Rev. bras. farmacogn. vol.26 número3

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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

Phytochemical

study

and

anti-inflammatory

and

antioxidant

potential

of

Spondias

mombin

leaves

Bárbara

Cabral

a

_,

_Emerson

_M.S.

_Siqueira

a

_,

_Mariana

_A.O.

_Bitencourt

b

_,

_Maíra

_C.J.S.

_Lima

b

_,

_Ana

_K.

_Lima

c

_,

Caroline

F. Ortmann

d

_,

_Vitor

_C.

_Chaves

d

_,

_Matheus

_F.

_{Fernandes-Pedrosa}

b

_,

_Hugo

_A.O.

_Rocha

c

_,

Katia

C. Scortecci

c

_,

_Flávio

_H.

_Reginatto

d

_,

_Raquel

_B.

_Giordani

a

_,

_Silvana

_M.

_Zucolotto

a,∗ a_{Pharmacognosy}_Laboratory,_Department_of_Pharmacy,_Universidade_Federal_do_Rio_Grande_do_Norte,_Natal,_RN,_Brazil

b_Technology_and_{Pharmaceutical}_{Biotechnology}_Laboratory,_Programa_de_{Pós-graduac¸}_ão_em_Ciências_{Farmacêuticas,}_Universidade_Federal_do_Rio_Grande_do_Norte,_Natal,_RN,_Brazil c_Natural_Polymers_{Biotechnology}_Laboratory,_Department_of_{Biochemistry,}_Centro_de_{Biociências,}_Universidade_Federal_do_Rio_Grande_do_Norte,_Natal,_RN,_Brazil

d_{Pharmacognosy}_Laboratory,_Programa_de_{Pós-graduac¸}_ão_em_Farmácia,_Centro_de_Ciências_da_Saúde,_Universidade_Federal_de_Santa_Catarina,_Campus_{Universitário}_–_Trindade, Florianópolis,SC,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20October2015

Accepted12January2016

Availableonline15March2016

Keywords: Spondiasmombin

Chemicalmarker

Anti-inflammatoryandantioxidant

a

b

s

t

r

a

c

t

SpondiasmombinL.,Anacardiaceae,isaplantnativeofBrazil,whereitisknownas“cajá”.Inorderto

findapotentialapplicationforthisnativespecies,theanti-inflammatoryandantioxidanteffectswere

investigated.Theanti-inflammatoryactivitywasevaluatedusingtheinvivomodelcarrageenan-induced

peritonitisinmice.Theinvitroantioxidantpotentialaswellthecytotoxicityagainst3T3fibroblastcells

alsowereevaluated.ThroughHighPerformanceLiquidChromatography-diodearraydetectoranalysis,

ananalyticmethodwasdevelopedandvalidated.Itallowedtheidentificationandquantificationof

ellagicacidandchlorogenicacidinhydroethanolicextractofS.mombinleaves.Thisextractshowed

anti-inflammatoryeffectat100,200,300and500mg/kg,however,theethylacetatefraction,at200mg/kg,

showedthehighlightedresults.Ellagicacidandchlorogenicacid(2.5,5and10mg/kg)alsoinhibitedthe

leukocytemigrationtothesiteofinflammation.Theextract,fractionsandcompoundsshowedsignificant

antioxidantpotentialwhenevaluatedindifferentassays.Theresultsshowninthisworksuggestthe

anti-inflammatorypotentialoftheleafextractofS.mombimonperitonitismodelinducedbycarrageenan,

itwasalsoobservedantioxidantpropertiesassociatedwithanabsenceofcytotoxicityincellculture.

Furtherinvivostudiesarerequiredtoconfirmtheanti-inflammatoriesactionofS.mombinanditspossible

anti-inflammatorymechanismsofaction.

Introduction

Theethnopharmacologyassociatedwiththechemicalstudyhas becomeanimportanttoolinbioprospecting.Studieshave associ-atedinformationonthetraditionaluseofmedicinalplantswith phytochemical and pharmacological studies, searching for new drugsandherbalmedicines(Medeirosetal.,2013).Brazilhasoneof theworld’shighestlevelsofbiodiversity,includingseveralplantsof economicinterest(Albuquerqueetal.,2007).TheNorthand North-eastregionsarethosewheremostoftheexistingbiodiversityis concentrated,whichallowsaccesstonumerouskindsofplantsand fruitspecie(Mattiettoetal.,2010).

∗ _{Corresponding}_author.

E-mail:silvanazucolotto@ufrnet.br(S.M.Zucolotto).

