE 600 (Bruker).
of therapeutic use in diabetic complications. During a Chemical shifts were represented as parts per million
search for aldose reductase inhibitors from plant (ppm) referenced to the residual solvent signal. Column
sources, an
n
-BuOH-soluble fraction of the stem barks chromatography was carried out using Kieselgel 60,
00-230 mesh (Merck) and YMC gel ODS-A, 150
YMC). TLC was performed on glass-backed Kieselgel
0 F254 and RP F254s plates. HPLC analysis was per-
4
(
6
µm
*
These authors contributed equally to this work.
Correspondence to: Yong Soo Kwon, College of Pharmacy, Kang-
won National University, Chuncheon 200-701, Korea
Tel: 82-33-250-6921, Fax: 82-33-255-7865
formed using a Spectra system (Thermo Separation
Products).
E-mail: yskwon@kangwon.ac.kr
1165

1166
H. Y. Park et al.
Plant material
give compounds
9
(50 mg) and 10 (66.7 mg), respec-
The stem bark of S. japonica was collected from the tively. The purity of isolated compounds (> 95%) were
Herbal garden of Kangwon National University (June, determined by HPLC using a Intersil column (ODS-2,
2
007) in Korea and identified by Prof. Y. S. Kwon, 150
College of Pharmacy, Kangwon National University.
A voucher specimen (KNUH-B-0701) was deposited in Acid hydrolysis of 10
× 4.5 mm) with acetonitrile : water (20 : 70).
the herbarium of the College of Pharmacy, Kangwon
National University, Korea.
Compound 10 (10 mg) was refluxed with 5% HCl (2
mL) in water for 1 h. The reaction mixture was diluted
with water and fractionated by EtOAc. The EtOAc-
soluble fraction was concentrated and column-chrom-
Extraction and isolation
The dried stem bark of S. japonica (1.7 kg) was ex- tographed on silica gel (CHCl : MeOH = 19 : 1) gave
3
tracted with MeOH at room temperature for a week. compound 10a (2.3 mg). The remaining aqueous layer
The MeOH extract (288 g) was suspended in water was adjusted to pH 7 with NaHCO and filtered. The
3
and then partitioned with
n
-hexane, CHCl , and
n-
filtrate was concentrated and examined by TLC with
3
BuOH, successively, leaving a residual water-soluble authentic sugar.
fraction. Each fraction was evaporated in vacuo to
yield the residues of
fraction (7 g), and -BuOH fraction (70 g). Among the Mp : 147~150 C; UV (MeOH)
solvent fractions, a -BuOH soluble fraction exhibited (MeOH + NaOAc) λmax 261, 345 nm; UV (MeOH +
5% inhibition on aldose reductase at 100 g/mL. NaOH) λmax 258, 348 nm; IR (ATR)max 3371 (OH), 1620
Column chromatography was performed to isolate the (C=O), 1597, 1509, 1473 (C=C), 1163, 1069, 1034 (C-O)
n-hexane fraction (40 g), CHCl3 Compound 10
o
n
n
λmax 257, 324 nm; UV
6
µ
-
1
1
active compounds from the
n
-BuOH soluble fraction,. cm ; H-NMR (400 MHz, MeOH-d4
)
δ
: See Table I;
13
The -BuOH soluble fraction (60 g) was column chro-
n
C-NMR (100 MHz, MeOH-d4) δ : See Table I; FAB-
matographed on a silica gel (500 g, 9 × 60 cm) using
an isocratic elution with CHCl : MeOH (4 : 1), in order
Table I. 1H- and 13C-NMR data for 10 (in MeOH-
)a
3
d
4
to divide the fraction into six fractions (Fr. 01 - Fr. 06).
