以高通量方法筛选红桑叶的抗菌活性并进行活性组分的分离研究

1、以高通量方法筛选红桑叶的抗菌活性并进行活性组分的分离研究【摘要】目的通过一种简单快速的高通量方法筛选具有抗菌活性的植物提取物，并通过此种寓方法从有活性的粗提物中纯窜化活性组分。方法首先建立渣植物提取物库和抗菌的高通卧量筛选方法。然后针对转糖链球菌Streptoco屠ccusmutans和伴黝放线放线杆菌Actino疰bacillusacti臊nomycetemcomitans进行高通量筛选孺。结果通过筛选发现红桑叶椰具有很强的抑菌活性。为了禧进一步研究其活性，将其粗使提物通过HPLC进行分离辣得到71个组分，再通过同毋样的高通量筛选方法进行抗菌活性测定，对有活性的组佚分通过血琼脂法进行验证。实

2、验数据表明红桑叶的一个组分#31对两种细菌有几鸵乎完全的抑制，而另外两个愿组分对伴放线放线杆菌Ac引tinobacillus荨actinomycete氖mcomitans有部分滞抑制。结论所建立的高通量圈筛选方法简便快捷，筛选结嶙果稳定可靠，可用于大规模的植物提取物的抗菌活性筛杳选。红桑叶具有很强的抗菌隧活性，且可分离得到活性组匙分，红桑叶具有进一步开发为抗菌药物和保健品的潜力壤。【关键词】红桑叶；高通量抗菌筛选；抗菌活性弓；抑制率Identif鹳icationofthe懵Antibacteria媒lActivityofM蚌orusrubraLea鞅vesbyaHigh-T莼hroughput

3、Scr桥eeningMethod键Abstract:Ob桦jectiveThisr文esearchinten漉dedtotestthe吮antibacteria描lactivityofa鹎plantextract旖libraryusingasimpleandfa材stcolorimetr闹icdetectionmethodandiden乓tifythosepla妥ntextractswi姗thbioactivit谌y,andtofurtherpurifythosebioactivefractionsofthe裉selectedplantextracts.Me羧thodsWefirstcreat

4、edaplan鲜textractlibr翠aryanddevelo效pedanantimicrobialHighTh踯roughputScre斩ening(HTS)me芑thod.Then,th蹇eplantextrac榕tsweretested玲forantibacte擎rialactivity偈againsttwosp亦eciesofbacte锥ria,S.mutans搅andA.actinom锤ycestemcomit冂ans.ResultsT骈heMorusrubra鹁leafextractw蜜asselectedbe舌causeithadth泛estrongestbi衿ologica

5、lactivityagainstt舞heoralbacteriatested.Tof淞urtherstudytheMorusrubraleaves,theex驸tractwassepa茇ratedintosma懦llfractionsviahigh-performanceliquid烘chromatography(HPLC).Thefractionswer啮etestedagain骣stbacteriaS.匕mutansandA.actinomyceste镧mcomitansusi箪ngHTSandthea绎ntibacterial纹activityofth欲oseselectedb娄ioac

6、tivefrac眢tionswasfurt尚herconfirmed犁bybloodagarplatingmethod.Onefractio啷nofMorusrubr陈aleaves,#31,桁exhibitednearcompleteinh七ibiton(99%or骘better)forbo龀thbacteria,w谵hiletwoother矩fractionsexh鸭ibitedsome,b埏utnotcomplet西e,inhibitionofA.actinomy峥cestemcomita！ns.Conclusio战nTheHTScanbe蓠usedtoscreen慕foralargep

7、la屐ntextractlib阎raryduetoitsfastandsimpleprocedure.M湿orusrubraleavesexhibited犁thestrongestantibacterialactivityand锵theactivefra橹ctionwasobta菸ined.TheMoru壅srubraleaves览havepotentia缅lusagetotrea岌tbacterialin芭fectioninthefuture.Keyw苜ordsMorusrub荚raleaves；Hi核gh-Throughpu馑tantibacteri濉alScreening,奄Antiba

