Chemical characterization, antioxidant and anti-HIV activities of a Brazilian propolis from Ceará state

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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

Chemical characterization, antioxidant and anti-HIV activities of a Brazilian propolis from Ceará state

Caroline Cristina Fernandes da Silva

^a

, Antonio Salatino

^a,∗

, Lucimar Barbosa da Motta

^b

, Giuseppina Negri

^a,b

, Maria Luiza Faria Salatino

^a

aDepartamentodeBotânica,InstitutodeBiociências,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil

bUniversidadePaulista,SãoPaulo,SP,Brazil

a r t i c l e i n f o

Articlehistory:

Received28September2018 Accepted4April2019 Availableonline14May2019

Keywords:

Anti-HIV Antioxidant Brazilianpropolis Flavonoids Botanicalsource HIV-1reversetranscriptase

a b s t r a c t

Propolis(beeglue)aproductofApismelliferaL.isaresinousmixturecontainingchieflybeeswaxand resinharvestedbybeesfromplantleaves,budsandexudates.ExtractsofapropolissamplefromSal- itre,amunicipalityofCearástate(northeastBrazil)wereobtainedwithsolventsofincreasingpolarity (hexane,chloroform,ethylacetateandmethanol).AchemicalprofilewascarriedoutbyGC–EI-MSand HPLC–DAD–ESI-MS/MS.Lupenone,lupeol,octanoicacidtetracosylesterandoctanoicacidhexacosyl esterwereidentifiedbyGC–EI-MS.AntioxidantactivitywasevaluatedbytheDPPHand␤-carotene discoloringmethods,andanti-HIVactivitybytheinvitroinhibitionofHIV-1reversetranscriptase.

Theethylacetateextractexhibitedthehighestantioxidantandanti-HIVactivityandwasfractioned bycolumnchromatographyusingsilicagelandsevendifferenteluents.Theactivefractionsweresub- mittedtosemipreparativeHPLCandthefollowingcompoundswereisolated:caffeicacid,p-coumaric acid, diprenylcinnamic acid, quercetin, naringenin, isorhamnetin, quercetin-3-O-diglucoside,4,2,4- trihydroxy-2-methoxychalcone,gossypetin-3,3,4,7-tetramethylether,myricetin-3,7,3-trimethylether and5-hydroxy-3,6,7,8,4-pentamethoxyﬂavone. Theethyl acetateextractanditsfractionsF5-F7,as wellasquercetin,isorhamnetin,myricetin-3,7,3-trimethyletherandp-coumaricacidexhibitedhigh antioxidantactivityonbothDPPHand␤-caroteneantioxidantmethods.Isorhamnetinexhibitedmod- erate inhibitory effect against HIV-1 reversetranscriptase (56.99±3.91%), followed bynaringenin (44.22±1.71%),quercetin(43.41±4.56%)anddiprenylcinnamicacid(41.59±2.59%).Theseresultsagree withpreviousauthorswhoreportedanti-HIVactivityofﬂavonoids.

Introduction

Propolis(beeglue)isaresinousmixtureproducedbyApismel- liferaL.Itcontainsbeesecretions(includingbeeswax)andresins, whichbeescollectfromvariousbotanicalsources,suchasbuds, leaves,andexudates.Otherpropolisconstituentsarepollen,sugars, aminoacidsandminerals.Propolishasbeenshowntoexertmany biological activities (wound-healing, anti-inﬂammatory, gastro- protective,hepatoprotective,immunomodulatory,antineoplastic, antidiabetic,amongmanyothers)(Hashemi,2016;Berrettaetal., 2017).Honeybees,independently ofthegeographicalzone,can locateappropriate propolisplantresinsources forproduction a propolis possessing biological activity (Bittencourt et al., 2015;

Machado et al.,2016; Berretta et al.,2017; Kocot etal., 2018).

∗ Correspondingauthor.

E-mail:asalatin@ib.usp.br(A.Salatino).

Propolischemicalcompositionvariessubstantially, accordingto geographyandlocalﬂora(Bertellietal.,2012;Petelincetal.,2013;

Toretietal.,2013).Propolistypesarecharacterizedbygeography, chemicalproﬁleandsourceplants(Berrettaetal.,2017;Kocotetal., 2018).

Propolisfromdistinctregionsexhibitedcharacteristicchemi- calprofilesandsourceplants (Berrettaetal.,2017;Kocotetal., 2018). In temperate zones (Europe, nontropic regions of Asia, NorthAmerica,andcontinentalAustralia),thepropolisknownas poplar typepropolisis originatedmainlyfrombudexudates of Populusnigra,Salicaceae,and possessflavonoidswithnoB-ring oxygenation,phenolicacidsandtheirestersasmainconstituents (Ristivojevi ćetal.,2015;Al-Anietal.,2018).However,inRussiaa distinctpropolistypeisproduced,derivedfrombirch(Betulaverru- cosa,Betulaceae)containingflavonesandflavonolsdifferentfrom those ofpoplar propolis(MiguelandAntunes, 2011;Martinotti andRanzato,2015).MediterraneanorItaliancypresspropolisis originatedfromtheresinofCupressussempervirens,Cupressaceae.

https://doi.org/10.1016/j.bjp.2019.04.001

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

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ThistypeofpropolisisproducedincountriessuchasGreece,Sicily, Malta,Cyprus,Croatia,andAlgeria,andcontainschieﬂyditerpenes (Popovaetal.,2010,2011).Atleastthree plantspeciesprovide resinforOmanipropolis:Azadirachtaindica,Meliaceae,Acaciaspp.

(probablyA.nilotica,Fabaceae,andMangiferaindica,Anacardiaceae.

OmanipropoliscontainsC₅-prenylflavanonesasmainconstituents (Popovaetal.,2013).TropicalpropolisfromCubaandVenezuela originatesfromresinexudedbyflowersofClusiaspp.,Clusiaceae, andcontainsbenzophenonesasmainconstituents(Cuesta-Rubio etal.,2002).ThepropolisfromtropicalPacificOceanislands(Tai- wan,Okinawa,andIndonesia)originatedfromMacarangatanarius, Euphorbiaceae,ischaracterizedbyC-geranylflavanones(Trusheva etal.,2011;Kumazawaet al.,2014).Bolivianpropolisfromthe valleys(Cochabamba,ChuquisacaandTarija)isrichinprenylated phenylpropanoids, while samples from La Paz and Santa Cruz exhibited cycloartane and pentacyclictriterpenes as main constituents(Ninaetal.,2016).

InBrazil,severaltypesofpropolishavebeencharacterized,each derivingfromadistinctresinsource(Berrettaetal.,2017).Among Brazilianpropolis,thegreen andred typeshavebeenthemost frequentlystudiedamongBrazilianpropolis(Huangetal.,2014).

