Rev. bras. farmacogn. vol.27 número5

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Original

Article

Anatomical

characterization

of

ultra-structures,

biominerals

and

histolocalization

of

metabolites

in

leaves

of

Genipa

americana

Alex

L. Vasconcelos

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_,

_Andréa

_V.

_Santos

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_,

_Rafael

_J.R.

_Padilha

b

_,

_Luiz

_C.

_Alves

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_,

_Karina

_P.

_Randau

a,∗

a_Laboratório_de_{Farmacognosia,}_Departamento_de_Ciências_{Farmacêuticas,}_Universidade_Federal_de_Pernambuco,_Recife,_PE,_Brazil b_Setor_de_Microscopia_Eletrônica,_Laboratório_de_{Imunopatologia}_Keizo_Asami,_Universidade_Federal_de_Pernambuco,_Recife,_PE,_Brazil

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Articlehistory:

Received13April2017 Accepted19May2017 Availableonline13June2017

Keywords:

Crystalsmorphotype Histochemistry Histolocalization Jenipapo Leafanatomy

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InsertedinoneofthelargestfamiliesamongtheAngiosperms,GenipaamericanaL.,Rubiaceae,canbe foundinallBrazilianterritory,presentinggreatmedicinalimportance,whereseveraluseshavebeen attributed.Inviewofthis,thisworkhasthepurposeofanalyzingtheultrastructural,biomineral, phyto-chemicalandhistochemicalcharacteristicsoftheleavesofthisnativespeciesfromBrazil.Forthis,light microscopy,polarizationandscanningelectronmicroscopytechniqueswereusedwithX-rayscattering energy,associatedtochromatographicandhistochemicaltests.Theanatomicalultrastructural charac-teristicsoftheleavesdetailedinformationaboutthetypeandarrangementofthecuticle,trichomes, surfaceandarrangementofthetissuesthatdeterminethebotanicalidentityofthisspecies.The phyto-chemicaltestsalloweddeterminingtheirchromatographicpatternandhistochemistrytodeterminethe exactstoragesiteofthesesubstancesintheleaf.Itwasobservedthatthecharacterizationofthe crys-tallinemacro-patternpresentintheanalyzedspecies,aswellasitsexactelementalcomposition,canbe consideredanimportantdifferentialdiagnosisfactor.Theresultscharacterizetheleavesofthisspecies indifferentaspects,beinganativespeciesandpharmacologicallypromising,withdifferentpopularuses andprovenpharmacologicalactivities,andmoreindepthstudiesisneeded.

Introduction

Rubiaceaeisconsideredasoneofthelargestfamiliesamong Angiosperms,itcomprisesthefourthlargestnumberofspecies, withapproximately13,000speciesdistributedin550genera.The

Genipagenusischaracterizedbytwospecies:Genipaamericana

L.andG.infundibuliformisZappi&Semir(Delpreteetal.,2005). Amongthestudiescarried outonthegenus,theresearchesare concentratedontheG.americanaspecies.

Popularlyknownasjenipapo,thisspeciescanbefoundallover Brazilianterritory.Itis anativeplantofBrazil,of arborealsize, reachingupto30minheight(Corrêa,1984;Delpreteetal.,2005;

Zappi,2015).Naturalists,whovisitedBrazilinthecolonialperiod,

havelongdescribedtheuseofG.americanaasabodydye(Alves,

2010,2013).

Severalmedicinaluseshavetraditionallybeenattributedtothis species.Theleafdecoctionhasanantidiarrheal orantisyphilitic

∗ _{Corresponding}_author.

E-mail:karina.prandau@ufpe.br(K.P.Randau).

action, whereas theleaf infusion is used against liver diseases

(Corrêa,1984).Basedonethnobotanicalsurveys,Souzaetal.(2013)

point outthetraditionaluseofthis speciesin thetreatmentof cough,anemia,contusions,dislocations;asdepurativeand associ-atedwithpopularbeliefs.Amongthesecondarymetabolitesfound inthisspecies,ithighlightsthepresenceoftannins(Revilla,2001), monoterpenes(Onoetal.,2007),steroids(Conceic¸ãoetal.,2011) andmostlyiridoidswhicharecharacteristicoftheRubiaceaefamily

(Jensen,1983;UedaandIwahashi,1991;Onoetal.,2005).

