Rev. bras. farmacogn. vol.27 número3

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

Original

Article

Anatomy

and

histochemistry

of

leaves

and

stems

of

Sapium

glandulosum

Evelyn

Assis

de

Andrade

_,

_Daniela

_Gaspardo

_Folquitto

_,

_Lívia

_Eidam

_Camargo

_Luz

_,

Kátia

Sabrina

Paludo

_,

_Paulo

_Vitor

_Farago

_,

_Jane

_Manfron

_Budel

b_{DepartamentodeCiênciasFarmacêuticas,UniversidadeEstadualdePontaGrossa,PontaGrossa,PR,Brazil} c_{DepartamentodeFarmácia,UniversidadeEstadualdeMaringá,Maringá,PR,Brazil}

d_{DepartamentodeBiologiaEstrutural,MoleculareGenética,UniversidadeEstadualdePontaGrossa,PontaGrossa,PR,Brazil}

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t

i

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f

o

Articlehistory:

Received10August2016 Accepted10January2017 Availableonline16February2017

Keywords:

Druses Euphorbiaceae Pau-leiteiro

Pharmacobotanicalstudy Latex

Qualitycontrol

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SapiumbelongstoEuphorbiaceaefamilyandcomprises23species.Sapiumglandulosum(L.)Morongis popularlyknowninBrazilas“pau-leiteiro”and“leitosinha”anditisusedintraditionalmedicineto cicatrisation.Itsleafextractshaveshownanalgesic,anti-inflammatoryandantibacterialactivities.The preliminarysetofpharmacognostictoolsusedforqualityassessmentofmedicinalplantpartsis macro-andmicro-anatomyandS.glandulosumhasnotanatomicalandhistochemicaldescription.Thustheaim ofthisstudywastoinvestigatetheanatomicalandhistochemicalcharacteristicsoftheleafandstem ofS.glandulosumasameansofprovidinginformationforqualityassessmentofherbalindustry.The leavesandstemswereinvestigatedbyemployingfieldemissionscanningelectronmicroscopy,light microscopy,andhistochemistrytechniques.TheanalysisshowedthatS.glandulosumhadthefollowing anatomicalfeatures:dorsiventralandamphistomaticleaves;paracyticstomata;tabularcrystaldruses; non-articulatedandbranchedlaticifers;midrib’sbiconvexshapewithvascularsystemsinopenarcwith invaginatedends;petiolewitharoundshapeandslightconcavityontheadaxialside;sixcollateral vascularbundlesinU-shapedorganisation;acircularstemshapeandasclerenchymatousring.Inthe histochemicaltestslipophiliccomponentswerefoundincuticleandinthelatex;phenoliccompounds weremetinthemesophyllandinthelatex;starchgrainswerefoundintheparenchymatoussheath; lignifiedelementsweremetinthesclerenchymatousringinthecortexandintheperivascular scle-renchymatouscaps,beyondinthevesselelements.Thesefeaturesarehelpfulwhenconductingaquality controlprocess.

©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

SapiumJacq. isone ofthemostimportant genusof Euphor-biaceae.It consistsof23acceptedspecies(ThePlantList,2015) anddeserves consideration becauseof thecomplexityinvolved indelimitingitsspecies(Seccoetal.,2012).It isformedmainly byneotropicalspeciesandisdistributedinfields,savannas, sea-sonalforests,rainforestsandwoodlands(SátiroandRoque,2008;

PscheidtandCordeiro,2012).

This genus presents several species that are used in popu-larmedicine,suchasS.chihsinianumS.K.Lee,S.discolor(Champ. ex Benth) Muell. Arg.,S. rotundifolium Hemsl., and S. sebiferum

(L.)Roxb,whichareusedmainlytocicatrisation(AlMuqarrabun

∗ Correspondingauthor.

E-mail:jane@uepg.br(J.M.Budel).

etal.,2014).Somespeciesof Sapiumhavebeenchemicallyand pharmacologicallystudied.Extractsandsinglecomponentsfrom thisgenuswerereportedtohavepromisingbiologicalactivities suchasantioxidant,antimicrobial,andcytotoxiceffects(Hajduand

Hohmann,2012;AlMuqarrabunetal.,2014).

Sapiumglandulosum(L.)Morong,whichispopularlyknownin Brazilas“pau-leiteiro”and“leiteiro”,isatreethatcanreach3–8m inheightandisamongthemostpolymorphicspeciesofSapium.Itis usedintraditionalmedicinetotreathernias(HajduandHohmann,

2012;AlMuqarrabunetal.,2014)anditsusehasbeenpotentially

recommendedfortherecoveryofdegradedareas(Ferreiraetal.,

2009).