GreenpropolisfromsoutheastBrazilisderived fromyoungtis- suesofBaccharisdracunculifolia,Asteraceae,andcontainsmainly prenylatedphenylpropanoids,caffeoylquinicacidsandditerpenes (Salatino et al., 2005; Fernandes-Silva et al., 2013; Righi et al., 2013).Redpropolis,producedinthelittoralofBraziliannortheast, isoriginatedfromDalbergiaecastaphyllum,Fabaceae,andpossesses chalconesandisoﬂavonesasmainconstituents(Righietal.,2011;

Mendonçaetal.,2015;Rufattoetal.,2017).Severalothertypesof Brazilianpropolishavebeencharacterized.Agreenpropolispro- ducedinthenortheastofthecountrycontainschieflyflavonoids andis derivedfromMimosatenuiflora,Fabaceae(Ferreiraetal., 2017).Adark-coloredpropolisfromtheAmazonisoriginatedfrom Clusiaspp., Clusiaceae,and contains prenylated benzophenones (CastroIshidaetal.,2011).Somepropolistypeshavebeenchar- acterizedchemically,butstillnotbotanically.Thus,therearetwo propolistypes in Piauí state, one containingcycloartane triter- penoids (Silva et al., 2005) and another containing flavanones andglycosylflavones(Righietal.,2013);ayellowpropolisfrom Mato Grosso State (central west, Brazil) possesses triterpenes (Machado et al., 2016) and a propolis type from Pirenópolis (GoiásState,centralwestBrazil)containspredominantlypreny- latedflavonoidsandtriterpenes(Righietal.,2013).Brownpropolis from Campo Grande, Mato Grosso do Suland Paraná contain, mainly,prenylatedphenylpropanoicacids,flavonoidsandchloro- genicacids(Fernandesetal.,2015;Andradeetal.,2017;deOliveira Dembogurskietal.,2018).Ontheotherhand,thebrownpropolis fromBahiaState,possessessaturatedhydrocarbons,methylcinna- mate,sitosterolcinnamateandananixanthonesmainconstituents (Santosetal.,2017).

Indevelopingcountries,acquiredimmunodeficiencysyndrome (AIDS)is stilla great causeofdeath. Humanimmunodeficiency virus-1(HIV-1)leadstoAIDSviadestructionofT-cellsafterinvasion andreplicationinsidethehostcells.Mostconventionaldrugsused asanti-HIV(anti-humanimmunodeficiencyvirus)arenucleoside- based.Theirusehasseriouslimitationsbecauseofadverseeffects, resistanceandtoxicity.Theyundergofirstpassmetabolism,lead- ingtoinactivationandgenerationoftoxicmetabolites(Singhetal., 2010;Pasettoetal.,2014;Saravananetal.,2015).Naturalprod- uctshavebeenusedasleadcompoundsaimingtoobtaineffective drugsagainstmanyailments,includingAIDS.Severalflavonoids havebeenshowntobeactiveagainstHIV(Pasettoetal.,2014).

Althoughﬂavonoidsarecommonconstituentsofseveralpropolis types,notmuch hasbeeninvestigatedaboutpropolisantiretro- viralactivity.Amongthemethodsusedtotestanti-HIVactivity, theanti-reversetranscriptasemethodhasbeenwidelyusedfor

bioassayguidedfractionationanddeterminationofIC₅₀ofisolated compounds(Reutrakuletal.,2007;Zhangetal.,2017).

Theobjectiveofthepresentstudyistodeterminethechemical compositionofapropolisproducedinthemunicipalityofSalitre (stateofCeará,northeastBrazil).Inaddition,itisintendedtoisolate constituentsofthispropoliswithantioxidantandanti-HIVactiv- ity,aimingtodetectconstituentsaccountingfortheactivityofthe extracts.

Materialandmethods Propolissampleandextraction

ThepropolissamplewascollectedinthemunicipalityofSalitre (7^◦1656S,40^◦2721W),Cearástate(northeastBrazil).Approx- imately50gofpropoliswassubmittedtoextractioninSohxlet for 6h, using 500ml of solvents withincreasing polarity (hexane,chloroform,ethylacetateandmethanol).Theextractswere concentratedunderreducedpressure,re-suspendedinchloroform (hexaneandchloroformextracts)ormethanol(ethylacetateand methanolextracts)andﬁltered.

CharacterizationofconstituentsbyGC–EI-MS

Analyses of 1␮l of chloroform and methanol solutions at 1mg/ml were carried out by GC–MS in split mode, using a 6850 Agilent gas chromatograph operating at the split mode andequippedwithacapillarycolumnDB-5HT(30m×0.32mm, 0.25␮m)coupledtoaninjectorusingapulsedsplitratio1:10.The chromatographwascoupledtoa5975CVLMSDAgilentmassspec- trometeroperatingwithelectronimpactionizationat70eV.The temperatureofbothinjectoranddetectorwas300^◦C.Heliumwas usedascarriergasat1.5cm³minflowrate.Oventemperatures rangedfrom100to300^◦Cat10^◦C/min,finishingwitha15min isothermalperiod. The massspectrometer wassetto detectin them/zrange50–650inthescanmodeusingasourcetempera- tureof250^◦C,aquadrupoletemperatureof110^◦Candafilament emissioncurrent of35␮A.Characterizationof constituentswas achievedbymatchingqueryspectratospectrapresentinarefer- encelibrary.Thus,thefragmentationpatternoftheEImassspectra ofconstituentswascomparedviaspectrummatchingwiththeref- erencemassspectrainthelibraryNIST08andWiley-275(Hewlett Packard,Wiley/NBS,Kooetal.,2013),andalistofmostprobable identitieswasproducedbasedonthebestmassspectrummatching score.Comparisonwithdatareportedinliteraturealsowasused forcharacterization,aswellascomparisonwithauthenticstandard oflupeol.Relativeamountsofconstituentswereassumed tobe proportionaltotheareasunderthecorrespondingchromatogram peaks.

