w w w . s b f g n o s i a . o r g . b r / r e v i s t a
Original
Article
Essential
oil
from
Ageratum
fastigiatum
reduces
expression
of
the
pro-inflammatory
cytokine
tumor
necrosis
factor-alpha
in
peripheral
blood
leukocytes
subjected
to
in
vitro
stimulation
with
phorbol
myristate
acetate
Bethânia
A.
Avelar-Freitas
a,b,∗,
Valéria
G.
Almeida
a,
Michaelle
G.
Santos
a,
Josué
A.T.
Santos
a,
Poliana
R.
Barroso
c,
Cristiane
F.F.
Grael
c,
Luiz
E.
Gregório
d,
Etel
Rocha-Vieira
a,
Gustavo
E.A.
Brito-Melo
aaLaboratóriodeImunologia,UniversidadeFederaldosValesdoJequitinhonhaeMucuri,Diamantina,MG,Brazil
bLaboratóriodeBiologiaCelularInstitutodeCiênciaeTecnologia,UniversidadeFederaldosValesdoJequitinhonhaeMucuri,Diamantina,MG,Brazil
cLaboratóriodeFarmacognosiaeFitoquímica,UniversidadeFederaldosValesdoJequitinhonhaeMucuri,Diamantina,MG,Brazil
dLaboratóriodeInsumosNaturaiseSintéticos,UniversidadeFederaldeSãoPaulo,Diadema,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received28October2014 Accepted11March2015 Availableonline30March2015
Keywords:
Ageratumfastigiatum
Asteraceae Medicinalplant Anti-inflammatory
a
b
s
t
r
a
c
t
Ageratumfastigiatum(Gardner)R.M.King&H.Rob.,amemberoftheAsteraceaefamilypopularlyknownin
Brazilas“matapasto”,isindicatedinfolkmedicineasanti-inflammatoryandanalgesic.Despiteitspopular use,littleisknownaboutitspotentialeffectontheparametersinvolvedinaninflammatoryresponse.The objectiveofthisstudywastocharacterizethechemicalcompositionoftheessentialoilfromA.fastigiatum andtoevaluatethefrequencyoftumornecrosisfactoralphaandinterferongammaproducingcellsin peripheralbloodlymphocytesstimulatedwithphorbolmyristateacetateinthepresenceofessentialoil
fromA.fastigiatum.Non-toxicconcentrationsofessentialoilfromA.fastigiatumwereevaluatedincultures
ofperipheralbloodleucocytesusingthetrypanblueexclusionassaybyflowcytometry.GC–MSanalysis revealedthattheprevalentcompoundsidentifiedintheessentialoilfromA.fastigiatumsamplewere␣ -pinene,limonene,trans-caryophyllene,␣-humulene,caryophylleneoxide,1,2-humulene-epoxide, 1,6-humulanodien-3-ol,and␣-cadinol.ResultsshowedthatexposuretoessentialoilfromA.fastigiatumat concentrationsof0.5×10−2and1×10−2l/mlcausednoalterationsinleukocyteviabilityascompared tothecontrolgroup.Bothconcentrationsloweredthepercentageoftumornecrosisfactoralpha (+)-lymphocytesandneutrophils.Therewerenochangesinthepercentageoflymphocytespositiveforthe interferongammacytokine.Ourresultssuggestthatpartoftheanti-inflammatoryactivityattributedto
A.fastigiatummaybeduetotheeffectofsomeofitscomponentsindecreasingthenumberofcellsthat
producethepro-inflammatorycytokinetumornecrosisfactoralpha.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Ageratum fastigiatum (Gardner) R.M. King & H. Rob., Aster-aceae,popularlyknownin Brazilas“matapasto”or“enxota”,is indicatedinfolkmedicineasanti-inflammatoryandanalgesic( Del-Vechio-Vieiraetal.,2009a).Localhealerspreparetheextractfrom freshly choppedleavesand branches(withor without inflores-cences), which are then subjected to maceration or decoction. Followingfiltration, theaqueous extract is applied topicallyto
∗ Correspondingauthor.
