Rev. bras. farmacogn. vol.25 número4

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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

Cytotoxicity

of

latex

and

pharmacobotanical

study

of

leaves

and

stem

of

Euphorbia

umbellata

(Janaúba)

Lívia

E.C.

Luz

a

_,

_Katia

_S.

_Paludo

b

_,

_Vera

_L.P.

_Santos

c

_,

_Celia

_R.C.

_Franco

d

_,

_Traudi

_Klein

a

_,

_Rozi

_Z.

_Silva

a

_,

Flávio

L. Beltrame

a

_,

_Jane

_M.

_Budel

a,∗

a_Departamento_de_Ciências_{Farmacêuticas,}_Universidade_Estadual_de_Ponta_Grossa,_Ponta_Grossa,_PR,_Brazil

b_Departamento_de_Biologia_Estrutural,_Molecular_e_Genética,_Universidade_Estadual_de_Ponta_Grossa,_Ponta_Grossa,_PR,_Brazil c_Departamento_de_Engenharia_Saúde_e_Meio_Ambiente,_Centro_{Universitário}_{Internacional,}_Curitiba,_PR,_Brazil

d_Departamento_de_Biologia_Celular,_Universidade_Federal_do_Paraná,_Curitiba,_PR,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4May2015 Accepted7July2015 Availableonline30July2015

Keywords:

HeLa HRT-18 Morfo-anatomy

Synadeniumgrantii Synadeniumumbellatum

a

b

s

t

r

a

c

t

InsouthernBrazil,thebottledlatexofSynadeniumgrantiiHookf.,Euphorbiaceae,ispopularlyusedas atreatmentofalltypesofcancer.Similarly,SynadeniumumbellatumPax.isusedinthecentralwestern regionofBrazilforthesamepurposeandinthesamemannerofuse.Bothplantsarepopularlyknown asjanaúbaorleitosinha.Theobjectivesofthisstudyweretousepharmacobotanicalanalysisto ver-ifywhetherthesetwospecies,whichareconsideredtobedistinct,areactuallythesametodetermine anatomicalmarkers;toassistintheidentificationanddifferentiationofotherEuphorbia;andtoevaluate thecytotoxicactivityofthelatexinrelationtoHeLaandHRT-18cells.Leavesandstemsofthespecies werecollectedinGoiâniaandPontaGrossaandwereinvestigatedusingscanningelectronmicroscopy andopticalmicroscopytechniques.Thelatexwasalsocollectedandanalyzedinrelationtoitscytotoxic effectbyemployingMTTandNRtechniques.Thepharmacobotanicalstudyofthespecimensinboth localitiesshowedthattheywerethesamespecies,namelyEuphorbiaumbellata(Pax)Bruyns,whichis thescientificnomenclatureacceptedandconfirmedbyanexperttaxonomistwhospecializesin Euphor-bia.Thepharmacobotanicalcharacteristicshighlightedinthisstudycanassistintheidentificationof thetaxonandcontributetothecontrolofthequalityofthisplantdrug.Theevaluationofthelatexin relationtoHRT-18cellsdemonstratedactionafter48hofexperiment.Incontrast,inrelationtoHeLa cellsitsinducedcytotoxicityinalltimesandadose-dependentmanner.TheIC50values(72h)observed

were252.58±18.51␮g/mland263.42±15.92␮g/mltoMTTexperimentand250.18±19.48␮g/mland

430.56±19.71␮g/mltoNRexperimentfortheHeLaandHRT-18cells,respectively.

Introduction

Cancerisoneofthemostcommondiseasesaroundtheword. Statisticsshowthatin 2011itwasthediseasewiththehighest mortalityrates(WHO,2014).InBrazil,differentkindofcancers affectthepopulation,butcancerofthecolon/rectum,cervixand leukemiaarethemostprevalent(INCA,2014).

Tofindnewtreatmentsforthisdisease,studiesofmedicinal plants canbe anefficient tool. Manosroi et al.(2012) consider medicinalplantsasthemainsourcestoobtainnewdrugsforthe treatmentofcancer.Inthesameway,despitetheadvanceofnew processesoforganicsynthesisandbiotechnology,medicinalplants

∗ Correspondingauthor.

E-mail:[email protected](J.M.Budel).

playanimportantroleinmedicaltherapyandclearlyrepresenta windowofopportunityforthepharmaceutical/chemicalindustry

(BrazFilhoetal.,2010).