CharacterizationofconstituentsbyHPLC–DADand HPLC–DAD–ESI-MS/MS

RPHPLC–DAD–ESI-MS/MSanalysesofethylacetateextractand itsfractionswereconductedusingaDADSPD-M10AVPShimadzu systemequippedaphotodiodearraydetectorcoupledtoEsquire 3000Plusmassspectrometer,BrukerDaltonics,whichconsisted oftwoLC-20ADpumps,SPD-20Adiodearraydetector,CTO-20A column ovenand SIL20ACautoinjector (Shimadzu Corporation Kyoto,Japan). Alltheoperations,acquisitionsand dataanalyses werecontrolledbytheShimadzuCBM-20Acontroller.Separations werecarriedoutusingaC18RPLunaPhenomenexreversephase column(4.6×250mmi.d.,5␮m),protectedwithasecurityguard cartridge(GeminiC18,4.0×2.0mmi.d.).Themassspectrometer wasaquadrupoleiontrapwithatmosphericpressureionization sourcethroughelectrosprayionizationinterface(ESI)operatingin

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thescanMSmodefromm/z100to1500.Theethylacetateextract anditsfractions(2mg/ml)werefilteredthrougha0.45␮mpoly- tetrafluoroethylene(PTFE)membranefilters,anda10␮laliquot wasinjected intothe chromatograph. Thecolumn temperature wasmaintainedat40^◦Candtheflowratewas1ml/min.TheDAD acquisitionwavelengthwassetintherangeof240–400nmand chromatogramswererecordedat250,300and352nm.Afterpass- ingthroughtheflowcelloftheDAD,thecolumneluatewassplit, andaflowof100␮l/minwasdivertedtothemassspectrometer.

ThemobilephasewascomposedbyeluentA(0.1%aqueousacetic acid) and eluent B (methanol). The following elution program, basedonconcentrationsoftheBsolvent,wasused:0min,20%;

10min,40%;20min,60%;30min,80%;40min,95%;50min,95%.

Heliumwasusedasthecollision,andnitrogenasthenebulizing gas,respectively.Nebulizationwasaidedwithacoaxialnitrogen sheath gas provided at pressure of 27psi. Mass spectra were acquired in both negative and positive modes with ion spray voltageat3.0kV,capillarytemperatureat300^◦C,capillaryvoltage at45Vanddryinggasﬂow6l/min.Collisioninduceddissociation spectrawereobtainedintheiontrapusingheliumasthecollision gas,withvoltagerampingcyclesfrom0.5to1.3V.Theconstituents werecharacterized by ultravioletand mass spectral data (MS).

The MS data were compared with mass spectral databases Phenol-Explorer (www.phenol-explorer.eu), ChemSpider (http://www.chemspider.com),Metlin(http://metlin.scripps.edu) andHMDB(www.hmdb.ca),besidesMSdatareportedinliterature (seeTable2).Quercetin,naringenin,isorhamnetin,caffeicacidand p-coumaricacidfromSigma-Aldrichwereusedasstandardsusing thesamechromatographicconditions.

Bioguidedisolationofactiveconstituents

Theextractswithhigherantioxidantactivity(evaluatedaccord- ingtosectionAntioxidantactivity;seebelow)werefractionated bycolumnchromatographyusingsilicagelandthefollowingsol- vents:F1:hexane;F2:hexane:chloroform(50:50);F3:chloroform;

F4:chloroform:ethylacetate(50:50);F5:ethylacetate;F6:ethyl acetate:methanol(50:50)andF7:methanol.Fractionsthatexhib- itedhigherantioxidantandanti-HIVactivitiesweresubmittedto semi-preparativeHPLCtoisolatethemainactiveconstituents.The isolationwasperformedwithareversephaseZorbaxC18RP-18 column(HewlettPackard;9.6×250mm,5␮m)andmobilephase composedbyeluentA(0.1%aq.aceticacid)andeluentB(methanol) at2.5ml/minconstantﬂowrateand40^◦Cconstanttemperature.

Thefollowing elutionprogramwasused,based ontheconcen- trationofB:0min:20%;10min:40%;20min:60%;30min:80%;

40min:90%;50min:95%.Theantioxidantandanti-HIVactivities ofisolatedcompoundswereevaluatedandthoseobtainedinhigh contentswereanalyzedby¹HNMRand¹³CNMR.

NMRspectraofisolatedcompounds

NMRspectraofisolatedcompoundswererecordedinMeOD withaBrukerAdvanceIII(300MHz).Chemicalshiftsaregivenin ppmandwerereferencedtothesolvent(MeOD)signalat3.31ppm for¹Hand49.3ppmfor¹³C.

Antioxidantactivity

Antioxidant activities were measured by the DPPH (1,1- diphenyl-2-picryl-hydrazyl)freeradicalscavenging(Morenoetal., 2000) and the ␤-carotene discoloring methods (Koleva et al., 2002),withminormodiﬁcations.FortheDPPHmethod,samples at10,15and30␮g/mlweremaintainedinthedarkfor30min.

TheabsorbancewasmeasuredinaSynergy^TMNeo2Multi-Mode

MicroplateReaderat517nm.Thepercentageoffreeradicalscav- engingwascalculatedbythefollowingformula:

%Freeradicalscavenging

=DPPHabsorbance−sampleabsorbance DPPHabsorbance ×100

Quercetininmethanolsolutions(5–30␮g/ml)wasusedasref- erencecontrol anda solutionof DDPHinmethanolasnegative control. The antioxidant activity of the samples was calcu- latedaccordingtotheregressionequationy=13.043x(R²=0.96), obtainedwiththequercetinsolutions.Theresultsoffreeradical scavengingcapacityaregivenasmgofquercetinequivalentperg ofpropolis(mgQE/g).Testsforallsampleswererunintriplicates.

Forthe␤-carotenediscoloringmethod,areactivesolutioncon- taining200␮gof␤-carotene,25␮loflinoleicacidand200␮lof Tween40wassolubilizedin50mlofdistilledwatersaturatedwith oxygen.Theabsorbanceofthissolutionwassettoliebetween0.7 and0.8at470nm(Righietal.,2013).Fortheoxidationreaction, 1mlofthereactivesolutionwasaddedto120␮lofmethanolsolu- tionsofthesamplesatthe120␮g/mlconcentration.Thesolutions wereleftstandinginthedarkfor2h.Then,atevery30min,the absorbanceofthesolutionswasreadat470nm,usingaSynergy^TM Neo2Multi-ModeMicroplate.Theresultsareexpressedaspercent ofinhibition,comparingthedecreaseinabsorbanceofthesam- pleswiththedecreaseoftheabsorbanceofthecontrol(reactive solution+methanol),accordingtothefollowingformula:

%Antioxidantactivity

=1−controlabsorbance−sampleabsorbance controlabsorbance ×100

Quercetininmethanolsolutions(20–120␮g/ml)wasusedas referencecontrol.Theantioxidantactivityofthesampleswascal- culated accordingto theregression equation y=102.14x+13.92 (R²=0.92),obtainedwiththequercetinsolutions.Resultsofantiox- idantactivitiesaregivenasmgofquercetinequivalentpergof propolis(mgQE/g).Allsamplesweretestedintriplicates.

