Antileishmanial activity and chemical composition from Brazilian geopropolis produced by stingless bee Melipona fasciculata

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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

Antileishmanial activity and chemical composition from Brazilian geopropolis produced by stingless bee Melipona fasciculata

Richard Pereira Dutra

^a

, Jeamile Lima Bezerra

^a

, Mayara Cristina Pinto da Silva

^b

, Marisa Cristina Aranha Batista

^a

, Fernando José Brito Patrício

^b

,

Flavia Raquel Fernandes Nascimento

^b

, Maria Nilce Sousa Ribeiro

^a,∗

, Rosane Nassar Meireles Guerra

^b

aLaboratóriodeFarmacognosia,CentrodeCiênciasBiológicasedaSaúde,UniversidadeFederaldoMaranhão,SãoLuís,MA,Brazil

bLaboratóriodeImunoﬁsiologia,CentrodeCiênciasBiológicasedaSaúde,UniversidadeFederaldoMaranhão,SãoLuís,MA,Brazil

a r t i c l e i n f o

Articlehistory:

Received30September2018 Accepted18February2019 Availableonline11May2019

Keywords:

Apidae Meliponini Geopropolis Leishmaniasis Gallicacid Ellagicacid

a b s t r a c t

GeopropolisisproducedbysomestinglessbeespeciessuchasMeliponafasciculataandconsistsofa mixtureofplantresins,salivarysecretionsofthebee,wax,andsoil.Thisstudyevaluatedtheantileish- manialactivityinvitro,cytotoxicityandchemicalcompositionofgeopropolisproducedbyM.fasciculata inthesavannahregionofMaranhão,Brazil.Thegeopropolisextractwasobtainedthroughmaceration usingin70%ethanol.Thehydroalcoholicextractofgeopropolisafterliquid–liquidpartitionyieldedthe hexane,chloroformic,ethylacetateandhydroalcoholicfractions.Antileishmanialactivitywasevaluated againstpromastigoteandintracellularamastigoteofLeishmaniaamazonensis.Cytotoxicwasrealizedin BALB/cmiceperitonealmacrophages.Chemicalanalysiswasperformedbygaschromatography–mass spectrometryandhighperformanceliquidchromatographycoupledtoanultraviolet–visibledetector.

ThegeopropolisinhibitedtheL.amazonensispromastigotesgrowthandwaseffectiveinreducingthe infectionofmurinemacrophagessincethenumberofinternalizedamastigotesweresmallerincells treatedwiththegeopropolisextractinrelationtotheuntreatedgroup.Theethylacetatefractionwas themostactiveandshowedthehighestindexofselectivityasantileishmanialproduct.Thegeopropolis fromM.fasciculatahadanantileishmanialeffect,mainlyaftertheobtentionoftheethylacetatefraction thatimprovedtheactivitywithoutincreasingthecytotoxicityagainstmurinemacrophages.Analysisfor gaschromatography-massspectrometeridentiﬁedasmaincompoundsthegallicandellagicphenolic acids,eitherintheextractorintheactivefraction.Theresultsobtainedbyhighperformanceliquidchro- matographyitwaspossibletoconﬁrmthepresenceandquantifytheconcentrationgallicandellagic acideitherintheextractorintheactivefraction.Theseresultssuggestthattheantileishmanialactivity ofgeopropolisisrelatedtothepresenceofderivativesofthesephenolicacids,mainlygallicandellagic acids.

Introduction

Leishmaniasisisaninfectiousdiseasethatoccursincountries with tropical and temperate climates and it is transmitted to humansbythebiteofsandﬂiesinfectedwithprotozoaofthegenus Leishmania.Thisdiseaseaffectsapproximately2millionpeoplein theworldeveryyear(WHO,2018).

The severity of disease varies ranging from cutaneous or mucosal to visceral or diffuse cutaneous infection. Leishmania amazonensis,aspeciestransmittedmainlyintheAmazonregion,

∗ Correspondingauthor.

