Alkaloids from Chelidonium majus and their inhibitory effects on LPS-inducedin RAW264.7 cells

Alkaloids from Chelidonium majus and their inhibitory effects on LPS-induced NO production in RAW264.7cellsJi Eun Park a , ,To Dao Cuong a , ,Tran Manh Hung b ,IkSoo Lee c ,MinKyun Na d ,Jin Cheol Kim e ,SungWoo Ryoo f ,Jeong Hyung Lee f ,Jae Sue Choi g ,Mi Hee Woo a ,Byung Sun Min a ,⇑aCollege of Pharmacy,Catholic University of Daegu,Gyeongbuk 712-702,Republic of KoreabDivision of Pharmacognosy and Natural Products Chemistry,Faculty of Medicine and Pharmacy,Duy Tan University,K7/25Quang Trung,Danang City,Viet Nam cKorea Institute of Oriental Medicine,Daejeon 305-811,Republic of Korea dCollege of Pharmacy,Yeungnam University,Gyeongbuk 712-764,Republic of Korea eKorea Research Institute of Chemical Technology,Daejeon 305-600,Republic of Korea fCollege of Natural Sciences,Kangwon National University,Kangwon 200-701,Republic of Korea gFaculty of Food Science and Biotechnolgy,Pukyoung National University,Busan 608-737,Republic of Koreaa r t i c l e i n f o Article history:Received 1July 2011Revised 23September 2011Accepted 30September 2011Available online xxxx Keywords:Chelidonium majus Papaveraceae AlkaloidAnti-inflammatory activitya b s t r a c tA new alkaloid,methyl 20-(7,8-dihydrosanguinarine-8-yl)acetate (1),together with six known alkaloids,stylopine (2),protopine (3),norchelidonine (4),chelidonine (5),berberine (6),and 8-hydroxydihydrosan-guinarine (7),were isolated from Chelidonium majus .Their chemical structures were primarily estab-lished using 1D and 2D NMR techniques and mass spectrometry.The anti-inflammatory activity of the isolates was examined for their inhibitory effects on LPS-induced NO production in macrophage RAW264.7cells.Among them,compounds 5and 7showed strong inhibitory activities toward the LPS-induced NO production in macrophage RAW264.7cells with IC 50values of 7.3and 4.5l M,respectively.In addition,compounds 5and 7inhibited the inductions of COX-2and iNOS mRNA in dose-dependent manners,indicating that these compounds attenuated the syntheses of these transcripts at the transcriptional level.Ó2011Elsevier Ltd.All rights reserved.Inflammation is a central feature of many pathophysiological conditions in response to tissue injury and host defenses against invading microbes.Chronic inflammations and infections lead to the upregulation of a series of enzymes and signaling proteins in affected tissues and cells.Among these pro-inflammatory en-zymes,the inducible forms of nitric oxide synthase (NOS),and cyclooxygenase (COX),which are responsible for increasing the levels of NO and prostaglandins (PGs),respectively,are known to be involved in the pathogenesis of many chronic diseases including multiple sclerosis,Parkinson 0s,Alzheimer 0s diseases,and colon cancer.1,2NO is produced by iNOS in macrophages,hepatocytes and renal cells,under the stimulation of lipopolysaccharide (LPS),tumor necrosis factor-alpha (TNF-a ),interleukin-1or interferon-gamma,3meanwhile,COX is the enzyme that converts arachidonic acid to PGs.Like NOS,COX has been found to exist in two isoforms and one of these,COX-2,is an inducible form which is responsible for the production of large amounts of pro-inflammatory PGs at the inflammatory site.4Furthermore,TNF-a is one of the most impor-tant pro-inflammatory cytokines and is mainly produced by monocytes and macrophages.It is secreted during the early phase of acute and chronic inflammatory diseases such as asthma,rheu-matoid arthritis,septic shock,and other allergic diseases,as well as the activation of T cells.5During a screening procedure on higher plants to find novel candidates as anti-inflammatory agents,the methanol extract of Chelidonium majus L.