Synergistic Antifungal Activity of Berberine Derivative B-7b and Fluconazole

20 Pages·2015·4.56 MB·English

RESEARCHARTICLE
Synergistic Antifungal Activity of Berberine
Derivative B-7b and Fluconazole
LiPingLi1,WeiLiu1,HongLiu2,FangZhu1,DaZhiZhang2,HuiShen1,ZhengXu2,Yun
PengQi2,ShiQunZhang1,SiMinChen1,LiJuanHe2,XinJuCao2,XinHuang1,Jun
DongZhang1,LanYan2*,MaoMaoAn1*,YuanYingJiang1,2*
1 ShanghaiTenthPeople'sHospital,andDepartmentofPharmacology,TongjiUniversitySchoolof
Medicine,1239SipingRoad,Shanghai,200092,PRChina,2 NewDrugResearchandDevelopmentCenter,
SchoolofPharmacy,SecondMilitaryMedicalUniversity,Shanghai,China
* [email protected](LY); [email protected](MMA); [email protected](YYJ)
Abstract
Ourpreviousstudydemonstratedberberine(BBR)andfluconazole(FLC)usedconcomi-
tantlyexhibitedasynergismagainstFLC-resistantCandidaalbicansinvitro.Wealsosug-
OPENACCESS
gestedBBRplayedamajorantifungalroleinthesynergismofFLCandBBR,whileFLC
Citation:LiLP,LiuW,LiuH,ZhuF,ZhangDZ,Shen increasedintracellularBBRconcentrations.Ourfollowingsystematicstructuralmodification
H,etal.(2015)SynergisticAntifungalActivityof
andreconstructionofBBRcoreidentifiedthenovelscaffoldofN-(2-(benzo[d][1,3]dioxol-5-
BerberineDerivativeB-7bandFluconazole.PLoS
ONE10(5):e0126393.doi:10.1371/journal. yl)ethyl)-2-(substitutedphenyl)acet-amidederivatives7a-i,includingB-7bandB-7dexhibit-
pone.0126393 ingremarkablesynergisticantifungalactivityandlowcytotoxicity.Here,thestudymainlyin-
AcademicEditor:JoySturtevant,LouisianaState vestigatedthesynergisticactivityofFLCandB-7bandtheunderlyingmechanism.Invitro
University,UNITEDSTATES interactionofFLCandB-7bwasinvestigatedagainst30FLC-resistantclinicalisolatesofC.
Received:November28,2014 albicansandnon-C.albicansspecies,includingCandidatropicalis,Candidaparapsilosis,
Candidaglabrata,CandidakruseiandCryptococcusneoformans.Thepotentsynergistic
Accepted:April1,2015
activityofB-7bincombinationwithFLCagainstFLC-resistantC.albicanswasfound
Published:May19,2015
throughthecheckerboardmicrodilutionassay.Thefindingsofagardiffusiontestsandtime-
Copyright:©2015Lietal.Thisisanopenaccess killcurvesconfirmeditsbettersynergismwithFLC.Andasexpected,B-7bexhibitedmuch
articledistributedunderthetermsoftheCreative
lowercytotoxicitythanBBRtohumanumbilicalveinendothelialcells.IncontrasttoBBR,
CommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany wefoundthatendogenousROSaugmentationwasnotinvolvedinthesynergismofFLC
medium,providedtheoriginalauthorandsourceare andB-7b.Accordingtotheresultsfromourpresentcomparativeproteomicstudy,itseemed
credited. thatthedisruptionofproteinfoldingandprocessingandtheweakeningofcells’self-defen-
DataAvailabilityStatement:Allrelevantdataare siveabilitycontributedtothesynergismofFLCandB-7b.Together,theseresultssuggested
withinthepaperanditsSupportingInformationfiles.
novelscaffoldBBRderivativeB-7bcouldbeapromisingsynergistincombinationwithFLC
Funding:ThisstudywassupportedbytheNational forthetreatmentofinvasivefungalinfections.