Theleavesof S. glandulosumcontainanthracene derivatives, monoterpenes,tanninsandflavonoids(daSilvaetal.,2011,2012). Thisspeciesislatex-bearingandthelatexhasproteinswith con-siderableproteolyticactivity.Thisactivityisnotablyinhibitedby aserineproteaseinhibitor(Sobottkaetal.,2014).Theleafextracts

http://dx.doi.org/10.1016/j.bjp.2017.01.001

Classifying medicinalplants isa serious problembecauseof theircommonnames.Asinglemedicinalspeciesfrequentlyhas anumberofpopularnamesandapopularnamecanoccasionally beusedforarangeofplants(Uptonetal.,2011).Somespeciesof

Sapium,suchasS.glandulosum,S.arbutum(Müll.Arg.)Huberand

S.sellowianum(Müll.Arg.)KlotzschexBaill(Agraetal.,2008),are popularlyknowninBrazilas“pau-leiteiro”,“leiteiro”or “burra-leiteira”.Inthiscontextthemostimportantconsequenceinregard totheuseofinappropriatefolknamesisthesubstitutionof ther-apeuticand safeherbsby toxicvegetable species(Uptonetal.,

2011).

Thepreliminarysetofpharmacognostictoolsusedforquality assessmentofmedicinalplantpartsismacro-andmicro-anatomy

(Uptonetal.,2011).Consequently,theaimofthisstudywasto

investigatetheanatomicalandhistochemicalcharacteristicsofthe leafandstemofS.glandulosumasameansofprovidinginformation forqualitycontrolintheherbalindustry.Furthermore,thereare nopreviouspapersintheliteratureaboutthepharmacobotanical characteristicsofthistaxon.

Materialsandmethods

Plantmaterial

The leaves and stems of Sapium glandulosum (L.) Morong, Euphorbiaceae,werecollectedfromgrownspecimensinopenand sunnyareasintheCamposGeraisregionofParaná(24◦₁₈′_Sand

49◦₃₇′_{W),BrazilinOctober2013.Matureleavesandstems(atleast}

tensamples)obtainedfromthesixthnodeandbelow(median, intercostalandmarginregions),aswellasstemfragmentsfrom 5to15cmfromtheshootwerepreparedforthe pharmacobotan-icalassays.Theplantmaterialcontaininginflorescenceswasused toprepareavoucherspecimen,whichwasidentifiedbyOsmardos SantosRibasandstoredattheMuseuBotânicodeCuritibaunder thenumber390589MBM.

Pharmacobotanicalassays

TheleavesandstemsofS.glandulosumwereplacedinasolution

ofFAA70(Johansen,1940),andstoredin70%ethanol(Berlynand

Miksche,1976).Fortheexaminationofleafandstemmaterial

free-handlongitudinalandcross-sectionswereprepared.Intheleaves itwasincludedthemidrib,interneuralregions,andlateralveins. ThesematerialswerestainedusingAstrablueandbasicfuchsine

(Roeser,1972)andtoluidineblue(O’Brienetal.,1964)toobtain

semi-permanentslides.Thediaphanisationoftheleaveswas per-formedbyfollowingthetechniqueofFuchs(1963).Forthecrystals descriptionsHeetal.(2012)wereused.

Histochemicaltests

Thefollowingstandardsolutionswereemployedinthe histo-chemicaltests:methylenebluetotestformucilage(Oliveiraetal., 2005);hydrochloricphloroglucintorevealtracesoflignin(Sass, 1951);SudanIIIfortestinglipophiliccompounds(Foster,1949); Hoepfner–Vorsatztest, modifiedby Reeve(1951)(aqueous 10% sodiumnitrate,aqueous10%aceticacid,aqueous10%ureaand,2N NaOH)andferricchloridetotestforphenolicsubstances(Johansen, 1940);Bouchardatreactivefornitrogencompounds(Borio,1959); methylenebluetotestmucilage(Oliveiraetal.,2005)and iodine-iodidetorevealstarch(BerlynandMiksche,1976).

