Original Papers
671
Authors
Di Hu 1, 2, Na Han 1, Xuechun Yao 3, Zhihui Liu 1, Yu Wang 4, Jingyu Yang 3, Jun Yin 1
Affiliations
1
2 3 4
Key words " Schisandra chinensis l " Schisandraceae l " dibenzocyclooctadiene l lignans " microglia l " nitric oxide l " structure‑activity l relationship
Development and Utilization Key Laboratory of Northeast Plant Materials of Liaoning Province, Department of Pharmacognosy, Shenyang Pharmaceutical University, Shenyang, China College of Pharmacy, Harbin University of Commerce, Harbin, Heilongjiang Province, China Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China The Chinese Peopleʼs Liberation Army 463 Hospital, Shenyang, China
Abstract
Abbreviations
!
!
To explore the relationship of the dibenzocyclooctadiene lignans from Schisandra chinensis to their anti-inflammatory activities, series of dibenzocyclooctadiene lignans were isolated and assessed by testing their inhibitory effects on nitric oxide production in lipopolysaccharide-induced BV2 mouse microglia. It was found, for the first time, that dibenzocyclooctadiene lignans which have S-biphenyl and methylenedioxy groups strongly inhibited LPS-induced microglia activation. The methoxy group on the cyclooctadiene introduced more effectiveness, but the presence of an acetyl group on the cyclooctadiene or hydroxyl group on C-7 decreased the inhibitory activity.
Aβ: AD: CNS: IMDM: L-NAME: LPS: NO: PD: TNF-α:
Introduction
nificantly inhibited LPS-induced activation of microglia and NO production [8]. Therefore, in the present work, we investigated the inhibitory effects of the lignans of the fruits of S. chinensis on NO release in LPS-activated microglial cells, in order to discover their structure-activity relationships to anti-inflammatory effect.
!
received revised accepted
January 22, 2014 May 7, 2014 May 20, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368592 Planta Med 2014; 80: 671–675 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Jun Yin Department of Pharmacognosy, College of Chinese Herbs 48# Shenyang Pharmaceutical University 103 Wenhua Road Shenyang 110016 China Phone: + 86 24 23 98 64 91 Fax: + 86 24 23 98 64 60 [email protected]
Microglia is the main resident monocyte cell in CNS [1] and is quiescent in the normal brain. However, it can be activated by cytokines produced by infiltrating immune effector cells after CNS injury or by LPS during bacterial infection [2, 3]. Activated microglia produce inflammatory mediators including NO, reactive oxygen species, and cytokines, such as TNF-α [4], which contribute to the pathogenesis of several neurodegenerative diseases such as AD and PD [3]. Therefore, decreasing inflammatory damage by inhibiting the microglial activation is a crucial strategy to prevent the further deterioration in the neurodegenerative diseases. The fruits of Schisandra chinensis (Turcz.) Baill (Schisandraceae) have long been used in traditional Chinese medicines as a tonic, antitussive, and sedative herbal medicine. Its main bioactive components are dibenzocyclooctadiene lignans which improve memory performance [5–7]. In our previous research, we found that the main lignan component of these fruits, schizandrin, sig-
amyloid-beta Alzheimerʼs disease central nervous system Iscoveʼs modified Dulbeccoʼs medium L-nitro-arginine methyl ester lipopolysaccharide nitric oxide Parkinsonʼs disease tumor necrosis factor-α
Supporting information available online at http://www.thieme-connect.de/products
Results and Discussion !