ScienceFoundationofChina
(81330083,81202563,81471924),NationalKeyBasic
ResearchProgramofChina(No2013CB531602),
NationalScienceandTechnologyMajorProjectfor
theCreationofNewDrugs(No2013ZX09103001-
014),theShanghaiScienceandTechnologySupport Introduction
Program(No14401902200,No14431902200),
Candidaalbicans,oneofprevalenthumanfungalpathogens,mainlycausingsuperficialmyco-
NationalScienceandTechnologyMajorProjectfor
ses,invasivemucosalinfections,anddisseminatedsystemicdisease,isstillthemostcommon
theCreationofNewDrugs(No2013ZX09J13108-
03B);theShanghaiManufacture-Education- pathogenicfungus,andtheinfectedpatientshaveamortalityrateofapproximately40%,
PLOSONE|DOI:10.1371/journal.pone.0126393 May19,2015 1/20
SynergismofBerberineDerivativeB-7bandFluconazole
Research-MedicalCooperativeProject(No althoughanincreaseinthefrequencyofinfectionsduetonon-C.albicansspecies,including
12DZ1930505). Candidatropicalis,Candidaparapsilosis,Candidaglabrata,CandidakruseiandCryptococcus
CompetingInterests:Theauthorshavedeclared neoformans[1–11].Inspiteoftheneedforeffectiveantifungaltherapyisincreasing,theavail-
thatnocompetinginterestsexist. ableantifungalagentsarestilllimited.Fluconazole(FLC)ismostwidelyusedduetoitshigh
bioavailabilityandlowtoxicity[12,13].However,withtheincreasingclinicaluseofFLC,drug-
resistantisolatesareemergingrapidly,whichhavesignificantlylimitedtheeffectivenessofFLC
andcontributedtothefailureofitstreatmentforC.albicansinfectionsintheclinic[14,15].
Berberine(BBR),analkaloidwidelyfoundinplantfamiliesincludingHydrastiscanadensis
(goldenseal),Berberisaquifolium(Oregongrape),andBerberisvulgaris(barberry),iscurrently
demonstratedtohaveantimicrobialactivityagainstdifferentkindsoforganismssuchasbacte-
ria,viruses,protozoansandfungi,andhavemultipleclinicalusesincludingantidiarrheic,anti-
inflammatory,antiarrhythmicandanticancer[16–21].Itssynergisticantifungalpropertiesin
combinationwithsomeknownantifunalagents(suchasFLC,amphotericinBandmiconazole)
havealsobeenreported[22–24].Thebetter-establishedsynergisticcombinationsofBBRwith
azoleshelptoenhancetheantifungalactivitiesofazoles,especiallyforFLCusedasfirst-line
drugagainstcandidiasis,andthereforetheinvestigationoftheinvitrointeractionbetweennat-
uralantimicrobial(e.g.BBR)andsyntheticchemicaltherapeuticagent(e.g.FLC)contributeto
thedevelopmentofnewantifungaltherapeutics[25,26].
WehavedemonstratedthatBBRandFLCusedconcomitantlyishighlyefficaciousinkilling
FLC-resistantC.albicansclinicalstrains[27],andBBRplaysacrucialroleinthesynergistican-
tifungalactivityofFLCandBBR,whiletheroleofFLCistoassistBBRinaccumulatinginC.
albicanscells,especiallyinthenucleus,whereBBRprobablybindstoDNA,causingcellcycle
arrestandDNAdamage,asdescribedindetailpreviously[28].Ourfurtherproteomicstudy
suggestedthatincreasedgenerationofendogenousreactiveoxygenspecies(ROS)andmito-
chondrialaerobicrespirationshiftcontributedtothesynergisticactivityofFLCandBBR
againstFLC-resistantC.albicans[29].
