Journal of Ethnopharmacology 153 (2014) 737–743
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Lignans from the stems of Clematis armandii (“Chuan-Mu-Tong”) and their anti-neuroinflammatory activities Juan Xiong a, Van-Binh Bui c,d, Xin-Hua Liu b,n, Zhi-Lai Hong a, Guo-Xun Yang a, Jin-Feng Hu a,c,n a
Department of Natural Products Chemistry, Fudan University, Shanghai 201203, China Department of Pharmacology, School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, China c Department of Chemistry, School of Science & Engineering, No. 3663 Zhongshan Road N, East China Normal University, Shanghai 200062, China d Department of Chemistry, Hoa Lu University, No. 491C Xuanthanh Road, Ninh Binh 40000, Vietnam b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 October 2013 Received in revised form 13 February 2014 Accepted 15 March 2014 Available online 21 March 2014
Ethnopharmacological relevance: The dried stems of Clematis armandii (Caulis clematidis armandii), named “Chuan-Mu-Tong” in Chinese Pharmacopoeia, have been traditionally used as an herbal remedy mainly for inflammation-associated diseases. The Aim of the study is to identify the potential antineuroinflammatory components from Clematis armandii. Materials and methods: The ethanol extract of “Chuan-Mu-Tong” was suspended in H2O and exhaustively extracted with CH2Cl2. The CH2Cl2 fraction was successively subjected to column chromatography (CC) over silica gel, Sephadex LH-20, and semi-preparative HPLC. The structures of the isolated compounds were identified by spectroscopic methods and by comparison with those reported in the literature. Their anti-neuroinflammatory activities were evaluated by inhibitory effects on pro-inflammatory mediators [e.g. nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α)] in lipopolysaccharide (LPS)-activated BV-2 cells. Results: One new and sixteen known lignans were isolated and characterized. The absolute configuration of the new lignan, (7R,8S)-9-acetyl-dehydrodiconiferyl alcohol (1), was elucidated by a combination of 1D/2D NMR techniques and the Electronic Circular Dichroism (ECD) spectroscopy based on the empirical helicity rules. The anti-neuroinflammatory bioassay showed that compounds 1, (7R,8S)-dehydrodiconiferyl alcohol (2), erythro-guaiacylglycerol-β-coniferyl ether (5), and threo-guaiacylglycerol-β-coniferyl ether (6) displayed significant inhibitory effects on NO production. Among them, neolignans 1 and 2 exhibited more potent activities than the positive control (NG-monomethyl-L-arginine, L-NMMA), with an IC50 value of 9.3 and 3.9 μM, respectively. Moreover, both 1 and 2 were also found to concentrationdependently suppress the TNF-α release in LPS-stimulated BV-2 cells. Conclusion: The results revealed that lignans are the major components of “Chuan-Mu-Tong”, and their anti-neuroinflammatory activities strongly support the traditional application of this herb medicine on inflammation. Moreover, the dihydrobenzo[b]furan neolignans 1 and 2 as well as Caulis clematidis armandii could be further exploited as new therapeutic agents to treat inflammation-mediated neurodegenerative and aging-associated diseases. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Clamatis armandii Ranunculaceae Lignans Anti-neuroinflammation NO TNF-α
1. Introduction Neuroinflammation has proven to be implicated in the pathogenesis of neuronal damage in many neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) (Wyss-Coray and Mucke, 2002; Hirsch and Hunot, 2009). Microglial cells, the resident immune cells of the central nervous system,
n
Corresponding author. Tel./fax: þ 86 21 51980172. E-mail addresses: [email protected] (X.-H. Liu), [email protected], [email protected] (J.-F. Hu). http://dx.doi.org/10.1016/j.jep.2014.03.036 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
play a decisive role in immune surveillance and host defense under physiological conditions (Perry and Gordon, 1988). However, the unregulated activation of microglia with overproduction of nitric oxide (NO) and pro-inflammatory cytokines [e.g. Tumor necrosis factoralpha (TNF-α), interleukin (IL)-1β, and IL-6] has been considered to cause neuronal cell death and finally results in neurodegenerative diseases (Block and Hong, 2005). Therefore, inhibition of these inflammatory mediators produced by activated microglia emerges as a potential therapeutic approach for neurodegenerative diseases. Clematis, a large genus (ca. 300 species) in the family Ranunculaceae, is widely distributed in the Northern hemisphere and has been used extensively as traditional herb medicines
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around the world (Chawla et al., 2012). Clematis armandii Franch is a flowering climbing plant, which is often found in southwestern China, especially in Si-Chuan (Szechwan) Province. The dried stem of Clematis armandii (Caulis clematidis armandii) is listed as a popular herb medicine named “Chuan-Mu-Tong” in Chinese Pharmacopoeia Committee (2010). In fact, “Mu-Tong” is regarded as one of the medium-grade drugs in Shennong's Herbal. Its see-trough hole in both ends of the stem accounts for its name “Mu-Tong”, meaning “pierced wood”. Meanwhile, “Chuan” represents its growing area (Si-Chuan) of this plant. In China, “ChuanMu-Tong” has long been used for the treatment of inflammation conditions such as rheumatism and urinary tract infection (Chinese Pharmacopoeia Committee, 2010; Chawla et al., 2012). Previous phytochemical and pharmacological studies on Clematis armandii were quite limited, and only a few steroids (Huang et al., 2004; Yan et al., 2007) and lignan glycosides (Huang et al., 2003) have been reported. During our ongoing effort to search for new anti-neuroinflammatory and anti-aging agents (Wang et al., 2011; Tang et al., 2013), the 95% EtOH extract of the stems of the title plant was preliminarily found to decrease LPS-stimulated NO production in BV-2 cells (IC50 ¼ 129.7 μg/mL). Further studies showed that the CH2Cl2 fraction of the crude extract was responsible for the activity, with an IC50 value of 72.0 μg/mL. Therefore, the chemical components of the above CH2Cl2 fraction were investigated. The anti-neuroinflammatory activities of the isolated compounds (one new and 16 known ligans, Fig. 1) were evaluated for their inhibitory effects on LPS-induced NO and TNF-α production in BV-2 cells.
2. Material and methods 2.1. General procedures NMR spectra were taken on a Bruker Avance II 400 MHz spectrometer. Chemical shifts were expressed in δ (ppm), and referenced to the residual solvent signals. IR and ECD spectra were recorded on a Thermo Nicolet NEXUS-670 FT-IR spectrophotometer and a Jasco 810 spectrometer, respectively. Optical rotations were measured on a PerkinElmer 341 polarimeter. ESI–MS spectra were measured on a Waters UPLC H Class-SQD and an Agilent 1100 series mass spectrometer; HR-ESI–MS spectra were measured on a Bruker Daltonics micrOTOF-QII mass spectrometer. Semi-preparative HPLC was performed on a Waters e2695 system with a Waters 2998 Photodiode Array Detector (PAD) and a Waters 2424 Evaporative Light-scattering Detector (ELSD); a SunFire ODS column (5 μm, 250 10 mm) was utilized. Column chromatography (CC) was performed using silica gel (200–300 mesh, Ji-Yi-Da Silysia Chemical Ltd., Qingdao, China), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Silica gel-precoated plates (GF254, 0.25 mm, Kang-Bi-Nuo Silysia Chemical Ltd, Yantai, China) were used for TLC. Spots were visualized using UV light (254 and/or 366 nm) and by spraying with 5% (v/v) H2SO4–EtOH followed by heating to 120 1C.
2.2. Plant material The dried stems of Clematis armandii were purchased from Shanghai Jiu-Zhou-Tong Medicine Co. Ltd, and were originally collected in November 2009 from Sichuan Province of China. The plant was identified by Prof. Bao-Kang Huang (Department of Pharmacognosy, The second Military Medical University, Shanghai, China). A voucher specimen (No. 091125) was deposited at the Herbarium of the School of Pharmacy, Fudan University.
