Rev. bras. farmacogn. vol.27 número4

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

Original

Article

Determination

of

phenolic

profile

by

HPLC-ESI-MS/MS

and

anti-inflammatory

activity

of

crude

hydroalcoholic

extract

and

ethyl

acetate

fraction

from

leaves

of

Eugenia

brasiliensis

Diogo

A. Siebert

a

_,

_Juliana

_Bastos

b

_,

_Daniel

_A.

_Spudeit

b

_,

_Gustavo

_A.

_Micke

b

_,

_Michele

_D.

_Alberton

a,∗

a_Laboratório_de_Pesquisa_em_Produtos_Naturais,_Departamento_de_Ciências_{Farmacêuticas,}_Universidade_Regional_de_Blumenau,_Blumenau,_SC,_Brazil b_Laboratório_de_Eletroforese_Capilar,_Departamento_de_Química,_Universidade_Federal_de_Santa_Catarina,_{Florianópolis,}_SC,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3August2016 Accepted31January2017 Availableonline30March2017

Keywords:

Hydroalcoholicextract Flavonoids

Inflammation Pleurisy Nitricoxide

a

b

s

t

r

a

c

t

EugeniabrasiliensisLam.,Myrtaceae,isusedinfolkmedicineforanti-inflammatorydiseasessuchas

arthritisandrheumatism.Thisstudyinvestigatedtheanti-inflammatoryactivityandphenolicprofileof thecrudehydroalcoholicextractandethylacetatefractionfromE.brasiliensisleaves.Crude hydroal-coholicextractandtheethylacetatefractionwereanalyzedbyHPLC-ESI-MS/MSincomparisonto standardphenoliccompounds.Theanti-inflammatoryactivityofthecrudehydroalcoholicextract(1, 10and25mgkg−1₎_and_the_ethyl_acetate_fraction_(10,₂₅_and₅₀_mg_kg−1₎_was_evaluated_in_a_swiss

mousemodelofacutepleurisyinducedbycarrageenan,beingthetotalcellcount,exudationandanalysis ofnitrite/nitratetheinflammationparameters.HPLC-ESI-MS/MSanalysisrevealedapigenin,catechin, galangin,isoquercetin,myricetin,quercetinandrutin.Crudehydroalcoholicextractandethylacetate fractionwereeffectiveininhibitingcellmigrationinalltesteddoses.Crudehydroalcoholicextractwas effectiveininhibitingexudationonlyatthe10mgkg−1_dose;_ethyl_acetate_fraction_was_effective_in_all

testeddoses.Resultsfornitrite/nitratelevelsrevealsthatonlytheethylacetatefractionwaseffective atthetesteddoses.Thisisthefirstreportofthepresenceofisoquercetin,galanginandapigenininthis species.Resultsfromthephytochemicalanalysisenhancethechemicalknowledgeofthisspecies.Inthe future,togetherwithmorestudies,validationofitspopularuseininflammatorydiseasesispossible.

Introduction

Eugenia brasiliensis Lam., Myrtaceae, popularly known in

Brazilas“grumixama”,“grumixameira”and“brazilian-cherry”,is endemictotheAtlanticRainforestin Brazil(Legrandand Klein, 1969). This plant is used in folk medicine as diuretic, for the treatmentofdiarrhea, arthritisand rheumatism (Revilla, 2002). Some articles in the literature have reported biological activi-ties from extracts, essential oil and isolated compounds from this species.The essential oilhaveshown promising antibacte-rialactivityagainstStaphylococcusaureus(Maginaetal.,2009a). Thecrudeextractfromtheleaveshavedemonstratedan interest-ingantioxidantactivity,whichisprobablyrelatedtothephenolic andflavonoidcontent(Maginaetal.,2010), andalsoexertedan antidepressant-likeeffectinthetailsuspensiontestinmice(Colla etal.,2012).Regardingpreviousanti-inflammatorystudies,this

∗ Correspondingauthor.