However,BBRitselfisnotagoodsynergisttobeusedincombinationwithFLCbecauseof
itshightoxicity[30,31].Asdescribedindetailpreviously[32],wecarriedoutaseriesofsys-
tematicstructuralmodificationandreconstructionofBBRcore,aimingtoseekingnovelsyner-
gisticagentswithlowercytotoxicitytoimprovetheeffectivenessofFLCagainstFLC-resistant
C.albicans,andidentifiedthenovelscaffoldofN-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-2-
(substitutedphenyl)acetamidessuchasB-8,B-7bandB-7d.
ItishypothesizedthatthenovelscaffoldespeciallyB-7b,whencombinedwithFLC,exerts
potentsynergisticantifungalactivityagainstFLC-resistantC.albicansandotheryeastfungi.In
thisstudy,selectedBBRderivativesweretestedfortheirabilitytoenhancetheantifungaleffica-
cyofFLCbytime-killcurves,agardiffusiontestandcheckerboardmicrodilutionassay.Inad-
dition,acomprehensivecomparativeproteomicanalysiswasperformedtoinvestigatethe
synergisticmechanismbetweenFLCandB-7b.
MaterialsandMethods
Strains
ThirtyclinicalisolatesofFLC-resistantC.albicans,oneFLC-sensitiveC.albicansSC5314,one
C.neoformans56992,C.tropicalisATCC20026,C.parapsilosisATCC22010,C.krusei
ATCC2340andC.glabrataATCC1182providedbyprofessorChangzhongWang(Schoolof
integratedtraditionalandwesternmedicine,AnhuiuniversityoftraditionalChinesemedicine,
Hefei,China)wereusedinthisstudy.AllstrainsweremaintainedonSDAagar(1%peptone,
4%dextrose,and1.8%agar)platesandre-culturedatleastmonthlyfrom-80°Cstock.Foruse
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SynergismofBerberineDerivativeB-7bandFluconazole
Fig1.ThestructuresofBBRandBBRderivatives(B-8,B-7b,B-7d).
doi:10.1371/journal.pone.0126393.g001
intheexperiments,yeast-phasecellsofthevariousstrainsweregrownYPDbrothovernightin
arotaryshakerat30°C.
Agents
Drugspreparedindimethylsulfoxide(DMSO)includedFLC(Pfizer-RoerigPharmaceuticals,
NewYork,NY),BBR(Sigma-Aldrich,St.Louis,MO)andBBRderivativesB-8,B-7bandB-7d
(Fig1)structuredandidentifiedbymethodsshownin[32],andtheirinitialstoredconcentra-
tionwas6.4mg/mlinDMSO[27].
Checkerboardmicrodilutionassay
TheinvitroMICsofthecompoundsagainstall30clinicalisolatesofC.albicansweredeter-
minedbythemicrobrothdilutionmethodaccordingtotheClinicalandLaboratoryStandards
Institute(formerlytheNationalCommitteeforClinicalLaboratoryStandards)asdescribed
previously[27].TheinitialconcentrationoffungalsuspensioninRPMI1640mediumwas
103CFU/ml,andthefinalconcentrationsrangedfrom0.125to64μg/mlforFLCand1to
32μg/mlforB-7b.ThefinalconcentrationforFLCorB-7balonerangedfrom0.125to
64μg/ml.96-wellflat-bottomedmicrotitrationplateswereincubatedat35°Cfor24hor72h.
Opticaldensitywasmeasuredat630nm.MIC weredeterminedasthelowestconcentration
80
ofthedrugs(aloneorincombination)thatinhibitedgrowthby80%,comparedwiththatof
drug-freewells.
Thedataobtainedbythecheckerboardmicrodilutionassayswereanalyzedusingthe
model-fractionalinhibitoryconcentrationindex(FICI)methodbasedontheLoeweadditivity
theory.Thefractionalinhibitoryconcentrationindex(FICI)isdefinedasthesumoftheMIC
ofeachdrugwhenusedincombinationdividedbytheMICofthedrugusedalone.Synergy
andantagonismweredefinedbyFICIsof(cid:1)0.5and>4,respectively.AnFICIresultof>0.5
but(cid:1)4wasconsideredindifferent[33].