PhotomicrographswerecapturedusingaOlympusCX31light microscopethatwasequippedwithaC7070digitalcamera.The semi-permanentandhistochemicaltestslideswerethenanalysed

Fieldemissionscanningelectronmicroscopy(FESEM)and energy-dispersiveX-rayspectroscopy(EDS)

Forthefieldemissionscanningelectronmicroscopy(Mira3 Tes-can)freshleavesandstemswereused.Thesamplesweresubmitted inhighvacuumwithhighacceleratingvoltage(15kV).Thismethod requiredthesamplestobepreviouslydehydratedusingincreasing amountsofethanolthendriedinacriticalpointdryer.Afterwards, theyweresubmittedtometallisationwithgold(Quorum,modelo SC7620).QualitativeX-raymicroanalyseswereperformedon cer-taincrystalsandincellswithoutcrystals(control)usinganEDS machine (Mira 3Tescan)onthe samevariable-pressure micro-scope.Thisprocedurewascarriedoutatthemulti-userlaboratory (LABMU)ofUEPG.

Resultsanddiscussion

TheleavesofS.glandulosum(Fig.1A,B),infrontalview,showed epidermalcellswithstraighttoslightwavyanticlinalwalls(Fig.1C, F), which were relatively thin on both sides. The leaves were amphistomaticandtheparacyticstomatawereobserved predom-inantlyontheabaxialside(Fig.1C–E).Ontheadaxialside,they appeared only near themidrib as observed in Fig. 1F, G. They measured35␮minlengthonaverageandthestriatecuticlewas

tangentiallypositionedinthesubsidiarycells(Fig.1C–E).Metcalfe

andChalk(1950)reportedparacyticstomataintheEuphorbieae

tribe.ValleandKaplan(2000)reportedthatS.glandulosumhad amphistomaticleaves,whileS.sellowianum(Müll.Arg.)Klotzschex Baill.hadhypostomaticleaves.Theseauthorsaffirmedthatthe dis-tributionofstomatawasataxonomicfeaturethathelpstoseparate thesetwospecies.

Incross-section,theepidermiswasuniseriateandthecellswere largerontheadaxialside.Thecuticlewassmoothandthinand reactedwithSudanIIIinthehistochemicaltest(Fig.1H).Druses werefoundintheepidermalcells(Fig.1H).Themesophyllwas dor-siventralandwasformedbyonelayerofpalisadeparenchymaand abouteightlayersofspongyparenchyma.Smallcollateralvascular bundles were immersed in the mesophyll and they were sur-roundedbyaparenchymatoussheath.Druseswerealsoobserved inthemesophyll(Fig.1H).

Phenoliccompoundsaresecondarymetabolitesresponsiblefor adaptationandresistancetohostileenvironmentfactors.Theyare implicatednotonlyinthedefensemechanismsofplantsagainst fungalpathogensbutalsoagainstinsectherbivores(Lattanzioetal., 2006).Inthepresentstudy,phenoliccompoundsreactedpositively withferricchlorideandHoepfner–Vorsatztestandtheyarefound inthemesophyll.

Themidrib,intransection,wasbiconvex;however,the convex-ity wasmore conspicuousontheabaxial surface(Fig.2A).The epidermisis uniseriate and it is covered bya striate and thick cuticle.ThecuticlereactedwithSudanIII(Fig.2C).Thecuticleis themostimportantbarrieragainstuncontrolledwaterlossfrom leaves,stems,fruitsandotherpartsofhigherplants(Riedererand

Schreiber,2001).Cutinisthemaincomponentofthecuticleandis

alipophilicpolymerthatisdepositedinandthetopoftheouter wallepidermalcells(Uptonetal.,2011).Cuticleornamentationis oneofthemostusefultaxonomiccharacteristicsofepidermisin leavesappearingasstriations,ridges,orpapillae(Barthlottetal.,

1998;Uptonetal.,2011).

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Fig.1. Sapiumglandulosum(L.)Morong,Euphorbiaceae.(A)Aspectofaerialvegetativeorgans,inhabit.(B)Leaves,showingabaxial(ab)andadaxial(ad)sides.(C)Abaxial sideofepidermisinfrontalview,showingstomata(st)andstriatecuticle(ct).(D)Abaxialsideofepidermisinfrontalview,showingstomata(st)andstriatecuticle(ct) (FESEM–fieldemissionscanningelectronmicroscope).(E)Detailofthestomata(st),epicuticularwaxes(wa)andstriatecuticle(st),showingmeasureofthestomatainthe abaxialepidermis–FESEM.(F)Adaxialsideofepidermisinsurfaceview,indicatingstomata(st).(G)Adaxialsideofepidermisinsurfaceview,indicatingstomata(st)near themidrib(md)–FESEM.(H)Leafincross-sectionindicatingcuticle(ct),druses(dr),epidermis(ep),palisadeparenchyma(pp),spongyparenchyma(sp).Scalebar=1cm (A,B),10␮m(E),50␮m(C,D,F,H),and100␮m(G).