E-mail:mnribeiro@ufma.br(M.N.Ribeiro).

which is associated with localized cutaneous lesions. In Brazil occur both visceral leishmaniasis (VL) and tegumentary leishmaniasis (TL), endemic disease in Northern and Northeastern Brazil,mainlyinthestatesofBahia,Ceará,Maranhão,andPiauí (Marzochietal.,2009).

Currently,thedrugsusedtotreatleishmaniasisincludepentava- lentantimonial,amphotericinB,andpentamidine,presentvariable efﬁcacyagainstdifferentLeishmaniaspecies,elevatedtoxicityand caninduce resistanceoftheparasite (Croft and Coombs,2003;

Silva-López,2010).Therefore,thereisanurgentneedfornewdrugs thatcanbeusedbothfortheprophylaxisandforthetreatment ofleishmaniasis.Inthisrespect,beeproductsarepromisingcan- didatessincetheyareimportantsourcesofbioactivecompounds (BankovaandPopova,2007;Lavinasetal.,2019).

https://doi.org/10.1016/j.bjp.2019.02.009

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

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IntheStateofMaranhão,northeastBrazil,Meliponafasciculata Smith,popularlyknownas“tiúba”or“tiúba-do-maranhão”,isthe mostwidelycultivated stinglessbeespecies becauseof itshigh productivityofhoneyandgeopropolis.Thisspeciesisthereforeone ofthemainsourcesofincomeformanyfamiliesinthestate(Dutra etal.,2008)andthegeopropolisproducedispopularlyusedfor thetreatmentofinﬂammation,weakness,hemorrhoids,gastritis, cough,andalsoasahealingagent(Kerr,1987;Araújoetal.,2016b).

Geopropolisisproducedfromplantresinsthataremixedwith pollen,wax,salivarysecretions,andsoil.Inthebeehive,thisprod- uct is used to ﬁll small cracks, to seal entry holes, and as an antimicrobialagent(Araújoetal.,2016b;Sanchesetal.,2017).

Thechemicalcompositionofgeopropolisproducedbydiffer- ent species of Melipona has shown phenolic acids, ﬂavonoids, coumarins,hydrolysabletannins,prenylatedbenzophenone,ter- penes,steroids,saponins,fattyacidsandsugars(Dutraetal.,2008, 2014;DaSilvaetal.,2013;Souzaetal.,2013,2014;Araújoetal., 2015;Batistaetal.,2016;Cunhaetal.,2016).

Thefollowingactivitieshavebeenattributedtothis product:

antimicrobial(Mulietal.,2008;Liberioetal.,2011;Araújoetal., 2016b;Santosetal.,2017),antioxidant(DaSilvaetal.,2013;Souza etal.,2013;Dutraetal.,2014;Batistaetal.,2016;Ferreiraetal., 2017),anti-inﬂammatory(Liberioetal.,2011;Franchinetal.,2013), antiproliferative,cytotoxic(Cunhaetal.,2013,2016;Camposetal., 2014), anti-nociceptive(Souzaet al.,2014), immunomodulatory (Liberioetal.,2011;Araújoetal.,2015),antitumor(Cinegagliaetal., 2013;Bartolomeuetal.,2016)andgastroprotective(Ribeiro-Junior etal.,2015).

There are studies investigating the antileishmanial activity ofpropolis produced by Apis melliferabees (Ayreset al., 2007;

Machadoetal.,2007;Duranetal.,2008,2011;Pontinetal.,2008).

However,therearenoreportsdescribingtheantileishmanialactiv- ityoftheM.fasciculatageopropolis.

Therefore,thepresent studyinvestigated theantileishmanial activity, cytotoxicity and chemical composition of geopropolis producedbyM.fasciculataSmithinordertocontributethebio- prospection of new products with activity against Leishmania infection.