Fr. 01 (1 g) was re-chromatographed on silica gel by
elution with benzene : EtOAc (2 : 1) and divided into
six sub-fractions (Fr. 011-Fr. 016). Fr. 016 was further
10
Pposition
H (J
= Hz)
C
2
3
4
5
6
7
8
9
10
1'
2'
3'
4'
5'
6'
1''
2''
3''
4''
5''
8.08 (s)
7.47 (s)
6.83 (s)
154.45
124.81
177.50
105.06
149.26
156.76
103.97
154.74
117.06
127.66
131.38
117.64
158.92
117.64
131.38
102.27
074.94
077.98
071.39
078.16
purified by silica gel column chromatography [3.8
cm, CHCl : MeOH (13 : 1)] to give compounds (51 mg)
and (9 mg), respectively. Fr. 2 (7 g) was re-chromato-
× 50
1
2
graphed on a silica gel column (500 g, 6.5 × 50 cm) by
elution with EtOAc : MeOH (9 : 1) and divided into five
sub-fractions (Fr. 021-Fr. 025). Fr. 023 was recrystallized
from MeOH to give compound
3
(355 mg). Fr. 022 was
re-chromatographed on a silica gel column (80 g, 2.5
×
45 cm) by elution with benzene : EtOAc (2:1) to give
compound
4
(42.8 mg). Fr. 024 was re-chromatograph-
7.45 (d, 8.8)
7.15 (d, 8.8)
ed on a silica gel column (80 g, 2.5 × 50 cm) and ODS
column (70 g, 2.5 × 50 cm) by elution with water sat-
urated-EtOAc : MeOH (19 : 1) and MeOH:H
2
O (60 :
7.15 (d, 8.8)
7.45 (d, 8.8)
4.95 (d, 7.5)
4
0) to give compounds (100 mg) and (12 mg),
5
6
respectively. F03 (6.5 g) was subjected to MPLC on a
Combiflash Retrieve using Redisep C-18 column (130
g), eluting with MeOH : H O (40 : 60) to yield five sub-
2
fractions (Fr.031 - Fr.035). Fr. 032 was re-chromato-
graphed on a silica gel column (80 g, 2.5 × 50 cm) by
⎫
⎪
⎬
⎪
⎭
3.42 ~ 3.51 (m)
elution with CHCl
3
: MeOH (6 : 1) to give compounds
3.73 (dd, 5.1, 12.0)
3.50 (m)
6''
062.54
7
(2.7 mg) and (54 mg). Fr. 034 was re-chromato-
8
graphed on a silica gel column (80 g, 2.5 × 50 cm) and
OCH
3
3.92 (s)
056.53
Sephadex LH20 (50 g, 2.5 × 50 cm) by elution with aThe assignments were based on DEPT, COSY, HSQC,
EtOAc : MeOH (15 : 1) and MeOH : H O (60 : 40), to and HMBC experiments.
2

A New Isoflavone Glycoside from the Stem Bark of Sophora japonica
1167
+
MS m/z : 447 [M+H] ; HR-FAB-MS m/z : 447.1178 al., 2002), ononin (
5
) (Hwang et al., 1997), 5-hydroxypse-
+
[
M+H] (calcd for 447.1291).
udobaptigenin-7-
O
-glucoside (
6) (Laman and Volynets,
1975), glycitin (7) (Park et al., 1999), calycosin-7-O-
Compound 10a
H-NMR (600 MHz, MeOH-
glucoside ( ) (Lee et al., 2008), and paratensein-7-
), glucoside (9) (Ahut et al., 1984), respectively.
8
O-
1
4
d )
δ
: 3.97 (3H, s, OCH
3
6
7
8
.87 (2H, d,
.39 (2H, d,
J
J
= 8.5 Hz, H-3’, H-5’), 6.89 (1H, s, H-8),
= 8.5 Hz, H-2’, H-6’), 7.56 (1H, s, H-5), and produced a quassi molecular ion [M + H] at m/z
Compound 10 was obtained as a brownish powder
+
+
.14 (1H, s, H-2); EI-MS m/z : 284 [M] , 166, 118.
