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A new coumarin derivative, 4-acetatecoumarin, with antifungal activity and association study against Aspergillus spp.

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h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Clinical

Microbiology

A

new

coumarin

derivative,

4-acetatecoumarin,

with

antifungal

activity

and

association

study

against

Aspergillus

spp.

Felipe

Q.S.

Guerra

a,∗

,

Rodrigo

S.A.

Araújo

b

,

Janiere

P.

Sousa

a

,

Viviane

A.

Silva

a

,

Fillipe

O.

Pereira

c

,

Francisco

J.B.

Mendonc¸a-Junior

b

,

José

M.

Barbosa-Filho

b

,

Julio

Abrantes

Pereira

a

,

Edeltrudes

O.

Lima

a

aUniversidadeFederaldaParaíba,DepartamentodeciênciasFarmacêuticas,JoãoPessoa,PB,Brazil bUniversidadeEstadualdaParaíba,DepartamentodeCiênciasBiológicas,JoãoPessoa,PB,Brazil cUniversidadeFederaldeCampinaGrande,Centrodeeducac¸ãoesaúde,Cuité,PB,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21June2016 Accepted30June2017

Availableonline12October2017 AssociateEditor:LuisHenrique Guimarães Keywords: Coumarin Antifungalactivity A.flavus A.fumigatus Modeofaction

a

b

s

t

r

a

c

t

Fungalinfectionshavebecomeaconcernforhealthprofessionals,andtheemergenceof resistantstrainshasbeenreportedforallknownclassesofantifungaldrugs.Amongthe fungicausingdisease,wehighlightthosethatbelongtothegenusAspergillus.Forthese reasons,thesearchfornewantifungalsisimportant.Thisstudyexaminestheeffectsofa coumarinderivative,4-acetatecoumarin(Cou-UMB16)bothaloneandtogetherwith antifun-galdrugs,anditsmodeofactionagainstAspergillusspp.Cou-UMB16wastestedtoevaluate itseffectsonmyceliagrowth,andgerminationofAspergillusspp.fungalconidia.We inves-tigateditspossibleactiononcellwalls,onthecellmembrane,andalsothecapacityof thiscoumarinderivativetoenhancetheactivityofantifungaldrugs.Ourresultssuggest thatCou-UMB16inhibitsAspergillusspp.virulencefactors(myceliagrowthandgermination ofconidia)andaffectsthestructureofthefungalcellwall.WhenapplyingCou-UMB16in combinationwithazoles,bothsynergisticandadditiveeffectswereobserved.Thisstudy concludesthatCou-UMB16inhibitsmycelialgrowthandsporegermination,andthatthe activityisduetoitsactiononthefungalcellwall,andthatCou-UMB16couldactasan antifungalmodifier.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Correspondingauthor.

E-mail:felipeqsguerra@gmail.com(F.Q.Guerra).

https://doi.org/10.1016/j.bjm.2017.06.009

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Introduction

Inrecentyears,fungalinfectionshavebecomeaconcernfor healthprofessionals,andtheemergenceofresistantstrains hasbeenreportedforallknownclassesofantifungaldrugs. Asaresult,especiallyinimmunocompromisedpatients, mor-tality, morbidity, and the cost of treatment against fungal pathogens have all increased. Among the disease-causing fungi,wehighlightthosethatbelongtothegenusAspergillus. Aspergillusfumigatusisthemostcommonspeciesisolatedin casesofinvasiveaspergillosis,thespeciesAspergillusflavus, Aspergillusniger,andAspergillusterreusfollowclosebehind.In certainlocations,A.flavusandA.terreusspeciesarefrequently isolated.1,2

A. fumigatus,being saprotrophic, playsan essential role inrecyclingcarbonandnitrogenworldwide.Thefungushas ahighcapacitytosporulatewhichresultsinanubiquitous presence,both indoors and outdoors, ofhigh atmospheric conidia concentrations(1–100conidia/m3).A. fumigatusis a

prevalentairbornefungal pathogen,andcauses severeand usually fatal invasive infections in immunocompromised hosts.3

A. flavus is the second leading cause of invasive aspergillosisand is the most common cause ofsuperficial fungal infections. Particularly common clinical syndromes associated with A. flavus include chronic granulomatous sinusitis, keratitis, cutaneous aspergillosis, wound infec-tions,andosteomyelitis(followingtraumaandinoculation). In addition, A. flavus produces aflatoxins, the most toxic and potent natural hepatocarcinogenic compounds ever characterized.4