openarcwithinvaginatedends,whichwassurroundedbya scle-renchymaticsheath(Fig.2A,B).Theorganisationofvascularsystem isrelevantfeatureofspeciescharacterisationanddifferentiation

(Almeidaetal.,2017;Bobeketal.,2016).Inthemidriboftheleaf

ofEuphorbiaceae,theorganisationofthevasculartissueswas

vari-able(Gaucher,1902).ThevascularsystemorganisationcanhelpS.

glandulosumidentification.

Severaldruseswereevidentinthegroundparenchyma,mainly nearthesclerenchymaticsheath(Fig.2B,E,G).Drusesare consid-eredclustercrystalsandareformedbyaggregateshavingseveral sidesand sharppoints(Uptonet al.,2011)asblockys,tabulars, styloids,andtetrahedralcrystals(Heetal.,2012).Inthepresent

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Fig.2.Sapiumglandulosum(L.)Morong,Euphorbiaceae–Midrib.(A)Generalaspectincross-section(FESEM).(B)Detailofthevascularsysteminreactionwithhydrochloric phloroglucin,indicatingcrystal(cr),sclerenchyma(sc),phloem(ph)andxylem(xy).(C)Detailoftheabaxialside,showingcollenchyma(co),cuticle(cu)inreactionwith SudanIII,epidermis(ep),groundparenchyma(gp).(D)LaticiferinFESEM.(E)Phloem(ph)region,evidencingcrystals(cr),sclerenchyma(sc),laticifers(la)withlatex(lx). (F)LaticiferinlongitudinalsectioninreactionwithHoepfner–Vorsatzmodifiedreagent,crystals(cr)andstarchgrains(sg).(G)Tabularcrystaldruse(cr)-FESEM.Bar=2␮m

(G),10␮m(D),50␮m(C,E,F),100␮m(B),and200␮m(A).

Pompert(1989)reportedthatthelaticifersweresmallerandless

frequentinS.haematospermumthaninS.longifolium.

Accordingto Demarcoet al. (2013), Euphorbiaceae presents non-articulatedbranchingandarticulatedanastomosinglaticifers. These authorsreportedthat S. haematospermumpresented two articulatedlaticiferssystemsin theleafandstem,onethatwas formedof narrowlaticifersand theother shapedby wide lati-cifers.Non-articulatedlaticifersareinitialisedfromsinglecellsat anearlyperiodinseedlinggrowth;articulatedlaticifersareformed

bychainsofcells whose adjoiningwallscanoccasionallybreak down,formingvessels(Rudall,1987).

ep

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Fig.3.Sapiumglandulosum(L.)Morong,Euphorbiaceae–Petiole.(A)Generalaspect.(B)Collenchyma(co)andepidermis(ep).(C)Vascularsystem,showingvascularbundle (vb),phloem(ph),xylem(xy)andcrystals(cr).(D)Detailofthestarchgrainsinreactionwithiodine-iodide.(E)Longitudinalsection,showinglaticifer(la)inreactionwith ferricchloride.(F)Longitudinalsectionindicatinglaticifer(la)inreactionwithSudanIII.Bar=25␮m(D),50␮m(B,C,E,F),and200␮m(A).

insectherbivores(Konno,2011).Itisfoundinthevacuoleof secre-torycellsknownaslaticifers,whichincludeacomplexcombination ofcompoundssuchasphenolics,enzymes,terpenes,alkaloids, vita-mins,mucilage,andlipids(Hageletal.,2008;Folquittoetal.,2014; Luzet al.,2015).Latexhasbeenattributedwithcytotoxic(Luz etal.,2015),anti-tumour(Biscaroetal.,2013),anti-ulcer(Costa etal.,2012),andproteinase(Sobottkaetal.,2014)activities,among others.