SpondiasmombinL.isaplantbelongingtotheAnacardiaceae family.ThisspeciesisnativeofBrazilandtheirfruitsarecommonly knownas“cajá”.Itoccurswidespreadthroughtropicalregionsin America,AfricaandAsia.InBrazil,itismainlyfoundintheNorth andNortheast(Soares,2005).Thisnativefruithaspotentialuse forprocessingtomakejelly,juice,jamsandicecreammainlyin northeasternBrazilandtheirleavesareusedinfolkmedicinefor thetreatmentofseveraltopicandsystemicdiseaseslike inflam-mationofthemouth and throatand incases ofprostatitis and herpeslabialis(LorenziandMatos,2008).Despitethis therapeu-ticapproach,thechemicalstudiesarescarceandsomephenolic acids,flavonoids,tanninsandtriterpeneshavebeenisolatedfrom

S. mombinleaves so far(Corthout et al., 1991; Fred-Jaiyesimia etal.,2009).Asurveyoftherelevantliteraturerevealedthatthe leavesof S.mombinexhibit antimicrobial,leishmanicide, antivi-ral,antiedematogenic, hypoglycemicand antioxidantproperties

(Corthoutetal.,1994;Fred-Jaiyesimiaetal.,2009;Nworuetal.,

http://dx.doi.org/10.1016/j.bjp.2016.02.002

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2011;Silvaetal.,2011,2012).Regardingtheanti-inflammatory effectonlyonereportsupportitspopularuse(Nworuetal.,2011). Thechemicalcharacterizationoftheleafextractfromthisspecies aswellastheidentificationofchemicalmarkersthatcouldbe use-fulinextractstandardizationforapplyingitinherbaldrugswasnot developed.Inaddition,itisimportanttohighlightthattheextract standardizationenableustoguaranteereliableresultsregarding biologicalactivities.

Takentogether,theaimsofthisstudywere:(i)todevelopan analyticmethodologybyHPLC-DADin ordertoquantify chemi-calmarkersinextractfromS.mombinleavesand(ii)toevaluate theiranti-inflammatorypotentialinvivomodel,(iii)toinvestigate theantioxidantpotentialsincetheoxidativestressisan impor-tantaspectassociatedtoinflammationand(iv)andtoevaluatethe cytotoxicityagainst3T3cells.

Materialsandmethods

Phytochemicalprocedures

Plantmaterialandextraction

LeavesofSpondiasmombinL.,Anacardiaceae,werecollectedin DomMarcolinoDantas,RioGrandedoNorte,Brazil(SS-RN◦₂₈′₁₄′′

W35◦₂₇′₃₇′′_),_on_November_2011._The_plant_material_was_identified

byAlandeAraújoRoqueandavoucherspecimenwasdeposited atHerbariumofFederalUniversityofRioGrandedoNorte,Brazil, underthereferencenumber12252.S.mombinleavesweredriedat roomtemperatureandtrituratedmechanically.Theextractfrom

S.mombinleaveswaspreparedbymacerationinethanol:water (70:30,v/v),forsevendays,filteredandlaterlyophilized.Itwas obtainedthehidroethanolicextractfromS.mombinleaves(HE).

InordertocharacterizetheactivecompoundsfromHE,aportion oftheextractwasresuspendedinwaterandsubjectedto liquid-liquidpartitionwithsolventsofincreasingpolarity:hexane(Hex) (3×300ml),dichloromethane(DCM) (3× 300ml),ethylacetate (EtOAc)(3×300ml)andbutanol(ButOH)(3×300ml).Five frac-tionswereobtained(Hex,DCM,EtOAc,ButOH)andanaqueous residualfraction(ARF).Allfractionsweredriedunderreduced pres-sureat45◦_C.

Chromatographicprocedures

Theanalyticalstandardswerepurchasedfrom:chlorogenicacid hemihydrate(Sigma–Aldrich®_,

≥98%),ellagicacid(Roth®_,

≥98%) andisoquercitrin(HwiAnalytikGmbH®

,94.16%).Thesolventsused inextractpreparationwereAnalyticalGradeandinHPLCanalysis wereusedHPLCgradesolvents.Thewaterwaspurifiedwitha Milli-Qsystem(Millipore®_,_Bedford, _USA)._All _solutions_prepared _for

HPLCanalysiswerefilteredthrougha0.45mmmembranebefore use.

