Seed and peel essential oils obtained from Campomanesia adamantium fruit inhibit inflammatory

15 Pages·2017·1.65 MB·English

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].
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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.
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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)
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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
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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