RESEARCHARTICLE Seed and peel essential oils obtained from Campomanesia adamantium fruit inhibit inflammatory and pain parameters in rodents DanieliZuntiniViscardi1*,Jucicle´iadaSilvaArrigo1,CamiladeAzevedoChavesCorreia1, CaˆndidaAparecidaLeiteKassuya2,ClaudiaAndreaLimaCardoso3,Iriani RodriguesMaldonade4,ElianaJanetSanjinezArgandoña5 1 CollegeofExactandTechnologicalSciences,FederalUniversityofGrandeDourados,Dourados,Mato GrossodoSul,Brazil,2 CollegeofHealthSciences,FederalUniversityofGrandeDourados,Dourados, a1111111111 MatoGrossodoSul,Brazil,3 CourseofChemistry,StateUniversityofMatoGrossodoSul,Dourados,Mato a1111111111 GrossodoSul,Brazil,4 BrazilianAgriculturalResearchAgency(Embrapa),Bras´ılia,FederalDistrict,Brazil, a1111111111 5 CollegeofFoodEngineering,FederalUniversityofGrandeDourados,Dourados,MatoGrossodoSul, a1111111111 Brazil a1111111111 *[email protected] Abstract OPENACCESS Campomanesiaadamantium(Myrtaceae)ispopularlyknownas“gabiroba”andhasbeen Citation:ZuntiniViscardiD,ArrigoJdS,Correia CdAC,KassuyaCAL,CardosoCAL,MaldonadeIR, usedinfolkmedicineasantirheumatic,antidiarrheal,hypocholesterolemicandanti-inflam- etal.(2017)Seedandpeelessentialoilsobtained matory.Thisstudyevaluatedtheanti-inflammatoryandantinociceptiveactivitiesandtoxi- fromCampomanesiaadamantiumfruitinhibit cologyofessentialoilsfrompeel(EOP)andseed(EOS)ofC.adamantiumfruitsinanimal inflammatoryandpainparametersinrodents. models.Differentgroupsweretreatedwithdosesof100and300mg/kgandtheinflamma- PLoSONE12(2):e0157107.doi:10.1371/journal. pone.0157107 toryparameterswereevaluatedincarrageenaninducedpawoedemaandleukocytemigra- tioninpleurisymodel,whileantinociceptiveactivitywasevaluatedusingformalinmethodin Editor:FranciscoJ.Schopfer,Universityof Pittsburgh,UNITEDSTATES rodents.ThemajorconstituentofEOPandEOSwaslimonenewith13.07%and20.89%, respectively.Noclinicalsignsoftoxicityhavebeenobservedinanimals.Itwasobserveda Received:March1,2016 significantdecreased(P<0.01)inleukocytemigrationatthedoseof300mg/kgofEOPand Accepted:January30,2017 EOS,withmaximalinhibitionof89±3%forEOPand80±6%forEOS.Pawoedemawas Published:February21,2017 inhibitedatalltimes,andmaximalinhibitionwasatthedoseof100mg/kgat2haftercarra- Copyright:©2017ZuntiniViscardietal.Thisisan geenaninjectionwith72±2%forEOPand74±2%forEOS.EOSandEOPalsoreducedthe openaccessarticledistributedunderthetermsof firstandsecondphasesofformalin-inducednociceptiontest.Inthefirstformalin-phase, theCreativeCommonsAttributionLicense,which maximalinhibitionswereat48±5%forEOPand66±4%forEOS(300mg/kg).Attheinflam- permitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginal matoryphaseinducedbyformalin,maximalinhibitionswere72±2%forEOPand80±2%for authorandsourcearecredited. EOSatthedoseof100mg/kg.SeedandpeelessentialoilsfromC.adamantiumfruitinhib- DataAvailabilityStatement:Allrelevantdataare itedleukocytemigration,inflammatoryandneurogenicpainandoedemasuggestingtheir withinthepaper. useasnutraceuticalorpharmacologicalagent. Funding:Theauthorsalsowishtothankthe Fundac¸ãodeAmparoàPesquisadoEstadodo