, Raquel Rodríguez-Borges

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w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original Article

Partial characterization of ethanolic extract of Melipona beecheii propolis and in vitro evaluation of its antifungal activity

Jesús Ramón-Sierra

^a,1

, Enrique Peraza-López

^a,1

, Raquel Rodríguez-Borges

^a

, Alejandro Yam-Puc

^a

, Tomás Madera-Santana

^b

, Elizabeth Ortiz-Vázquez

^a,∗

aTecnológicoNacionaldeMéxico,InstitutoTecnológicodeMérida,Mérida,Yucatán,México

bCentrodeInvestigaciónenAlimentaciónyDesarrollo,Hermosillo,Sonora,México

a r t i c l e i n f o

Articlehistory:

Received1October2018 Accepted17April2019 Availableonline11May2019

Keywords:

Stinglessbee Phenoliccontent Antifungal Candidaalbicans Antioxidantactivity Propolis

a b s t r a c t

TheethanolicextractofMeliponabeecheiiBennett1831,propolisfromYucatánMexicowasevaluated invitroforthedeterminationofitsphenoliccompoundcontent,antioxidantcapacityandantifungal activity.TheresultswerecomparedagainstthoseoftheethanolicextractofApismelliferapropolis.The totalphenoliccontent,flavonoidcontentandflavanones–dihydroflavonolscontentwereassessedbycol- orimetricmethods.TheantifungalactivitywasassessedinvitroagainstCandidaalbicans.Fortheethanolic extractofM.beecheiipropolis;totalphenoliccontent,was263.25±8.78␮g/ml,totalflavonoidcontent was768±204␮g/mlandflavanones–dihydroflavonolscontentwas335.42±15.08␮g/ml.Forantiox- idantactivityassessedasDPPHscavengingandironreducingpowerethanolicextractofM.beecheii propolisreportedIC50of32.47and1.60␮g/mlofgallicacidequivalentrespectively.Regardingantifungal activityagainstC.albicans,theminimalinhibitoryconcentrationandminimalfungicidalconcentration forethanolicextractofM.beecheiipropoliswere1.62±0.33and2.50±0.22␮g/mlofdryextract;Forboth minimalinhibitoryconcentrationandminimalfungicidalconcentration,ethanolicextractofM.beecheii propolisrequired30%lessconcentrationofdryextractthanethanolextractofA.melliferapropolisto exertthesameantifungalactionsagainstC.albicans.Tothebestofourknowledge,thisisthefirstreport abouttheflavonoidandflavanones–dihydroflavonolscontentofM.beecheiipropolis.Duetothelack ofinformationavailableaboutthestinglessbee’shoneycombproducts,thestudyandconservationof endemichoneybeesshouldremainasanactivefocusofresearch.

Introduction

ThegenusCandidarepresentsasetofyeast-likefungibelong- ingtotheascomycetephylum.ThespeciesofthegenusCandidaare mostlyfree-livingorganisms,andmayexistascommensalsinthe genitourinaryandgastrointestinaltractsofvariousspeciesofani- mals,includinghumans.Undercertainconditions,severalspecies ofCandidabehaveasopportunisticpathogens,both forhumans andotheranimals(Rodriguesetal.,2014).Thepathologiesgener- atedbyCandidarangefromsuperﬁcialmycosestolife-threatening systemicinfections(deOliveiraSantosetal.,2018).

ThefactthataparticularspeciesofCandidabehavesasanoppor- tunisticpathogenorcommensaldependsonacomplexseriesof interactionsbetweenthemicroorganism,thehostandthemicro-

∗ Correspondingauthor.

E-mail:eortiz@itmerida.mx(E.Ortiz-Vázquez).

1Theseauthorsequallycontributedtothepaper.

bial ﬂora present at the interaction site. Infections caused by Candidaaremorefrequentinimmunocompromisedpopulations (e.g.children, theelderlyandpatientswithimmunodeﬁciency), individualswithskinormucousmembranesbreaches(e.g.intra- venousdrugusers,intravenouscatheters),orthoseinwhomthe usualmicrobiotaisfoundtobealtered(e.g.hormonalchanges,use ofbroad-spectrumantibiotics)(TurnerandButler,2014).

