Monoamine oxidase inhibitory components from the roots of Sophora flavescens.

Abstract

In our search for monoamine oxidase (MAO) inhibitors from natural resources, we found that the methanol extract of the roots of Sophora flavescens showed an inhibitory effect on mouse brain monoamine oxidase (MAO). Bioactivity-guided isolation of the extract yielded two known flavonoids, formononetin (1) and kushenol F (2), as active compounds along with three inactive compounds, oxymatrine (3), trifolirhizin (4), and beta-sitosterol (5). Formononetin (1) and kushenol F (2) showed significant inhibitory effects on MAO in a dose-dependent manner with IC50 values of 13.2 and 69.9 microM, respectively. Formononetin (1) showed a slightly more potent inhibitory effect against MAO-B (IC50: 11.0 microM) than MAO-A (IC50: 21.2 microM). Kushenol F (2) also preferentially inhibited the MAO-B activity than MAO-A activity with the IC50 values of 63.1 and 103.7 microM, respectively.

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Arch Pharm Res Vol 28, No 2, 190-194, 2005
190
http://apr.psk.or.kr
Monoamine Oxidase Inhibitory Components from the Roots of
Sophora flavescens
Ji-Sang Hwang1, Seon A Lee1, Seong Su Hong1, Kyong Soon Lee1, Myung Koo Lee1,2, Bang Yeon Hwang1,
and Jai Seup Ro1
1College of Pharmacy, Chungbuk National University, Cheongju 361-763, Korea and 2Research Center for Biore-
source and Health, Chungbuk National University, Cheongju 361-763, Korea
(Received November 6, 2004)
In our search for monoamine oxidase (MAO) inhibitors from natural resources, we found that
the methanol extract of the roots of Sophora flavescens showed an inhibitory effect on mouse
brain monoamine oxidase (MAO). Bioactivity-guided isolation of the extract yielded two known
flavonoids, formononetin (1) and kushenol F (2), as active compounds along with three inac-
tive compounds, oxymatrine (3), trifolirhizin (4), and β-sitosterol (5). Formononetin (1) and
kushenol F (2) showed significant inhibitory effects on MAO in a dose-dependent manner with
IC50 values of 13.2 and 69.9 µM, respectively. Formononetin (1) showed a slightly more potent
inhibitory effect against MAO-B (IC50: 11.0 µM) than MAO-A (IC50: 21.2 µM). Kushenol F (2)
also preferentially inhibited the MAO-B activity than MAO-A activity with the IC50 values of 63.1
and 103.7 µM, respectively.
Key words: Sophora flavescens, Leguminosae, Formononetin, Kushenol F, Monoamine oxi-
dase inhibitor
INTRODUCTION
Monoamine oxidase (MAO, EC 1.4.3.4) is a FAD-con-
taining enzyme located in the outer mitochondrial membrane
of neuronal, glial, and other cells. MAO catalyzes the
oxidative deamination of a number of neurotransmitters,
such as norepinephrine, dopamine, and serotonin, as well
as exogenous and endogenous amines to their corre-
sponding aldehydes (Bach et al., 1988; Benedetti and
Dostert, 1992). Two different subtypes, MAO-A and MAO-
B, have been identified based on their substrate selectivity,
inhibitor sensitivity, and amino acid sequence (Abell and
Kwan, 2001; Shih et al., 1999). MAO-A preferentially
oxidizes norepinephrine and serotonin, and is selectively
inhibited by clorgyline. However, MAO-B preferentially
deaminates β-phenylethylamine and benzylamine, and is
selectively inhibited by l-deprenyl and pargyline (Kalgutkar
et al., 1995).
Due to the key role played by the two MAO forms in the
metabolism of monoamine neurotransmitters, inhibitors of
MAO are of considerable pharmacological and therapeutic
interest in the area of several neurological diseases.
MAO-A inhibitors are useful in the therapy of mental
disorders, mainly as antidepressants, whereas MAO-B
inhibitors expected to be useful in the therapy of
Parkinson's and Alzheimer's disease (Thomas, 2000;
Yamada and Yasuhara, 2004). A number of MAO inhibitors
such as coumarins (Jo et al., 2002), xanthones (Suzuki et
al., 1981; Thull et al., 1993), and alkaloids (Lee et al.,
2003; Naoi and Nagatsu, 1987), have been isolated from
natural products or synthesized for the development of
medicines.