Quantitativehigh-performanceliquidchromatographyanalysis

Thequantificationofthephenoliccompoundswasperformedin ahighperformanceliquidchromatography(HPLC)usingaPerkin Elmer200seriesequippedwitha diodearraydetector(DAD),a quaternarypump,adegasseronlineandanautosampler.AllHPLC datawereprocessedusingtheTotalChrom®

Workstationsoftware. Chromatographicanalyzeswereperformedusingareversedphase column(Phenomenex®₎_C-18_(4.6_mm

×250mm,5␮m).An elu-tionsystemwithacetonitrile100%(solventA)andaceticacid1%, adjustedtopH3(solventB)atthefollowgradientflowwasused: 0–30min,alinearchangefromA:B(13:87,v/v)toA:B(15:85,v/v); 30–40min,anisocraticelutionwithA:B(15:85,v/v);40–45min, alinearvariationuptoA:B(20:80,v/v).Theflowratewaskept constantat1ml/min,andthechromatogramswererecordedat 340nm,whereastheUVspectraweremonitoredatwavelength of450–200nm.Thepeakswerecharacterizedbycomparisonof

theirretentiontimesandUVspectrawiththereferencestandards andbyaco-injection(extract+referencestandard).Thereference standardsolutions werepreparedinH2O:methanol(3:2,v/v)at

differentconcentrations: chlorogenicacid– 10, 20,30, 40, and 50␮g/ml;ellagicacid–20,30,40,50,and60␮g/ml.TheHEfromS. mombinleaveswasanalyzedat2.5mg/ml.Thereferencestandards wereanalyzedintriplicateandtheaveragepeakareasmeasured.

Validationofanalyticalprocedure

Thevalidationofanalyticalprocedureswasperformedin accor-dancewithICHguidelines(ICH,2005).Thevalidatedparameters werespecificity, linearity,accuracy,precision(repeatabilityand intermediateprecision),limitofquantification(LOQ)andlimitof detection(LOD).

Pharmacologicalassays

Anti-inflammatoryactivity

Animals. Experiments werecarriedout usingmale BALB/cmice (6–8weeksold).Allmicewerehoused, 5–6percageata room temperatureof22±2◦_C,_and_a₁₂_h:12_h_light/dark_cycle,_with_ad libitumaccesstowaterandfood.Groupsofsixanimalswereusedin eachtestgroupandcontrolanimalsreceivedsalineonly.Allinvivo

experimentswereapprovedbytheethicalcommitteeofthe Fed-eralUniversityofRio Grandedo Norte,Brazil(protocolnumber 013/2013)andwerecarriedoutaccordingtothecurrentguidelines forlaboratoryanimalcare.Eachanimalwasusedonlyonce.

Carrageenan-inducedperitonitis. Theinflammationwasinducedin micesbyadministrationof1%carrageenan,intraperitoneally(i.p.). The animals(N=5)werei.p.pretreated with(i)vehicle(saline, 0.1ml/10g)or(ii)dexamethasone(0.5mg/kg)or(iii)HEfromS. mombinleaves(100,200,300or500mg/kg)or(iv)fractionsDCM, EtOAc,ButOHand ARF (200mg/kg) or(v) chlorogenicacidand ellagicacid(2.5,5or10mg/kg).Thirtyminutesaftertreatmentwith thesamples,theanimalsreceivedaninjectionintotheperitoneal cavityof50␮lof1%carrageenan.After4h,theanimalswere sac-rificedwithanoverdoseofthiopentalat80mg/kg,i.p.,followedby cervicaldislocation.Peritonealexudateswerecollectedwith2ml ofice-coldPBSforabdominallaparoscopy.Theexudateswere cen-trifugedat250×gfor5minat4◦_C._The_cell_pellet_was_diluted_1:10

inTürksolutionandthetotalleukocytenumberwasdetermined inaNeubauerchamber.ThesedosesofHEarebasedonprevious workpublishedinliterature(Nworuetal.,2011)andpilotstudiesin ourlaboratory(datanotshown).Chlorogenicacid(Sigma–Aldrich®_,

≥98%)andellagicacid(Roth®_,_≥_98%)_used_in_experiment_in_vivo_and invitromodelwerepurchasedcommercially.

Antioxidantactivity. Theantioxidantactivitywasperformedwith HEfromS.mombinleaves,DCM,AcOEt,ButOHandARFfractions andthecompoundsellagicacidandchlorogenicacid. TheDPPH radicalscavenging,Scavengerofsuperoxideion(O2−),Reducing

powerandSequestrationofthehydroxylradical(OH−₎_activity_was

evaluated.Theextractsandfractionsweretestedat60,125,250and 500␮g/mlandthecompoundsat5,15,30and60␮g/ml.