2.3. Extraction and isolation of compounds The dried stems of Clematis armandii (10 kg) were pulverized and extracted with 95% EtOH (20 L 3) at room temperature to give a brown crude extract (500 g), which was suspended in H2O (2 L) and then extracted with CH2Cl2 (1.5 L 3). After removal of the solvent under reduced pressure, the CH2Cl2 extract (200 g) was chromatographed over a silica gel column with a gradient elution of CH2Cl2–MeOH (90:1 to 0:1) to afford seven fractions (Fr. 1–Fr. 7). Fr. 2 (25.8 g) was subjected to silica gel CC with petroleum ether (PE)–acetone (5:1 to 1:1), yielding four subfractions (Fr. 2.1–Fr. 2.4). Purification of Fr. 2.4 by semi-preparative HPLC with MeOH– H2O (45:55, v/v; 3.0 mL/min) furnished compounds 7 (32.0 mg, tR ¼24.2 min) and 9 (8.1 mg, tR ¼32.9 min). Fr. 3 (21.4 g) was applied to a silica gel column, using PE–acetone (3:1 to 0:1) to give subfractions Fr. 3.1–Fr. 3.4. Compounds 14 (29.0 mg) and 15 (3.5 mg) were obtained from Fr. 3-2 and Fr. 3-3, respectively, by silica gel CC (CHCl3–MeOH, 80:1), and both were further purified by gel permeation chromatography (GPC) on Sephadex LH-20 with CH2Cl2–MeOH (v/v, 1:1). Fr. 4 (15.0 g) was subjected to silica gel CC with PE–acetone (3:1 to 0:1) to yield five subfractions (Fr. 4.1–Fr. 4.5). Fr. 4.1 was purified by silica gel CC (CHCl3–MeOH, 70:1) and Sephadex LH-20 (MeOH) to afford compound 8 (9.6 mg). Fr. 4.2 and Fr. 4.3 were purified by semi-preparative HPLC eluted with MeCN–H2O (65:35) at 3.0 mL/min to furnish compounds 1 (5.7 mg, tR ¼15.0 min) and 16 (20.0 mg, tR ¼13.9 min). Compounds 2 (24.0 mg) and 17 (36.0 mg) were isolated from Fr. 4.4 by repeated silica gel CC. Fr.5 (5.1 g) was eluted by a silica gel column with CHCl3–MeOH (70:1) to generate four subfractions, and each was further purified by semi-preparative HPLC (flow rate: 3.0 mL/min) to afford compounds 4 (MeOH–H2O 40:60; 15.0 mg, tR ¼22.4 min), 10 (MeOH–H2O 36:64; 3.1 mg, tR ¼17.7 min), 11 (MeOH–H2O 20:80; 3.2 mg, tR ¼ 34.7 min), and 13 (MeOH–H2O 30:70; 6.2 mg, tR ¼29.7 min). Fr.6 (10.8 g) was loaded on a silica gel column with a CHCl3–MeOH gradient (50:1–20:1) to give four subfractions (Fr. 6.1–Fr. 6.4). Compound 3 (98.0 mg) was obtained from Fr. 6.1 by CC over silica gel with CH2Cl2–EtOAc (3:1). Fr. 6.2 was applied to silica gel CC (CHCl3–MeOH 20:1), and then semi-preparative HPLC with MeOH–H2O (35:65, 3.0 mL/min) to afford compounds 5 (3.5 mg, tR ¼ 23.2 min) and 6 (7.5 mg, tR ¼20.5 min). Compound 12 (8.6 mg) was purified from Fr. 6.3 by silica gel CC with CHCl3–MeOH (40:1). 2.4. Physical and spectroscopic data of compounds (7R,8S)-9-Acetyl-dehydrodiconiferyl alcohol (1): yellow amorphous 4 powder, [α]22 M, MeOH) D 20.0 (c 0.06, CHCl3); ECD (c 5.25 10 λmax (Δε): 213 nm ( 3.14), 234 nm (þ 1.42), 286 nm (5.41); IR (KBr) νmax (cm 1): 3397, 2915, 2361, 1742, 1601, 1495, 1384, 1260, 1128 and 1035; 1H (400 MHz) and 13C NMR (100 MHz) spectrascopic data, see Table 1. ESIMS: m/z 423 [MþNa] þ , HR-ESIMS: m/z 423.1410 [MþNa] þ (calcd. for C22H24O7Na: 423.1414, Δ ¼1.1 ppm). ( 7)-Syringaresinol (8): yellow oil, [α]22 D 0 (c 0.12, CHCl3) [ref. optically inactive] (Nawwar et al., 1982). 1H NMR (400 MHz, CDCl3): δ 6.59 (4H, br s, H-2, H-6, H-20 , H-60 ), 5.59 (2H, s, 4-OH, 40 -OH), 4.74 (2H, d, J¼ 4.1 Hz, H-7, H-70 ), 4.29 (2H, dd, J ¼8.9, 6.7 Hz, Ha-9, Ha-90 ), 3.92 (2H, m, Hb-9, Hb-90 ), 3.89 (12H, s, OCH33,5,30 ,50 ), 3.10 (2H, m, H-8, H-80 ); ESIMS: m/z 441 [M þNa] þ . Erythro-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-methoxypropanol (10): white amorphous powder, [α]22 D þ 63.3 (c 0.092, CHCl3); IR (KBr) νmax (cm 1): 3442, 2916, 2360, 1602, 1457, 1384, 1271, 1153 and 1032; for 1H (500 MHz, CD3OD) and 13C NMR (125 MHz, CD3OD) spectroscopic data see lit (Huang et al., 2012); 1H NMR (400 MHz, CDCl3): δ 6.85 (2H, d, J¼8.0 Hz, H-5 and H-50 ), 6.69 (1H, d, J¼7.6, 2.0 Hz, H-60 ), 6.67 (1H, d, J¼7.6, 2.0 Hz, H-6), 6.58 (2H, d, J¼ 2.0 Hz, H-2, H-20 ), 5.60 (1H, s, 4-OH), 5.54 (1H, s, 40 -OH), 4.40 (1H, d, J¼ 6.5 Hz, H-7), 3.81 (3H, s, 3-OCH3), 3.77 (3H, s, 30 -OCH3), 3.76 (1H, dd, overlapped, Ha-9),
J. Xiong et al. / Journal of Ethnopharmacology 153 (2014) 737–743
739
Fig. 1. Chemical structures of lignans 1–17.
3.70 (1H, dd, J¼10.7, 4.3 Hz, Hb-9), 3.16 (3H, s, 7-OCH3), 3.08 (1H, q-like, J¼6.5 Hz, H-8); 13C NMR (100 MHz, CDCl3): δ 131.3 (C-1), 109.4 (C-2), 146.5 (C-3), 145.2 (C-4), 114.1 (C-5), 120.8 (C-6), 85.2 (C-7), 54.4 (C-8), 64.3 (C-9), 130.9 (C-10 ), 111.8 (C-20 ), 146.2 (C-30 ), 144.5 (C-40 ), 113.7 (C-50 ), 121.5 (C-60 ), 55.9 (3-OCH3, 30 -OCH3), 56.9 (7-OCH3); ESIMS: m/z 357 [MþNa] þ , HR-ESIMS: m/z 357.1310 [MþNa] þ (calcd. for C18H22O6Na, 357.1309, Δ ¼1.1 ppm). 2.5. Measurement of NO production and cell viability in LPSactivated BV-2 cells The mouse microglia BV-2 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA), and
maintained in Dulbecco's modified Eagle's medium containing 1800 mg/L NaHCO3, supplemented with 10% fetal bovine serum, 100 U/mL penicilin and 100 μg/mL streptomycin at 37 1C in a humidified atmosphere with 5% CO2. The anti-neuroinflammatory activity in BV-2 cells was evaluated according to the reported protocol with modification (Kim et al., 2013). NO production was quantified by nitrite accumulation in the culture medium using the Griess reaction kit (Beyotime Biotechnology, China) according to the manufacturer's instructions. Briefly, BV-2 cells were pretreated with different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 μM) of indicated compounds for 4 h, and then stimulated with or without lipopolysaccharide (LPS) (1 μg/mL, Sigma-Aldrich) for 24 h. The isolated supernatants were mixed with an equal volume of Griess
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Table 1 1 H (400 MHz) and
13
C (100 MHz) NMR dataa of compound 1.