8、cter泞ialactivity,妾InhibitionratePlant-b衩asedantibioticsproduct肀scouldbeimpo兴rtantalterna褊tivestothetr浯aditionalant叫ibioticstocombattherecen蜜tdevelopment吹ofantibiotic佶s-resistantb须acterialstra苍insduetotheoveruseofover鳓-the-counter缥antibiotics掊1，2.Plantmaterialsandp供lantpartshav攵ebeenusedasm艉edicinein

9、Chi碍natotreatdis溯easesforcent桀uries.Plant此saretherichs庶ourcefordrugdiscoveryduetotheseconda骊rymetabolite业sproduced.P祯lant-baseda诊ntibioticsp饽roductscanserveasanimpor宠tantsourcefo血rfuturethera忾peutictreatmentofinfecti厕ousdiseases3，4.Scree聘ningforantib擢ioticsfromav粗astplantsour些cecanbeadaun摊tingtaskbe

10、ca鹂usethereareh裟undredsofplantspeciesandhundredsofpl腌antsecondary徭metabolitese橇venwithinasingleplant5，碉6.Usingthet嫖raditionalscreeningmetho焐dstoidentify懿antibacteria斓lphytochemic跎alsfromaplantextractlibr沉arycanbetime蒜-consumingan阌dveryexpensi狲ve.Thus,asi鲭mple,cost-ef幕fective,andf宦asthigh-thro逖ughput

11、antimi鬲crobialscree走ningmethodis掬neededforusi膑ngtheplantex抡tractlibraryfordrugdisco玄verysourcean慑dpurificatio裹nprocess.Inthisstudy,wer足eportafastandsimplecolor峥imetricHTSfo疳rscreeningpl鞣antextractli鸠braryforplan侈t-basedantib孙ioticsusingr涉esazurindyea靠sanindicator氘.Resazurinde官tectionmethodwasusedfor

12、d艏etectingbact惟erialcontami海nationinfood椤industryandi躞nfectiousdis逋easedetection7，8.Resazurindyeismuchsimpleandl后esstime-consuming.Theliv豸ebacteriacouldmetabolize易theblueresaz酝urinintopinkcolor,whilet珲hedeadbacter链iacouldnot.襻Afterincubat蠓ioninthepres裳enceofresazurin,bacteriacouldbejudge葬dbydeadoral

13、i猩vesimplybylo蛤okingattheco泷loroftheresa徼zurindye.T胁osumup,wedev胞elopedananti原microbialHig癌hThroughputScreening(HTS舾)methodforpl窬antdrugdiscovery.Wetest绎edaplantextr擘actlibraryco鲺nsistingof16垫3plantspecie素sforantimicr媸obialactivit後yagainsttwos壕peciesoforalbacteria:St属reptococcusm椐utans(abacte螳riainv

14、olvedi娉ndentalcariesformation)a棹ndActinobaci懂llusactinomy瀣cestemcomita嘿ns(stronglya熟ssociatedwit间hperiodontit簪is).Morusru噘braleavesistheextracttha祢tindicatedst蹊rongbiologic筠alactivityag何ainstthethreespeciesofba措cteriatested滋.However,th娄erewerefewst炯udiesontheMo蕲rusantimicrobialpropertiesandtheexis孥t

15、ingresearch酶isEithervery怊generalordoe憾sntfocusont贱hequalities共9，10.Inthis砜study,weexpa肺ndedourresea朵rchontheMorusrubraleavesextractbysep涮aratingoutit驭spropertiesv当iaHPLC.Thisallowedustoisolatetheact疵ivefractioni倜nthecrudeext垒ractandgiveu廷stheopportun惊itytofurther犹characterizeitsproperties.1MATERIA洫L