E-mail:[email protected](B.A.Avelar-Freitas).
treat pain and inflammation(Gonc¸alves et al.,2011).The anti-inflammatory effectof theessential oilofA. fastigiatum (EOAF) wasdemonstratedinvivo bythereductionof bothpawedema and leukocytemigration induced by carrageenan in an animal model(Del-Vechio-Vieiraetal.,2009a).However,themechanisms involved in the anti-inflammatory effect of the plant have yet tobeelucidated; componentsoftheessential oilmay interfere withmediatorsof theimmune response, suchas inflammatory cytokines.Inflammationaimstofightinfectionandpromotetissue repair,buttheexaggeratedinflammationcausesharmfuleffects tothebody,asoccursinsomeautoimmunediseases(Sedgerand McDermott, 2014).Cytokines are released in response to vari-ous formsof cellularstressincludinginflammation.Among the
http://dx.doi.org/10.1016/j.bjp.2015.03.002
pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-␣)plays a key role inleukocyte migration,central mediator of inflammation.Othercytokines,suchasinterferongamma(IFN-␥), activateleukocytepopulations.TNF-␣isapleiotropiccytokinethat canamplifyinflammatoryresponsesbystimulatingthereleaseof chemotacticfactorsfrommacrophagesandepithelialcellsandby upregulatingtheexpressionofleukocyteandendothelialadhesion molecules(Braeggeretal.,1992;Wildermanetal.,2006;Bibikova etal.,2014).IFN-␥isamajorcytokineresponsibleforactivating macrophagesandstimulatinggreaterhistocompatibilitycomplex expression,increasesantigenpresentationandactivationofcells oftheadaptiveimmuneresponse(Keesetal.,1984;Amaldietal., 1989;Alasandaguttietal.,2014).Consideringthepopularbelief around the anti-inflammatory activity of A. fastigiatum and its potentialeffectonimmuneresponse,theobjectiveofthisstudywas toinvestigatetheinvitroprofileofcytokinesinhumanleukocyte culturestreatedwithEOAFandstimulatedwithphorbolmyristate acetate(PMA).
Methodology
Plantmaterial
Theaerialparts(brancheswithleavesand inflorescences)of Ageratumfastigiatum(Gardner)R.M.King&H.Rob.,Asteraceae, werecollectedonDecember 2010at theJuscelino Kubitscheck Campus – UFVJM (18◦12.125′S 43◦34.367′W,altitude 1392m),
locatedinthetownofDiamantina,MinasGerais.Avoucher spec-imen wasdeposited inthe Herbarium DIAM-UFVJM,underthe registration number 1300. A technically proficient expert per-formedspeciesidentification.AccordingtoNormativeInstruction No. 154/2007 by the BiodiversityInformation System (SISBIO), properauthorizationwasobtainedforthecollectionofplant mate-rial,withregistrationno.5141849issuedbytheBrazilianInstitute ofEnvironmentandRenewableNaturalResources.Authorization toaccessandshippingsamplecomponentofgeneticheritageno. 010476/2012-1(ConselhoNacionaldeDesenvolvimentoCientífico eTecnológico).
Extractionofessentialoil
Theoilwasobtainedfromfreshplantmaterial,accordingto MethodIoftheBrazilianPharmacopeia,5thEdition(2010).This processisbasedonhydro-distillationwiththeextraction extend-ingovera4-hperiod.Theessentialoilwasobtainedandstored inflasksat−20◦C,protectedfromlight.From70goffreshplant
material,0.06mlofoilwasobtained(ml/70g×100)givingaratio of0.086%.
AnalysisoftheEOAFcomponentsbygaschromatography–mass spectrometry(GC/–MS)
GC/–MS analysis wasperformed using a Shimadzu GC–MS-QP2010equippedwithaDB-5-MSAgilentJandWcapillarycolumn (30m×0.25mm×0.25m).Highpurityheliumwasusedasthe carriergasatapressureof57.5kPawithacolumnflowof1ml/min. Theinjector temperaturewas240◦C,andtheoventemperature
increasedfrom60◦Cto240◦Cat3◦C/min.Electronionizationat
70eVwastheselectedionizationmethod.Underthesame experi-mentalconditions,theoilwasco-injectedwithahomologousseries oflinearhydrocarbons(AlltechC9–C24),tocalculatetheretention index(RI)ofeachconstituentoftheessentialoil(VanDenDool etal.,1963).Compoundswereidentifiedbyanalysisand compar-isonofthemassspectrawiththeWiley7,NIST 62, andFFNSC 1.3libraries and bycomparison of theRItothose in literature (Adams,1995)andwebsites(<http://webbook.nist.gov/quimica>
and<http://pherobase.com>).Therelativeareas(%)ofeach indi-vidualpeakfromtheGC/MSchromatogramwereproportionalto thetotalioncurrentwithoutstandardization.