(greater celandine)was shown to exhibit con-siderable inhibitory activity. C.majus belongs to the family Papaveraceae and is a rich source of various biologically active substances.Anti-inflammatory,anti-microbial,antiviral,and antispasmodic activities are attributed to greater celandine.6–9Fur-thermore,the C.majus herbal drug has been traditionally used for its choleretic activity and for the treatment of liver disorders.10The main chemical components of C.majus are as follows:benzo[c ]phenanthridines both quaternary (chelerythrine,sanguin-arine)and tertiary (chelidonine),protopine and its derivatives (protopine,allocryptopine),and protoberberines (berberine,copti-sine).11Other previously isolated constituents included flavonoids and various acids (ferulic,cumaric,caffeic,and chelidonic acids).12Few clinical trials have been conducted to gain a better under-standing of the efficacy of C.majus ,which is often used with other herbal ingredients,in the treatment of functional gastric disorders or upper abdominal pain due to biliary system dysfunction.120960-894X/$-see front matter Ó2011Elsevier Ltd.All rights reserved.doi:10.1016/j.bmcl.2011.09.128⇑Corresponding author.Tel.:+82538503613;fax:+82538503602.E-mail address:bsmin@cu.ac.kr (B.S.Min).These authors contributed equally to this work.These studies have provided evidence of a statistically significant improvement in patients treated with greater celandine.13From the continuing investigation on the discovery of the inhibitory ef-fect on LPS-induced NO production in macrophage RAW264.7cells, the subsequent isolation of C.majus from the EtOAc-soluble frac-tion resulted in the isolation of a new alkaloid(1),as well as six previously known isolates(2–7).14On comparing the spectral data with those in the literature and of authentic samples,the known compounds were identified as stylopine(2),15c protopine(3),15b norchelidonine(4),15d chelidonine(5),15c berberine(6),15b and 8-hydroxydihydrosanguinarine(7).15a In the present paper,the isolation and structural elucidation of the isolates,as well as the evaluation of their inhibitory effects on LPS-induced NO produc-tion in macrophage RAW264.7cells are described.Compound1was obtained as yellow powder,and shown posi-tive reactions with Dragendorff’s reagent.The molecular formula of 1was found to be C23H19NO6,on the basis of the molecular ion at m/z405.1215for the[M]+(calcd for C23H19NO6,405.1212)in the HR-EI-MS.16The IR spectrum of1showed the presence of2922 (C–C),1720[(C@O)–O],1650(C@O),and1478(aromatic absorp-tion)cmÀ1.The1H NMR spectrum displayed,in the aromatic re-gion,two one-proton singlets at[d H7.14(1H,s,H-1)and7.47 (1H,s,H-4)],two pairs of AB-type ortho-coupling quartets at[d H7.52(1H,d,J=8.4Hz,H-5)and7.74(1H,d,J=8.4Hz,H-6)],and[d H6.90(1H,d,J=8.0Hz,H-11)and7.42(1H,d,J=8.0Hz,H-12)],as well as one N-methyl group in the higher-field region[d H 2.61(3H,s,N–CH3)],and two methylenedioxy protons at[d H 6.08and6.07(each1H,d,J=1.2Hz,–OCH2O–),6.05and6.04(each 1H,d,J=1.2Hz,–OCH2O–)].All of the1H NMR spectra also exhib-ited a double doublet[d H4.80(1H,dd,J=4.8,10.8Hz)],the typicalsignal of H-8from an8-hydroxydihydrosanguinarine skeleton iso-lated from Dactylicapnos torulosa,15a substituted at C-8by a directly connected methine(Fig.1).The13C NMR spectroscopic data cou-pled with an HMQC experiment established the presence of23sig-nals including sixteen aromatic carbons,one carboxylic carbon signal at d C173.6,two methylenedioxy carbons at(d C102.6and 103.1),one methine carbon signal at d C56.2,one methylene carbon signal at d C39.8and one N-methyl carbon signal at d C43.4and one methoxy carbon signal at d C52.2.The NMR spectroscopic data,to-gether with the molecular formula,suggested that1had a methyl acetate group,which was confirmed by a COSY experiment.In the 1H–1H COSY spectrum,H-8showed a clear correlation with H-20 (Fig.2).With the further aid of the HMBC spectrum of1,which notably contained cross-peaks