AmongtheCandidaspeciesC.albicans,C.glabrata,C.tropicalis, C.parapsilosisandC.kruseistandoutasbeingfrequentlyidentiﬁed asopportunisticpathogens.Theseﬁvespeciesaregroupedinwhat isknownastheCandidapathogenicspeciescomplex(Turnerand Butler,2014).Althoughtherearedrugswithactivityagainstseveral speciesofCandida,theiruseislimitedduetotheemergenceof resistantstrainsandthenarrowsafetymarginformanyofthese compounds.Thismakescandidiasisanimportanthealthconcern, especiallyforhospitalizedpopulations(Srivastavaetal.,2018).

Duetothecontinuousemergenceofstrainsresistanttoclassical antifungalagents,it isnecessarytoinvestigatenewcompounds withactivityagainstthisopportunisticpathogen,preferablywith

https://doi.org/10.1016/j.bjp.2019.04.002

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

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fewersideeffectsthanthosecurrentlyavailable(Srivastavaetal., 2018).

Propolis,togetherwithwax,pollenandhoneyisaproductofthe beehive.Itisproducedbybeesbymixingplantresinswithsecre- tionsfromthebee’sdigestivetract.Propolisispresentedatroom temperatureasamalleablesolidandisusedbybeesasamaterial forsealingandwaterproofingtheinteriorofthehive(Pasupuleti etal.,2017).Propoliscontainsdiversecompoundswithbiological activity,inparticularanthocyanins,flavonoids,tannins,saponins, terpenoids,polypeptidesandlecithins.Amongthebiologicalactiv- itiesattributedtopropolisareantibacterial,antifungal,antioxidant andanti-inflammatorycapacity(Pazinetal.,2017).

Mostoftheavailableinformationaboutthepropertiesofpropo- liscomesfromtheanalysisoftheoneproducedbytheEuropeanbee (Apismellifera)(FarooquiandFarooqui,2012),withrelativelylittle informationregardingthoseproducedbystinglessbees(Oliveira etal.,2006).

Inthepresentinvestigationwecarriedoutapartialcharacter- izationofthepropolisproducedbyastinglessbeeendemicofthe YucatanPeninsulainMexico(Meliponabeecheii)throughtheeval- uationof anethanolicextract elaboratedfromit.Themeasured parametersconcernedtwoofthemostrelevantbiologicalactiv- itiesofthepropolis; theantioxidantactivityand theantifungal activity.Theantifungal activitywasevaluatedinvitrousingthe opportunisticpathogenC.albicansasabiologicalmodel.Asapoint ofcomparison,thepropolisgeneratedbyA.melliferawasevaluated bythesameprocedure.

Materialsandmethods Biologicalsamples

ThepropolisofMeliponabeecheiiwascollecteddirectlyfromthe hive.ThecollectionwascarriedoutinApril2015inthemunicipality ofManiYucatanMexico(20.3931^◦N,89.3918^◦W).Thesameproce- durewascarriedoutwiththepropolisofApismellifera,whichwas collectedinMarch2015fromthemunicipalityofTiziminYucatan Mexico(21.1455^◦N,88.1496^◦W).Propoliswasprotectedfromlight andimmediatelytransferredtothelaboratorywhereitwasstored at−80^◦Cuntilfurtheranalysis.

ThestandardizedCandidaalbicansstrainATCC10231andaclin- icalisolatefromthesamespecies(Y01),wereused.Theclinical isolatewasidentiﬁedaccordingtothestandardizedprotocolsof O’HoranHospitalin thestateof Yucatan,Mexico.Themicroor- ganismswerestoredat−80^◦Candactivatedimmediatelypriorto use.

Reagentsandlaboratoryequipment

Thefollowingreagentswereused:ethanol,methanol,distilled water(Hycel),gallicacid,NaNO2,AlCl3·6H2O(Fermont),catechin, naringenin,trichloroaceticacid(Sigma-Aldrich),2,4-dinitrophenyl hydrazine(Baker)andSabouraudDextroseAgarculturemedium (SDA),SabouraudDextrose broth(SDB) (Bioxon).Spectrometric measurementswerecarriedoutinaPerkinElmerLamb-biospec- trophotometer,eachevaluationwasperformedintriplicate.

