Biological properties of volatile oil from Brazilian brown propolis

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RevistaBrasileiradeFarmacognosia29(2019)807–810

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

Short communication

Biological properties of volatile oil from Brazilian brown propolis

Vitor Hugo Melo de Lima

^a

, Karoliny de Cassia Rodrigues Almeida

^a

, Cassia Cristina Fernandes Alves

^b

, Matheus Leandro Rodrigues

^c

,

Antônio Eduardo Miller Crotti

^c

, João Matias de Souza

^d

, Arthur Barcelos Ribeiro

^d

, Iara Silva Squarisi

^d

, Denise Crispim Tavares

^d

, Carlos Henrique Gomes Martins

^e

, Mayker Lazaro Dantas Miranda

^f,∗

aInstitutoFederaldeEducac¸ão,CiênciaeTecnologiadoSuldeMinasGerais,CampusPousoAlegre,PousoAlegre,MG,Brazil

bInstitutoFederaldeEducac¸ão,CiênciaeTecnologiaGoiano,CampusRioVerde,RioVerde,GO,Brazil

cDepartamentodeQuímica,FaculdadedeFilosoﬁa,CiênciaseLetrasdeRibeirãoPreto,UniversidadedeSãoPaulo,RibeirãoPreto,SP,Brazil

dNúcleodePesquisasemCiênciasExataseTecnológicas,UniversidadedeFranca,Franca,SP,Brazil

eDepartamentodeMicrobiologia,InstitutodeCiênciasBiomédicas,UniversidadeFederaldeUberlândia,Uberlândia,MG,Brazil

fInstitutoFederaldeEducac¸ão,CiênciaeTecnologiadoTriânguloMineiro,CampusUberlândiaCentro,Uberlândia,MG,Brazil

a r t i c l e i n f o

Articlehistory:

Received7May2019 Accepted27July2019

Availableonline5September2019

Keywords:

Apismellifera Resin

Balsamicsubstances Volatiles

Apitherapy

a b s t r a c t

Propolis,isabeeproductcollectedfromexudatesandﬂowerbudsofseveralplants,hasstrongaroma andseveralbiologicalapplications.Thisstudyaimedatevaluatingthechemicalcompositionandin vitroantioxidant,antibacterialandcytotoxicpropertiesofvolatileoilfromBrazilianbrownpropolis.It wasextractedbyhydrodistillationandanalyzedbygaschromatography-ﬂameionizationdetectionand gaschromatography-massspectrometry.Volatileoilfrombrownpropolisexhibitedstrongantibacterial activityagainstH.pylori(MIC3.25␮g/ml),Mycobacteriumtuberculosis(MIC50␮g/ml)andM.avium(MIC 62.5␮g/ml).ItwasevaluatedinvitroforantioxidantactivitybyDPPH(IC5025.0␮g/ml)andABTS(IC50

30.1␮g/ml)methods.Itscytotoxicpropertywasevaluatedinnormal(humanﬁbroblasts,GM07429A) andtumor(MCF-7-humanbreastadenocarcinoma;HeLa-humancervicaladenocarcinomaandM059J- humanglioblastoma)celllines.IC50valueswere81.32␮g/mlforGM07429Aand85.00,129.40and84.12

␮g/mlforMCF-7,HeLaandM059Jcells,respectively.Threemajordereplicatedcomponentsofvolatileoil frombrownpropoliswereacetophenone(15.2%),nerolidol(13.3%),andspathulenol(11.6%).Ourresults contributetoabetterunderstandingofthechemicalandbiologicalpropertiesofBrazilianbrownpropolis andprovideevidenceforitspotentialmedicinaluse.

Introduction

Propolisisaresinoussubstancethatbeesproducebygather- ingmaterialfromdifferenttypesofplants.Theyuseitasasealant toprotecttheirhivesfrominvasiveorganisms.Majorconstituents ofpropolisincludephenoliccompoundswhicharerepresentedby ﬂavonoids,phenolicacidsandtheiresters;theyattributepromis- ingbiologicalactivities,suchasantimicrobial,anti-inﬂammatory andantioxidantones,topropolis.Eventhoughthechemicalcom- positionofpropolismayvary,itusuallycomprises50%ofresinand plantbalm,30%ofwax,10%ofvolatileoils,5%ofpollenand5%of othersubstances,suchasorganicresidues(Wagh,2013).

∗ Correspondingauthor.

E-mail:maykermiranda@iftm.edu.br(M.L.Miranda).