Anti-HIVactivity

AmongthemethodsusedtotestantiviralactivityagainstHIV, thequantitativedeterminationofretroviralreversetranscriptase activity is used for bioassay guided fractionation and determi- nation of IC50 of isolated compounds (Reutrakul et al., 2007;

Zhang et al., 2017).The colorimetric enzyme immunoassayfor thequantitativedeterminationofretroviralreversetranscriptase activitywasevaluatedwithrecombinantHIV-1enzyme,usinga non-radioactiveHIV-1RTcolorimetric ELISAkit(Roche Applied Sciences,Mannheim,Germany),Roche^®,followingthegeneralrec- ommendationsofthemanufacturer.Samplesweredilutedin10%

dimethylsulfoxide(DMSO)toaﬁnalconcentrationof200␮g/ml.

Aliquotsof20␮lofthesampleswereappliedon96wellplates.

Foscarnetwasusedaspositivecontrolat10␮g/ml.Controlsused werecontrolA(withoutsampleandwithoutreversetranscriptase) and controlB(without sample,but withreversetranscriptase).

TheabsorbancewasreadonaThermoScientiﬁc^TMMultiskan^TM FCMicroplatePhotometerat405nm.Thepercentageofinhibition wascalculatedbythefollowingformula(Meragelmanetal.,2001;

Onoetal.,1990):

%Inhibition

=1− sampleabsorbance−controlAabsorbance controlBabsorbance−controlAabsorbance×100

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Results

CharacterizationofisolatedconstituentsbyNMR

Naringenin(9):¹HNMR(300MHz,MeOD,TMS),ıH(ppm)J (Hz)5.43(1H,dd,J=13,2.8Hz,H-2),3.26(1H,dd,J=13,16.5Hz, H-3a),2.65(1H,dd,J=16.5,2.8Hz,H-3b),5.93(1H,d,J=2.1,H-6), 5.94(1H,d,J=2.1,H-8),6.85(2H,d,J=7.8Hz,H-3,5),7.35(2H,d, J=7.8Hz,H-2,6).¹HNMRchemicalshiftsareinaccordancewith literaturedatareportedbyLemosdaSilvaetal.(2015).

4,2,4-Trihydroxy-2-methoxychalcone(12):¹HNMR(300MHz, MeOD,TMS),ıH(ppm)J(Hz)7.68(1H,d,J=14Hz,H-␤),7.66(1H, d,J=14Hz,H-␣),7.63(1H,d,J=6.5Hz,H-6),7.62(1H,d,J=7Hz, H-6),7.09(1H,dd,J=3,6.5Hz,H-5),7.07(1H,dd,J=2.8,7Hz,H-5), 6.6(1H,d,J=3Hz,H-3),6.7(1H,d,J=2.8Hz,H-3),3.96(3H,s).The identiﬁcationwascarriedoutthroughcomparisonwith¹HNMR andMSdatareportedbyKhamsanetal.(2012)andKajiyamaetal.

(1992).

Myricetin-3,3,7,-trimethyl ether (14): ¹H NMR (300MHz, MeOD,TMS)ı7.56(2H,s,H-2,6), 6.52(1H,d, J=2.0Hz, H-8), 6.40(1H,d,J=2.0Hz,H-6),3.96(3H,s,OMe),3.94(3H,s,OMe), 3.83(3H,s,OMe).¹³CNMR(150MHz,CD3OD)ı180.8(C-4),163.3 (C-7),160.8(C-5),157.4(C-9),154.1(C-2),152.0(C-3,5),140.0 (C-3),122.3(C-4),116.4(C-1),112.5(C-2,6),104.7(C-10),92.2 (C-8,6),61.2(OMe),60.7(OMe),57.1(OMe).¹HNMRand¹³CNMR chemicalshiftsareinaccordancewithliteraturedatareportedby Kranjcetal.(2016).

5-Hydroxy-3,6,7,8,4-pentamethoxyﬂavone (15): ¹H NMR (400MHz,MeOD) ı 8.13(2H, d, J=8.8Hz, H-2,6), 7.10 (2H, d, J=8.8,H-3,5),3.97(3H,s, OMe),4.10(3H,s,OMe),3.96(3H,s, OMe),3.90(3H,s,OMe),3.87(3H,s,OMe).The¹HNMRchemical shiftsareinaccordancewithliteraturedatareportedbyPaulaetal.

(2002)andZouetal.(2010).

CharacterizationofconstituentsbyGC–EI-MS

Fig.1representsthechromatogramoftheGCanalysesofthe hexaneextract.Themainconstituentsdetectedinhexaneandchlo- roformextractsarelistedinTable1.Theywerecharacterizedbased onmassspectra(Kooetal.,2013)comparisonswiththelibraryNIST 08andWiley-275,withsimilaritypercentagehigherthan95%and NISTMatchFactor–850.Twomainconstituentsweredetected andcharacterizedinbothextracts,namely,octanoicacidtetracosylester(3)andoctanoicacidhexacosylester(4),withmolecular ions(M⁺)atm/z480(C₃₂H₆₄O₂)andm/z508(C₃₄H₆₈O₂),respectively.Thebasepeakatm/z143correspondstotheprotonatedacid moiety((C8H15O2)⁺)(Palaciosetal.,2015).Saturatedstraight-chain estercontainsproductionsderivedfromthefattyacidmoietyof theester([R₁COO]⁺)(Tadaetal.,2014).Thetriterpenoidlupenone 1exhibitedmolecularionM⁺atm/z424(30)andabasepeakat m/z205(100),agreeingwithdataofXuetal.(2018),whilelupeol (2)hadM⁺atm/z426(1)andbasepeakatm/z189(100)(Table1), agreeingwithdataofPereiraBeserraetal.(2018).

CharacterizationofconstituentsbyRPHPLC–DAD–ESI-MS/MS Fig. 2 represents the chromatogram obtained by RP HPLC–DAD–ESI-MS/MS analysis of the ethyl acetate extract.

CompoundscharacterizedarelistedinTable2,includingretention times,[M+H]⁺and[M−H]⁻ionscorrespondingtoprotonatedand deprotonatedmolecules,respectively, andrelevantMS/MSions.

Caffeic acid (5), p-coumaric acid (6), quercetin (8), naringenin (9) and isorhamnetin (10)were identiﬁed by comparison with authenticstandards(Sigma-Aldrich).Naringenin(9)wasidentiﬁed alsoby ¹H NMR spectrum and comparison withdata reported by Lemos da Silva et al. (2015). Isorhamnetin (10) exhibited

5 10 15 20 25

4 3

1 2

30 35 min

Abundance

Fig.1. ChromatogramobtainedbyGC–EI-MSanalysisofthehexaneextractofa propolissamplefromthemunicipalityofSalitre(stateofCeará,northeastBrazil).