16、SANDMETHODSPlantmateri滹alPlantmaterialswerecoll辙ectedinthepa锅rkandsurroun楦dingsofLexin冷gton,Kentuck符yandidentifi咣edbytheProf.汞HuanginUniversityofKentu末cky,Bacter嗪ialstrainand绚growthcondit翱ionTwooralbacterialspeci以eswereusedin蹁thisstudy.A.呖actinomycete唯mcomitans(ATCC714)andStr窬eptococcusmu圉tans(ATCC2

17、51火75),werepurc圣hasedfromATC桴C.TSBYEmedia蜈andAnaerobeB镭rothwerepurc孬hasedfromOxo陪idLtd.Growth爱conditionswe垛reat37inanaerobiccondit鲜ion.Instru莉mentsHPLCColumn:Phenomen铜exGemini5lC舱18,System:Waters510pumps蛑(2),Waters48蹄4TunableAbsorbanceDetect酚or;Microplat铒ereader:MRXp理latereader(DynatechLabor缨atories

18、).Evaporationequi蝽pment:Savant郇SC-110SpeedV剑ac,SavantRT-胎100Refrigera撬tedCondensationwithaSava荬ntVP100oilpu在mp;waterpurificationequi眚pment:Milli-莠Q.Chemical圭sMethanol,Re狠sazurinandEt模hanolwerepur斟chasedfromSigma,USA.Bloodagarplateswereobtained脊fromRemelCom嗡pany,USA.P裉lantextractl劐ibraryPlante摸xtract

19、swerep射reparedfromp鳏lantmaterial翌sbycrushing2衾gramsofplant靠materialusin下gamortarandp鹅estleandfollowedbyadding湮8mlof50%EtOH巧.Eachindividualextracti裎sallowedtosh名akefor24hour看sina6-wellti夯ssueculturep翅latebeforebE缸Ingfilteredthroughfilter医paper.Filter顸edextractsar鹩ethenlyophil谳ized16to24ho洌ursintodryp

20、owder.Thedrie余dextractsobt董ainedthroughthelyophiliz蜣ationprocess禀arere-suspen楝dedin100lof虹50%EtOH.Atot螗alof163plantextractswere怎preparedthis奈way.HTSscr耢eeningofextr憎actlibraryLy灭ophilizedext啸ractsweretes琏tedforactivitythroughthe锊HTSprocedure僖byadding1lo轸feachextract掺toselectedwe琐llsofa96wellplatecon

21、tain铬ing100lofTS榇BYEmediumand孤10%ofbacteriafromtheover馅nightculture.Theplateswe惜rethenincuba彖tedinananaer涎obicchamberbetween16and18hours.After眄incubation,3lofthecolor帑imetricindic戎atorresazurinwasaddedtoeachwell.Thel潞ivebacteriacouldmetaboli煳zetheblueres蜘azurinintopi单nkcolor,whil桅ethedeadbacteriacou

22、ldnot肃.Theplatewas确thenallowedt疱oincubatefor帻theresazurin惕tobemetabolizedbythebact邕eriaprovidin诗gthechangein逄color.Wellsc椭ontainingthe篚S.mutansrequ粥ired1hrofinc滑ubationandA.胧actinomycest崃emcomitansre馀quired2hours骑.AreadingusingaDynex96w娣ellplateread瞬eristakenfor稼eachbacteria酾at600nm.Typ萼icallywellsw

23、喂ithviablecel筚lwillrangein碰pinkcolorgiv於inganODreadi瓞ngoftoWells裙withnonviabl阮ebacteriawil燹lbebluegivin主ganODreading妫aboveTheresa熳zurinscreeningmethodissimpleandfast.昧HPLCproce爝dureEachfrac跑tionfromtheH盛PLCwastested糖againstS.mut桫ansandA.acti饶nomycetemcom碎itansusingth饧eHTSprocedur回elistedabove嘉.Thepl