In order to clarify the lignans of S. chinensis and the structure-activity relationships underlying their inhibitory effects on LPS-induced NO production in BV2 microglia cells, the relevant pure lignans were extracted by separation and purification. Twenty-five dibenzocyclooctadiene lignans were obtained. By the comparison of their spectral data with those of the references, they were identified as tigloylgomisin P (1) [9], (−)-tigloyl-deangeloyl-gomisin F (2) [10], gomisin F (3) [11], gomisin B (4), gomisin G (5) [12], deoxyschizandrin (6) [13], (±)-γ-schizandrin (7) [14], gomisin A (8), schizandrin (9) [12], (−)-gomisin M1
Hu D et al. Structure-Activity Relationship Study …
Planta Med 2014; 80: 671–675
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Structure-Activity Relationship Study of Dibenzocyclooctadiene Lignans Isolated from Schisandra chinensis on LipopolysaccharideInduced Microglia Activation
Original Papers
(10), (−)-gomisin L1 (11), (+)-gomisin M2 (12) [14], (+)-gomisin k3 (13) [15], schisantherin D (14) [16], gomisin N (15) [17], schizandrin C (16) [16], (±)-gomisin M1 (17) [14], gomisin J (18) [18], kadsulignan N (19) [19], methylgomisin O (20) [20], methylisogomisin O (21) [21], 7(18)-dehydroschisandro A (22) [22], rubschisantherin (23) [23], gomisin D (24) [24], and gomisin E (25) [18]. To avoid any false-positive results deriving from the reduced viability of cells, the cytotoxicity of the lignan compounds on BV2 microglial cells in the presence or absence of LPS (1 µg/mL) was examined by MTT assays. Results showed that co-treatment of non-stimulated or stimulated microglial cells with all the isolated compounds did not affect the cell viability at the tested concentrations (data not shown). Their inhibitory effects at different concentrations on NO release in LPS-stimulated BV2 microglia cells were evaluated by the Griess reaction. Ten compounds showed moderate to strong inhibitory effects on LPS-induced NO release in BV2 microglia cells, with IC50 values ranging from 1.91 to 72.05 µM, when compared with the positive control L-NAME (IC50 value 35.71 µM). Treatment of non-stimulated cells with L-NAME or isolated compounds (0.1–100 µM) for 48 h did not cause any change of NO release (data not shown). In general, compounds 3, 11, 14, 15, 20, " Fig. 1). 21, 23, and 25 had the most promising activity (l Comparison of the inhibitory effects on LPS-induced microglia activation of compounds 3, 8, 11, 14, 15, 19, 20, 21, 23, and 25 indicated that most of the active compounds belong to the S-biphenyl dibenzocyclooctadiene lignans, while the lignans with Rbiphenyl conformation are only 16.7 %. Similar structure-activity relationships had already been reported for lignans with S-biphenyl configuration, such as gomisin J, gomisin N, and schisandrin C, which showed more potent inhibitory activity than those with R-biphenyl configuration (such as schisandrol A, schisandrol B, tigloylgomisin H, angeloylgomisin H, schisandrin A, and γ-schisandrin) on NO release in Raw 264.7 cells [25]. Recently, there were many researches on the neuroprotective effect of schisandrin B both in vitro and in vivo [26–29]. However, schisandrin B in the fruit of S. chinensis is a mixture composed of (±)-γ-schizandrin and gomisin N [30, 31]. The present study showed that gomisin N (IC50 = 19.44 µM) is the effective constituent of schisandrin B on the inhibition of LPS-induced microglia activation. Whether a comparable result would be obtained in vivo should be further investigated. In the previous study, the methylenedioxy group was considered to be important for dibenzocyclooctadiene lignans to display their neuroprotective effects against Aβ toxicity [32]. In our study, the structure-activity relationship on anti-inflammatory activity was consistent with the previous researches. The results revealed that: (1) Dibenzocyclooctadiene lignans with methylenedioxy groups (for example, compound 8) exhibited inhibitory effects on LPS-induced microglia activation, while no effects were observed on lignans without those groups (compound 9). (2) Compound 3, with one methylenedioxy group, exhibited more potent activity than 14 with two methylenedioxy groups. A similar result could also been found when comparing compounds 15 to 16, but this result was in disagreement with a previous research [32]. (3) The compounds with the methylenedioxy group at C-2 and C-3 were more active than those that showed this group at C-12 and C-13, as seen in the comparison between compounds 11 and 10, and compounds 21 and 20. The present study also revealed the importance and influence of the substituent group on the cyclooctadiene for the inhibition on LPS-induced microglia activation. Compared with compound 15,
Hu D et al. Structure-Activity Relationship Study …
Planta Med 2014; 80: 671–675
compound 20, with a methoxy group, displayed a more potent effect, however, the presence of an acetyl group at this position decreased the inhibitory activity on NO release (compound 23). In addition, compound 25 exhibited inhibition on LPS-induced microglia activation, but no effect was observed for 24, may be due to the presence of a hydroxyl group on C-7. In conclusion, the present work provided the structure-activity relationships of dibenzocyclooctadiene lignans of S. chinensis on the inhibition of LPS-induced NO release from microglial cells. The results might be helpful for future synthetic and pharmacological studies in order to obtain therapeutic drugs for the treatment of neurodegenerative diseases accompanied with microglial activation.
Materials and Methods !
Plant material The fruits of S. chinensis were collected in September 2009 in Liaoning province in China and identified by Professor Qishi Sun (Department of Chinese medicine, Shenyang Pharmaceutical University, Shenyang, China). A voucher specimen (SPU 1201) was deposited in the Pharmacognosy Department of the Shenyang Pharmaceutical University.