Materialandmethods

Geopropolissample

The geopropolis sample was collected from beehive in meliponary in the municipality of Fernando Falcão, Maranhão, Brazil(2^◦3730S44^◦5230W), asavannah regionfrom Maran- hãoState,northeastBrazilinNovemberof2008.Geopropoliswas removedfromthehiveswithaspatula,storedinsterilecontainer, andtransportedtothelaboratoryforpreparationoftheextractand chemicalandbiologicalanalysis.

Extractionandfractionationofgeopropolisextract

Thegeopropolissample(500g)wasseparatelymaceratedwith 1:2 (w/v) in 70% ethanol for 48h and ﬁlteredto separate the inorganic part(soil). The hydroalcoholic extract of geopropolis (HEG)wasfractionatedbyliquid–liquidpartitionusingsolventsof increasingpolarity,obtaininghexane(HF),chloroform(CF),ethyl acetate(EAF)andhydroalcoholic(HAF)fractions,asdescribedby Dutraetal.(2014).

Parasites

Leishmaniaamazonensis(MHOM/BR/1987/BA-125)promastig- otesweremaintainedin axeniccultures containing Schneider’s

medium(Sigma,St.Louis,MO,USA)supplementedwith10%inacti- vatedfetalbovineserum(Gibco,Carlsbad,CA,USA)andgentamicin (50␮g/ml;Sigma),incubatedat24^◦C.Thegrowthofthecultures wasmonitoreddailyandparasites werecountedinaNeubauer chamber.OnlyL.amazonensispromastigoteformsinthestationary phasewereusedintheassays.

Obtentionofmurineperitonealmacrophages

Thecellswereobtainedbywashingtheperitonealcavityfrom Balb/cmice withcoldsterilephosphate-bufferedsaline(PBS) 5 daysafterintraperitonealinjectionof 1mlof3%sterilethiogly- colate.The cellsuspensionswere placedin 96-wellﬂat-bottom microplatesandafter3hofadherence,thenonadherentcellswere removedbywashingwithPBSat37^◦C.Theadherentcellsin100␮l ofcompletemediumwereusedinmostoftheexperiments.The cellswerecentrifuged(160×g,10min,4^◦C)andresuspendedin completemediumRPMIand10%fetalbovineserum(FBS)(Ribeiro- Diasetal.,1999).Allexperimentsinvolvingtheuseofexperimental animalswereperformedinaccordancetotheethicalstandardsof FederalUniversityofMaranhãoandwereapprovedbytheethics committee(Protocol:23115-012975/2008-43).

Evaluationofextract,fractionsandphenolicacidsagainst Leishmaniaamazonensispromastigotes

Theantileishmanialactivityoftheextract,fractionsandphe- nolicacidsgallic andellagic(SigmaAldrich,StLouis,MO, USA) wereevaluated against promastigotes of L.amazonensis in cul- ture using microplates. The HEG, fractions and phenolic acids were resuspended in PBS and diluted in complete RPMI 1640 medium to final concentrations of 500, 250, 125, 62.5, 31.25, and15.62␮g/ml.Aliquotsof 10␮lof thesuspensioncontaining 5×10⁶/mlpromastigoteforms ofL.amazonensiswereaddedto thewells.AmphotericinB(SigmaAldrich,StLouis,MO,USA),the referencedrugforthetreatmentofleishmaniasis,wasusedaspos- itivecontrolinconcentrationsvaryingfrom0.07to10␮g/ml,and comparedtothenegativecontrol(culturemedium).After24hof incubation,thetotalnumberoflivepromastigoteswasdetermined byflagellamotilityinaNeubauerchamber,undera bright-field lightmicroscopy.Theconcentrationthatinhibitedculturegrowth by50% (IC₅₀)wasdeterminedby nonlinearregression (Bezerra etal.,2006).