Viabilitycell. The3T3cellsweregrownin75cm2_flasks_with₉_ml_of

mediumDulbeccomodifiedEagleculturemedium(DMEM).Then, fortheassaycells,theyweretransferredto96wellplatesandit wasgrownat5×103_cell/well._These_cells_were_grown_overnight

inordertoattachin200␮lmediumat37◦_C_and_5%_CO

2.Afterthat,

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20

15

10

5

0

HO₂C

OH OH

OH

HO

OH

OH HO

HO OH

OH OH

OH OH CH2OH

OH

1

2

3

HO

O O O

O

O O

O 5

1

2

3

10 15 20

Time (min)

25 30 35 40

Absorbance (mA

U)

Fig.1. Chromatogramat340nmobtainedbyHPLC-DADofhydroethanolicextractfromSpondiasmombinleaves.Chlorogenicacid(1),ellagicacid(2)andisoquercetrin(3).

wereremovedand cells were washedwith200␮lPBS(twice), afterthatitwasadded100␮loffreshmedium.Then,itwasadded 100␮lofMTTreagent(5mg/ml)(Sigma,M5655)dissolvedinfresh DMEM.Thecellswerethenincubatedfor4hat37◦_C_and_5%_CO

2.

Tosolubilizetheformazancrystalsitwasused100␮lofethanol whichwasaddedtoeachwellandafter20mintheabsorbancewas readat570nmusingtheELISAreader(Biotec␮Quant).Asa con-trol,untreatednormalcellswereculturedonlyinthepresenceof DMEM.Thecellproliferation(%)wasexpressedas:[(SampleAbs 570nm×100)/ControlAbs570nm].

Statisticalanalysis

Resultswereexpressedas means±standard device.Statistic analysiswasconductedbyStudent’s t-test or one-wayanalysis ofvariance(ANOVA)followedbytheTukeyposthoctest. Statis-ticalsignificancewasconsideredof95%(p<0.05).Thestatistical programINSTAT,Graph-Pad,SanDiego,CA,wasused.

Resultsanddiscussion

PhytochemicalanalysisofS.mombin

AnalysisbyHPLC-DADthoughUVanalysisofeachpeakshowed thattheHEofS.mombinleavespresentsalargeamountofphenolic andflavonoidderivatives.ThequalitativeanalysisofHEenabled ustoindicatetheoccurrenceofthreephenolicmajorcompounds: chlorogenicacid,ellagicacidandisoquercetinandonenoidentified peakatUV340nm,bytheretentiontime,theUVspectraandby co-injectionoftheHEandreferencestandard(Fig.1).

Intheliteraturethereisnoreportabouttheidentificationof chlorogenicacidandisoquercetininS.mombinspecies,however, ellagicacidhasalreadybeenidentifiedinmethanolextractfrom theleaves(Silvaetal.,2012).Duetothis,ananalyticalmethodology wasdevelopedandvalidatedforthequantificationofchlorogenic acidandellagicacidbyHPLCforHEfromS.mombinleaves.Since thepresenceofflavonoidsandphenolicacidsinspeciesofSpondias

Table1

PhenolicacidscontentinhydroethanolicextractfromSpondiasmombinleaves.a

Species Compound Content/gextract

Spondiasmombin

leaves(2.5mg/ml)

Chlorogenicacid (Rt=8.28min)

12.0mg/gextract

Ellagicacid (Rt=34.21min)

19.4mg/gextract

a_Expressed_as_mg/g_of_extract_±_SD₍_n₌_3)/_Rt₌_retention_time.

genusisdescribedinliterature(Corthoutetal.,1992;Satpathyetal.,

2011;Silvaetal.,2012;Engelsetal.,2012),thesecompoundscould

behighlightedaschemicalmarkersforthisgenus.Theflavonoid isoquercetincannotbequantifiedin HEduetounavailabilityof suitableamountoftheirreferencestandard. Themethod devel-opedshowedachromatogramoftheHEfromS.mombinleaves withsymmetricalpeaks(tailingfactor:0.9and0.75,chlorogenic acidandellagicacid,respectively)andappropriatedresolution(2.0 tobothcompounds).

Standard solutionsof chlorogenicacidand ellagicacidwere prepared,ataconcentrationrangefrom10to60␮g/ml,andthe chromatographic method showeda suitable linear relationship (r2_>_0.995)_for_both_standard_solutions._This_enabled_us_to_measure

thecontentofthechlorogenicacidandellagicacidinHE(Table1). Thelimitofquantification(LOQ)andthelimitofdetection(LOD) weredefinedbyrelative standarddeviation(RSD>5%)andbya signal:noiseratioof3:1respectively(Table2).

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Table2

Calibration,LODandLOQdataofphenolicacidstandards.