DifferentstudieshaveindicatedthepotentialofG.americanafor useinthetreatmentofasthma(Dengetal.,2013),ophthalmic dis-eases(Koriyamaetal.,2013;Songetal.,2013),antiviralaction(Lin etal.,2013),cardiovasculardisorders(Zhangetal.,2013), antide-pressantactivity(Tianetal.,2010),inthetreatmentofAlzheimer’s disease(Nametal.,2013;Gaoetal.,2014;Wangetal.,2012;Zhang etal.,2012),anti-inflammatory(Fuetal.,2012;Lietal.,2012)(Kim

etal.,2012;Dandoetal.,2013;Yangetal.,2013).

In view of the recognized popular use of this species and distinct pharmacological studies demonstrating its therapeutic potential,thisworkaimsatanalysingtheanatomical ultrastruc-tural,biomineral,phytochemicalandhistochemicalcharacteristics ofG.americana,nativetoBrazil.

http://dx.doi.org/10.1016/j.bjp.2017.05.002

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Materialsandmethods

Plantmaterial

GenipaamericanaL.,Rubiaceae,matureleaveswerecollected fromadultspeciesonthecampusoftheUniversidadeFederalde Pernambuco(−8,0464168,−34,9451315),Recife,NortheastBrazil,

atOctober12,2015,inthefruitingseason.Thevoucherspecimen wasdepositedat theHerbariumDárdanode AndradeLima,for botanicallegitimation,bythenumber89999.

Anatomicalcharacterization

Matureleaveswerecollectedfromsecondorthirdnode,from uptofiveindividuals.ThesampleswerefixedinFAA50(Johansen, 1940). Crossand paradermicsections of themiddle portion of theleaflets werefreehandobtained, submittedtodouble stain-ingprocesswithastrablueandsafranin(Johansen,1940;Kraus

andArduin,1997)andmountedinglycerineonsemi-permanent

slides.Analyzeofthestructureswasperformedusingopticaland polarizedlightmicroscopy.

Phytochemistrydetermination

The methanolicextract from fresh leaveswas processed by infusion (0.1%) and subsequently analyzed by thin-layer chro-matography(TLC)(Kieselgel 60, 0.2mm, Merck),withdifferent eluents systems and reagents to search alkaloids, mono- and sesquiterpenes,triterpenesandsteroids,saponins,flavonoids, cin-namicderivativesandphenylpropanoidglycosides,hydrolyzable andcondensedtannins.Phytochemicalscreeningoftheextractwas carriedoutusingaconventionalprotocol(Harborne,1998;Randau

etal.,2004).

Histochemicalanalyses

Freehandcrosssectionsfromthemiddleportionoffreshleaves weresubjectedtoreactionwithspecificreagents,accordingtothe differentgroupsofmetabolitestobeinvestigated:Lugolreagent

(Johansen, 1940)for starch,Sudan IV (Krausand Arduin, 1997)

fordetectionofepicuticlewax,acidphloroglucin(Jensen, 1962) forlignins,potassiumdichromate(Gabe,1968)forphenolic com-pounds,NADIreagent(DavidandCarde,1964)foressentialoilsand oil-resins,vanillin-sulphuricacid(WagnerandBladt,1996)for iri-doids,antimonytrichloride(MaceandHowell,1974)forsteroids, vanillin-hydrochloricacid(Maceand Howell,1974)fortannins, Dragendorffreagent(YoderandMahlberg,1976)foralkaloidsand hydrochloricacid(Johansen,1940)for calciumoxalatecrystals. Foreachprocedureemployedonthesamplesslideswithoutany treatment(blanks)werealsomounted.Imagesdocumentationwas performedwithalightmicroscope(Alltion)equippedwithadigital camera.

Scanningelectronmicroscopy

Crosssectionsobtainedbyhandwerefixedwith2.5% glutaralde-hyde(GA)+4%formaldehydebuffer.Afterfixationandwashing, thedehydratedsamplesbyincreasingethanolicseries(Johansen, 1940)weresubmittedtoCO2CriticalPoint(HCP-2).Afterwards,the samplesweremountedonmetalbracketsandcoatedwithagold layermetalizerQuorumQ150TES(Haddadetal.,1998).Theimages obtainedwereusedin theanatomical characterizationof ultra-structureswithaQUANTA200FEGscanningelectronmicroscope (SEM).