Evaluationofextractandfractioninmacrophagesinfectedwith Leishmaniaamazonensisamastigotes

PeritonealmacrophagesfromBalb/cmicewereharvestedand plated at 2×10⁶cell/mlin a 24-well plate containinga cover- slipof13mmdiameter,allowedtoadherefor2hat37^◦Cin5%

CO2. Afterthis periodthewells werewashed threetimes with unsupplementedRPMI1640toremovenon-adherentcells.Then the macrophages were infected with promastigotes at ratio of promastigote/macrophage(10:1),andthecellswereincubatedat 37^◦C in 5% CO₂. After4h incubation, free promastigotes were removedandcounted(Silvaetal.,2018).Theinfectedmacrophages weretreatedduring24hwithHEG(47␮g/ml)andEAF(29␮g/ml) accordingtheinhibitoryconcentrationdetectedattheinvitroanti- promastigote assay. Afterincubation, thecells wereﬁxed with methanol,Giemsastainedandexaminedbylightmicroscopy.The numberofamastigotes/100macrophagecellsandthepercentage ofinfectedcellsweredetermined.Theinfectionindexwascalcu- latedbymultiplyingthepercentageofinfectedmacrophagesbythe meannumberofamastigotesperinfectedcells(LimaJunioretal., 2014;Silvaetal.,2018).

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Cytotoxicityassay

Peritoneal macrophages wereplated at 2×10⁵cells/well on coverslipsin96-wellplatefor24hinthepresenceorabsenceofthe HEGandfractionsindifferentconcentrations(15.62,31.25,62.5, 125,250and500␮g/ml).Attheendoftheperiodwereadded10␮l of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT,Sigma Aldrich,St Louis, MO, USA) 5mg/mland the cells wereincubatedfor 3hat37^◦Cin5%CO₂.Thesupernatantwas removedandtheplateswereincubatedovernightatroomtemper- aturewith100␮lof10%solutionofsodiumdodecylsulfate–HCl todissolution offormazancrystals.Absorbancewasdetermined in a spectrophotometer (MR 5000, Dynatech Laboratories Inc., Gainesville,VA,USA)at540nm.The50%cytotoxicconcentration (CC50)wasdeterminedbyregressionanalysis.Theselectivityindex (SI)wasdeterminedastheratioofCC₅₀forthemacrophagesto IC₅₀forthepromastigote.Eachassaywascarriedoutintriplicate inthreeindependentexperiments(Ribeiro-Diasetal.,1999).

Samplepreparationforgaschromatography–massspectrometry (GC–MS)analysis

The HEG and fractions of geopropolis (10mg) were solu- bilized in pyridine (300␮l) and bis-(trimethylsilyl) triﬂuoroac- etamide(BSTFA,SigmaAldrich,StLouis,MO, USA)including1%

trimethylchlorosilane(TMCS)(BSTFA/TMCS),100␮l,andheatedat 80^◦Cfor20minaccordingtoBatistaetal.(2016).

AnalysisoftheextractandfractionsbyGC/MS

AnalysisoftheHEGandfractionswereperformedongaschro- matograph(GC)(Agilent6890) coupledtoamass spectrometer (MS)(Agilent5973NMSD),equippedwithaHP-5MSfusedsilica (30m×0.25mmi.d.,ﬁlmthickness0.25␮m)andoperatinginthe electronimpactionizationmode(70eV),wasusedfor analyses.

Thetemperaturesoftheinjectoranddetectorweremaintainedat 230^◦Cand250^◦C,respectively.Heliumwasusedasthecarriergas ataﬂowrateof1ml/min,andtheinjectionvolumewas1␮linsplit ratio10:1.Analysisconsistedof5minofisothermalheating(70^◦C), atemperaturegradientof5^◦C/minat310^◦C,andalastminuteof heatingat310^◦C.Theinterfacewasmaintainedat200^◦Candthe detectorwasoperatedinthescanningmode(m/z50–650)anda scanintervalof2scans⁻¹.Theidentiﬁcationofgeopropoliscom- poundswasbasedonthepercentageofsimilaritypluscomparison ofmassspectra(MS)usingsoftwareNISTAMIDSversion2.0data library,withthepercentageoftotalionchromatograms(TIC%).