Untilrecently,theonlydrugsavailabletotreataspergillosis wereamphotericinBanditraconazole,thelatterinoraland intravenousformulations.Recentlyvoriconazole, posacona-zole,andcaspofunginhavealsobeenapprovedfortreatment. Althoughresistancetoantifungal drugsis notascommon asresistanceto antibacterialagents, therehas been a sig-nificantincreaseinthenumberofreportedcasesoffungal resistance.SomeinvestigatorshaveisolatedstrainsofA.flavus

frompatientsamplesthatareresistanttoamphotericinBand itraconazole.1,4

For these reasons, the search for new antifungals is important.Inworkrecentlyreportedbyourgroup,the anti-fungalactivityof24coumarinderivativesagainstA.fumigatus

and A. flavus were described. Certain derivatives showed significantantifungalactivitywithMinimumInhibition Con-centration (MIC) values ranging from 16 to 1024␮g/mL, including 4-acetatecoumarin (Fig. 1) with an MIC value of 16␮g/mL.6

Theseresultsmotivatedustoassessmorethoroughlythe activityofthiscoumarinderivativeonthefungalcell.Another promisingalternativeisthepossibilityofcombiningcoumarin derivativeswithantifungalsagainststrainsofAspergillusspp. Combinationtherapyofthe availableantifungaldrugswith naturalproductsandtheirderivativesisevenlessexplored, especiallyinthecaseofcoumarinderivatives.6,7Theaimof

thisstudywastoexaminetheeffectsofcoumarinderivative, 4-acetetatecoumarin,bothaloneandtogetherwithantifungal drugsanditsmodeofactionagainstAspergillusspp.

O O

O

O

Fig.1–Chemicalstructurefor4-acetatecoumarin (Cou-UMB16).

Material

and

methods

Microorganisms

Aspergillusspp.usedintheantifungalassaywasobtainedfrom thearchivalcollectionoftheFederalUniversityofParaíba Lab-oratory ofMycology(LM).Theyincluded A.fumigatusATCC 46913 and A. flavusATCC 16013.Stock inoculates (suspen-sions)ofAspergillusspp.werepreparedfrom8-dayoldpotato dextroseagar(DifcoLab.,USA);the culturesgrownatroom temperature.Fungalcolonieswerecoveredwith5mLof ster-ilesalinesolution(0.9%),thesurfacewasgentlyagitatedwith vortexes.Thefungalelementswithsalinesolutionwere trans-ferredtosteriletubes.Inoculatewasstandardizedat0.5tube ofMcFarlandscale(106CFU/mL).Thefinalconcentration

con-firmation was done by counting the microorganisms in a Neubauerchamber.8,9

Chemicals

The product tested was the coumarin derivative,

4-acetatecoumarin (Cou-UMB16), obtained by biosynthesis.5 Amphotericin B,fluconazole,itraconazole andvoriconazole were obtainedfromSigmaAldrich®,Brazil.Thedrugswere dissolved inDMSO(dimethylsulfoxide),and steriledistilled waterwas usedtoobtain solutions of1024␮g/mLfor each antifungal.TheconcentrationofDMSOdidnotexceed0.5% intheassays.

Culturemedia

Totestthebiologicalactivityoftheproducts,PotatoAgar(AP), Sabourauddextroseagar(SDA)(purchasedfromDifco Labora-tories,Detroit,MI,USA),andRPMI-1640-Lglutamine(without sodiumbicarbonate)(Sigma–Aldrich,SãoPaulo,SP,Brazil) cul-turemediawereused.Theywerepreparedandusedaccording tothemanufacturers’instructions.

Effectsonmyceliagrowth

Analysis of the interference of the coumarin derivative, 4-acetatecoumarin, on mycelia growth was performed by

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determiningthedry myceliaweightsofA. fumigatus(ATCC 46913)andA.flavus(ATCC16013).11,12 FlaskscontainingMIC

(16␮g/mL)andMIC×2(32␮g/mL) ofcoumarinderivativein RPMI-1640medium,wereinoculatedwithsuspensionofthe