between H-8/H-20/10-OCH3and C-10(d C173.6),established that a methyl acetate group was substituted at C-8through C-20(Fig.2).The relative configuration of position C-8was not solved due to the lack of information in the NMR spectra and the failure to crystallize1.Based on the above data analyses,compound1was established as a new alkaloid, namely methyl20-(7,8-dihydrosanguinarine-8-yl)acetate.Inflammation is an essential aspect of host response to infection and injury,and it is characterized by the abundant productions ofTable1Inhibition of NO production in macrophage RAW264.7cells by compounds1–7Compound IC50value a(l m)1>502>503>50416.5±4.557.3±0.66>307 4.5±0.5Celastrol b 1.0±0.1a The inhibitory effects are represented as the molar concentration(l M)giving50%inhibition(IC50)relative to the vehicle control,the results are the mean of threereplications.b Positive control.2J.E.Park et al./Bioorg.Med.Chem.Lett.xxx(2011)xxx–xxxNO,PGE2,and of cytokines such as IL-6and TNF-a,and thus,these pro-inflammatory mediators are important anti-inflammatory tar-gets.Lipopolysaccharide(LPS)is an endotoxin,one of the constitu-ent of the outer membrane of gram-negative bacteria,17it stimulates innate immunity by regulating the productions of inflammatory mediators,like NO,TNF-a,IL-6,prostanoids and other leukotrienes.17Several reports showed that murine RAW 264.7cells stimulated by LPS offered an excellent model for the screening and subsequent evaluation of the effects of candidate drugs on the inflammatory pathway.In our experiments,the cyto-toxic effects of isolates1–7were evaluated in the presence or ab-sence of LPS using MTT assay,18and these compounds did not affect the cell viability of RAW264.7cells at doses of50l M after 24h(data not shown).The amount of NO produced was deter-mined by the amount of nitrite,a stable metabolite of NO.To assess the effects of these compounds on the LPS-induced NO production in RAW264.7cells,the cell culture medium was harvested,with the amount of nitrite measured using the Griess reaction.19As shown in Table1,compounds5and7showed strong inhibitory ef-fects with IC50values of7.3and4.5l M,respectively,compound4 showed a weak inhibitory effect with and IC50value of16.5l M, while the other compounds exhibited no activities.The effects of 5and7on the LPS-induced COX-2and iNOS expressions were also investigated.20RAW264.7cells were stimulated on exposure to 1l g/ml of LPS for18h in the presence of increasing concentrations of5and7,and the expression levels of COX-2and iNOS proteins were determined using immunoblot analyses.Western blot was performed to determine whether the inhibitory effects of com-pounds5and7on the pro-inflammatory mediator(NO)are related to the modulations of the expressions of iNOS,and COX-2.In unstimulated RAW264.7cells,iNOS and COX-2protein levels were almost undetectable.However,in response to LPS,the expression of iNOS and COX-2was markedly augmented,compounds5and 7significantly inhibited this iNOS and COX-2protein induction in a concentration-dependent manner(Fig.3).Moreover,preincu-bation with compounds5and7at3l M resulted in a marked reduction in LPS-induced COX-2,and at3l M of compound5and at1l M of compound7resulted in a marked reduction in LPS-in-duced iNOS expression(Fig.3).These compounds suppressed the production of NO as well as the expressions of iNOS and COX-2 in LPS-stimulated pounds5and7were also shown to inhibit not only the expressions of iNOS and COX-2 mRNA,but also the iNOS and COX-2promoter activities in LPS stimulated RAW264.7cells,suggesting that these compounds could suppress LPS-induced iNOS and COX-2expressions at the transcription level.In accordance with our study,it is unclear tofind out the signif-icant differences in the relationship between activity and chemical structures,however,the compounds with hydroxy methine