AnalysisoftheextractandfractionsbyHPLC/UV

Theidentificationandquantificationofgallic(GA)andellagic acids(EA)intheHEGandEAFfractionwereperformedbyhighper- formanceliquidchromatographycoupledtoanultraviolet–visible detector(HPLC–UV).Theanalysiswasproceededinchromatograph VarianHPLCsystemequippedwithaProStarAutosampler410,2 ProStar215pumps,ProStar325UV-Visdetector(Varian,PaloAlto, USA)controlledwithGalaxiesoftware(Varianv1.9.3.2),usinga PhenomenexRP-GeminiC18column(250mm×4.6mm,5␮m)at roomtemperature.Themobilephasewascomprisedof0.1%(v/v) formicacidacidifiedultrapurewater(assolventA)andacetoni- trile(assolventB).Thegradientprogramwasasfollows:90–10%B (5min),80–20%B(10min),70–30%B(25min),65–35%B(30min).

Thedetectionofcompoundsofinterestwasperformedatawave- length()of254nm,andthevolumeinjectedwas20␮l,withthe mobilephaseﬂowof1.0ml/min.

TheconcentrationofGAandEAinthesampleswereobtained fromtheregression-equationresultedbythelinearregressionof

Table1

Antileishmanicidalactivityoftheextractandfractionsofgeopropolis,gallicacid, andellagicacidagainstpromastigoteforms,cytotoxicityandselectivityindexfor Leishmaniaamazonensis.

Sample L.amazonensis Macrophage

IC50(␮g/ml)^a CC50(␮g/ml)^a SIprob

HEG 47.00±2.70^a 292.70±35.49^a 6.22

HF Noactivity Nd Nd

CF 43.21±2.32^a 78.34±32.64^b 1.81

EAF 29.90±2.93^b 244.90±22.60^c 8.19

HAF 49.48±1.40^a 381.70±9.29^d 7.71

Gallicacid 14.48±1.87 Nd Nd

Ellagicacid 151.7±19.50 Nd Nd

AmphotericinB 1.0±0.46^c Nd Nd

aValuesrepresentthemeanoftriplicatemeasurements±standarddeviation.

Differentlettersinthesamecolumnindicateasigniﬁcantdifference(Tukeytest, p<0.05).HEG,hydroalcoholicextractofgeopropolis;HF,hexanefraction;CF,chloroformfraction;EAF,ethylacetatefraction;HAF,hydroalcoholicfraction;Nd,not determined.

bSIpro(selectivityindex)=CC50macrophages/IC50promastigoteforms.

IC50=concentrationproducing50%inhibitionofL.amazonensispromastigotes.

CC50=concentrationproducing50%inhibitionofperitonealmacrophages.

a calibrationcurve, correlating the chromatographic peak area referredtothegallicacidandellagicacidpurchasedfromSigma- Aldrich(St.Louis,MO,USA)andtheirconcentrationinthesample (1mg/ml). Calibration curve (ﬁve points)was used toquantify eachstandardwiththerangeof5–100␮g/mlforgallicacid,and 2.5–40␮g/mlforellagicacid.

Statisticalanalysis

Allassayswererepeatedthreetimesandtheresultsexpressed areexpressedasthemean±standarddeviationandwereanalyzed.

Alldatawerecomparedbyanalysisofvariance(one-wayANOVA) followedbytheTukeytest.Differenceswereconsideredsigniﬁcant whenp<0.05.AlldatawereanalyzedwiththeGraphPadPrism 7.0software(SanDiego,CA,USA).Allanalyseswereperformedin triplicate.

Results

TheHEG,CF,EAF,andHAFshowedanti-leishmaniaactivitysince theywereabletoreducethenumberoflivepromastigotes.HEG reducedthenumberofviablepromastigotesofL.amazonensisin adose-dependentmannerwithIC₅₀of47␮g/ml,andatthecon- centrationof250␮g/mlwasabletoinhibit100%ofpromastigote forms(Table1).Afterfractionation,theEAFfractionwasthemost active (IC₅₀ 29.89␮g/ml),inhibiting 100% of thepromastigotes from62.5␮g/ml.Theotherfractionsalsoshowinhibitoryeffectfor L.amazonensispromastigotesatthetestedconcentrations(CF:IC50

43.21␮g/mlandHAF:IC5049.48␮g/ml).FHshowednoantileish- manialactivity(Table1).