General experimental procedures A Perkin-Elmer 241MC polarimeter was used to record the optical rotations. HRESIMS were measured on a Bruker microTOF mass spectrometer. IR spectra were performed on a Bruker IFS55 infrared spectrometer. UV spectra were obtained using a UV2201 Shimadzu UV‑vis scanning spectrophotometer. CD spectra were taken in MeOH on a Jasco J-810 spectropolarimeter. 1H NMR (300 MHz and 600 MHz), 13C NMR (75 MHz), and 2D NMR were recorded on Bruker-ARX‑300 and Bruker-ARX‑600 spectrometers with TMS as an internal standard. Preparative TLC was conducted on plates (20 × 20 cm, 1 mm thick) coated with silica gel GF254 (Qingdao Marine Chemical Co., Ltd.). Column silica gel (100–200 or 200–300 mesh; Qingdao Marine Chemical Co., Ltd.) and Sephadex LH-20 gel (GE Healthcare) were used for column chromatography. Analytical HPLC was carried out on a Shimadzu LC10-A HPLC system with an LC-10ATVP pump, a Shimadzu LC-10AVP UV‑VIS detector, and an N-2000 chromatographic work station (Intelligent Information Engineering Co., Ltd.) using a YMC-Pack ODS‑A column (5 µm, 250 × 6.0 mm). Preparative HPLC was carried out on a YMC-Pack ODS‑A column (5 µm, 250 × 30 mm, flow rate 12 mL/min, UV detection at 254 nm) equipped with a pump. All chemical reagents were purchased from Yuwang Chemicals Industries, Ltd.
Biological materials IMDM and FBS were purchased from Gibco BRL; LPS (E5: 055), MTT, and positive control L-NAME (purity > 98%) were from Sigma-Aldrich Co. The compounds with purity greater than 95 % identified by HPLC were initially dissolved in DMSO and then diluted with PBS for experiments. DMSO at the highest concentration possibly under the experimental conditions (0.1%) was not toxic to cells.
Microglial cell culture BV2 mouse microglia cell line was a kind gift from Dr. Young Choong Kim (Seoul National University). The cells were grown in IMDM supplemented with 10 % FBS and 50 µM 2-mercaptoe-
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
672
Original Papers
673
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Fig. 1 Effects of lignan compounds on nitric oxide production induced by lipopolysaccharide in BV2 microglial cells. BV2 microglial cells were treated with serial dilutions of compounds in the presence of LPS (1 µg/mL) and then incubated for 48 h. NO production was measured by Griess reagent, and viability was detected by MTT assay. L-NAME was used as a positive control.
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Original Papers
thanol and incubated at 37 °C in a humidified atmosphere (5 % CO2). Cells at density of 3 × 104 cells/well were plated onto 96-well microtiter plates for MTT and nitrite assay. Compounds with or without LPS (1 µg/mL) were added to the culture medium of BV2 microglia cells for 24 h for the experiments. The doses of all the tested compounds were chosen according to our preliminary study as those which would not influence the viability of BV2 cells.
Cytotoxicity assay Cell viability was evaluated in microglial cells by the MTT reduction assay [33]. In brief, cells were seeded onto 96-well microtiter plates and treated with various test sample solutions with or without LPS (1 µg/mL) for 48 h. After treatments, medium was removed and the cells were incubated with MTT (0.25 mg/mL) at 37 °C for 3 h. The formazan crystals in the cells were solubilized with the solution containing 50% DMF and 20% sodium dodecyl sulfate (pH 4.7). The absorbance of formazan was assayed by a Spectra (shell) reader (Tecan) at a wavelength of 490 nm.
Nitrite assays Microglial cultures were placed into IMDM medium and incubated with CoCl2 as indicated. Nitrite concentration (as a measure of NO production) in the supernatant was determined by the Griess reaction as described previously [34]. Cells (5 × 104 cells/well) were seeded onto 96-well microtiter plates and treated with various test sample solutions in the presence or absence of LPS (1 µg/mL) for 48 h. 50 µL culture supernatants were mixed with 50 µL Griess reagent (part I: 1 % sulfanilamide; part II: 0.1 % naphthylethylene diamide dihydrochloride and 2 % phosphoric acid) at room temperature. Fifteen minutes later, the absorbance was determined at 540 nm using Spectra (shell) reader. Nitrite concentration was calculated according to a standard curve of sodium nitrite generated by known concentrations.
Data analysis Results were expressed as mean ± SEM with triplicate samples. Statistical significance (p < 0.05) was assessed by one-way ANOVA followed by Dunnettʼs t-test (SPSS 13.0 software).
