Rev. bras. farmacogn. vol.26 número4

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

screening

of

the

dichloromethane–ethanolic

extract

of

Eriosema

campestre

var.

macrophylum

roots

and

its

antiproliferative

effect

on

human

peripheral

blood

lymphocytes

Michaelle

G.

Santos

_,

_Valéria

_G.

_Almeida

_,

_Bethânia

_A.

_{Avelar-Freitas}

_,

_Cristiane

_F.F.

_Grael

_,

Luiz

E.

Gregório

_,

_Wagner

_F.

_Pereira

_,

_Gustavo

_E.A.

_Brito-Melo

a_{LaboratóriodeImunologia,DepartamentodeFarmácia,UniversidadeFederaldosValesJequitinhonhaeMucuri,Diamantina,MG,Brazil} b_{InstitutodeCiênciaeTecnologia,UniversidadeFederaldosValesdoJequitinhonhaeMucuri,Diamantina,MG,Brazil}

c_{LaboratóriodeFarmacognosia,UniversidadeFederaldosValesdoJequitinhonhaeMucuri,Diamantina,MG,Brazil} d_{LaboratóriodeCiênciasAmbientais,QuímicaseFarmacêuticas,UniversidadeFederaldeSãoPaulo,Diadema,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19February2015 Accepted13August2015 Availableonline9October2015

Keywords: Eriosemacampestre

Flavonoids Cellularproliferation IL-2

a

b

s

t

r

a

c

t

Eriosemacampestrevar.macrophylum(Grear)Fortunato,Fabaceae,isanativeplantoftheBrazilian

Cer-radoandthedecoctionofitsrootshasbeenusedbyfolkmedicineforthetherapyofinflammatory diseases.Inthisstudyweaimedtoinvestigatetheeffectofthedichloromethane–ethanolicextract

ofE.campestrerootsontheproliferativeresponseoflymphocytesandtoexaminetheprofileof

IL-2production.Theeffectofdichloromethane–ethanolicextractofE.campestreontheproliferationof phytohemagglutinin-stimulatedlymphocyteswasevaluatedbyusingflowcytometryandthecell super-natantswereassayedforIL-2concentrationsbyusinganenzyme-linkedimmunosorbentassay.The phytochemicalscreeningofE.campestrerootswasperformedtodeterminethemainsecondary metabo-litesthroughchromogenicandprecipitationreactionsandbyusingHPLC-PAD.Inadditiontothepresence ofsubclassesofflavonoids(flavonesandflavonols)indichloromethane–ethanolicextractofE.campestre, weobservedthattheextractinducedaconcentration-dependentdecreaseinIL-2levelsonthe super-natantofthecellculturesaswellasanantiproliferativeeffectonTlymphocytes,includingCD4+andCD8+ cells.Theanti-inflammatoryeffectsattributedtoE.campestrebyfolkmedicinemaypartlybeexplained byitsantiproliferativeactiononTlymphocytes.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Inflammationisacompleximmuneresponsethatinvolves mul-tiplefactors.Theinitialeventsresultintheactivationof innate immuneresponsesandinvolvementofenzymesystems(Nathan,

2002;Medzhitov,2008).Inlateevents,cooperationbetweenTand

Blymphocytescontributes toantigen-specificantibody produc-tion;Tlymphocytesinitiateacell-mediatedimmune-inflammation processinsituandmaintaintheactivationofphagocyticcellsby transformingthemintotissue-destructiveeffector cells(Barton, 2008).In chronicinflammatory diseases,thepermanent activa-tionandproliferationofTlymphocytesamplifytissuedamageand contributetotheclinicaloutcomeobservedinthesediseases,in additiontothecellularinteractionsoccurringintheaffectedsites (Caietal.,2012;Chimentietal.,2013).

∗ Correspondingauthor.

E-mail:gustavomelo@ufvjm.edu.br(G.E.A.Brito-Melo).

Sincechronicinflammatorydisordersarecharacterizedbythe highactivationandproliferationofTlymphocytes,most immuno-suppressivedrugsaimtoblockthecellcycleprogressionofthese cells(Macián,2005).Inadditiontothedrugsregisteredforfirst-line therapy,whichmayhavemanyandsometimesseveresideeffects

(De Mattos etal., 2000), there are numerousalternative herbal

treatmentswithpromising,butnot yetproven,efficacy(Reuter etal.,2010).

