Chemical composition, immunostimulatory, cytotoxic and antiparasitic activities of the essential oil

16 Pages·2017·2.18 MB·English

RESEARCHARTICLE
Chemical composition, immunostimulatory,
cytotoxic and antiparasitic activities of the
essential oil from Brazilian red propolis
AˆngelaSena-Lopes1☯,FranciscoSilvestreBrilhanteBezerra1☯,RaquelNascimentodas
Neves1,RodrigoBarrosdePinho1,MaraThaisdeOliveiraSilva1,LucielliSavegnago1,
TiagoCollares1,FabianaSeixas1,KarineBegnini1,JoãoAntonioPêgasHenriques2,
MarianaRoeschEly2,LucianeC.Rufatto2,SidneiMoura2,ThiagoBarcellos2,
FrancinePadilha3,OdirDellagostin1,SibeleBorsuk1*
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1 CentrodeDesenvolvimentoTecnolo´gico(CDTEc),UniversidadeFederaldePelotas(UFPel),Campus
a1111111111
CapãodoLeão,CapãodoLeão,RioGrandedoSul,Brazil,2 DepartamentodeTecnologia,Institutode
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Biotecnologia,UniversidadedeCaxiasdoSul,CaxiasdoSul,RioGrandedoSul,Brazil,3 Institutode
a1111111111
TecnologiaePesquisa(ITP),UniversidadedeTiradente,Aracaju,Sergipe,Brazil
a1111111111
☯Theseauthorscontributedequallytothiswork.
*[email protected]
Abstract
OPENACCESS
Citation:Sena-LopesAˆ,BezerraFSB,dasNeves
MoststudiesofBrazilianredpropolishaveexploredthecompositionandbiologicalprop-
RN,dePinhoRB,SilvaMTdO,SavegnagoL,etal.
(2018)Chemicalcomposition,immunostimulatory, ertiesofitsethanolicextracts.Inthiswork,wechemicallyextractedandcharacterizedthe
cytotoxicandantiparasiticactivitiesoftheessential essentialoilofBrazilianredpropolis(EOP)andassesseditsadjuvant,antiparasiticand
oilfromBrazilianredpropolis.PLoSONE13(2):
cytotoxicactivities.ThechemicalcompositionofEOPwasanalyzedusinggaschroma-
e0191797.https://doi.org/10.1371/journal.
pone.0191797 tographywithmassspectrometry(GC-MS).EOPwastestedforinvitroactivityagainst
Trichomonasvaginalis(ATCC30236isolate);trophozoitesweretreatedwithdifferent
Editor:Ing-FengChang,NationalTaiwan
University,TAIWAN concentrationsofEOP(rangingfrom25to500μg/mL)inordertoestablishtheMICand
IC values.AcytotoxicityassaywasperformedinCHO-K1cellssubmittedtodifferent
Received:August1,2017 50
EOPconcentrations.BALB/cmicewereusedtotesttheadjuvanteffectofEOP.Theani-
Accepted:January11,2018
malsweredividedin3groupsandinoculatedasfollows:0.4ng/kgBWEOP(G1);50μgof
Published:February1,2018 rCP40protein(G2);oracombinationof0.4ng/kgBWEOPand50μgofrCP40(G3).
Copyright:©2018Sena-Lopesetal.Thisisan TotalIgG,IgG1andIgG2alevelswereassessedbyELISA.Themajorconstituentcom-
openaccessarticledistributedunderthetermsof poundsofEOPweremethyleugenol(13.1%),(E)-β-farnesene(2.50%),andδ-amor-
theCreativeCommonsAttributionLicense,which
phene(2.3%).ExposuretoEOPinhibitedthegrowthofT.vaginalis,withanIC valueof
permitsunrestricteduse,distribution,and 50
reproductioninanymedium,providedtheoriginal 100μg/mLofEOP.AnEOPconcentrationof500μg/mLwasabletokill100%oftheT.
authorandsourcearecredited. vaginalistrophozoites.TheEOPkineticgrowthcurveshoweda36%decreaseintropho-
DataAvailabilityStatement:Allrelevantdataare zoitegrowthaftera12hexposureto500μg/mLofEOP,whilecompleteparasitedeath
withinthepaper. wasinducedat24h.WithregardtoCHO-K1cells,theCC was266μg/mL,and92%
50
Funding:ThisworkwassupportedbyGrant cytotoxicitywasobservedafterexposureto500μg/mLofEOP.Otherwise,aconcentra-
#1608/2011fromCoordenac¸ãode tionof200μg/mLofEOPwasabletoreduceparasiteproliferationby70%andwasnot
Aperfeic¸oamentodePessoaldeN´ıvelSuperior
cytotoxictoCHO-K1cells.Asanadjuvant,asynergisticeffectwasobservedwhenEOP
(Capes)toJoãoAntonioPêgasHenriques.URL:
wascombinedwiththerCP40protein(G3)incomparisontotheadministrationofeach
http://www.capes.gov.br/.Thefundershadnorole
instudydesign,datacollectionandanalysis, componentalone(G1andG2),resultinginhigherconcentrationsofIgG,IgG1andIgG2a.
decisiontopublish,orpreparationofthe EOPisconstitutedbybiologicallyactivecomponentswithpromisingantiparasiticand
manuscript.