Supporting information The detailed compound isolation procedure is available as Supporting Information.
Acknowledgments !
This work was financially supported by a grant from the Natural Science Foundation of China (81202891).
Conflict of Interest !
There is no conflict of interest. The authors alone are responsible for the content and writing of the paper.
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References 1 Kreutzberg GW. Microglia: a sensor for pathological events in the CNS. Trends Neurosci 1996; 19: 312–318 2 Gonzalez-Scarano F, Baltuch G. Microglia as mediators of inflammatory and degenerative diseases. Annu Rev Neurosci 1999; 22: 219–240 3 Stoll G, Jander S. The role of microglia and macrophages in the pathophysiology of the CNS. Prog Neurobiol 1999; 58: 233–247 4 Amor S, Puentes F, Baker D, van der Valk P. Inflammation in neurodegenerative diseases. Immunology 2010; 129: 154–169 5 Egashira N, Kurauchi K, Iwasaki K, Mishima K, Orito K, Oishi R, Fujiwara M. Schizandrin reverses memory impairment in rats. Phytother Res 2008; 22: 49–52 6 Kim DH, Jung WY, Park SJ, Kim JM, Lee S, Kim YC, Ryu JH. Anti-amnesic effect of ESP‑102 on Aβ1–42-induced memory impairment in mice. Pharmacol Biochem Behav 2010; 97: 239–248 7 Hu D, Li CX, Han N, Miao LJ, Wang D, Liu ZH, Wang H, Yin J. Deoxyschizandrin isolated from the fruits of Schisandra chinensis ameliorates Aβ1–42-induced memory impairment in mice. Planta Med 2012; 78: 1332–1336 8 Zhang N, Hu D, Yang JY, Cai XD, Yin J, Wu CF. Studies on chemical constituents of leaf of Schisandra chinensis and inhibitory effect of schizandrin on activation of microglia induced by LPS. Chin J Med Chem 2010; 20: 110–115 9 Chen YG, Qin GW, Xie YY. [Studies on chemical constituents of Schisandra propinqua (Wall.) Hook. f. et Thoms]. Zhongguo Zhong Yao Za Zhi 2001; 26: 694–699 10 Smejkal K, Slapetová T, Krmenčík P, Babula P, DallʼAcqua S, Innocenti G, Vančo J, Casarin E, Carrara M, Kalvarová K, Dvorská M, Slanina J, Kramářová E, Julínek O, Urbanová M. Evaluation of cytotoxic activity of Schisandra chinensis lignans. Planta Med 2010; 76: 1672–1677 11 Taguchi H, Ikeya Y. The constituents of Schizandra chinensis Baill. The structures of two new lignans, gomisins F, G, and the absolute structures of gomisins A, B, and C. Chem Pharm Bull 1977; 25: 364–366 12 Ikeya Y, Taguchi H, Yosioka I, Kobayashi H. The constituents of Schisandra chinensis Baill. I. Isolation and structure determination of five new lignans, gomisin A, B, C, F and G, and the absolute structure of schisandrin. Chem Pharm Bull 1979; 27: 1383–1394 13 Ikeya Y, Taguchi H, Sasaki H, Nakajima K, Yosioka I. The constituents of Schisandra chinensis Baill. VI. C‑13 nuclear magnetic resonance spectroscopy of dibenzocyclooctadiene lignans. Chem Pharm Bull 1980; 28: 2414–2421 14 Ikeya Y, Taguchi H, Yosioka I. The constituents of Schizandra chinensis Baill. X. The structure of γ-schisandrin and four new lignans, (−)-gomisin L1 and L2, (±)-gomisin M1 and (+)-gomisin M2. Chem Pharm Bull 1982; 30: 132–139 15 Ikeya Y, Taguchi H, Yosioka I. The constituents of Schizandra chinensis Baill. VII. The structures of three new lignans, (−)-gomisin K1 and (+)-gomisins K2 and K3. Chem Pharm Bull 1980; 28: 2422–2427 16 Ikeya Y, Taguchi H, Yosioka I. The constituents of Schisandra chinensis Baill. XII. Isolation and structure of a new lignan, gomisin R, the absolute structure of wuweizisu C and isolation of schisantherin D. Chem Pharm Bull 1982; 30: 3207–3211 17 Ikeya Y, Taguchi H, Yosioka I. The constituents of Schizandra chinensis Baill. V. The structures of four new lignans, gomisin N and gomisin O, epigomisin O and gomisin E, and transformation of gomisin N to deangeloylgomisin B. Chem Pharm Bull 1979; 27: 2659–2709 18 Yukinobu I, Heihachiro T, Itiro Y, Hiroshi K. The constituents of Schisandra chinensis Baill. IV. The structures of two new lignans, pre-gomisin and gomisin J. Chem Pharm Bull 