EuphorbiaceaeisoneofthelargestfamiliesoftheAngiosperms groupandhasapproximatelytenspecieswithrecognized promis-inganticanceractivity(Bhanotetal.,2011).Euphorbiahasabout 2000species,whicharepredominantlyfoundinaridorsemi-arid regionsofthetropicsandsubtropics(Hornetal.,2012).

Euphorbiaumbellata(Pax)Bruyns,whichiscommonlyknown

asjanaúbaorleitosinhainBrazil,istheacceptednamefor

Synade-niumgrantiiHookf.andSynadeniumumbellatumPax(ThePlantList,

2014).Thelatexofthisspeciesisusedinfolkmedicinetotreatall typesofcancer,allergy,Chagasdisease,internalbleeding,sexual impotence,leprosy,obesity,nervousulcerandmenstrualcramps. AccordingtoOrtêncio(1997),peoplepreparethegarrafada dissolv-ing18dropsoflatexin1lofwateranddrinktherecommended

http://dx.doi.org/10.1016/j.bjp.2015.07.005

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dosage(aglass)three timesa day,although this speciesis not includedintheBrazilianPharmacopeiaanddonothaveindication ofuseinBrazilianNationalForm.

Despitethepopularuseofmedicinalplants,anti-canceraction needstobeevaluated,inthefirstinstance,byinvitroassays(suchas MTT,NRandothers)regardingscreeningandtoevaluate cytotox-icity.PreviousstudieshaveshownthatE.umbellatahasacytotoxic effectinrelationtoEhrlich’s(Motaetal.,2012a),K-562(Nogueira etal.,2008)andB16F10(DeOliveiraetal.,2013)tumorcells.

InBrazil,alargenumberofnativeplantsareusedasmedicines and sometimes there are problems in identifying these plants

(Beltrameetal.,2006).Severalstudieshavedescribedtheuseofthe

wrongmedicinalplantsduetosimilarmorphological characteris-ticsbetweenvegetablespeciesandforthisreasonthefrequencyof theadulterationisaseriousproblem(Youssefetal.,2013;Amorin

etal.,2014;Camilottietal.,2014;Araujoetal.,2015).

For thisreason, adequatequalitycontrol methods,including morpho-anatomicalidentification,needtobeperformedformany plantsthatarenotcurrentlydescribedintheBrazilian Pharma-copeia.Consequently,thisstudyisintendedtoproposeastandard ofqualitycontrolinordertopromoteaparameterofauthenticity

forE.umbellata,andalsotoevaluatethepreliminarycytotoxicity

ofthelatexofthisspeciesinrelationtoHRT-18andHeLacells.

Materialsandmethods

Plantmaterial

AerialpartsofEuphorbiaumbellata(Pax)Bruyns,Euphorbiaceae, werecollectedfromspecimensgrowninopenandsunnyareasin PontaGrossa,Paraná,Brazil(altitude:975meters,latitude:25◦₀₅′

38′′_S_and _longitude:₅₀◦₀₉′₃₀′′_W)_in _August_2013,_and _in

Goiâ-nia,Goiás,Brazil(altitude:787m,latitude:16◦₄₀′_S_and_longitude:

49◦₁₄′_W)_in_September_2013._The_plant_material_containing

flow-erswasusedtoprepareavoucherspecimenthatwasidentifiedby ataxonomistwhoisaspecialistinEuphorbia,InêsCordeiro (Botan-icalInstituteofSãoPaulo),anditwasstoredattheMariaEneida P.KauffmannFidalgoHerbarium(BotanicalInstituteHerbariumof SãoPaulo)underthenumberSP453920.Matureleavesobtained fromthesixthnodeandbelow,aswellasstemfragmentsfrom5to 15cmfromtheshoot,werechemicallyfixedinFAA70(Johansen, 1940)andstoredin70%ethanol(v/v)(BerlynandMiksche,1976). Thefreshlatexwascollectedthroughtransversalcutsinbarkof

theE.umbellata.Thislatex(1g,density:1.22g/ml)wasaccurately

weightand dissolvedinwater usinga volumetric flask(10ml). Aliquotsofthissolutionweredilutedtogivethefollowingstock solutions:1000,750,500,250,100,39and19.45␮g/mlthatwere usedintheexperiments.