MatoGrossodoSul(FUNDECT)forfinancial support,throughtheprojectsFUNDECT/CAPESn˚ 45/2014-PAPOSREDEPRO´-CENTRO-OESTE– FASEII-Nº23/200.622/2014andtheConselho NacionaldeDesenvolvimentoCient´ıficoe Tecnolo´gico(CNPq).Thefundershadnorolein PLOSONE|DOI:10.1371/journal.pone.0157107 February21,2017 1/15 PharmacologicaleffectsofCampomanesiaadamantiumessentialoilinvivo studydesign,datacollectionandanalysis,decision Introduction topublish,orpreparationofthemanuscript. CerradoregionisthesecondlargestbiomainBrazil,afterAmazonia.Thisecosystemcom- Competinginterests:Theauthorshavedeclared prisesmorethan7,000plantspecies[1].Duetoitsdiverseflora,researchinteresthasbeen thatnocompetinginterestsexist. increasedandendemicmedicinalplantsfromCerradohavebeenasourceofbioactivecom- pounds.Gabirobaorguabiroba-do-campoorguavira,thefruitofCampomanesiaadamantium (Cambess.)O.Berg,iswidelyfoundandusedinareasofCerrado,mainlyintheMidwest regionandAtlanticForestintheSoutheastandSouthregionsofBrazil[2]. C.adamantiumbelongstoMyrtaceaefamilyandtheirfruits,aswellasfruitsfromother speciesofCampomanesia,aretraditionallyusedintheproductionofhomemadeliqueurs, juices,andsweets[3]andalsoemployedinfolkmedicineasantirheumatic,antidiarrheal, hypocholesterolemic,anti-inflammatory[4],andforthetreatmentofcystitis,andurethritis [5]. OurgrouphaspreviouslyshownthathydroalcoholicextractfromC.adamantiumfruit barksexhibitedanti-inflammatory,antihyperalgesic,andantidepressiveactivityinrodents, withnoevidenceoftoxicologicalsigns[6].Studieswithethylacetateandaqueousextracts fromleavesC.adamantiumhavedemonstratedantinociceptiveandanti-inflammatoryeffects supportingfolkmedicineuseoftheseplants[7].Phytochemicalanalyseshaveattributedthe anti-inflammatory,antiproliferativeandantimicrobialactivitiesofC.adamantiumtothepres- enceofflavonoidsandchalcones,whicharethemajorconstituentsintheextract[5,6,7,8]. TheessentialoilfromC.adamantiumleaveshavebeenpreviouslydescribedasantioxidant andantimicrobial,havingasmajorconstituentsinessentialoillimonene,α-pineneandβ- pinene[9].Despitethisevidence,therearenoreportsontheanti-inflammatory,antinocicep- tiveandtoxicologicalparametersoftheessentialoilofpeelandseedofthisfruit,whichjustify thepresentstudy. Therefore,weaimedtoevaluatethechemicalcompositionofseedsandpeelsofessentialoil fromC.adamantiumfruitandtotheirrelationtoacutetoxicity,leukocytemigration,inflam- matoryandneurogenicpainandpawoedemainanimalmodels. Materialsandmethods Plantmaterial ThevegetablematerialcollectedwasregisteredintheAuthorizationSystemandInformation ofBiodiversity(SISBIO),N˚39462.Thefieldstudiesdidnotinvolveendangeredorprotected species.Theguavira’sfruitswereproducedbysmallfarmerslocatedatPontaPorã-MS,Brazil, (23˚32010@S,55˚37033@W),onNovember2013. AvoucherspecimenwasdepositedintheherbariumoftheFacultyofBiologicalSciencesof UFGD(DDMS4602).Fruitsweresanitizedandpulp,peelandseedswereseparated.Peeland seedsweredriedat40˚Cfor62and72h,respectively,protectingthemfromdirectlightuntil