The antileishmanialactivityagainst promastigotesand cytotoxicity toHEGand theactivefractions(CF, EAF,andHAF) was comparedusingtheselectivityindex.TheHEGdemonstratedCC₅₀ of292.7␮g/mland EAFCC₅₀of 244.9␮g/ml, whileHAFand CF 381.7␮g/mland78.34␮g/mlrespectively.Theselectivityindexfor promastigoteshasshownthatthemostactivefraction(EAF)also haslowercytotoxicactivityformacrophages(Table1).

The extract (HEG) and the active fraction (EAF) induced a signiﬁcant reduction in the number of amastigotes within the macrophagescomparedtothecontrolgroup,intheexperimen- talinfectioninvitrowithperitonealmacrophagesinfectedwithL.

amazonensis.

Treatment of infection of macrophages with HEG and EAF reduced number of amastigotes inside macrophages. This

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A B

10

* *

8 6 4 2 0

10 8 6 4 2

Control 47 µg/ml 0 Control

HEG

47 µg/ml HEG 29 µg/ml

EAF

29 µg/ml EAF

Amastigotes / 100 macrophages Infection index

Fig.1. Invitroeffectofgeopropolisextract(HEG)andfraction(EAF)onLeishmaniaamazonensisintracellularamastigotes.Thenumberofamastigotesininfectedmacrophages (A),andinfectionindex(B).ThenegativecontrolisL.amazonensisintracellularamastigotesnottreated.Theresultsrepresentmean±standarddeviationofindividualsamples testedintriplicate(*):p<0.05incomparisontothenegativecontrol.

antileishmanialeffectwasassociatedwithareductionontheinfec- tionindex.Thepercentagereductionininfectionwas37%inthe macrophagestreatedwithHEG,whereasthosetreatedwithEAF reducedby33%(Fig.1).

TheanalysisofHEGandfractionsbyGC/MS,identifiedtwenty threecompoundsbycomparisonofretentiontimes,massspectra usingNISTLibraryandliteraturedata.Thecompoundsidentified belongtotheclassoffattyacids,organicacids,phenolicacids,triterpenes,steroids,sugarsandalcohols.Fattyacidswereidentifiedin thesamples,withtheexceptionoftheEAFfraction,withahigher concentrationofpalmiticandstearicacids.Inthehexanefraction thetriterpenes␤-amyrin,lupenoneandsteroidscampesterol,stig- masteroland␤-sitosterolwereidentified,whileinthechloroform fractionoleanolicacidand␤-sitosterol.Phenolicacids,sugarsand alcoholswereidentifiedmainlyinHEGandEAFfraction(Table2).

AccordingtheresultsobtainedbyHPLC/UVitwaspossibleto confirmthepresenceofgallicandellagicacid(Fig.2).Thecalibra- tioncurves obtainedtoquantifytheconcentrationofgallicacid andellagicacidpresentedcorrelationcoefficient≥0.998,confirm- ingthelinearityofthemethod.Thedetectedconcentrationofgallic acidwas2.66␮g/mgand9.85␮g/mgforHEGandEAF,respectively, whiletheellagicacidconcentrationwas20.32␮g/mgforHEGand 27.73␮g/mgfortheEAFfraction.

The fractionation of the extract by liquid–liquid partition increasedtheconcentrationofellagicandgallicacidsinEAF,mainly gallicacidsincetheconcentrationwasabout3.7timeshigherthan HEG.

Thegeopropolissampleswithantileishmanialactivityshowed thepresenceandhigherconcentrationsofphenolicacids,mainly EAF fraction with gallic acid (40.5%) and ellagic acid (31.6%) (Table2).ThemassspectraobtainedbytheGC–MSanalysisafter silylationwithBSTFAfromtheextractandfractionsofthegeo- propolisshowedtheionsm/z458,443,281,73forgallicacid(4TMS) andionsm/z590,575,487,281,73forellagicacid(4TMS).