Ethanolicextractfrompropolis

Eachtypeofpropolis(100g)wasmilledatroomtemperature untilauniformparticlesizewasachieved.Subsequently,10gofthe samplewassuspendedin100mlof80%(v/v)ethanol.Thesamples wereincubatedunderagitationfor48hat37^◦Cprotectedfrom light,followedbycentrifugationfor10minat5000×gat4^◦C.The supernatantwasrecoveredandﬁlteredwithaporesizeof0.45␮m.

TheresultoftheextractionwaslabeledasethanolextractofM.

beecheii (EEMB)and ethanol extract ofA. mellifera (EEAM).The extractswerestoredat−20^◦Cuntiluse.Theyieldoftheextrac- tionwasevaluatedbycomparingthedryweightoftheextractwith respecttotheinitialamountofpropolisusedfortheextraction.In ordertoavoidtheinterferenceofethanolinthedeterminationof theantifungalactivity,theextractwasdriedat45^◦Cfor24hfol- lowedbyresuspensioninDMSO1%ina1:1ratiowithrespectto theinitialvolume.

Determinationoftotalphenoliccontent(TPC),totalflavonoids content(TFC),totalflavanonesanddihydroflavonolscontent (TFDC)

Thequantificationoftotalphenolswascarriedoutaccording tothemethodologydescribedby(Bratetal.,2006)usinggallic acidasstandard.500␮loftheFolin–Ciocalteureagent(1:10)was addedto100␮lofeachoftheextractsunderstudy.Thesamples werehomogenizedfor30sandincubatedatroomtemperature for2min.Theabsorbancewasdeterminedat760nm.Thecontent offlavonoidswasdeterminedaccordingtothemethodologypro- posedbyLeeetal.(2003)usingcatechin asstandard.250␮lof NaNO₂(5%,w/v)wasaddedto250␮lofeachoftheextracts.The sampleswereincubatedatroomtemperaturefor5min,followed bytheaddition of125␮lof anaqueoussolution ofAlCl₃·6H₂O (10%,w/v).Thesampleswereincubatedfor10minatroomtem- perature, followed bythe additionof 125␮l of1MNaOH.The determinationoftheabsorbancewasperformedat490nm.The totalcontentofflavanonesanddihydroflavonolswasdetermined usingthemethodologydescribed byPopovaetal.(2004),using naringeninasstandard.2,4-Dinitrophenylhydrazine(500␮l,1%, w/v)wasaddedto200␮lofeachoftheextractsfollowedbyincu- bationat50^◦Cfor50min.Thesampleswereallowedtocoolat roomtemperaturefor10min,followedbytheadditionofpotas- siumhydroxideinmethanol(10%,w/v)intheamountneededto reachafinalvolumeof2.5ml.Theformer solution(200␮l)was mixedwithmethanol(800␮l)toobtaina finalvolumeof 1ml.

Spectroscopicreadingswereperformedatawavelengthof486nm.

Antioxidantcapacity

TheantioxidantcapacitywasevaluatedbyDPPHfreeradical scavengingactivityandthereducingpowerofiron(III).DPPHfree radicalscavengingactivitywasevaluatedaccordingtothemethod- ology proposedby da Silva et al. (2013). In this assay, 1ml of 0.1mMsolutionofDPPHinethanolwasaddedto100␮lofeachof thesamplesfollowedbyhomogenizationandincubationatroom temperaturefor30min.Thecontrolconsistedofdistilledwaterfol- lowingthesamestepsasforthesamples.Gallicacidwasusedasa standard.Theabsorbancewasmeasuredat517nm.