E-mail:[email protected](M.D.Alberton).

plantdemonstratedeffectinancrotonoil-inducedandarachidonic acid-inducedearedemainmicemodelofinflammation(Pietrovski etal.,2008).Finally,recentworkwithethanolicextractsofleaves, pulp,andseedsofE.brasiliensis,evaluatedthatoraladministration of theseextractsreduced theinvivocarrageenan-induced neu-trophilmigrationby47,41,and50%,respectively(Infanteetal., 2016).

Monoterpenes, such as ␣-pinene and ␤-pinene, oxygenated monoterpenesas␣-terpineol,sesquiterpenesascaryophylleneand oxygenatedsesquiterpenesasspathulenol,␣-cadinoland␶-cadinol havebeenidentifiedintheessentialoil(Fischeretal.,2005;Ramos etal.,2006;Morenoetal.,2007;Limaetal.,2008;Maginaetal., 2009a;Siebertet al.,2015).Someflavonoidssuchasmyricetin, quercetinand rutin,as wellasotherphenolic compoundssuch ascatechin,gallocatechinandgallicacid(Pietrovskietal.,2008), alsotriterpenessuchas␣-amyrin,␤-amyrin,betulin, 29-hydroxy-oleanolicacid,ursolicacid,betulinicacidandoleanolicacidhave beenidentifiedfromtheirleaves(Frighettoetal.,2005;Magina etal.,2012;Limaetal.,2014).Moreover,instudiesregardingthe specie’sfruits,somephenolic compoundssuchasanthocyanins,

http://dx.doi.org/10.1016/j.bjp.2017.01.008

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flavanols,flavonols,ellagitanninsandcarotenoidswereidentified, usingtechniquesthatincludeHPLC-ESI-MS/MS(Reynertsonetal., 2008;Floresetal.,2012;Silvaetal.,2014;Teixeiraetal.,2015).Most ofthesubstancesabovementionedhaveprovenanti-inflammatory andotherbiologicalactivities(DiCarloetal.,1999;Huguetetal., 2000;Maginaetal.,2009b).

ConsideringthepopularuseofthisEugeniaspeciesand previ-ousreportregardingtheanti-inflammatoryactivity,thisstudyaims toinvestigatetheeffectofE.brasiliensisleavesinamousemodel ofpleurisyinducedbycarrageenan.Therefore,weevaluatedthe effectofthecrudehydroalcoholicextract(CHE)andethylacetate fraction(EAF)onleukocytemigration,exudateconcentrationsand nitrate/nitrite(NOx)levels.Moreover,analysisandidentification ofphenoliccompoundsbyhigh-performanceliquid chromatogra-phytandemmassspectrometryusingtheelectrosprayionization (HPLC-ESI-MS/MS)wereperformed.

Materialsandmethods

Plantmaterial

LeavesfromEugeniabrasiliensisLam.,Myrtaceae,werecollected inFlorianópolis,SantaCatarinastate(27◦₃₆′_13.65′′_S,₄₈◦₃₁′_14.75′′ W),in March2012.Plant material wasidentifiedby Dr.Daniel deBarcellosFalkenbergfromtheBotanyDepartmentof Univer-sidadeFederaldeSantaCatarina(UFSC),andavoucherspecimen wasdepositedintheherbariumFLORofthesameinstitutionunder registrynumber34675.

PreparationoftheCHE

Theplantmaterialwasdriedandmilled,totaling1813gof mate-rial.Thismaterialwasmaceratedinhydroalcoholicsolution(92.8%, ww−1₎_for_seven_days._The_extract_was_filtered_and_solvent evapo-ratedinrotatoryevaporator(below60◦_C)_coupled_with_a_vacuum condenser,andconcentratedtoareducedvolume.Aftertotal evap-orationofthesolvent,192.5gofcrudeextractwasobtained,which representayieldof10.62%ofplantmaterial.