Morphologicalanalyzeandelementalcompositionofcrystals

The chemical microanalyses by EDS were done with X-ray

detectorattachedtotheZeiss-EVO-LS15scanningelectron micro-scope,underthesameoperationalconditions.Absorbancepeaks indicatingrelativeelementalchemicalconcentration≥0.5%were

consideredsignificant.

Results

Theepidermalcells,whichconstitutetheadaxialface,present polygonalcontourscontainingamorphousinclusionswithinthem thatvaryinsize(Fig.1A).Ontheabaxialsidethecellsaresmaller anditcanbenoticedthepresence ofparacyticandanomocytic stomata,restrictedtothisface,characterizingtheleafas hypostom-atic.Severalinclusionsarealsoobservedontheabaxialfaceofthe leafblade,insidetheepidermalcellsandonstomatalguardcells (Fig.1B).TheimagesobtainedbyScanningElectronMicroscopy (SEM)addressthatthecuticlepresentsintheformatofcrystalline projections,suchasanextensivelayerofnanofilamentsthatcover theentireepidermalsurfaceonbothfaces(Fig.1CandD),aswell asonthestomata(Fig.1E).

In cross-section the epidermisis covered by a thick cuticu-larlayer.Theepidermisisunstratified,formedbylargercellson the adaxial side, more elongated in the anticlinal direction,as opposed totheabaxialepidermal cells, smallerand more elon-gatedinthepericlinaldirection.Themesophyllhasadorsiventral organizationandpresentsapalisadeparenchymafacingthe adax-ialface,constitutedbytwo-threelayersofelongatedcells(Fig.1F). Thelacunarparenchymaismulti-stratifiedcomposedofloosely organizedroundedcells,withsmallintercellularspacesbetween them,comprisingtheabaxialregionofthemesophyll.Some sec-ondaryvascularbundlescanbeseendippedintothemesophyll. Thepresenceofscarcemulticellularandglandulartectortrichomes (Fig.1GandH)canbeobservedontheabaxialsurface. Further-more,thepresenceofthesetrichomeswasalsodetectedbyelectron microscopy,revealingsmallandscarceglandulartrichomesonthe abaxialsideofthebladethatareinsertedbetweenthestomataand theepicuticularlayer(Fig.1I).

Themidribpresentsbiconvexsymmetry,beingprominenton theabaxialsurface,withvascularbundlesofconcentric arrange-mentwithnoperivascular sclerenchymalfibers (Fig.1Jand K). At the center of the main vascular cylinder, in the medullary parenchymaregion,thereareaccessoryvascularcaps,ofwhichthe presenceofdrusescanbeconfirmed(Fig.1LandM).Underlyingthe epidermalmonolayerthereareseveralstrataofangular-type col-lenchymacellswherethepresenceofcrystallineinclusionsofdruse typeisrecognized(Fig.1N).Throughpolarizedlightmicroscopy, thepresenceof thesecrystalsthroughout theleafbladecanbe observed,presentingahigherincidenceinthecorticalparenchyma ofthemidrib(Fig.1O).

Phytochemistrydeterminationandhistochemicalcharacterization

Accordingtotheresults, thepresenceofiridoids,mono and sesquiterpenes and of triterpenes and steroids in the extract obtainedfromtheleaveswasobserved. Thetestsalsorevealed thepresenceofhydrolyzabletannins,proanthocyanidins,cinnamic derivativesandphenylpropanoglycosides.Theanalyzesfor alka-loidsandsaponinswerenegative.Theresultsobtainedformedthe basisforperformingthehistochemicaltests.

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ad ab

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pcd pp

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Fig.2. HistolocalizationofmetabolitespresentinleavesofGenipaamericana.Controlsample:(A)Midrib.(B)Mesophyll.Histochemicaltests:(C)Starchgrainsinparenchyma cells.(D)Coveringcuticularepidermalcells.(E)Presenceofligninsinwoodyvascularbundles.(F)Polyphenolspresentinepidermalcellsandpalisadeparenchyma.(G)Volatile oilsinsideabaxialepidermalcells.(H)Iridoidsinsidethepalisadeparenchymacells.(I)Steroidsinperivascularidioblastsinthemidrib.(J)Proanthocyanidinsinsidethe palisadeparenchymacells.cp,cortexparenchyma;ep,epidermis;ew,epicuticularwax;ir,iridoids;lg,lignin;pa,parenchyma;pcd,proanthocyanidins;ph,phloem;phc, phenoliccompounds;pp,palisadeparenchyma;sp,spongyparenchyma;st,starchgrain;std,steroids;vo,volatileoil;xy,xylem.Bars:A=100␮m.B,C,D,E,F,G,H,I,J=25␮m.