Eriosema campestre var. macrophylum (Grear) Fortunato,

Fabaceae,isaplantpopularlyknownas“pustemeira”andisnative totheBrazilianCerrado(Grear,1970;Fortunato,1999).Thisspecies consistsofsmallanderectherbsrangingfrom14to17.5cm/high, withyellowflowersandfusiformroots(RogalskiandMiotto,2011). ThedecoctionofE.campestrerootshasbeendescribedandusedby folkmedicineforthetherapyofinflammatorydiseasesincluding inflammatoryskindisorderssuchaspsoriasis.However,nostudy ofthechemicalcompositionofthisplantoranybiologicalanalysis hasyetprovidedevidenceregardingtheeffectivenessofthisplant asananti-inflammatory.

http://dx.doi.org/10.1016/j.bjp.2015.08.009

Wedecidedtoperformaphytochemicalscreeningto charac-terizethemainclasses ofsecondary metabolitespresent inthe plantextractaswellastoinvestigatetheprofileofitschemical composition.Moreover,inadditiontotheroleofTlymphocytesin thepathologicalprocessofchronicinflammatorydiseases,wealso investigatedtheeffectofthedichloromethane–ethanolicextract

ofE.campestreroots(DEEC)ontheproliferativeresponseof

lym-phocytesaswellastheprofileof IL-2production, thecytokine essential for the expansion of these cells during the adaptive immuneresponse.

Materialsandmethods

Plantmaterial

FreshEriosemacampestrevar.macrophylum(Grear)Fortunato, Fabaceae,plantswerecollectedinthecityofDatas,MinasGerais, Brazil (S 18◦_27.318′_{, W 43}◦_39.764′_{, 1223m altitude). Botanical}

identification was performed by Dr. Ana Paula Fortuna Perez, Curator ofHerbario BOTU, Department ofBotany, Universidade Estadual Paulista, Botucatu, SP, Brazil, and a specimen was depositedundervouchernumber881atJeanineFelfiliDendrologic HerbariumoftheFederalUniversityofJequitinhonhaandMucuri Valleys.

Preparationofplantextracts

TherootsofE.campestreweredriedtoaconstantweightatroom temperature.Thedriedmaterial(450g)wasgroundandmacerated inamixtureofdichloromethaneandethanol(1:1v/v)for72hat roomtemperature.Themaceratewasseparatedbyfiltrationand concentrated undervacuum ona rotatoryevaporator(Fisatom, model801,Brazil)at40◦_{Ctofurnishabrownresidue(12.1g).A}

5.0mg/mlsolutionoftheresidue(DEEC)inDMSO(Sigma,USA) wasprepared.Smallstockaliquotswerekeptat−20◦_Cuntilthe

timeofuse.NewdilutionsoftheextractinDMSOwereperformed toobtaintherequiredconcentrationinthecellculturemedium.

Chromatographicprofileusinghighperformanceliquid

chromatography(HPLC)

Reagents

HPLC-grademethanolwasobtainedfromJ.T.Baker(Ecatepec, Mexico),HPLC-grade acetonitrilewasobtainedfromTediaHigh PuritySolvents(Fairfield,USA),trifluoroaceticacidwaspurchased fromSynth(SãoPaulo,Brazil)andHPLC-qualityultrapurewater waspreparedbyusingaMilliporeMilli-QDirect-8System (Biller-ica,USA).

Chromatographicanalysis

SamplesofDEECdilutedto10mg/mlweredissolvedin HPLC-grademethanol,filteredthrougha0.2␮mmembraneandanalyzed ona ThermoAccelaHPLCsystem(ThermoFisherScientificInc., Waltham, USA) with a solvent delivery unit, on-line degasser, columnovenandautosampler,andequippedwithaphotodiode array detector (PAD). For data analysis, we used ChromQuest software (Version 5.0, Thermo Fisher Scientific Inc., Waltham, USA).ALunaC18(2)analyticalcolumn(150×4.6mm;particlesize 3␮m;Phenomenex,USA)wasused.Thesamplewaselutedbya gradientelutionemployingultrapurewater(SolventA)and ace-tonitrile(SolventB),bothcontaining0.1%v/vtrifluoroaceticacid: 0–5min,90%ofA,50min,100%B;after10minthecolumnwas re-equilibratedwith90%A.Thecolumntemperaturewasmaintained at40◦_{C.Analysiswasperformedataflowrateof1.0ml/minand}

wasmonitoredat288nm.

Biologicalsamplesandpreparationofperipheralblood

mononuclearcells(PBMC)

Peripheralbloodwasobtainedfrom22healthyadultdonors. Volunteerswithanyinfectious,autoimmunediseasesormaking useofantibiotics,anti-inflammatorymedication,corticosteroids orotherimmunosuppressivedrugswerenotconsideredforblood donation. Informedwritten consentwasobtainedfromall par-ticipants. The study was approved by the Ethical Committee at the UFVJM, Diamantina, Minas Gerais, Brazil (register code 569.313/2014).