PreparationofEAFfraction

Analiquot of117gfromtheCHEwasresuspendedin water andthemixturewasstoredunderrefrigeration(2–8◦_C)_over_night andfiltered.Theaqueoussolutionwasdefattedbywashingwith dichloromethane. Thus, ethyl acetate fractionwas prepared by liquid-liquidpartition,yielding23.87g.

DeterminationofphenoliccompoundsintheCHEextractandEAF fractionofEugeniabrasiliensisbyHPLC-ESI-MS/MS

TheCHEand theEAFwere analyzedby HPLC-ESI-MS/MS in LabEC/INCT-Catalise(UFSC).AnalysiswereconductedinaAgilent® 1200(AgilentTechnologies,Germany)liquidchromatograph sys-tem, with a Phenomenex Synergi®

4␮ Polar-RP 80A column (150mm×2mm,particlesizeof4␮m)atthetemperatureof30◦C.

TheeluentswereformedbymixingsolventsA(H2O+0.1%formic acid)andB(acetonitrile/H2O,95:5)asfollows:1ststage–80% sol-ventA(isocraticmode)for10min;2nd_stage_–_linear_gradient_of solventsAandB(from80to5%ofA)for15min;3rd_stage_–_5% solventAand95%B(isocraticmode)for5minwithaflowrateof 200␮lmin−1_of_mobile_phase._In_all_analyses,_the_injected_volume was5␮l.

Theliquidchromatographwascoupledtoamassspectrometry systemconsistingofahybridtriplequadrupole/lineariontrapmass spectrometerQtrap®

3200(AppliedBiosystems/MDSSCIEX,USA) withTurboIonSpray®

asionizationsource,inpositiveionization

mode,withthefollowingsourceparameters:ionsprayinterface at400◦_C;_ion_spray_voltage_of₄₅₀₀_V;_curtain_gas,₁₀_psi;_nebulizer gas,45psi;auxiliarygas,45psi;collisiongas,medium.TheAnalyst® (version1.5.1)softwarewasusedforrecordingandprocessingthe data.PairsofionsweremonitoredinMRM(MultipleReaction Mon-itoring)mode.

For the identification of the compounds, 29 standard phe-nolic compounds (4-methylumbelliferone, apigenin, apigenin 7-glucoside, aromadendrine, caffeic acid, catechin, chlorogenic acid,cinnamicacid, coniferaldehyde,coumarin,epicatechin, fer-ulicacid,galangin,hispidulin,isoquercetin,kaempferol,luteolin, myricetin,naringin,p-coumaricacid,quercetin,resveratrol,rutin, scopoletin,sinapaldehyde, sinapicacid, syringaldehyde,syringic acid,vanillicacid)dissolvedinmethanol(1mgl−1₎_were_analyzed insameconditionsasdescribedabove(Bertinetal.,2014).

Evaluationofanti-inflammatoryactivityofEugeniabrasiliensis

Animals

Experiments were carried out using 30-day-old Swiss mice of both sexes(18–25g). Animalswerekept atcontrolledroom temperature(22±3◦_C)_under_a₁₂_h_light-dark_cycle,_with_access towater and foodadlibitum.Allprocedures usedin thestudy wereapprovedbytheEthicsCommitteeonAnimalUse(protocol number008/12)fromUniversidadeRegionaldeBlumenau(FURB). Thenumberofanimals(n=5–6)wastheminimumnecessaryto demonstratetheconsistenteffectsoftreatment.

Carrageenan-inducedpleurisy

The pleurisy procedure was performed according to the methodologydescribedbySalehetal.(1999,1996).Accordingto theexperimentalprotocol,differentgroupsofanimalswere pre-treated(30min)withdifferentconcentrationsoftheCHE(1,10 and25mgkg−1₎_and_the_EAF_(10,₂₅_and₅₀_mg_kg−1_),_administered intraperitoneally(i.p.).