δH (J values in Hz) No 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 OH 3-OCH3 30 -OCH3 OAc
a b c
δC
1b
1c
6.89 (1H, brs)
6.97 (1H, brs)
6.88 (1H, d, overlapped) 6.88 (1H, brd, overlapped) 5.47 (1H, d, 7.4) 3.78 (1H, ddd, 7.4, 7.4, 5.8) 4.44 (1H, dd, 11.2, 5.6) 4.32 (1H, dd, 11.2, 7.5)
6.81 (1H, d, 8.0) 6.86 (1H, dd, 8.0, 1.7) 5.51 (1H, d, 7.4) 3.78 (1H, ddd, 7.4, 7.4, 5.2) 4.46 (1H, dd, 11.1, 5.2) 4.34 (1H, dd, 11.1, 7.4)
6.89 (1H, brs)
6.99 (1H, brs)
6.88 6.56 6.24 4.32 5.65 3.87 3.91 2.03
brs) brd, 15.6) dt, 15.6, 5.8) brd, 5.8)
6.97 6.58 6.24 4.23
s) s) s)
3.86 (3H, s) 3.91(3H, s) 2.03 (3H, s)
(1H, (1H, (1H, (2H, (s) (3H, (3H, (3H,
1b
(1H, (1H, (1H, (2H,
brs) dt, 15.6, 1.4) dt, 15.6, 5.8) dd, 5.8, 1.4)
132.3 108.6 147.9 145.8 114.3 119.6 88.8 50.3 65.4 131.0 115.1 144.4 146.7 127.7 110.5 131.3 126.6 63.8 56.0 56.0 20.8 170.8
Assignments were made by a combination of 1D and 2D NMR experiments. Measured in CDCl3. Measured in CD3OD.
reagent. NaNO2 was used to generate a standard curve, and NO production was determined by measuring the optical density at 540 nm by a microplate reader (M200, TECAN, Austria GmbH, Austria). Cell viability was measured using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Wu et al., 2010; 2011). NG-monomethyl-L-arginine (L-NMMA, Beyotime, purity Z99%), a well-known NO synthase inhibitor, served as a positive control. The IC50 values were determined by GraphPad Prism 5.
2.6. Measurement of TNF-α level by enzyme-linked immunosorbent assay (ELISA) The levels of TNF-α in the culture medium were measured with a commercially available kit (R&D Systems Inc., MN, USA). Briefly, BV2 cells were pretreated with different concentrations (12.5, 25, and 50 μM) of indicated compounds for 4 h, and then stimulated with or without LPS (1 μg/mL) for 24 h. The culture medium (200 μL per well) was collected and centrifuged at 16,000 rpm for 5 min. Then, 100 μL of the supernatants were used for measuring the level of TNF-α by ELISA according to the manufacturer's instructions. Optical densities were read on a microplate reader at 450 nm. Results were presented with pg/mL.
3. Results 3.1. Compounds isolated from the title plant One new (1) and sixteen known (2–17) lignans (Fig. 1) were obtained from the CH2Cl2 fraction of the EtOH extract of the stems of Clematis armandii. By comparison of their spectroscopic data and physicochemical properties with those reported in the literature, the known compounds were identified to be (7R,8S)-dehydrodiconiferyl alcohol (2) (Yuen et al., 1998), (7R,8S,80 S)-3,30 -dimethoxy-7,
90 -epoxylignane-4,40 ,9-triol [3, ( )-lariciresinol] (Yamauchi et al., 2007), (2R,3R)-secoisolariciresinol (4) (Moon et al., 2008), erythroguaiacylglycerol-β-coniferyl ether (5) (Katayama and Kado, 1998), threo-guaiacylglycerol-β-coniferyl ether (6) (Katayama and Kado, 1998), (þ)-pinoresinol (7) (Xie et al., 2003; Yeo et al., 2004), (7)syringaresinol (8) (Nawwar et al., 1982), (þ )-epipinoresinol (9) (Rahman et al., 1990), erythro-2,3-bis(4-hydroxy-3-methoxy-phenyl)-3-methoxypropanol (10) (Huang et al., 2012), (þ)-erythro-1,2bis(4-hydroxy-3-methoxyphenyl)-1,3-propandiol (11) (Yoshikawa et al., 1998; Warashina et al., 2006), threo-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-methoxypropanol (12) (Hsiao and Chiang, 1995), threo1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propandiol (13) (Kishimoto et al., 2004; Warashina et al., 2006), boehmenan (14) (Seca et al., 2001), threo-carolignan H (15) (Seca et al., 2001), erythro-carolignan E (16) (Seca et al., 2001; Rudiyansyah and Garson 2010), and threocarolignan E (17) (Seca et al., 2001; Rudiyansyah and Garson 2010). Among the isolates, the dimeric lignans 14–17 were reported from the clematis genus for the first time. 3.2. Structure elucidation of the new compound Compound 1 was obtained as a yellow amorphous powder. Its HR-ESI–MS exhibited a psudo-molecular ion peak at m/z 423.1410, corresponding to the molecular formula C22H24O7 (calcd for C22H24O7Na, 423.1414). The IR spectrum showed absorption bands (ν max) at 3397 (–OH), 1742 (–CO), and 1601 (aromatic ring) cm 1. The 1H NMR spectrum (Table 1) of 1 displayed signals of five aromatic protons (δ 6.88, 5H), a trans double bond [δ 6.56 (1H, br d, J ¼15.6 Hz, H-7), 6.24 (1H, dt, J ¼15.6, 5.8 Hz, H-8)], two oxymethylene groups [δ 4.44 (1H, dd, J¼ 11.2, 5.6 Hz, H-9a), 4.32 (1H, dd, J ¼11.2, 7.5 Hz, H-9b); 4.32 (2H, br d, J ¼5.8 Hz, H2-90 )], two methoxy groups [δ 3.87 (3H, s, OMe-3) and 3.91 (3H, s, OMe-30 )], and an acetyl methyl at δ 2.03 (3H, s). In addition to the characteristic signals for two methoxy (δ 56.0, Ar-OMe 2) and one acetoxyl (δ 170.8, 20.8) substituents, the 13C NMR spectrum of 1 exhibited eighteen resonances attributed to the core structure, which included fourteen olefinic carbons (δ 108.6–147.9) due to two benzene rings and a double bond, two sp3 methines (δ 50.3, 88.8) and two sp3 oxygen-bearing methylenes (δ 63.8, 65.4). The above data suggested that compound 1 was a dihydrobenzo[b] furan neolignan (Valcic et al., 1998; Su et al., 2002) similar to dehydrodiconiferyl alcohol (2) (Yuen et al., 1998), except for the observation of an additional acetyl group in 1. The planar structure of 1 was verified by detailed 2D (COSY, HSQC and HMBC) NMR experiments (Fig. 2). The acetoxyl group was unambiguously positioned at C-9 according to the obvious 3J correlations between H2-9 (δ 4.32, 4.44) and the carbonyl carbon at δ 170.8 in the HMBC NMR spectrum. Moreover, clear HMBC correlations were observed from OMe-3 (δ 3.87) to C-3 (δ 147.9), from OMe-30 (δ 3.91) to C-30 (δ 144.4), and from the D2O-exchangeable hydroxy group at δ 5.65 (1H, s, OH-4) to C-2 (δ 108.6)/C-3/C-4 (δ 145.8). These observations unambiguously allowed the assignment of two methoxy groups at C-3 and C-30 , and a hydroxy group at C-4. The trans configuration of H-7 and H-8 was determined by the smaller coupling constant of J7,8 (7.4 Hz) (Li et al., 1997), which was further confirmed by the diagnostic NOE correlations between H-7 (δ 5.47) and H2-9 (δ 4.32, 4.44) (Fig. 2). The absolute configuration of 1 was then established to be 7R,8S by the observed negative Cotton effect at 286 nm in its ECD spectrum, which is assignable to the 1Lb band for the dihydrobenzo[b]furan chromtophore (Antus et al., 2001). This was also supported by the negative optical rotation value of 1 ([α]22 D ¼ 20.0 (c 0.06, CHCl3)), which was comparable with those of (7R,8S)-dehydrodiconiferyl alcohol [2, [α]23 D ¼ 11 (c 0.8, CHCl3)] (Yuen et al. 1998) and related analogues (Yuen et al. 1998; Xiong et al., 2011). Thus, compound 1 was identified to be (7R,8S)-9-acetyl-dehydrodiconiferyl alcohol.