1979; 27: 1583–1588 19 Chen YG, Qin GW, Xie YY, Cheng KF, Lin ZW, Sun HD, Kang YH, Han BH. Lignans from Kadsura angustifolia. J Asian Nat Prod Res 1998; 1: 125– 131 20 Ren R, Ci XX, Li HZ, Li HM, Luo GJ, Li RT, Deng XM. New dibenzocyclooctadiene lignans from Schisandra sphenanthera and their proinflammatory cytokine inhibitory activities. Z Naturforsch B 2010; 65: 211–218 21 Chun L, Huang HX, Chen JJ, Pu JX, Yang LB, Zhao Y, Liu JP, Gao XM, Xiao WL, Sun HD. Lignans from Schisandra propinqua var. propinqua. Chem Pharm Bull 2007; 55: 1281–1283 22 Ikeya Y, Sugama K, Okada M, Mitsuhashi H. Two lignans from Schisandra sphenanthera. Phytochemistry 1991; 30: 975–980 23 Wang HJ, Chen YY. [Studies of lignans from Schisandra rubriflora Rhed et Wils]. Yao Xue Xue Bao 1985; 20: 832–841 24 Ikeya Y, Taguchi H, Yosioka I, Iitaka Y, Kobayashi H. The constituents of Schizandra chinensis Baill. II. The structure of a new lignan, gomisin D. Chem Pharm Bull 1979; 27: 1395–1401
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30 Nakajima K, Taguch, H, Ikeya Y, Endo T, Yosioka I. [The constituents of Schizandra chinensis Baill. XIII. Quantitative analysis of lignans in the fruits of Schizandra chinensis Baill. by high performance liquid chromatography]. Yakugaku Zasshi 1983; 103: 743–749 31 Ka FL, Kam MK, Ka MN. Separation and purification of schisandrin B from Fructus Schisandrae. Ind Eng Chem Res 2008; 47: 4193–4201 32 Song JX, Lin X, Wong RN, Sze SC, Tong Y, Shaw PC, Zhang YB. Protective effects of dibenzocyclooctadiene lignans from Schisandra chinensis against beta-amyloid and homocysteine neurotoxicity in PC12 cells. Phytother Res 2011; 25: 435–443 33 Chang JY, Chavis JA, Liu LZ, Drew PD. Cholesterol oxides induce programmed cell death in microglial cells. Biochem Biophys Res Commun 1998; 249: 817–821 34 Barger SW, Harmon AD. Microglial activation by Alzheimer amyloid precursor protein and modulation by apolipoprotein E. Nature 1997; 388: 878–881
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25 Oh SY, Kim YH, Bae DS, Um BH, Pan CH, Kim CY, Lee HJ, Lee JK. Anti-inflammatory effects of gomisin N, gomisin J, and schisandrin C isolated from the fruit of Schisandra chinensis. Biosci Biotechnol Biochem 2010; 74: 285–291 26 Pan SY, Han YF, Carlier PR, Pang YP, Mak DH, Lam BY, Ko KM. Schisandrin B protects against tacrine- and bis(7)-tacrine-induced hepatotoxicity and enhances cognitive function in mice. Planta Med 2002; 68: 217– 220 27 Wang B, Wang XM. Schisandrin B protects rat cortical neurons against Aβ1–42-induced neurotoxicity. Pharmazie 2009; 64: 450–454 28 Giridharan VV, Thandavarayan RA, Sato S, Ko KM, Konishi T. Prevention of scopolamine-induced memory deficits by schisandrin B, an antioxidant lignan from Schisandra chinensis in mice. Free Radic Res 2011; 45: 950–958 29 Zeng KW, Zhang T, Fu H, Liu GX, Wang XM. Schisandrin B exerts antineuroinflammatory activity by inhibiting the Toll-like receptor 4-dependent MyD88/IKK/NF-κB signaling pathway in lipopolysaccharideinduced microglia. Eur J Pharmacol 2012; 692: 29–37
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Hu D et al. Structure-Activity Relationship Study …
Planta Med 2014; 80: 671–675
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671
Authors
Di Hu 1, 2, Na Han 1, Xuechun Yao 3, Zhihui Liu 1, Yu Wang 4, Jingyu Yang 3, Jun Yin 1
Affiliations
1
2 3 4
Key words " Schisandra chinensis l " Schisandraceae l " dibenzocyclooctadiene l lignans " microglia l " nitric oxide l " structure‑activity l relationship
Development and Utilization Key Laboratory of Northeast Plant Materials of Liaoning Province, Department of Pharmacognosy, Shenyang Pharmaceutical University, Shenyang, China College of Pharmacy, Harbin University of Commerce, Harbin, Heilongjiang Province, China Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China The Chinese Peopleʼs Liberation Army 463 Hospital, Shenyang, China
Abstract
Abbreviations
!