Cellculture

Humancervical adenocarcinoma– HeLa(ATCCCCL-2)– and

humanileocecalcolorectaladenocarcinoma–HRT-18(ATCC CCL-244)cells–wereobtainedfromcommercialsources(cellbank,Rio deJaneiro,BCRJ).Thecellswereculturedinahumidity-controlled environmentcontaining5%CO2andatemperatureof37◦C,andfed

withRPMIgrowthmedia(RoswellParkMemorialInstitute, Culti-lab,SãoPaulo,Brazil)supplementedwith10%fetalserumbovine, penicillinandstreptomycin(GIBCO,Lifetechnologies,SãoPaulo, Brazil).

Cytotoxicityassays

ThecytotoxiceffectofE.umbellatalatexwasdeterminedby a colorimetric assay using MTT ((3-[4,5 dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazoliumbromide(Amresco,Solon,USA)and

neutralred(NR)(Inlab,SãoPaulo).MTTassaymeasuresthe activ-ityoflivingcellsviamitochondrialdehydrogenases.Solutionsof MTTareyellowishincolor.Mitochondrialdehydrogenasesofviable cellscleavethetetrazoliumring,yieldingpurpleformazancrystals, whichareinsolubleinaqueoussolutions(Mossman,1983).TheNR isavitaldye(BasicRed5,Toluylenered).Viablecellswilltakeup thedyebyactivetransportandincorporatethedyeintolysosomes, whereasnon-viablecells willnottakeupthedye(Borenfreund

andPuerner,1985).BrieflytheHeLaandHRTcellswereseeded

(8×104_cells_per_well)_into_96-well_culture_plates_and_maintained

inRPMIcontaining10%fetalbovineserum,at37◦_C_in_a_5%_CO₂

atmospherefor24h.Then,thecellculturemediafromeachwell wasremovedandreplacedwith190␮loffreshmediaand10␮l

ofE.umbellatalatexstocksolutions(finalconcentrations):1000,

750,500,250,100,39and19.45␮g/ml.Thecellswereincubated at37◦_C_in_a_5%_CO

2atmospherefor24,48and72h.Afterthese

times,themediawasremoved.FortheMTT,thecellswererinsed withphosphate-bufferedsaline(PBS) and70␮lofMTT solution (0.5mg/ml)andincubatedfor2hat37◦_C._{Subsequently,}₁₀₀_␮_l_of

0.1MHClinanhydrousisopropanolwasaddedandtheabsorbance wasmeasuredat570nm.FortheNRtest,themediumfromeach wellwasaspiratedandwellswererinsedwithPBS.Immediately 200␮lofworkingNRsolution(40mg/mlofNRinRPMImedium) wereaddedand theplates incubatedfor2hat37◦_C._After_this

time,NRsolutionwasremovedandthecellsweretreatedwith 0.5%formaldehyde/1%CaCl2inPBSfor1minfollowedby100␮l ofNRsolubilizationreagent(50%ethanol,1%aceticacidinPBS). Theabsorbancewasmeasuredat520nm.Negativecontrolwas performedusingcellsincubatedonlywithRPMImedium.Thecell viabilitywasexpressedin%comparedwithnegativecontrol.Each latexconcentrationwasassayedintriplicateandrepeatedthree times.TheIC50valueswerecalculatedtoMTTandNR72h

exper-imentthatpresentedthehighercytotoxicityforbothcellslines

(Döll-Boscardinetal.,2012;Assafetal.,2013;Taoetal.,2013).

Pro-bitregressionandtheleastsquaresmethodusingStatPlusversion 5.8.4statisticalsoftwarewereusedtodothedetermination.

Morphologicalanalysis

HeLa and HRTcells (20×104₎_were_added _in_24-well _plates

containingonecover slip(eachwell)and thesewereincubated withRPMImediumfor24hat37◦_C_and_5%_CO

2.Thecellculture

mediafromeachwellwasremovedandreplacedwith380␮lof freshmediaand20␮lofE.umbellatalatexstocksolutions(final concentrations):500and750␮g/ml(forHeLacells)and750and 1000␮g/ml(forHRT-18)andincubatedat37◦Cina5%CO2

atmo-spherefor24and48h.After,thecellswerewashedwithPBSthey werefixedwith2%formaldehydefor2minandstainedwith May-Grünwaldstain.Thecytotoxiceffects(vacuolization,rounding,loss ofadhesion,blebbing,nuclearcondensationand fragmentation) wereanalyzedusingaphotomicroscope(OlympusBX41coupled toaOlympusDP71camera).