Then,thepleurisywasinducedbyadministrationof0.1mlof gradeIV␭carrageenan(Cg)(1%)intherightpleuralcavity(i.pl.). After4h,theanimalsweresacrificedwithanoverdoseof pento-barbitalandthepleuralcavityexposedandwashedwith1mlof asolutionofheparin(20IUml−1₎_diluted_with_phosphate_buffered saline(PBS,pH7.6).Fortheindirectquantificationofexudation, 10minaftertheadministrationofCg,theanimalsreceived0.2ml ofEvansbluedyesolutionat25mgkg−1_{intraorbitally}₍_i.o_.)._To ver-ifythelevelsofnitrite/nitrate(NOx),groupsofanimalswerecarried outseparately,withthebestdoseofeachtestsamplewithoutthe administrationofEvansbluedyeinordertoavoidinterferencewith theresults.Theexperimentswerealwaysaccompaniedbypositive controlgroups(animalsthatreceivedonlyCg1%,0.1ml,i.pl.)and negativecontrolgroups(animalsthatreceivedonly0.1mlsaline i.pl.).Inaddition,experimentswereperformedusing dexametha-sone(4mgkg−1_,_i.p_.)_as_{anti-inflammatory}_reference_drug.

Totalanddifferentialleukocytecount

Totalcell counts wereperformed in Neubauer chambersby meansofanopticalmicroscope(magnification400×)after

dilut-ingasampleofthecollectedfluidfromthepleuralspacewithTürk solution(1:200). Cellularsmearswerepreparedwithanaliquot ofpleurallavagetodeterminethedifferentialleukocytecount.The slideswerestainedwithMay–Grünwald–Giemsadye,andthe anal-ysiswascarriedoutunderoilimmersionobjective(Salehetal., 1996,1999).

Indirectquantificationofexudation

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Table1

PhenoliccompoundsidentifiedinEugeniabrasiliensisbyHPLC-ESI-MS/MS.

Compounds Rt(min) Calculatedmass(M) Experimentalmass[M+H]+ _MS/MS₍_m_/_z₎

1 Catechin 10.94 290.26 291.1 139.0

2 Myricetin 11.85 318.24 319.1 153.0

3 Isoquercetin 12.34 464.38 465.1 303.1

4 Rutin 12.46 610.52 611.1 303.1

5 Quercetin 12.47 302.23 303.0 153.1

6 Galangin 17.35 270.24 271.1 77.1

7 Apigenin 17.79 270.24 271.0 153.1

curveofthedyeintherangeof0.05–50␮gml−1_,_according_to_the

methodologydescribedbySalehetal.(1996,1999).Resultswere expressedas␮gml−1_.

Quantitativeanalysisofnitrite/nitrate(NOx)

Toverifythelevelsofnitrite/nitrate(NOx),groupsofanimals withthebestdoseofeachtestsamplewithouttheadministration ofEvansbluedyewereperformedtoavoidinterferencewiththe results.Basedontheresultsofcellmigrationandexudation,were testedtheCHEatthedoseof10mgkg−1_,_and_the_EAF_at_the_dose of10mgkg−1_.

Nitricoxide(NO)wasmeasuredindirectlybyitsbreakdown productsnitrite(NO2−)andnitrate(NO3−),usingtheGriess reac-tion (Green et al., 1982), and the pre-processing proposed by (Miranda etal.,2001).Nitriteconcentrationswereestimatedby interpolationfromastandardcurveofsodiumnitrite(0–150mM) bycolorimetricmeasurementsat540nminanELISAplatereader. ResultswereexpressedasmM.

Statisticalanalysis

Statisticalanalysis of theresultsof anti-inflammatory activ-ity,wereconductedbytheANOVAparametrictestsupplemented whennecessarybyDunnett’stest,orttest(nonparametrictest)by meansoftheGraphPadPRISM®

(version3.0)statisticalsoftware.p values<0.05wereconsideredsignificant.