For thedetermination of the reducing power of Fe(III),the methodology proposed by Coelho et al. (2017) was used. In this test 250␮lof phosphate buffer(0.2M pH6.6) and 250␮l K4Fe(CN)6·3H2O(1%,w/v)wereaddedto250␮lofeachextract followedbyhomogenizationandincubationfor20minat50^◦C, then250␮lof10%CCl₃CO₂Hwereadded.Subsequently,themix- tures werecentrifuged at4000×g for 10min.The supernatant (500␮l)wastransferredtoa newvialfollowed bytheaddition of400␮lofFeCl₃.Thesampleswereincubatedfor10minat50^◦C.

Thesampleswereallowedtocoolfor10minatroomtempera- ture.Theabsorbanceat700nmwasdetermined.Thedetermination ofthepercentageofuptakeoffreeradicalsandthereductionof Fe(III)toFe(II)wasmadeusingtheformula:((TA−SA)/TA)×100, where TA=target absorbance, SA=sample absorbance (Coelho etal.,2017).

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Table1

ComparisonbetweentheamountofcompoundsofphenolicoriginandantioxidantactivityoftheethanolextractofthepropolisoftwodifferentspeciesofbeesApismellifera (EEAM)andMeliponabeecheii(EEMB).

Ethanolicextractofpropolis

EEAM EEMB

Totalphenols(␮g/mlgallicacid) 798.74±20.76 263.25±8.58

Totalﬂavonoids(␮gcatechin/ml) 1723.47±531 768±204

Totalﬂavanones–dihydroﬂavonols(␮gnaringenin/ml) 382.38±86.23 335.42±15.08

IC50ofDPPH(␮gequivalentsofgallicacid/ml) 49.53±3.91 32.47±3.13

IC50ofreduceFe(III)(␮gequivalentsofgallicacid/ml) 4.3±.03 1.671±.02

IC50:Half-maximalinhibitoryconcentration;DPPH:2,2-diphenyl-1-picrylhydrazyl-hydrate.

EvaluationoftheantifungalactivityofEEMBandEEAMpropolis

The antifungal activity of ethanolic extract against C. albicans was evaluated in vitro for both the ATCC 10231 strain andaclinicalisolate(Y01).Diskdiffusionantibiogramwasper- formedasaqualitativetest.Forthis,PetridishescontainingSDA were massively sowed withC. albicans inoculum standardized at 1×10⁶colony-formingunits/ml (CFU). Disks for antibiogram (13mm)wereimpregnatedwiththesubstances tobeevaluated andplacedontheagarsurface.Arangeofconcentrationsofthe ethanolicextractofpropoliswereevaluated(1–15␮g/mlofdry base), the negative control being carried out with the solvent (DMSO1%),thepositivecontrolconsistingof1.35␮gofnystatin.

Allsampleswereincubatedat37^◦Candevaluatedafter24h.

Thedetermination of theminimuminhibitoryconcentration (MIC)andtheminimumfungicidalconcentration(MFC)wascar- riedoutusingthemicrodilutionmethodin1.5mlmicrocentrifuge tubes(Silicietal.,2005;Espinel-Ingroffetal.,2007;Keskinetal., 2017).Startingfrom0.5McFarlandinoculum,thenecessaryvol- umewasaddedto250␮lofSabouraudDextroseBroth(2×)toreach aﬁnalconcentrationof10⁶CFUinavolumeof500␮l.Immediately, thenecessaryamountofpropolisextractresuspendedin1%DMSO wasaddedtoreachtheconcentrationtobeevaluated(1–15␮g/ml ofdryextract),followedbytheadditionofdistilledwaterinthe amountnecessarytoreachanendvolumeof500␮l.Thesamples wereincubatedunderagitationat150rpmfor48hat37^◦C.Fungal growthwasassessedbymeasuringabsorbanceat600nmat12,24, 36and48h(Silicietal.,2005;Espinel-Ingroffetal.,2007;Keskin etal.,2017).Thesolvent(1%DMSO)wasusedasacontrol(total growth)and 2␮gofnystatinaninhibition control.Thepercent inhibitionwascalculatedbytheformula:((SA−NCA)/PCA)×100, whereSA=sampleabsorbance,NCA=negativecontrolabsorbance andPCA=positivecontrolabsorbance(Alsopetal.,1980;Kayaand Özbi˙lge,2014).MICwasconsideredasthatconcentrationofpropo- lisextractthatshowedan80%reduction ingrowthat24hand MFCtheconcentrationthatproduceda99%reductioningrowth comparedtocontrolat48h.