use.Humiditywascalculatedonadrybasis(UBS)at70˚Cfor24hours[10]. Thecalculationoftheyieldwasobtainedtakingintoaccountthemoisturecontentona dry,basis,accordingtothefollowingequation: V Yield ¼ oilessential (cid:3)100 W (cid:0) Wsample(cid:3)H sample 100 WhereV isoilessentialvolumeobtained(mL),w isinitialsamplemass(g) oilessential sample andHisthemoisturecontent. Animals. MaleWistarrats(200–230g)andmaleandfemaleSwissmice(20–25g)were obtainedfromFederalUniversityofGrandeDourados(UFGD)biotherium.Animalswere PLOSONE|DOI:10.1371/journal.pone.0157107 February21,2017 2/15 PharmacologicaleffectsofCampomanesiaadamantiumessentialoilinvivo keptincollectivecages—(6animals/cage)atacontrolledtemperature(23±1˚C)withlight cycle(12hlight/dark),drinkingwaterandacommercialdietadlibitum.The23/2014protocol wasapprovedbythe“EthicsCommitteeonAnimalUse(CEUA)ofFederalUniversityof GrandeDourados”. Extractionandcompositionofessentialoil Essentialoilwasobtainedfrom200gofdriedpeeland200gofdriedseedsfromC.adaman- tiumbyHydrodistillation(3replicates)usingaClevenger-typeapparatusfor3hours.Atthe endofeachdistillation,oilswerecollectedandtransferredtoglassflasks,andkeptatatemper- atureof-18˚Cforfurtheranalyses.Samplesobtainedbyhydrodistillationwereanalyzedby GC/qMS(ShimadzuP2010plus.ShimadzuTokyo,Japan)equippedwithanautoinjectorsplit/ splitless.ThechromatographicseparationwasperformedonaDB-5column(J&W.Folsom, California)5%phenyl-dimethylpolysiloxane(30mlong×0.25mmdiameter×0.25mmof filmthickness)underthefollowingconditions:carriergashelium(99.999%)ataflowrateof1 mL/min;1μLofinjectionvolumesplitratio(1:20).Thetemperatureprogramofthefirstcol- umnstartedat50˚Cfor5min,heatingat3˚C/minuntil250˚C.Theinjectortransferlineand detectortemperatureusedweremaintainedat250˚C.TheMSscanparametersincludedelec- tronimpactionizationvoltageat70V,amassrangefrom50to550Daltonsandascaninterval of0.5s.Samples(1mgoftheessentialoils)wasdilutedin1mLofn-hexanebeforeinjection. Temperature-programmedretentionindexes[11]werecalculatedusingamixtureofnor- malalkanes(C -C )asexternalreferences.Identificationofcompoundswasperformedusing 6 30 retentiontime[12]andcomparingwithinterpretationofmassspectraofunknowncompo- nentsaccordingtoWileymassspectralibrary,WileyMS6thEdition. Relativepeakareasforeachchromatographicpeakwereusedtoevaluatethecontribution ofeachcompoundtothetotalareaandforcomparisonsbetweensamples.Thesumofallpeak areaswasconsidered100%ofthesampleandforeachpeakapercentagewasassignedcorre- spondingtoitsarea[11]. Anti-inflammatorytests Pleurisy. DifferentgroupsoffemaleSwissmice(n=5)wereorallyadministeredwith EOP(essentialoilfrompeel)andEOS(essentialoilfromseed)atthedosesof100and300mg/ kg,respectively,in0,9%ofsalinesolution,whichwasadministeredasacontrolinathird groupofanimals.Positivecontrolgroupreceiveddexamethasonesubcutaneouslyatdoseof1 mg/kg.Pleurisywasinducedintheexperimentalgroupsbyintrapleuralinjectionof100μLof 1%carrageenandilutedinsaline,afteronehouroftreatment(withoutanesthetization),aspre- viouslydescribed[13].Negativecontrolreceived100μLofsterilesalinebyintrapleuralinjec- tion.After4h,animalswereeuthanizedandthepleuralcavitywaswashedwith1mL