PLOSONE|https://doi.org/10.1371/journal.pone.0191797 February1,2018 1/16
ChemicalcompositionandbiologicalpropertiesoftheessentialoilfromBrazilianredpropolis
Competinginterests:Theauthorshavedeclared immunostimulatoryactivitiesandcanbeinvestigatedfortheformulationofnewvaccines
thatnocompetinginterestsexist. ortrichomonacidaldrugs.
Introduction
Currently,theBrazilianpropolisisclassifiedin13differenttypes,accordingtoitsphysical-
chemicalproperties,botanicaloriginandgeographicareawherepropoliscanbecollected.Ini-
tially,Parketal.[1]classifiedpropolissamplesfromdifferentBrazilianregionsin12groups.
Afterwards,a13thtypeofpropoliswithpeculiarchemicalcompositionwasfoundinhives
locatedalongsidethecoastandmangrovesintheBraziliannortheastandwascalledBrazilian
redpropolis(BRP)[2].Inthelastdecade,thechemicalcompositionofBRPhasbeenrevealed
andrelatedtoitsbioactivities.ThemainchemicalcomponentsfoundinBRPareclassifiedas
isoflavones,flavones,flavonols,aurones,chalcones,pterocarpansandxanthonesgroups[3–6].
ThebotanicaloriginofBRPwasidentifiedasresinousexudatesofDalbergiaecastophyllum[7],
however,likelyasecondplantspeciesparticipatesasoneofthemainsourcesofresinsforBRP
[5].
DuetothevarietyofchemicalcompoundsofBRPincorporatedwithintheresinmixture,
especiallyphenoliccompounds,manybiologicalactivitieshavebeenreportedlikeantioxidant
[2,8],antibacterial[9,10],fungicide[11],antiparasitic[10,12],antineoplasic[13,14],anti-caries
[15],cytotoxic[2,10],anti-inflamatory[16,17],antinociceptive[18]andwasalsobeneficialto
thewoundhealingprocess[19].
Severalexperimentsandclinicalinvestigationshaveshowntheantiparasiticandadjuvant
propertiesofthehydroalcoholicextractofpropolis(HEP).HEPofdifferenttypesofpropolis
showedantiparasitaryefficacyagainstGiardialamblia[20,21],Toxoplasma gondiiandTricho-
monasvaginalis[22].ItisimportanttohighlightthatT.vaginalisistheetiologicalagentof
trichomoniasis,themostcommonnon-viral,sexuallytransmitteddisease(STD)intheworld
[23].Inaddition,metronidazole-resistantT.vaginalishasbeenimplicatedinanincreasing
numberofrefractorycases,whichareusuallytreatedwithincreaseddosesofthedrug,leading
toanincreaseintheoccurrenceofsideeffects[24].Clearly,alternativecurativetherapiesare
needed,andpropolisemergesasanaturalproductforalternativedrugs.
Moreover,HEPhasbeenusedtoformulateveterinaryvaccinesagainstbacterialorviraldis-
eases,oncesomephenoliccompoundshaspresentedimmunomodulatoryactivity[25–27].As
adjuvant,propolisconferstothevaccinalformulationdesirableproperties,suchasreduced
viscosity,goodstability,lowreactogenicity,lowtoxicity,andisabletoinducearobusthumoral
andcellularresponse[26].
Asaforementioned,mostofthepreviousstudiesdescribingthebiologicalactivitiesofBRP
usesethanolastheextractionsolvent,andtheextractionmethodinfluencesdirectlytheyield
andselectivityforsomecompounds[10,28].Volatilecompoundslikethosefoundinessential
oilsareinlowconcentrationsinpropolis,butplayanimportantrolebycontributingtopropo-
lispleasantaromaanditsbiologicalactivities.Researchershaveappliedvariousmethodsto
obtainpropolisvolatilessuchasdistilllation-extractionorhydrodistillation[29].