Resultsanddiscussion

DeterminationofphenoliccompoundsintheCHEandEAF fractionofEugeniabrasiliensis

Inordertorecordaprofileofphenoliccompoundswhichare presentintheCHEandEAF,thesesampleswereanalyzedbythe hyphenatetechniqueHPLC-ESI-MS/MS,thatcombinesthe versatil-ityoftheliquidchromatographyandthesensibilityandspecificity ofatandemmassspectrometer,thereforebeingextremelyreliable (Wuetal.,2013).

Thefingerprintof theextractsand fractionsof E.brasiliensis ledtotheidentification ofphenolic compounds based ontheir molecularformula,fragmentation patternand comparison with theretentiontimesof standardscommerciallyavailable.In this method,thesampleswerecomparedqualitativelywithstandards of29phenoliccompounds.Table1summarizesthesevenphenolic compoundsidentifiedinCHEorEAFfraction,theirretentiontime, protonation[M+H]+_and_molecular_weight.

Theresultsrevealedthepresenceoftheflavonoidscatechin(1),

myricetin(2),isoquercetin(3),rutin(4),quercetin(5)andgalangin (6)intheCHE.IntheEAF,thecompoundscatechin(1),isoquercetin (3),galangin(6)andapigenin(7)havebeenidentified.

O

OH HO

O R4

R1

R2

R3

O

OH HO

OH OH

OH

1 2_R₁_=R₂_=R₃_=R₄_=OH

3 R₁=R₂=OH; R₃=H; R₄=O_-glu 4_R₁_=R₂_{=OH; R}₃_{=H; R}₄₌O_-rut 5_R₁_=R₂_{=OH; R}₃_{=H; R}₄_=OH 6_R₁_=R₂_=R₃_{=H; R}₄_=OH 7 R1=R3=R4=H; R2=OH

Althoughsomeofthesubstancesidentifiedhavealreadybeen describedintheleavesofthisspecies,likecatechin,myricetin,rutin andquercetin,thisisthefirstreportofthepresenceofisoquercetin, galanginandapigenininE.brasiliensis.

Anti-inflammatoryactivityofCHEandEAFfractionfromEugenia brasiliensis

The resultsfor cell migration and exudation (Figs. 1 and 2) showedthat theCHEwaseffective ininhibiting cellmigration, whichincreaseswithhigherdoses,alongwiththeEAF,however withthesame intensity(no significantdifference) at alltested doses. CHE inhibited 43±12% (10mgkg−1 _dose) _and ₅₈_±_11% (25mgkg−1 _dose), _and _the _EAF _inhibited ₄₃_±_5% ₍₁₀_mg_kg−1 dose), 43±12% (25mgkg−1 dose) and 60±11% (50mgkg−1)

the total cell count, compared with the non-treated group (p<0.001).

Onexudationlevels,CHEwaseffectiveininhibitingthemonly atthe10mgkg−1_dose₍₅₅_±_10%),_and_the_EAF_inhibited exuda-tionsignificantlyinalltesteddoses(38±9%,10mgkg−1_;₄₆_±_7%, 25mgkg−1_; ₄₂_±_9%, ₅₀_mg_kg−1 _dose) _compared _with_the non-treatedgroup(p<0.001).

TheresultsforNOxlevels(Fig.3)revealsthatEAFwaseffectivein inhibitingtheproductionofthisinflammatorymediator(76±19%)

even more than the standard drug, dexamethasone(41±25%).

However,theCHEwasnotabletoinhibitsignificantlythe produc-tionofNOinthepleuralcavity,atthetesteddose.

Although pleurisy inducedby carrageenan is not anasthma model,theadministrationofcarrageenanintothepleuralcavity providesaninflammatoryenvironmentsimilartothatobservedin patientswithneutrophilicasthmaphenotype.Thesampleswere evaluatedontheearlyphaseofinflammation(4hafter administra-tionofcarrageenan1%),whichischaracterizedbyanenhancement inthetotal numberofcells (dueprimarilytoneutrophilinflux, rather than mononuclear)exudationand increase ofNO (Saleh etal.,1996).