24、antext冕ractwastosep那aratewiththeHPLCasfollow滥ing.Column:浈PhenomenexGe笄mini5lC18；S鹏ystem:Waters营510pumps(2),垌Waters484Tun膦ableAbsorbanceDetector；捏Detector:UV2管80nm；Injecti灿onVolume：20孛l；Flowrate：m剿l/min.Table陔1HPLCGradie祸ntTwenty20擗gofaselected霆plantextractwereinjected鼗intoHPLCand71fractions(1mlperfr

25、actio颁n)werecollec诀tedroughly1fraction/minu渫tewherethefr糙actionnumber箐correlateswiththeminutei蕾nwhichitwass哧eparated.Th杏isallowsusto双identifythep貘eakandeachfr廊actionwascol萆lectedas1mls遛olution.Eac隽hfractionwas雕driedusingaS羡avantdryingm尬achine.Each獯fractionwasr覆e-suspendedi伛n25lof50%et缝hanolsolutio汤n

26、.Bloodag阋arplatingFra螺ctionsinthe9艘6-wellplatet宙hatshowedbiologicalactiv哳ityviatheHTS弩werediluted10000xandplat弥edinduplicat撬eonbloodagarplates(Remel).Controls螫werealsoplat昆edalongwithw藐ellsthatdidnotshowactivi锆tyaroundtheactiveones(If般fraction31sh儆owedactivity膈wewouldplatefraction30,31,and32.)Th屋epl

27、ateswerei棠ncubatedat37苤inaninanaer捃obicconditio蓼nfor48hours.骁Thecolonies轰(CFU)werecou卒ntedoneachag膂arplateafter扒theincubatio胤n.2RESULTS胭AsimpleHTSs弥creeningmethodcouldbesuc免cessfullyusedtoscreenant嚅ibacterialac留tivityofalargeplantextractlibrary.Am牢ongthe163plantextract,th窜eMorusrubrae瘵xtractshowed螯t

28、hesignificantanti-bacte遣rialactivity.TheMorusru镏braextractwasfurtherpuri粱fiedbyHPLCin前to71fractionsandtestedag娟ainforantiba纟cterialactiv带ity.TheHPLCp入rofilewassho插wninFigure1.HPLCpurificationTheresultoftheHPLCw向asshowedinFi讪gure1,where2冱0gMorusrubr夕aextractsolu毙tionwasinjected.71fracti硕onswerecollec

29、tedroughly1fraction/minute.TheHPLCranfor80minut鹊esat1mlperml瓒.Amethanol/watergradien酥twasused.F粪igure1TheHP宾LCChromatogr萜aphofMorusru哲braleavesext拇ractfractionsHPLCfractionHTSresultsTheHPLCfrac动tionsweretes诸tedagainfora侨ntibacterial骅activity(Figure2a).Ther迮ewerethreefr拯actions(frac俯tion4,20,and猕31)w

30、hichshow买edsignifican疳tanti-bacterialactivity,乌withfraction滠31showingthe悖mostbioactiv凝ity.Theresu芾ltsofeachfractionsanti-渠bacterialact而ivityinvitro沂couldbeseesinFigure2aand醇Figure2b.Figure2aTheHT俦Sresultsofth咬eMorusrubral梃eavesHPLCfractions(1-71)腮againstS.mutans.Onelofe玎achfractionw宜asaddedintothewe

31、llofthe96-wellplate.券Figure2bTh夏eHTSresultso裼ftheMorusrub陷raleavesMULH茁PLCfractions(1-71)agains禚tS.mutans.T第hewellsinthi牢splatehavebe城entreatedwit阔hS.mutansand蜗theMorusrubr蟑aHPLCfractio裟ns(1-71readinglefttorigh勖t).ThisplatehavebeentreatedwithS.muta鹱nsandtheHPLC悉fractions(1-71readinglef泥tt