Statisticalanalysis

Multivariateregressionanalysis,onewayANOVAwithTukey rangetest wasperformedusingtheIBMSPSSstatisticsV23for assessingthedifferencesintheantifungaleffectofbothpropolis.

Results

Determinationoftotalphenoliccontent(TPC),totalflavonoids content(TFC),andtotalflavanonesanddihydroflavonolsContent (TFDC).

Theyieldoftheextractionwas4.70(0.47%)and5.10(0.51%) mgdryweightofextractper1gofpropolisfromM.beecheiiandA.

melliferarespectively.EEMBandEEAMhadatotalphenoliccontent (measuredasgallicacidequivalents)of263.25±8.78␮g/mland 798.74±20.76␮g/ml,respectively.Theaboverepresentsayieldof

2.76mg/gofgallicacidequivalentspergramofrawpropolisinthe caseofM.beecheii7.80 andmg/gofrawpropolisofA.mellifera (Table1).

The total ﬂavonoids content expressed as catechin equiva- lentswere768±204␮g/mland1723.47±531␮g/mlforEEMBand EEAM,respectively.Theﬂavonoidextractionyieldwas7.68mg/g and17.23mg/ginthecaseofpropolisofM.beecheiiandA.mellifera respectively(Table1).

Thetotal ﬂavanonesand dihydroﬂavonolscontentexpressed as equivalents of naringenin was 335.42±15.08␮g/ml and 382.38±86.23␮g/mlforEEMBandEEAM,respectively.Theyield oftheextractionwas3.35mg/gofcrudepropolisinthecaseofM.

beecheiiand3.82mg/gforA.mellifera(Table1).

Antioxidantcapacity

IntheDPPHfreeradicalscavengingactivity,theEEMBandEEAM showedanIC₅₀of32.47␮g/mland49.53␮g/mlofgallicacidequiv- alentsrespectively.WithrespecttothereducingpowerofIronIII, anIC50of1.60␮g/mlingallicacidequivalentsforM.beecheiiand 4.30␮g/mlforA.mellifera(Table1)wasobtained.

Antifungalactivity

Diskdiffusionantibiogram.UsingATCC10231strain50␮lof EEMBgenerated aninhibition haloof 21.12mm,50␮lof EEAM generatedaninhibitionhaloof16.16mm.Usingtheclinicaliso- late(Y01)50␮lofEEMBgenerateda20mminhibitionhalo,EEAM showednoapparentinhibition.Thesolvent(1%DMSO)showedno apparentantifungalactivity(Fig.1).

DeterminationoftheMICandMFC

Based onthe dryweight of theextract, theMIC forC. albi- cansATCC10231wasdeterminedas1.62±0.33␮g/mlforEEMB and 2.30±0.15␮g/ml for EEAM. The MFC for the same strain was2.50±0.22␮g/mland3.31±0.28␮g/mlforEEMBandEEAM, respectively (Fig. 2 and Table 2). In the case of the clinical isolate (Y01) the MIC was determined at 2.25±0.2␮g/ml and 2.75±0.15␮g/ml for EEMB and EEAM, respectively. The MFC for the same isolate was determined in 2.90±0.55␮g/ml and 3.75±0.11␮g/mlforEEMBandEEAM,respectively.Thesolvent(1%

DMSO)showednoinhibitionoffungalgrowth(Fig.2andTable2).

Multipleregressionanalysiswascarriedouttoassessthedoses- response effectoftheEEMBand EEAMpropolisagainst C.albicans (ATCC 10231 and Y01). For each of tested concentration theinhibitoryeffectofEEMBpropoliswashigherthanthesame concentrationofEEAMpropolisuntilbothreachedMFC,thesedif- ferencesshowedstatisticalsigniﬁcance(p=0.031andp=0.011for C.albicansATCC10231yY01,respectively(Fig.2).OnewayANOVA withTukeyrangetestwasusedtoevaluatetheexertedeffectsby bothEEMBandEEAMpropolisresultinginsigniﬁcantdifferences (p<0.05),exceptforconcentrationsabove3.50␮g/ml.