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A

6

5

4

3

2

1

0

100

75

50

25

0 S

S Cg Dexa

Monon

uclear

, %

1 10 25

Cg Dexa

T

otal leucocyte (x10

6)

∗∗∗

∗∗ ∗∗∗ ∗∗∗

∗∗

1 10 25

CHE Eugenia brasiliensis (mg kg–1, i.p.)

C

100

75

50

25

0

12

10

8

6

4

2

0

P

olymor

phon

uclear

, %

Ev

ans b

lue dy

e (

µ

g ml

–1)

∗∗∗

∗∗∗ ∗∗

∗∗∗

S

S Cg Dexa 1 10 25

Cg Dexa 1 10 25

CHE Eugenia brasiliensis (mg kg–1, i.p.)

D

B

Fig.1.EffectofcrudehydroalcoholicextractofEugeniabrasiliensis(1,10,25mgkg−1_,_i.p_.)_on_the_number_of_total_leukocytes_(A),_percentage_of_{polymorphonuclear}_cells_(B),

percentageofmononuclearcells(C)andexudation(D)onthepleuralcavityinthemousemodelofpleurisy(4h)inducedbycarrageenan.Controlanimalsweretreatedonly withsaline(S),carrageenan(0.1mlof2%,i.pl.)(Cg)anddexamethasone(4mgkg−1_,_i.p_.)_(Dexa)._Each_group_represents_the_mean_of_4–6_animals._***_p_<_0.001;_**_p_<_0.01_and

*p<0.05.

6

A

C

B

D

100

75

50

25

0 5

4

3

2

1

0

100 _10.0

7.5

5.0

2.5

0.0 75

50

25

0

S _S

S Cg Dexa 10 25 50 S Cg Dexa 10 25 50

Cg Dexa _Cg

T

otal leucocyte (x10

6)

P

olymor

phon

uclear

, %

Monon

uclear

, %

Ev

ans b

lue dy

e (

µ

g ml

–1)

EAF Eugenia brasiliensis (mg kg–1_{, i.p.)}

EAF Eugenia brasiliensis

(mg kg–1_{, i.p.)} EAF Eugenia brasiliensis

(mg kg–1_{, i.p.)}

EAF Eugenia brasiliensis (mg kg–1_{, i.p.)} 10

∗∗∗

∗∗∗ ∗∗∗

∗

∗∗∗ ∗∗∗ ∗∗∗

∗∗∗ ∗∗∗ ∗

∗∗∗

25 50 _Dexa ₁₀ ₂₅ ₅₀

Fig.2. EffectofethylacetatefractionofEugeniabrasiliensis(10,25and50mgkg−1_,_i.p_.)_on_the_number_of_total_leukocytes_(A),_percentage_of_{polymorphonuclear}_cells_(B),

percentageofmononuclearcells(C)andexudation(D)onthepleuralcavityinthemousemodelofpleurisy(4h)inducedbycarrageenan.Controlanimalsweretreatedonly withsaline(S),carrageenan(0.1mlat2%,i.pl.)(Cg)anddexamethasone(4mgkg−1_,_i.p_.)_(Dexa)._Each_group_represents_the_mean_of_4–6_animals._***_p_<_0.001;_**_p_<_0.01_and

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18

15

12

9

6

3

0

S Cg

NO

x

(mM)

Dexa ∗∗

∗∗∗

CHE EAF

E. brasiliensis

Fig.3.Effectofcrudehydroalcoholicextract(10mgkg−1_,_i.p_.)_and_ethyl_acetate

fractionofEugeniabrasiliensis(10mgkg−1_,_i.p_.)_on_NO

xlevelsinthepleuralcavity

inthemousemodelofpleurisy(4h)inducedbycarrageenan.Controlanimalswere treatedonlywithsaline(S),carrageenan(0.1mlat2%,i.pl.)(Cg)anddexamethasone (4mgkg−1_,_i.p_.)_(Dexa)._Each_group_represents_the_mean_of_4–6_animals._***_p_<_0.001;