As a part of our ongoing research for MAO inhibitors
from botanical resources, it was found that the methanol
extract from the roots of Sophora flavescens significantly
inhibited the mouse brain MAO activity. S. flavescens Ait.
(Leguminosae) is a perennial herb and is widely distributed
in Korea, Chinese, and Japan. Sophorae Radix, the dried
root of S. flavescens has been used in traditional medicine
as antibacterial, anti-inflammatory, antipyretic, antiasthmatic,
and anthelmintic (Jung and Shin, 1989). Phytochemical
studies of S. flavescens have been reported the isolation
of quinolizidine alkaloids, triterpenoids, and flavonoids
(Tang and Eisenbrand, 1992).
In this study, we identified MAO inhibitors in the dried
root of S. flavescens and characterized their inhibitory
Correspondence to: Jai Seup Ro, College of Pharmacy, Chung-
buk National University, Cheongju 361-763, Korea
Tel: 82-43-261-2818, Fax: 82-43-268-2732
E-mail: jsroh@chungbuk.ac.kr

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Monoamine Oxidase Inhibitory Components from the Roots of Sophora flavescens
191
activities on MAO-A and MAO-B.
MATERIALS AND METHODS
General experimental procedures
Melting points were measured on a Büchi model B-540
without correction. Optical rotations were determined on
JASCO DIP-370 polarimeter at 25 oC. UV and IR spectra
were obtained on a JASCO UV-550 and Perkin-Elmer
model LE599 spectrometer, respectively. 1H-NMR (300
MHz) and 13C-NMR (75 MHz) spectra were recorded
using a Bruker DRX 300 MHz NMR spectrometers using
CDCl3 or DMSO-d6 as a solvent. EI-MS was recorded on
Hewlett-Packard MS 5988 mass spectrometer. Open
column chromatography was performed using a silica gel
(Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck),
and thin layer chromatography (TLC) using a pre-coated
silica gel 60 F254 (0.25 mm, Merck).
Kynuramine, clorgyline, l-depreyl, 4-hydroxyquinoline,
and iproniazid were purchased from Sigma Chemical Co.
(St. Louis, MO, USA).
Plant materials
The dried roots of S. flavescens were collected from the
herb garden at Chungbuk National University, Cheongju,
Korea, in October, 2001 and identified by Prof. K. S. Lee,
a plant taxonomist at Chungbuk National University. A
voucher specimen (CBNU 01020) has been deposited at
the Herbarium of College of Pharmacy, Chungbuk National
University, Korea.
Animals
The ICR male mice were purchased from Samyook
Animal Center (Soowon, Korea) and maintained in accor-
dance with the guidelines for animal care and use of
laboratory animals, Chungbuk National University, Korea.
Extraction and activity-guided isolation
The dried roots of S. flavescens (3 kg) were extracted
with 80% MeOH three times at room temperature to yield
a dark-brown residue (450 g, 78.3% MAO inhibitory
activity at the concentration of 250 µg/mL). The methanol
extract suspended in water, and then partitioned in turn
with CH2Cl2, EtOAc, BuOH, and water. When evaluated at
200 µg/mL, the MAO inhibitory activities for these four
extracts were 82.9, 76.2, 14.0, and 9.2%, respectively. The
most active CH2Cl2 extract (78 g) was chromatographed
over silica gel column with a gradient of MeOH in CH2Cl2
(1, 2, 5, 10, 20, 50, 100%, 1 L each) as the solvent system,
and afforded seven combined fractions (SR1-SR7). SR-2
fraction (5.6 g, 90.5% MAO inhibitory activity at the
concentration of 150 µg/mL) was further purified over
silica gel column with CH2Cl2-MeOH (20:1) gave for-
mononetin (1) (25 mg) and β-sitosterol (5) (150 mg).
Repeated silica gel column chromatography of SR-5
fraction (7.2 g, 85.0% MAO inhibitory activity at the
concentration of 150 µg/mL) using CH2Cl2-MeOH (15:1,
10:1, 5:1) gave kushenol F (2) (12 mg), oxymatrine (3) (21
mg), and trifolirhizin (4) (9 mg).
