Decursin and decursinol angelate selectively inhibit NADH-fumarate reductase of Ascaris suum

Article abstract of DOI:10.1055/s-2007-990245

NADH-fumarate reductase (NFRD) is a key enzyme in many anaerobic helminths. Decursin and decursinol angelate have been isolated from the roots of Angelica gigas Nakai (Apiaceae) as NFRD inhibitors. They inhibited Ascaris suum NFRD with IC₅₀ values of 1.1 and 2.7 μM, respectively. Their target is the electron transport enzyme complex I. Since the inhibitory activities of decursin against bovine heart complexes are weak, it is a selective inhibitor of the nematode complex I. In contrast, decursinol angelate moderately inhibits bovine heart complexes II and III. Decursinol inhibits A. suum NFRD to a similar extent, but its target is complex II. It also inhibits bovine heart complexes II and III. Georg Thieme Verlag KG Stuttgart.

Full text of DOI:10.1055/s-2007-990245

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Decursin and Decursinol Angelate
Selectively Inhibit NADH-Fumarate
Reductase of Ascaris suum
Kazuro Shiomi1, 2, Hiroko Hatano2, Hiromi Morimoto3, Hideaki Ui1,
Kimitoshi Sakamoto4, Kiyoshi Kita4, Hiroshi Tomoda3, Eun Woo Lee5,
±
Tae Ryeon Heo6, Hirokazu Kawagishi7, Satoshi Omura1, 2
Abstract
NADH-fumarate reductase (NFRD) is a key enzyme in many an-
aerobic helminths. Decursin and decursinol angelate have been
isolated from the roots of Angelica gigas Nakai (Apiaceae) as
NFRD inhibitors. They inhibited Ascaris suum NFRD with IC50 val-
ues of 1.1 and 2.7 mM, respectively. Their target is the electron
transport enzyme complex I. Since the inhibitory activities of de-
cursin against bovine heart complexes are weak, it is a selective
inhibitor of the nematode complex I. In contrast, decursinol an-
gelate moderately inhibits bovine heart complexes II and III. De-
cursinol inhibits A. suum NFRD to a similar extent, but its target
is complex II. It also inhibits bovine heart complexes II and III.
In the course of screening for anthelmintic antibiotics, we have
been interested in the differences in energy metabolisms between
the host and helminths [1]. The NADH-fumarate reductase (NFRD)
system is part of a special respiratory system in parasitic hel-
minths, which is found in many anaerobic organisms. The system
is composed of complex I (NADH-rhodoquinone oxidoreductase)
and complex II (rhodoquinol-fumarate reductase). Electrons from
NADHare accepted by rhodoquinone through complex I, and then
transferred to fumarate through complex II. We have screened
fungal metabolites for inhibitors of NFRD using Ascaris suum
(roundworm) mitochondria, and obtained nafuredin and atpenins.
Nafuredin is a selective inhibitor of helminth complex I and
showed anthelmintic activity in vivo [2], [3], [4]. Atpenins are
non-selective complex II inhibitors, but they are the most potent
complex II inhibitors ever reported [5]. Atpenin A5 is bound to a
ubiquinone binding site of complex II, which was revealed by the
co-crystal structure of atpenin A5 and the E. coli enzyme [6].
1478
Affiliation: 1 Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Ja-
2
3
pan ´ The Kitasato Institute, Tokyo, Japan ´ School of Pharmacy, Kitasato
4
University, Tokyo Japan ´ Graduate School of Medicine, The University of
Tokyo, Tokyo, Japan ´ PNE Co. Ltd., 907±12 Daechidong, Kangnam-Gu,
Seoul, Korea ´ Faculty of Biological Engineering, Inha University, Incheon,
Korea ´ Graduate School of Science and Technology, Shizuoka University,
5
6
7
Shizuoka, Japan
Correspondence: Prof. Kazuro Shiomi ´ Kitasato Institute for Life Sciences ´
Kitasato University ´ 5-9-1 Shirokane ´ Minato-ku ´ Tokyo 108±8641 ´ Japan ´
Phone: +81-3-5791-6439 ´ Fax: +81-3-5791-6131 ´ E-mail: shiomi@lisci.kitasato-
u.ac.jp
±
Prof. Satoshi Omura ´ The Kitasato Institute ´ 5-9-1 Shirokane ´ Minato-ku ´
Tokyo 108±8642 ´ Japan ´ Phone: +81-3-5791-6101 ´ Fax: +81-3-3444-8360 ´
E-mail: omura-s@kitasato.or.jp
Received August 24, 2006 ´ Revised May 8, 2007 ´ Accepted July 6, 2007
Bibliography: Planta Med 2007; 73: 1478±1481 ꢀ Georg Thieme Verlag KG
Stuttgart ´ New York ´ DOI 10.1055/s-2007-990245 ´ Published online October
18, 2007 ´ ISSN 0032-0943