J. Xiong et al. / Journal of Ethnopharmacology 153 (2014) 737–743
741
Fig. 2. Key COSY, HMBC and NOE correlations for compound 1.
Table 2 Inhibitory effects on NO production in LPS-stimulated BV-2 cells. Compound/biomass
IC50a,b (μM)
Cell viabilityb,c (%)
95% EtOH extract CH2Cl2 fraction 1 2 5 6 e L-NMMA
129.7 7 3.5d 72.0 7 2.4d 9.3 7 0.7 3.9 7 0.5 31.4 7 1.3 17.8 7 0.9 22.7 7 1.1
93.2 7 2.3 95.3 7 3.2 100.0 7 6.6 106.47 2.7 94.17 3.6 99.7 7 6.1 99.6 7 2.5
a IC50 value of each compound was defined as the concentration of indicated compound that caused 50% inhibition of NO production in LPS-stimulated BV-2 cells. b The results are averages of three independent experiments, and the data were expressed as mean 7 SD. c Cell viability after treatment with 50 μM of each compound (100 μg/mL for crude extracts) was expressed as a percentage (%) of untreated control cells. d Values in μg/mL. e L-NMMA: positive control.
anti-neuroinflammatory effects. Moreover, compound 1, the new lignan bearing an additional acetyl group related to compound 2, exhibited lower potency than 2, indicating that a free hydroxy group at C-9 is benefit for decreasing NO production. Additionally, the inhibitory effects on LPS-induced TNF-α production in BV-2 cells of the aforementioned active lignans (1, 2, 5, and 6) were also tested. As shown in Fig. 3, LPS evoked a significant increase in TNF-α release compared with the control cells. In agreement with their inhibitory effects on NO production, compounds 1 and 2 both suppressed TNF-α release in LPS-stimulated BV-2 cells in a dose-dependent manner (Fig. 3). These two compounds at a concentration of either 25 or 50 μM could attenuate the secretion of TNF-α when compared to the LPS-treated alone (Fig. 3). However, no such an effect on the level of TNF-α release was found for 5 or 6 (data not shown). Taken together, the above data suggested that neolignans 1 and 2 could suppress the inflammatory response stimulated by LPS in BV-2 cells.
4. Discussion and conclusions 3.3. Inhibitory effects on LPS-induced NO and TNF-α production in BV-2 cells Microglial activation and subsequent neuroinflammation-mediated neurotoxicity have been suggested as a potential therapeutic target to alleviate the progression of neurodegenerative diseases (Liu and Hong, 2003). Lipopolysaccharide (LPS), a major constituent of the Gramnegative bacterial cell wall, plays a pivotal role in the initiation of inflammation mediated by releasing inflammatory factors (e.g. NO, TNF-α), which are thought to be responsible for neuroglia-mediated neuroinflammation (Gao et al., 2003). Among various inflammatory substances, excessive accumulation of NO has been known to be toxic to neurons and NO is regarded as an important mediator for inflammation and neuronal cell death (Iadecola, 1997). Meanwhile, TNF-α, a key pro-inflammatory cytokine, plays a central role in inflammation-associated diseases (Bradley, 2008). The 95% EtOH extract and its non-polar fraction (CH2Cl2 portion) of the stems of the title plant were preliminarily found to decrease NO production in LPS-activated murine microglia BV-2 cells (Table 2). Seventeen lignans (1–17) were thereafter isolated from the CH2Cl2 fraction and were subsequently evaluated for their antineuroinflammatory effects. As shown in Table 2, compounds 1, 2, 5 and 6 could significantly decrease NO production in BV-2 cells without influence on cell viability at concentrations up to 50 μM, while others were inactive (IC50 4200 μM). Among them, the dihydrobenzo[b] furan neolignans 1 and 2 exerted the most potent activities on the inhibition of NO release with IC50 values of 9.3 and 3.9 μM, respectively. The significant potencies of 1, 2 and 6 (IC50 ¼17.8 μM) each was found to be much higher than that of the positive control, NGmonomethyl-L-arginine (L-NMMA, IC50 ¼22.7 μM). Interestingly, the active lignans 1, 2, 5, and 6 all possess a 3-phenylprop-2-en-1-ol moiety, which might be a necessary unit contributed to their potent
Lignans are widely distributed in vascular plants and possess various pharmacological activities including anti-inflammation (Saleem et al., 2005). A number of lignans have been previously reported to attenuate the microglia-mediated neuroinflammatory responses by inhibiting the overproduction of inflammatory factors. For example, schisandrin B (a dibenzocyclooctadiene lignan) isolated from the fruits of Schisandra chinesnesis, was found to significantly down-regulate some pro-inflammatory substances including NO and TNF-α (Zeng et al., 2012). Macelignan, a dibenzylbutane lignan isolated from Myristica fragrans, suppressed the production of NO and TNF-α triggered by LPS in BV-2 cells. Interestingly, this lignan could even cross the hematoencephalic barrier [CLuptake (mL/min ¼0.79)] (Jin et al., 2005). Therefore, the potent anti-neuroinflammatory activity of naturally occurring lignans and their possible ability to cross blood–brain barrier would make them promising neuroprotective agents for treatment of neurodegenerative diseases. In the present study, one (1) new and sixteen (2–17) known lignans were successfully isolated and characterized from the bioactive fraction (CH2Cl2 portion). Among them, the dihydrobenzo[b] furan neolignans 1 and 2 showed significant anti-neuroinflammatory activities through suppression of NO and TNF-α production in activated microglial cells. The above findings would stimulate further exploration of these lignans and Caulis clematidis armandii itself for their therapeutic potential in the treatment of inflammationmediated neurodegenerative and aging-related diseases.
Acknowledgments This work was supported by NSFC grants (Nos. 81273401, 81202420), a STCSM grant (No. 11DZ1921203), grants from the
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Fig. 3. Inhibitory effects of compounds 1 and 2 on TNF-α release in LPS-stimulated BV-2 cells. Data are the mean 7SEM of results from at least three independent experiments. #p o 0.01 vs. unstimulated control; *p o0.05 vs. LPS-stimulated control.
Ph.D. Programs Foundation of Ministry of Education (MOE) of China (Nos. 20120071110049, 20120071120049), a MOST grant (No. 2011ZX09307-002-01), and the National Basic Research Program of China (973 Program, Grant no. 2013CB530700).