Compound Linearityrange(␮g/ml) Calibrationequationa _Correlation_factor₍_r2₎ _LODb₍_␮_g/ml) _LOQb₍_␮_g/ml)

Chlorogenicacid 10–50 y=9727.8x−6129 0.9936 0.625 5.0

Ellagicacid 20–60 y=13976x−28,598 0.9901 1.25 5.0

a_Six_data_point₍_n₌_3).

b_LOD₌_limit_of_detection;_LOQ₌_limit_of_{quantification.}

Table3

Repeatability,intermediateprecisionandaccuracydataofphenolicacidsstandards.

Compound Repeatability Intermediateprecision Recoverya

Concentration(␮g/ml) R.S.D.(%) Concentration(␮g/ml) R.S.D.(%) Mean% R.S.D.(%)

Chlorogenicacid 10 1.0 10 1.0 110 1.0

30 2.4 30 0.3

50 0.4 50 1.0

Ellagicacid 20 0.8 20 2.5 102 0.8

30 1.0 30 1.2

50 4.5 50 0.8

a_Recovery_was_determined_by_injection_of_spiked_samples,_in_triplicate,_with_reference_standard.

theliterature(CassandDegani,2001;ICH,2005).Concerning accu-racy,animportantrecoverywasobservedforthestandardsinboth extracts,which wasdeterminedbyspikingsamples(2.5mg/ml) withthestandardsolutionsofellagicacid(50␮g/ml)and chloro-genicacid(10␮g/ml)(1:1,v/v)(Table3).

The quantification of individual compounds was performed using an analytical regression curve (r2_>_0.991). _The _standards

wereanalyzedintriplicateandtheaverageofthemeasuredpeak areas.Afterobtainingtheequationofthestraightthecompounds werequantifiedaccordingtotheareaunderthecurve.Itwasfound 19.4mg/gofellagicacidand12mg/gofchlorogenicacidintheHEof

S.mombinleaves(Table1).Apreviousreportaboutquantification ofellagicacidinS.mombinshowedacontentof41.56±0.01mg/g inthemethanol:water(80:20,v/v)leavesextract(Silvaetal.,2012) buttheenvironmentalconditionswerenotdescribed.This differ-encecanbeattributedtothesolventusedintheextractionprocess aswellastotheedafoclimaticfeaturesinBraziliannortheast,where phenolicderivativesareparticularlysusceptibleto environmen-talchangesinplantingthebehavior(Araújoetal.,2012).Another possibilityisthebiosynthesisofsecondarymetabolites,sincethe biogenesisofphenolicacids,flavonoidsandtanninsareverysimilar andhavethesameprecursors,whichcangenerateacompensation mechanismfortheproductionofaparticularsecondarymetabolite. Inthisworktheharvestofplantmaterialwascarriedoutin sum-merandinrainforest,thesolventusedforextractionwascomposed ofethanol:water(70:30,v/v).Therearenoreportsintheliterature aboutthequantificationofchlorogenicacidinthespeciesS. mom-binthusithighlightsourresultssinceitwasquantifiedas12mg metabolite/gextract.

Assessmentofanti-inflammatoryactivity

PreviousstudieswithspeciesofSpondiasgenusshowed anti-inflammatoryactivity.AccordingtoliteraturestudieswithSpondias mangiferausingbutanol andethyl acetate fractionsobtainedof ethanolicextractadministeredorallyatdoses75,150,300mg/kg b.w.showedasignificantreduction inpawvolumewhen com-paredwiththerespectivecontrolgroupchallengedbycarrageenan

(Sachanetal.,2011).Itwasalsonotedthestudyofethylacetate

extractofSpondiaspinnatahadanti-inflammatoryeffectsatdoses of 200, 400mg/kg, b.w. (p<0.001) in reducing rat paw edema inducedbycarrageenanassay(Raoetal.,2009).

Additionallywasreportedtheanti-inflammatoryactivityofS. mombinleavesinpawedemamodel(Nworuetal.,2011).Thestudy showedthattreatmentwiththemethanolextractfromS.mombin

leavesat 100, 200and 400mg/kg inhibitedby a dose depend-entwaythepawedemainducedbycarrageenanbesidescausing reductionoftheLPS-inducedbyTNF-␣andinproductionofnitric oxide(Nworuetal.,2011).Basedonthesestudiesandinpopular useofS.mombinininflammatoryprocesses,ourstudyevaluated theinvivoanti-inflammatoryextractpotentialbyacuteperitonitis modelusingcarrageenan-induceddexamethasone(5mg/kg,i.p.)as referencecompound.

