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346
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
Constituents of Asarum sieboldii with Inhibitory Activity on
Lipopolysaccharide (LPS)-Induced NO Production in BV-2 Microglial Cells
by Ah-Reum Han, Hye Jeoung Kim, Minkyu Shin, Moochang Hong, Yang Seok Kim, and Hyunsu Bae*
BK21 Oriental Medical Science Center, College of Oriental Medicine, Kyung Hee University,
1 Hoeki-dong, Dongdaemun-gu, Seoul 130-701, Korea
(phone: þ 82-2-961-0323; fax: þ 82-2-967-2080; e-mail: [email protected])
Bioassay-guided fractionation of the root extract of Asarum sieboldii led to the isolation of the four
active compounds ()-sesamin (1), (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide
(2), kakuol (3), and 13,4,5-trimethoxytoluene3 ( ¼ 1,2,3-trimethoxy-5-methylbenzene; 4), in terms of
inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Compounds 1 – 4 showed
potent inhibition of NO production, with IC50 values in the low nanomolar-to-micromolar range. Also
isolated were the known compounds methylkakuol (5), 13,5-dimethoxytoluene3, safrole, asaricin,
methyleugenol, and ()-asarinin, which were found to be inactive in the above assay. Among the ten
known isolates, compounds 1, 2, and 5 were found for the first time in this plant.
Introduction. – The Asarum species (Aristolochiaceae) are herbal plants distributed through North America, Europe, and Asia [1]. The roots of these species were
used in traditional medicine as antitussive [2], anti-allergic [3], antihyperlipemic [4],
expectorant [5] [6], anti-inflammatory [7] [8], anesthetic [9], and antifungal agents
[10] [11]. Previous phytochemical work on Asarum species resulted in the isolation of
various types of essential oils [5] [12] [13], amides [14] [15], lignans [14] [16], flavonoids
[17] [18], terpenoids [19], and alkaloids [20].
As a part of the search for new therapeutic agents from medicinal plants against
inflammation and neurodegenerative diseases, the spray-dried extracts of 270 herbal
medicines in the PhytoLibraryTM kit were primarily tested for their inhibitory activities
towards lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV-2
microglial cells. As a result, the roots of A. sieboldii, which showed considerable
inhibitory activity (70.4% inhibition at 1 mg/ml), were selected for activity-guided
fractionation.
NO is a simple, inorganic, gaseous free radical that is produced by the oxidation of
l-arginine catalyzed by NO synthase (NOS), with a wide range of physiological and
pathological actions [21]. Among the NOS family, inducible NOS (iNOS), in
particular, is involved in overproduction of NO, which is associated with oxidative
stress and with pathophysiological responses, including circulatory shock, inflammation, and carcinogenesis [22]. Also, it can be expressed in response to pro-inflammatory
agents such as interleukin 1b (IL-1b), tumor necrosis factor a (TNF-a), and LPS in
various cell types, including macrophages, endothelial cells, and smooth-muscle cells
[23]. In addition, overproduction of NO in the brain released by microglial cells
contributes to neuronal damages accelerating various neurological disorders such as
G 2008 Verlag Helvetica Chimica Acta AG, ZJrich
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
347
Parkinson and Alzheimer [24]. Therefore, any treatment for the pathological symptoms
related to NO is considered as a clinical approach either in preventing or in treating
inflammatory and neurodegenerative disorders [24] [25].
Herein, we describe the isolation, structural identification, and biological properties
of ten known isolates from A. sieboldii, including the biologically active constituents
()-sesamin (1) [14], (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (2) [14], kakuol (3) [10], and 13,4,5-trimethoxytoluene3 ( ¼ 1,2,3-trimethoxy-5methylbenzene; 4) [26], as well as the inactive compounds methylkakuol (5) [27], 13,5dimethoxytoluene3 ( ¼ 1,3-dimethoxy-5-methylbenzene) [28], safrole [29], asaricin
[30], methyleugenol [12], and ()-asarinin [14].
Results and Discussion. – 1. Chemistry. The hexane-soluble fraction of the MeOH
extract of the roots of Asarum sieboldii Miquel showed inhibitory activity, with 68.9%
inhibition at a concentration of 1 mg/ml. This fractions was, thus, subjected to detailed
phytochemical analysis, which resulted in the isolation of the above ten compounds,
which were structurally elucidated by physical and spectroscopic methods, as well as by
comparison of their data with those published in the literature. In addition, the
configuration of ()-sesamin (1) and ()-asarinin were confirmed by comparison of
their optical rotations with published values [14]. Compounds 1, 2, and 5 were found in
this species for the first time, and compound 5 has not been isolated before from the
genus Asarum.