PBMCwasisolatedfromheparinizedhumanperipheralblood samples(15ml)byusingtheFicoll-Paque(specificgravity1.077) gradientdensitymethod,asdescribedpreviouslybyBicalhoetal.

(1981). Peripheral blood was diluted with phosphate-buffered

saline(PBS;pH7.2)andcentrifugedinaFicoll-Histopaque(Sigma, USA) discontinuous gradient at 400×g at room temperature for 30minfor obtaining thecharacteristic layer containing the mononuclear cells. PBMCwas collected, washed with PBS and centrifuged(240×gatroomtemperaturefor7min).Cellswere sus-pendedataconcentrationof1×107_{cells/mlinPBSorRPMI-1640}

medium (Sigma, USA) supplemented with10%heat-inactivated fetalcalfserum(FCS;Gibco,InvitrogenCorporation,USA),2mM

l_{-glutamine(Sigma, USA)and anantibiotic/antimycoticcocktail}

(100UI/ml penicillinG, 100␮g/ml streptomycinand 250ng/ml amphotericinB–Sigma,USA).ThePBMCfromeachresearch sub-ject wasobtained separately and used for cell culture analysis individually.

Cellviabilityanalysis

PBMC (5×105_{) were cultured with 0.5% dimethyl sulfoxide}

(DMSO),assolventcontrol,or DEEC(100,50,25or12.5␮g/ml) at37◦_{Cinahumidifiedincubatorwitha5%CO2-airatmosphere}

for24hor5days.UntreatedPBMCwasusedastheunstimulated cellculturecontrol(Ctrl).Intheendoftheincubation,cellswere washedwithPBS,centrifuged(240×g,roomtemperature,7min) andresuspendedwith0.5mlPBS.Then,10␮lofcellsuspension wasmixedwithanequalvolumeof0.4%trypanblue(Sigma,USA). Total,viableandnonviablecellnumberswerecountedunderthe microscopebyusingahemocytometerNeubauerchamber.

Lymphocyteproliferativeresponse

PBMC (1×107 _{cells) were resuspended in PBS and labeled}

with 1␮M of BD HorizonTM Violet Proliferation Dye 450 (VPD 450,BDBiosciences,USA)for15minat37◦_{C(Lyons,1999).The}

VPD450-stainedPBMC(5×105 _{cells/wellin24-wellplate)were}

culturedinRPMI-1640containing10%FCS(Gibco,Invitrogen Cor-poration,USA),2mMl-glutamineandtheantibiotic–antimycotic

cocktail(Sigma,USA),withorwithoutphytohemagglutinin(PHA 5␮g/ml). Cells were also stimulated with PHA in combination with 0.5% DMSO (solvent control) or different concentrations of DEEC (25,12.5 or6.25␮g/ml). Cells stimulatedwithPHA in combinationwithdexamethasoneat8␮g/ml(Aché,Brazil)were used as the inhibition control (DEXA). The plate was kept in a humidified incubator with a 5% CO2 air atmosphere for five

days at 37◦_{C. After incubation, cells were harvested, washed}

102

0 500

M1 1000

1500

0

500 _B

C

D A

400

300

200

100

0 500

350 300 250 200 150 100 50 0 400

300

200

100

103 ₁₀4 ₁₀5

102 ₁₀3 ₁₀4 ₁₀5

102 ₁₀3 ₁₀4 ₁₀5

102 ₁₀3

VPD450 (FI)

M5 M4

M3

M2 M1

M5

M6

M4

M3

M2 _M1

M5 M4

M6

Count

M3

M2 M1

104 ₁₀5

Fig.1.AnalysisofcellproliferationusingVioletProliferationDye(VPD)450inflow cytometry.A,VPD450-stainedcellsderivedfromunstimulatedcultures;B,VPD 450-stainedcellsinculturesstimulatedwithphytohemagglutinin(PHA),C,VPD 450-stainedcellsinculturesstimulatedwithPHAandtreatedwithDMSO(solvent control),D,VPD450-stainedcellsinculturesstimulatedwithPHAandtreatedwith DEEC25␮g/ml.

fluorescencehistograms(Fig.1)byusingthefollowing formula, asdescribedbyAnguloandFulcher(1998):PI=(100−Y)/Y,where

Y (%)=X0+X1/2+X2/4+X3/8+X4/16+X5/32; X0 represents the

percentageofT-cellsthatdidnotdivide(locatedinM1),andX1–X5 representsthepeaksofgradualdivision(locatedinM2–M6).