phosphatebuffer-saline(PBS).Analiquotof20μLoflavage(exudate)wascollectedfromthe pleuralcavity,anddilutedwithTurck(1:20)andusedfortotalleukocytecountinaNeubauer chamber[14]. Carrageenan-inducedpawoedema. DifferentgroupsofmaleSwissmice(n=5)were orallytreatedwithEOPandEOS(100and300mg/kg),orvehicle(controlgroup).Another groupwastreatedsubcutaneouslywithdexamethasone(1mg/kg).After1h,animalsreceived asolutionof50μLcarrageenaninjection(300μg/paw)inthelefthindpaw.Theotherpaw receivedthesamevolumeofsterilesaline0.9%.Thepawvolumewasmeasuredat1,2,3and4 hoursaftercarrageenaninjectionwithapletysmometer.Resultswereexpressedasthediffer- encebetweentheleftandrightpawsateachtime[4]. PLOSONE|DOI:10.1371/journal.pone.0157107 February21,2017 3/15 PharmacologicaleffectsofCampomanesiaadamantiumessentialoilinvivo Antinociceptivetests Formalin-inducedspontaneouspainmodel. Nociceptionwasevaluatedafterinjection offormalin[15,16].Sixtyminutesbefore,maleWistarrats(n=5)weredividedingroups:dex- ametasone(1mg/kg.s.c.),EOS(100and300mg/kg),EOP(100and300mg/kg)andvehicle (salinesolution(0.9%),treatedbyoralroute.Onehouraftertreatmentsitwasinjected20μL ofsalinecontaining2.5%offormalinintherighthindpaw.Painreactiontime(pawlicking)in secondswasevaluatedfrom0to5min(phase1—neurogenicpain)andfrom15to30min (phase2—inflammatoryresponse)afterinjectionofformalininthepaw[14],whichrepresents thetonicresponsetopain,accompaniedbyaninflammatoryresponse.Following,animals weresubmittedtopawoedemameasurement,coldsensitivityandopenfieldtests. Coldsensitivity. Coldhyperalgesiawasmeasuredbytheacetonetestasdescribedby DecosterdandWoolf[17].Aneedleconnectedtoasyringewasusedtodrop30μLofacetone onthepawandtheduration(inseconds)ofthepawwithdrawalwasrecorded.Minimaland maximalcut-offswereassignedat0.5and20sec,respectively.Pawwithdrawalsduetoloco- motionorweightshiftingwerenotcountedandsuchtrialswererepeated[7]. Open-fieldtest. Analysesoflocomotionactivitieswereperformedafterformalinexperi- ment.Theratswereacclimatedinlocomotormeasurementchambersbeforetesting.Forthe test,ratswerepositionedinthecenterofanopen-fieldapparatus,consistedofwhitesquare arena(80×80cm)surroundedbywalls(40cmheight)withitsfloordividedbylinesinto16 squares(20×20cm).Locomotoractivitywasdeterminedbythenumberofsquarescrossed during5minutes.Theapparatuswascleanedwith10%ethanolsolutionandpapertowels betweeneachsection[18]. Formalin-inducedpawoedema. Animalsreceivedanintraplantarinjectionofformalin (20μLofasolution2.5%v/v)intherighthindpaw.Thicknesspawoedemawasassessedusing apletysmometer30minbeforeanytreatmentandafteronehourofformalininjection.Results wereexpressedinμLasthedifferencebetweenthebaselineandpost-injectionoedemavalues, withmodificationsofKassuyaetal.