Thisstudyaimedtotesttheextracttoanotherinflammation modelinordertoassessotherinflammatoryparameterssuchas leukocytemigration.Thecarrageenan-inducedperitonitisisawell characterizedexperimentalmodelofacuteinflammationwidely usedtotestnewanti-inflammatorytherapies,itenablethe quan-tificationandthecorrelationofcellularmigrationoninflammatory exudate(SedgwickandLees,1986;Paulinoetal.,2008;Bitencourt

etal.,2014;Limaetal.,2014).

Carrageenanisanindirectphlogisticagentthat inrats’ peri-toneum induces neutrophil migration which is dependent of residentmacrophageactivation(Souzaetal.,1988).Furthermore, studiesshowed thatcarrageenan injectioninto theperitoneum ofratsinducestheexpressionofnitricoxidesynthaseand COX-2resultingsoinliberationofalargeamountofnitricoxideand prostaglandins(Tomlinsonetal.,1994;Hatanakaetal.,1999).We havefoundthatintraperitonealinjectionofcarrageenanwasable toinduceaninflammatoryreactionwhichisrevealedbyhighcell migrationtotheperitonealcavity.

Ourresultsdemonstratedthattheanti-inflammatorypotential of HE of theS.mombin leavesat100, 200, 300 and 500mg/kg reduced thenumber ofleukocytesinfluxtoperitonealcavityof treated animals. In comparison with thecontrol group treated onlywithcarrageenan,theHEat300and500mg/kgshowedthe mostsignificanteffectwithaninhibitionofleukocytemigration of 51.75% and55.54%, respectively (Fig. 2A).Thesigns ofacute inflammationarevasodilatation,edemaandleukocyteinfiltration

(SherwoodandToliver-Kinsky,2004).Thisstudyshowedthatthe

extractofS.mombincouldactinoneoftheinflammation mech-anisminhibitingsignificantly theleukocytemigration tosite of inflammation.

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500 300 200 100 Dexa Sal

0 10 20 30

*** _***

***

* _*

Carrageenan 1% (1 mg/ml)

HE Spondias mombin (mg/kg)

A

Peritoneal leukocytes (x 10

6/ml)

ARF ButOH EtOAc DCM Dexa Sal

0 10 20 30

Carrageenan 1% (1 mg/ml)

***

*** *** ***

***

B

10 5 2.5 10 5 2.5 Dexa Sal

0 10 20 30

Carrageenan 1% (1 mg/ml) Elargic acid

(mg/kg)

Chlorogenic acid (mg/kg)

***

*** ***

C

Fig.2. EffectofhydroethanolicextractoftheSpondiasmombinleavesonleukocytemigrationinducedbycarrageenaninanacuteperitonitismodel.a_HE_tested_at

concen-trationsof100,200,300and500mg/kg,i.p.b_Fractions:_DCM:_{dichloromethane,}_EtOAc:_ethyl_acetate,_ButOH:_n_-butanol_and_ARF:_aqueous_residual_fraction_at₂₀₀_mg/kg,

i.p.c_ellagic_acid,_chlorogenic_acid_tested_at_{concentrations}_of_2.5,₅_and₁₀_mg/kg._Sal:_saline._Dexa:_{dexamethasone}_(0.5_mg/kg,_i.p.)._Data_are_expressed_as_mean_±_standard

deviation.*p<0.05,***p<0.001versusthepositivecontrolgroup(treatedwithcarrageenanonly).N=6.

withseveralpolaritysolventscouldbeconsideredusefulsinceone fraction(EtOAc)showedabetterpotentialincomparisontoHE.

HEand fractions wereanalyzedby HPLC-DAD(340nm)and showthepresenceofphenoliccompounds.Thisisanimportant finding,sincemanyphenolicarerelatedtotheanti-inflammatory activityofvarious plants(Rotelliet al.,2003; Santangeloet al., 2007).InEtOAcfractionwereidentifiedandquantifiedtwo phe-noliccompoundsellagicacidandchlorogenicacid,andthesewere evaluatedyourpotentialanti-inflammatory,inordertoverifythe contributionofthesecompoundsforthebioactivityoftheextract. These compounds were assayedat 2.5, 5 and 10mg/kg, i.p. Nostatisticaldifferencesbetweenthetreatmentgroups(Fig.2C) were observed. It is important to emphasize that the dose of 10mg/kgshowedthebestinhibitionprofiletotheellagicacidand chlorogenicacidthatwasdemonstratingbyinhibitionofleukocyte migrationof82and70%,respectively,tothesiteofinflammation.

Besidestheinhibitionofleukocytemigrationtheellagicacidand chlorogenicacidithasactivityinotherways,asreportedinother studies.