IL-2production

PBMC (5×105 _{cells/well on 24-well plates) were cultured}

in RPMI-1640 containing 10% FCS (Gibco, Invitrogen Corpora-tion,USA),2mMl_{-glutamine,theantibiotic–antimycoticcocktail}

(Sigma,USA) andPHA (5␮g/ml)with(i)PBS(control),(ii)0.5% DMSO(solventcontrol)or(iii)differentconcentrationsof DEEC (25,12.5or6.25␮g/ml).CellsstimulatedwithPHAin combina-tionwithdexamethasoneat8␮g/mlwereusedastheinhibition control(DEXA).Theplatewasmaintainedat37◦_{C ina}

humidi-fiedincubatorwitha5%CO2airatmospherefor36h,followedby

centrifugationfor10min,at240×gandatroomtemperature.The cellsupernatantswerecollectedandassayedforIL-2 concentra-tionsbyusinganenzyme-linkedimmunosorbentassay(ELISA;R&D Systems,USA)accordingtothemanufacturer’sinstructions.

Inbrief,plates werecoatedwithmousecaptureantibodyto humanIL-2andincubatedovernightatroomtemperature.Wells werewashedfivetimeswithPBScontaining0.05%Tween20(PBST) and incubated with 1% bovine serum albumin in PBS contain-ing0.05%sodiumazide.Differentconcentrationsofrecombinant humanIL-2dilutedinPBS(standardcurve)orcellsupernatants wereaddedandincubatedatroomtemperaturefor2h.After wash-ingwithPBST,goatbiotinylatedantibodiestohumananti-IL-2were added,andtheplatewasincubatedatroomtemperaturefor2h priortoaddingstreptavidinhorseradishperoxidase.Afterdetection withtetramethylbenzidine(Sigma,USA),sulfuricacidwasadded toblocktheenzymaticreaction.Theabsorbanceat450nmwas determinedinamicrotiterplatereader(SpectraMax190, Molecu-larDevices,USA).ConcentrationsofIL-2insampleswereestimated bycomparisonwithastandardcurve.

Statisticalanalysis

GraphPadPrism,version5.0forWindows(GraphPadSoftware, USA)wasusedforthestatisticalanalysis,andp-values0.05were consideredtobestatisticallysignificant.Dataarereportedasthe mean±standarddeviation(SD).TheShapiro–Wilktestwasused toevaluatethenormalityofthedata.Analysisofvariance (one-wayANOVA)wasemployed,followedbyTukeyposthocpairwise comparisons.

Resultsanddiscussion

E.campestreisaplantnativetoBrazilanditstraditionaluseas

atherapyagainstchronicinflammatorydisordersiswidespreadin theBrazilianCerrado.However,therearenoreportsofany chem-icalorbiological-toxicologicalstudiesforthisplantthusfar.Thus, thecurrentstudyprovidedthefirstinvestigativecontributionto thechemicalcharacterizationandbiologicalanalysisofthisplant species.

The phytochemical analysis (Farnsworth, 1966; Bustamante etal.,2010)ofDEECshowedthepresenceofflavonoidsand ter-penes.

A complex chemical profile for DEEC was observed in the HPLC-PADanalysis,indicatingthepresenceofsubstancesofhigh, mediumandlowpolarity(Fig.2A).Highpolaritycompoundsare representedbypeakswithshorterretentiontimes,elutedbetween 1and20min,becausethesesubstanceshaveahigheraffinityforthe mobilephase.Somepeakswereelutedbetween20and35min,a factthatdemonstratesthepresenceofmediumpolaritymolecules. Thelateelutingpeaksobservedbetween35and47min,which rep-resentahigheraffinityforthenon-polarstationaryphase(C18), aredistinguishedbyahigherintensityandtheyseemtoconstitute majoritycompoundscomparedwiththeotherpeaks.This obser-vationindicatesthatthesubstancesoflowpolaritypredominatein theextract.

Flavonoidshavetwoabsorptionbandsintheultraviolet–visible region:bandIIwithamaximumabsorptionbetween240nmand 285nm,correspondingtothebenzoylgroup,andbandIwitha max-imumabsorptionregionfrom300to550nm,whichcorrespondsto thecinnamoylgroup.TheanalysisofbandsIandII(absorption max-imumandintensity)canassistinidentifyingthetypeofflavonoid

(MarstonandHostettmann,2006).TheHPLC-PADspectrashowed

1750

A

C

B

1500 Retention time

1250

1000

750

500

250

0

1750

1500

1250

1000

750

500

250

0

0 5 10 15 20 25 30

Minutes

204

232

364

Rt: 41.94 min

190 600190 600

Wavelength (nm)