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SynergismofBerberineDerivativeB-7bandFluconazole
Agardiffusiontest
C.albicans103(oneFLC-resistantisolatewithaMICof>64μg/mlforB-7b)wastestedby
agardiffusionassayasdescribedpreviously[27].A3-mlaliquotof106-CFU/mlsuspension
wasspreaduniformlyontotheyeastextract-peptone-dextroseagarplatewithorwithout
4μg/mlFLC.Then,6-mmpaperdisksimpregnatedwithFLCandB-7baloneorincombina-
tionwereplacedontotheagarsurface.Therewas5μlofDMSOincontroldisks.Inhibition
zonesweremeasuredafterincubationat35°Cfor48h.
Time-killtest
C.albicans103inRPMI1640mediumwaspreparedatthestartinginoculumof103CFU/ml
or105CFU/ml.Theconcentrationswere4μg/mlofB-7band8μg/mlofFLC.DMSOcom-
prised<1%ofthetotaltestvolume.Atpredeterminedtimepoints(0,4,8,12,16,24,36and
48h)afterincubationwithagitationat35°Cinashakingincubator,100-μlaliquotwasre-
movedfromeverysolutionandseriallydiluted10-foldinsterilewater.A100-μlaliquotfrom
eachdilutionwasspreadonthesabourauddextroseagarplate.Colonycountsweredetermined
afterincubationat35°Cfor48h.Synergismandantagonismweredefinedasarespectivein-
creaseordecreaseof(cid:3)2log CFU/mlinantifungalactivityproducedbythecombination
10
comparedwiththatbythemoreactiveagentaloneafter24h,whileachangeof<2log CFU/
10
mlwasconsideredindifferent[27].
CytotoxicityevaluationofBBRderivativesusingXTTassay
Humanumbilicalveinendothelialcell(HUVEC)wasculturedinDMEMmedium(HyClone)
supplementedwith10%fetalbovineserum(HyClone).ThecytotoxiceffectofB-7bon
HUVECviabilitywasassessedbytheXTTassay[34].Briefly,cells(3×103cells/well)werecul-
turedin96-wellmicrotiterplatesandtreatedwithdifferentconcentrationsofB-7bfor24h.At
theendofincubation,50μLofPMS-XTTsolution(finalconcentration,50μgofXTTand
0.38μgofPMSperwell)wasaddedtoeachwellandincubatedat37°Cfor4h.Absorbanceat
450nmwasmeasuredusingtheElizaPlateReader.
ProteinsamplepreparationandNanoLC-MS/MS
FLC-resistantC.albicans103cells(OD =0.1)weretreatedoruntreatedwithFLC(64μg/ml)
600
and/orB-7b(16μg/ml)for9hat30°C.Cellswereharvestedandwashedwithphosphate-buff-
eredsaline(PBS,pH7.4)buffer.Next,thecellpelletwaslysedin10mloflysisbuffer[50mM
Tris,1.5mMEDTA,1%(v/v)Triton×100,0.4%(w/v)SDS,pH7.5]and20mlof0.5mmdiam-
eterglassbeads(Biospec,Bartlesville,OK)byvortexingfor30secondsandcoolingonicefor
30secondsrepeatedlyinaMiniBead-beater(Biospec)untilatleast80%ofthecellshadbeen
lysedasdeterminedbyphase-contrastmicroscopicexamination.Aftercentrifugationat13000
gfor5min,theclarifiedproteinsupernatantwasdeterminedusingtheRCDCProteinAssay
Kit(Bio-Rad,Herclues,CA)andaccordingtothemanufacturer’sdirections.
TheaboveobtainedproteinsupernatantwasappliedtoanEASY-nanoliquidchromatogra-
phy(LC)system(ProxeonBiosystem)coupledonlinetoanESI-LTQ-OrbitrapVelosmass
spectrometer(ThermoFisherScientific)asapreviouslydescribedmethod[35].Thedatabase
searcheswereperformedwiththefollowingparameters:CandidaGenomeDatabase(CGD),C.
albicansSC5314_version_A22-s04-m01-r04_orf_trans_all.fasta.Proteinabundancewascalcu-
latedfromallqualifiedcorrespondingpeptidesmatchedtothatproteinandfinalresultswere
filteredusinga1%falsediscoveryrate(FDR).