2. Biological Activity. All compounds obtained in the present study were evaluated
for their inhibitory activity on NO production in LPS-activated BV-2 microglial cells.
Among the isolates, compounds 1 – 4 were found to be significantly active. ()-Sesamin
(1) exhibited the most potent inhibition with an IC50 value of 6.26 10 10 m. However,
it was cytotoxic at the tested concentrations of 2.82 10 7 to 2.82 10 3 m in the
MTT 1) assay. Therefore, its iNOS inhibitory activity seems to be related to its
concomitant cytotoxic effect. There have been some other reports on the inhibitory
1)
MTT ¼ 3-(4,5-Dimethyl-1,3-thiazol-2-yl)-2,5-diphenyltetrazolium bromide.
348
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
activity of sesamin on LPS-induced NO production in BV-2 microglial cells [31] [32], so
that compound 1 was considered as a positive control in the present study.
Compounds 2, 3, and 4 inhibited the LPS-induced NO production in a dosedependent manner, with IC50 values of 1.52 10 9, 3.6 10 7, and 1.4 10 6 m,
respectively (Figure). These compounds did not show cytotoxicity at the test
concentrations, indicating their true iNOS inhibitory activity. Especially, compound
4, which exhibited the lowest IC50 value, inhibited significant LPS-induced NO
production, i.e., 75.9% inhibition at the maximum test concentration of 1 mg/ml (5.49 10 3 m) .
Figure. Effects of compounds 1 – 4 on LPS-induced NO production in BV-2 microglial cells. Inhibition of
NO production was calculated relative to negative control, and values represent mean S.D. of three
experiments performed in triplicate.
Although compounds 3 and 5 have very similar structures, the only difference being
the presence of a hydroxy (OH) vs. a methoxy (MeO) function ortho to the acyl group,
only 3 showed inhibitory activity on LPS-induced NO production, compound 5 being
inactive. Therefore, we can assume that the MeO group in 14,5-(methylenedioxy)propiophenones3 plays an important role in terms of inhibitory activity.
The remaining five known compounds, 13,5-dimethoxytoluene3, safrole, asaricin,
methyleugenol, and ()-asarinin, showed negligible inhibitory activities, i.e., less than
50% inhibition at the maximum test concentration of 1 mg/ml. Hence, these
compounds were regarded as inactive.
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
349
In conclusion, compounds 2 – 4 showed significant activities in our assay. Thus,
further study on their mechanism of action can be encouraged to derive novel
therapeutic agents against inflammation and neurodegenerative diseases. The effect of
sesamin (1) on LPS-induced NO production in BV-2 microglial cells has been reported
previously. Hou et al. [31], and Jeng et al. [32] reported that this compound decreased
nitrite accumulation with 50% inhibition at 20 mm in BV-2 microglial cells, and
inhibited LPS-induced cytokine production by suppression of p38 mitogen-activated
protein kinase and nuclear factor-kB, respectively, although the configuration of this
compound was not indicated. (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10tetraenamide (2) was reported to inhibit NO production in LPS-stimulated RAW
264.7 macrophage cells, with an ID50 value of 6 mg/ml [33]. Therefore, this is the first
report on the evaluation of inhibitory activity on NO production of all isolates, except
for 1, in LPS-activated BV-2 microglial cells.
This work was supported by a grant (R13-2007-019-00000-0) from the Medical Science and
Engineering Research Center program of the Ministry of Science and Technology (MOST) and the Korea
Science and Engineering Foundation (KOSEF), and by a grant (15024) from the Creation of Geriatric
Natural-MediCluser program of the Korean Government, Seoul City.
Experimental Part
General. Flash column chromatography (FC): silica gel 60 (70 – 230 mesh; Merck). TLC: precoated
Kieselgel 60 F254 plates (0.25 mm; Merck); visualization under UV light (254 and 365 nm) and by spraying
with 10% (v/v) H2SO4 , followed by heating at 1208 for 5 min. The PhytoLibraryTM kit was purchased
from PURIMED Co. (Seoul, Korea). Optical rotations: Jasco P-1010 polarimeter at 258. 1D- and 2DNMR experiments: Bruker Avance-400 FT-NMR instrument; with Me4Si as internal standard. HR-EIMS and ESI-MS: Jeol JMS-700-M mass spectrometer and an API 3200-Q-TRAP LC/MS/MS system,
resp.