[14] Toxicitytest Acuteoraltoxicity. Treatmentswereperformedbysingleoraladministrationatdoses0, 175,560,1792,or2000mg/kgofbodyweightofEOPandEOS.FemaleSwissmice(n=8) wereobservedforsignsoftoxicityduringthefirst0.5,1,2,4,8,12and24hoursandatevery 48hoursfor14days.Duringtheexperimentalperiodanimalswereobserveddailyforclinical aspects,includingposture,seizures/tremors,consistencyandappearanceofthefeces,eyelid closure,piloerection,appearanceofskinandhair,stress,salivation,behavior,bodyweightand consumptionoffoodandwater[19]. Statisticalanalyses Resultswereexpressedasmean±standarderrorofthemean(S.E.M.).Forcomparisonof resultsamongexperimentalgroupsitwasusedanalysisofvariance(one-wayANOVA)fol- lowedbyNewman-Keulstest.Thenumberofanimalspergroupisindicatedinthelegends. Statisticaldifferenceswereconsideredsignificantatp<0.05. Results ThemoisturecontentdeterminedfordriedpeelandseedofC.adamantiumwere26.07± 3.80%(wt,massofwaterpermassofdriedmatter)and7.29±0.31%(wt),whiletheobtained essentialoilsyieldswereforpeel0.32%andseed0.98%(w/w). PLOSONE|DOI:10.1371/journal.pone.0157107 February21,2017 4/15 PharmacologicaleffectsofCampomanesiaadamantiumessentialoilinvivo Thelistofcompoundsidentifiedwithacompositionhigherthan1%inoilsisshowninthe Table1.Theretentiontimes,whichweredeterminedfromthreeindependentexperiments, showedacoefficientofvariationlessthan2%.Themajorconstituentsinpeeloilswerelimo- nene(13.07%)andthujopsene(6.96%)whileseedoilswerelimonene(20.89%)andβ-pinene (11.48%)(Table1). OraladministrationofEOPandEOSsignificantlyinhibitedtheleukocytemigrationatall dosestested(100and300mg/kg).Asignificantdecreasewasobservedinleukocytemigration, atthedoseof300mg/kgofEOPandEOS,withmaximuminhibitionat89±3%forEOPand 80±6%forEOS(Fig1). EOPandEOScausedareductioninpawoedemainducedbycarrageenan(Fig2)atall times,andmaximuminhibitionwasatthedoseof100mg/kgafter2hofcarrageenaninjection (72±2%forEOPand74±2%forEOS),respectively. EOPandEOS(Fig3,PhaseI)producedsignificantantinociceptiveeffectsinthefirstphase whencomparedtocontrolgroup.EOPandEOS,respectivelyatthedosesof100and300mg/ kgsignificantlyreducedlickingtimeinthesecondphaseofformalintestinrats(Fig3Phase II).Dexametasone(1mg/kg)producedsignificantantinociceptiveactivityinbothphasesof theformalinmethod.ItwasalsofoundthatorallyadministrationofEOPandEOSinhibited formalininducedpawoedema. EOSadministration(100and300mg/kg)significantlyattenuatedcoldhypersensitivity durationafterformalininjection.Animalsalmostdidnotmoveandraisedtheirpawsafew timesafteracetoneapplication(Fig4A).Furthermore,hypersensitiveresponsetocoldwaslon- gerthan10seconds(Fig4B). Fig4AshowsthatoraltreatmentwiththeEOPandEOSwithdosesvaryingfrom100mg/ kgand300mg/kg,theformalin-inducedincreasedinsensitivitytoacoldstimulushassignifi- cantlyreduced,withamaximalinhibitionat65±3%,68±2%,75±6%,77±2%,respectively, comparedtothecontrolgroup.Treatmentwithdexamethasonesignificantlyinhibitedthe coldsensitivityinducedbyformalinat85±3%(Fig4A). EOP(100mg/kgand300mg/kg)andEOS(100mg/kgand300mg/kg)alsocausedareduc- tioninpawoedemainducedbyformalin.Fig4Bshowspawoedemawithreductionof 49±2%,53±2%,72±4%,79±3%,respectively,comparedtothecontrolgroup.Treatment withdexamethasonesignificantlyinhibitedthepawoedemainducedbyformalinby87±3% (Fig4B).TheoraltreatmentwithessentialoilfromseedandpeelofC.adamantiumfruitdid