Ontheotherhand,studiesfocusingonessentialoilsofpropolisvolatilesarerelatively
scarce,mostofthemdealingwithantimicrobialpropertiesagainstfungus,Gram-positiveand
Gram-negativebacteria[29].Volatilesofpropolisfromstinglessbeeswereabletostimulate
theimmunesystemofelderlypatientsbyincreasingtheirnaturalkillercellactivity[30].Essen-
tialoilsofChinesepropolisinhibitedtheproliferationofhumancolorectalcancercellsby
PLOSONE|https://doi.org/10.1371/journal.pone.0191797 February1,2018 2/16
ChemicalcompositionandbiologicalpropertiesoftheessentialoilfromBrazilianredpropolis
inducingcellcyclearrestandapoptosis[31].However,tothebestofourknowledge,thereare
nostudiesdescribingthechemicalcompositionorthebiologicalactivitiesoftheessentialoil
derivedfromtheBrazilianredpropolis(EOP).Herein,thechemicalcompositionoftheEOP
isshowed.Still,invitrotrichomonacidalandcytotoxicactivities,andtheadjuvantactionof
EOPinarecombinantvaccineareassessed.
Materialsandmethods
Redpropolissamplecollectionandessentialoilpreparation
RedpropoliswascollectedfromthemunicipalityofBrejoGrande,locatedinthestateofSer-
gipe,innortheasternBrazil(S10˚28’25”andW36˚26’12”).SampleswerecollectedinSeptem-
ber2011andweremaintainedfrozenat−20˚Cuntiluse.Forthepreparationoftheessential
oil,conventionalhydrodistillationwascarriedoutusingaClevenger-typeapparatus.Briefly,
380gofBRPwastransferredtoavolumetricflask,and2Lofdistilledwaterwasadded.A
heatingmantlewasplacedontotheClevengerapparatus.Theessentialoilwasextractedafter
boilingthemixtureforatimeperiodofonehour.Theessentialoilobtainedwascollectedin
ambervialsandstoredat4˚Cuntiluse.Thetotalyieldobtainedwasapproximately0.25%.
Gaschromatography–massspectrometry(GC-MS). Chemicalcompositionanalysis
wasperformedusingagaschromatographcoupledwithamass-selectivedetector(Hewlett
Packard6890/MSD5973,PaloAlto,USA)equippedwiththeHPChemStationsoftware
andtheWiley275library.AcapillarycolumncomposedofHP-INNOWaxfusedsilica(30
m×250μm)of0.50μmfilmthickness(Hewlett-Packard,PaloAlto,USA)wasusedforthe
study.Thetemperatureprogramusedwasasfollows:thecolumntemperaturestartedat40˚C
(heldfor8min)andwasincreasedto180˚Cat3˚C/minandthento230˚C(heldfor20min)
at20˚C/min;theinjectorwasatatemperatureof250˚Candtheinterfaceat280˚C.Thesplit
ratiowas1:100,heliumwasthecarriergas(56kPa),theflowratewas1mL/min,theionization
energywas70eV,andtheinjectedvolumewas0.4mL.
Anti-Trichomonasvaginalisactivityassay
TheT.vaginalis30236isolateusedforevaluatingantiparasiticactivityinthisstudywas
obtainedfromtheAmericanTypeCultureCollection(ATCC).Trophozoiteswereaxenically
culturedintrypticase-yeastextract-maltose(TYM)medium(withoutagar;pH=6.0)supple-
mentedwith10%sterilebovineserum(inactivatedat56˚C)andincubatedat37˚C[32].
Cultureswith95%viability,asconfirmedthroughobservationofmotility,morphology,and
trypan-blueexclusion(0.4%)assayunderthelightmicroscopeat400Xmagnification,were
consideredasaliveandwereusedforevaluatingtheactivityofEOP.
Allassayswereperformedin96-wellmicrotiterplates(Cral1).Parasiteswereseededatan
initialdensityof2.6×105trophozoites/mLofTYMandincubatedinthepresenceofEOP.
Threeexperimentalcontrolswerealsoperformed:parasitesonly,thevehicleusedforsolubili-
zationofEOP(0.6%DMSO)and100μMMTZ(metronidazole;Sigma-Aldrich)(asapositive
control).Themicrocultureplateswereincubatedat37˚Cwith5%CO for24h.Subsequent
2
totheincubation,apreparationcontainingtrophozoitesandtrypanblue(0.4%)ina1:1ratio
waspreparedandcountedinaNeubauerchamber.Cultureswith95%viabilitywereutilized
fortheassay.
Todeterminetheminimuminhibitoryconcentration(MIC),differentconcentrationsof
EOP(25,50,100,200,300,400,and500μg/mL)weretestedasdescribedbyHu¨bneretal.[33],
withsomemodification.ParasitepelletswereusedtoestablishtheMICaswellasconcentra-
tionsbelowandabovetheMICvalue.SamplesusedascontrolswereinoculatedinfreshTYM
mediumat37˚C.ParasiteswerecountedinaNeubauerchamberwithtrypanblueevery24h
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ChemicalcompositionandbiologicalpropertiesoftheessentialoilfromBrazilianredpropolis
for96htoconfirmMIC.Theviabilityoftrophozoiteswasassessedbytheexclusionofthetry-
panbluedyeaswellasbymotilityandmorphology.TheIC (halfthemaximuminhibitory
50
concentration)valuesweredeterminedatthesameconcentrationsusedfortheMICtest.