Plant Material. The roots of Asarum sieboldii Miquel (Aristolochiaceae) were purchased from Sun
Ten Pharmaceutical Co., Ltd. (Taipei, Taiwan). A voucher specimen (D10) was deposited at the
Herbarium of College of Oriental Medicine, Kyung Hee University, Korea.
Extraction and Isolation. The roots of A. sieboldii (3 kg) were extracted with MeOH (5 10 l) for
24 h by percolation. The solvent was evaporated in vacuo to afford a MeOH extract (300 g), which was
suspended in H2O (1 l), and extracted successively with hexane (5 1 l), AcOEt (5 1 l), and BuOH
(3 1 l). Compound 1 (2.3 g, 0.077%) [14] was isolated from the hexane layer by precipitation. The
soluble hexane extract (100 g) was fractionated by FC (1 kg SiO2 ; hexane/CHCl3 10 : 0, 19 : 1, 9 : 1, 4 : 1,
1 : 1, then neat MeOH, 5 l each) to afford 15 fractions: Fr. I – Fr. XV. Safrole (3.7 g, 0.12%) [29],
methyleugenol (14.3 g, 0.47% w/w) [12], and 1 (1.1 g, 0.036%) [14] were also isolated from the first
separation. Fr. VII (6 g), eluted with hexane/CHCl3 9 : 1, was subjected to FC (150 g SiO2 ; hexane/AcOEt
99 : 1, 98 : 2, 95 : 5, then neat MeOH, 2 l each) to provide asaricin (459 mg, 0.015%) [30]. Fr. VIII (15 g)
was purified by FC (300 g SiO2 ; hexane/AcOEt 99 : 1, 98 : 2, 95 : 5, then neat MeOH, 3 l each) to afford
13,5-dimethoxytoluene3 (189 mg, 0.0063%) [28] and 3 (59 mg, 0.0019%) [10]. Fr. XI and Fr. XII (12 g),
eluted with hexane/CHCl3 1 : 1, were further fractionated by FC (300 g SiO2 ; hexane/AcOEt 99 : 1, 98 : 2,
95 : 5, 9 : 1. 4 : 1, then neat MeOH, 3 l each) to yield 4 (154 mg, 0.0051%) [26] and 5 (389 mg, 0.013%) [27].
Fr. XIII (17 g), eluted with hexane/CHCl3 1 : 1, was further separated by FC (400 g SiO2 ; linear gradient
of 0, 0.1, and 0.2% MeOH in CHCl3 , then neat MeOH, 4 l each) to afford 2 (1.0 g, 0.033%) [14].
Measurement of NO Production in LPS-Activated BV-2 Microglial Cells. Measurement of NO
formation by iNOS was performed in cultured BV-2 microglial cells. The cells were maintained in
Dulbecco3s Modified Eagle Medium (DMEM) supplemented with penicillin-streptomycin and 10% fetal
bovine serum (FBS) at 378 in humidified air containing 5% of CO2 . To evaluate the inhibitory activity of
the test materials on LPS-induced NO production, the cells in 10% FBS-DMEM, without Phenol Red,
350
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
were plated in 24-well plates (5 105 cells/ml), and then incubated for 24 h. After incubation, the cells
were washed with PBS, replaced with new medium, and then incubated in the medium with 1 mg/ml of
LPS in the presence or absence of test sample. After an additional 20-h incubation, the media were
collected and analyzed for nitrite accumulation as an indicator of NO production by the Griess reaction.
Briefly, 100 ml of Griess reagent (0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in H2O and 1%
sulfanilamide in 5% H3PO4 ) were added to 100 ml of each supernatant from LPS-treated or LPS-andsample-treated cells in 96-well plates. The UV/VIS absorbance was measured at 540 nm using a
microplate reader, and the nitrite concentration was determined by comparison with a NaNO3 standard
curve. The percentage inhibition was expressed as [1 – (NO level of test samples/NO level of vehicletreated control)] 100. IC50 Values, the sample concentration resulting in 50% inhibition of NO
production, were determined by non-linear regression analysis (% inhibition vs. concentration).
MTT Assay for Cell Viability. Cytotoxicity was measured, after 24 h of continuous exposure to the
various concentrations of test compounds, by means of a colorimetric assay, based on the ability of
mitochondria in viable cells to reduce MTT1).
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Received June 7, 2007
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