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Candida albicans ATCC 10231 Candida albicans Y01

EEAP

EEMB

EEAM C-

C-

C+ C+

C-

A B

Fig.1. QualitativeassayoftheantifungalactivityoftheEEMBandEEAMpropolisbydiskdiffusionassayagainstCandidaalbicansATCC10231andaclinicalisolate(Y01).

EEMB,ethanolextractofMeliponabeecheiipropolis;EEAM,ethanolextractofApismelliferapropolis;C+−,positivecontrol(nystatin);C−−,negativecontrol(DMSO1%).

A B

100 100

80

60

40

20

0

1.5 2.0 2.5 3.0 3.5

90

80

70

60

50

1.5 2.0 2.5 3.0 3.5

Propolis concentration (in µg/ml) Propolis concentration (in µg/ml)

Propolis origin

Melipona beecheii Apis mellifera

Inhibition percentage Inhibition percentage

Fig.2.Dose–responsecurveoftheantifungalactivityofpropolisagainstC.albicans.Left(A)comparisonoftheactivityofEEMBandEEAMpropolisagainstCandidaalbicans ATCC10231.Right(B)comparisonoftheactivityofEEMBandEEAMpropolisagainstaclinicalisolateofC.albicans(Y01).Noticethatinbothcasesagivenconcentration ofMeliponabeecheiipropolisextractinducedamoreintensesuppressionofgrowthofC.albicansthanthesameconcentrationofApismelliferapropolisextractuntilboth reachedtheMFC.Thedifferencebetweenthetreatmentswasregardedasstatisticallysigniﬁcant(p=0.031forATCC10231andp=0.011forY01).EEMB,ethanolextractof M.beecheiipropolis;EEAM,ethanolextractofA.melliferapropolis.

Table2

CompressionoftheantifungalactivityagainstCandidaalbicansATCC10231oftheethanolextractofpropolisfromMeliponabeecheiiandApismelliferaanditsrelationship withthecontentofphenoliccompoundspresentinthesample.

Propolisorigin ␮gdryextract/ml ␮lextract(1%

DMSO)/ml

Totalphenols (␮g/mlgallicacid)

Totalﬂavonoids (␮gcatechin/ml)

Total

ﬂavanones–dihydroﬂavonols (␮gnaringenin/ml)

M.beecheii MIC 1.62 3.44 0.90 2.64 1.15

MFC 2.5 5.31 1.39 4.07 1.78

A.mellifera MIC 2.2 4.3 3.43 7.4 1.44

MFC 3.3 6.49 5.18 8.92 2.17

DMSO:DimethylSulfoxide;MIC:MinimumInhibitoryConcentration;MFC:MinimumFungicidalConcentration.

Discussion

Phenoliccompoundsareadiversegroupofsecondarymetabo- lites produced by multiple plant species and represent the mostdiverseclassofcompoundspresentinpropolis.Substances identified as phenolic compounds include phenolic acids, tannins,flavonoids,curcuminoids,coumarins,lignans,amongothers (Huangetal.,2010).Inplantspecies,thesesubstancesfulfillvar- iousfunctions:intracellular signaling,defense againstpredators (e.g. toxins, antinutritional factorsetc.), protectionagainst phy- topathogens,andattractionofpollinatingspecies,amongothers

(Huangetal.,2010).Antioxidantcapacityisoneofthemostrelevant biologicalactivitiesattributedtopropolis.Ithasbeenproposedthat variousantioxidantsofnaturalorigincanmodifythenaturalhis- toryofdiversepathologiessuchashypertension,diabetesmellitus, obesity,systemic inﬂammatorydiseases, amongothers (Sforcin, 2016).Ithasbeendocumentedthatmuchoftheantioxidantactivity ofpropolisoccursduetoitsphenoliccompoundscontent(Huang etal.,2010;Libenetal.,2018).