Akineticgrowthcurvewasconstructedinordertoobtainamorecomparableactivitypro-
fileofEOPactivityagainstT.vaginalis.Theviabilityoftrophozoiteswasobservedundera
lightmicroscope96hafterincubationwithEOPattherequiredMIC.Growthanalysiswas
performedat1,6,12,24,48,72,and96hbythetrypanblue(0.4%)exclusionmethodandby
theevaluationofmotilityandmorphology.Allassayswereperformedindependentlyatleast
threetimesintriplicate,andtheresultswereexpressedasthepercentageofviabletrophozoites
comparedtountreatedparasites.
FortheAnti-Trichomonas vaginalisactivityassay,statisticalanalysiswasconductedusing
one-wayanalysisofvariance(ANOVA),andaprobabilityvalueofp<0.05wasconsidered
significant.Tukey’stestwasutilizedtoidentifysignificantdifferencesbetweenthemeansof
differenttreatments.GraphPadPrismversion5.0forWindows(GraphPadSoftware,USA)
wasusedforstatisticalanalysis.
Cytotoxicityassay. Chinesehamsterovary(CHO-K1)cellswereobtainedfromtheRio
deJaneiroCellBank(PABCAM,FederalUniversityofRiodeJaneiro,RJ,Brazil)andcultured
asamonolayerinDulbecco’sModifiedEagle’sMedium(DMEM)(VitrocellEmbriolife)sup-
plementedwith10%fetalbovineserum(FBS)(VitrocellEmbriolife),1%L-glutamine,and1%
penicillin/streptomycin.Thecellsweregrownat37˚Cinanatmosphereof95%humidified
airand5%CO .
2
Thepost-treatmentproliferationoftheCHO-K1celllinewasdeterminedbymeasuringthe
reductionofsoluble3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT,
Sigma-Aldrich)towater-insolubleformazan.Cellswereseededin96-wellplatesatadensityof
2×104cellsperwellin100μLvolumesandgrownat37˚Cina5%CO atmospherefor24h
2
beforetheywereusedinthecellviabilityassay.Thecellswerethentreatedwith31.25,62.5,
125,250and500μgofEOPfor24h.DMSOwasusedasvehiclecontrol(VC),untreatedcells
wereusedasanegativecontroland100μMofmetronidazole(MTZ)wasusedasapositive
control.Followingtheincubation,20μLofMTT(Sigma-Aldrich)wasaddedtoeachwelland
thecellswereincubatedforanadditionalperiodof3hat37˚C.Differencesinthetotalcellular
metabolismweredetectedatawavelengthof492nmbyusingamicroplatereader.Theinhibi-
tion(%)ofcellproliferationwasdeterminedasfollows:inhibitorygrowth=(1−Abs treated
492
cells/Abs controlcells)×100%.TheIC wascalculatedusingGraphPadPrismversion5.0
492 50
forWindows(GraphPadSoftware,USA).Allobservationswerevalidatedbyatleastthree
independentexperimentsintriplicate.
Adjuvantassay
Experimentalanimals. Six-toeight-week-oldfemaleBALB/cmicewereobtainedfrom
theCentralAnimalFacilityoftheFederalUniversityofPelotas,wheretheimmunizationassays
werealsoconducted.Allmicewerekeptincagescontainingwoodshavingsandbeddingand
hadfreeaccesstowaterandamaintenancedietadlibitum.A12-hlight/darkcycleatroomtem-
perature(21±2˚C)wasalsomaintained.Allexperimentswereperformedincompliancewith
theproceduresoftheBrazilianCollegeofAnimalExperimentation(COBEA).TheEthicsCom-
missiononAnimalExperimentation(CEEA)ofPelotasFederalUniversity(UFPel)approved
theproject(number2422).Alleffortsweremadetominimizeanimalsuffering.
Antigenexpressionandpurification. TherecombinantendoglycosidaseCP40(rCP40),
derivedfromC.pseudotuberculosis,wasusedasanantigenfortheadjuvantassay.ThepAE/cp40
recombinantplasmidpreviouslyconstructedbyourgroup[34]wastransformedintothe
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ChemicalcompositionandbiologicalpropertiesoftheessentialoilfromBrazilianredpropolis
EscherichiacoliBL21(DE3)Starstrainbytheheatshockmethod.Theinductionofexpression
wasperformedbyadding1mMofIPTGfollowedbyincubationat37˚Cfor3hwithconstant
agitation.Westernblottingusingamonoclonalanti-6x-His-tagantibodyconjugatedtoperoxi-
dase(Sigma-Aldrich)wasusedtoconfirmrCP40expression.Asepharose-nickelcolumn,His-
TrapTM(GEHealthcare),wasusedforpurificationoftherecombinantprotein.Puritywas
determinedby12%SDS-PAGE,andtheconcentrationwasmeasuredusingaBCAkit(PIERCE).