Analysisoftheviabilityofperipheralbloodleucocytesfollowing exposuretodifferentconcentrationsofessentialoil
Bloodsampleswereobtainedfromsixhealthyvolunteers. Vol-unteersusingcorticosteroidsorotherimmunosuppressantdrugs werenotconsideredforblooddonation.Thestudywasapproved bytheEthicsCommitteeoftheUFVJMunderregistrationnumber 146/10.Bloodsampleswerecollectedinvacuumtubescontaining heparin(Vacutainer;BectonDickinson,SanJose,CA,USA).Forcell viabilityanalysis,10mlofvenousbloodobtainedfromsixyoung blooddonorswasincubatedfor10mininthepresenceof10mlof redbloodcelllysissolution(NH4Cl8.3mg/ml,NaHCO31mg/ml,
ethylenediaminetetraaceticacid[EDTA]1mg/ml)followedbytwo washingstepsandcentrifugation(250×gfor7min)ofthe leuco-cytesinthepresenceofphosphatebufferedsaline(PBS,0.015M, pH7.4).Cellularconcentrationwasadjustedto1×107cells/ml.A
50-laliquot ofthecellsuspension(5×105cells)wasaddedto
eachculturecontainingRPMImediumanddifferentconcentrations ofEOAFsolubilizedindimethylsulfoxide(DMSO;Sigma–Aldrich Corporation):5×10−3,1×10−2,and2.5×10−2l/ml.Cellcultures
treatedwithsalineonlyorDMSOwereusedasthenegativecontrol andsolventcontrol,respectively.After24hofincubationat37◦C
and5%CO2,theleukocyteswerewashedwithPBS(centrifugation
at250×gfor7min)andresuspendedin50lsaline.A10laliquot ofthecellsuspensionwasincubatedinthepresenceof190lof 0.4%trypanblueforcellcountingwithaNeubauerchamber.For flowcytometryanalysis,trypanbluewasusedataconcentration of0.002%(Avelar-Freitasetal.,2014).Cellviabilitywascalculated asthenumberofviablecellsdividedbythetotalnumberofcells.
InvitroeffectofEOAFontheproductionofcytokinesbyhuman lymphocytes
Bloodsampleswerecollectedinvacuumtubescontaining hep-arin(Vacutainer;BectonDickinson,SanJose,CA,USA),and500-l aliquotswereplacedinpolypropylenetubes(Falcon2059,Becton Dickinson,SanJose,CA,USA).Sampleswereincubatedinthe pres-enceof500lofRPMI-1640medium(Gibco,GrandIsland,NY,USA) and10g/mlBrefeldin(SigmaChemicalCo.,St.Louis,MO,USA)in ahumidCO2incubator(5%CO2)at37◦Cfor4h.
ToanalyzetheeffectofEOAFonthecytokineprofileofleukocyte populations,leukocytecultureswereincubatedinthepresenceof EOAFatconcentrationsof5×10−3and1×10−2l/ml,and
stimu-latedwith25ng/mlPMA(Sigma,St.Louis,MO,USA).PMAactivates leucocytesandinducesproductionofpro-inflammatorycytokines (BishayiandSamanta,2002).Hence,weevaluatedtheinhibitory effectofEOAFoncytokineproductionbyhumanleucocytes.Cells stimulatedwithPMAin combination with8g/ml dexametha-sone(disodiumphosphatedexamethasone–decadroninjection, 4mg/ml,Aché,Brazil)wereusedasaninhibitioncontrol.
0 1000
800
600
400
200
0
200 400
FSC-H
A
B
C
FL1-H
FL2-H FL2-H
FL1-H
SSC-H
Neutrophils
48.56% 17.31%
Lymphocytes
600 800 1000 10100
0
101
102
103
10
100
101
102
103
10
101 102 103 104
100 101 102 103 104
Fig.1. Strategyforflowcytometryanalysis.Sequenceofproceduresusedforanalysisofthepercentageofcellpopulationsbyflowcytometry.Pointdistributiongraph showingthevariables,FSC(size)×SSC(granulosity),usedfortheselectionofthelymphocyteandneutrophilpopulations(A),followedbythepointdistributiongraph, FL1×FL2,usedtoevaluatecytokineexpressioninleucocytes.Thegraphshownin(B)wasobtainedfromaculturestimulatedwithphorbolmyristateacetate(PMA)and labeledwithmonoclonalantibodiesfortumornecrosisfactoralpha(TNF-␣).Thegraphshownin(C)wasobtainedfromaculturestimulatedwithPMAinthepresenceof theessentialoilofAgeratumfastigiatum,alsolabeledwithmonoclonalantibodiesforTNF-␣.