Rt: 13.03 min Rt: 38.70 min

Rt: 42.25 min

254 214

274

354 365 222 264

Rt: 9.72 min Rt: 9.86 min

Rt: 12.50 min

Rt: 16.91 min

Rt: 41.56 min Rt: 10.35 min

Rt: 11.72 min

Rt: 12.88 min

Rt: 17.87 min

Rt: 11.33 min 209

270

270 335

190 600 600

204

203

265 331

260 208

337

268

346 217 190

nm nm

mA

u

mA

U

nm nm

190

203

268

204

267

204 261

331 333

600

190 nm

600

190 nm

600

190 nm

332

190 600

nm

190 600

nm

190 600

nm

190 600

600

203 203

267

269 333

333 338

265 366

35 40 45 50 55 60

Fig.2.ChemicalanalysisofDEEC.HPLC-PADchromatogramofDEEC,monitoredat288nm(A)andUV/VISspectra(190–600nm),indicatingthepresenceofflavones(B)and flavonols(C).

SeveralstudieswithotherspeciesoftheEriosemagenushave reportedthatflavonoidsareresponsibleforthebiologicalactivity attributedtotheseplants.Insomestudies,authorshavereported thepresenceofseveralnovelflavonoidssuchasKraussianones1–7, observedintherootsofE.kraussianum(Drewesetal.,2002,2004), ErioschalconesAandB,isolatedfromE.glomerata(Awouafacketal., 2008),RobusflavonesAandB,extractedfromtheaerialpartsofE.

robustum(Awouafacketal.,2013)andKhonklonginolsA–H,

iso-latedfromtherootsofE.chinense(Sutthivaiyakitetal.,2009).It ispossiblethatE.campestrecontainsnovelmolecules.Thisidea supportsthenecessityforfurtherchemicalstudiesofitsbioactive compounds.

Thesubstancespresentinplantextractsmightincludethose withcytotoxicactivities(Veiga-Júnioretal.,2005),andthe tox-icityof thesecompounds maydefine theuseof theplantas a phytomedicine. To investigate this issue, the first trials in our study aimedto evaluate the toxicity of DEEC in human PBMC by using the trypan blue exclusion test. After 24h of culture in the presence of different concentrations of the extract, we observed that DEEC at 12.5, 25 and 50␮g/ml didnot decrease cellviability(12.5␮g/ml=97.56±0.32%;25␮g/ml=95.09±1.71%; 50␮g/ml=93.88±1.46%) compared with the control cultures (96.75±1.49%)andsolventcontrol(DMSO=95.66±1.56%).Data showedcytotoxicityonlywhenPBMCwastreatedwithDEECat 100␮g/ml(56.60±7.24%)(Fig.3A).

After five days of cell culture, DEEC in concentrations lower than 25␮g/ml showed no cytotoxic effect on PBMC (12.5␮g/ml=93.56±3.03%; 25␮g/ml=83.75±6.67%) compared withtheuntreated(Ctrl=91.50±2.86%)andsolventcontrol cul-tures(DMSO=85.47±7.75%).However,asignificantreductionin cellviabilitywas observedin cultures treatedwithDEEC at 50

(65.47±10.37%)and100␮g/ml(54.70±2.90%)comparedwiththe DMSO-treatedanduntreatedcellcultures(Fig.3B).

TheseresultsallowedustochooseconcentrationsofDEEClower than25␮g/mlastheoptimumconcentrationsforuseincell pro-liferationassayssincethecellsarekeptincontactwithanextract forfivedays.

Ctrl 0 20 40 60 80 100

0 20 40 60 80 100

DMSO 12.5 25 50 100 _{Ctrl DMSO 12.5 25 50 100}

a,b

a,b a

DEEC (μg/ml)

A

B

DEEC (μg/ml)

Cell viability

, %

Cell viability

, %

Fig.3.EffectofDEEConcellviabilityafter24h(A)andfivedays(B)ofculture.PercentagesofviablePBMCinPBS-treated(Ctrl),DMSO-treatedandDEEC-treatedcellswere assessedbyusingthetrypanblueexclusiontest(n=8).Theresultsareexpressedasthemean±SDofthepercentageofcellviabilityindifferentgroups.Significantdifferences (p0.05,ANOVAwithposthocTukeytest)areidentifiedbytheletters“a”and“b”forcomparisonsrelativetothecontrolandDMSOcellcultures,respectively.