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The screening for NFRD inhibitors from herbal medicines led to Compounds 1 and 2 are pentenoic acid esters of decursinol (3),
the isolation of two compounds, decursin (1) [7], [8] and decur- which has been also isolated from A. gigas [7]. It has been report-
sinol angelate (2) [9], [10] (Fig.1), from the roots of Angelica gigas ed that some biological activities of 1 and 2 were correlated with
Nakai, which have been used for the treatment of anemia and as those of 3 but other activities were not correlated [11], [16], [17],
analgesic, sedative, and tonic agents. Some biological and bio- [18]. Therefore, we prepared 3 from 1 and evaluated the enzyme
chemical activities of 1 and 2 have been reported, such as anti- inhibitory activities of 3. Compound 3 inhibited A. suum NFRD
bacterial activity against Helicobacter pylori [11], antitumor ac- and bovine NADHoxidase at concentrations similar to 1 and 2
tivity [12], activation of protein kinase C [13], [14], and inhibition (Table 1). However, 3 also potently inhibited A. suum complex II
of nitric oxide synthesis [15].
and bovine heart complexes II and III (Table 2). Its inhibitions
against A. suum and bovine heart complex I were both weak. It
Compounds 1 and 2 inhibited A. suum NFRD in the micromolar is interesting that the selective inhibition against A. suum com-
range (Table 1). However, their IC50 values against bovine NADH plex I or II depends on whether the ester moiety exists or not. H.
oxidase (complexes I + III + IV) were more than 10 times higher. pylori is known to have NFRD [19], and 1 and 2 were reported to
Therefore, 1 and 2 showed selectivity against the nematode NFRD inhibit the growth of H. pylori, whereas the inhibition by 3 was
(Turkey's test; IC50 values between complexes I and II, p < 0.05).
weak [11]. It is suggested that the growth inhibition of H. pylori
by 1 and 2 may be due to the selective inhibition of complex I.
Next, we evaluated inhibitory activities of 1 and 2 against each
complex using submitochondrial particles (SMP) of A. suum and It is also interesting that the inhibition of bovine complex III is
bovine heart (Table 2). Compound 1 inhibited complex I about 15 affected by the side-chain ester structure. Compound 3 with no
times more potently than complex II in A. suum (IC50 values be- side-chain ester is the most favorable for complex III inhibition.
tween complexes I and II, p < 0.05). Its inhibitory activities The extension of angelic acid in 2 caused an eight-fold increase of
against bovine heart complexes I and II, and III were very weak the IC50 value. The isomer, senecioic acid, in 1 hampered complex
(IC50 values between A. suum and bovine complex I, p < 0.05). III inhibition, which may be due to the steric hindrance of the
Therefore, 1 inhibits A. suum complex I selectively. The inhibitory methyl group. A similar tendency was also observed for the inhi-
activities of 2 against A. suum complexes I and II are similar to bition of bovine complex II.
those of 1. Its inhibitions against bovine heart complexes I, II,
and III were moderate. Therefore, 2 is a inhibitor of A. suum com- As for the effects on electron transport enzymes, 1 and 3 were re-
plex I, but it inhibits mammalian enzymes more potently than 1. ported to inhibit chloroplast-mediated electron transport only in
the dark [16]. They inhibited the photophosphorylation activities
of chloroplasts, but the inhibition exhibited by 3 was more po-
Fig. 1 Structures of
decursin (1), decur-
sinol angelate (2),
and decursinol (3).
tent than that of 1. The correlation between electron transport
enzymes and photophosphorylation requires further study.
1479
Materials and Methods
NMR spectra were recorded on Varian Inova 600 or Mercury 300
spectrometers. Mass spectra were obtained on a JEOL JMS-AX505
HA spectrometer. Optical rotations were recorded on a JASCO
model DIP-181 polarimeter.
The roots of Angelica gigas Nakai (Apiaceae) were purchased at a
Korean registered market, and the quality was controlled accord-
ing to the Korean Pharmacopoeia. The roots were collected in
Chungcheongbuk-do, Korea, in 2002.
The dried root powder of Angelica gigas Nakai (200 g) was treated
with 2000 mL of acetone twice. The extracts were combined,
concentrated, and partitioned between EtOAc (2000 mL) and
water (2000 mL). The EtOAc layer was concentrated to give
17.8 g of dark green oil. Subsequently, 1.7 g of the oil were
charged on a silica gel column (63±200 mm, 4.1”12 cm, Merck
KGaA; Darmstadt, Germany) and eluted stepwise with hexane-
EtOAc 80:20 (500 mL), 70:30 (1000 mL), 60:40 (500 mL), and
50:50 (500 mL). Fractions of 50 mL were collected and moni-
tored by NFRD inhibition and TLC (Merck No. 105715, hexane-
EtOAc 50:50). Fraction 16 (750±800 mL), which had potent
NFRD inhibitory activity and showed a single spot on silica gel
TLC, was concentrated to yield 524 mg of yellow oil. Of this oil,
49.0 mg were applied to HPLC (Senshu pak Pegasil ODS,
Table 1 Inhibitory activities of NFRD and NADH oxidase by 1±3
Compound
IC50 (mM)
NFRD (A. suum)
NADH oxidase (bovine heart)
1
2
3
1.09  0.03a
2.71  1.00b
4.80  0.63c
65.0  7.1a
43.0  4.5b
35.7  9.1c
a,b,c Items with the same letters were significantly different (p < 0.05).
Letter¼ Planta Med 2007; 73: 1478±1481