A. fumigatus (ATCC 46913) and A. flavus (ATCC 16013) test strains.Inthecorrespondingcontrol,theamountofcoumarin derivative was substituted by distilled water. The system wasincubatedat28◦Cfor8days.Flaskscontainingmycelia werefilteredthroughWhatman®Grade1QualitativeFiltration Paper(particleretention:11␮m),andthenwashedwith dis-tilledwater.Themyceliaweredriedat60◦Cfor6h,andkeptat 40◦Covernight.Thefilterpapercontainingdrymyceliafrom twoindependentassayswereweighed,andthemeanvalues obtained.Thepercentagegrowthinhibitionbasedonthedry weightofeachattimeofanalysis,wascalculatedaccording toSharmaandTripathi.10

Conidialgerminationassay

The coumarin derivative, 4-acetatecoumarin (Cou-UMB16), and amphotericinBwere testedto evaluateeffectson the germinationoffungalconidiaofA.fumigatus(ATCC46913), andA.flavus(ATCC16013).FlaskscontainingMIC(16␮g/mL) andMIC×2(32␮g/mL)ofcoumarinderivativeandacontrol withdistilledwater,were used.In steriletesttubes, 500␮L ofRPMI-1640plusthe Cou-UMB16were evenlymixed with 50␮L offungal conidia suspension and immediately incu-batedat28◦C.Samplesofthismixtureweretakenafter24h ofincubationforanalysis.Theexperimentwasperformedin duplicate,wherethe200conidiawasanalyzedinaNeubauer chamber,andtheinhibitionpercentageofsporegermination ateachtimepointwascalculatedbycomparingtheresults obtainedinthetestexperimentswiththeresultsofthe con-trolexperiment.Theanalysiswasconductedunderanoptical microscope(Zeiss®PrimoStar).13,14

Theeffectsofsorbitolongrowthafterexposureto antifungalagents

Theassaywasperformedusing mediumwithandwithout sorbitoltoevaluatepossiblemechanismsinvolvedinthe anti-fungalactivityofthetestproductontheAspergillusspp.cell wall.Thesorbitolwasaddedtotheculturemediuminafinal concentrationof0.8M.Theassaywasperformedby microdi-lutionmethodin96-wellplates ina“U”(Alamar,Diadema, SP,Brazil).8,9Theplatesweresealedaseptically,incubatedat

28◦C,andreadingsweretakenat3days.Basedonthe abil-ityofsorbitoltoactasafungalcellwallosmoticprotective agent,thehigherMICvaluesobservedinthemediumwith sorbitoladdedascomparedtothestandardmediumsuggest thatthecellwallisoneofthepossiblecelltargetsforthe prod-ucttested.15,16AmphotericinBwasusedasthecontroldrug.

Theassaywasperformedinduplicateandexpressedasthe geometricmeanoftheresults.

ErgosterolbindingassayMICvaluedeterminationinthe presenceofergosterol

Toassessif theproductbindstofungal membranesterols,

an experiment was performed according to the method

described by Escalante,16 with some modifications. The

ergosterolwaspreparedaswasdescribedbyLeiteetal.15The

MICofcoumarinderivative,4-acetatecoumarin(Cou-UMB16) against Aspergillus spp. was determined by using broth microdilutiontechniques,8,9 inthepresenceandabsenceof

exogenous ergosterol (Sigma–Aldrich, São Paulo, SP, Brazil) added totheassay medium,indifferent linesofthesame micro-plate. Briefly, a solution of Cou-UMB16 was doubly dilutedseriallywithRPMI-1640(volume=100␮L)containing ergosterolataconcentrationof400␮g/mL.Avolumeof10␮L yeast suspension(0,5McFarland) wasadded toeach well. ThesameprocedurewasrealizedforamphotericinB,whose interactionwithmembraneergosterolisalreadyknown, serv-ingasacontroldrug.Theplatesweresealedandincubated at28◦C.Theplateswerereadafter3daysofincubation,and theMICwasdeterminedasthelowestconcentrationoftest agentinhibitingthevisiblegrowth.Theassaywascarriedout induplicateandthegeometricmeanvalueswerecalculated. Thus,thisbindingassayreflectedtheabilityofthecompound tobindwithergosterol.