Digitsonthechromatogramspeakscorrespondtocompoundscharacterizedand listedinTable1.

UV/vismaximumabsorptionat270,295sh,and360nm,andalso [M+H]⁺atm/z317(Table2).Themassspectrumofdeprotonated moleculeat[M-H]⁻ atm/z 315exhibitedbasepeakat m/z300, produced by a stable radical anion [M−H−15]^•− (Engels et al., 2012;Caoetal.,2009).

Compound 7, tentatively characterized as diprenylcinnamic acid,exhibitedUV/vismaximumabsorptionat310nm,typicalof phenylpropanoids,and[M+H]⁺atm/z285.Thebasepeakatm/z147 isproducedbythelossoftwoprenylgroups(138Da)(Righietal., 2013;Taddeoetal.,2016).Compound11exhibitedUV/vismaxi- mumabsorptionat260and360nm,characteristicofﬂavonols,as wellas[M−H]⁻atm/z625.Itsbasepeakwasm/z301(characteristic ofquercetin)andtheobservedlossesof324Daareconsistentwith O-glycoside(hexoside)fragmentation(KararandKuhnert,2015;

Kumaretal.,2017).Compound11wascharacterizedasquercetin- 3-O-diglucoside.UV/vismaximumabsorptionofchalconesliesin therange340–390nm.Themaximumabsorptionofcompound12 was370nm.ItsMSspectrumprovidedadeprotonatedmoleculeat m/z285andanadduct[2M−H]⁻ionatm/z570(Table2andFig.3).

TheESIMSunderpositivemodeprovidedadominantproduction atm/z257,correspondingtothelossof30Daandaneutralloss ofCH₂O(methanal).AccordingtoGeorgeetal.(2009)ESImeth- odsprotonatechalconesatthecarbonyloxygen,producingspecies thatundergoNazarovtypecyclizations,capableofcatalyzingpro- tonmigrationsinthegasphase.SubstituentsuchasOCH₃atthe 2-positioncanbesufﬁcientlybasictoassistinprotontransport.

The¹HNMRspectrumfrom12exhibitedapairofdoublettrans protonsatı7.68(1H,d,J=14.0Hz)and7.66(1H,d,J=14.0Hz).The signalsatı7.62(1H,d,J=7Hz,H-6),7.07(1H,dd,J=2.8,7Hz,H-5) andı6.7(1H,d,J=2.8Hz,H-3)areconsistentwithsubstitutionat 2and4positionoftheBring.Thesignalsat7.63(1H,d,J=6.5Hz, H-6),7.09(1H,dd,J=3,6.5Hz,H-5)andatı6.6(1H,d,J=3Hz,H- 3)isconsistentwithAringpossessinghydroxylgroupsat2and 4positions.Themethoxylgroupappearedatı3.96(3H,s,OCH3).

Basedon¹HNMRandMSdatareportedbyKajiyamaetal.(1992), Georgeetal.(2009),Khamsanetal.(2012),Lietal.(2017),andSun etal.(2017),compound12wasassignedas4,2,4-trihydroxy-2- methoxychalcone(2-methoxyisoliquiritigenin).

Compound 13, tentatively identiﬁed as gossypetin-3,3,4,7- tetramethyl ether, exhibited UV maximum absorption at 260–360nm, a sodium adduct at m/z 397 and [M+H]⁺ at m/z 375. The characterization was based on the mass spectral database HMDB (www.hmdb.ca) and MS data reported by Ali Khan et al. (2018) and Kranjc et al. (2016). Compound 14, assigned asmyricetin-3,3,7-trimethyl ether, exhibited UV maximum absorption at 260–355nm, [M−H]⁻ at m/z 359 and [M+H]⁺atm/z361,respectively(Table2andFig.3).TheMS/MS experiments at m/z 359 produced a fragment ion at m/z 344 arisingfromthelossofthemethylradical(Husseinetal.,2003;

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Table1

CompoundscharacterizedbyGC–EI-MSanalysesfromthen-hexaneandchloroformextractsofapropolissamplefromSalitre(stateofCeará,northeastBrazil).

Comp.number RT(min) Proposedcharacterization MSdata(M⁺)andfragmentions(m/z)^a

1 26.48 Lupenone 424(30)205(100),189(60),218(50),245(40),313(40),409(30),109(90)

2 26.65 Lupeol 426(1),218(50),207(80),189(100),121(80),95(100)

3 27.58 Octanoicacidtetracosylester 480(1),207(13),143(100),125(51),95(16)

4 29.62 Octanoicacidhexacosylester 508(1),207(13),143(100),125(51),95(16)

GC,gaschromatography;EI,electronimpact;MS,massspectrumdata;RT,retentiontime;NIST,NationalInstituteofStandardsandTechnology.

aNISTMatchFactor:850.

mAU 200

6

5

7 8

10 9 12

11 1314 150 15

100

50

0

0 5 10 15 20 25 30 35 40 45 min

Fig.2. ChromatogramobtainedbyCLAE–DAD-MS/MSanalysisoftheethylacetate extractofapropolissamplefromthemunicipalityofSalitre(stateofCeará,northeast Brazil).Digitsonthechromatogrampeakscorrespondtocompoundscharacterized andlistedinTable2.

Kranjc et al., 2016). Methoxylated ﬂavonoid structures lead to stableradicalanions[M−H−15]^•−(Engelsetal.,2012).Inpositive ionization mode fragment ions at m/z 345 and m/z 328 were observed,correspondingtothesequentiallossesofmethylgroup andwater.The¹HNMRspectrumexhibitedthreedistinctmethoxy protonresonancesatı3.96,3.94and3.83ppm.Inthearomatic region,thespectrumexhibitedasingletatı7.56ppmcorrespond- ing tothe two protons (H-2 and H-6)and two meta coupled protondoublets(J=2Hz)atı6.52and6.40ppm,corresponding to theH-6 and H-8 protons of chromone moiety. In ¹³C NMR spectrum,themostupﬁeldsignalsatı61.2,60.7and57.1were

assignedtothecarbonsofthemethoxygroups.Etheriﬁcationat position3, ledtothedownﬁeld shiftofC-3toı 140.6ppm,as reportedbyKranjcetal.(2016).