Wideapplicationofpropolistomodernmedicinehasdrawn increasing attention to its chemical composition. Its composi- tionincludeschemicalconstituentsthatprotectorganismsagainst chronicdiseases causedby oxidativestress,suchascancerand metabolic disorders. Thus, this natural product has promising antioxidantpotentialsinceitactsasabodydefenseagentagainst freeradicalsthatarefoundinallorganisms(Calegarietal.,2017).In addition,previousinvitrostudieshavedemonstratedthatextracts ofpropoliscouldinhibitthegrowthofMycobacteriumtuberculosis aswellassynergisetheeffectofestablishedantituberculardrugs suchasisoniazid(Alietal.,2018).

Thecytotoxicactivityofpropolishasalsodrawnresearchers’

interest worldwide, since studies have proven that it may be appliedasanutritionalsupplementduringcancertreatments.Sev- eralreportshaveshowncytotoxiceffectsofpropolisfromdifferent

https://doi.org/10.1016/j.bjp.2019.07.004

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

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808 V.H.Limaetal./RevistaBrasileiradeFarmacognosia29(2019)807–810

originsandtheirfractionsinseveralcancercelllines(Pratsinisetal., 2010;Borgesetal.,2011).

Literature hasreported advancesin theuse of propolis and itscomponents,mainlyregardingitspromisingandspeciﬁcanti- Helicobacterpyloriactivity.Somestudiesshowthatgastriccancer isstronglyrelatedtoinfectioncausedbytheHelicobacterpylori;

itleadstoaninﬂammatoryprocess,withconsequentinductionof oxidativestress,andtopre-neoplasticconditions(Boyanovaetal., 2005).Itshouldbeemphasizedthatseveralbiologicalactivities describedbytheliteraturerefertoextractsfrompropolis,rather thanfromitsvolatileoils.Asaresult,itshouldalsobehighlighted thatthisstudyistheﬁrstreportofantioxidant,anti-Helicobacter pylori,antimycobacterial andcytotoxicproperties of volatileoil extractedfromatypeofBrazilianbrownpropolis.

Considering the well-known pharmacological potential of volatileoilfromBrazilianbrownpropolis(Fernandesetal.,2015), thisstudyaimedtoevaluatethechemicalcompositionandinvitro antioxidant,antibacterialandcytotoxicpropertiesofsamplescol- lectedinBordadaMata,inthesouthofMinasGerais(MG)state, Brazil.

Materialandmethods

Freshbrownpropolis(500g)producedbyApismelliferaL.inthe AtlanticForestregioninBordadaMata,MG,waspurchasedfrom ApiárioKarina(BordadaMata,MG,Brazil)onJune15th,2017.

Volatile oil from brown propolis (EOP) was obtained by hydrodistillationfor3hinaClevenger-typeapparatusandstored at4^◦CuptoGC–MS,GC-FIDandbioassays.Gaschromatography- ﬂameionizationdetectionand gaschromatography–massspec- trometryanalyseswereperformedbyShimadzuQP2010Plusand GCMS2010 Plus(Shimadzu Corporation,Kyoto, Japan) systems.

GC–MSand GC-FID conditions and theidentiﬁcation of chemi- calconstituentsofEOPwerecarried outinagreementwiththe methodologyproposedbyLemesetal.(2018).

Free radical scavenging activity of 2,2- diphenyl-1-picrylhydrazyl (DPPH^•) and azino-bis (ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) were determined by a spectrophotometric method (Justus et al., 2018), withmodiﬁcations.In the DPPHassay,different concentrations ofEOPinmethanol(10–100␮g/ml)wereaddedto2ml 0.1mM solution of DPPH that was previously prepared and incubated inthedarkfor30min.Absorbancewasrecordedat517nmbya UVspectrophotometer.IntheABTSassay,1980mldilutedABTS⁺ solutionwasaddedto20␮lEOPpreviouslydilutedinethanoland absorbanceat734nmwasmeasured6minafterinitialmixing.BHT wasusedaspositivecontrol.Assayswerecarriedoutintriplicate.

Inhibitionpercentage was calculated as (I%)=(A0−A/A0)×100, whereA0istheabsorbanceofthecontrolandAistheabsorbance ofthesamples.IC50valuewascalculatedastheconcentrationof samplerequiredtoscavenge50%offreeradicalsbygraphingthe I%versusvolatileoilconcentration.