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SynergismofBerberineDerivativeB-7bandFluconazole
Bioinformaticanalysis
Toinvestigatebiologicalsignificance,theidentifieddifferentialproteinsineachcomparison
wereinputintoDAVIDbioinformaticsresources6.7(theDatabaseforAnnotation,Visualiza-
tion,andIntegratedDiscovery,http://david.abcc.ncifcrf.gov/)forfunctionalannotationand
KEGG(theKyotoEncylopediaofGenesandGenomes)pathwayanalysis[36].Briefly,DAVID
functionalannotationandKEGGpathwayanalysiswereperformedonthelistofdifferential
proteinswithdifferentialratioofover±1.2andanominalp-valueoflessthan0.05ineach
comparison.FortheGeneOntology(GO)enrichmentinDAVID,theGOtermsof“Biological
Process”,“CellularComponent”and“MolecularFunction”wereused,andonlythetermsthat
reportedap-valueof(cid:1)0.05andcountnumber(cid:3)5proteinswereselected.Thesignalingpath-
wayswereidentifiedandmappedusingDAVIDandtheKEGGAutomaticAnnotationServer
(KAAS)[37].TheKEGGpathwayswithatleast5proteinsandp-valueof<0.05wereidentified
asenriched.
Further,protein-proteininteraction(PPI)networksofthedifferentialproteinswerepre-
dictedandanalyzedusingSTRINGv9.1(SearchToolfortheRetrievalofInteractingGenes)
database[38].STRING,awebresource(http://string-db.org/)dedicatedtoprotein-proteinin-
teractionsincludingdirect(physical)andindirect(functional)associations,quantitativelyinte-
gratesinformationfromnumeroussourcesincludinggenomiccontext,high-throughput
experiments,(Conserved)co-expressionandpreviousknowledge.Knownandpredictedpro-
tein-proteininteractionsarescoredandintegrated,resultingincomprehensiveproteinnet-
workscurrentlycovering5,214,234proteinsfrom1133organismsintheSTRINGdatabase.
Thisanalysisgaveuniquelycomprehensivecoverageofbothexperimentalandpredictedinter-
actioninformationwitharelativeconfidencescore,implyingthatonlyinteractionswithhigh
levelofconfidencewereextractedandconsideredasvalidlinksforthePPInetwork[38–40].In
agivennetwork,proteinsarerepresentedasnodesandtheinteractionsaredefinedasedges
(lines),andthickeredgesrepresentstrongerassociations.ForaPPInetwork,themajorityof
thenodeswerelinkedwitheachother,whilesomeofalteredproteinswereisolatedwithout
partners.The“hub”proteinswereproteinswithanumberofedges,meaningthecapabilityof
physicallyinteractingwithmanypartners.
Statisticalanalysis
Atleastthreebiologicalreplicateswereperformedforallexperimentsunlessotherwiseindicat-
ed.Analysisofvariance(ANOVA)wasusedwhenmultiplegroupswereanalyzedandthe
two-tailedStudent’st-testwasusedforanalysisoftwogroups,withpairedanalysiswhenap-
(cid:4) (cid:4)(cid:4)
propriate.Statisticalsignificancewassetatapvalueof0.05,0.01or0.001,indicatedby , ,
(cid:4)(cid:4)(cid:4)
,respectively.