Formononetin (1)
Colorless crystal; mp 260 oC; EI-MS m/z 268 [M]+; 1H-
NMR (300 MHz, CDCl3) δ: 8.07 (1H, d, J = 8.8 Hz, H-5),
7.94 (1H, s, H-2), 7.44 (2H, d, J = 8.7 Hz, H-2' and H-6'),
6.95 (2H, d, J = 8.7 Hz, H-3' and H-5'), 6.92 (1H, dd, J =
8.8, 2.1 Hz, H-6), 6.83 (1H, d, J = 2.1 Hz, H-8), 3.83 (3H,
s, 4'-OCH3); 13C-NMR (75 MHz, CDCl3) δ: 55.7 (4'-OCH3),
102.6 (C-8), 114.4 (C-3' and C-5'), 115.7 (C-6), 117.3 (C-
4a), 124.5 (C-1'), 124.8 (C-3), 127.8 (C-5), 130.6 (C-2'
and C-6'), 153.7 (C-2), 158.0 (C-8a), 159.5 (C-4), 163.1
(C-7), 177.1 (C-4).
Kushenol F (2)
Yellow pale powder; [α]D
424 [M]+; 1H-NMR (300 MHz, DMSO-d6) δ: 7.37 (1H, d, J
= 8.3 Hz, H-6'), 6.41 (1H, dd, J = 8.3, 2.4 Hz, H-5'), 6.37
(1H, d, J = 2.4 Hz, H-3'), 6.15 (1H, s, H-8'), 5.60 (1H, dd, J
= 11.1, 5.0 Hz, H-2), 5.05 (1H, t, J = 6.7 Hz, H-4"), 4.62
(1H, d, J = 2.1 Hz, H-9"a), 4.60 (1H, d, J = 2.1 Hz, H-9"b),
1.61 (3H, s, 10"-CH3), 1.56 (3H, s, 6"-CH3), 1.45 (3H, s,
7"-CH3); 13C-NMR (75 MHz, DMSO-d6) δ: 17.8 (C-7"), 19.1
(C-10"), 25.8 (C-6"), 27.1 (C-1"), 31.9 (C-3"), 42.8 (C-3),
47.8 (C-2"), 75.3 (C-2), 95.1 (C-8), 102.7 (C-10), 103.4
(C-3'), 107.8 (C-5'), 108.2 (C-6), 111.2 (C-9"), 117.8 (C-1'),
124.4 (C-4"), 128.6 (C-6'), 131.6 (C-5"), 149.1 (C-8"),
156.1 (C-4'), 159.4 (C-2'), 162.1 (C-5), 163.0 (C-9), 165.3
(C-7), 197.8 (C-4).
25 65o (c, 0.2 in MeOH); EI-MS m/z
Oxymatrin (3)
Colorless powder; mp 207oC; [α]D
EI-MS m/z 264 [M]+; 1H-NMR (300 MHz, CDCl3) δ: 5.32 (1H,
m, H-11), 4.43 (1H, dd, J = 12.6, 5.1 Hz, H-17e), 4.21 (1H, t,
J = 12.6 Hz, H-17a); 13C-NMR (75 MHz, CDCl3) δ: 17.9 (C-
8), 18.0 (C-9), 19.4 (C-3), 25.5 (C-4), 26.9 (C-12), 29.3 (C-
13), 33.7 (C-14), 35.3 (C-5), 42.4 (C-17), 43.5 (C-7), 53.7 (C-
11), 67.9 (C-6), 69.9 (C-2), 70.4 (C-10), 170.8 (C-15).
25 +45o (c, 0.1 in MeOH);
Trifolirhizin (4)
Colorless powder; mp 142-144 oC; EI-MS m/z 446 [M]+;
1H-NMR (300 MHz, DMSO-d6) δ: 7.30 (1H, d, J = 8.5 Hz,
H-1), 6.99 (1H, s, H-7), 6.70 (1H, dd, J = 8.5, 1.8 Hz, H-2),
6.50 (1H, d, J = 1.8 Hz, H-4), 6.49 (1H, s, H-10), 5.90 (2H,
d, J = 12.8 Hz, -OCH2O-), 5.52 (1H, d, J = 6.8 Hz, H-11a),
5.02 (1H, d, J = 7.2 Hz, H-1); 13C-NMR (75 MHz, CDCl3)
δ: 41.3 (C-6a), 61.5 (C-6), 66.7 (C-6), 70.5 (C-4), 74.0 (C-
2), 77.4 (C-5), 77.9 (C-3), 78.5 (C-11a), 94.1 (C-10), 101.1

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192J.-S. Hwang et al.