!
To explore the relationship of the dibenzocyclooctadiene lignans from Schisandra chinensis to their anti-inflammatory activities, series of dibenzocyclooctadiene lignans were isolated and assessed by testing their inhibitory effects on nitric oxide production in lipopolysaccharide-induced BV2 mouse microglia. It was found, for the first time, that dibenzocyclooctadiene lignans which have S-biphenyl and methylenedioxy groups strongly inhibited LPS-induced microglia activation. The methoxy group on the cyclooctadiene introduced more effectiveness, but the presence of an acetyl group on the cyclooctadiene or hydroxyl group on C-7 decreased the inhibitory activity.
Aβ: AD: CNS: IMDM: L-NAME: LPS: NO: PD: TNF-α:
Introduction
nificantly inhibited LPS-induced activation of microglia and NO production [8]. Therefore, in the present work, we investigated the inhibitory effects of the lignans of the fruits of S. chinensis on NO release in LPS-activated microglial cells, in order to discover their structure-activity relationships to anti-inflammatory effect.
!
received revised accepted
January 22, 2014 May 7, 2014 May 20, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368592 Planta Med 2014; 80: 671–675 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Jun Yin Department of Pharmacognosy, College of Chinese Herbs 48# Shenyang Pharmaceutical University 103 Wenhua Road Shenyang 110016 China Phone: + 86 24 23 98 64 91 Fax: + 86 24 23 98 64 60 [email protected]
Microglia is the main resident monocyte cell in CNS [1] and is quiescent in the normal brain. However, it can be activated by cytokines produced by infiltrating immune effector cells after CNS injury or by LPS during bacterial infection [2, 3]. Activated microglia produce inflammatory mediators including NO, reactive oxygen species, and cytokines, such as TNF-α [4], which contribute to the pathogenesis of several neurodegenerative diseases such as AD and PD [3]. Therefore, decreasing inflammatory damage by inhibiting the microglial activation is a crucial strategy to prevent the further deterioration in the neurodegenerative diseases. The fruits of Schisandra chinensis (Turcz.) Baill (Schisandraceae) have long been used in traditional Chinese medicines as a tonic, antitussive, and sedative herbal medicine. Its main bioactive components are dibenzocyclooctadiene lignans which improve memory performance [5–7]. In our previous research, we found that the main lignan component of these fruits, schizandrin, sig-
amyloid-beta Alzheimerʼs disease central nervous system Iscoveʼs modified Dulbeccoʼs medium L-nitro-arginine methyl ester lipopolysaccharide nitric oxide Parkinsonʼs disease tumor necrosis factor-α
Supporting information available online at http://www.thieme-connect.de/products
Results and Discussion !
In order to clarify the lignans of S. chinensis and the structure-activity relationships underlying their inhibitory effects on LPS-induced NO production in BV2 microglia cells, the relevant pure lignans were extracted by separation and purification. Twenty-five dibenzocyclooctadiene lignans were obtained. By the comparison of their spectral data with those of the references, they were identified as tigloylgomisin P (1) [9], (−)-tigloyl-deangeloyl-gomisin F (2) [10], gomisin F (3) [11], gomisin B (4), gomisin G (5) [12], deoxyschizandrin (6) [13], (±)-γ-schizandrin (7) [14], gomisin A (8), schizandrin (9) [12], (−)-gomisin M1
Hu D et al. Structure-Activity Relationship Study …
Planta Med 2014; 80: 671–675
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Structure-Activity Relationship Study of Dibenzocyclooctadiene Lignans Isolated from Schisandra chinensis on LipopolysaccharideInduced Microglia Activation
Original Papers