cross-sectionsofthemidribandmesophyllwithouttreatmentwith thereagents(Fig.2AandB).Theresultsdemonstratethepresence of starch granules present in the mesophyll, both in the pal-isadeparenchymaandintheepidermalcells(Fig.2C).Theintense accumulationofamyloplastscanalsobeobservedinthemedullary parenchymaregion,inthecenterofthevascularcylinder.

FromthereactionwithSudamIII,thepresenceofathick epi-cuticularwaxylayercanbeobserved,coveringbothsidesofthe blade.Thecuticlepresentsasanorangelayer(Fig.2D).The pres-enceofligninscanalsobeverifiedinthewoodyvascularbundles, characterizedbythepinkcolorconferredbyfloroglucinol(Fig.2E). After reaction with potassium dichromate, the presence of phenoliccompoundscanbeverified,characterizedbyreddish col-oration,distributedthroughouttheleafbladeregion,mostlyinthe

prismaticcellsthatmakeupthepalisadeparenchymaand epider-malcellsoftheadaxialface(Fig.2F).Thevolatileoilspresentin theleavesofG.americanawereevidencedbythereactionwith theNADIreagent,conferringbluecolortothesphericaldroplets describedabove,accumulatedintheepidermalandsubsidiarycells andabaxialepidermalcells(Fig.2G).

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0.15

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O C

Ca

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0 2 4 6 8 10 12

KeV Full scale 261 cts cursor: 0.000

C O Ca

Fig.3.ScanningelectronmicrographandelementalcompositionofthecrystalsobservedinGenipaamericana.(A)Presenceofdruseinthemidriboftheleafincross-section. (B)Detailoftherosette-likedruse(whewellitetype)coveredbythemembraneoftheidioblast.(C)Analysisofelementalcompositionofthecrystal.(D)Percentageofthe chemicalconstituentsofthecrystal.Bars:A=20␮m.B=2␮m.

Thepresenceofproanthocyanidinswascharacterizedbypoor red coloration as wellas theresults found in phytochemistry. Theaccumulationofthesesubstanceswasverifiedinsomecells ofthepalisadeparenchyma,afterreactionwithsulfuricvanillin (Fig.2J).Thereactionwithhydrochloricaciddemonstratedthe sol-ublenatureofthecrystallineinclusions,confirmingthechemical compositionofcalciumoxalateofthesecrystals.

Thedominantcrystallinemacro-patternintheleavesofG. amer-icanaisdrusetypeofcrystalsinrosette.Thistypeofcrystalcanbe foundalongthemesophyll,intheparenchymalcortexandinthe medullaryparenchymaofthemidrib(Fig.3AandB).

Themicroanalysisofdruses,performedbyscanningelectron microscopy (SEM), by energy dispersive X-ray analyze (SEM), revealed three peaks of absorbance for calcium (0.37, 3.7 and 4.0keV),apeakforoxygen(0.37keV)andoneforcarbon(0.25keV). Accordingtotheresultsabove,wecanstatethatthedruseis com-posedmainlyofcalcium(0.13%),followedbyoxygen(0.06%)and carbon(0.02%)(Fig.3CandD).

Discussion

Characteristicssuchashypoestomaticleaf,epidermalcellswith straight anticline walls, paracyticand anomocyticstomata, uni andmulticellulartectortrichomesanddorsiventralmesophyllare commonoccurrencesforthefamily, among therepresentatives ofRubiaceae(Metcalfeand Chalk,1979),asverifiedinTocoyena

formosa(ChamandSchlitd.)K.Schum.(Coelhoetal.,2006)and

PsychotriaviridisRuiz&Pav.(Quinteiroetal.,2006).