Consideringthehighconcentrationofgallicandellagicacids in themost active fraction(EAF), analytical standards of these compoundswereevaluatedforantileishmanialactivityagainstL.

amazonensispromastigotes.Thephenolicacidsexhibitedantileish- manialactivity,however,gallicacidwasmoreeffectiveininhibit againstpromastigotesforms(IC5014.48␮g/ml)thanellagicacid (IC₅₀151.7␮g/ml)(Table2).

Discussion

Theextractandfractionsofthegeopropolispresentedananti- Leishmaniaactivity,withtheEAFfractionbeingmoreeffectivethan theextract(HEG)inreducethepromastigotesnumbers,andalsoto

reducethenumberofamastigotesininfectedmacrophages,despite thelowcytotoxicityforuninfectedmurinemacrophages.

The selectivity index (SI) foundfor geopropolis extract and itsfractionsindicatehighertoxicitytopromastigoteformsanda moderatetoxicitytouninfectedmacrophages(SI<10)(Bringmann etal.,2013).Theseresultsareimportantinthesearchforeffec- tivenatural productsfor the treatmentof leishmaniasis (Alves etal.,2017).AccordingtoVeigaetal.(2017)extractswithIC50

≤100␮g/mlforpromastigoteformsareconsideredpromisingfor thebioprospectionofproductswithanti-Leishmaniaactivity,espe- ciallywhentheyshowlowtoxicity(CC50>200␮g/ml)tonoinfected cellsvalues,asshowedheretotheEHGandEAF.

Aftertheliquidchromatography tandem–massspectrometry (LC–MS/MS)itwaspossibletoidentifyelevencompoundspresent inHEGandEAF,includinggallicandellagicphenolicacids,aswell asthehydrolysabletanninsas:corilagin,HHDP-glucose,peduncu- lagin,trigalloylglucose,tellimagrandinI,valoneicaciddilactone, casuarictin, tellimagrandinIIetrisgalloyl-HHDP-glucose)(Dutra etal.,2014).

The presence of gallic acid in M. fasciculata geopropolis is expectedsincethiscompoundhasbeenidentifiedasoneofthe maincompoundsingeopropolisfromotherspeciesofthegenus Melipona as: M. quadrifasciata, M. scutellaris, and M. marginata (Velikovaetal.,2000;Dutraetal.,2014;Batistaetal.,2016).Few studieshaveidentifiedellagicacidinpropolisandgeopropolissam- ples(Dutraetal.,2014;Araújoetal.,2016a;Batistaetal.,2016).The qualitativeandquantitativevariationsinphenoliccompoundsseen betweendifferentbeeproductsaremainlyinfluencedbytheplant speciesvisitedbythebees,typeofbeespecies,geographicregion, andclimaticfactors(Gómez-Caravacaetal.,2006).

Itispossiblethattheanti-Leishmaniaactivityisrelatedtothe presenceofthegallicand ellagicacids,identiﬁedintheextract and EAF, as conﬁrmed by the mass spectra (Zoechling et al., 2009;Deganietal.,2014)sincethesetwophenolic acidsexhib- itedantileishmanialactivityagainstpromastigotes(Tasdemiretal., 2006;Shuaibuetal.,2008),butitisimportanttoemphasizesthat accordingourresultsthegallicacidwasmoreeffective.

Previous results also showed a lower anti-Leishmania effect againstthepromastigotesofL.majorforthegallicacidincompar- isontotheellagicacid(IC5016.4and9.8␮g/ml,respectively).The bothphenolicacidswerealsoeffectiveagainstamastigotes,since theywereabletoreducetheinfectionandinfectivityonBalb/c macrophagesinfectedbyL.major(IC50values5.0and0.9␮g/ml, respectively),withselectivityindexhigherthan20(Alvesetal., 2017).

