Constituents of Asarum sieboldii with Inhibitory Activity on Lipopolysaccharide (LPS)‐Induced NO Production in BV‐2 Microglial Cells

Constituents of Asarum sieboldii with Inhibitory Activity onLipopolysaccharide LPS-Induced NO Production in BV-2 Microglial Cells by Ah-Reum Han, Hye Jeoung Kim, Minkyu Shin, Moochang Ho

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: hbae@khu.ac.kr)

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 IC50values 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 distrib-uted 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 frommedicinal plants against inflammation and neurodegenerative diseases, the spray-dried extracts of 270 herbal medicines in the PhytoLibraryTMkit 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, inflamma-tion, 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

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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-tetraena-mide (2) [14], kakuol (3) [10], and 13,4,5-trimethoxytoluene3 ( ¼ 1,2,3-trimethoxy-5-methylbenzene; 4) [26], as well as the inactive compounds methylkakuol (5) [27], 13,5-dimethoxytoluene3 (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 fromthe 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 IC50value of 6.26  10 10m However,

it was cytotoxic at the tested concentrations of 2.82 10 7 to 2.82 10 3m in the MTT1) assay Therefore, its iNOS inhibitory activity seems to be related to its concomitant cytotoxic effect There have been some other reports on the inhibitory

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1) MTT¼ 3-(4,5-Dimethyl-1,3-thiazol-2-yl)-2,5-diphenyltetrazolium bromide

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 dose-dependent manner, with IC50 values of 1.52 10 9, 3.6  10 7, and 1.4  10 6m , 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 3m)

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)pro-piophenones3 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.

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

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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-2,4,8,10-tetraenamide (2) was reported to inhibit NO production in LPS-stimulated RAW 264.7 macrophage cells, with an ID50value 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) fromthe Medical Science and Engineering Research Center programof the Ministry ofScience and Technology (MOST) and the Korea Science and Engineering Foundation (KOSEF), and by a grant (15024) fromthe Creation of Geriatric Natural-MediCluser programof the Korean Government, Seoul City

Experimental Part

General Flash column chromatography (FC): silica gel 60 (70 – 230 mesh; Merck) TLC: precoated Kieselgel 60 F254plates (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 PhytoLibraryTMkit was purchased from PURIMED Co (Seoul, Korea) Optical rotations: Jasco P-1010 polarimeter at 258 1D- and 2D-NMR experiments: Bruker Avance-400 FT-2D-NMR instrument; with Me4Si as internal standard

HR-EI-MS and ESI-HR-EI-MS: Jeol JHR-EI-MS-700-M mass spectrometer and an API 3200-Q-TRAP LC/HR-EI-MS/HR-EI-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 Herbariumof 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 fromthe hexane layer by precipitation The soluble hexane extract (100 g) was fractionated by FC (1 kg SiO2; hexane/CHCl310 : 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 fromthe first separation Fr VII (6 g), eluted with hexane/CHCl39 : 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/CHCl31 : 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/CHCl31 : 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 ofNO 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,

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were plated in 24-well plates (5 105cells/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 fromLPS-treated or LPS-and-sample-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 NaNO3standard curve The percentage inhibition was expressed as [1 – (NO level of test samples/NO level of vehicle-treated 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 CHEMISTRY & BIODIVERSITY – Vol 5 (2008) 351