Immunizationassayandbloodsamplecollection. Fortheimmunizationassay,themice
weredividedintothefollowingthreegroupsofsixanimalseach:(a)G1:anegativecontrol
groupimmunizedwith0,4ng/kgBWEOP;(b)G2:inoculatedwithonly50μgofrCP40pro-
tein;and(c)G3:thetestgroupinoculatedwith0,4ng/kgBWEOPplus50μgofrCP40.All
doseswerepreparedinafinalvolumeof200μLusingsterilesalinesolution(0.9%NaCl)asa
vehicleandadministeredviatheintramuscularroute.Theanimalswereimmunizedwithtwo
dosesadministered21daysapart.Animalswerebledatdays0,21,and42post-immunization
todeterminetheantibodylevels.Bloodwascollectedfromtheretro-orbitalsinuswithPasteur
pipettes.Allbloodcollectionswereperformedunderanesthesiawith10%ketamine(1mL/kg)
and2%xylazine(0.1mg/kg)(AgenerUniãoSau´deAnimal,Brazil).Aftercoagulation,the
bloodwascentrifugedat1,500gfor15min.Theserumwasremovedandstoredat−20˚C.
Assessmentofantibodyproduction. Serumsamplesfromtheimmunizedmicewere
usedinimmunoassays(indirectELISA)forthequantificationoftotalIgG,IgG1andIgG2a
anti-rCP40antibodies.Polystyrene96-wellplates(Maxisorp-Nunc)werecoatedwith100μL
ofabicarbonate-carbonatebuffer(pH9.8)containing0.1μg/wellofrCP40protein.Plates
wereincubatedfor18hat4˚Candwashed3timeswithPBS-T(PBS1X,pH7.4,0.1%Tween
20).Theplateswerethenincubatedwith200μL/wellof5%skimmilkinPBSfor2hat37˚C,
forblockingpurposes.Athree-stepwashwasthenperformed.Allserumsamplesobtained
frommiceweredilutedinPBS(v/v1:50),and100μLofeachdilutedsamplewasaddedtothe
platesinduplicate.Plateswereincubatedfor1hat37˚C.Afterwashingthreetimeswith
PBS-T,100μL/wellofanti-mouseIgGconjugatedtohorseradishperoxidase(Sigma-Aldrich)
wasadded(1:5,000inPBS-T)totheplatesinordertodetecttotalIgGpriortotherevelation
step.InplatesdesignatedforthedetectionofIgG1andIgG2a,100μL/wellofeithergoatanti-
mouseIgG1(1:5,000inPBS-T)orgoatanti-mouseIgG2a(1:2,000inPBS-T)wasadded.The
plateswereincubatedat37˚Cfor1handthenwashedthreetimeswithPBS-T,subsequentto
which100μL/wellofanti-goatIgGconjugatedtohorseradishperoxidase(Sigma-Aldrich)was
added(1:5,000inPBS-T).Theplateswerere-incubatedfor1hat37˚Candthenwashedfive
timeswithPBS-T.Thereactionwasdevelopedbytheadditionof100μL/wellofthesubstrate-
chromogensolution[o-phenylenediaminedihydrochloride;OPDtablets(Sigma-Aldrich)in
0.4mg/mLphosphate-citratebuffercontaining0.04%of30%hydrogenperoxide,pH=5.0]
andincubationatroomtemperatureinthedarkfor15min,anditwasstoppedbytheaddition
of50μL/wellofH SO solution(4N).Opticaldensity(OD)wasdeterminedusingamicroti-
2 4
ter-platereader(MicroplateReaderMindrayMR–96A)setat492nm.
DatawereanalyzedusingthesoftwareGraphPadPrismversion5.0forWindows(Graph-
PadSoftware,USA).DifferencesinIgGlevelsbetweenthegroupswereanalyzedusingone-
wayANOVAfollowedbyTukey’stestformultiplecomparisons.Theresultswereconsidered
statisticallysignificantifp-value<0.05.
Results
EOPchemicalcomponents
GC-MSanalysisoftheredpropolisextractenabledtheidentificationofcertaincompounds
presentintheessentialoil.AlistoftheseconstituentsispresentedinTable1.GC-MSisa
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ChemicalcompositionandbiologicalpropertiesoftheessentialoilfromBrazilianredpropolis
Table1. Volatilecompoundsidentifiedbygaschromatographyfollowedbymassspectrometry(GC-MS)ofthe
Brazilianredpropolisessentialoil.