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Lignans from the stems of Clematis armandii (“Chuan-Mu-Tong”) and their anti-neuroinflammatory activities Juan Xiong a, Van-Binh Bui c,d, Xin-Hua Liu b,n, Zhi-Lai Hong a, Guo-Xun Yang a, Jin-Feng Hu a,c,n a
Department of Natural Products Chemistry, Fudan University, Shanghai 201203, China Department of Pharmacology, School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, China c Department of Chemistry, School of Science & Engineering, No. 3663 Zhongshan Road N, East China Normal University, Shanghai 200062, China d Department of Chemistry, Hoa Lu University, No. 491C Xuanthanh Road, Ninh Binh 40000, Vietnam b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 October 2013 Received in revised form 13 February 2014 Accepted 15 March 2014 Available online 21 March 2014
Ethnopharmacological relevance: The dried stems of Clematis armandii (Caulis clematidis armandii), named “Chuan-Mu-Tong” in Chinese Pharmacopoeia, have been traditionally used as an herbal remedy mainly for inflammation-associated diseases. The Aim of the study is to identify the potential antineuroinflammatory components from Clematis armandii. Materials and methods: The ethanol extract of “Chuan-Mu-Tong” was suspended in H2O and exhaustively extracted with CH2Cl2. The CH2Cl2 fraction was successively subjected to column chromatography (CC) over silica gel, Sephadex LH-20, and semi-preparative HPLC. The structures of the isolated compounds were identified by spectroscopic methods and by comparison with those reported in the literature. Their anti-neuroinflammatory activities were evaluated by inhibitory effects on pro-inflammatory mediators [e.g. nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α)] in lipopolysaccharide (LPS)-activated BV-2 cells. Results: One new and sixteen known lignans were isolated and characterized. The absolute configuration of the new lignan, (7R,8S)-9-acetyl-dehydrodiconiferyl alcohol (1), was elucidated by a combination of 1D/2D NMR techniques and the Electronic Circular Dichroism (ECD) spectroscopy based on the empirical helicity rules. The anti-neuroinflammatory bioassay showed that compounds 1, (7R,8S)-dehydrodiconiferyl alcohol (2), erythro-guaiacylglycerol-β-coniferyl ether (5), and threo-guaiacylglycerol-β-coniferyl ether (6) displayed significant inhibitory effects on NO production. Among them, neolignans 1 and 2 exhibited more potent activities than the positive control (NG-monomethyl-L-arginine, L-NMMA), with an IC50 value of 9.3 and 3.9 μM, respectively. Moreover, both 1 and 2 were also found to concentrationdependently suppress the TNF-α release in LPS-stimulated BV-2 cells. Conclusion: The results revealed that lignans are the major components of “Chuan-Mu-Tong”, and their anti-neuroinflammatory activities strongly support the traditional application of this herb medicine on inflammation. Moreover, the dihydrobenzo[b]furan neolignans 1 and 2 as well as Caulis clematidis armandii could be further exploited as new therapeutic agents to treat inflammation-mediated neurodegenerative and aging-associated diseases. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Clamatis armandii Ranunculaceae Lignans Anti-neuroinflammation NO TNF-α
1. Introduction Neuroinflammation has proven to be implicated in the pathogenesis of neuronal damage in many neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) (Wyss-Coray and Mucke, 2002; Hirsch and Hunot, 2009). Microglial cells, the resident immune cells of the central nervous system,
n
Corresponding author. Tel./fax: þ 86 21 51980172. E-mail addresses: [email protected] (X.-H. Liu), [email protected], [email protected] (J.-F. Hu). http://dx.doi.org/10.1016/j.jep.2014.03.036 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
play a decisive role in immune surveillance and host defense under physiological conditions (Perry and Gordon, 1988). However, the unregulated activation of microglia with overproduction of nitric oxide (NO) and pro-inflammatory cytokines [e.g. Tumor necrosis factoralpha (TNF-α), interleukin (IL)-1β, and IL-6] has been considered to cause neuronal cell death and finally results in neurodegenerative diseases (Block and Hong, 2005). Therefore, inhibition of these inflammatory mediators produced by activated microglia emerges as a potential therapeutic approach for neurodegenerative diseases. Clematis, a large genus (ca. 300 species) in the family Ranunculaceae, is widely distributed in the Northern hemisphere and has been used extensively as traditional herb medicines
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around the world (Chawla et al., 2012). Clematis armandii Franch is a flowering climbing plant, which is often found in southwestern China, especially in Si-Chuan (Szechwan) Province. The dried stem of Clematis armandii (Caulis clematidis armandii) is listed as a popular herb medicine named “Chuan-Mu-Tong” in Chinese Pharmacopoeia Committee (2010). In fact, “Mu-Tong” is regarded as one of the medium-grade drugs in Shennong's Herbal. Its see-trough hole in both ends of the stem accounts for its name “Mu-Tong”, meaning “pierced wood”. Meanwhile, “Chuan” represents its growing area (Si-Chuan) of this plant. In China, “ChuanMu-Tong” has long been used for the treatment of inflammation conditions such as rheumatism and urinary tract infection (Chinese Pharmacopoeia Committee, 2010; Chawla et al., 2012). Previous phytochemical and pharmacological studies on Clematis armandii were quite limited, and only a few steroids (Huang et al., 2004; Yan et al., 2007) and lignan glycosides (Huang et al., 2003) have been reported. During our ongoing effort to search for new anti-neuroinflammatory and anti-aging agents (Wang et al., 2011; Tang et al., 2013), the 95% EtOH extract of the stems of the title plant was preliminarily found to decrease LPS-stimulated NO production in BV-2 cells (IC50 ¼ 129.7 μg/mL). Further studies showed that the CH2Cl2 fraction of the crude extract was responsible for the activity, with an IC50 value of 72.0 μg/mL. Therefore, the chemical components of the above CH2Cl2 fraction were investigated. The anti-neuroinflammatory activities of the isolated compounds (one new and 16 known ligans, Fig. 1) were evaluated for their inhibitory effects on LPS-induced NO and TNF-α production in BV-2 cells.
2. Material and methods 2.1. General procedures NMR spectra were taken on a Bruker Avance II 400 MHz spectrometer. Chemical shifts were expressed in δ (ppm), and referenced to the residual solvent signals. IR and ECD spectra were recorded on a Thermo Nicolet NEXUS-670 FT-IR spectrophotometer and a Jasco 810 spectrometer, respectively. Optical rotations were measured on a PerkinElmer 341 polarimeter. ESI–MS spectra were measured on a Waters UPLC H Class-SQD and an Agilent 1100 series mass spectrometer; HR-ESI–MS spectra were measured on a Bruker Daltonics micrOTOF-QII mass spectrometer. Semi-preparative HPLC was performed on a Waters e2695 system with a Waters 2998 Photodiode Array Detector (PAD) and a Waters 2424 Evaporative Light-scattering Detector (ELSD); a SunFire ODS column (5 μm, 250 10 mm) was utilized. Column chromatography (CC) was performed using silica gel (200–300 mesh, Ji-Yi-Da Silysia Chemical Ltd., Qingdao, China), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Silica gel-precoated plates (GF254, 0.25 mm, Kang-Bi-Nuo Silysia Chemical Ltd, Yantai, China) were used for TLC. Spots were visualized using UV light (254 and/or 366 nm) and by spraying with 5% (v/v) H2SO4–EtOH followed by heating to 120 1C.