The influence of DEEC on the proliferation of human lymphocytesbyusingcell-basedassayswascomparedwith phar-macologicaldosesofdexamethasone,whichisthebenchmarkof immunosuppressive pharmaceuticalsto defineits immunother-apeutic activity. DEEC exhibited a concentration-dependent

inhibitionofthecelldivisionoftheactivatedTlymphocytes, includ-ingCD4andCD8T-cells.Incomparisonwithdexamethasone,the inhibitionpromotedbyDEECat25␮g/mlonTlymphocytesand CD4T-cellspresentedefficacylevelsof71.65%and53.14%, respec-tively.In theCD8T-cell analysis,DEEC exhibited anefficacyof

4

1000 ₃₀₀

500 450

400 350 300 250 200 150 100 50 0

350 300 250 200 150 100 50 0 350

300 250 200 150 100 50 0 400 300 200 100 0

250 200 150 100 50

102 103 104 105 102 103 104 105

102 103 104 105

102 103 104 105

102 103 104 105

102 103 104 105 0

750 500 250 0

CD4+ cells

Proliferation index (PI)

CD8+ cells Lymphocytes

A

B

Untreated

PI=0.01

PI=3.55

PI=0.30 PI=0.49

PI=2.61

PI=1.11

PHA+DEEC 6.25μg/ml

PHA+DEEC 12.5μg/ml

Count

PHA+DEEC 25μg/ml PHA+DEXA (8μg/ml)

VPD450 (FI)

PHA

**

***

_***

***

***

***

*

***

3

2

1

0 4

3

2

1

0 5

4

3

2

1

0

PHA (5 μg/ml) –

–

–

– +

–

–

– +

–

+

–

+

12.5

–

– +

25

–

– +

–

–

+ +

6.25

–

– DEEC (μg/ml)

DMSO (0.5%)

DEXA (8 μg/ml)

Fig.4.InfluenceofDEEContheproliferationofactivatedlymphocytes.VPD450-labeledhumanlymphocytes(5×105_{cells)wereculturedinthepresenceofmedium,} dexamethasone(DEXA,8␮g/ml)ordifferentconcentrationsofDEEC(6.25,12.5and25␮g/ml)andactivatedwithPHA(5␮g/ml)forfivedays.Thelymphocyteproliferation

PHA (5 μg/ml)

**

*** ***

–

–

–

– +

–

–

– +

–

+

–

+

12.5

–

– +

25

–

– +

–

–

+ +

6.25

–

–

IL-2 (pg/ml)

80

60

40

20

0

DEEC (μg/ml)

DMSO (0.5%)

DEXA (8 μg/ml)

Fig.5.AnalysisofIL-2productioninthesupernatanthumanPBMCcultures acti-vatedbyPHAandtreatedwithDEEC.HumanPBMC(5×105_{cells)wereculturedin} thepresenceofmedium,DMSO(0.5%),dexamethasone(DEXA,8␮g/ml)ordifferent

concentrationsofDEEC(6.25,12.5and25␮g/ml)andactivatedwithPHA(5␮g/ml)

for36h.TheamountofIL-2wasmeasuredinthesupernatantofthecellculturesby usinganELISA-basedcytokinedetectionmethod.Theresultsarepresentedasthe mean±SDofsixindependentexperiments.ThestatisticalmethodusedwasANOVA followedbytheTukeytest.

167.94%,anantiproliferativeeffecthigherthanthosepresentedby dexamethasone.T-CD8lymphocytesplayanimportantroleinviral infections,cancerandtransplantrejectionaswellasinskinlesion and tissue damagemaintenance in autoimmuneand degenera-tiveinflammatorydiseasescharacterizedbyaheightenedcellular adaptiveimmuneresponse(Gaoetal.,2009).Theinhibitoryeffect ofDEECcouldthusbeapromisingtherapeuticstrategy inthese inflammatorysituations,openingnewperspectivesforresearch.

Tlymphocyteproliferation is initiatedby bondingtheT-cell receptor to external stimuli that initiate the secretion of the autocrinegrowthfactorIL-2,which,inturn,promotesthe inter-actionwiththeCD25receptorthatisupregulatedonthesurface ofactivatedT-cells(Gangulyetal.,2001;Malek,2008).Theeffect ofDEEConIL-2productioninthesupernatantofPBMCcultures wasanalyzedbyusingELISA(Fig.5).DEECinhibitedtheproduction ofIL-2inthePHA-stimulatedculturestreatedwiththeextractat 12.5␮g/ml(28.01±7.95pg/ml)and25␮g/ml(17.02±4.04pg/ml) comparedwiththecellculturesstimulatedonlywithPHAcultures (58.77±15.70pg/ml).Onceagain,DMSOwasnotabletoinhibitthe productionofIL-2(DMSO=39.79±15.7pg/ml).