atroomtemperatureprotectedfromlightfor30min.The mon-oclonal antibodieswereobtainedfrom Pharmingen(SanDiego, CA,USA)and wereconjugatedwithphycoerythrin(PE). Follow-ingincubation,thecellswerewashedwithPBS-Wforevaluation ofintracytoplasmiccytokineproductionbyflowcytometry (FAC-Scan–BectonDickinson)atanacquisitionrateof30,000eventsper tube.
ThesoftwareCellQuestTMwasusedfordatacollectionand
anal-ysis.Two-dimensionalplotswithpointdistributionweregenerated toshowtherelationshipbetweenvariablescellsize(FSC)and inter-nalcomplexity(SSC).Basedonthesetwovariables,thelymphocyte andneutrophilcellpopulationswasselectedandpercentageofcells positiveforeachevaluatedcytokinewasobtainedfromthiscell population(Fig.1).
Statisticalanalysis
StatisticalanalysiswasperformedusingtheGraphPadPrism software(GraphPadSoftwareInc.,SanDiego,CA,USA),version5.0. pvalues<0.05wereconsideredstatisticallysignificant.Dataare presentedinthegraphsasmean±standarderror.One-way anal-ysisofvariance(one-wayANOVA)andTukey’sposthoctestwere thestatisticaltestsusedforanalysis.
Resultsanddiscussion
Table1showsthecomponentsidentifiedin theessential oil alongwiththeirretentionindicesandpercentagevalues(relative areasofpeaksinchromatogram).Atotalof26compoundswere identified,correspondingtoapproximately70%oftheoil’s com-position.Fifteenoftheidentifiedcompoundsweresesquiterpenes and11monoterpenes.Theunidentifiedcompoundsdidnotpresent massspectrathatwerecompatiblewiththosefoundinthelibraries (theirmassspectrahadalowsimilarityindex[SI]whencompared tothosefromthemassspectrallibraries).Moreover,their reten-tionindiceswerenotcompatiblewiththosefoundintheliterature orfromwebsites;hence,preciseidentificationofthesecompounds wasnotpossible.
The monoterpenes, limonene, ␣- and -pinene, and the sesquiterpenestrans-caryophyllene, ␣-copaene,and spathulenol were detected in previous studies performed with EOAF ( Del-Vechio-Vieira etal., 2009a,b; Gonc¸alves et al.,2011).However, there were qualitative differences in the monoterpene and sesquiterpenecomponentsofEOAFwhencomparedtothe find-ingsfrompreviousstudies.Thesedifferencesreflectthevariability
Table1
Retentionindex(RI)andrelativeareas(%)ofeachindividualpeak(GC/MS chro-matogram)ofthecompoundsoftheessentialoilfromaerialpartsofAgeratum
fastigiatum.
Compounds RIa RI
calc %b
␣-pinene 939 932 7.5
sabinene 976 971 0.3
-pinene 980 977 0.6
myrcene 991 988 0.2
limonene 1031 1028 5.9
␣-campholenicaldehyde 1127 1126 0.3
trans-pinocarveol 1139 1140 0.3
trans-verbenol 1144 1145 0.5
pinocarvone 1162 1161 0.2
verbenone 1204 1206 0.6
carvone 1242 1243 0.5
␣-copaene 1376 1372 1.0
-elemene 1391 1387 0.6
trans-caryophyllene 1418 1415 2.0
␣-humulene 1454 1451 3.5
␥-muurolene 1477 1471 0.6
␣-curcumane 1483 1478 0.4
␣-muurolene 1499 1495 0.3
␥-cadinene 1513 1509 0.4
spathulenol 1576 1572 0.5
caryophylleneoxide 1581 1577 13.6
humulene1,2-epoxide 1606 1604 8.4
1,6-humulanedien-3-ol 1619 1617 17.7
epi-␣-cadinol 1640 1639 1.2
␣-muurolol 1645 1642 0.5
␣-cadinol 1653 1651 3.4
aRetentionindexesreportedintheliteratureoronwebsites.
bPercentagepeakareaproportionaltothetotalioncurrentwithout standardiza-tion;RIcalc:retentionindexexperimental/calculated.
amongessentialoilsobtainedfromplantsofthesamespeciesdueto genetic,ontogenetic,andenvironmentalfactors(Limaetal.,2003; Gobbo-NetoandLopes,2007).