Compound 15, assigned as 5-hydroxy-3,6,7,8,4- pentamethoxyﬂavone,providedUV/visspectrumwithmaximum absorptionat 255, 267sh and 350nm,[M+H]⁺ at m/z 389and sodium adduct at m/z 411. The mass spectrum was compared withdatareportedbyZhangetal.(2011).The¹HNMRspectrum showedsignalsatı8.13(2H,d,J=8.8Hz,H-2,6)andı7.10(2H,d, J=8.8,H-3,5)correspondingtotheprotonsofringBandmethoxy groupsatı3.97(3H,s),4.10(3H,s),3.96(3H,s),3.90(3H,s),3.87 (3H,s),whichisinaccordancewithdatareportedbyPaulaetal.

(2002)andZouetal.(2010).

Antioxidantactivities

Theantioxidant activityof theextractscarried out withthe DPPH method varied widely, for example5.32±1.08 (hexane extract)and240.20±4.90mgEQg⁻¹(ethylacetateextract);chlo- roformand methanol extractsshowedactivitiesof 30.46±5.82 and102.94±2.95mgEQg⁻¹,respectively.Ontheotherhand,with the ␤-carotene method, the antioxidant activities of extracts rangedfrom4.4±0.31 (hexaneextract)to37.95±1.3mgEQg⁻¹ (ethylacetateextract).Chloroformandmethanolextractsshowed activitiesof12.76±0.16and17.76±1.22mgEQg⁻¹,respectively.

Ethylacetateextractexhibited greaterantioxidantactivitywith bothevaluatedmethods,thus,bioassay-guidedfractionationwas

Table2

ConstituentsfromtheethylacetateextractofapropolissamplefromSalitre(stateofCeará,northeastBrazil),characterizedbyHPLC–DAD–ESI-MS/MSanalysis.

Comp.number RT(min) UVmax(nm) MS,negativemode (m/z)^a

MS,positivemode (m/z)^a

Proposed characterization

Meansusedfor characterization

5 16.2 300,330 [M−H]⁻179 [M+H]⁺181 Caffeicacid Standard

6 21.1 310 [M−H]⁻163 [M+H]⁺165 p-Coumaricacid Standard

7 25.8 310 ND [M+Na]⁺307

[M+H]⁺285 MS/MS147

Diprenylcinnamicacid Taddeoetal.(2016)andRighi etal.(2013)

8 30.3 256,300sh,360 [M−H]⁺301 [M+H]⁺303 Quercetin Standard

9 30.9 290,337sh [M−H]⁻271

MS/MS253,225

[M+H]⁺273 MS/MS153,147

Naringenin Standard;¹HNMRanalysis, LemosdaSilvaetal.(2015)

10 32.1 270,295sh,360 [M−H]⁻315

MS/MS300

[M+H]⁺317 Isorhamnetin Standard;Engelsetal.(2012) andCaoetal.(2009)

11 34.2 260,360 [M−H]⁻625

MS/MS301

ND Quercetin

3-O-diglucoside

KararandKuhnert(2015)and Kumaretal.(2017)

12 34.3 260sh,370 [M−H]⁻285 [M+Na]⁺309

[M+H]⁺287 MS/MS257,147, 137

4,2,4-Trihydroxy-2- methoxychalcone

1HNMRanalysis,Lietal.

(2017)

13 36.9 260,360 ND [M+Na]⁺397

[M+H]⁺375

Gossypetin-3,3,4,7- tetramethyl ether

MSdatabaseHMDB,AliKhan etal.(2018)andKranjcetal.

(2016)

14 37.4 255,265sh,352 [M−H]⁻359

MS/MS344

[M+H]⁺361 MS/MS345,328

Myricetin-3,7,3- trimethyl ether

NMR(¹Hand¹³C)analyses, Engelsetal.(2012)and Husseinetal.(2003)

15 39.9 255,267sh,350 ND [M+Na]⁺411

[M+H]⁺389

5-Hydroxy-3,6,7,8,4- pentamethoxyﬂavone

1HNMRanalysis,Zhangetal.

(2011),Paulaetal.(2002)and Zouetal.(2010)

HPLC,HighPerformanceLiquidChromatography;DAD,diodoarraydetecter;ESI,electrosprayionization;MS,massspectradata;RT,retentiontime;UV,ultravioletspectrum;

ND,notdetected.

aND:notdetermined.

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IntensIntensIntensIntens mAUmAU

250

287.0

308.9

100

200

300

300 358.7

719.4 743.7 361.1

741.5 400

400

500

600

700

150 200 250 300 350 400 450 500 550 600

200

284.6

2’,4,4’-trihydroxy-2-methoxychalcone (12)

Myricetin-3,7,3’-trimethyether (14)

570.8

300 400 500 600 m/z

m/z

m/z 300 350^nm

250 300 350^nm

C

A

B

A

B

Fig.3. UV/visspectroscopyandMSspectrometryofﬂavonoidsisolatedfromtheethylacetateextractofapropolissamplefromthemunicipalityofSalitre(stateofCeará, northeastBrazil:2,4,4-trihydroxy-2-methoxychalcone(12)andmyricetin-3,7,3-trimethyether(14),bothisolatedfromtheethylacetateextractofpropolissamplefrom themunicipalityofSalitre(stateofCeará,northeastBrazil).A:MS,positivemode;B:MS,negativemode;C:UV/visabsorptionspectrum.

achieved by column chromatography for isolation of its constituents.

Theantioxidantpropertiesoffractionsisolatedfromtheethyl acetateextractareshowninTable3.AntioxidantactivityonDPPH methodrangedfrom14.95±1.11(F3)to112.12±2.78mgEQg⁻¹ (F7).Ontheotherhand,theactivityofthefractionsrangedfrom 31.13±0.00(F6)to36.28±0.29mgEQg⁻¹(F5)withthe␤-carotene method,whileF1–F4wereinactive(Table3).

The main constituents of the most actives fractions F5–F7 derived from the ethyl acetate extract were isolated and their antioxidant properties were evaluated. The results are shown in Table 3. With the DPPH method, the antioxidant activity ranged from 2.51±2.20 for diprenylcinnamic acid (9) to 572.86±2.89mgEQg⁻¹forisorhamnetin(10).Ontheotherhand, with the ␤-carotene method, the antioxidant activities ranged

from13.94±0.64for4,2,4-trihydroxy-2-methoxychalcone(12)to 49.35±0.00mgEQg⁻¹forquercetin(8).

Anti-HIVactivity:HIV-1reversetranscriptasecolorimetricassay ThepreliminaryscreeningforHIV-1reversetranscriptaseinhi- bitionwasperformedusing200␮g/mlofconstituentsandextracts (Woradulayapinijetal.,2005).Atthisconcentration,hexane,chlo- roformandethylacetateextractswereinactiveandcrudemethanol extractshowedweakactivity(2.56±0.06%)regardingtheinhibi- tionoftheHIV-1reverse transcriptase.Fractions F2–F6didnot inhibittheHIVreversetranscriptase,andF1andF7showedweak activity(3.64±0.73%and22.54±1.71%,respectively).