notdecreaselocomotoractivityinopen-fieldtest(datanotshown). Toxicityofextractsandcompoundswereevaluatedasrequiredbyregulatoryagencies. Mortalityisthemostevidentsignoftoxicity.However,otheraspectsprovidemoresubtledata oftheadverseeffects,suchaslossofbodymassduringthestudyperiodandclinicalsignsof toxicity(diarrhea,piloerectionandbehaviorchanges).Animalsusedinthepresentstudy receivedessentialoils(EOSandEOP)ofC.adamantiumanddidnotexhibitanyclinicalsigns oftoxicityatalldosesadministeredandnosignificantchangeswereobservedinwaterand fooduptake.Furthermore,theabsoluteandrelativeweightoftheorgans(liver,kidneysand lungs)presentednostatisticallysignificantdifference(Tables2and3). Discussion Thepresentstudydemonstratedtheanti-inflammatory,antinociceptiveandtoxicological analysesofEOSandEOPfromC.adamantiumfruits.Experimentaldatademonstratedthat EOSandEOPinhibitedleukocytemigration,inflammatoryandneurogenicpainandoedema, suggestingtheiruseasanutraceuticalorpharmacologicalagent. PLOSONE|DOI:10.1371/journal.pone.0157107 February21,2017 5/15 PharmacologicaleffectsofCampomanesiaadamantiumessentialoilinvivo Table1. CompositionofthevolatilecompoundsindentifiedintheessentialoilsofthepeelandseedofCampomanesiaadamantiumfruits. Compounds R RI LI Composition(%) t Peel Seed Cumene 7.378 925 924 0.49 0.85 α-Pinene 7.653 932 932 4.67 8.50 Camphene 8.132 946 946 0.12 0.29 β-Pinene 9.190 976 974 4.82 11.48 Myrcene 9.667 989 988 1.07 1.85 α-Phellandrene 10.206 1003 1002 2.13 3.96 δ-Carene 10.425 1009 1008 0.19 0.11 α-Terpinene 10.679 1015 1014 0.39 0.79 o-Cymene 11.019 1023 1022 1.49 1.05 Limonene 11.186 1025 1024 13.07 20.89 1.8-Cineole 11.232 1026 1026 3.56 0.18 Z-β-Ocimene 11.449 1033 1032 0.12 - E-β-Ocimene 12.001 1045 1044 3.24 4.12 U-Terpinene 12.249 1054 1054 1.00 1.66 Terpinolene 13.724 1091 1086 1.82 2.09 Linalool 14.154 1096 1095 3.17 2.32 endo-Fenchol 14.816 1116 1114 0.11 0.21 Borneol 17.155 1168 1165 0.18 0.38 Terpinen-4-ol 17.477 1175 1174 0.79 0.89 α-Terpineol 18.101 1186 1186 2.40 2.61 Myrtenal 18.507 1196 1195 0.17 0.18 trans-Piperitol 19.007 1208 1207 0.12 0.14 trans-Carveol 19.238 1215 1215 0.12 0.13 Nerol 19.842 1228 1227 0.12 - Carvone 20.468 1240 1239 0.12 - Geraniol 21.020 1250 1249 0.15 - Perillaaldehyde 22.012 1270 1269 0.13 0.10 Carvacrol 23.133 1299 1298 0.12 - Methylgeranate 24.075 1323 1322 0.13 0.16 δ-Elemene 24.568 1335 1335 0.10 0.11 α-Ylangene 26.248 1375 1373 0.11 0.13 Isoledene 26.313 1376 1374 0.10 - α-Copaene 26.350 1375 1374 0.97 0.94 β-Cubebene 27.006 1388 1387 0.10 - β-Elemene 27.024 1389 1389 0.14 0.10 Sibirene 27.319 1400 1400 0.11 0.10 α-Gurjunene 27.697 1409 1409 0.33 0.33 Thujopsene 28.444 1429 1429 6.96 6.82 β-Copaene 28.497 1430 1430 0.41 0.38 Aromadendrene 29.005 1440 1439 2.78 2.15 Cedrane 29.088 1441 1441 0.23 0.13 trans-muurola-3,5-diene 29.421 1451 1451 0.10 0.10 α-Humulene 29.495 1452 1452 2.68 2.37 allo-Aromadendrene 29.830 1459 1458 0.83 0.52 trans-Cadina-1(6),4-diene 30.327 1475 1475 0.35 0.24 U-Muurolene 30.528 1478 1478 1.53 1.35 (Continued) PLOSONE|DOI:10.1371/journal.pone.0157107 February21,2017 6/15 PharmacologicaleffectsofCampomanesiaadamantiumessentialoilinvivo Table1. (Continued) Compounds R RI LI Composition(%) t Peel Seed GermacreneD 30.777 1485 1484 2.28 1.43 Widdra-2,4(14)-diene 30.575 1481 1481 0.69 0.53 α-Amorphene 30.660 1483 1483 0.23 0.05 β-Guaiene 31.091 1492 1492 0.35 0.16 Bicyclogermacrene 31.368 1500 1500 4.91 3.83 α-Muurolene 31.404 1501 1500 0.13 0.41 δ-Amorphene 31.903 1512 1511 0.51 0.13 U-Cadinene 32.002 1513 1513 1.31 1.84 δ-Cadinene 32.292 1522 1521 3.55 3.82 Trans-Cadina-1,4-diene 32.692 1533 1533 0.23 0.27 α-Cadinene 32.910 1537 1537 0.17 0.27 Selina-3,7(11)-diene 33.216 1546 1545 0.36 0.16 GermacreneB 33.676 1560 1559 0.47 0.14 Nerolidol 33.760 1562 1561 0.40 - Palustrol 34.072 1569 1567 0.63 0.10 Spathulenol 34.428 1578 1577 1.95 0.14 Globulol 34.993 1591 1590 4.00 - Cubeban-11-ols 35.163 1595 1595 1.18 0.10 Guaiol 35.365 1600 1600 0.60 0.28 Humuleneepo´xiII 35.637 1608 1608 1.04 0.11 Junenol 36.033 1620 1619 0.24 - 1-epi-Cubenol 36.319 1627 1627 1.00 0.23 α-acorenol 36.404 1632 1632 0.66 0.13 epi-α-Cadinol 36.750 1639 1638 0.71 0.18 α-muurolol 37.000 1645 1644 0.11 - β-Eudesmol 37.087 1648 1649 0.69 0.56 α-Cadinol 37.285 1653 1652 0.39 0.10 Valerianol 37.437 1656 1656 0.50 0.18 Allohimachalol 37.585 1661 1661 - 0.50 7-epi-α-Eudesmol 37.603 1662 1662 1.85 - Bulnesol 38.002 1671 1670 0.19 0.10 Eudesm-7(11)-em-4-ol 39.005 1701 1700 0.13 - CompoundslistedinorderofelutionfromaDB-5column.Rt,retentiontime(min);RI,retentionindicesonDB-5capillarycolumn;LI,literatureindices. doi:10.1371/journal.pone.0157107.t001 TheacutetreatmentwithEOPandEOSdidnotinducechangesinfoodandwaterintakeor behavior(irritability,contortion,tremors,convulsions,tearingandfur).Furthermore,the absoluteandrelativeweightofvitalorgansshowednostatisticallysignificantdifferencesin anyofthetesteddoses.Thus,theorallethaldose(LD50)ofEOPandEOSisgreaterthan2000 mg/kgandbothcanbeclassifiedaslowtoxicityoilsaccordingtotheOrganizationforEco- nomicCooperationandDevelopment[19].TheseresultscorroboratedwithSouzaetal.[6] andextendedworkwhopreviouslyhasshownthathydroalcoholicextractofC.adamantium didnotexhibittoxicityinmice,aswellastheessentialoil. Acuteinflammationischaracterizedbyoedema,fever,rednessandpain.Oedemaisan effectivemeasureofinflammationandisusefultoquantifyinducedcutaneousinflammation [20].Carrageenaninducedoedemaisabiphasicmodelwithmultiplemediatorsactingin PLOSONE|DOI:10.1371/journal.pone.0157107 February21,2017 7/15 PharmacologicaleffectsofCampomanesiaadamantiumessentialoilinvivo Fig1.Effectoforaladministrationofessentialoilfrompeel(EOP)andseed(EOS)ininhibition ofleukocytemigrationatbothdosestestedinpleurisytest.Miceweretreatedonehourbeforean intrapleuralinjectionofcarrageenan,withEOSandEOP(100or300mg/kg),dexamethasone(dexa,1mg/kg, s.c.),orsalinesolution(Vehicle).Naivegroup,alsotreatedwithsalinep.o.,receivedanintrapleuralinjection ofsterilesaline.Thebarsexpressthemean±SEMof5animals.