Thescarcepresenceofuniandmulticellulartectortrichomes canbeverified,besidesglandulartrichomes.Thepresenceof glan-dular trichomesis a characteristic that differsfromthe results achievedbySantosetal.(2006)andErbanoandDuarte(2010)inG. americanaleaves.Somestudieshaveindicatedthatexogenous fac-torssuchasherbivory,temperature,luminosity,wateravailability andseasonalityareabletoinfluencetheformationandfunctioning ofglandulartrichomesindifferentplantgroups(Viljoenetal.,2005;

Gonzálesetal.,2008;Martínez-Natarénetal.,2011).Thepresence

ofidioblastsisdetectedintheparenchymaticregion,externaland internallytothevascularcylinder,whichpresentcrystalline inclu-sions in theformof druses.Superior plantscommonly present crystalsstoredintheirtissues,beingrelatedtophysical protec-tion, removalof oxalate fromthe metabolicsystem,storage of calcium,andregulationoflightduringphotosynthesis(Franceschi

andNakata,2005).

Thecrystaltypeisanimportantdiagnosticfeatureforgenusand speciesofRubiaceae.Thepresenceofcrystalslikeraphide,druse, styloid,prismaticorcrystallinesandcanbeconfirmedinspecies belonging tothis family, beingcommon thepresence of druse andcrystallinesandforGenipagenus(MetcalfeandChalk,1979). RaphideswerenoticedinPalicourealongepedunculata(Pereiraetal., 2003), crystallinesandin somespecies of thegenusRondeletia

L. (Kocsis et al., 2004) and druse in T. formosa (Coelho et al.,

2006).ErbanoandDuarte(2010)confirmedthepresenceof

cal-ciumoxalatedruse onleavesandstemofG.americana.Moraes

et al.(2011) reportthepresence ofstyloid prismaticand rusty

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ananisotropiccharacteristicofcrystalsthathaveachemical com-positionof asingleelement.Crystals thathavemixed chemical compositionarereferredtoasnon-birefringent,makingit impos-sibletoevaluatethedifferentrefractiveindexesthattheypossess, sincetheypresentalterationsinopticalpropertiesinrelationto purecrystals(Hammond,2009;Heetal.,2012).

Phytochemistrydeterminationandhistochemicalcharacterization

Thecuticleshapeisdependentonthecompoundspresentin thewax.Asymmetricsecondary alcoholsand ␤-diketonesform waxspaces intheformofnanotubes,whereasprimaryalcohols andsymmetricalsecondaryalcoholsformflatplates(Hallam,1967;

Jeffreeetal.,1975).Differencesincuticlepatternscanbe

taxono-micallyusefulingenerictermsandevenbetweenspecies(Metcalfe

andChalk,1979;Cutter,1986).Thefibersareassociatedwiththe

vascularsystem,ascitedbyMetcalfeandChalk(1979)for Rubia-ceae.

Analyses revealed phenolic compounds intensely visible

throughoutthepalisadeparenchyma,beingalsoobservedinthe adaxial epidermal cells of less intense form. Studies have also reportedthepresenceofphenoliccompoundsindifferentspecies oftheRubiaceaefamily,suchasChomeliaobtusaCham.&Schltdl.

(Barrosetal.,2008),PalicourearigidaKunth(Rosaetal.,2010)and

G.americana(Portoetal.,2014).

Moststudiesonessentialoilsinthisspeciesarelimitedtofruits only.Somestudiesindicatethatthesemetabolitesarecomposed mainlyofcarboxylicacids,estersandalcohols(BorgesandRezende,

2000;Pinoetal.,2005;Pintoetal.,2006).Intheanalyzedleaves,

theessentialoilspresentagreaterincidenceofdistributioninthe abaxialface,beingfoundintheformofgranuleswithstoragesite intheepidermalandsubsidiarycells.

Theiridoidsarepresent inmostofthespeciesofRubiaceae, constitutinganimportantchemicalandpharmacologicalmarkerin

G.americana(AlvesandMing,2015).Accordingtotheanalyses,the accumulationsiteofthesesubstanceswasobservedinthephloem cellsandinsomepalisadecells,nearbythesevessels.Thepresence ofthesecompoundsisobservedinEucommiaulmoidesOliv.(Hirata

etal.,2011;Nametal.,2013;Zhangetal.,2013),Belliatrixago(L.)