Minimum inhibitory concentration (MIC in ␮g/ml) of EOP was calculated by the broth microdilution method on 96-well microplates.ThefollowingATCCstandardstrainwasused:Heli- cobacterpylori(ATCC43526).Anevaluationoftheactivityofthe volatileoilwithreferencedrugswasmadebycomparingbacte- rialgrowthoneach plateofH.pylori.EOP wasdissolvedin 5%

dimethylsulfoxidetoreachﬁnalconcentrationsrangingbetween 0.195and400␮g/ml.Theinoculumwasadjustedat625nmina spectrophotometertoproducecellconcentrationequalto5×10⁵ CFU/ml.PlateswereincubatedinaCO2 incubatorat37^◦C for3 daysundermicroaerobicconditions.Tetracyclineatconcentrations rangingfrom0.115to59.0␮g/mlwasemployedasthestandard drug,incubatedunderthesameconditionspreviouslymentioned.

Table1

ChemicalcompositionofvolatileoilfrombrownpropoliscollectedintheAtlantic Forestbiome,insoutheasternBrazil.

Compounds RIexp RIlit %RA

Benzaldehyde 962 961 0.4

␤-Pinene 980 981 0.1

Limonene 1035 1036 0.2

Acetophenone 1077 1078 15.2

trans-Linalooloxide 1087 1088 0.4

2-Methoxy-phenol 1089 1090 0.1

Linalool 1097 1098 2.0

Ho-trienol 1109 1110 0.1

Benzylnitrile 1142 1143 0.6

␣-Terpineol 1182 1183 1.4

Thymol 1287 1288 0.2

Carvacrol 1299 1299 0.5

Bicycloelemene 1315 1316 0.2

Eugenol 1347 1348 0.2

␣-Copaene 1365 1367 4.5

␤-Elemene 1380 1381 0.9

␣-Yalangene 1383 1383 0.2

␣-Gurjinene 1400 1401 2.1

␤-Caryophyllene 1416 1418 5.9

Aromadendrene 1435 1436 4.5

␣-Humullene 1450 1451 2.7

allo-Aromadedrene 1454 1455 2.6

␥-Muurolene 1476 1477 1.5

␤-Selinene 1483 1484 1.1

Ledene 1490 1490 3.4

␦-Cadinene 1511 1513 5.2

␣-Cadinene 1523 1524 2.4

Cadina-1.4-diene 1531 1533 2.9

Nerolidol 1565 1566 13.3

Spathulenol 1582 1582 11.6

Viridiﬂorol 1592 1593 1.0

Caryophylleneoxide 1611 1613 1.0

␣-Muurolol 1642 1643 2.9

␣-Cadinol 1656 1656 2.2

Total 93.5

RIexp:Retentionindexrelativeton-alkanes(C8–C20)ontheRtx-5MScolumn;RIlit: Retentionindex.%RA:relativearea.

Afterincubation,30␮l0.01%aqueousresazurinsolutionwasadded toeachwellinordertoevaluatedmicrobialgrowth.

MycobacteriaMycobacteriumtuberculosisH37Rv(ATCC27294) and M. avium(ATCC25291)were obtainedfrom theAmerican TypeCollection (ATCC)andmaintainedat−80^◦C.Antimycobac- terialactivityofEOPwasevaluatedbytheMICbrothmicrodilution methodconductedon96-wellmicroplates.Themethodologyused for evaluating antimycobacterial activity of EOP was the one describedbyMeloetal.(2017).

Inthisstudy,threedifferenthumantumorcelllineswereused:

breastadenocarcinoma(MCF-7),cervicaladenocarcinoma(HeLa) and glioblastoma (M059J) (Table 3).A normal humancell line (ﬁbroblasts,GM07492A)wasincludedtoevaluatewhetherEOPhas selectiveactivity.Themethodologyusedforevaluatingcytotoxic activityofEOPwastheonedescribedbySilvaetal.(2019).

Resultsanddiscussion

BrownpropoliscollectedinsoutheasternBrazil,yieldedhigh contentofvolatileoil(0.8%)byhydrodistillation.Toidentifythe constituentsofEOP,GC-FIDandGC-MSanalyseswereperformed and Table 1 shows the 34 constituents with relative amounts above0.1%,whichcorrespondsto93.5%ofidentiﬁedconstituents.

Acetophenone(15.2%),nerolidol(13.3%),␤-caryophyllene(5.9%), spathulenol(11.6%)and␦-cadinene(5.2%)werethemajorcompo- nentsofEOP.Onlynerolidolandspathulenolwerefoundinhigh amountsinEOPinMatoGrossodoSulstate,Brazil;acetophenone wasnotidentiﬁed(Fernandesetal.,2015).Ontheotherhand,only three majorconstituentswereidentiﬁed inEOPfoundin Piauí,

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V.H.Limaetal./RevistaBrasileiradeFarmacognosia29(2019)807–810 809

Table2

Invitroantioxidantandantibacterialactivitiesofvolatileoilfrombrownpropolis (IC50andMIC).