(10), (−)-gomisin L1 (11), (+)-gomisin M2 (12) [14], (+)-gomisin k3 (13) [15], schisantherin D (14) [16], gomisin N (15) [17], schizandrin C (16) [16], (±)-gomisin M1 (17) [14], gomisin J (18) [18], kadsulignan N (19) [19], methylgomisin O (20) [20], methylisogomisin O (21) [21], 7(18)-dehydroschisandro A (22) [22], rubschisantherin (23) [23], gomisin D (24) [24], and gomisin E (25) [18]. To avoid any false-positive results deriving from the reduced viability of cells, the cytotoxicity of the lignan compounds on BV2 microglial cells in the presence or absence of LPS (1 µg/mL) was examined by MTT assays. Results showed that co-treatment of non-stimulated or stimulated microglial cells with all the isolated compounds did not affect the cell viability at the tested concentrations (data not shown). Their inhibitory effects at different concentrations on NO release in LPS-stimulated BV2 microglia cells were evaluated by the Griess reaction. Ten compounds showed moderate to strong inhibitory effects on LPS-induced NO release in BV2 microglia cells, with IC50 values ranging from 1.91 to 72.05 µM, when compared with the positive control L-NAME (IC50 value 35.71 µM). Treatment of non-stimulated cells with L-NAME or isolated compounds (0.1–100 µM) for 48 h did not cause any change of NO release (data not shown). In general, compounds 3, 11, 14, 15, 20, " Fig. 1). 21, 23, and 25 had the most promising activity (l Comparison of the inhibitory effects on LPS-induced microglia activation of compounds 3, 8, 11, 14, 15, 19, 20, 21, 23, and 25 indicated that most of the active compounds belong to the S-biphenyl dibenzocyclooctadiene lignans, while the lignans with Rbiphenyl conformation are only 16.7 %. Similar structure-activity relationships had already been reported for lignans with S-biphenyl configuration, such as gomisin J, gomisin N, and schisandrin C, which showed more potent inhibitory activity than those with R-biphenyl configuration (such as schisandrol A, schisandrol B, tigloylgomisin H, angeloylgomisin H, schisandrin A, and γ-schisandrin) on NO release in Raw 264.7 cells [25]. Recently, there were many researches on the neuroprotective effect of schisandrin B both in vitro and in vivo [26–29]. However, schisandrin B in the fruit of S. chinensis is a mixture composed of (±)-γ-schizandrin and gomisin N [30, 31]. The present study showed that gomisin N (IC50 = 19.44 µM) is the effective constituent of schisandrin B on the inhibition of LPS-induced microglia activation. Whether a comparable result would be obtained in vivo should be further investigated. In the previous study, the methylenedioxy group was considered to be important for dibenzocyclooctadiene lignans to display their neuroprotective effects against Aβ toxicity [32]. In our study, the structure-activity relationship on anti-inflammatory activity was consistent with the previous researches. The results revealed that: (1) Dibenzocyclooctadiene lignans with methylenedioxy groups (for example, compound 8) exhibited inhibitory effects on LPS-induced microglia activation, while no effects were observed on lignans without those groups (compound 9). (2) Compound 3, with one methylenedioxy group, exhibited more potent activity than 14 with two methylenedioxy groups. A similar result could also been found when comparing compounds 15 to 16, but this result was in disagreement with a previous research [32]. (3) The compounds with the methylenedioxy group at C-2 and C-3 were more active than those that showed this group at C-12 and C-13, as seen in the comparison between compounds 11 and 10, and compounds 21 and 20. The present study also revealed the importance and influence of the substituent group on the cyclooctadiene for the inhibition on LPS-induced microglia activation. Compared with compound 15,
Hu D et al. Structure-Activity Relationship Study …
Planta Med 2014; 80: 671–675
compound 20, with a methoxy group, displayed a more potent effect, however, the presence of an acetyl group at this position decreased the inhibitory activity on NO release (compound 23). In addition, compound 25 exhibited inhibition on LPS-induced microglia activation, but no effect was observed for 24, may be due to the presence of a hydroxyl group on C-7. In conclusion, the present work provided the structure-activity relationships of dibenzocyclooctadiene lignans of S. chinensis on the inhibition of LPS-induced NO release from microglial cells. The results might be helpful for future synthetic and pharmacological studies in order to obtain therapeutic drugs for the treatment of neurodegenerative diseases accompanied with microglial activation.
Materials and Methods !
Plant material The fruits of S. chinensis were collected in September 2009 in Liaoning province in China and identified by Professor Qishi Sun (Department of Chinese medicine, Shenyang Pharmaceutical University, Shenyang, China). A voucher specimen (SPU 1201) was deposited in the Pharmacognosy Department of the Shenyang Pharmaceutical University.