32、oright).T豚heresazurini霞ndicatesthat跛fraction31ex劫hibitsthemos殓tinhibition,睃followedbyfraction20andf尻raction4.The坟bluecolorofr昶esazurinindi贸catedthefrac鲲tion31significantlyinhib计itedthegrowt诞hS.mutans.T埸enlofacontrolwellwithou羡tMorusrubraf驳raction(Right)orbluewell旗withMorusrub亍raHPLCfracti粉on#31(Left

33、)w彘asdilutedto1椒0-4and50lwa窘sdistributed腙ontoabloodag廿arplate.Thel革eftplatecont乎ainedsignificantlessS.mutansCFUsthan罱therightplat炎e.Bloodaga蛰rplatingresu槛ltsThe3wells暾(fraction4,2蒉0,and31)from励the96well-pl翩atethatshowe蹄dantibacteri唤alactivityinpresenceofre钟sazurinwerep铺latedonblood讲agarplatesin伽10-3

34、to10-4dilution.Afte褰rincubation,馁thecfu(colonyformingunit慷s)oneachplat邋ewerecounted卫andplotted.盎Fraction31(thedarkestcol蚜orwell)allow真ednobacteria馈lcolonies,100%inhibitionagainstoralb佴acteriaA.act柴inomycetemco滚mitans(Figur利e3a).Thiscf迹udataconfirm巽edthedataofH潮TSdata.Agai链n,fraction31纹showedasigni另f

35、icantinhibi蹋toryactivity含againsttheS.mutans.Thep俯lateoffracti奕on31hadanaverageof41cfuscomparedtoov噎er3800cfusof郾thecontrolpl劓ate.Thisindicated99%oft兔heS.mutanswa眢sinhibited(Figure3b).Fr雷action31ofMo在rusrubrahadabroadbioacti枪vityandshowe醴dinhibitorya棹ctivityagain艴stbothoralpa剌thogens,S.mutansandA.act

36、愣inomycetemco牖mitans.Figu歧re3aTheBloo噼dAgarPlatingresultsofthe冁fractionswit碘htheanti-bac叔terialactivi动tyinHTS(againstA.aetinom宋ycetemcomi-t掣ans).C=cont矍rolsplatedafterincubation.Figure3b宽TheBloodAgar飙Platingresul崎tsofthefract馈ionswiththea租nti-bacteria礓lactivityinHTS(against).躏C=controlsp山latedafteri

37、n责cubation.3替DISCUSSIONOurstudydemon旖stratedthato坦ursimpleandf鞲astHTSmethodcouldbeausef缙ultoolforscreeningforpla郭nt-basedanti榜bioticsasthe蛊methodenabledustoscreena贲plantextract浦libraryquick鸦ly.TheHTSidentifiedMoru坫srubraplantl魁eafasapotent蔺ialsourcefor嗄futureplant-basedantibio猿ticsasitexhi钿bitedasig

38、nif帱icantantibacterialactivity.Thescreeningmethodwa秘ssensitivean笾dcost-effect沈ivefortheini蝴tialscreening.Resazurind槛etectionmeth恨odhasbeenusedfordetectin璩gbacterialcontaminationi乌nfoodindustr挚y.Resazurind趵yeismuchsimpleandlesstime-consuming.赐Thelivebact塔eriacouldmet佩abolizethebl揪ueresazurinintopinkc

39、olor妫,whilethedea睾dbacteriacou垲ldnot.After叟incubationin数thepresenceo抒fresazurin,b赇acteriacould叛bejudgedbyde饺adoralivesim奚plybylooking钐atthecolorof醇theresaurindye.Ourstudydemonstrated设thatourHTSmethodwasausef哥ultoolforplant-basedanti烙bioticsasour半HTSmethodena妨bledustoscre僚enaplantextr芷actlibraryan靠di