Given that plant compounds and solvents are external fac-tors capableofinducing celldeath, ourinitialapproach wasto evaluatetheeffectofdifferentconcentrationsofEOAFonthe via-bilityofleucocytes.ResultsshowedthatanEOAF concentration of2.5×10−2l/mlreducedtheviabilityofperipheralblood
leu-cocytesto 87.9±2.9%(Fig.2), whereas EOAF concentrations of 0.5×10−2 and 1
×10−2l/ml did not affect leukocyte viability
(95.6±1.4%and95.1±1.2%,respectively)comparedtothe con-trolgroup(96.5±2.1%).Basedontheseresults,itwasconcluded thatEOAFconcentrationsof0.5×10−2and1
×10−2l/mlwerenot
85 90 95 100
Control DMSO 0.5 x 10–2 1 x 10–2 2.5 x 10–2
Ce
ll viab
ili
ty (%)
EOAF μl/ml
*
Fig.2. Percentageofviableleucocytesasobtainedforthenegativecontrol, sol-ventcontrol(dimethylsulfoxide[DMSO]1%),andforcellculturesincubatedin thepresenceoftheessentialoilofAgeratumfastigiatum(EOAF)at concentra-tionsof0.5×10−2,1
×10−2,and2.5
×10−2l/ml.Theanalysiswasperformed usingthetrypanblueexclusionassaybyflowcytometry.Resultsareexpressed asmean±standarderror.*Significantdifferenceoncomparisonwiththecontrol. Statisticalmethodsused:ANOVAfollowedbyTukey’stest.
usingtheNeubauerchambercellcountswereconsistentwiththose obtainedbyflowcytometry,asdescribedbyAvelar-Freitasetal. (2014).Thevaluesshowninthegraphandmentionedinthetext arerelatedtotheflowcytometryanalysis.
ThepercentageofneutrophilspositiveforTNF-␣wasreduced in the presence of EOAF at concentrations of 0.5×10−2 and
1×10−2l/ml(9.6.
±3.2%and7.3±2.5%,respectively),when com-paredtothecontrolgroup,whichwasonlysubjectedtostimulation withPMA(25.3±3.3%)(Fig.3A).GiventhatNeutrophils(Issekutz, 1981;Tayloretal.,2014)arethefirstcellstoreachthesiteof inflam-mation,inhibitionofthiscytokineleukocytepopulationmeansthe inhibitionof acuteinflammation.Accordingtotheresults,PMA wasnotagoodstimulusforIFNproductioninneutrophilsand pre-ventedassessmentoftheOEAFinhibitoryeffectontheproduction ofthiscytokine(Fig.3B).
Similartoneutrophils,thepercentageoflymphocytespositive forTNF-␣wasreducedinthepresenceofEOAFatconcentrations of0.5×10−2and1
×10−2l/ml(23.28
±3.07%and20.49±4.24%, respectively),whencomparedtothecontrolgroup,whichwasonly subjectedtostimulationwithPMA(40.78±3.46%)(Fig.4A).The compoundspresentintheA.fastigiatumessentialoilhadnoeffect ontheproductionofIFN-␥bylymphocytes,showingthattheeffect oftheessentialoilseemstobemoreefficientontheexpression ofimmunemediatorsobservedintheacuteinflammation,instead chronicprocesses(Fig.4B).