Compound Area(%) LRIobtained LRIreference
α-Pinene 0.2 908 907[37]
α-Cubebene 1.9 1351 1351[37,38]
(-)-Cyperene 0.2 1401 1400[39]
Methyleugenol 13.1 1407 1408[40]
α-Gurjunene 0.5 1412 1412[41]
β-Caryophyllene 1.5 1422 1422[42]
trans-α-Bergamotene 0.6 1438 1438[43]
(E)-β-Farnesene 2.5 1459 1459[37]
γ-Muurolene 0.2 1479 1479[44]
α-Curcumene 0.1 1485 1484[45]
Selinene 0.7 1490 1490[46]
β-Bisabolene 0.9 1506 1506[47]
δ-Amorphene 2.3 1514 1514[48]
https://doi.org/10.1371/journal.pone.0191797.t001
reliablemethod,indispensableinseveralareaswheretheanalysisofcomplexmixturesand
unambiguousidentificationisrequired,andsincevolatilecompoundsarewellidentifiedwith
GC-MS,thismethodissuitableforanalysisofessentialoils[35,36].UsingGC-MS,ourstudy
wasabletohighlightmethyleugenol(13.1%),(E)-β-farnesene(2.5%),δ-amorphene(2.3%),α-
cubebene(1.9%),andβ-caryophyllene(1.5%)asthemajorcomponentsoftheextract.
Anti-Trichomonasvaginalisactivityassay
AnalysisoftheEOPMICdatarevealedthatata500μg/mLconcentration,100%deathoftro-
phozoiteswasobserved.After24hofexposure,EOPdemonstratedoptimalanti-T.vaginalis
activityataconcentrationof500μg/mLwithanIC valueof100μg/mL(Fig1).Thenegative
50
controlandDMSOcontrolshowedpositivemotilityanddidnotstainwithTrypanblue
(0.4%),whereasthepositivecontrolstainedblueandexhibitednegativemotilityatthe24h
timepointinallassaysperformed.Whenthekineticgrowthcurvewasanalyzed,itwas
Fig1.Trichomonasvaginalis30236isolate;MICandIC50after24-hour-treatmentswithEOPat25,50,100,200,
300,400,500μg/mL.Datarepresentmean±standarddeviationofatleastthreeexperiments,allintriplicate.Different
lettersindicateasignificantdifference(p<0.05).
https://doi.org/10.1371/journal.pone.0191797.g001
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ChemicalcompositionandbiologicalpropertiesoftheessentialoilfromBrazilianredpropolis
Fig2.KineticgrowthcurveoftheTrichomonasvaginalis30236isolateafterexposuretoEOPat500μg/mLfor1
h,6h,12h,24h,48h,72hand96h.Datarepresentmean±standarddeviationofatleastthreeexperiments,allin
triplicate.Differentlettersindicateasignificantdifference(p<0.05).
https://doi.org/10.1371/journal.pone.0191797.g002
observedthattheexposureto500μg/mLofEOPreducedthegrowthoftrophozoitesby36%at
12handinducedcompleteparasitedeathat24h(Fig2).
Cytotoxicityassay
EOPtreatmentalsoinhibitedproliferationoftheCHO-K1cellline;however,thiscytotoxicity
wasonlysignificantat500μg/mL.Nosignificantcytotoxicitywasobservedupto250μg/mL
ingrowthinhibitionofCHO-K1cellsafter24hoftreatment(Fig3).Theinvitrocytotoxicity
activityofEOPshowedIC valuesof266and100μg/mLinCHO-K1cellsandtrophozoites,
50
respectively,whichmayindicateselectivityoftheEOPtreatmentatconcentrationsupto
250μg/mL.TheresultsenumeratedabovedemonstratethatEOPisanimportantresourcefor
futuredrugdevelopment,anditstherapeuticpotentialagainstvariousinfectiousentitiesmer-
itsfurtherstudyandanalysis.
Adjuvantassay
Fig4illustratestheresultsobtainedfromtheELISAassaydesignedforanalyzingtheproduc-
tionofanti-rCP40totalIgG,IgG1,andIgG2aantibodies.Theresultsdemonstratethatanimals
fromgroupG1wereunabletoinduceproductionofIgGanditsisotypes.IngroupG2,
increasedtotalIgG,IgG1,andIgG2alevelsweredetectedonday42,subsequenttotheadmin-
istrationoftwodosesofrCP40.Additionally,itwasobservedthattheassociationofEOPwith
rCP40(G3)leadstotheinductionofhigherlevelsofIgGanditsisotypes.Whencompared
withgroupsG1andG2,groupG3wasobservedtopresentasignificantlyhigherincrease
(p<0.05)intotalIgGtotalaswellasisotypesIgG1andIgG2aatdays21and42afterthefirst
immunization(Fig4A,4Band4C);thisisdemonstrativeoftheadjuvanteffectofEOP.The
day-42valuesclearlydemonstratethattheassociationofEOPwithrCP40wasabletoincrease
thelevelsoftotalIgG,IgG1andIgG2abyapproximately7.4,4.4and10.8fold,respectively,
incomparisontotheadministrationofrCP40alone.Moreover,groupG3showsmorepro-
nouncedIgG2aproductioncomparedtotheproductionofIgG1atday42.