IC50(␮g/ml) Bacteria MIC(␮g/ml)

DPPH 25.0±10.20 H.pylori(ATCC43526)^a 3.25 ABTS 30.1±8.11 M.avium(ATCC25291)^b 62.5 BHT 19.3±0.09 M.tuberculosis(ATCC27294)^b 50

Resultsareexpressedasmean±SD,n=3.IC50:concentrationofdrugin␮g/mlthat caused50%ofDPPH^•andABTS⁺inhibition.BHT:Positivecontrol.

aPositivecontrolused:tetracycline(MIC=1.0␮g/ml).

bPositivecontrolused:isoniazid(MIC=1.47␮g/ml).

Table3

Cytotoxicactivity(IC50values)ofvolatileoilfrombrownpropolisagainstdifferent humancelllines.

Cellline

Treatment(␮g/ml)

Volatileoilfrombrownpropolis DXR

IC50 IC50

GM07492A 81.32±2.48 0.50±0.20

MCF-7 85.00±2.67 62.10±2.00

HeLa 129.40±4.90 5.30±1.30

M059J 84.12±5.39 16.20±2.50

Doxorubicin(DXR)wasusedaspositivecontrol.GM07492A,humanlungﬁbroblasts;

MCF-7,humanbreastadenocarcinoma;HeLa,humancervicaladenocarcinoma;

M059J,humanglioblastoma.Valuesaremean±SD,n=3.

innortheasternBrazil:␤-caryophyllene,caryophylleneoxideand viridiﬂorol(Sousaetal.,2006).

Concentrationsofvolatilecompoundswerefoundtobequite differentfromtheonesfoundintheliterature.Itmaybeexplained bythehighvariabilityfoundinplantspeciesthatgrowinregions closetobeehives,sincebeescollectplantexudates.Propoliscom- positiondependsonseasonality,illumination,altitude,collector type,foodavailabilityandtheactivitydevelopedduringpropolis exploration.Thesefactorsinfluencethechemicalcompositionof oilextractedallovertheworlddirectly;asaresult,thesediffer- encesarebelievedtocontributesignificantlytochemicalproperties andbeneficialeffectsofalltypesofpropolis(Toretietal.,2013).

Speciﬁcally,propolisexploredfromthehivemaycontainamix- tureofresinsfromvariousplantsourcesandbeeswax.Ifindividual sourcesofresinareneededforchemicalanalysis,itmaybeneces- sarytocollecttheresinfromplanttissueorfromthehindlegsof returningresinforagers(Bankovaetal.,2016).

Regardingantioxidantactivity,resultsshowthat EOPexhib- ited signiﬁcant DPPH^• free radicalactivity, with IC₅₀ values of 25.0␮g/ml.Whentestedbytheazino-bis(ethylbenzothiazoline- 6-sulfonicacid)(ABTS⁺)method,thevalueofIC50=30.1␮g/mlwas alittlehigher.ThepositivecontrolwasBHT(butylatedhydroxy- toluene),whoseIC₅₀=19.3␮g/ml(Table2).

Antioxidant activity of EOP determined by the DPPH^• (2,2- diphenyl-1-picrylhydrazyl) and azino-bis (ethylbenzothiazoline- 6-sulfonicacid)(ABTS⁺)methodswereconsideredsigniﬁcantby comparison with the positive control BHT (butylated hydroxy- toluene),whoseIC50was19.3␮g/ml.BHTwaschosentobethe positivecontrolbecauseitinhibitsbothfreeradicalformationand lipidperoxidationeffectively(Bajpaietal.,2017).Thegoodantiox- idantactivity exhibited byEOP maybe explained by itsmajor constituents,mainlythesesquiterpenespathulenol(Nascimento etal.,2018).

AntibacterialactivityofEOPwasevaluatedagainstH.pylori,M.

tuberculosisandM.aviumstrains;itsMICvaluesweremeasuredby thebrothmicrodilutionmethod.EOPprovedhighlyactiveagainst H. pylori, M. tuberculosis and M. avium with MIC=3.25␮g/ml, 50␮g/ml and 62.5␮g/ml, respectively (Table 2). Positive con- trolsweretetracycline(MIC=1.0␮g/ml)foranti-Helicobacterpylori activity and isoniazid (MIC=1.47␮g/ml) for antimycobacterial

one.Thisresultisrelevant,sincepreviousresultsreinforcedthat volatileoilswhoseMIC=250␮g/mlwereconsideredmoderately activeagainst H.pylori(Kirmizibekmezetal., 2017).EOPhad a greater against Helicobacter pylori activity than the volatile oil from Apium nodiﬂorumleaves, whose MIC=12.5␮g/ml enabled it to be deﬁned as having appreciable value (Menghini et al., 2010).EOP hasalso shown promising antimycobacterial activity against Mycobacterium tuberculosis (MIC=50␮g/ml) and M.

avium(MIC=62.5␮g/ml).AccordingtoHoletzetal.(2002),nat- uralproductswithMICvaluesbelow100␮g/ml,between100and 500␮g/ml,from500to1000␮g/mlandabove1000␮g/mlexhibit good,moderate,weakandabsenceofantibacterialactivity,respectively.