Pharmacobotanicalstudy

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150

_A

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HeLa HRT-18

(V

iability %) 50

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iability %) 50

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39

LAtex (µg/ml) LAtex (µg/ml)

100 250 500 750 1000

CTRL 19.45 39

LAtex (µg/ml) LAtex (µg/ml)

100 250 500 750 1000 CTRL 19.45 39 100 250 500 750 1000 CTRL 19.45 39 100 250 500 750 1000 24 h

48 h 72 h

24 h 48 h 72 h

Fig.1.CytotoxicityinducedbylatexofEuphorbiaumbellata(Pax)BruynsforHeLaandHRT-18cellsat24,48and72h(MTTassay–A,andNR–B).RPMImediumwasusedas anegativecontrolonly.Resultswereexpressedasmeanandstandarderrorofthemean.One-wayANOVAfollowedbyTukey’spost-test.*p<0.05,**p<0.01and***p<0.001.

formucilage(DeOliveiraetal.,2005),andiodine-iodidetotestfor starch(BerlynandMiksche,1976).Someofthephotomicrographs werecapturedusingaOlympusCX31lightmicroscopeequipped withaC7070controlunit.Forthescanningelectronmicroscopy (SEM)analysis (Souza,1998), thesamplesfixedin FAA70were dehydratedina seriesof gradedethanoland CO2 critical point

dryinginaBal-TecCPD-030,coatedwithgoldinBalzersSCD-030 equipmentandexaminedusingaJeolJSM-6360LVmicroscope.

Resultsanddiscussion

Cytotoxicitystudy

TheHeLacellswereresponsivetotreatmentwiththelatexofE.

umbellatathatinducedadoseandtime-dependentcytotoxicity.

HRT-18 cells showedan initialresistancetolatex treatmentin 24h, but at 48h it was possible to observe reducing cell via-bility in the concentrations from 100 to 1000␮g/ml and after 72hatconcentrationof 1000␮g/ml, latexeffectively presented a toxic profile similar to that of the HeLa cells (Fig. 1). The 72hexperiment time waschosentoevaluatethedeathof50% of the cells (IC50), according to described by Döll-Boscardin

et al. (2012), Assaf et al. (2013) and Tao et al. (2013)

result-ing in values of 252.58±18.51␮g/ml and 263.42±15.92␮g/ml

for the HeLa and HRT-18 cells respectively to MTT

experi-mentand 250.18±19.48␮g/mland 430.56±19.71␮g/mltoNR experiment.

Previous studies have reported thecytotoxicity of the latex ofotherspeciesofEuphorbiaceaewhichcorroboratetheresults obtainedinthisstudy.Hsiehetal.(2011)showedthetoxicityof

Control

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20.0 µm

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x500 50 µm x1, 500 10 µm

x500 50 µm x5, 000 5 µm x400 50 µm

x10,000 1 µm st

ad

cu

st

Fig.3. Euphorbiaumbellata(Pax)Bruyns.A.Aspectofaerialvegetativeorgans.B.Aspectofleaves.C–J–Epidermisinsurfaceview.C.Adaxialside,showinganticlinal epidermalcellwalls,cuticleandstomata.D.Adaxialside,exhibitingcuticleandstomatum(SEM–scanningelectronmicroscopy).E.Detailofthepreviousfigure,showing cuticle,stomatumandepicuticularwax(SEM).F.Detailofepicuticularwax(SEM).G.Abaxialside,showinganticlinalepidermalcellwalls.H.Abaxialside,exhibitingcuticle andepicuticularwax(SEM).I.Abaxialside,presentingcuticleandstomata(SEM).J.Non-glandulartrichome(SEM).K.Non-glandulartrichome.L.Bladeorganization,in cross-section,revealinghomogeneousmesophyll,minorcollateralvascularbundleandparenchymaticsheath.M.Detailofthepreviousfigure,showingstomata,epidermis andspongyparenchyma.ab:abaxialside;ad:adaxialside;cu:cuticle;ep:epidermis;nt:non-glandulartrichome;ps:parenchymaticsheath,sp:spongyparenchyma;st: stomatum;vb:vascularbundle;wa:epicuticularwax.Bar=4cm(A);1cm(B);25␮m(G);50␮m(C,K,M);200␮m(L).

thelatexofEuphorbiaantiquorumL.inrelationtoHeLacells,which causedareductionofcellviabilityatconcentrationsof0.5–3mg/ml. Similarly, De Oliveira et al. (2013) showed a 64% inhibition of growthofB16F10cellsafterincubationwiththelatexofE. umbel-lata.