Control PMA DMSO0.5 x 10–21 x 10–2
EOAF μl/ml EOAF μl/ml
Dexa Control PMA DMSO0.5 x 10–21 x 10–2Dexa
PMA 25 ng/ml PMA 25 ng/ml
*
*
% Neutrophils
TNF-α
+
% Neutrophils
IFN-γ
+
0.0 0.2 0.4 0.6 0.8 1.0
0 10 20 30 40
A
B
*
Fig.3.Analysisofthepercentageoftumornecrosisfactoralpha(TNF-␣)-positive(A)andinterferongamma(IFN-␥)-positive(B)neutrophilsinleukocyteculturesstimulated withphorbolmyristateacetate(PMA)inthepresenceorabsenceoftheessentialoilofAgeratumfastigiatumatconcentrationsof5×10−3and1×10−2l/ml.dimethyl sulfoxide(DMSO)1%(1%,v/v)wasusedasthesolventcontrolanddexamethasone(8g/ml)wasusedasaninhibitioncontrolforcytokineproduction.Resultsareexpressed asmean±standarderror.*SignificantdifferenceincomparisonwiththePMAgroup.Statisticalmethodsused:ANOVAfollowedbyTukey’stest.
0 10 20 30 40
0 10 20 30 40 50
*
*
*
*
Control PMA DMSO0.5 x 10–21 x 10–2
EOAF μl/ml EOAF μl/ml
Dexa Control PMA DMSO0.5 x 10–21 x 10–2Dexa
PMA 25 ng/ml PMA 25 ng/ml
% lymphocytes
TNF-α
+
% lymphocytes
IFN-γ
+
A
B
TNF-␣isanimportantmediatorofinflammatoryresponseandis abletopromotetheexpressionofadhesionmoleculeson endothe-lialcells, inducingtherecruitment ofcells totheinflammation site(Braeggeretal.,1992;Wajant,2009;Apostolakietal.,2010). Thus,adecreaseinTNF-␣levelsmayreduceleukocytemigration and leadtoa lower number ofleucocytes atthe infectionsite. Further,TNF induces theexpressionof acute phase proteinsof inflammationsecretedbytheliver,whichenhancesinflammation. Thiseffectcanbebeneficialwhenthere issevereinflammation andinsomeautoimmunediseases.Infact,Del-Vechio-Vieiraetal. (2009a)showedreducedleukocytemigrationtothesiteof inflam-mationinducedbycarrageenaninanimals.Althoughthecytokines werenotevaluatedinthestudyofDel-Vechio-Vieiraetal.(2009a), cytokinemodulationmaybeinvolvedinthiseffect.
Caryophyllene oxide, one of the compounds present in the essentialoil(13.59%),alsoinhibitedpawedemainducedby car-rageenan;inthiscase,cytokineswerenotshowntobeinvolved, but it is possiblethat theyacted by loweringthe permeability oftheendotheliumintheinflamedtissue(Chavanetal.,2010). Othercomponentsoftheoilsuchas␣-pinene(7.51%),limonene (5.9%),trans-caryophyllene(2.04%),and␣-humulene(3.52%) iso-latedfromotherplantsinhibitedtheTNF-␣cytokineindifferent models (Bae et al., 2012; Yoon et al., 2010; Fernandes et al., 2007; Medeiroset al.,2007).Limonenemay interferewiththe p38 signalingpathway (Hirota et al., 2010), whereas the com-poundstrans-caryophylleneand␣-humulenemayinterferewith thenuclearfactor-kappaB(NF-B)pathway(Medeirosetal.,2007). Ourresultssuggestthat partof theanti-inflammatory activ-ity attributedtoEOAF may bedue tothe effectof someof its componentsindecreasingtheproductionofthepro-inflammatory cytokineTNF-␣inactivatedlymphocytesandneutrophils.
Authors’contributions
BAA(PhDstudent)contributedincollectingplantsampleand identification, confection of herbarium, running the laboratory work,analyzingthedataanddraftedthepaperandcontributed tobiologicalstudies.VGA,MGSandJATScontributedtobiological studies.PRB,CFFGandLEGcontributedinplantidentificationand herbariumconfectionandtochromatographicanalysis.ERVand GEBAMdesignedthestudy,supervisedthelaboratoryworkand contributedtocriticalreadingofthemanuscript.Alltheauthors havereadthefinalmanuscriptandapprovedthesubmission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
We are grateful to the Brazilian funding agencies FAPEMIG (APQ-01626-13)andCNPq(Processon.476367/2011-5)andCAPES forfinancialsupport,aswellastotheMulticentricPost-graduate PrograminPhysiologicalScienceandCenterforResearchin Nat-uralandSyntheticProducts,FacultyofPharmaceuticalSciencesof RibeirãoPreto,UniversityofSãoPauloforprovidinginfrastructure toperformtheanalysisinGC–MS.
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