Rev. bras. farmacogn. vol.26 número6

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RevistaBrasileiradeFarmacognosia26(2016)665–672

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

Original

Article

Comparative

analytical

micrographs

of

“vassouras”

(

Baccharis

,

Asteraceae)

Vanessa

B. Bobek

a

_,

_Gustavo

_Heiden

b

_,

_Camila

_Freitas

_de

_Oliveira

a

_,

_Valter

_Paes

_de

_Almeida

c

_,

Josiane

Padilha

de

Paula

d

_,

_Paulo

_Vitor

_Farago

d

_,

_Tomoe

_Nakashima

a

_,

_Jane

_Manfron

_Budel

d,∗

a_Programa_de_{Pós-graduac¸}_ão_em_Ciências_{Farmacêuticas,}_Universidade_Federal_do_Paraná,_Curitiba,_PR,_Brazil b_Embrapa_Clima_Temperado,_Pelotas,_RS,_Brazil

c_Curso_de_Farmácia,_Universidade_Estadual_de_Ponta_Grossa,_Ponta_Grossa,_PR,_Brazil

d_Programa_de_{Pós-graduac¸}_ão_em_Ciências_{Farmacêuticas,}_Universidade_Estadual_de_Ponta_Grossa,_Ponta_Grossa,_PR,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11March2016 Accepted3May2016 Availableonline14June2016

Keywords: Baccharisbrevifolia Baccharismicrodonta Baccharispauciflosculosa Baccharistrilobata Morpho-anatomy

a

b

s

t

r

a

c

t

BaccharisbelongstotheAsteraceaefamilyandcomprisesanumberofmedicinalspecies.Baccharis bre-vifoliaDC.,B.microdontaDC.,B.pauciflosculosaDC.,andB.trilobataA.S.Oliveira&Marchiori,whichare popularlyknowninBrazilas“vassouras”(“broom”),areallfoundinSouthernBrazil.Theanatomical featuresoftheleafandstemwereinvestigatedbyemployingtheusuallightandscanningelectron microtechniques,asameansofdifferentiatingthetaxa.Thefollowinganatomicalcharacteristicscanbe consideredtobediagnostic:theoccurrenceandtypeofstomata,midrib,stemandcrystalshapes,and thepresenceofthepetiole.

Introduction

Baccharis L., Asteraceae, includes about 354 species, which range fromthe USAtoArgentina (90% occurin South America and about178species are foundin Brazil) (Müller, 2013; BFG, 2015). However, further pharmacobotanical, genetic, chemical, biological,pharmacologicalandtoxicologicalstudiesarerequired. Variousethnobotanicalresearchstudieshavebeencarriedoutin SouthAmericancommunitiesthatusetheseplantsforthe treat-mentofseveraldiseases.Thesecommunitiesmostlymakeuseof severalspeciesofBaccharisasanalgesic,diuretic,spasmolytic, anti-diabetic,anti-infectiveandstomachicmedication(Zardini,1984; Mentzetal.,1997;AbadandBermejo,2007).Manyofthese proper-tiescanbeattributedtothepresenceofvolatileoilsthatarefound inBaccharisspecies(Lagoetal.,2008;Budeletal.,2012;Onofre etal.,2013;Lageetal.,2015;Valarezoetal.,2015).

PreviousdatahaverevealedthatBaccharispauciflosculosaDC. and B. microdonta DC. have antimicrobial activities (Perez and Anesini,1993,1994)andthevolatileoilofBaccharismicrodonta showsahighconcentrationofoxygenatedsesquiterpenes(49.91%), which are mainly caryophyllene oxide (24.06%), ␣-cadinole

∗ Correspondingauthor.

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

(8.44%) and viridiflorol (7.67%) (Lago et al., 2008). Thereis no pharmacobotanical,pharmacologicalorchemicalcharacterization available for B. brevifolia DC. and B. trilobata A.S. Oliveira & Marchiori.