Checkerboardassay

Acheckerboardmicro-titertestwasperformedtoevaluatethe interaction ofcoumarin derivative 4-acetatecoumarin with theantifungaldrugs(azolesandamphotericinB)againstA. fumigatusATCC46913,andA.flavusATCC16013.Aseriesof 2-folddilutionsineightforeachcoumarinderivativeand anti-fungal drug were made in RPMI-1640 toobtain four times the final concentration in micro-titerwells. Then, 50␮Lof eachdilutionofcoumarinderivativewasaddedtothe96-well micro-titerplatesintheverticaldirection,while50␮Lofeach dilutionoftheantifungaldrugswasaddedinthehorizontal direction,sothatvariouscombinationsofcoumarinderivative andantifungaldrugscouldbeachieved.Inaddition,10␮Lof inoculumfromthesporesuspension(1.5×105CFU/mL)was

added toeach well,andthe plateswere incubated at30◦C for3days.Inordertoevaluatetheactivityofthe combina-tionsofdrugs,fractionalinhibitoryconcentration(FIC)indices werecalculatedasFICA+FICB,whereFICAandFICBrepresent

theminimumconcentrationsinhibitingthefungalgrowthfor drugs Aand B,respectively: FICA=MICAcombination/MICA

aloneandFICB=MICBcombination/MICBalone.AmeanFIC

indexwascalculatedbasedonthefollowingequation: FIC index=FICA+FICB.

Inaddition,theinterpretationwasmadeasfollows: syner-gistic(<0.5),additive(0.5–1.),indifferent(>1),orantagonistic (>4).5,17–19

Dataanalysis

Theresultswereexpressedasmean±SE.Statisticalanalyses were performed witht-test. p<0.05 was considered signifi-cant.

Results

The coumarinderivative, 4-acetatecoumarin, Fig. 1, witha minimuminhibitoryconcentration(MIC)(16␮g/mL)hasbeen

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A

B

100 100 80 80 60 60 40 40 20 20 0 0 a,b a,b a,b a a a a a Conidia ger minated (%) Conidia ger minated (%) Control MIC (16µg/mL) MIC x 2 (32µg/mL) Amph B (2µg/mL) Amph B (4µg/mL)

Fig.2–PercentageofconidialgerminationofA.fumigatus(ATCC46913),A,andA.flavus(ATCC16013),B,intheabsence (control)andpresenceof4-acetatecoumarin(MIC:16␮g/mL;2×MIC:32␮g/mL)andamphotericinB(MIC:2␮g/mL;2×MIC: 4␮g/mL).a:p<0.05comparedtocontrol.b:p<0.05comparedtoamphotericinBwithrespectiveconcentration.

80 80 100 100 60 60 40 40 20 20 0 0 Dr y m ycelial w e ight (%) Dr y m ycelial w e ight (%) a a a,b a a a a a Control MIC (16µg/mL) MIC x 2 (32µg/mL) Amph B (2µg/mL) Amph B (4µg/mL)

A

B

Fig.3–PercentageofdrymyceliaweightproducedbyA.fumigatus(ATCC46913),A,andA.flavus(ATCC16013),B,inthe absence(control)andpresenceof4-acetatecoumarin(MIC:16␮g/mL;2×MIC:32␮g/mL)andamphotericinB(MIC:2␮g/mL; 2×MIC:4␮g/mL).Controlproduced100%ofdrymyceliaweight.a:p<0.05comparedtocontrol.b:p<0.05comparedto amphotericinBwithrespectiveconcentration.

Table1–MICvalues(␮g/mL)ofcoumarinderivative,4-acetatecoumarin(Cou-UMB16),intheabsenceandpresenceof sorbitol(0.8M)andergosterol(400␮g/mL)againstAspergillusspp.

MICvalues(␮g/mL) Microorganisms A.fumigatus ATCC(46913) A.flavus ATCC(16013) A.fumigatus ATCC(46913) A.flavus ATCC(16013) A.fumigatus ATCC(46913) A.flavus ATCC(16013)

Drugs −Sterols +Sorbitol +Ergosterol

Cou-UMB16 16 16 128 128 16 32

AmphotericinB 2 2 – – 16 16

Flucanozole 256 512 512 512 256 512

previouslyobservedbyourgroup,6anddisplayedinhibitory

activityagainstsporegerminationandmycelialgrowthofA. fumigatusATCC46913andA.flavusATCC16013.

ThepercentageofconidiagerminatedbyA.fumigatusATCC 46913andA.flavusATCC16013inthepresenceandabsence (control)oftestproductsisshowninFig.2.Inthetestwith two concentrations(MIC and MIC×2), Cou-UMB16showed significantinhibitoryactionagainstAspergillusspp.spore ger-mination(p<0.05)whencomparedtothecontrol.However, theactivitywaslesspotent(p<0.05)comparedwithrespective concentrationsofamphotericinB.