**p<0.01and*p<0.05.

NOproduction.Theseresultscorroboratewithstudyfromthesame speciesusingthecrotonoil-inducedandarachidonicacid-induced earedemainmice(Pietrovskietal.,2008),thatalsoobserveda promisingtopicalanti-inflammatoryactivityfromthe hidroalco-holicextract,hexane,dichloromethaneandethylacetatefractions, aswellastheisolatedcompoundsquercetin,catechinand gallocat-echin.Also,stayinagreementwithstudybyInfanteetal.(2016), whichshowedthatethanolicextractsofleaves,pulp,andseedsof E.brasiliensis,reducedtheinvivocarrageenan-inducedneutrophil migration.ExtractsfromotherspeciesoftheEugeniagenushave alreadydemonstratedanti-inflammatoryeffect,thereforeagreeing withthepresentstudy(Slowingetal.,1994;Ramirezetal.,2012; Bastingetal.,2014;Soaresetal.,2014).

Nitric oxide is a soluble gas produced from enzymescalled nitricoxidesynthases(NOS).ThreeNOSisoformsare described (NathanandXie,1994),ofwhichtwoareconstitutive(cNOS)and expressedunderphysiologicalconditions,andanotherisinduced (iNOS).Anumberofagents,suchasinflammatorycytokinesand oxidants,inducetheirexpressionindifferentcelltypessuchas macrophages(MorrisandBilliar,1994),neutrophils(Eiserichetal., 1998),mononuclearcells(Salveminietal.,1989),eosinophilsand vascularsmoothmusclecells(Guoetal.,1995).

IncreaseofNOinthecarrageenan-inducedpleurisymodelin micewaspreviouslydemonstratedandsuggeststheinductionof cNOSand iNOSenzymesin thepleural cavity cells ofinflamed animals.ThefactthatNOconcentrationsarenotrelatedeitherto neutrophilsormononuclearcellsisindicativethatthiscompound comesfrom differentsources besidesleukocytes (Barneset al., 1998).Theexudationlevelsaredirectlylinkedtotheincreasein theamountofNOproduced,andthereleasedNOplaysan impor-tantroleasvasodilatorandcontributestoexudation(Luzetal., 2016).

Manyphenoliccompoundsarepresentintheextractand frac-tionoftheplant,asevidencedbyHPLC-ESI-MS/MSanalysis.The anti-inflammatoryeffectofphenoliccompounds,suchasapigenin, quercetinandmyricetinaswellastheircapabilitytoinhibitNO productionfrommacrophages,isknownforsometime(Formica andRegelson,1995;Kimetal.,1999).However,thepossibilitythat otherclassesofcompoundsarecontributingtotheobservedresult, cannotbeexcluded.

Similar results with anti-inflammatory activity of phenolic compoundswereobservedbyZhuetal.(2009),whichshoweda sig-nificantinhibitionofleukocyteinfiltrationintotheinflammatory site in the air pouch model with N -formylmethionyl-leucyl-phenylalanine bacterial in mice when they were treated with catechins extracted from green tea (Camellia sinensis). Further-more, Wanget al.(2011) foundthat theextract enriched with greenteacatechinswaseffectiveinreducingpawedemainduced bycarrageenaninrats,andtheearedemainducedbyxylenein mice.Furthermore,quercetin,anotheridentifiedcompoundfromE. brasiliensis,haspreviouslydemonstratedanti-inflammatoryeffect in work conducted by Morikawa et al. (2003), when a signifi-cantlydecreaseinthetotalnumberofleukocytesandexudation, in the air pouch model induced by carrageenan in rats, was showed.