con-stituents as norchelidonine(4),chelidonine(5)and8-hydroxydihydrosanguinarine(7)reduced the NO production after LPS stimulation in a dose-dependent manner(Table1).Interest-ingly,the N-methyl-substituted skeletons as chelidonine(5)and 8-hydroxydihydrosanguinarine(7)showed somewhat more potent inhibitory activity than the other compounds(Fig.3).The anti-inflammatory effects of these compounds is due to their regulation of inflamatory mediators such as COX-2and iNOS.In summary,the present results demonstrated that these alkaloids might be important anti-inflammatory constituents of this plant. AcknowledgmentsThis research was supported by the National Research Founda-tion of Korea Grant funded by the Korean Government(MEST) (KRF-2009-0084675)and(NRF-C1ABA001-2010-0020484). References and notes1.Heiss,E.;Herhaus,C.;Klimo,K.;Bartsch,H.;Gerhauser,C.J.Biol.Chem.2001,276,32008.2.Kundu,J.K.;Surh,Y.J.Mutat.Res.2008,659,15.3.Kuo,P.C.;Schroeder,R.A.Ann.Surg.1995,221,220.4.Weisz,A.;Cicatiello,I.;Esumi,H.Biochem.J.1996,316,209.5.Palladino,M.A.;Bahjat,F.R.;Theodorakis,E.A.;Moldawer,L.L.Nat.Rev.DrugDisc.2003,2,736.6.Chung,H.S.;An,H.J.;Jeong,H.J.;Won,J.H.;Hong,S.H.;Kim,H.M.J.Pharm.Pharmacol.2004,56,129.7.Kokosca,L.;Polesny,Z.;Rada,V.;Nepovim,A.;Vanek,T.J.Ethnopharmacol.2002,82,51.8.Colombo,M.L.;Bosisio,E.Pharm.Res.1996,33,127.9.Hiller,K.O.;Ghorbani,M.;Schilcher,H.Planta Med.1998,64,758.10.Vahlensieck,U.;Hahn,R.;Winterhoff,H.;Gumbinger,H.G.;Nahrsted,A.;Kemper,F.H.Planta Med.1995,61,267.11.Barreto,M.C.;Pinto,R.E.;Arrabaca,J.D.;Pavão,M.L.Toxicol.Lett.2003,146,37.12.Barnes,J.;Anderson,L. A.;Phillipson,J. D.In Herbal Medicines,3rd ed.;Pharmaceutical Press:London,2007.13.Ritter,R.;Schatton,W.F.H.;Gessner,B.;Willems,plement.Ther.Med.1993,1,189.14.The air-dried whole plant of C.majus was purchased from a folk medicinemarket‘Yak-ryoung-si’in Daegu,Korea,in May2009.Botanical identification was performed by Professor Byung Sun Min,and the voucher specimen CUD-1523was deposited at the Herbarium of the College of Pharmacy,Catholic University of Daegu.The air-dried whole plant of C.majus(6kg)was extracted with MeOH(15LÂ3times)at room temperature for72h and the combined extract was removed under vacuum to afford a viscous residue358.17g.The MeOH extract was suspended in hot water and successively partitioned with n-hexane,EtOAc,and n-BuOH to afford n-hexane—(61.45g),EtOAc—(33.74g), and n-BuOH-soluble(26.85g)fractions.The EtOAc-soluble fraction(33.74g) was applied to a silica gel column,eluted with CHCl3–MeOH(50:1–0:1)to yielded11subfractions(E1–E11)according to their TLC profiles.Subfraction E1(1.06g)was applied to MPLC and eluted with MeOH–H2O(5:1–20:1)to obtain7(4mg).Re-chromatography of subfraction E5(2.32g)on a silica gel column and eluted with CHCl3–EtOAc(10:1–1:1)to afford six subfractions(E5.1–E5.6).Compound6(5.7mg)was isolated by applying subfraction E5.1(340.5mg) over a silica gel column and eluted with hexane–acetone(20:1–1:1).Further purification of subfraction E5.4(514mg)using silica gel column with a gradient of CHCl3–acetone(20:1–1:1)resulted in the isolation of5(151mg).Subfraction E6(1.04g)was purified by silica gel column using a gradient of CHCl3–MeOH(30:1–1:1)resulted in the isolation of1(23.6mg)and2(17.5mg)and six subfractions(E6.1–E6.6).Compound4(4.5mg)wascrystallized from subfraction E6.4(55mg)with hexane–EtOAc(5:1).Figure3.Inhibition of compounds5(A)and7(B)in LPS-induced iNOS and COX-2expressions in RAW264.7cells.Furthermore,subfraction E10(1.25g)was also purified using silica gel column, eluting with a gradient of CHCl3–MeOH(20:1–3:1)to afford3(62.9mg), respectively.15.