Results
ComparisonoftheactivitiesofthreeBBRderivativesandBBRagainst
FLC-resistantC.albicans
ThehighlyeffectiveinvitrosynergismofFLCandBBRagainstFLC-resistantclinicalisolates
ofC.albicanshasbeenverified[27].ToseekwhetherN-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-2-
(substitutedphenyl)acetamidederivativeshavethesameantifungaleffectivenessasBBR,We
performedacomparisonofBBRandthethreeBBRderivatives(B-8,B-7bandB-7d)against
FLC-resistantC.albicans103bydifferentmethods.Ataconcentrationof16μg/mlforBBR
andBBRderivativesinthetimekillingassay,asshowninFig2A,theclarityofsuspensioncul-
turetreatedwithB-7bandFLC(10μg/ml)after24h,wasobviouslyclearerthanthoseofthe
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SynergismofBerberineDerivativeB-7bandFluconazole
Fig2.GrowthconditionofC.albicans103treatedwithFLC,BBRanddifferentBBRderivatives.
ExponetiallygrowingFLC-resistantC.albicans103weretreatedwithorwithoutFLC(10μg/ml),BBR
(16μg/ml)anddifferentBBRderivatives(16μg/mlB-8,B-7b,B-7d)aloneorthecombinationsofFLCand
BBRorBBRderivativesinashakingincubator.(A)PicturesofthegrowthconditionofC.albicanswithan
initialinoculumof105CFU/mlweretakenafter24hincubation.(B)ThegrowthchangeofC.albicans103
withaninitialinoculumof105CFU/mlafter24h.Aliquotswereobtainedat24handseriallydilutionswere
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SynergismofBerberineDerivativeB-7bandFluconazole
spreadedonagarplates.Colonycountsweredeterminedafter48hincubation.(C)Agardiskdiffusionassay
ofdifferentcompounds(BBR,B-8,B-7bandB-7d)combinedwithFLCagainstC.albicans103.Panels1and
3showagarplates,andpanel2showsanagarplatecontaining4μg/mlFLC.Panel4describestheimages
forpanels1and2,whichcontaining4μgofBBR,B-8,B-7b,B-7dand2μgofFLCorjustDMSOascontrol
perdisc.Panel5describestheimageforpanel3,thecombinationof4μgcompounds(BBR,B-8,B-7band
B-7d)withFLC(2μg)orFLC(2μg)aloneascontrolwerecontainedineachdisc.
doi:10.1371/journal.pone.0126393.g002
othertwocompounds.ThestatisticalanalysisforcolonycountsindicatedthatB-7bhadbetter
synergisticactivitythantheothertwocompoundsincombinationwithFLC(10μg/ml)against
C.albicans103withtheinitialinoculumof105CFU/ml,thoughweakerthanthatofBBR
(Fig2B).
Furthertovisualizetheirsynergisticantifungaleffect,allcompoundsandthecombination
withFLC(4μg/ml)wereanalyzedbyagardiffusiontests(Fig2C).Allcompounds(BBR,B-8,
B-7bandB-7d)hadnoantifungalactivityinsmallamountsat4μgalone(Fig2C1).At4μg,
BBRcombiningwithFLCshowednosynergisticantifungalactivity.Incontrast,thethreeBBR
derivativesshowedpotentsynergisticantifungaleffectsontheagarplatewith4μg/mlFLC
(Fig2C2).ThemeandiametersoftheinhibitoryzonesforBBR,B-8,B-7bandB-7dwere0,
25.2,27.0and26.3mm,respectively.Inaddition,asshowninFig2C3,thesizesoftheinhibito-
ryzoneswere0,17.1,17.6and17.6mmarounddisksimpregnatedwith2μgFLCpluscom-
pounds(BBR,B-8,B-7bandB-7d),respectively.Insmallamounts(suchas4μg),B-7bhad
slightlybetter(orthesame)synergisticantifungalactivitycomparedwithB-8andB-7d,but
muchbetterthanBBR.Therefore,weconductedallsubsequentstudieswithB-7basarepresen-
tativeexample.
IncombinationwithFLC,B-7batlowerconcentrationsexhibitsbetter
synergisticactivitythanBBRagainstFLC-resistantC.albicans
Astheabovedescription,insmallamountsat4μg,B-7bhadmorepowerfulsynergisticeffect
thanBBR(Fig2C).InordertodeterminewhetherB-7batlowerconcentrationsincombina-
tionwithFLCshowedbettersynergisticactivitythanthatofBBRagainstFLC-resistantC.albi-
cans103,theirsynergismwasalsoconμrmedbytime-killingtest(Fig3).BBR(4μg/ml),B-7b
(4μg/ml)orFLC(8μg/ml)alonehadnoeffectonthegrowthofC.albicans103after48h.
