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
aaUniversidadeFederaldaParaíba,DepartamentodeciênciasFarmacêuticas,JoãoPessoa,PB,Brazil bUniversidadeEstadualdaParaíba,DepartamentodeCiênciasBiológicas,JoãoPessoa,PB,Brazil cUniversidadeFederaldeCampinaGrande,Centrodeeducac¸ãoesaúde,Cuité,PB,Brazil
a
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t
i
c
l
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o
Articlehistory:
Received21June2016 Accepted30June2017
Availableonline12October2017 AssociateEditor:LuisHenrique Guimarães Keywords: Coumarin Antifungalactivity A.flavus A.fumigatus Modeofaction
a
b
s
t
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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/).
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 1024g/mL, including 4-acetatecoumarin (Fig. 1) with an MIC value of 16g/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 of1024g/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
determiningthedry myceliaweightsofA. fumigatus(ATCC 46913)andA.flavus(ATCC16013).11,12 FlaskscontainingMIC
(16g/mL)andMIC×2(32g/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:11m),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(16g/mL) andMIC×2(32g/mL)ofcoumarinderivativeandacontrol withdistilledwater,were used.In steriletesttubes, 500L ofRPMI-1640plusthe Cou-UMB16were evenlymixed with 50L 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.
Ergosterolbindingassay–MICvaluedeterminationinthe 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=100L)containing ergosterolataconcentrationof400g/mL.Avolumeof10L 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, 50Lof eachdilutionofcoumarinderivativewasaddedtothe96-well micro-titerplatesintheverticaldirection,while50Lofeach dilutionoftheantifungaldrugswasaddedinthehorizontal direction,sothatvariouscombinationsofcoumarinderivative andantifungaldrugscouldbeachieved.Inaddition,10Lof 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)(16g/mL)hasbeen
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:16g/mL;2×MIC:32g/mL)andamphotericinB(MIC:2g/mL;2×MIC: 4g/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:16g/mL;2×MIC:32g/mL)andamphotericinB(MIC:2g/mL; 2×MIC:4g/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(400g/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-UMB16at16g/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
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-tilledwaterwasusedtoobtaininitialsolutionof1024g/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
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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