447.1178 by HR-FAB-MS, consistent with an elemental
formula of C22 10. The IR spectrum of 10 showed
Assay for rat lens aldose reductase inhibitory absorption bands at 3371 (OH), 1620 ( -unstaturat-
activity
ed C=O), 1597, 1509, 1473 (aromatic C=C), 1163, 1069,
According to method modified from Hayman and 1034 (C-O) cm . The presence of a singlet at
22
H O
α, β
-1
δ
8.08 in
1
Kinoshita (Hayman and Kinoshita, 1965), rat lens the H-NMR spectrum and UV absorption band at 254
homogenate was prepared. In summary, the lenses and 324 nm suggested that it was an isoflavonoid. The
1
were removed from the eyes of Sprague-Dawley rats
(
H-NMR spectrum of 10 exhibited signals from the H-
7.47 (1H,
lenses were homogenized in 100 mM sodium phosphate s) and 6.83 (1H, s), respectively. Two AB - type dou-
buffer (pH 6.2). The supernatant was obtained by cen- blets at 7.15 (2H, d, = 8.8 Hz) and 7.45 (2H, d,
trifugation of the homogenate at 10,000 rpm at 4 C for 8.8 Hz) indicated the presence of H-3’, H-5’ and H-2’,
Daehan Biolink Co. Ltd) weighing 320-350 g. The 5 and H-8 position of the isoflavone ring at δ
δ
J
J =
o
o
2
0 min and was divided into 1 mL, and stored at
Enzyme activities were assayed spectrophotome- the presence of a methoxyl group. In addition, an
tically on a JASCO V-530 spectrophotometer. The anomeric proton at 4.95 (1H, d, = 7.5 Hz) was
activities of rat lens aldose reductase (RLAR) were assigned. The signals of methoxyl and anomeric proton
measured according to the procedure of Nishimura et showed an HMBC correlation with the C-6' ( 149.26)
al. (1991). The reaction mixture contained 0.15 mM and C-4’ ( 158.92) signals, respectively, in the HMBC
NADPH, 10 mM DL-glyceraldehyde, 100 L of RLAR spectrum (Fig. 1), respectively. Upon acid hydrolysis
and 10 L of the test sample or dimethylsulfoxide of 10, glycitein (10a) (Park et al., 1999) and glucose
DMSO) in a total volume of 1 mL of 100 mM sodium were identified by H-NMR and TLC, respectively.
phosphate buffer (pH 6.2). After the reaction mixtures The anomeric configuration of glucose in 10 was con-
−40 C. H-6’, respectively. A singlet at δ 3.92 (3H) indicated
δ
J
δ
δ
µ
µ
1
(
o
were incubated in advance at 25 C for 3 min, the cluded to be of
reaction was started by addition of the enzyme, and Hz) of its anomeric proton. Consequently, the structure
then the decrease in absorbance was measured at 340 of 10 was established as glycitein-4’- -glucoside.
nm for 6 min using a JASCO V-530 spectrophoto- All of the isolated compounds were examined for rat
meter. lens aldose reducatase inhibitory activity. Daidzein
The inhibition activity (%) was estimated as follows :
A sample/min − ∆A blank/min)/( A control/min
A blank/min)] × 100.
A sample/min showed a decrease in absorbance for
min with a sample, A blank/min with DMSO and
β-form because of the large J value (7.5
O-β-D
[
∆
1
−
(∆
∆
−
∆
6
∆
water instead of a sample and a substrate, respectively,
and A control/min with DMSO instead of a sample.
∆
The 50% inhibition concentrations of the RLAR inhi-
bitory activity were calculated from the log-dose inhi-
bition curve against test concentrations.
RESULTS AND DISCUSSION
Repeated silica gel, ODS, and Sephadex LH 20 column
chromatography led to the isolation of compounds
. Compounds were identified by comparison
with spectral data from the literature of daidzein (
Lee et al., 2008), calycosin ( ) (Park et al., 2002),
trifolirhizin ( ) (Park et al., 2002), puerol A (
) (Park et Fig. 1. The structures of 1-10 isolated from S. japonica
1-
10
1-9
1)
(
2
3
4

1168
H. Y. Park et al.
Table II. The inhibitory effects 1-10 of S. japonica on aldose sources, it was showed mild inhibitory activity with
reductase
an IC50 value of 19.4 µM.
Inhibitory effect (IC50 values)a
Compound
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15.1
34.6
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3.2
29.2
15.4
6.4
78.5
95.7
34.5
77.6
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), puerol A (
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.2, 6.4, and 1.9 M, respectively, as shown Table II.
Calycosin ( ), trifolirhizin ( ), glycitin ( ), and glycitein-
'- -glucoside (10) showed mild inhibitory activity
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spectively. Ononin ( ), 5-hydroxypseudobaptigenin-7-
-glucoside ( ), and calycosin-7- -glucoside ( ) showed
very weak inhibitory activity in the present study. In
conclusion, daidzein ( ), puerol A (
-glucoside ( ) were isolated from the bark of S.
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-glucoside (10) was newly isolated from plant
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