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ChemicalcompositionandbiologicalpropertiesoftheessentialoilfromBrazilianredpropolis
Fig3.CHO-K1cellviabilityaftertreatmentwithdifferentconcentrationsofEOP.CellproliferationinCHO-K1
wasinvestigatedbytheMTTassay,andmetronidazolewasusedasapositivecontrol.Dataareexpressedasthe
mean±SEMoftheviabilitiesofCHO-K1cellsinthreeindependentexperiments.((cid:3))indicatesadifferencebetween
thetreatmentsinCHO-K1cells.Thedifferenceswereconsideredsignificantatp<0.05.VC=vehiclecontrol;
MTZ=metronidazole.
https://doi.org/10.1371/journal.pone.0191797.g003
Discussion
AlthoughsomeBrazilianpropolisessentialoils,mainlythosederivedfromgreenpropolis,
havebeencharacterizedpreviously[36,49,50], thisisthefirststudytoundertakethechar-
acterizationofBRPessentialoil.OnlytheethanolicandmethanolicextractsofBRPhave
beentestedbeingisoflavonesthemainisolatedcompounds[18,51,52]. Thus,wehypothe-
sizedthattheessentialoil(EO)ofBRPcouldprovidesomevolatilecompoundswithimpor-
tantbiologicalproperties,whichareprobablylostoreliminatedintheotherextraction
methods.Infact,methyleugenol,aphenylpropene,wasthemajorvolatilecomponentiso-
latedfromEOPandmostoftheotherisolateswereclassifiedassesquiterpenes.Along
similarlines,itisknownthattheBraziliangreenpropolisalsohasapredominanceofses-
quiterpenes.Nerolidol,β-caryophyllene,spathulenol,andδ-cadinenehavebeenidentified
asothermajorvolatilecomponentsofgreenpropolis[29].StudieshaverevealedthatEO
constituentsmakeupadiversefamilyoflow-molecular-weightorganiccompoundswith
extremevariationsinbiologicalactivity.Basedontheirchemicalstructure,theactivecom-
poundsofEOscanbedividedintofourmajorgroups:terpenes,terpenoids,phenylpro-
penes,and“others”[53].
SomeofthevolatilecompoundspresentinEOPhavealreadybeencreditedwithhaving
antiparasiticactivity.Thesechemicalentitiesincludeβ-caryophyllene,α-pinene,α-bergamo-
tene,and(E)-β-farnesene[54,55].Additionally,methyleugenolisolatedfromtheessentialoil
extractedfromtheplantAgastacherugosashowedsignificantnematicidalactivityagainst
Meloidogyneincognita[56].MethyleugenolisaspecificconstituentofBrazilianredpropolis,
amonganothertypesofpropolis,whichwasrevealedforthefirsttimein2008[57].Therefore,
inthiswork,wehypothesizedthatmethyleugenol,aphenylpropenewiththemajorconcentra-
tioninEOPandthesesquiterpenesmayberesponsiblefortheantiparasiticactivityagainst
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ChemicalcompositionandbiologicalpropertiesoftheessentialoilfromBrazilianredpropolis
Fig4.Anti-rCP40totalIgG(4A),IgG1(4B)andIgG2a(4C)levelsinimmunizedmicetreatedwithEOPand
recombinantendoglycosidaseCP40fromC.pseudotuberculosis(rCP40),eitherseparatelyortogether.
Measurementsweretakenonday0andthen21and42dayspost-immunization.Datarepresentmeans±standard
deviationsofIgGlevelsintheseracollectedfrom6animals/group.Differentlettersinthesameexperimentalday
indicateasignificantdifference(p<0.05).
https://doi.org/10.1371/journal.pone.0191797.g004
T.vaginalisexhibitedhere.However,furtherstudiesshouldbeperformedwiththeisolated
constituentsofEOPtoidentifythespecificantiparasiticagents.
Inspiteoftheanti-T.vaginalisactivity,ahighercytotoxicitylevelwasobservedattheEOP
concentrationthatpresented0%viabilityoftrophozoitesafter24hofexposure(500μg/mL)
comparedtoalowerconcentration(200μg/mL),whichwasnottoxicforCHO-K1cellsbut
reducedparasiteproliferationby70%.Therefore,itishypothesizedthatcytotoxicitycouldbea
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ChemicalcompositionandbiologicalpropertiesoftheessentialoilfromBrazilianredpropolis
limitingfactorfortheparenteraluseofEOP,butitsuseintopicalformsforthetreatmentof
trichomoniasis,throughtopicalvaginalapplication,appearstobeaninterestingalternative.It
iswellknownthattopicalapplicationallowsforthedeliveryofhigherdrugconcentrationsto
thevaginalepitheliumatlowertreatmentdoses[58].