*p<0.001whencomparedtocontrolgroup. Differencesbetweengroupswereanalyzedbyanalysisofvariance(one-wayANOVA)followedbythe Newman-Keulstest. doi:10.1371/journal.pone.0157107.g001 Fig2.Effectoforaladministrationofessentialoilfrompeel(EOP)andseed(EOS)fromCampomanesia adamantiunincarrageenan-inducedpawoedemainmice.AnimalsreceivedEOPandEOS(100or300 mg/kg,p.o.)orcontrol(vehicle)ordexamethasone(DEXA,1mg/kg,s.c.)andafter1hanintraplantarinjectionof carrageenan(300μg/paw)forevaluationofpawoedemafor(A)1,(B)2,(C)3,and(D)4haftercarrageenan injection.Eachbarrepresentsthemean±SEMof5animals.*p<0.001whencomparedwiththecontrol group.Differencesbetweengroupswereanalyzedbyanalysisofvariance(one-wayANOVA)followedbythe Newman-Keulstest. doi:10.1371/journal.pone.0157107.g002 PLOSONE|DOI:10.1371/journal.pone.0157107 February21,2017 8/15 PharmacologicaleffectsofCampomanesiaadamantiumessentialoilinvivo Fig3.Effectoforaladministrationofessentialoilfrompeel(EOP)andseed(EOS)fromC. adamantiumfruitsinformalin-inducedpawlicking.EssentialoilofEOSandEOPatdosesof100and 300mg/kgshowsantinociceptiveeffectsinphaseIandIItest.Resultsrepresentmean±S.E.M.(n=6).* P<0.05,whencomparedtocontrolgroup.Eachbarrepresentsthemean±SEMof5animals.*p<0.001 whencomparedtocontrolgroup.Differencesamonggroupswereanalyzedbyanalysisofvariance(one-way ANOVA)followedbytheNewman-Keulstest. doi:10.1371/journal.pone.0157107.g003 sequencetoproduceaninflammatoryresponse.Intheearlyphase,itwasobservedthatEOP andEOSdecreasedcarrageenan-inducedpawoedema.Therewassignificantreductionin oedemaformationafter3hoursofadministrationoftheEOPandEOSwhencomparedwith controlgroup.PrevioustestsperformedwithC.adamantiumleavesextractbyFerreiraetal. [7]alsodemonstratedreducedoedemawhencomparedtothecontrolgroup.Accordingtothe resultsofthisstudy,EOPandEOSseemtoactmainlyontheinitialphaseofthecarrageenan inducedinflammatoryresponse. PlantsfromtheMyrtaceaeFamilyaredistributedacrosstheCerradoregionofBraziland somespecieshavebeenusedtotreatpainandinflammation,amongotherdiseases[6].The PLOSONE|DOI:10.1371/journal.pone.0157107 February21,2017 9/15 PharmacologicaleffectsofCampomanesiaadamantiumessentialoilinvivo Fig4.Effectoforaladministrationofessentialoilfrompeel(EOP)andseed(EOS)fromC. adamantiumfruitsinformalin-inducedcoldsensitivityandoedemainducedbyformalin.(A)Effectof essentialoilsofC.AdamantiumfruitoncoldsensitivityaftertreatmentwithEOPandEOS.(B)Oedema inducedbyformalin.Eachbarrepresentsthemean±SEMof5animals.*P<0.001whencomparedtothe controlgroup.Differencesamonggroupswereanalyzedbyanalysisofvariance(one-wayANOVA)followed bytheNewman-Keulstest. doi:10.1371/journal.pone.0157107.g004 anti-inflammatoryactivityofEOPandEOSinacuteinflammationwasassessedbycarra- geenan-inducedpleurisymodel.Thisisaclassicaltesttoevaluatethistypeofinflammation, forminganexudateinthepleuralcavitycharacterizedbyinfiltrationofpolymorphonuclear leukocytes,andthereleaseofvariouschemicalmediators,whichareimportantintheinflam- matoryprocess[21]. PLOSONE|DOI:10.1371/journal.pone.0157107 February21,2017 10/15
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