2.2. Plant material The dried stems of Clematis armandii were purchased from Shanghai Jiu-Zhou-Tong Medicine Co. Ltd, and were originally collected in November 2009 from Sichuan Province of China. The plant was identified by Prof. Bao-Kang Huang (Department of Pharmacognosy, The second Military Medical University, Shanghai, China). A voucher specimen (No. 091125) was deposited at the Herbarium of the School of Pharmacy, Fudan University.
2.3. Extraction and isolation of compounds The dried stems of Clematis armandii (10 kg) were pulverized and extracted with 95% EtOH (20 L 3) at room temperature to give a brown crude extract (500 g), which was suspended in H2O (2 L) and then extracted with CH2Cl2 (1.5 L 3). After removal of the solvent under reduced pressure, the CH2Cl2 extract (200 g) was chromatographed over a silica gel column with a gradient elution of CH2Cl2–MeOH (90:1 to 0:1) to afford seven fractions (Fr. 1–Fr. 7). Fr. 2 (25.8 g) was subjected to silica gel CC with petroleum ether (PE)–acetone (5:1 to 1:1), yielding four subfractions (Fr. 2.1–Fr. 2.4). Purification of Fr. 2.4 by semi-preparative HPLC with MeOH– H2O (45:55, v/v; 3.0 mL/min) furnished compounds 7 (32.0 mg, tR ¼24.2 min) and 9 (8.1 mg, tR ¼32.9 min). Fr. 3 (21.4 g) was applied to a silica gel column, using PE–acetone (3:1 to 0:1) to give subfractions Fr. 3.1–Fr. 3.4. Compounds 14 (29.0 mg) and 15 (3.5 mg) were obtained from Fr. 3-2 and Fr. 3-3, respectively, by silica gel CC (CHCl3–MeOH, 80:1), and both were further purified by gel permeation chromatography (GPC) on Sephadex LH-20 with CH2Cl2–MeOH (v/v, 1:1). Fr. 4 (15.0 g) was subjected to silica gel CC with PE–acetone (3:1 to 0:1) to yield five subfractions (Fr. 4.1–Fr. 4.5). Fr. 4.1 was purified by silica gel CC (CHCl3–MeOH, 70:1) and Sephadex LH-20 (MeOH) to afford compound 8 (9.6 mg). Fr. 4.2 and Fr. 4.3 were purified by semi-preparative HPLC eluted with MeCN–H2O (65:35) at 3.0 mL/min to furnish compounds 1 (5.7 mg, tR ¼15.0 min) and 16 (20.0 mg, tR ¼13.9 min). Compounds 2 (24.0 mg) and 17 (36.0 mg) were isolated from Fr. 4.4 by repeated silica gel CC. Fr.5 (5.1 g) was eluted by a silica gel column with CHCl3–MeOH (70:1) to generate four subfractions, and each was further purified by semi-preparative HPLC (flow rate: 3.0 mL/min) to afford compounds 4 (MeOH–H2O 40:60; 15.0 mg, tR ¼22.4 min), 10 (MeOH–H2O 36:64; 3.1 mg, tR ¼17.7 min), 11 (MeOH–H2O 20:80; 3.2 mg, tR ¼ 34.7 min), and 13 (MeOH–H2O 30:70; 6.2 mg, tR ¼29.7 min). Fr.6 (10.8 g) was loaded on a silica gel column with a CHCl3–MeOH gradient (50:1–20:1) to give four subfractions (Fr. 6.1–Fr. 6.4). Compound 3 (98.0 mg) was obtained from Fr. 6.1 by CC over silica gel with CH2Cl2–EtOAc (3:1). Fr. 6.2 was applied to silica gel CC (CHCl3–MeOH 20:1), and then semi-preparative HPLC with MeOH–H2O (35:65, 3.0 mL/min) to afford compounds 5 (3.5 mg, tR ¼ 23.2 min) and 6 (7.5 mg, tR ¼20.5 min). Compound 12 (8.6 mg) was purified from Fr. 6.3 by silica gel CC with CHCl3–MeOH (40:1). 2.4. Physical and spectroscopic data of compounds (7R,8S)-9-Acetyl-dehydrodiconiferyl alcohol (1): yellow amorphous 4 powder, [α]22 M, MeOH) D 20.0 (c 0.06, CHCl3); ECD (c 5.25 10 λmax (Δε): 213 nm ( 3.14), 234 nm (þ 1.42), 286 nm (5.41); IR (KBr) νmax (cm 1): 3397, 2915, 2361, 1742, 1601, 1495, 1384, 1260, 1128 and 1035; 1H (400 MHz) and 13C NMR (100 MHz) spectrascopic data, see Table 1. ESIMS: m/z 423 [MþNa] þ , HR-ESIMS: m/z 423.1410 [MþNa] þ (calcd. for C22H24O7Na: 423.1414, Δ ¼1.1 ppm). ( 7)-Syringaresinol (8): yellow oil, [α]22 D 0 (c 0.12, CHCl3) [ref. optically inactive] (Nawwar et al., 1982). 1H NMR (400 MHz, CDCl3): δ 6.59 (4H, br s, H-2, H-6, H-20 , H-60 ), 5.59 (2H, s, 4-OH, 40 -OH), 4.74 (2H, d, J¼ 4.1 Hz, H-7, H-70 ), 4.29 (2H, dd, J ¼8.9, 6.7 Hz, Ha-9, Ha-90 ), 3.92 (2H, m, Hb-9, Hb-90 ), 3.89 (12H, s, OCH33,5,30 ,50 ), 3.10 (2H, m, H-8, H-80 ); ESIMS: m/z 441 [M þNa] þ . Erythro-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-methoxypropanol (10): white amorphous powder, [α]22 D þ 63.3 (c 0.092, CHCl3); IR (KBr) νmax (cm 1): 3442, 2916, 2360, 1602, 1457, 1384, 1271, 1153 and 1032; for 1H (500 MHz, CD3OD) and 13C NMR (125 MHz, CD3OD) spectroscopic data see lit (Huang et al., 2012); 1H NMR (400 MHz, CDCl3): δ 6.85 (2H, d, J¼8.0 Hz, H-5 and H-50 ), 6.69 (1H, d, J¼7.6, 2.0 Hz, H-60 ), 6.67 (1H, d, J¼7.6, 2.0 Hz, H-6), 6.58 (2H, d, J¼ 2.0 Hz, H-2, H-20 ), 5.60 (1H, s, 4-OH), 5.54 (1H, s, 40 -OH), 4.40 (1H, d, J¼ 6.5 Hz, H-7), 3.81 (3H, s, 3-OCH3), 3.77 (3H, s, 30 -OCH3), 3.76 (1H, dd, overlapped, Ha-9),
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Fig. 1. Chemical structures of lignans 1–17.
3.70 (1H, dd, J¼10.7, 4.3 Hz, Hb-9), 3.16 (3H, s, 7-OCH3), 3.08 (1H, q-like, J¼6.5 Hz, H-8); 13C NMR (100 MHz, CDCl3): δ 131.3 (C-1), 109.4 (C-2), 146.5 (C-3), 145.2 (C-4), 114.1 (C-5), 120.8 (C-6), 85.2 (C-7), 54.4 (C-8), 64.3 (C-9), 130.9 (C-10 ), 111.8 (C-20 ), 146.2 (C-30 ), 144.5 (C-40 ), 113.7 (C-50 ), 121.5 (C-60 ), 55.9 (3-OCH3, 30 -OCH3), 56.9 (7-OCH3); ESIMS: m/z 357 [MþNa] þ , HR-ESIMS: m/z 357.1310 [MþNa] þ (calcd. for C18H22O6Na, 357.1309, Δ ¼1.1 ppm). 2.5. Measurement of NO production and cell viability in LPSactivated BV-2 cells The mouse microglia BV-2 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA), and
maintained in Dulbecco's modified Eagle's medium containing 1800 mg/L NaHCO3, supplemented with 10% fetal bovine serum, 100 U/mL penicilin and 100 μg/mL streptomycin at 37 1C in a humidified atmosphere with 5% CO2. The anti-neuroinflammatory activity in BV-2 cells was evaluated according to the reported protocol with modification (Kim et al., 2013). NO production was quantified by nitrite accumulation in the culture medium using the Griess reaction kit (Beyotime Biotechnology, China) according to the manufacturer's instructions. Briefly, BV-2 cells were pretreated with different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 μM) of indicated compounds for 4 h, and then stimulated with or without lipopolysaccharide (LPS) (1 μg/mL, Sigma-Aldrich) for 24 h. The isolated supernatants were mixed with an equal volume of Griess
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Table 1 1 H (400 MHz) and
13
C (100 MHz) NMR dataa of compound 1.