The effect of different concentrations of test drugs (at MICandMIC×2)onmycelialgrowthwasdeterminedbydry mycelialmassquantification;theresultsareshowninFig.3. Withrespecttoeffects onA. fumigatusATCC 46913and A. flavusATCC16013,concentrationsofCou-UMB16at16␮g/mL

(MIC),and32mL−1(MIC×2)inhibitedmycelialgrowthof nor-malstrains(p<0.05)whencomparedtothecontrol.Further analysis demonstrated that Cou-UMB16 at its MIC×2 was more potentthan amphotericin Batits respective MIC×2 (p<0.05).

Inthisstudy,theMICvaluesofCou-UMB16inculturemedia withorwithoutexogenousergosterolwereidenticalinboth experiments,suggestingthatthecoumarinderivativetested does not act via binding to plasma membrane ergosterol; resultsshowninTable1.

Toinvestigatetheactiononthecellwallofthecoumarin derivativeCou-UMB16wecarriedoutatestwithsorbitol(as anosmoticshield);thetestresultsareshowninTable1.The Cou-UMB16MICvaluesincreased8timesinthepresenceof culturemediumwithsorbitolcomparedtoculturemedium withoutsorbitol,suggestingthatCou-UMB16indeedactson

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Table2–MICofantifungaldrugsandeffectof combinationwithcoumarinderivative,

4-acetatecoumarin(Cou-UMB16),againstA.flavus,ATCC 16013.

Antifungal+Cou-UMB16 MIC(␮g/mL) FICindex (typeof interaction) Cou-UMB16 16 AmphotericinB 2 Flucanozole 256 Itraconazole 128 Voriconazole 2 Cou-UMB16/AmphotericinB 16/2 2(Indifferent) Cou-UMB16/Flucanozole 16/256 2(Indifferent) Cou-UMB16/Itraconazole 2/64 0.63(Additivity) Cou-UMB16/Voriconazole 2/0.5 0.38(Synergism)

Note:Cou-UMB16,4-acetatecoumarin;FIC,fractionalinhibitory con-centration;MIC,minimalconcentrationinhibitory.

Table3–MICofantifungaldrugsandeffectof combinationwithcoumarinderivative,

4-acetatecoumarin(Cou-UMB16),againstA.fumigatus,

ATCC46913.

Cou-UMB16 MIC(␮g/mL) FICindex (typeof interaction) Cou-UMB16 16 AmphotericinB 2 Flucanozole 256 Itraconazole 128 Voriconazole 2 Cou-UMB16/AmphotericinB 16/4 1.5(Indifferent) Cou-UMB16/Flucanozole 16/2 2(Indifferent) Cou-UMB16/Itraconazole 4/256 1.25(Indifferent) Cou-UMB16/Voriconazole 8/128 1.0(Additivity)

Note:Cou-UMB16,4-acetatecoumarin;FIC,fractionalinhibitory con-centration;MIC:minimalconcentrationinhibitory.

thestructureofthefungalcellwall,resultssimilartoother coumarinsanalyzedbyourgroup.20

Tables2and3showtheresultsofCou-UMB16in

combi-nationwiththeantifungaldrugs(amphotericinB,andazole derivatives), againstA. flavusATCC 16013 and A. fumigatus

ATCC46913.ThecombinationofCou-UMB16with voricona-zoleagainstA.flavusATCC16013revealedsynergisticeffects. AgainstA.flavusATCC16013additiveeffectswereobserved forthecombinationofCou-UMB16withitraconazole;the frac-tionalinhibitoryconcentrationindex(FIC)beingequalto0.63. WithvoriconazoleagainstA. fumigatusATCC46913,the FIC indexwas1.0.However,thecombinationofCou-UMB16and amphotericinBandfluconazolerevealedanFICindexgreater than1.0forallstrainstested.