40、dentifyMor榫usrubraplant厍leafasapotentialsourcefo虑rfutureplant舶-basedantibiotics.Theexperimentalda蚯tashowedthat纷theMorusrubraleaveshadst淬rongantibact釜erialeffecta碉gainstoralpa某thogensS.mut丕ansandA.acti槔nomycetemcom铴itans.Theda框taindicatedt蕾hatthefracti料on31couldcom庀pleteornearc霾ompleteinhib猬itionagai

41、nst颃bothofthebac笳terialspecie筒s,S.mutansan骼dA.actinomyc濯etemcomitans意.Thefractionappearstobeb培actericidala楸ndhadabroadk翔illingcapabi嫠lity.Morus蟑rubraiscommo喙nlyknownasredmulberrytre钜e.Morusrubra澌isMorusrubra鱼L.species,Mo疆rusL.genus,Moraceaefamil浓y.Morusisbro钱adlydistribu踣tedontheearth.Morusrubraison

42、eofthedozenspeciesof倘MorusLgenus.冗Themainchemi垓calcomponent骀sinMorusleav苻esareflavanones,flavonoi崾dglycosides,卿alkaloids,Polysaccharide秫,sterol,vola踅tileoilandvi酰tamins.Thepharmacologica鲛lstudiesshow旆edthattheMor傧usrubraleave蕈shadmultifunctions,suchashypoglycemiceffect,lipi档ds-decreasin响geffects,ant

43、胆ihyperlipide鄯miaeffect,an站ti-inflammat煮ory,anti-can断cerandanti-v劣irus1120.盆Littleresea濯rchhasbeenonitsantibacterialactivityagainstoralpathogens.Pe军riodontaldis蕻easehasbeencalledasilentepidemic.Nearly25%ofadu徜ltsfromtheag拾es33to45suff嗖erfromdestructiveperiodo叵ntaldisease躏21.Manymor奕esufferfromg呶ingi

44、vitis,an餍dotherbacter濯iarelatedinf磨lammatoryora僮ldiseases.T携hus,identify丐inganddevelopingmoreeffe已ctivetherapeuticregimens碉targetingasp刽ectsofthemic雏robialinfect共ionandinflam涯matoryrespon狐sesinoralcav粒itywouldprov觜ideasignific哈antbenefitto康theseindivid炮uals.Gingivi鲤tisandperiod象ontitisaffec碚taslarge

45、apro瘃portionofthe谒globalpopula缦tionasanydis莞easeknowntomankind.Thus艽,approachestoidentifyand号characterize荻novelnatural结productsthatcanbeprovidedtothepopula获tionthroughoralmedicamen铴tswouldconst舍ituteasignif奸icantcontrib笛utiontothehe茬althcareente酞rprise.Natu钉ralpharmaceu薛ticalswithth残eabilitytoim磕pro

46、veoralhea优lthwithouttheadverseeffe霍ctofantibiot沮ics,suchasth槐eMorusrubra,绘couldbeexplo噫itedforfutur漆enewantimicrobialmedicin荠eandnewusage敞oftheMorusle岬aves.【参考文贯献】1Corna徂gliaG,Fontan剿aR.Epidemiol蕃ogicalsurvey决ofbacterialr娣esistanceinupperrespirat腭orytractinfe钭ctionsinItal呐yJ.IntJAnt簿imicrobAgent瓦s.2

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51、cro嗵titerassayfo寥rrapiddetect嗌ionofofloxacinresistance素inM.tubercul奂osisJ.IntJ堍TubercLungDi瓞s.XXJul;10(7)：808.9Ha币nsawasdiC,Ka圣wabataJ.Alph您a-glucosidas反einhibitorye盹ffectofmulbe嘭rry(Morusalb虢a)leavesonCaco-2J.Fito渤terapia.XXDec;77(7-8):56丞8-73.EpubXX：倪22.10OkuT缕,YamadaM,NakamuraM,SadamoriN,Nakamur廿aS.Inhibitor犀yeffectsofextractivesfro没mleavesofMor罟usalbaonhuma浙nandratsmallin