In the literature chlorogenic acid is able to inhibit nitric oxideproduction,inhibittheexpressionofbothCOX-2and pro-inflammatory cytokines (including IL-1␤ and TNF-␣) (Esposito etal.,2014),aswellasitcanalsoactbyinhibitingthenuclear translocationandactivationofNF-␬B,amasterregulatorof inflam-mation(Franciscoetal.,2013;Hwangetal.,2014).Also,invivo

studiesshowedthatchlorogenicacidat50and100mg/kg inhib-ited therat pawedema induced by carrageenan (Santoset al., 2006).Inaddition,importantanti-inflammatoryand antinocicep-tiveactivitiesbychlorogenicacidweredemonstratedsince this compoundhastakinggreateractionthansalicylicacid(Yonathan

etal.,2006).

Regardingellagicacid,thiscompoundwasabletoinhibitCOX-2

andNF-␬B(Marinetal.,2013)andpresentedanti-inflammatory

activity at 10 and 50mg/kg when tested in induced arthritis animalmodel and didnot showanyeffectondisease develop-mentinalowerdose,butinhibitedthepawvolume(p<0.05)with thehigherdose.Inouranalysis,ellagicacidandchlorogenicacid inhibitedaleukocytemigration inacuteperitonitismodel,thus providinganewandadditionalevidenceofitsanti-inflammatory potential.Takentogether,theseresultssuggestthatellagicacidand chlorogenicacidcouldbeinvolvedintheanti-inflammatoryaction observedforHEextract.

Theinhibitionofleukocytemigrationintotheperitoneal cav-itymaybebytwomechanisms:byinhibitingtheproductionof chemotacticsubstancesand/orexpressionofadhesionmolecules, thisbecausetheleukocytesrequirechemotacticsubstancesthat facilitate your migration to theplace of injury (Sherwood and

Toliver-Kinsky,2004).

Insummary,cansaidthattheHEofSmombinhastheabilityto inhibitleukocytemigrationtotheperitonealcavitybyacute peri-tonitismodelandthatthecompoundsellagicacidandchlorogenic acidmaybeinvolvedwiththisactivity.Howeverittakesmore stud-iestojustifytheuseoftheleafextractfromS.mombintotreat inflammatoryproblemsandtoelucidatethemechanismsinvolved inthisaction.

Assessmentofantioxidantactivity

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100

A

B

C

D

80 c

a a a a a

d

a _a

c b

b

d

b 100

80

60

20 40

0

100

80

60

20 40

0 c

b

60

40

20

0

1.0

a b

b b

b

c b

b 0.8

0.6

0.4

0.2

0.0

Control g

roup DCM

EtO

Ac

ButOH ARF

Ellagic acid

Chlorogenic acid

HE

Spondias mombin

Control g

roup DCM

EtO

Ac

ButOH ARF

Ellagic acid

Chlorogenic acid

HE

Spondias mombin

Control g

roup DCM

EtO

Ac

ButOH ARF

Ellagic acid

Chlorogenic acid

HE

Spondias mombin

Control g

roup DCM

EtO

Ac

ButOH ARF

Ellagic acid

Chlorogenic acid

HE

Spondias mombin

Sca

venging DPPH, %

Sca

venging supero

xide r

adical, %

Sca

venging h

ydro

xyl r

a

dicals

, %

Absorbance (nm)

Fig.3.AntioxidantactivityofhydroethanolicextractoftheSpondiasmombinleaves.XaxiscorrespondstohydroethanolicextractfromS.mombinleaves(HE);fractions:

DCM:dichloromethane,EtOAc:ethylacetate,ButOH:n-butanol,ARF:aqueousresidualfractionandellagicacidandchlorogenicacid.Thegraphsrepresentthedataobtained

forscavengingDPPHradical(controlgroup:gallicacid30mg/ml)a;superoxideradicals(controlgroup:gallicacid30mg/ml)b;reducingpower(controlgroup:ascorbic

acid0.2mg/ml)c;scavenginghydroxylradicals(controlgroup:gallicacid15mg/ml)d;expressedasmean±standarddeviationofpercentageofsequestrationDPPHata

concentrationof60␮g/mloftheHEandfractionsand30␮g/mlofchlorogenicacidandellagicacid.a,b,c.Differentlettersmeansignificantdifferencebetweenthecrude

extractfractionsandcompoundsidentified(p≤0.05).

capacity associated with an anti-inflammatory potential are desirablefeaturesforabioactivecompound.Consideringthe anti-inflammatorypotentialdemonstratedbyHE,DCM,EtOAc,ButOH, ARFfractions,andellagicacidandchlorogenicacidcompounds,it isimportanttoevaluatetheirantioxidantpotentials.This inves-tigationwasperformedinseveralapproaches:(i)scavengingfree radicalDPPHassay,(ii)superoxideradicalscavengingactivity,(iii) reducingpower,and(iv)hydroxylradicalscavengingactivity.