The petiole in the middle part and in cross-section had an almostroundshape,however,withaslightconcavityonthe adax-ialside (Fig.3A).Theepidermishad thesamecharacteristicsas theleafbladeandreactedwithSudanIIIinthemicrochemicaltest. Beneaththeepidermis,therewereabouteightlayersofangular col-lenchyma(Fig.3B).Laticifers(Fig.3E,F)aspreviouslydescribedfor themidrib,couldbeobservednearthevascularbundles.Druses werefoundinthegroundparenchyma.Thevascularsystemwas formedbyaboutsixcollateralvascularbundles(Fig.3C)ina U-shapedorganisation.Almeidaetal.(2016)andBobeketal.(2016)

indicatedtheimportanceoftheshapeandvascularpatternofthe petioleincross-sectionandaffirmedthatthesecharacteristicscan beusedasgoodmarkersinplants.

Starch is widelydistributed throughout plant tissues, but is commonlyfoundinhighestconcentrationsinroots,rhizomes,and fruits(Uptonetal.,2011).Inthepresentstudystarchgrainswere metinthegroundparenchymaofthemidrib(Fig.2F)andinthe

vascularbundlesheathofpetioleandreactedwithiodine-iodide in the histochemical test (Fig. 3D). Not only the presence but alsothestructureofstarchgranulescanbeimportantfortaxon identification(Uptonet al., 2011).In S. glandulosumtheywere very smalland rounded and/orovate and appeared compound aggregatesoftwoormoregranules.

Intransection,thestempresenteda circularshape (Fig.4A). The epidermisappeared in a singleseries withthickened cuti-cle.Therewereseverallayersof cellsin thecortex(Fig.4B,C). Thesclerenchymawasformedbythickenedcellscontaininglignin, leadingtoasclerenchymatousringinthecortex(Fig.4C)andinthe perivascularsclerenchymatouscaps(Fig.4B).Theyreactedwith hydrochloricphloroglucinreagent(Fig.4C)andwithmethylene blue(Fig.4B).Ligninispolymerhighphenolicswhichconferswater resistance,strength,andelasticity.Itisdepositedamongthe cel-lulosemicrofibrilsofprimaryand/orsecondarycellwalls(Upton etal.,2011).Thepresenceofperivascularsclerenchymatouscaps helpedtheS.glandulosumidentification.

Theendodermiswasformedbyalayerofcells.Thevascular cylinderpresented cambia,formingphloemoutwardand xylem inward. As expected,lignin was also found in vessel elements

(Figs.2B,3C,4B).Laticifers,aspreviouslyreportedforthemidrib

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Fig.4.Sapiumglandulosum(L.)Morong,Euphorbiaceae–Stem.(A)GeneralaspectinFESEM.(B)Cross-section,showingcortex(cx),phloem(ph),sclerenchyma(sc)and xylem(xy).(C)Detailofthecellsofthesclerenchymatousring,cortex(cx)andepidermis(ep).(D)Longitudinalsection,showinglaticifers(la).(E)Tabularcrystaldruses(cr) inFESEM.(F)Cortexshowingseveralcrystals(cr).ScaleBar=2␮m(E),50␮m(B–D,F),and200␮m(A).

Crystalidioblasts were gathered in the stem (Fig.4E,F), as observedinthemesophyll,midribandpetiole.Inthepresentstudy, thecrystalswereanalysed fortheirelementalcomposition and thespectrashowedprominentpeaksforcalcium(27.74%),carbon (16.7%)andoxygen(55.56%),ascanbeseeninFig.5,indicatingthat thesecrystalswereformedofcalciumoxalate.Crystalshavebeen

identifiedinsomestudiesascalciumoxalatebyusingEDS(Heetal.,

2012;Almeidaetal.,2016;Swiechetal.,2016).

The functionsof crystalsin plants are toact as an internal reservoirforcalcium,toprovidetissuerigidity,ionicbalance,to remove calcium, magnesium, oxalicacid, aluminium and other heavymetals,andalsotoactasaprotectivedeviceagainst

for-20

10

0

Ca

O

C

Ca

Spectrum 2

0 2 4 6 8 10 12 14 16 18 keV

cps/eV

aginganimals(FranceschiandNakata,2005;Heetal.,2012;Silva etal.,2014).Thepresenceorabsenceofcrystals,theirtype and theirchemicalcomposition,canbecharacterisedastaxonomic fea-tures(Meric,2009).Withreferencetothechemicalcomposition, excesscalciumishabituallyprecipitatedincalciumsaltssuchas carbonate,citrate,malate,oxalate,phosphate,silicateandsulphate

(WeinerandDove,2003).