Morphologicalanalysis

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ep

sp

sg ph

la xy

cx

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lx

la

ph xy

xy

la la

ep

ph

Fig.4. Euphorbiaumbellata(Pax)Bruyns–A.Transectionofmidrib,showingepidermis,vascularsystem,andlaticifers.B.Transectionofmidrib,indicatingvascularsystem andlaticifers.C.Detailofthelaticifersshowingmucilage,stainedblue.D.Detailofvascularsystemandlaticifersofthemidrib.E.Midribincross-section,showingstarch grainsinreactionwithiodine-iodide.F.Petioleincross-section,exhibitinglaticifersnearthevascularbundle.ep:epidermis;gp:groundparenchyma;la:laticifer;lx:latex; ph:phloem;sg:starchgrains;sp:spongyparenchyma;xy:xylem.Bar=50␮m(C,D,E);100␮m(B);200␮m(A,F).

andchromatincondensation.After48h,moreseveretoxicitywas observed,inadditiontotheabovecitedeffects(datanotshown). ThelatexalsoinducedmorphologicalchangesintheHRT-18cells suchasintensevacuolization,cellroundingandchromatin con-densationin highconcentrations(Fig.2).Severalspecies ofthe genusEuphorbiahavebeenextensivelystudiedfortheirantitumor and cytotoxic activities (Betancur-Galvis et al., 2002;

Sadeghi-Aliabadietal.,2009;Amirghofranetal.,2011;Hsiehetal.,2011;De

Oliveiraetal.,2013),andapoptosisisalwaysthepreferredpathway

fortherapeutictumorkillingasaformofnon-inflammatorycell death.

Eventssuchastheformationofblebbingandchromatin con-densationwerealsoobservedinK-562leukemiccellsthatwere treatedwithethanolextractofaerialpartsofS.umbellatumand stainedwithGiemsa(Motaet al.,2012b).Sumathiet al.(2011)

found morphological alterations, such as chromatin condensa-tion, which characterized apoptosis in chick embryo fibloblast cellsexposedtoEuphorbiaantiquorumlatex.Anotherstudyfound

thatE.antiquorumlatexalsoinducedapoptosis,whichwas

char-acterizedby morphological changes in HeLa cells; an increase

in the sub-G1 population and in the levels of caspase-8, 9

and 3, and the suppression of Bcl-2 expression (Hsieh et al.,

2011).

Pharmacobotanicalstudy

SynadeniumgrantiicollectedinPontaGrossa(PR)and

Synade-nium umbellatum in Goiânia (GO) – both samples considered

twoindependentspeciesbysomescientificbotanicalwebsites– showedthesamemorphological andanatomicalcharacteristics. Therefore, morpho-anatomical description and the figures cor-respond tothe two copies, which in turn belong to Euphorbia

umbellataasconfirmedbythepharmacobotanicalstudiesandby

thetaxonomist.

Euphorbia umbellata (Fig. 3A) had leaves which measured

approximately12.4cmlongand4.7cmwide(Fig.3AandB).They weresimple,green,petiolate,pinnate,spatulate-lanceolateinform withfromobtusetoacuteapex,attenuatebaseandentiremargins (Fig.3Aand B).Thephyllotaxyrevealedalternatearrangements (Fig.3A).

InseveralEuphorbiatheshapeoftheanticlinalepidermalcell wallsvaryfromstraighttosinuous(KakkarandPaliwal,1974;Gales

andToma,2006;Essietetal.,2012;Zahraetal.,2014).Infrontal

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ep

cx

fi

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pi

fi la

fi

ph

la cx

pi

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st

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X1, 000 10µm

2mm

A

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Fig.5.Euphorbiaumbellata(Pax)Bruyns–Stem–A.Generalaspectincross-section(SEM).B.Cortexregion,incross-section.C.Stomatainfrontalview(SEM).D. Cross-section,showingcortex,laticifers,fibers,phloem,xylemandpith.E.Detailofthefibersandlaticifers.F.Detailofthevascularsystemandfibers.cx:cortex,ep:epidermis,st: stomatum,fi:fiber,la:laticifer,pi:pith,ph:phloem,xy:xylem.Bar=50␮m(B,E,F);100␮m(D).

thickness,which wasstriateand withwax thatwasdenser on theadaxialside(Fig.3EandF).Thestriatecuticleshowedvariable thicknessandappearedinconnectedstripsthatformedpolygonal shapes(Fig.3D,E,HandI).Theepicuticularwaxtypewascrystalloid inrosetteshapes(Fig.3EandF).