General experimental procedures A Perkin-Elmer 241MC polarimeter was used to record the optical rotations. HRESIMS were measured on a Bruker microTOF mass spectrometer. IR spectra were performed on a Bruker IFS55 infrared spectrometer. UV spectra were obtained using a UV2201 Shimadzu UV‑vis scanning spectrophotometer. CD spectra were taken in MeOH on a Jasco J-810 spectropolarimeter. 1H NMR (300 MHz and 600 MHz), 13C NMR (75 MHz), and 2D NMR were recorded on Bruker-ARX‑300 and Bruker-ARX‑600 spectrometers with TMS as an internal standard. Preparative TLC was conducted on plates (20 × 20 cm, 1 mm thick) coated with silica gel GF254 (Qingdao Marine Chemical Co., Ltd.). Column silica gel (100–200 or 200–300 mesh; Qingdao Marine Chemical Co., Ltd.) and Sephadex LH-20 gel (GE Healthcare) were used for column chromatography. Analytical HPLC was carried out on a Shimadzu LC10-A HPLC system with an LC-10ATVP pump, a Shimadzu LC-10AVP UV‑VIS detector, and an N-2000 chromatographic work station (Intelligent Information Engineering Co., Ltd.) using a YMC-Pack ODS‑A column (5 µm, 250 × 6.0 mm). Preparative HPLC was carried out on a YMC-Pack ODS‑A column (5 µm, 250 × 30 mm, flow rate 12 mL/min, UV detection at 254 nm) equipped with a pump. All chemical reagents were purchased from Yuwang Chemicals Industries, Ltd.
Biological materials IMDM and FBS were purchased from Gibco BRL; LPS (E5: 055), MTT, and positive control L-NAME (purity > 98%) were from Sigma-Aldrich Co. The compounds with purity greater than 95 % identified by HPLC were initially dissolved in DMSO and then diluted with PBS for experiments. DMSO at the highest concentration possibly under the experimental conditions (0.1%) was not toxic to cells.
Microglial cell culture BV2 mouse microglia cell line was a kind gift from Dr. Young Choong Kim (Seoul National University). The cells were grown in IMDM supplemented with 10 % FBS and 50 µM 2-mercaptoe-
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Fig. 1 Effects of lignan compounds on nitric oxide production induced by lipopolysaccharide in BV2 microglial cells. BV2 microglial cells were treated with serial dilutions of compounds in the presence of LPS (1 µg/mL) and then incubated for 48 h. NO production was measured by Griess reagent, and viability was detected by MTT assay. L-NAME was used as a positive control.
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Original Papers
thanol and incubated at 37 °C in a humidified atmosphere (5 % CO2). Cells at density of 3 × 104 cells/well were plated onto 96-well microtiter plates for MTT and nitrite assay. Compounds with or without LPS (1 µg/mL) were added to the culture medium of BV2 microglia cells for 24 h for the experiments. The doses of all the tested compounds were chosen according to our preliminary study as those which would not influence the viability of BV2 cells.
Cytotoxicity assay Cell viability was evaluated in microglial cells by the MTT reduction assay [33]. In brief, cells were seeded onto 96-well microtiter plates and treated with various test sample solutions with or without LPS (1 µg/mL) for 48 h. After treatments, medium was removed and the cells were incubated with MTT (0.25 mg/mL) at 37 °C for 3 h. The formazan crystals in the cells were solubilized with the solution containing 50% DMF and 20% sodium dodecyl sulfate (pH 4.7). The absorbance of formazan was assayed by a Spectra (shell) reader (Tecan) at a wavelength of 490 nm.
Nitrite assays Microglial cultures were placed into IMDM medium and incubated with CoCl2 as indicated. Nitrite concentration (as a measure of NO production) in the supernatant was determined by the Griess reaction as described previously [34]. Cells (5 × 104 cells/well) were seeded onto 96-well microtiter plates and treated with various test sample solutions in the presence or absence of LPS (1 µg/mL) for 48 h. 50 µL culture supernatants were mixed with 50 µL Griess reagent (part I: 1 % sulfanilamide; part II: 0.1 % naphthylethylene diamide dihydrochloride and 2 % phosphoric acid) at room temperature. Fifteen minutes later, the absorbance was determined at 540 nm using Spectra (shell) reader. Nitrite concentration was calculated according to a standard curve of sodium nitrite generated by known concentrations.
Data analysis Results were expressed as mean ± SEM with triplicate samples. Statistical significance (p < 0.05) was assessed by one-way ANOVA followed by Dunnettʼs t-test (SPSS 13.0 software).
Supporting information The detailed compound isolation procedure is available as Supporting Information.
Acknowledgments !
This work was financially supported by a grant from the Natural Science Foundation of China (81202891).
Conflict of Interest !
There is no conflict of interest. The authors alone are responsible for the content and writing of the paper.
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