IntheDPPHassay(Fig.3)itwasobservedapotentialdonation ofelectronsorH+_ions_with_values_ranging_from_scavenging_66%

to76%forallsamplestested.TheHE,ButOHandchlorogenicacid wereabletosequester74.53,73.71and91.47%,ofthefree rad-ical,respectively.Intheassayofreducingpowertoevaluatethe reductivecapacityitwasobservedapositiveresultforallsamples analyzed(Fig.3).Furthermore,it wasobservedthat allsamples testedforthehydroxylradicalscavengingactivityshowed seques-trationpercentagehigherthan100%.Theantioxidantcapacityof theleavescanbeassociatedtotheirbioactivecompounds,mainly antioxidantpolyphenols,becauseoftheirabilitytoscavengefree radicals(Mandicetal.,2008).Onthisway,wesuggestthatthe phe-nolicacidspresentintheextractandfractionsofS.mombincould contributefortheextractantioxidantactivity.

Finally,apreliminaryevaluationofthepotentialcytotoxicityof HE,fractions(DCM,AcOEt,ButOH,ARF),andtheellagicacidand chlorogenicacidcompoundswascarriedout.Itwasobservedthat

Table4

Viabilitycelleffectsofthehidroethanolicextract,fractionsandchlorogenicacidand

ellagicacidfromSpondiasmombinleaves.HEandfractionswereassayedat60,125

and250␮g/mlandcompoundsat5,15and60␮g/mlon3T3fibroblasts.Resultsare

expressedaspercentageofproliferationmedia±standarddeviationfromtriplicate

assays.

Fibroblasts3T3(24h)

Extractandfractions 60␮g/ml 125␮g/ml 500␮g/ml HE 125±0.0205 129±0.0095 181±0.185 DCM 253±0 128±0.0005 142±0.0025 EtOAc 168±0.0205 204±0.034 128±0.0445 ButOH 141±0.0195 93±0.0005 158±0.048 ARF 100±0.01 108±0.0025 67±0.022

Compounds 5␮g/ml 15␮g/ml 60␮g/ml Chlorogenicacid 122±0.0355 106±0.002 105±0.0025 Ellagicacid 154±0.0535 127±0.0015 181±0.1205 HE=hidroethanolicextract ofSpondiasmombin leaves,DCM=dichloromethane fractionEtOAc=ethylacetatefraction.ButOH=n-butanolfraction.ARF=aqueous residualfraction.

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AcOEtandButOH(Table4).Itwasobservedacytotoxiceffectonly atthemaximalconcentration500␮g/mlfortheresidualfraction (ARF).

Insummary,tothebestofourknowledge,thisisthefirsttime thatisreportedantioxidant/anti-inflammatoryproperty(invitro/in vivo)totheleafextractfromS.mombinalongwith characteriza-tionoftheirchemicalcomposition.Theevidencefoundedinthis work,makesitasuitablecandidateforfurtherconsiderationasan alternativetreatmenttoreduceoxidativestressandinflammation.

Conclusions

Theresultsshowninthisworksuggesttheanti-inflammatory potentialof theleafextract ofS. mombimon peritonitismodel inducedbycarrageenan,Itwasalsoobservedantioxidant prop-ertiesassociatedwithanabsenceofcytotoxicityincellculture. Furthermore,itwasfoundthatchlorogenicacidandellagicacid contributetoactionpharmacologicallyofS.mombin.

Therefore,theseresultsindicatethepotentialoftheleafofS. mombinasasourceofnewanti-inflammatoryherbaldrugand/or molecules,and seemtojustifypartof itsmainpopularuses in traditionalmedicine.Furtherstudiesarerequiredtoconfirmthe anti-inflammatories action of S. mombin and its possible anti-inflammatorymechanismsofaction.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

BECVFparticipatedofphytochemicalanalysis,interpretationof dataanddraftedthemanuscript.MMMparticipatedinthe evalua-tionofanti-inflammatoryactivity,acquisitionandinterpretationof data.AHKparticipatedinantioxidantanalysisandrevisedit criti-callythemanuscript.RSparticipatedinitsdesignandcoordination, andhelpedtodraftthemanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Theauthorsacknowledgeallparticipantsfortheirvaluabletime andcommitmenttothestudy.WealsothankCNPqforfinancial support(478661/2010-0).TheauthorsarealsogratefultoMScAlan deAraújoRoqueresponsibleforspeciesidentification.

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