Ontheotherhand,theisolatedcompoundsweremoreeffective, varying from17.88±4.04 [p-coumaric acid(6)] to56.99±3.91%

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Scheme1. StructuresofconstituentsofapropolissamplefromSalitre(stateofParaná,northeastBrazil).DigitscorrespondtocompoundslistedinTables1and2.

Table3

Antioxidantactivityoffractionsisolatedbycolumnchromatographyoftheethyl acetateextractandconstituentsisolatedfromthesefractionsintheethylacetate extractofasampleofpropolisfromSalitre(stateofCeará,northeastBrazil).The resultsareexpressedasmgofequivalentquercetinpergram(EQg⁻¹).

Fraction ␤-Carotenemethod DPPHmethod

F1 Inactive 17.83±1.20

F5 36.28±0.29 28.33±3.34

F6 31.13±0.00 98.75±1.67

F7 32.47±1.02 112.12±2.78

Isolatedcompounds

p-Coumaricacid(6) 31.84±3.12 374.96±2.33 Diprenylcinnamicacid(7) Inactive 2.51±2.20

Naringenin(9) 28.74±2.56 35.44±3.70

Isorhamnetin(10) 42.38±2.16 572.86±2.89

4,2,4-Trihydroxy-2-methoxychalcone(12) 13.94±0.64 32.69±3.47 Myricetin-3,3,4-trimethylether(14) 41.87±0.48 559.97±1.64

Table4

HIV-1reversetranscriptaseinhibitionofisolatedconstituentsfromtheethylacetate ofasampleofpropolisfromSalitre(stateofCeará,northeastBrazil).

Compound %Inhibition

p-Coumaricacid(6) 17.88±4.04

Diprenyl-cinnamicacid(7) 41.59±2.59

Quercetin(8) 43.41±4.56

Naringenin(9) 44.22±1.71

Isorhamnetin(10) 56.99±3.91

4,2,4-Trihydroxy-2-methoxychalcone(12) 34.01±4.12 Myricetin-3,3,4-trimethylether(14) 35.35±3.99

[isorhamnetin(10)],ascanbeseeninTable4.Theinhibitoryeffect onHIV reversetranscriptasecan beclassifiedasstrong(>90%), moderate(>50–90%)andweaklyactive(<50%).Accordingtothis classification,themostactivecompoundwasisorhamnetin(10, Table2)withamoderateactivity(56.99±3.91%,Table4).Accord- ingtoa studycarried outbyColeetal.,flavonoidsthatcontain eithera␣-or␤-hydroxy-carbonylmotifwithintheirstructurecan exhibithighanti-HIVactivity.

Discussion

There are many studies about green propolis from south- eastregionofBrazilandredpropolisfromthenortheastregion.

However, there is little information aboutpropolis from Ceará State(Albuquerqueetal.,2007;Gutierrez-Gonc¸alvesandMarcucci, 2009).Itisknownthatthechemicalcompositionofpropolisvaries

accordingtophytogeographicregions(Kasiotisetal.,2017).Onthe otherhand,thechemicalcompositionofpropolisisoftensimilar inadeterminedgeographicregion(Pasupuletietal.,2017;Kocot etal.,2018).

Thepropolissampleanalyzedinthisstudywasharvestedin the municipality of Salitre, in the Atlantic forest regionof the Araripeplateau,CearáState.Albuquerqueetal.(2007)analyzed thechemicalcompositionofapropolisfromCeará,harvestedin AltoSanto,locatedwithina Caatingaregion.TheAtlanticforest regionoftheAraripeplateaupossessesdistinctsoil,weatherpat- ternsandflora,incomparisonwiththeCaatingaregion.Inpropolis fromAltoSanto,canaricacid,thetriterpeneslupeol,lupenone,ger- maniconeandtheflavonoidsquercetin,kaempferolandacacetin wereidentifiedasmainconstituents.Thebotanicalresin source ofpropolisfromAltoSanto,althoughunknown,probablycontains canaricacidasoneofthemainconstituents(Albuquerqueetal., 2007).AscanbeseeninTables1and2,lupenone(1),lupeol(2) andquercetin(8)weredetectedinSalitrepropolis.However,these compoundsarewidelydistributedinplants.Amongtheothercon- stituentsdetected,chalcones(forexample12)havebeendetected alsoinBrazilianredpropolis(Bueno-Silvaetal.,2017).Themain polarconstituentsdetectedinpropolisfromSalitreweremethoxy- latedflavonols,suchasmyricetin-3,3,7-trimethylether.Anewtype ofgreenpropolisharvestedfromRioGrandedoNorteStatepos- sessesmethoxylatedflavonolsandchalconesasmainconstituents (Ferreiraetal.,2017).However,myricetin-3,3,7-trimethylether (14)and5-hydroxy-3,3,4,7,8-pentamethoxyflavone(15)suggest thatplantssofarunreportedcontributeasresinsourcesforSalitre propolis.Thepresenceofachalcone(compound12,Table2,and Scheme1)asarelevantconstituentinSalitrepropolissuggeststhat aplantfromtheFabaceaefamilymightbeoneofitsresinsources (Ferreiraet al.,2017), although not belongingto thesubfamily Faboideae.Plants of thelatter groupof Fabaceae often contain isoflavonoids,asisthecaseofD.castaphyllum,theresinsourceof Brazilianredpropolis(Rufattoetal.,2017;SalatinoandSalatino, 2018).Isoflavonoidswerenotdetectedinthepresentstudy.

The biological activityof propolis hasbeen assigned to the antioxidantactivityofphenoliccompounds.Freeradicalsandother oxidativeagentscanproducemanycelltoxinsthatinduceoxida- tivedamageinbiomolecules(Silva-Carvalhoetal.,2015;Zheng et al.,2017).Flavonoidsand otherphenolic compoundsprotect cells against thedamage caused by oxidation, acting as potent inhibitorsofoxidativestress,whichisinvolvedinthepathogene- sisofneurodegenerativedisorders(Zhengetal.,2017).Inaddition, theintracellularfreeradicalscavengingcapacitiesofphenoliccom- pounds can protect cell membranes against lipid peroxidation (DalepraneandAbdalla,2013;Galeottietal.,2018).Phenoliccom- poundsexertantioxidantactivitythroughthedonationofhydrogen

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atomsfromanaromatichydroxylgroup,leadingtosequestration offreeradicals(Zaccariaetal.,2017;Amoratietal.,2017).