Vegetablerawmaterialsareameansofinitiallydeterminingthe qualityofamedicinalplantoranherbaldrug.Theherbal indus-tryexperiences seriousproblemsduetothesubstitutionand/or adulterationofplantspeciesbyalternativesthataresimilar.Such tamperingimpairstheeffectivenessofherbaldrugsandinsome casesmaycauseintoxication(WHO,2003).InthecaseofBaccharis, theproblemisevenworsebecausealotofitsspecieshaveasimilar morphology.

An additional problem is the inappropriate use of popular names,which cancausemistakesin theidentificationofherbal drugs. The samespecies often hasseveral folknames and fur-thermore,asingularcommonnamemaydesignateseveralspecies (Uptonetal.,2011).B.brevifolia,B.microdonta,B.pauciflosculosa andB.trilobata,popularlyknowninBrazilas“vassouras”(“broom”) occurinSouthernBrazilandtheirmorphologyissimilar.Asaresult, someconfusionand/ormistakescanresultfromthepopularuseof thisterm.

For these reasons, the purpose of this paper was to study anatomicaldataregardingtheleavesandstemsofB.brevifolia,B. microdonta,B.pauciflosculosaandB.trilobataasameansof provid-ingadditionalsupportfordifferentiatingthesespecies.

http://dx.doi.org/10.1016/j.bjp.2016.05.001

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666 V.B.Bobeketal./RevistaBrasileiradeFarmacognosia26(2016)665–672

Fig.1.(A,E,I,M)BaccharisbrevifoliaDC.;(B,F,J,N)BaccharismicrodontaDC.,(C,G,K,O)BaccharispauciflosculosaDC.,(D,H,L,P)BaccharistrilobataA.S.Oliveira&Marchiori; (A,B,C,D)vegetativeandreproductivebranch.(E,F,G,H)Abaxialsideofleafepidermisand(I,J,K,L)adaxialsideofleafepidermis,showingepidermalcellwallsand stomatum(st).(M,N,O,P)Viewoftheleafsurface,stomatum(st),striatedcuticle(ct),non-glandulartrichome(nt),andglandulartrichome(gt)bySEM;Scalebar=5cm(A, B,C,D),50␮m(E,F,G,H,I,J,K,L),20␮m(M,N,O).

Materialsandmethods

Plantmaterial

Aerialpartsof atleastfour specimensof Baccharisbrevifolia DC.,B.microdontaDC.,B.pauciflosculosaDC.andB.trilobataA.S. Oliveira&Marchiori,Asteraceae,werecollectedintheregionof CamposGerais,PontaGrossa,Paraná,SouthernBrazil(coordinates 25◦ ₀₈′_S_and₅₀◦ ₂₇′ _W)_during_the_summer_of_2013._A_voucher

wasidentifiedbytaxonomistsandregisteredundertheregistration numbersHUPG20411(B.brevifolia),HUPG20406(B.microdonta),

HUPG20413(B.trilobata)andHUPG20409(B.pauciflosculosa)in theherbariumattheStateUniversityofPontaGrossa.

Anatomicalstudy

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V.B.Bobeketal./RevistaBrasileiradeFarmacognosia26(2016)665–672 667

Fig.2. (A)BaccharisbrevifoliaDC.,(B)BaccharismicrodontaDC.,(C)BaccharispauciflosculosaDC.,(D)BaccharistrilobataA.S.Oliveira&Marchiori.Cross-sectionoftheleaves, showingcollenchyma(co),cuticle(ct),epidermis(ep),endodermis(en),fibers(fi),palisadeparenchyma(pp),phloem(ph),spongyparenchyma(sp),secretoryduct(sd), stomatum(st),trichomesincluster(tc),vascularbundle(vb),andxylem(xy).Scalebar=100␮m(B),50␮m(A,C,D).

(BerlynandMiksche,1976).Thesematerialswerealsodehydrated andembeddedinglycolmethacrylate(Leicahistoresin®

)(Feder andO’brien,1968).Afterinclusion,theblocksthatwereobtained weresectionedat7–9␮musingarotarymicrotome(Spencer820) witha type-Csteelknife.Longitudinal sectionswereplaced on slides andstainedwithbasicfuchsin combinedwithastrablue (Britoand AlquinI,1996).Theslidesweremountedinsynthetic resin(Entellan®_).