However,theantifungalactivityofFLCwasdramaticallyraisedbyadditionofBBRorB-7b.
Undertheinitialinoculumof103CFU/ml(Fig3A),thecombinationofFLCwithBBRorB-7b
respectivelyproduceda4.56or5.24-log -CFU/mldecreasecomparedwiththenumberof
10
CFUproducedby8μg/mlofFLCaloneat48h(Table1).Giventhebeginninginoculumof105
CFU/ml(Fig3B),theFLC+BBRorFLC+B-7bcombinationyielded2.22or3.12-log -CFU/ml
10
decreasecomparedwiththeFLCaloneat48h(Table1).Despiteofthestartinginoculum,the
combinationofFLCandB-7byieldedasignificantdecreaseofCFU/mlcomparedwiththe
combinationofFLCandBBRatboth24hand48h(Fig3Cand3D).Interestingly,thesignifi-
cancelevelforthesynergisticeffectofFLCandB-7bcomparedwiththatofBBRwasobserved
earlier(at24h)andmuchmoreprominent(p<0.001),whentheinitialinoculumdeclined
from105CFU/mlto103CFU/ml(Fig3Cand3D).
ToinvestigatethelowerdosageofB-7bwhichincombinationwithFLCstillexhibitsitssyn-
ergism,differentconcentrationsofB-7b,FLCandthecombinationofB-7bandFLC(2μg)
wereanalyzedbyagardiskdiffusionassayasdescribedindetailpreviously[27].B-7baloneat
8,4,2,1μgperdischadnoantifungalactivityagainsttheFLC-resistantC.albicans103(Fig
4A).WhileFLCat2μgperdiscshowedonlyweakinhibitioneffectagainstC.albicans,the
halosurroundingthediscwascloudywithcolony(Fig4Aand4C).B-7bat2μgstillshowedpo-
tentsynergisticantifungaleffectontheagarplatecontaining4μg/mlFLC(Fig4B).Themean
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Fig3.TimekillingcurvesofC.albicans103treatedwithdifferentconcentrationsofB-7b,BBRand
FLC.FLC-resistantC.albicans103weretreatedwithFLC(8μg/ml),B-7b(4μg/ml),BBR(4μg/ml),FLC+B-
7b(8+4)μg/mlandFLC+BBR(8+4)μg/mlbyusinginitialinoculumsof103CFU/ml(A,C)and105CFU/ml
(B,D).Aliquotswereobtainedattheindicatedtimepointsandseriallydilutionswerespreadonagarplates.
Colonycountsweredeterminedafter48hincubation.
doi:10.1371/journal.pone.0126393.g003
diametersoftheinhibitoryzonesfor1,2,4and8μgB-7bwere0,16.7,27.8and33.3mm,re-
spectively.Furthermore,whendifferentamountsofB-7b(1,2,4and8μg)wascombinedwith
FLC(2μg),theclearhalossurroundingthediscwereobserved,andthemeandiametersof
thesezoneswere15.0,15.6,17.8and18.3mm,respectively.
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Table1. Decreaseinlog CFU/mlofC.albicans103usingBBRorB-7bcombiningwithFLCat24hor
10
48h.
Mean(±SD)Decreaseinlog CFU/mlofC.albicans103usingBBRorB-
10
7bcombiningwithFLCat24hand48h.