δH (J values in Hz) No 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 OH 3-OCH3 30 -OCH3 OAc
a b c
δC
1b
1c
6.89 (1H, brs)
6.97 (1H, brs)
6.88 (1H, d, overlapped) 6.88 (1H, brd, overlapped) 5.47 (1H, d, 7.4) 3.78 (1H, ddd, 7.4, 7.4, 5.8) 4.44 (1H, dd, 11.2, 5.6) 4.32 (1H, dd, 11.2, 7.5)
6.81 (1H, d, 8.0) 6.86 (1H, dd, 8.0, 1.7) 5.51 (1H, d, 7.4) 3.78 (1H, ddd, 7.4, 7.4, 5.2) 4.46 (1H, dd, 11.1, 5.2) 4.34 (1H, dd, 11.1, 7.4)
6.89 (1H, brs)
6.99 (1H, brs)
6.88 6.56 6.24 4.32 5.65 3.87 3.91 2.03
brs) brd, 15.6) dt, 15.6, 5.8) brd, 5.8)
6.97 6.58 6.24 4.23
s) s) s)
3.86 (3H, s) 3.91(3H, s) 2.03 (3H, s)
(1H, (1H, (1H, (2H, (s) (3H, (3H, (3H,
1b
(1H, (1H, (1H, (2H,
brs) dt, 15.6, 1.4) dt, 15.6, 5.8) dd, 5.8, 1.4)
132.3 108.6 147.9 145.8 114.3 119.6 88.8 50.3 65.4 131.0 115.1 144.4 146.7 127.7 110.5 131.3 126.6 63.8 56.0 56.0 20.8 170.8
Assignments were made by a combination of 1D and 2D NMR experiments. Measured in CDCl3. Measured in CD3OD.
reagent. NaNO2 was used to generate a standard curve, and NO production was determined by measuring the optical density at 540 nm by a microplate reader (M200, TECAN, Austria GmbH, Austria). Cell viability was measured using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Wu et al., 2010; 2011). NG-monomethyl-L-arginine (L-NMMA, Beyotime, purity Z99%), a well-known NO synthase inhibitor, served as a positive control. The IC50 values were determined by GraphPad Prism 5.
2.6. Measurement of TNF-α level by enzyme-linked immunosorbent assay (ELISA) The levels of TNF-α in the culture medium were measured with a commercially available kit (R&D Systems Inc., MN, USA). Briefly, BV2 cells were pretreated with different concentrations (12.5, 25, and 50 μM) of indicated compounds for 4 h, and then stimulated with or without LPS (1 μg/mL) for 24 h. The culture medium (200 μL per well) was collected and centrifuged at 16,000 rpm for 5 min. Then, 100 μL of the supernatants were used for measuring the level of TNF-α by ELISA according to the manufacturer's instructions. Optical densities were read on a microplate reader at 450 nm. Results were presented with pg/mL.
3. Results 3.1. Compounds isolated from the title plant One new (1) and sixteen known (2–17) lignans (Fig. 1) were obtained from the CH2Cl2 fraction of the EtOH extract of the stems of Clematis armandii. By comparison of their spectroscopic data and physicochemical properties with those reported in the literature, the known compounds were identified to be (7R,8S)-dehydrodiconiferyl alcohol (2) (Yuen et al., 1998), (7R,8S,80 S)-3,30 -dimethoxy-7,
90 -epoxylignane-4,40 ,9-triol [3, ( )-lariciresinol] (Yamauchi et al., 2007), (2R,3R)-secoisolariciresinol (4) (Moon et al., 2008), erythroguaiacylglycerol-β-coniferyl ether (5) (Katayama and Kado, 1998), threo-guaiacylglycerol-β-coniferyl ether (6) (Katayama and Kado, 1998), (þ)-pinoresinol (7) (Xie et al., 2003; Yeo et al., 2004), (7)syringaresinol (8) (Nawwar et al., 1982), (þ )-epipinoresinol (9) (Rahman et al., 1990), erythro-2,3-bis(4-hydroxy-3-methoxy-phenyl)-3-methoxypropanol (10) (Huang et al., 2012), (þ)-erythro-1,2bis(4-hydroxy-3-methoxyphenyl)-1,3-propandiol (11) (Yoshikawa et al., 1998; Warashina et al., 2006), threo-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-methoxypropanol (12) (Hsiao and Chiang, 1995), threo1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propandiol (13) (Kishimoto et al., 2004; Warashina et al., 2006), boehmenan (14) (Seca et al., 2001), threo-carolignan H (15) (Seca et al., 2001), erythro-carolignan E (16) (Seca et al., 2001; Rudiyansyah and Garson 2010), and threocarolignan E (17) (Seca et al., 2001; Rudiyansyah and Garson 2010). Among the isolates, the dimeric lignans 14–17 were reported from the clematis genus for the first time. 3.2. Structure elucidation of the new compound Compound 1 was obtained as a yellow amorphous powder. Its HR-ESI–MS exhibited a psudo-molecular ion peak at m/z 423.1410, corresponding to the molecular formula C22H24O7 (calcd for C22H24O7Na, 423.1414). The IR spectrum showed absorption bands (ν max) at 3397 (–OH), 1742 (–CO), and 1601 (aromatic ring) cm 1. The 1H NMR spectrum (Table 1) of 1 displayed signals of five aromatic protons (δ 6.88, 5H), a trans double bond [δ 6.56 (1H, br d, J ¼15.6 Hz, H-7), 6.24 (1H, dt, J ¼15.6, 5.8 Hz, H-8)], two oxymethylene groups [δ 4.44 (1H, dd, J¼ 11.2, 5.6 Hz, H-9a), 4.32 (1H, dd, J ¼11.2, 7.5 Hz, H-9b); 4.32 (2H, br d, J ¼5.8 Hz, H2-90 )], two methoxy groups [δ 3.87 (3H, s, OMe-3) and 3.91 (3H, s, OMe-30 )], and an acetyl methyl at δ 2.03 (3H, s). In addition to the characteristic signals for two methoxy (δ 56.0, Ar-OMe 2) and one acetoxyl (δ 170.8, 20.8) substituents, the 13C NMR spectrum of 1 exhibited eighteen resonances attributed to the core structure, which included fourteen olefinic carbons (δ 108.6–147.9) due to two benzene rings and a double bond, two sp3 methines (δ 50.3, 88.8) and two sp3 oxygen-bearing methylenes (δ 63.8, 65.4). The above data suggested that compound 1 was a dihydrobenzo[b] furan neolignan (Valcic et al., 1998; Su et al., 2002) similar to dehydrodiconiferyl alcohol (2) (Yuen et al., 1998), except for the observation of an additional acetyl group in 1. The planar structure of 1 was verified by detailed 2D (COSY, HSQC and HMBC) NMR experiments (Fig. 2). The acetoxyl group was unambiguously positioned at C-9 according to the obvious 3J correlations between H2-9 (δ 4.32, 4.44) and the carbonyl carbon at δ 170.8 in the HMBC NMR spectrum. Moreover, clear HMBC correlations were observed from OMe-3 (δ 3.87) to C-3 (δ 147.9), from OMe-30 (δ 3.91) to C-30 (δ 144.4), and from the D2O-exchangeable hydroxy group at δ 5.65 (1H, s, OH-4) to C-2 (δ 108.6)/C-3/C-4 (δ 145.8). These observations unambiguously allowed the assignment of two methoxy groups at C-3 and C-30 , and a hydroxy group at C-4. The trans configuration of H-7 and H-8 was determined by the smaller coupling constant of J7,8 (7.4 Hz) (Li et al., 1997), which was further confirmed by the diagnostic NOE correlations between H-7 (δ 5.47) and H2-9 (δ 4.32, 4.44) (Fig. 2). The absolute configuration of 1 was then established to be 7R,8S by the observed negative Cotton effect at 286 nm in its ECD spectrum, which is assignable to the 1Lb band for the dihydrobenzo[b]furan chromtophore (Antus et al., 2001). This was also supported by the negative optical rotation value of 1 ([α]22 D ¼ 20.0 (c 0.06, CHCl3)), which was comparable with those of (7R,8S)-dehydrodiconiferyl alcohol [2, [α]23 D ¼ 11 (c 0.8, CHCl3)] (Yuen et al. 1998) and related analogues (Yuen et al. 1998; Xiong et al., 2011). Thus, compound 1 was identified to be (7R,8S)-9-acetyl-dehydrodiconiferyl alcohol.