VariousEuphorbia,such as E.pannonica Host, E.dobrogensis Prodán,E.myrsinitesL.,E.myrsinitessubsp.litardiereiFontQuer &GarciasFont, E.nicaeensisAll., E.characiasL.and E.bazargica Prodánhavedistinctpapillaeofvariousshapesandsizes(Kakkar

andPaliwal,1974;GalesandToma,2006;Lukovi ´cetal.,2009;Pinto

etal.,2014;Christodoulakisetal.,2015).InE.umbellatathepapillae

wasnotobserved.

AccordingtoKakkarandPaliwal(1974),thestomatain Euro-peanspeciesofEuphorbiaarepredominantlyanomocytic;however anisocyticand anomocytictypescanalsobefound(Kakkarand

Paliwal,1974;GalesandToma,2006;Lukovi ´cetal.,2009;Pinto

etal.,2014).Frequently,morethanonetypeofstomatamayoccur

onthesameleafside(KakkarandPaliwal,1974).Ontheotherhand,

diacytictypestomatahavebeenfoundinE.royleanaBoiss.,E. splen-densBojerexHook.,E.peplusL.(Zahraetal.,2014),whileparacityc, staurocytic,brachyparacyticandlaterocytictypeswereobserved onbothsurfacesofE.hirtaL.andE.heterophyllaL.(Essietetal., 2012).Inthepresentstudy,paracyticstomataoccurredonboth

sidesoftheleaves(Fig.3C,D,EandI)andtheywerelocatedatthe samelevelinthesurroundingcells(Fig.3M).

Thepresenceofthestomaintheleavesisanimportant fea-turetocharacterizeanddifferentiatethespecies.Amphistomatic leaveshavebeenobservedinseveralEuphorbia,suchasE.peplus

L.(Mendivelsoetal.,2003);E.nicaeensissubsp.glareosa(Lukovi ´c

etal.,2009);E.amygdaloidesL.,E.cyparissiasL.,E.falcataL.,E.

bazar-gicaProdán,E.carniolicaJacq.(GalesandToma,2006);E.buxifolia Lam.,E.corollataL.,E.radiansBenth(KakkarandPaliwal,1974);

andE.hirta(Pintoetal.,2014).However,hypostomaticleaveswere

foundinE.taurinensisAll.(GalesandToma,2006)andE.milli(Essiet etal.,2012).Inthepresentstudy,E.umbellatashowed amphistom-aticleaves.

GalesandToma(2006)investigatednineteenspeciesof

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Fig.6.Euphorbiaumbellata(Pax)Bruyns.Microchemicaltests–incross-section.A.BladeshowinglatexandcuticleinreactionwithSudanIII.B.Stemindicatingxylemand latexinreactionwithferricchloride.C.SteminreactionwithBouchardatreactive,showinglaticifers.D.SteminreactionwithBouchardatreactive,showingthelatexinthe laticifers.E.Starchgrainsreactedwithiodine-iodideinthecortex.F.StemshowingcuticleinreactionwithSudanIII.G.Stemexhibitingfiberandxyleminreactionwith phloroglucin.cu:cuticle,cx:cortex,ep:epidermis,fi:fiber,la:laticifer,lx:latex,pi:pith,ph:phloem,sg:starchgrain,xy:xylem.Bar=50␮m(A,C,D,E,F);200␮m(B).

Similartrichomeswereobservedin E.heterophylla(Kakkar and

Paliwal,1974;Essietetal.,2012),E.pulcherrimaWilld.exKlotzsch

(Zahraetal.,2014)andE.hirta(Essietetal.,2012;Pintoetal.,2014).

ManymembersofEuphorbiapresentisolateral(GalesandToma,

2006;Lukovi ´cetal.,2009)ordorsiventralmesophyll(Galesand

Toma,2006;Pintoetal.,2014;Christodoulakisetal.,2015).