Crystals of calcium oxalateare most commonly reported in higherfamiliesandtheyoccurinmostorgansandtissuesinthe vegetablespecies.However,theirsizeandnumberareresponsive tochanges intheconcentration ofcalcium intheenvironment

(Nakata,2003;FranceschiandNakata,2005).Crystalsofcalcium

oxalateareformedfromendogenouslysynthesisedoxalicacidand Catakenfromtheenvironment,andtheyareformedand accumu-latedinspecies-specificmorphologies(Meric,2009).

Anatomicalcharacterisationisaninherentpartofpracticallyall pharmacopoeiasandisoneoftheprimaryidentificationtests nec-essaryfor pharmacopoeialcompliance.Theindividualstructural elementsarecomparativelyfrequentwithinthesameplantorgans, butthewaysinwhichthetissues,elementsandcellsaresetwithin aplantorganpermitsdiagnosistobeperformedandlendsupport tothequalityassessmentofherbaldrugs(Uptonetal.,2011).

Themainanatomicalcharacterswerehighlightedinthis phar-macobotanical study, which was performed to provide more informationaboutthestandardisationoftheS.glandulosumspecies in orderto supportthequality control of this vegetable mate-rial.Thefollowingcharacteristicsarehelpfulwhenconductingthe qualitycontrolprocess:dorsiventraland amphistomaticleaves; paracyticstomata;calciumoxalate tabularcrystal druses; non-articulatedandbranchedlaticifers;biconvexwithvascularsystems inopenarcwithinvaginatedends;petiolewithroundshapeand slightconcavityontheadaxialside;sixcollateralvascularbundles inU-shapedorganisation;circularstemshape,sclerenchymatous ringinthecortexandperivascularsclerenchymatouscaps.

Thehistochemicaltestshowedthepresenceofthelipophilic andphenoliccompoundsinthelatex;phenoliccompoundsinthe mesophyll;starchgrainssmallandroundedandcompound aggre-gatesoftwoormoregranules;lignifiedelementsweremetinthe sclerenchymatousringinthecortexandintheperivascular scle-renchymatouscaps,beyondinthevesselelements.

Authors’contributions

EAA,DGF,LECL andKSPassisted in carryingout the labora-torywork.EAAcontributed incollectingtheplantmaterial and itsidentification.PVFperformedthescanningelectronmicroscopy (FESEM)analysis.JMBcreatedtheproject,supervisedthe labora-torywork,andwrotethepaper.Alltheauthorshavereadthefinal manuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorswouldliketothanktheElectronMicroscopyCentre oftheLABMUattheStateUniversityofPontaGrossaforproviding theFESEMimagesandEDSspectra.

References

Agra,M.F.,Silva,K.N.,Basílio,I.J.L.D.,deFreitas,P.F.,Barbosa-Filho,J.M.,2008.Survey ofmedicinalplantsusedintheregionNortheastofBrazil.Rev.Bras.Farmacogn. 18,472–508.

AlMuqarrabun,L.M.R.,Ahmat,N.,Aris,S.R.S.,2014.Areviewofthemedicinaluses, phytochemistryandpharmacologyofthegenusSapium.J.Ethnopharmacol.155, 9–20.

Almeida,V.P.,Hirt,A.A.,Raeski,P.A.,Mika,B.E.,Justus,B.,dosSantos,V.L.P.,Franco, C.R.C.,dePaula,J.P.,Farago,P.V.,Budel,J.M.,2017.Comparative morphoanatom-icalanalysisofMikaniaspecies.Rev.Bras.Farmacogn.27,9–19.

Barthlott,W.,Neinhuis,C.,Cutler,D.,Ditsch,F.,Meusel,I.,Theisen,I.,Wilhelmi,H., 1998.Classificationandterminologyofplantepicuticularwaxes.Bot.J.Linn. Soc.126,227–236.

Berlyn,G.P.,Miksche,J.P.,1976.BotanicalMicrotechniqueandCytochemistry.Iowa StateUniversity,Ames.

Biscaro,F.,Parisotto,E.B.,Zanette,V.C.,Günther,T.M.F.,Ferreira,E.A.,Gris,E.F., Cor-reia,J.F.G.,Pich,C.T.,Mattivi,F.,WilhelmFilho,D.,Pedrosa,R.C.,2013.Anticancer activityofflavonolandflavan-3-olrichextractsfromCrotonceltidifoliuslatex. Pharm.Biol.51,737–743.

Bobek,V.B.,Heiden,G.,Oliveira,C.F.,Almeida, V.P.,dePaula,J.P.,Farago,P.V., Nakashima,T.,Budel,J.M.,2016.Comparativeanalyticalmicrographsof vas-souras(Baccharis,Asteraceae).Rev.Bras.Farmacogn.26,665–672.