(-OCH2O-), 101.9 (C-1), 104.8 (C-4), 106.2 (C-7), 111.8
(C-2), 115.0 (C-11b), 119.1 (C-6b), 132.8 (C-1), 142.0 (C-
8), 148.3 (C-9), 154.5 (C-10a), 157.0 (C-4a), 159.3 (C-3).
MAO preparation and assay for MAO activity
A crude mitochondrial fraction from mouse brain was
isolated by the method of Naoi et al. with minor modifi-
cation (Naoi and Nagatsu, 1987; Ro et al., 2001). MAO
activity was measured fluorometrically using kynuramine
as a substrate according to the method of Kraml with a
slight modification (Kraml, 1965; Ro et al., 2001). The
samples (50 µL) were added to 0.2 M potassium phos-
phate buffer (750 µL, pH 7.4), which contained 30 µL of
mouse brain mitochondrial suspension. The reaction was
initiated by the addition of 200 µL of 500 mM kynuramine.
After incubation of 37 oC for 30 min, the reaction was
terminated by the addition of 250 µL of 10% ZnSO4 and
50 µL of 1 N NaOH, and the reaction mixture was
centrifuged at 3,000 × g for 5 min. 1.4 mL of 1 N NaOH
was added in 700 µL of assay mixture taken from the
supernatant, then the mixture was transferred into a fluoro
96-well plate. The fluorescence intensity of 4-hydroxyqui-
noline, which was formed from kynuramine by MAO, was
measured at an emission wavelength of 380 nm and an
excitation wavelength of 315 nm using a Perkin Elmer LS
50B fluorescence spectrometer. The suspension was pre-
incubated with either 1 µM of l-deprenyl (type-B inhibitor)
or clorgyline (type-A inhibitor) for 15 min to measure
MAO-A or MAO-B activity, respectively.
RESULTS AND DISCUSSION
In our ongoing search for naturally occurring MAO
inhibitors, a methanol extract from the roots of S.
flavescens exhibited strong inhibitory activity on mouse
brain MAO. Activity-guided isolation of the methylenechloide-
soluble fraction yielded two known flavonoids, formononetin
(1) and kushenol F (2), as active compounds along with
three inactive compounds, oxymatrine (3), trifolirhizin (4),
and β-sitosterol (5) (Fig. 1). The structures were identified
by physical and spectroscopic methods (mp, UV, IR, [α]D,
MS,
obtained with those of published values (Lee et al., 2003;
Park et al., 2003; Ryu et al., 1997; Sekine et al., 1993; Wu
et al., 1985).
MAO activity in mouse brain mitochondria was mea-
sured using the non-selective substrate kynuramine, and
clorgyline or l-deprenyl was added to define MAO-A or
MAO-B activity, respectively.
As shown in Table I, Formononetin (1) and kushenol F
(2) inhibited MAO activity in a concentration dependent
manner and the IC50 values were 13.2 and 69.9 µM,
respectively. Under the same experimental conditions, the
IC50 value of iproniazid, a MAO inhibitor as a positive
control, was 12.9 µM. In order to verify the selectivity of
the MAO activity, l-deprenyl-pretreated MAO preparation
was used for the measurement of MAO-A activity, and a
clorgyline-pretreated one was for MAO-B. This result
indicated that formononetin (1) showed a slightly potent
inhibitory effect against MAO-B (IC50: 11.0 µM) than MAO-
A (IC50: 21.2 µM) (Table II). Kushenol F (2) also preferen-
tially inhibited the MAO-B activity than MAO-A activity in a
concentration dependent manner with the IC50 values of
63.1 and 103.7 mM, respectively (Table III).