Differ-ingfromthisgeneralobservation,inE.umbellata,themesophyll

was homogeneous and composed of several layers of spongy

parenchyma(Fig.3LandM).Collateralvascularbundlestraversed

the spongy parenchyma and were encircled by parenchymatic

sheathwithoutvisibleCasparianstrips.Theywerelocatedinthe lowerthirdofthemesophyll(Fig.3L).

InthemidribofthebladeofEuphorbiaceae,theorganizationof thevasculartissuesisvariable,initiallyformingawholeringand anopenarcontheadaxialsurfaceattheends(Gaucher,1902).The midribpresentsasinglevascularbundleinthecenteroftheground parenchymainvariousEuphorbia(GalesandToma,2006;Lukovi ´c

etal.,2009;Pintoetal.,2014).Intransection,themidrib’sshape

wasdescribedasconcave-convexinE.hirta(Pintoetal.,2014)and asbiconvexinE.characias(Christodoulakisetal.,2015).InE.

umbel-lata,themidrib,incross-section,wasplano-convex(Fig.4A).This

speciesexhibitedseveralcollateralvascularbundles,whichwere organizedintriangleinthegroundparenchyma(Fig.4AandB), whereseveralstarchgrainswerefound(Fig.4E).

Laticifersoccurinovertwentyfamilies,includingsomegenusof Euphorbiaceae(Demarcoetal.,2013),Asteraceae,Moraceae(Esaú,

1974;Hageletal.,2008)andCampanulaceae(Folquittoetal.,2014).

Thelatexisresponsiblefortheantitumoractivityassessedinthe

E.umbellataspecies,whichpresentsflavonoids,saponins,phorbol

estersandterpenes(Bagalkotkaretal.,2006;Costaetal.,2012). Ter-penesaredescribedtopresentcytotoxicactivity(Bagavathietal., 1988)aswellasphenoliccompoundsthatinduceROSgeneration andhaveanti-tumoractivity(Biscaroetal.,2013).

Thelaticifersdifferinsize,inthethicknessofthewalls,inthe distributionandtheiroccurrenceinthevegetaltissuesandorgans, andintheirpresenceinspecificstructures(GalesandToma,2007). Theyarepresentnotonlyinthepithandprimarycortexoftheaxis, butalsonearthesheathbundleintheleafandmesophyllofmature plants(MetcalfeandChalk,1957).AccordingtoEsaú(1965)and

Hageletal.(2008),thegenusEuphorbiapresentsnon-articulated,

branchingandnon-anastomosinglaticifers.

InaccordancewiththestandardoftheEuphorbiagenus,inthe leavesofE.umbellata,thelaticiferswerenon-articulated,branching andnon-anastomosing;theywerealsopresentnearthevascular bundlesandinalltheotherpartsofthemesophyllandthemidrib (Fig.4A–D).Theyshowedapolygonalshapeinthetransverse sec-tionandthethickcellulosicwall(Fig.4BandD).Similarfeatures werefoundinE.virgataWaldst.&Kit.,however,thelaticifershave thinwallsandtheyareanalogoustosecretoryducts(Galesand

Toma,2007).

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previousdescribedfor theleaves.Therewereabout6 layersof angularcollenchymaadjacenttotheepidermis.Approximately12 free collateral vascular bundles formeda horizontal lineinthe centerofthegroundparenchyma;however,inthemiddleofthis linetherewasaconvexity.Laticiferscouldbeobservednearthe vascularsystem(Fig.4F).InE.hirta,thepetioleincross-section hasanalmostroundshape.Theuniseriateepidermishasshowed non-glandulartrichomesandthevascularsystemhavepresented 4collateralvascularbundlesdistributedinanopenarc(Pintoetal.,

2014).

ThestemofE.umbellata,intransection,presenteda circular

shape(Fig.5A).Theepidermiswasuniseriate(Fig.5B).The cuti-clewasthin,striateandreactedpositivelytolipophiliccompounds (Fig. 6F).In variousspecies ofEuphorbia,theepidermiscells of thestemhavethickpericlinalwallscoveredbyacuticleof vari-ablethickness(GalesandToma,2006).InE.umbellata,thestomata (Fig. 5C) were foundat the same level as theother epidermal cells.

Regardingthetrichomes,thestemofsomeEuphorbiataxaare pubescent.ThetrichomesareunicellularinE.helioscopiaL.,E.

amyg-daloides,E.agraria,andE.glareosaormulticellularuniseriateinE.

maculata(GalesandToma,2006)andE.hirta(Pintoetal.,2014).