Apreviousstudyhasevaluatedtheeffectsofcompoundswithguaiacylandsyringylstruc-
tures(asdegradationproductsoflignin)andnaturalaromaticacidsandtheiraldehydesonT.
vaginalisandfoundthatthetestedcompoundsexhibitedantiparasiticactivitywithMICvalues
rangingfrom10to200μg/mL.Isoeugenol,aphenylpropenegeneratedbyapropenyl-substi-
tutedguaiacol,wasthemostpotenteffectoragainstT.vaginaliswithaMICvalueof10μg/mL
[59],whichismuchlowerthanMICfoundhereforEOP.SinceEOPisacomplexmixture
containingmainlymethyleugenol,aphenylpropenewithsimilaritieswithisoeugenolinchem-
icalstructure,andsesquiterpenesinlowconcentrations,itisexpectedadifferentbiological
resultincomparisontotheuseofeachisolatedcompound.
Inaddition,somestudieshaverevealedthatredpropolisextractsfromdifferentgeographi-
calregionshavesignificantpotentialasantiparasiticagents.Theethanolicextractofredprop-
olisobtainedfromNigeria,whentestedagainstTrypanosomabrucei,demonstratedpotent
antiparasiticactivityinbothsensitiveandresistantstrains[60].Inanotherstudy,foursamples
ofethanolicextractsofBrazilianpropolisweretestedagainstLeishmaniaamazonensis.Three
extractsofgreenpropolisandoneofredpropolisweretested,andallofthemwerecapableof
reducingparasiteloadsininfectedmacrophages.Nevertheless,theextractobtainedfromBRP
showedmaximalactivityagainstL.amazonensiswhilebeingnon-toxictothemacrophagecul-
tures[61].
Recently,theantiparasiticactivityofBRPhasattractedtheattentionofresearchers.Poly-
mericnanoparticlesoftheBRPextractandtheethanolicextractofBRPhavedemonstrated
anti-leishmanialactivityagainstL.(V.)braziliensis,withIC valuesof31.3μg/mLand
50
38.0μg/mL,respectively,demonstratingthepotentialofBRPinthecompositionofpharma-
ceuticalsforleishmaniasistherapy[12].Additionally,whenethanolicextractsfromvarious
typesofgreen,brownandredpropolisfromdifferentregionsofBrazilweretestedagainstTry-
panosomacruziY-strainepimastigotes,aninhibitoryeffectonparasitegrowthwasobservedin
thefirst24hforallthetypesofpropolis.However,onlytheethanolicextractsofBRPpre-
sentedapersistentT.cruzi-inhibitoryeffectafter96h,suggestingredpropolisasapotential
alternativeforthetherapeutictreatmentofChagasdisease[10].
Inthisstudy,wealsoconfirmedthatEOPhasthepotentialtobeusedasanadjuvantina
veterinaryvaccineformulation.Inourstudy,arecombinantendoglycosidaseCP40(rCP40),
encodedbythecp40geneofC.pseudotuberculosis,wasusedastheantigenasitsantigenic
propertieshavebeenpreviouslyconfirmed[34,62].Ourresultsconfirmedthatassociationof
therCP40proteinwithEOPimprovestheimmuneresponsetothistargetcomparedtothe
administrationofeitherofthesecomponentsalone.Weobservedthatthecombinationofthe
rCP40withEOPledtoIgGabsorbanceshigherthanthosefoundbyotherresearchersusing
traditionaladjuvantssuchassaponinorFreund’scompleteadjuvant[34].Herein,weusedfor
thefirsttimeEOPasanadjuvant.Infact,thisisthefirststudytoevaluatetheeffectofBRPasa
vaccinaladjuvant.
ComparedtotheadministrationofrCP40alone,acombinationoftherecombinantprotein
withEOPwasabletoinduceincreasesof7.4-,4.4-and10.8-foldhigherlevelsoftotalIgG,
IgG1,andIgG2a,respectively.Oneofthemostimportantchallengescurrentlyfacingadjuvant
researchisthesearchforthe‘perfectmix’:anoptimal,safeformulationwhosedifferentcom-
ponentsarenotonlyadditivebutalsosynergisticinnature,andwhoseadministrationgives
risetothedesiredrobustimmuneresponse[63].Inthisstudy,weobservedasynergisticeffect
whenEOPwasassociatedwithrCP40.Alongsimilarlines,anotherrecentstudyhasreported
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