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Table 2 Inhibitory activities of 1±3 on electron transport complexes
Compound
IC50 (mM)
A. suum SMP
bovine heartSMP
complex II
complex I
complex II
complex I
complex III
1
2
3
6.42  0.19a
8.40  1.40b
> 100
112  13a
81  25b,c,d
3.86  0.73e
60.1  7.0a
45.9  3.7b
40.6  5.3e,f,g
> 100
> 100
35.2  1.4c
1.41  0.20f
17.0  1.2d
2.10  0.12g
a,b,c,d,e,f,g Items with the same letters were significantly different (p < 0.05).
2”25 cm; Senshu Scientific Co.; Tokyo, Japan), elued with tasato Institute and Dr. Achim Harder of Bayer HealthCare AG,
CH3CN-H2O 60:40; flow 8 mL/min, column temperature 408C; Animal Health-Research & Development-Parasiticides for valu-
detection of eluates by a multiwavelength detector MD-910 (JAS- able discussions. This work was supported in part by the Grant
CO). The major peak (tR = 25 min) was collected and concentra- of the 21st Century COE Program, Ministry of Education, Culture,
ted under vacuum to give 45.8 mg of colorless oil. It was further Sports, Science, and Technology, Japan.
purified by HPLC (Senshu pak Pegasil Silica, 2”25 cm, Senshu
Scientific Co.); eluted with hexane-EtOAc 85:15, flow 8 mL/min,
column temperature 408C; detection of eluates by MD-910. Two References
peaks were eluted at 55 min and 51 min, which were identified
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Decursin (1): colorless oil; HR-FAB-MS: m/z = 329.1393 [M + H]+
(calcd. for C19H21O4 :329.1389); [a]D22: + 144.2 (c 0.2, MeOH). The
1H- and 13C-NMR data were the same as those previously report-
ed [12].
±
2 Omura S, Miyadera H, Ui H, Shiomi K, Yamaguchi Y, Masuma R et al. An
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Decursinol angelate (2): colorless oil; HR-FAB-MS: m/z =
329.1393 [M + H]+ (calcd. for C19H21O4 :329.1389); [a]D22: + 114.6
(c 0.2, MeOH). The 1H- and 13C-NMR data were the same as pre-
viously reported [12].
1480
5 Miyadera H, Shiomi K, Ui H, Yamaguchi Y, Masuma R, Tomoda H et al.
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Decursinol (3) was prepared from 1 as reported previously [11].
Decursin (1, 5.0 mg) was dissolved in 0.5 mL of MeOH, and 2.0 mL
of 1 M NaOHwere added to the solution. After the solution had
been stirred at room temperature for 75 min, its pHwas adjusted
to 3 by 1 M HCl and extracted with EtOAc. The organic layer was
concentrated (4.0 mg) and purified by HPLC (Senshu pak Pegasil
ODS, 2”25 cm); 30% CH3CN, 8 mL/min, 408C; detection of elu-
ates by MD-910. The major peak eluted at 27 min was collected
to give 2.2 mg of 3.
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same as those previously reported [20].
10
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11
12
13
14
Acknowledgements
We thank Dr. Hideto Miyoshi of the Graduate School of Agricul-
ture, Kyoto University for providing substrate synthetic quinones
for the enzyme assay. We also thank Prof. HaruoTanaka of the Ki-
Letter¼ Planta Med 2007; 73: 1478±1481

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
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Letter¼ Planta Med 2007; 73: 1478±1481