All.(Vendittietal.,2013),GardeniajasminoidesJ.Ellis(Zhangetal.,

2013),CastillejatenuifloraBenth(Carrillo-Ocampoetal.,2013). InfruitsofG.americanawereidentifiedthepresenceofdifferent phytosteroids(Barbosa,2008).Conceic¸ãoetal.(2011)reportthat thepresenceofsteroidsinextractsofG.americana,beingrelatedto anticanceractivity.Therearefewstudiesthatindicatethepresence ofthesemetabolitesintheleaves. Theassaysperformedin this workrevealedthat,intheleaves,thesesubstancesarestoredin idioblastslocatedintheperivascularregions,closetothephloem. Thephytochemical,resultsinthesearchforproanthocyanidins, contributedwiththehistochemicaltests,wherelittlereddish col-orationwasobservedinsidesomecellsofthepalisadeparenchyma. AccordingtoPortoetal.(2014),thereisalowaccumulationofthese substancesinthisspecies,corroboratingthehistochemicalresults describedfortheleavesanalyzedinthisstudy.

Scanningelectronmicrographs,coupledwithspectrometricand histochemicalassays,revealedthepresence ofcrystalline inclu-sionsofthedrusetypeofrosettecrystals(whewellite)throughout theleafblade.Fromthesetests,thechemicalcompositionof cal-ciumoxalateofthesecrystalswasconfirmed.Thepresenceofthese inclusions,thedeterminationoftheirchemical natureandtheir morphology,aswellasthelocationsitewithinthemedicinalplant, canhelpin thecharacterizationoftaxonomicgroups,becoming

relevantintheidentificationandpurityofplantdrugs(Metcalfeand

Chalk,1979;MonjeandBaran,2002;AnithaandSandhiya,2014).

In the last decades, many plant crystals made of calcium

oxalate have not been accurately identified. This

biomin-eral occurs in two hydration states in plants: monohydrate

(whewellite) or dihydrate (weddellite) (Franceschi and Horner

Junior, 1980; Frey-Wyssling, 1981; Arnott, 1982). Monje and

Baran (2002), in their works distinguished the whewellite of

the weddellite druse from its star-like forms. Different crys-talline morphotypes can be observed in Rubiaceae as styloid crystals,raphideanddruses(MetcalfeandChalk,1979).Zinietal.

(2016)reportthepresenceofdrusesinBorrerialatifoliaK.Schum.,

B.orientalisE.L.Cabral,R.M.Salas&L.M.Miguel,B.palustris(Cham. &Schltdl.)Bacigalupo&E.L.Cabral.Styloidcrystalsareobservedin somespeciesofthegenusPsychotriaL.ErbanoandDuarte(2010)

reportingeneralthepresenceofdrusesintoleavesandstemofG. americana,butdetailsaboutthemorphologicalanalyze,elemental compositionandhistolocalizationoftheseinclusionsin medici-nallyimportantleavesarescarceintheliteraturereferringtothese species.Thesedrusescanbeconsideredrelevantstructurestothe ultrastructuralanatomiccharacterization.

Conclusion

The anatomical characteristics commonly attributed to the representativesofRubiaceaearenotconsideredrelevantin inter-specificdifferentiation,sincetheyareofgeneraloccurrenceforthe family.Inresponsetothis,theuseofSEMbecomesausefultool intheanatomicalcharacterizationoftheG.americanafoliar ultra-structures.Besidesthephytochemistry,thehistochemistryallowed toobtainmorespecificdata,determiningtheexactstoragesiteof thesesubstancesintheleaf.Thecharacterizationofthecrystalline macro-pattern,aswellasitsexactelementalcomposition,canbe consideredanimportantfactorofdifferentialdiagnosis.Theresults presentedallowedthecharacterizationofthisspeciesindifferent aspectsandpointtoG.americanaasapromisingnativespecieswith differentpopularusesandprovenpharmacologicalactivities,and furtherstudiesareneeded.

Authors’contributions

ALVandAVSassistedincarryingoutthelaboratorywork.RJRP andLCAhelpedinconductingthescanningelectronmicroscopy (SEM)analyses.ALVwrotethepaperandKPRprovidedacritical readingofthemanuscript.ALVandKPRplannedtheproject, col-lectedtheplantmaterialandwasresponsibleforitsidentification. KPRsupervisedthelaboratorywork.Alltheauthorshavereadthe finalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthankstoCentrode TecnologiasEstratégicas do

Nordeste (CETENE) and Laboratório de Imunopatologia Keizo

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