103CFU/ml 105CFU/ml
24h 48h 24h 48h
FLC8μg+BBR4μg 2.26(0.21) 4.56(0.07) 2.03(0.06) 2.22(0.02)
FLC8μg+B-7b4μg 2.95(0.17) 5.24(0.02) 2.28(0.07) 3.12(0.005)
doi:10.1371/journal.pone.0126393.t001
B-7bhasmuchlowercytotoxicitywhencomparedtoBBRinvitro
ItisnecessarytoassessthetoxicityofB-7bbecauseofthehightoxicityofBBRitself[30,31].In
thestudy,invitrocytotoxiceffectofB-7bagainstHUVECwasperformedviaXTTassays.As
showninFig5,B-7bhadnotoxiceffectonHUVECattheconcentrationof128μg/ml,while
BBRsignificantly(p<0.001)reducedcellviability(cellviability<50%)atconcentrations
(cid:3)128μg/ml.theresultsindicatedthatB-7bshowedmuchlowertoxicityandwasquitepromis-
ingwhencomparedtoBBR.
Fig4.AgardiskdiffusionassayofdifferentconcentrationsofBBRderivativescombinedwithFLC
againstC.albicans103.AgardiskdiffusionassayofdifferentconcentrationsofB-7bcombinedwithFLC
againstC.albicans103.PanelsAandCshowagarplates,andpanelBshowsanagarplatecontaining
4μg/mlFLC.PanelDdescribestheimagesforpanelsAandB,whichcontaining1,2,4,8μgofB-7band2μg
ofFLCorjustDMSOascontrolperdisc.PanelEdescribestheimageforpanelC,thecombinationofB-7b(1,
2,4,8μg)withFLC(2μg)orFLC(2μg)aloneascontrolwerecontainedineachdisc.
doi:10.1371/journal.pone.0126393.g004
PLOSONE|DOI:10.1371/journal.pone.0126393 May19,2015 9/20
SynergismofBerberineDerivativeB-7bandFluconazole
Fig5.TheinvitrocytotoxicityevaluationofB-7busingXTTassay.ThecytotoxiceffectofB-7btowards
HUVECviabilitywasassessedbyanXTTtestfollowinga4-htreatment,whencomparedtothatofFLCand
BBR.***,P<0.001versuscellstreatedwithBBR.
doi:10.1371/journal.pone.0126393.g005
ThecombinationofFLCandB-7bagainstclinicalFLC-resistantC.
albicans
DuetothestrongsynergisticactivityincombinationwithFLCwhenagainstFLC-resistantC.
albicans103,wefurtherconfirmedthesynergisticactivityofB-7bincombinationwithFLC
against30clinicalstrainsofFLC-resistantC.albicans.TheMICresultsofB-7baloneorin
combinationwithFLCagainst30clinicalFLC-resistantC.albicanstestedbyCheckerboard
microdilutionassayweresummarizedinTable2.TheMICsofB-7borFLCaloneagainstall
testedstrainswere>64μg/ml,respectively.However,thecombinationofFLCandB-7b
markedlyreducedtheMIC s,andthemedianFICIwas0.016(range,0.012to0.07),whilethe
80
medianFICIofBBR+FLCwas0.034(range,0.017to0.127)asshowninthepreviousreport
[27].B-7bincombinationwithFLCdisplayedpowerfulsynergisminall30FLC-resistantC.
albicans(100%)tested,accordingtotheanalysisofFICImethod.
ThecombinationofFLCandB-7bagainstvariedyeaststrains
WealsotestedantifungalactivityofB-7baloneorincombinationwithFLCinotheryeast
strains(FLC-sensitiveC.albicans,C.neoformans,C.tropicalis,C.krusei,C.parapsilosisandC.
Table2. MICsofB-7baloneandincombinationwithFLCagainst30clinicalC.albicans.
MIC80(μg/ml)
median range
FLC >64 >64
B-7b >64 >64
FLC/B-7ba 1/1 0.5-8/1-2
FICindex 0.016 0.012–0.07
interactioneffect(n/%)b Syn(30/100)
aMICincombinationexpressedas[FLC]/[B-7b].FLC:Fluconazole.
bSyn,synergism.Thenumberofstrainsandpercentagefortheinteractioneffectwereshown.
doi:10.1371/journal.pone.0126393.t002
PLOSONE|DOI:10.1371/journal.pone.0126393 May19,2015 10/20