Borio,E.B.L.,1959.LobelialangeanaDusén.Contribuic¸ãoparaoestudo farmacognós-tico.daFaculdadedeFarmáciadaUniversidadedoParaná,Brasil,86pp.Tesepara concursoàdocêncialivredacadeiradefarmacognosia,FaculdadedeFarmácia daUniversidadedoParaná.

Costa,L.L.G.,David,V.C.,Pinto,R.M.C.,Minozzo,B.R.,KozlowskiJr.,V.A.,Campos,L.A., Silva,R.Z.,Beltrame,F.L.,2012.Anti-ulceractivityofSynadeniumgrantiilatex. Rev.Bras.Farmacogn.22,1070–1078.

daSilva,C.H.T.P.,Sobrinho,T.J.S.P.,Saraiva,A.M.,Pisciottano,M.N.C.,deAmorim, E.L.C.,2012.Phytochemicalprofileandantibacterialactivityofbarkandleaves

ofCaesalpiniapyramidalisTul.andSapiumglandulosum(L.)Morong.J.Med.Plants

Res.6,4766–4771.

daSilva,C.H.T.P.,Sobrinho,T.J.S.P.,eCastro,V.T.N.A.,Lima,D.C.A.,deAmorim,E.L.C., 2011.AntioxidantcapacityandphenoliccontentofCaesalpiniapyramidalisTul.

AndSapiumglandulosum(L.)MorongfromNortheasternBrazil.Molecules16,

4728–4739.

Demarco,D.,Castro,M.M.,Ascensão,L.,2013.TwolaticifersystemsinSapium

haematospermum–newrecordsforEuphorbiaceae.Botany91,545–554.

Demarco,D.,Kinoshita,L.S.,Castro,M.M.,2006.Laticíferosarticulados anastomosa-dos:novosregistrosparaApocynaceae.Rev.Bras.Bot.29,133–144.

Ferreira, B.G.A., Zuffellato-Ribas, K.C., Carpanezzi, A.A., Tavares, F.R., Koehler, H.S.,2009.Metodologias deaplicac¸ãodeAIBnoenraizamento deestacas semilenhosasdeSapiumglandulatum(Vell.)Pax.Rev.Bras.PlantasMed.11, 196–201.

Folquitto,D.G.,Budel,J.M.,Pereira,C.B.,Brojan,L.E.F.,Folquitto,G.G.,Miguel,M.D., Silva,R.Z.,Miguel,O.G.,2014.Analyticalmicrographyandpreliminary phyto-chemistryoftheleavesandstemsofLobeliaexaltataPohl.(Campanulaceae). Lat.Am.J.Pharm.33,245–250.

Foster,A.S.,1949.PracticalPlantAnatomy,2nded.D.VanNostrand,Princeton. Franceschi,V.R.,Nakata,P.A.,2005.Calciumoxalateinplants:formationand

func-tion.Annu.Rev.PlantBiol.56,41–71.

Fuchs,C.H.,1963.FuchsinstainingwithNaOHclearingforlignifiedelementsof wholeplantsorplantsorgans.StainTechnol.38,141–144.

Gales,R.C.,Toma,C.,2007.Researchesregardingthemorphology,structureand dis-tributionofvegetativeorgansofsomeEuphorbiaspeciesfromRomania’sflora. Biol.Veg.1,40–45.

Gaucher,L.,1902.RecherchesanatomiquessurlesEuphorbiacées.Ann.Sci.Nat.Bot. 15,161–309.

Hagel,J.M.,Yeung,E.C.,Facchini,P.J.,2008.Gotmilk?Thesecretlifeoflaticifers. TrendsPlantSci.13,631–639.

Hajdu,Z.,Hohmann,J.,2012.Anethnopharmacologicalsurveyofthetraditional medicineutilizedinthecommunityofPorvenir,BajoParaguáIndian Reserva-tion,Bolivia.J.Ethnopharmacol.139,838–857.

He, H., Bleby, T.M., Veneklaas, E.J., Lambers, H., Kuo, J., 2012. Morpholo-gies and elemental compositions of calcium crystals in phyllodes and branchletsofAcaciarobeorum(Leguminosae:Mimosoideae).Ann.Bot.109, 887–896.