Inaddition,severalauthors(Kolodziejetal.,2001; Kolodziej andKinderlen,2005)showedlowtoxicitytothegallicaciddespite theanti-LeishmaniaactivitytoL.donovanipromastigotesdatathat

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Table2

ChemicalcompositionidentiﬁedintheextractandfractionsofthegeopropolisaftersilylationbyGC–MS.

Compoundclass Identiﬁedcompound^a HEG HF CF EAF HAF

TIC(%)^b

Fattyacids Palmiticacid 0.52 7.27 0.96 – 0.51

Oleicacid – 1.72 – – –

Stearicacid 1.53 2.12 5.29 – 2.47

Arachidicacid – 0.33 – – –

Behenicacid – 0.44 – – –

Lignocericacid – 0.25 – – –

Organicacid Quinicacid 1.68 – – – 1.01

Phenolicacids Gallicacid 22.30 – 4.80 40.5 5.76

Ellagicacid 14.70 – 1.53 31.6 21.60

Protocatechuicacid 0.10 – 0.14 – –

Triterpenes ␤-Amirin – 0.42 – – –

Lupenona – 0.43 – – –

Oleanolicacid – 0.13 2.35 – –

Steroids Campesterol – 0.07 – – –

Stigmasterol – 0.55 – – –

␤-Sitosterol – 1.47 0.39 – –

Sugars Frutose 0.32 – – – –

Glucose 21.0 – – 3.15 18.60

Mannose 12.8 – – – 10.33

Xylose 0.61 – – – 0.28

Alcohols Glycerol 0.22 – – – 0.35

Xylitol 0.92 – – – 1.22

Inositol 0.10 – – – 0.10

aNameofthecompoundswithoutthetrimethylsilyl(TMS)constituent.

bExpressedinpercentageoftotalionchromatograms(TIC%).

HEG,hydroalcoholicextractofgeopropolis;HF,hexanefraction;CF,chloroformfraction;EAF,ethylacetatefraction;HAF,hydroalcoholicfraction.

30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20

0 5,71 18,35

RT [min]

mAU

30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 450 400 350 300 250 200 150 100 50 0

5,73 18,37

RT [min]

mAU

30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -20

5,74 18,38

RT [min]

mAU

EAF

Standards

GA

EA

HEG

Fig.2.HPLCchromatogramsofhydroalcoholicextractofgeopropolis(HEG),ethylacetatefraction(EAF),standardsofgallicacid(GA)andellagicacid(EA)in254nm.

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corroboratewiththoseonesfoundinthepresentstudywiththe extractofgeopropolisandthegallicacid.

Conclusions

Theantileishmanial activityof the hydroalcoholic extract of geopropolis fromM.fasciculata and its ethyl acetate fractionis probablyrelatedtothepresenceof gallicacid, andellagic acid, withhigheffectonthepromastigotesandamastigotesformand withamoderatetoxicitytomurinemacrophageswhatmayopena newperspectiveintheresearchofnewdrugswithantileishmanial effect.

Authors’contributions

RPD,JLBandMCPScontributedtobiologicalstudies.RPDand MCABcontributedbycollectinggeopropolissamples,andperform- ingchromatographicanalysis.FJBPcontributedtothecytotoxicity test.FRFNcontributedtocriticalreadingofthemanuscript.MNSR andRNMGdesignedthestudy,supervisedthelaboratoryworkand contributedtocriticalreadingofthemanuscript.Alltheauthors havereadtheﬁnalmanuscriptandapprovedthesubmission.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

TheauthorsthankthefundingagencyCAPES,andFAPEMAfor thedoctoralfellowshipandforthegranttodevelopthepresent study.Wearealsoindebtedtothebeekeepersforprovidingthe geopropolissamples,andtoDr.AldinaBarral,CentrodePesquisas Gonc¸aloMoniz, FIOCRUZ,Bahia,Brazil,forkindly providingthe Leishmaniaamazonensis.

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