Theseresultsindicatedthattheantiproliferativeaction exhib-ited bytheextract onTlymphocytes wasdue toitsinhibitory action onthe production of IL-2. Theinhibitory effect of DEEC (25␮g/ml) on the secretion of IL-2 presented an efficacy of 38.33%incomparisontothoseobservedinculturestreatedwith dexamethasone. The expression of IL-2 involves the activation of transcription factors, mainly NF-kB. The recognition of an antigen bytheT lymphocytereceptor/CD3 complex, aswellas co-stimulatorystimuliinitiatestheactivationofthesignaling path-wayrelatedtoNF-kB.ThistranscriptionfactorisessentialforT-cell proliferation,cytokineproductionandthedevelopmentofa well-establishedcell-mediatedimmuneresponse,cytokineproduction andconsequentlyanincreaseofinflammatorymediatorssuchas prostaglandinsandleukotriene(Kaneetal.,2002;LiandVerma,

2002).

The inhibition of IL-2 production is the target for several immunosuppressive drugs (Schreiber and Crabtree, 1992). BecauseoftheinhibitoryactionofDEEConcellproliferationand itsinhibitory effect onthesecretion of IL-2, inaddition tothe

predominantpresence offlavonoids intheE.campestreextract, moleculesbelongingtothischemical groupmightbetheactive compounds responsible for the effects presented here. Several studies haveidentified flavonoids asthe agents responsiblefor the inhibition of lymphocyte proliferation, and this effect has been shown to be associated with lower levels of IL-2 in cell cultures(Gaoetal.,2009;Changetal.,2008;Gharagozlooetal., 2010).FurtherstudiesusingfractionatedDEECwillbeperformed toelucidate thestructure of thesubstances responsiblefor the effectdemonstratedhereaswellastheperformanceoftheactive fractionsininflammatorymodelsinvivo.

The results suggest that the anti-inflammatory activity attributedtoE.campestrebyfolkmedicinemaybe,atleastinpart, theresultoftheactionofitsflavonoidsontheactivationand pro-liferationofTlymphocytes.ThisfindingmeansthatE.campestre isapromisingnaturalsourceforthedevelopmentofnew effec-tivephytomedicinesforthetreatmentofthedamageobservedin autoimmuneandinflammatorydegenerativediseases,especially thosemediatedbyTlymphocytes.

Authors’contributions

MGS collected plant sample, performed the experiments, interpreted the results and drafted the manuscript. VGA and BAAF contributed to biological studies and to critical read-ing of the manuscript. CFFG contributed to prepare the dichloromethane–ethanolicextractofE.campestreroots.LEG con-tributedto chromatographicanalysis. WFP contributedto draft andcriticalreadingofthemanuscript.GEABMdesignedthestudy, supervisedthelaboratoryworkandcontributedtocriticalreading ofthemanuscript.Alltheauthorshavereadthefinalmanuscript andapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WearegratefultoDr.DavidLeeNelsonforthetechnicalreview ofthemanuscriptandtotheBrazilianfundingagenciesFAPEMIG (CBB-APQ-00581-11),CNPqandCAPESforfinancialsupport,aswell astothePost-GraduatePrograminPharmaceuticalScienceofthe UFVJM(PPGCF/UFVJM).

References

Angulo,R.,Fulcher,D.A.,1998.MeasurementofCandida-specificblastogenesis: com-parisonofcarboxyfluoresceinsuccinimidylesterlabellingofTcells,thymidine incorporation,andCD69expression.Cytometry34,143–151.

Awouafack,M.D.,Kouam,S.F.,Hussain,H.,Ngamga,D.,Tane,P.,Schulz,B.,Green, I.R.,Krohn,K.,2008.AntimicrobialprenylateddihydrochalconesfromEriosema glomerata.PlantaMed.74,50–54.

Awouafack,M.D.,Tane,P.,Eloff,J.N.,2013.Twonewantioxidantflavonesfromthe twigsofEriosemarobustum(Fabaceae).Phytochem.Lett.6,62–66.

Barton,G.M.,2008.Acalculatedresponse:controlofinflammationbytheinnate immunesystem.J.Clin.Invest.118,413–420.

Bicalho,H.M.S.,Gontijo,M.C.,Nogueira-Machado,J.A.,1981.Asimpletechniquefor simultaneoushumanleukocytesseparation.J.Immunol.Methods40,115–116. Bustamante,K.G.L.,Lima,A.D.F.,Soares,M.L.,Fiuza,T.S.,Tresvenzol,M.F.,Bara,M.T.F., Pimenta,F.C.,Paula,J.R.,2010.Avaliac¸ãodaatividadeantimicrobianadoextrato etanólicobrutodacascadasucupirabranca(PterodonemarginatusVogel)– Fabaceae.Rev.Bras.PlantasMed.13,341–345.

Cai,Y.,Fleming,C.,Yan,J.,2012.NewinsightsofTcellsinthepathogenesisof psoriasis.CellMol.Immunol.9,302–309.