Discussion

Current antifungal treatments against Aspergillus spp. are stilleffective,butresistantstrainsandintrinsicallyresistant species are emerging fast. Current antifungal drugs suffer from a number of limitations that can render their use

difficult; e.g.amphotericinB’sdose-limiting nephrotoxicity; andazoles’simplefungistaticmodeofaction(withthe devel-opmentoffungalresistance).Thisrevealstheurgentneedfor newantifungalswithbroadfungicidalspectrumsofaction, andwithfewerdose-limitingsideeffects.22,23

An important field of study for new antifungal drugs is plant biosynthesis of secondary metabolites (and their derivatives). The coumarin derivative, 4-acetatecoumarin (Cou-UMB16);abiosyntheticcompound,hasreported antifun-galactivity.6

Coumarins are a group of compounds are derived

from phenylalanine metabolism. They reveal a benzene

ring attached to a pyran ring, the simplest and the

most common representative found being coumarin (1,2-benzopyrone). Thesemetabolites are foundthroughoutthe world.Coumarinshavebeenisolatedinvariousplantparts; the leaves, roots, and fruits of several different families such as: Fabaceae, Asteraceae, Rutaceae, Saxifragaceae and

Thymelaceae.23

Various coumarins and their derivatives have already proventheirantifungalactivity.Itisbelievedthatthese prop-ertiesareusuallyareflectionoftheirinitialrolesinvegetables; suchasthephytoalexins.24–27Theinterestingbiological

activ-itiesofthecoumarinshavemadethemattractivetargetsfor organicsynthesis.6,27,28

Thisstudyshowsthat4-acetatecoumarin(Cou-UMB16)has activityagainstmycelialgrowthandsporegerminationforA. fumigatusATCC 46913and A. flavusATCC 16013.Aspergillus

spp.areubiquitoustothesoilandproducelargenumbersof asexualconidia(conveyedbytheenvironment),whichthen becomeimportantfactorsforthespread ofthefungusand consequenthostinfections.29

Thecoumarinderivative,Cou-UMB16,beforetheassays, wasdissolvedinDMSO(dimethylsulfoxide),andsterile dis-tilledwaterwasusedtoobtaininitialsolutionof1024␮g/mL. TheconcentrationofDMSOdidnotexceed0.5%intheassays, securedthatitdoesnotinterferewithantifungalactivityof coumarin.29,30

Thecoumarinderivative,Cou-UMB16displayedinhibitory actionagainstAspergillusspp.sporegermination,thus inter-feringinthisimportantvirulencefactor.Hyphaearedifficult to phagocytize, and can induce apoptosis in macrophages (afterphagocytosis)ifforminginside.Theresultingmycelium formation is an important virulence factor for Aspergillus

spp.31,32 Reductions in mycelial growth (produced by

Cou-UMB16)interferewithAspergillusspp.virulence.Thisresult wassimilar tothatfoundinourpreviousstudy withother coumarinderivatives.21

TheMIC valuesforCou-UMB16were multiplied4times in thepresence ofsorbitoldosedculturemediumas com-paredtonormalculturemedium,suggestingthatCou-UMB16 acts onthe structureofthe fungal cell wall. Studies show thatcoumarinactsviaformationofporesinthecellwall,a consequentreleaseofcytoplasmiccontents,andcelldeath; thiscorroboratestheresultsfoundinourstudy.21,24However, other studies showthat coumarinsalterthemitochondrial morphologyofthefungalcell,thusinducingapoptosis.23

ThesynergisticeffectobservedagainstA.flavusATCC16013 whencombiningCou-UMB16andvoriconazole;andthe addi-tiveeffectsagainstA.flavusATCC16013whencombinedwith

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itraconazole;andagainstA.fumigatusATCC46913when com-binedwithvoriconazole,suggestthatCou-UMB16maybeused infuturecombinationswiththesedrugs.Severalreportshave indicatedyetother antifungalcombinations,beingassayed

invitroandthenappliedinclinics,33–35andwithdifferingplant

derivatives,7butagainstAspergillusspp.,combinationsof

syn-theticdrugswithcoumarinderivativesarelittleexplored.

Conclusion

Our results suggest that the coumarin derivative

4-acetatecoumarin(Cou-UMB16)hasantifungalactivitywhich inhibits virulence factors in the Aspergillus species tested. Theresultsalsodemonstratethattheactionofthecoumarin derivative is against the structure of the fungal cell wall.

When applying Cou-UMB16 in combination with azoles

(voriconazoleanditraconazole),bothsynergisticandadditive effectswereobserved,dependingonthefungalstrainused.

Conflicts

of

interest

Theauthor(s)declare(s)thattherearenoconflictsofinterest regardingthepublicationofthispaper.

Acknowledgments

The authors are grateful to CAPES (Coordenac¸ão de

Aperfeic¸oamento Pessoal de Nível Superior), the Federal University of Paraíba (UFPB) for financial support, and to DavidHardingwhoeditedtheEnglishversion.

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