Inthepresentstudy,itwasconﬁrmedthatboththepropolis of M. beecheii and A. mellifera possess at least two mecha- nismsofantioxidantactivity:theuptakeoffreeradicalsandthe

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reductionofmetalionsinsolution.Itwasalsopossibletodemon- strate a signiﬁcant difference in the total content of phenolic compoundsandﬂavonoidsinpropoliselaboratedbyM.beecheii andA. mellifera (p<0.05).Nevertheless,EEMB propolisrequired lessconcentrationofgallicacidequivalentsthanEEAMpropolis toexertthesameantioxidanteffectbothinDPPHtest andiron reducingpower.Thiseffectcanbeexplainedduetothedifferences inthephenoliccompoundsineachpropolis.

Thecontentofphenoliccompoundspresentinpropolisishighly variablebecauseitdepends onthediversity ofplantspeciesto whichthebeeshaveaccessduringitselaboration(Zhangetal., 2016).Additionally,eachbeespecieshasauniquehabitinitsresin collectionthatincreasestheregionalandinterspeciﬁcvariationsin thechemicalcompositionofthepropolis(Bakchicheetal.,2017).

Theantioxidantcapacityisnotthesameforeachofthediverse phenoliccompounds(Tabartetal.,2009).Ithasbeenshownthat therearesigniﬁcantdifferencesinthecompositionofthepropolis generatedbyA.melliferawithrespecttothatproducedbydifferent speciesofstinglessbees(meliponinitribe)(Huangetal.,2010).

Theabilitytoreducemetalionsisespeciallyrelevantinfood productssincetheabsorptionofmanymineralsonlyoccursatcer- tain oxidationstates. The aboveis demonstratedin thecase of iron,whichcanonlybeabsorbedasiron(II),lackingasigniﬁcant bioavailabilityasiron(III).Theuseofantioxidantsrepresentsan alternativetoimprovethebioavailabilityofthisclassofminerals (Davidsson,2003;HurrellandEgli,2010).

Theﬁndingsofthisstudycoincidewiththatdescribedbythe literatureforpropolisofA.mellifera,aswellaswiththosereported forotherstinglessbeespecies(ManriqueandSantana, 2008;da Silvaetal.,2013).Thecompoundspresentintheethanolicextracts ofthepropolisofA.melliferaandM.beecheiidemonstratedactiv- ityagainstC.albicans.Thiscoincideswithreportsoftheliterature regardingthepropolisofA.melliferaandseveralspeciesofstingless bees(Camposetal.,2015;Capocietal.,2015;Shehuetal.,2016;

Maureiraetal.,2017;Dezmireanetal.,2017).However,informa- tionregardingthebiologicalactivitiesofpropolis ofM.beecheii continuestobescarce(Popovaetal.,2005;Fonte-Carballoetal., 2016;Ibrahimetal.,2016).

Asfarasweknow,thisisthefirstreportregardingthecontentof flavonoidsanddihydroflavonols,aswellastheantioxidantactivity oftheethanolicextractofthepropolisofM.beecheii.

Conclusion

TheethanolextractofthepropolisofA.melliferaandM.beecheii containssigniﬁcantamountsofphenoliccompounds,whichexert anantioxidantactivitybyatleasttwodifferentmechanisms.The totalcontentofphenoliccompoundswassuperiorinthecaseofthe ethanolextractofA.melliferatothatofM.beecheii.Theethanolic extractofM.beecheiipresentedahigherantifungalactivityagainst C.albicansthanthatofA.mellifera,reachingMICwithlessthan3␮g ofdryextractperml,containingaconcentrationless1␮g/mlgallic acidequivalents.

Authorscontributions

JRSandEPLequallycontributedtorunningthelaboratorywork, analysisofthedataanddraftingthepaper.RRBcontributedtothe collectionofpropolisandrunningthelaboratorywork.AYPcon- tributedinanalysisofdataanddraftedthepaper.TMScontributed incriticalreviewofthemanuscript.EOVdesignedthestudy,super- visedthelaboratoryworkandcontributedincriticalreviewofthe manuscript.Alltheauthorshave read theﬁnal manuscript and approvedthesubmission.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

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