ThepromisingantibacterialactivityexhibitedbyEOPmaybe explainedbysynergicorevenantagonisticeffectsamongitsdif- ferent compounds. Most constituents of volatile oils belong to thefamily terpenoids;mostofthem have apolarcharacterand mayeasilypermeatethephospholipidbilayerofbiologicalmem- branes. Theseconstituentsusuallyexhibit antimicrobialactivity because they are capable of breaking through the lipid struc- ture,causedamagetotheintegrityofthemembrane,unbalance intracellularpHand, consequently,leadtocelldeath(Rautand Karuppayil,2014).

EOPcytotoxicitywasassessedagainsttheGM07492Anormal cellline, whose IC50 was81.32␮g/ml andagainst MCF-7,HeLa andM059Jtumorcelllineswhose IC₅₀ were85.00,129.40and 84.12␮g/ml,respectively(Table3).Thepositivecontrolwasdox- orubicinandIC50valuesarealsoshowninTable3.

CytotoxicactivityofEOPwasobservedagainstnormalhuman andtumorcelllinesemployedbythisstudy,but withoutselec- tivity.Speciﬁcally,themainmechanismsthatmediatecytotoxic effectsofvolatileoilsincludetheinductionofcelldeathbyacti- vation of apoptosis and/or necrosis processes, cell cycle arrest andlossoffunctionofvolatileorganelles(Raut andKaruppayil, 2014). It is important to highlight that the cytotoxicity assay also showed that EOP exhibits antibacterial activity against H.

pylori, M. tuberculosis and M. avium at concentrations lower (MIC=3.25␮g/ml;MIC=50␮g/ml;MIC=62.5␮g/ml,respectively) thanthosewhichshowedcytotoxicityinnormalhumanﬁbroblasts cells(IC₅₀=81.32␮g/ml).Possibly,synergisticinteractionsamong volatileoilscomponentswhicharebeneﬁcialtotheirdifferentbio- logicalproperties(RautandKaruppayil,2014).

Conclusions

It is clear that studies of volatileoils from propolis are far frombeingexhaustive.Furtherresearchisneededtorevealtheir chemistry and to support their medicinal properties scientifi- cally. Differences in the chemical composition of volatile oils extractedfrompropolisareobservedastheresultoftheirbotanic andgeographicorigin.Acetophenone,nerolidol,␤-caryophyllene, spathulenoland ␦-cadinenewerethemajorconstituentsidenti- fiedinEOP.EvaluationoftheantibacterialactivityofEOPshowed thatitissignificantagainstthebacteriaH.pylori,M.tuberculosis andM.avium.Thepreliminaryresultoftheantioxidantpotential showedbyEOPcouldbepromisingforthedevelopmentofnews cosmeticswithantioxidantpotential.Inassaysofcytotoxicactivity, EOPprovedtobecytotoxicagainstallcelllinesunderstudy,butit showednoselectivitytotumorcelllines.Thehydrodistillationpro- cessunderstudywascapableofextractingbioactivesubstances frombrownpropolisandoriginatethevolatileoilwhichisrespon- sibleforbiologicalpropertiesfoundbythisstudy.Inshort,further invivoexperimentsareneededtoconfirmtheabsenceofpotential mutagenicrisksposedbyEOPfromtheBrazilianAtlanticForest biomeanditsmechanismofaction.

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Authors’contributions

VHML,KCRA,CCFA,MLR,JMS,ABRandISScontributedtothe design and implementationof theexperiment. AEMC, DCT and CHGMproofreadthemanuscript.MLDMcontributedtothestatis- ticalanalysesandalsoproofreadthemanuscript.MLDMandAEMC contributedtothechromatographicanalysis.CHGMandDCT,as professors,workedon overallplanning and experiment design.

Allauthorscontributed tobiological studies,laboratory experi- mentsanddataanalyses.Theyproofreadtheﬁnalmanuscriptand approvedthesubmission.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

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