Johansen,D.A.,1940.PlantMicrotechnique.McGrawHillBook,NewYork. Konno,K.,2011.Plantlatexandotherexudatesasplantdefensesystems:rolesof

variousdefensechemicalsandproteinscontainedtherein.Phytochemistry72, 1510–1530.

Lattanzio,V.,Lattanzio,V.M.T.,Cardinali,A.,2006.Roleofphenolicsintheresistance mechanismsofplantsagainstfungalpathogensandinsects.In:Imperato,F. (org),Phytochemistry:AdvancesinResearch.ResearchSignposts,Trivandrum, pp.23–67.

Luz,L.E.C.,Paludo,K.S.,Santos,V.L.P.,Franco,C.R.C.,Klein,T.,Silva,R.Z.,Beltrame, F.L.,Budel,J.M.,2015.Cytotoxicityoflatexandpharmacobotanicalstudyof leavesandstemofEuphorbiaumbellata(Janaúba).Rev.Bras.Farmacogn.25, 344–352.

Meric,C.,2009.CalciumoxalatecrystalsinsomespeciesofthetribeInuleae (Aster-aceae).ActaBiol.Crac.Ser.Bot.51,105–110.

Metcalfe,C.R.,Chalk,L.,1950.AnatomyoftheDicotyledons.ClarendonPress,Oxford. Nakata,P.A.,2003.Advancesinourunderstandingofcalciumoxalatecrystal

forma-tionandfunctioninplants.PlantSci.164,901–909.

O’Brien,T.P.,Feder,N.,McCully,M.E.,1964.Polychromaticstainingofplantcellwalls bytoluidineblueO.Protoplasma59,368–373.

Oliveira,F.,Akisue,G.,Akisue,M.K.,2005.Farmacognosia.Atheneu,SãoPaulo. Pompert,M.G.,1989.Estudiomorfo-anatomicodedosespeciesdeSapium

Reeve,R.M.,1951.Histochemicaltestsforpolyphenolsinplanttissues.StainTechnol. 26,91–96.

Riederer,M.,Schreiber,L.,2001.Protectingagainstwaterloss:analysisofthebarrier propertiesofplantcuticles.J.Exp.Bot.52,2023–2032.

Roeser,K.R.,1972.DieNadelderSchwarzkiefer-MassenproduktundKunstwerkder Natur.Mikrokosmos61,33–36.

Rudall,P.J.,1987.LaticifersinEuphorbiaceaeaconspectus.Bot.J.Linn.Soc.94, 143–163.

Sass,J.E.,1951.BotanicalMicrotechnique,2nded.IowaStateCollege,Ames. Sátiro,L.N.,Roque,N.,2008.AfamíliaEuphorbiaceaenascaatingasarenosasdo

médiorioSãoFrancisco,BA,Brasil.ActaBot.Bras.22,99–118.

Secco,R.S.,Cordeiro,I.,Senna-Vale,L.,Sales,M.F.,Lima,L.R.,Medeiros,D.,Haiad,B.S., Oliveira,A.S.,Caruzo,M.B.R.,Carneiro-Torres,D.,Bigio,N.C.,2012.Anoverview ofrecenttaxonomicstudiesonEuphorbiaceaes.l.inBrazil.Rodriguésia63, 227–242.

macropadrões.ActaAmazon.44,435–446.

Sobottka,A.M.,Tonial,F.,Sytwala,S.,Melzig,M.,2014.Proteinaseactivityinlatexof threeplantsofthefamilyEuphorbiaceae.Braz.J.Pharm.Sci.50,559–565. Swiech,J.N.D.,Bobek,V.B.,Folquitto,D.G.,Silva,R.Z.,Budel,J.M.,Farago,P.V.,Miguel,

M.D.,Miguel,O.G.,2016.Morpho-anatomyofthevegetativeorgansof

Philoden-dronmeridionale(Araceae).Lat.Am.J.Pharm.35,2142–2148.

ThePlantList,2015.Sapium,http://www.theplantlist.org/browse/A/Euphorbiaceae/ Sapium/(accessedOctober2015).

Upton,R.,Graff,A.,Jolliffe,G.,Länger,R.,Williamson,E.,2011.AmericanHerbal Pharmacopoeia:BotanicalPharmacognosy–MicroscopicCharacterizationof BotanicalMedicines.CRCPress,BocaRaton.

Valle,L.S.,Kaplan,M.A.C.,2000.Sapiumglandulatumcomplex(Euphorbiaceae).An. Acad.Bras.Cienc.72,293–294.