Another study evaluated the anti-inflammatory effect of quercetin using other models of inflammation related to atherosclerosis,suggestingapromisinganti-inflammatory activ-ity,thereforeagreeingwiththisstudy(Kleemannetal.,2011).In arecentreviewarticle(Leihereretal.,2013),theauthorslisteda numberofactivities,amongthemanti-inflammatory,forvarious phenolic compounds,includingcatechinsandquercetin(Suzuki etal.,2007;Qureshietal.,2011a;Yoonetal.,2011;AbdEl-Azizetal., 2012).Moreover,rutindemonstrated anti-inflammatoryactivity inamodelofintestinaloxidativedamageinducedby methotrex-atein ratsandwasalso abletoinhibit75.6, 81.0 and 80.4%of cyclooxygenase-1and2,and15-lipoxygenaserespectivelyinvitro (Gautametal.,2016).

PreviousworkdemonstratedasignificantreductioninNO lev-elsinembryonicmyogenicculturefromcellsoftheratheart,when theyweretreatedwiththeflavonoidquercetinbeforethe inflam-matory stimuli inducted with bacterial lipopolyssacaride (LPS) (Angeloni and Hrelia, 2012). Moreover, theprevious treatment withquercetindiminishedtheNOproductioninLPS-stimulated macrophages (Qureshi etal., 2011b).Theflavanolcatechin was alsoefficienttoreduceNOplasmaticlevelsintumorangiogenesis inducedmice,anddiminishtheNOproductioninLPS-stimulated macrophages(GuruvayoorappanandKuttan,2008).Morerecently, researchersdemonstratedthatleafextractsfromKorean

Chrysan-themum species, thathave in theircomposition many phenolic

compounds suchasapigenin,dose-dependentlysuppressedNO productionstimulatedbyLPS,inhibitedproductionofLPS-induced PGE2 and reducedtheLPS-inducedexpressions ofinducibleNO synthaseandcyclooxygenase-2(Kimetal.,2015).

Inanotherstudy,galanginwasabledecreaseNOproduction, reducemessengerRNAlevelsofcytokines,includingIL-1␤andIL-6, andproinflammatorygenes,suchasinduciblenitricoxidesynthase (iNOS),inadose-dependentmanner,decreasedtheprotein expres-sionlevelsofiNOSandinhibitIL-1␤productioninLPS-stimulated RAW264.7murinemacrophages.Galanginwasfoundtoelicit anti-inflammatory effectsbyinhibiting extracellularsignal-regulated kinasesandNF-␬B-p65phosphorylation(Jungetal.,2014).

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Conclusions

Resultsfromthephytochemicalstudystayinagreementwith theliteraturetotheEugeniagenus,beingthefirsteverreportof thepresenceofisoquercetin,galanginandapigenininthisspecies, enhancingthechemicalknowledgeofthisspecies.

OurresultsshownthatCHEandEAFfromE.brasiliensiswere effectiveininhibitingleucocytesmigration totheinflammatory site, and this effect was due to ability of this plant to inhibit theneutrophilinflux.Theothersignalofinflammationevaluated, theexudate,wasalsosignificantlyreducedbytreatmentwithE. brasiliensis.Theseresults,atleastfortheEAFfraction,aredirectly relatedwiththereductionofNOlevelsinthepleuralsamplesfrom treatedanimalswhichsuggestananti-inflammatoryactivityinthe modeltested.Thiseffectmaybe,atleastinpart,duetothepresence ofthephenoliccompoundsidentifiedbyHPLC-ESI-MS/MS. Further-more,thisfactaidsthevalidationoftheplantuseinfolkmedicine wereitis already beingusedinthetreatmentofinflammatory diseases.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata

appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Authors’contributions

DAS(MScstudent)collectedplantsample,performedthe exper-iments,interpretedtheresultsanddraftedthemanuscript.JBand DASconductedtheHPLC-ESI-MS/MSanalysis.GAMsupervisedthe HPLC-ESI-MS/MSanalysisandlaboratorywork.MDAdesignedthe study,supervisedthelaboratorywork,interpretedtheresultsand contributedtocriticalreadingofthemanuscript.Alltheauthors havereadthefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

The authors are grateful to CAPES, FURB, UFSC and INCT – Cataliseforthefinancialandtechnicalsupport.

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