TheantioxidantactivityofthepropolissamplefromSalitrewas evaluatedbytwoassays,DPPHand␤-carotene/linoleicacid.The DPPH(1,1-diphenyl-2-picrylhydrazyl)assaymeasuresthehydro- genatomdonationcapacityofphenoliccompoundtoscavengethe DPPHradical(Table3).The␤-carotenebleachingmethodevaluate theabilityofphenoliccompoundtopreventtheoxidationof␤- carotene,protectingitfromthefreeradicalsgeneratedduringthe peroxidationoflinoleicacid(Zhengetal.,2017).Theethylacetate extractexhibitedthebestantioxidantactivityinbothassays.Ethyl acetateextractsofpropolisfromseveralAlgerianregionsexhibited highantioxidantactivitybyscavengingfreeradicalsandpreventing lipidperoxidation(Boufadietal.,2014).

Theconfiguration,substitution,andtotalnumberofhydroxyl groups in flavonoids can influence the mechanisms of radical scavenging.TheBringhydroxylconfigurationdeterminesthescav- engingofreactiveoxygenspecies(ROS)throughthedonationof hydrogen,producingrelativelystableflavonoidradicals(Amorati etal.,2017).ThesuppressionofROSformationisperformedeither byinhibitionofenzymesorbychelatingtraceelementsinvolved in free radicalgeneration, scavengingROS and upregulation or protection of antioxidant defenses, involved in mechanisms of antioxidantaction(Amoratietal.,2017).Quercetinexhibitshigh antioxidantactivity,becausepossessanorthocatecholgroupon theBring,whichenablesthegenerationofintra-andintermolec- ularhydrogenbonds(Tremland ˇSmejkal,2016).Ascanbeseen inTable3,quercetin(8),isorhamnetin(10)andmyricetin-3,3,7- trimethylether(14)exhibitedhighantioxidantactivity,followed byp-coumaric acid(6).Quercetin, a constituentof many types ofpropolis(Zhengetal.,2017), revealedimportantcytotoxicity processesagainstculturedhumancells(Tremland ˇSmejkal,2016;

Amoratietal.,2017)andcanbeusedasanoveltherapeuticagent forneurodegenerativediseasesinducedbyoxidativestress(Bao etal.,2017).

ThepropolissamplefromSalitreexhibitedmoderateactivity relativetotheinhibitionofHIV-1reversetranscriptase(Table4).

ThisenzymeconvertstheviralRNAtoviralDNA,whichisinte- grated into thehost genome by HIV integrase, responsible for cleaving thetranslated viral proteinsrequired for formation of newHIVparticles,whicharereleasedtoinfectothershostcells (Pasetto et al., 2014; Saravanan et al., 2015; Li. et al., 2016).

Flavonoidsactasbactericidalandbacteriostaticagents bydam- agingcytoplasmicmembranes,inhibitingenergymetabolismand synthesis of nucleic acids. Thus, it can be used as antiretrovi- rals for HIV therapy, due to higher antiviral activity and low toxicity(Ahmadetal.,2015;Saravananetal.,2015).Theinhibi- tionoftheHIV-1reversetranscriptasedecreasedinthefollowing order:isorhamnetin(10),naringenin(9),quercetin(8),diprenylcinnamic acid (7), myricetin-3,3,7-trimethyl ether (14, Fig. 3) and2,4,4-trihydroxy-2-methoxychalcone(12,Fig.3).Ingeneral, thepresence ofprenylgroupsmayincreaseanti-HIVactivity.A ﬂavonoidcontaininga5,7-dihydroxy-6,8-diprenylsystemonthe Aringexhibitedhighanti-HIVactivity(Meragelmanetal.,2001;

Kurapatietal.,2016).AhighlyactiveﬂavonoidagainstHIV-1pro- teaseis6,8-diprenylgenistein,anisoﬂavonewithtwoprenylgroups ontheAringandonehydroxylgroupatthe4positionoftheB-ring (Leeetal.,2009).

Quercetin exhibited moderate activity against HIV-1, as reportedbyKurapatietal.(2016).Myricetin,withanadditional hydroxylgrouponthe5 position,isastrongerinhibitorofHIV- 1 reverse transcriptase, indicating that the presence of either the unsaturation between positions 2 and 3 of the ﬂavonoid pyrone ring and three hydroxyl groups are important requi- sitesfor inhibition of reverse transcriptaseactivity (Ono et al., 1990). Myricetin showed promising results against different

strainsofHIV-1,whilealsoshowedinsigniﬁcantcytotoxiceffects (Pasetto et al., 2014).Myricetin3-O-rhamnosideand myricetin 3-O-(6-rhamnosylgalactoside)inhibitedthereversetranscriptase activity.Theglycosylatedmoietyenhancedtheanti-HIV-1activ- ity of myricetin (Ortega et al., 2017). However, in propolis sample from Salitre, the hydroxyl groups of myricetin are in methoxylated form and myricetin-3,3,7-trimethyl ether exhibited weak activity as inhibitor of reverse transcriptase HIV-1 (35.35±3.99%). Coherently, the inhibitory activity against HIV- 1 reverse transcriptase was shown to decrease proportionally with the degree of methoxylation of ﬂavones (Ortega et al., 2017).

Conclusions

The studied propolis sample from Salitre, a locality in the AtlanticForestregionofthestateofCeará,possesseschemicalpro- ﬁledistinctfromallpropolistypesofBrazilianpropolisreported sofar.Theﬁndingofachalconeamongtherelevantconstituents suggestsa leguminousplantasresin source.Thepropolis sam- plepossesses high antioxidant activity,as revealed byanalysis of its ethyl acetate extract. Fractions F5–F7 and isolated compounds,suchasp-coumaricacid(6)quercetin(8),isorhamnetin (10)andmyricetin3,3,7-trimethylether (14)wereshown tobe effective antioxidants. In addition, isorhamnetin (10) exhibited moderateinhibitionofHIV-1reversetranscriptase,whileotheriso- latedconstituentswereactiveinlowerdegree.Theresultsobtained strengthentheviewthatpropolisresearchisaneffectivemeansto detectbioactivesubstancesofplantoriginandthatmuchfurther workisneededaimingthedeterminationofthebotanicaloriginof propolisproducedinBrazil.

Author’scontributions

ThisstudyispartofdoctoralthesisofCCFS;ASandMLFSwere thementorsandrevisethemanuscript;GNandLBMwerework collaborators. All theauthors made important contributions in theaccomplishmentofthework,readtheﬁnalmanuscript and approveditssubmission.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

TheauthorsthankCNPqandFAPESPforprovisionoffundsused inthedevelopmentofthisresearch;aswellasApiárioBoscofor provisionoftheSalitrepropolissample.

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