(a)Guo-Lin,Z.;Gerhard,R.;Eberhard,B.;Martin,N.;Ralf,M.;Christoph,S.Phytochemistry1995,40,299;(b)Kim,D.K.;Lee,K.T.;Baek,N.I.;Kim,S.H.;Park,H.W.;Lim,J.P.;Shin,T.Y.;Eom,D.O.;Yang,J.H.;Eun,J.S.Arch.Pharm.Res.2004,27,1127;(c)Lee,J.;Shon,M.Y.;Jang,D.S.;Ha,T.J.;Hwang,S.W.;Nam,S.H.;Seo,E.K.;Park,K.H.;Yang,M.S.Agric.Chem.Biotechnol.2005,48, 198;(d)Matthew,J.;Fleming,H.A.;McManus,A.R.;Chan,W.H.;Ruiz,J.;Dockendorff,C.;Lautens,M.Chem.Eur.J.2008,14,2112.16.Physical and spectroscopic data of new compound(1,methyl20-(7,8-dihydrosanguinarine-8-yl)acetate)):Yellow powder;mp192°C;½a 25D+23.0(c0.5,CHCl3);UV k max(MeOH)276nm;IR m max(KBr)2922,1720,1650,1602,1478,1320,1263,772,618cmÀ1;HR-EI-MS m/z405.1215[M]+(calcd for C23H19NO6,405.1212);1H NMR(400MHz,CD3OD)d:7.14(1H,s,H-1),7.47 (1H,s,H-4),7.52(1H,d,J=8.4Hz,H-5),7.74(1H,d,J=8.4Hz,H-6),2.61(3H,s, N-CH3),4.80(1H,dd,J=4.8,10.8Hz,H-8),2.41(1H,dd,J=4.8,14.0Hz,H-20a),2.32(1H,dd,J=10.8,14.0Hz,H-20b),6.90(1H,d,J=8.0Hz,H-11),7.42(1H,d,J=8.0Hz,H-12),6.08and6.07(each1H,d,J=1.2Hz,–OCH2O–),6.05and6.04 (each1H,d,J=1.2Hz,–OCH2O–),3.67(3H,s,20-OCH3).13C NMR(100MHz, CD3OD)d:105.3(C-1),149.7(C-2),149.2(C-3),101.5(C-4),132.7(C-4a),125.4 (C-5),121.1(C-6),56.2(C-8),116.6(C-8a),148.8(C-9),146.0(C-10),108.9(C-11),117.8(C-12),129.1(C-12a),127.1(C-13),124.7(C-14),139.4(C-14a),103.1(–OCH2O–),102.6(–OCH2O–),39.8(C-20),52.2(10-COO C H3),43.4(N–CH3),173.6(10-C OOCH3).17.Guha,M.;Mackman,N.Cell Signal2001,13,85.18.(a)Shin,K.M.;Kim,I.T.;Park,Y.M.;Ha,J.;Choi,J.W.;Park,H.J.;Lee,Y.S.;Lee,K.T.Biochem.Pharmacol.2004,68,2327;(b)Cell culture.The RAW264.7cells were maintained in Dulbecco’s Modified Essential.These cells were grown at 37°C in DMEM supplemented with10%heat-inactivated FBS,penicillin (100units/ml),and streptomycin sulfate(100l g/ml)in a humidified atmosphere of5%CO2.After pre-incubation of RAW264.7cells for4h,0–10l g/ml each compound was added;(c)MTT assay for cell viability.RAW264.7 viability after24h of continuous exposure to the compounds was measured with a colorimetric assay based on the ability of mitochondria in viable cells to reduce MTT.Briefly,1Â104cells/well treated for24h with vehicle or compounds were examined for cell viability.Viability of the macrophages treated with vehicle(0.5%DMSO)only was defined as100%viable.Survival of macrophage cells after treatment with compounds was calculated using the following formula:viable cell number(%)=OD570(treated cell culture)/OD570 (vehicle control)Â100.19.Determination of NO production and the cell viability assay:The level of NOproduction was determined by measuring the amount of nitric from the cell culture supernatants as described previously.Briefly,the RAW264.7cells (ATCC,Rockville,MD,USA,1Â105cells/well)were stimulated with or without 1l g/ml of LPS(Sigma Chemical Co.,St.Louis,MO)for24h in the presence or absence of the test compounds(0.5–25l M).The cell culture supernatant (100l l)was then reacted with100l l of Griess reagent.The remaining cells after the Griess assay were used to test their viability using a MTT(Sigma Chemical Co.,St.Louis,MO)-based colorimetric assay as previously described.Celastrol was used as the positive control.20.Immunoblot analysis:Proteins were extracted from cells in ice-cold lysis buffer(50mM Tris–HCl,pH7.5,1%Nonidet P-40,1mM EDTA,1mM phenylmethyl sulfonylfluoride,1l g/ml leupeptin,1mM sodium vanadate,150mM NaCl).Fifty microgram of protein per lane was separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis(PAGE)and followed by transferring to a polyvinylidene difluoride membrane(Millipore,Bedford,MA,USA).The membrane was blocked with5%skim milk,and then incubated with the corresponding antibody.Antibodies for COX-2,iNOS were obtained from Santa Cruz Biotechnology(Santa Cruz,CA,USA).Antibody for a-tubulin was purchased from Sigma.After binding of an appropriate secondary antibody coupled to horseradish peroxidase,proteins were visualized by enhanced chemiluminescence according to the instructions of the manufacturer (Amersham Pharmacia Biotec,Buckinghamshire,UK).4J.E.Park et al./Bioorg.Med.Chem.Lett.xxx(2011)xxx–xxx。