Inthispresentstudy,trichomeswerenotobservedinthestem.

InmostoftheEuphorbiainvestigatedbyGalesandToma(2006),

withtheexceptionofE.maculata,variablenumbersoflayersof collenchymajustbeneaththeepidermisweretangentiallypresent.

In E.plathyphyllos L.and E.helioscopia,thecortexis formedby

angularcollenchyma.Additionally,numerousauriferouscavities wereobservedinthecortexareaofE.myrsinitessubsp.litardierei,

E.myrsinitessubsp.myrsinitesandE.glareosa.Inthepresentstudy,

severalstrataofcorticalparenchymawerefoundinE.umbellata (Fig.5BandD).Thistissuewasrathercompactandcollenchyma wasnotfound.Inthecorticalparenchyma,severalstarchgrains wereobserved(Fig.6C–F).

Inthisstudy,thevascularcylinderwasformedbyphloem (out-side)andxylem(inside)andperivascularfibercapsthatsometimes adjoinedthephloem(Fig.5D–F).Thexylemandfibersreacted posi-tivelytohydrochloricphloroglucin(Fig.6G)andthexylemreacted withferricchloride(Fig.6B).Perivascularfibersarecommonin

Euphorbia,howevertheywerenotobservedinE.myrsinitessubsp.

litardierei,E.myrsinitessubsp.myrsinitesandE.dobrogensis(Gales

andToma,2006).Similarlaticifersthatwereencounteredinthe

leaveswerecommonlyobservednearthephloem(Fig.6B,C,Dand G)inthestem.

Aftermicrochemicaltreatmentwithvarious reagents,itwas observedthatphenolic compounds(Fig.6B),mucilage (Fig.4C), lipophiliccomponents(Fig.6A)andnitrogencompounds(Fig.6D) werepresentinthelatex.Inordertoconfirmthepresenceofthe nitrogencompounds,cross-sections ofthestems wereexposed tospecifictreatmentandtoBouchardatreactive,theresultswere respectivelynegative(Fig.6C)andpositive(Fig.6D).

In some Euphorbia, such as E. glareosa, E. myrsinites subsp.

litardierei,E.myrsinitessubsp.myrsinites,andE.cyparissias,thecells

ofthepithhavenumerousaeriferouscavities,whichareseparated byuniseriatelamellae.Conversely,inE.taurinensisandE.falcate subsp.acuminatathepithisrelativelycompact.InE.umbellata,the pithappearedinthecenterofthestem,surroundedbythexylem. Itwasformedbythin-walledparenchymaticcells(Figs.5Dand6E). These cells werequitecompacted and showedthepresence of starchgrains(Fig.6E).

It can be concluded from the present study that the main morpho-anatomical features were highlighted in this pharma-cobotanicalstudy helping toward thestandardization of the E.

umbellataspeciesinordertoimprovethequalitycontrolofthis

vegetablematerial.Resultsofthisstudyindicatedthatlatexhad cytotoxiceffects ontumoralcell lines(HeLa andHRT-18 cells).

MorphologicalchangescausedbythisphytocomplextoHeLacells areassociatedwithapoptosis.

Conflictofinterest

Theauthorsdeclarenoconflictsofinterest.

Authors’contributions

LECL(Postgraduatestudent)wasresponsibleforperformingthe testspresentedinthepaper.Shecontributedincollectingand iden-tifyingplantsamples,performedthevoucher,analyzedthedata, anddraftedthearticle.KSPwasresponsibleforanti-cancertests. analyzingthedataanddraftingthearticle.VLPSandCRCFwere responsibleforscanningelectronmicroscopy.TKcontributedto runningthelaboratoryworksand preparingmaterialfor analy-sis.RZSdonatedthevegetalmaterialandhelpedwithlaboratory support,aswellasanalysisofresults.FLBwasresponsiblefor finan-cialsupport,analysisofthedataanddraftingthepaper.JMBwas responsibleformorpho-anatomicaltests,analysisofthedataand draftingthearticle.

Acknowledgment

The authors are grateful to the Fundac¸ão Araucária (Grant 24/2012:ProgramaUniversal/PesquisaBásicaeAplicada),the Post-graduatePharmaceuticalSciencesProgram,andUEPG(C-LABMU laboratory)forfinancialsupportandfellowships.

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