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Fig. 2. Key COSY, HMBC and NOE correlations for compound 1.
Table 2 Inhibitory effects on NO production in LPS-stimulated BV-2 cells. Compound/biomass
IC50a,b (μM)
Cell viabilityb,c (%)
95% EtOH extract CH2Cl2 fraction 1 2 5 6 e L-NMMA
129.7 7 3.5d 72.0 7 2.4d 9.3 7 0.7 3.9 7 0.5 31.4 7 1.3 17.8 7 0.9 22.7 7 1.1
93.2 7 2.3 95.3 7 3.2 100.0 7 6.6 106.47 2.7 94.17 3.6 99.7 7 6.1 99.6 7 2.5
a IC50 value of each compound was defined as the concentration of indicated compound that caused 50% inhibition of NO production in LPS-stimulated BV-2 cells. b The results are averages of three independent experiments, and the data were expressed as mean 7 SD. c Cell viability after treatment with 50 μM of each compound (100 μg/mL for crude extracts) was expressed as a percentage (%) of untreated control cells. d Values in μg/mL. e L-NMMA: positive control.
anti-neuroinflammatory effects. Moreover, compound 1, the new lignan bearing an additional acetyl group related to compound 2, exhibited lower potency than 2, indicating that a free hydroxy group at C-9 is benefit for decreasing NO production. Additionally, the inhibitory effects on LPS-induced TNF-α production in BV-2 cells of the aforementioned active lignans (1, 2, 5, and 6) were also tested. As shown in Fig. 3, LPS evoked a significant increase in TNF-α release compared with the control cells. In agreement with their inhibitory effects on NO production, compounds 1 and 2 both suppressed TNF-α release in LPS-stimulated BV-2 cells in a dose-dependent manner (Fig. 3). These two compounds at a concentration of either 25 or 50 μM could attenuate the secretion of TNF-α when compared to the LPS-treated alone (Fig. 3). However, no such an effect on the level of TNF-α release was found for 5 or 6 (data not shown). Taken together, the above data suggested that neolignans 1 and 2 could suppress the inflammatory response stimulated by LPS in BV-2 cells.
4. Discussion and conclusions 3.3. Inhibitory effects on LPS-induced NO and TNF-α production in BV-2 cells Microglial activation and subsequent neuroinflammation-mediated neurotoxicity have been suggested as a potential therapeutic target to alleviate the progression of neurodegenerative diseases (Liu and Hong, 2003). Lipopolysaccharide (LPS), a major constituent of the Gramnegative bacterial cell wall, plays a pivotal role in the initiation of inflammation mediated by releasing inflammatory factors (e.g. NO, TNF-α), which are thought to be responsible for neuroglia-mediated neuroinflammation (Gao et al., 2003). Among various inflammatory substances, excessive accumulation of NO has been known to be toxic to neurons and NO is regarded as an important mediator for inflammation and neuronal cell death (Iadecola, 1997). Meanwhile, TNF-α, a key pro-inflammatory cytokine, plays a central role in inflammation-associated diseases (Bradley, 2008). The 95% EtOH extract and its non-polar fraction (CH2Cl2 portion) of the stems of the title plant were preliminarily found to decrease NO production in LPS-activated murine microglia BV-2 cells (Table 2). Seventeen lignans (1–17) were thereafter isolated from the CH2Cl2 fraction and were subsequently evaluated for their antineuroinflammatory effects. As shown in Table 2, compounds 1, 2, 5 and 6 could significantly decrease NO production in BV-2 cells without influence on cell viability at concentrations up to 50 μM, while others were inactive (IC50 4200 μM). Among them, the dihydrobenzo[b] furan neolignans 1 and 2 exerted the most potent activities on the inhibition of NO release with IC50 values of 9.3 and 3.9 μM, respectively. The significant potencies of 1, 2 and 6 (IC50 ¼17.8 μM) each was found to be much higher than that of the positive control, NGmonomethyl-L-arginine (L-NMMA, IC50 ¼22.7 μM). Interestingly, the active lignans 1, 2, 5, and 6 all possess a 3-phenylprop-2-en-1-ol moiety, which might be a necessary unit contributed to their potent
Lignans are widely distributed in vascular plants and possess various pharmacological activities including anti-inflammation (Saleem et al., 2005). A number of lignans have been previously reported to attenuate the microglia-mediated neuroinflammatory responses by inhibiting the overproduction of inflammatory factors. For example, schisandrin B (a dibenzocyclooctadiene lignan) isolated from the fruits of Schisandra chinesnesis, was found to significantly down-regulate some pro-inflammatory substances including NO and TNF-α (Zeng et al., 2012). Macelignan, a dibenzylbutane lignan isolated from Myristica fragrans, suppressed the production of NO and TNF-α triggered by LPS in BV-2 cells. Interestingly, this lignan could even cross the hematoencephalic barrier [CLuptake (mL/min ¼0.79)] (Jin et al., 2005). Therefore, the potent anti-neuroinflammatory activity of naturally occurring lignans and their possible ability to cross blood–brain barrier would make them promising neuroprotective agents for treatment of neurodegenerative diseases. In the present study, one (1) new and sixteen (2–17) known lignans were successfully isolated and characterized from the bioactive fraction (CH2Cl2 portion). Among them, the dihydrobenzo[b] furan neolignans 1 and 2 showed significant anti-neuroinflammatory activities through suppression of NO and TNF-α production in activated microglial cells. The above findings would stimulate further exploration of these lignans and Caulis clematidis armandii itself for their therapeutic potential in the treatment of inflammationmediated neurodegenerative and aging-related diseases.
Acknowledgments This work was supported by NSFC grants (Nos. 81273401, 81202420), a STCSM grant (No. 11DZ1921203), grants from the
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Fig. 3. Inhibitory effects of compounds 1 and 2 on TNF-α release in LPS-stimulated BV-2 cells. Data are the mean 7SEM of results from at least three independent experiments. #p o 0.01 vs. unstimulated control; *p o0.05 vs. LPS-stimulated control.
Ph.D. Programs Foundation of Ministry of Education (MOE) of China (Nos. 20120071110049, 20120071120049), a MOST grant (No. 2011ZX09307-002-01), and the National Basic Research Program of China (973 Program, Grant no. 2013CB530700).
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