Chimenti,M.S.,Ballanti,E.,Perricone,C.,Cipriani,P.,Giacomelli,R.,Perricone,R., 2013.Immunomodulationinpsoriaticarthritis:focusoncellularandmolecular pathways.Autoimmun.Rev.12,599–606.

DeMattos,A.M.,Olyaei,A.J.,Bennett,W.M.,2000.Nephrotoxicityof immunosup-pressivedrugs:long-termconsequencesandchallengesforthefuture.Am.J. KidneyDis.35,333–346.

Drewes,S.E.,Horn,M.M.,Munro,O.Q.,Dhlamini,J.T.B.,Meyer,J.J.M.,Rakuambo, N.C.,2002.Pyrano-isoflavoneswitherectile-dysfunctionactivityfromEriosema kraussianum.Phytochemistry59,739–747.

Drewes,S.E.,Horn,M.M.,Khan,F.,Munro,O.Q.,Dhlamini,J.T.B.,Rakuambo,C.,Meyer, J.J.M.,2004.Minorpyrano-isoflavonesfromEriosemakraussianum:activity-, structure-,andchemicalreactionstudies.Phytochemistry65,1955–1961. Farnsworth,N.R.,1966.Biologicalandphytochemicalscreeningofplants.J.Pharm.

Sci.55,225–276.

Fortunato,R.H.,1999.CâmbiosNomenclaturalesenEriosema(Fabaceae: Papil-ionoideae,Cajaninae)II.Kurtziana27,371–382.

Ganguly,T.,Bandheka,L.P.,Sainis,K.B.,2001.ImmunomodulatoryeffectofTylophora indicaonConAinducedlymphoproliferation.Phytomedicine8,431–437. Gao,X.,Deeb,D.,Liu,Y.,Dulchavsky,S.A.,Gautam,S.C.,2009.

Immunomodula-toryactivityofxanthohumol:inhibitionofTcellproliferation,cell-mediated cytotoxicity and Th1 cytokine production through suppression of NF-␬B. Immunopharmacol.Immunotoxicol.31,477–484.

Gharagozloo,M.,Velardi,E., Bruscoli,S.,Agostini,M.,Sante,M.D., Donato,V., Amirghofran,Z.,Riccardi,C.,2010.SilymarinsuppressCD4+Tcellactivationand proliferation:effectsonNF-kappaBactivityandIL-2production.Pharmacol.Res. 61,405–409.

Grear,J.W.,1970.ArevisionoftheAmericanspeciesofEriosema(Leguminosae– Lotoideae).Mem.NewYorkBot.Gard.20,1–98.

Kane,L.P.,Lin,J.,Weiss,A.,2002.It’sallRelative:NF-kappaBandCD28costimulation ofT-cellactivation.TrendsImmunol.23,413–420.

Li,Q.,Verma,I.M.,2002.NF-kappaBregulationintheimmunesystem.Nat.Rev. Immunol.2,725–734.

Lyons,A.B.,1999.Dividedwestand:Trackingcellproliferationwith carboxyfluo-resceindiacetatesuccinimidylester.Immunol.CellBiol.77,509–515. Macián,F.,2005.NFATproteins:keyregulatorsofT-celldevelopmentandfunction.

Nat.Rev.Immunol.5,472–484.

Malek,T.R.,2008.Thebiologyofinterleukin-2.Annu.Rev.Immunol.26,453–479. Marston,A.,Hostettmann,K.,2006.Separationandquantificationofflavonoids.In:

Andersen,O.M.,Markham,K.R.(Eds.),Flavonoids:Chemistry,Biochemistry,and Applications.Taylor&Francis,BocaRaton,pp.1–36.

Medzhitov,R.,2008.Originandphysiologicalrolesofinflammation.Nature454, 428–435.

Nathan,C.,2002.Pointsofcontrolininflammation.Nature420,846–852. Reuter,J.,Merfort,I.,Schempp,C.M.,2010.Botanicalsindermatology:an

evidence-basedreview.Am.J.Clin.Dermatol.11,247–267.

Rogalski,L.D.,Miotto,S.T.S.,2011.OgêneroEriosema(DC.)Desv. (Leguminosae-Papilionoideae)nosestadosdoParanáedeSantaCatarina.Brasil.Rev.Bras. Bioci.9,350–370.

Schreiber,S.L.,Crabtree,G.R.,1992.ThemechanismofactionofcyclosporinAand FK506.Immunol.Today13,136–142.

Sutthivaiyakit,S.,Thongnak,O.,Lhinhatrakool,T.,Yodchun,O.,Srimark,R., Dow-taisong,P.,Chuankamnerdkarn, M., 2009.Cytotoxic andantimycobacterial prenylatedflavonoidsfromtherootsofEriosemachinense.J.Nat.Prod.72, 1092–1096.