The regulation of the activity of MAO-B has been
thought to be an effective approach for the prevention of
1H- and
13C-NMR) and by comparing the data
Fig. 1. Structures of isolated com pounds from S. flavescens

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Monoamine Oxidase Inhibitory Components from the Roots of Sophora flavescens
193
Parkinson's disease and adjunct treatment of Alzheimer's
disease, while those of MAO-A expected to be useful for
the treatment of depression and anxiety (Thomas, 2000;
Yamada and Yasuhara, 2004). These results suggest that
S. flavescens which contains compounds with slightly
potent MAO-B inhibitory activity may be as a possible
therapeutic candidate for the Parkinson's and Alzheimer's
disease. However, further pharmacological investigations
and in vivo physiological functions of S. flavescens remain
to be elucidated.
ACKNOWLEDGMENTS
This work was supported by a grant (R05-2001-000-
00664-0) from the Basic Research Program of the Korea
Science & Engineering Foundation. We are grateful to
Korea Basic Science Institute for
spectral measurements.
1H- and
13C-NMR
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Table I. Inhibitory effects of com pounds 1-5 on MAO in m ouse brain
Concentration
(µM)
MAO activity (% of control)
(nm ol/m in/m g protein)
IC50
(µM)
Control
Iproniazid
Form ononetin (1)
1.259 ± 0.081 (100.0)
0.724 ± 0.020 (57.6)
0.999 ± 0.021 (79.4)
0.819 ± 0.051(65.1)* *
0.541 ± 0.045(42.9)* * *
0.358 ± 0.031(128.5)* * *
1.026 ± 0.031(181.5)
0.843 ± 0.061(167.0)*
0.550 ± 0.042(143.7)* * *
0.262 ± 0.012(120.8)* * *
1.288 ± 0.101(102.3)
1.217 ± 0.048(196.7)
1.192 ± 0.037(194.7)
1.024 ± 0.042(181.4)
1.436 ± 0.053(102.4)
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16
32
20
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50
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12.9
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Kushenol F (2)69.9
Oxym atrine (3) >100
Trifolirhizin (4)>100
β-Sitosterol (5) >100
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Concentration
(µM)(nm ol/m in/m g protein)
Control1.259 ± 0.131
MAO-A (Deprenyl-treated)
Control + Deprenyl0.469 ± 0.032(100.0)
Form ononetin (1)8 0.361 ± 0.025(177.0)
16 0.278 ± 0.031(159.2)*
320.177 ± 0.023(137.7)* *
640.090 ± 0.016(119.1)* * *
MAO-B (Clorgyline-treated)
Control + Clorgyline0.745 ± 0.082(100.0)
Form ononetin (1)4 0.575 ± 0.021(177.1)
8 0.420 ± 0.035(156.3)*
16 0.294 ± 0.034(139.5)* * *
320.183 ± 0.021(124.5)* * *
The activities of MAO-A and MAO-B in m ouse brain were m easured in
the presence of 1 µM l-deprenyl or clorgyline, respectively. The data
represent the m ean± S.E.M. of three independent experim ents
perform ed in triplicate. Significantly different from the control value: *
P<0.05; * * P<0.01; * * * P<0.001 (Student's t-test).
MAO activity (% of control) IC50
(µM)
21.2
11.0
Table III. Inhibitory effects of kushenol F (2) on MAO-A and MAO-B in
m ouse brain
Concentration
(µM)
MAO activity (% of control)
(nm ol/m in/m g protein)
IC50
(µM)
Control
MAO-A (Deprenyl-treated)
Control + Deprenyl
Kushenol F (2)
1.259 ± 0.131
103.7
0.469 ± 0.032(100.0)
0.396 ± 0.012(181.2)
0.321 ± 0.015(165.8)*
0.151 ± 0.024(130.9)* *
0.071 ± 0.012(114.5)* * *
20
40
80
160
MAO-B (Clorgyline-treated)
Control + Clorgyline
Kushenol F (2)
163.1
0.745 ± 0.082(100.0)
0.559 ± 0.052(180.4)
0.444 ± 0.033(163.8)*
0.326 ± 0.024(146.8)* *
0.206 ± 0.021(129.6)* * *
20
40
80
160
The activities of MAO-A and MAO-B in m ouse brain were m easured in
the presence of 1 µM l-deprenyl or clorgyline, respectively. The data
represent the m ean±S.E.M. of three independent experim ents
perform ed in triplicate. Significantly different from the control value:
* P<0.05; * * P<0.01; * * * P<0.001 (Student's t-test).

Page 5

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