Thediaphanizationoftheleaveswasperformedbyfollowingthe techniqueofFuchs(1963),andthatofPatel(1979)wasusedforthe stomataclassification.Theresultswereillustratedwiththeaidof photosthatweretakenwithanOlympusCX31opticalmicroscope, whichwasattachedtoaC7070digitalcamera.Inconductingthe ultra-structuralanalysis(Souza,2007),thesampleswerefixedin FAA70,dehydratedinagradedethanolseriesthroughthecritical pointprocedure(BalzersCPD030),coatedwithgold(Balzers Sput-teringSCD030)andanalyzedusingascanningelectronmicroscope (JeolJSM6360LV).

Resultsanddiscussion

Morphologicalcharacteristicsareoftenusedtoidentifyspecies, althoughwhenthemorphologyissimilarmistakescanoccur dur-ingclassification.Inthiscase,anatomicalfeaturescanhelpinthe identification,especially when they are powdered (Nodari and Guerra,2000).Similarmorphologicalfeaturestendtooccurwith Mikaniaspp.(Araújoetal.,2015),Passifloraspp.(Woschetal.,2015), Piperspp.(Gogoszetal.,2012),amongothergenera.

Inbotanicalproducts,adulterationsinvolvethewholeor par-tialreplacementofonespeciesforanother,especiallywhenthey aresimilar,belongtothesamegenus,orareusedforthesame popularusage (Uptonet al., 2011). Inthis context,B. brevifolia (Fig.1A),B.microdonta(Fig.1B),B. pauciflosculosa(Fig.1C)and B.trilobata (Fig.1D)showeda similarmorphologytowhat has beenobservedinrelationtootherBaccharisspecies(Barrosoand Bueno,2002;Oliveiraetal.,2011;Jasinskietal.,2014;Budeletal., 2015).

InseveralBaccharisspeciestheanatomicalshapeofthe anticli-nalepidermalcellwallsoftheleavesvariesfromstraighttowavy (Souzaetal.,2011;Oliveiraetal.,2011;Bobeketal.,2015;Barreto etal.,2015).Accordingtothepattern,allthespeciesanalyzedin thepresentstudyshowanticlinalepidermalcellwallsthatwere straighttoslightlywavyandwithrelativelythinanticlinalwallson bothsides(Fig.1E–L).Thecuticleisstriated(Fig.1M)forthefour species,especiallyaroundthestomata(Fig.1N–P).

Thepresence ofstomata,aswellastheamountand typeof stomatainleafepidermis,areimportantfeaturesforcharacterizing anddifferentiatingaspecies.Rodriguezetal.(2010)reportedthat thedensityofstomatacanhelptodifferentiatebetweenB. articu-lata(Lam.)Pers.andB.trimera(Less.)DC.Bothamphistomaticand hypostomaticleavesoccurinBaccharis(Molaresetal.,2009;Budel etal.,2013;Bobeketal.,2015;Barretoetal.,2015).

Inthepresentstudy,onlyB.microdontahashypostomaticleaves (Fig.1Fand J), theothersare amphistomatic(Fig.1Eand G–L).

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Fig.3.(A)BaccharisbrevifoliaDC.,(B)BaccharismicrodontaDC.,(C)BaccharispauciflosculosaDC.,(D)BaccharistrilobataA.S.Oliveira&Marchiori.Cross-sectionofthemidribs, showingcambium(ca),collenchyma(co),endodermis(en),fibers(fi),phloem(ph),secretoryduct(sd),stomatum(st),vascularbundle(vb),andxylem(xy).Scalebar=50␮m.

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Fig.5. (A)BaccharisbrevifoliaDC.,(B)BaccharismicrodontaDC.,(C)BaccharispauciflosculosaDC.,(D)BaccharistrilobataA.S.Oliveira&Marchiori.Generalappearanceofthe stemincross-section.Scalebar=50␮m.

anisocyticstomatahavebeendescribedformostspeciesof Bac-charis(BudelandDuarte,2008a,b;Souzaetal.,2011;Oliveiraetal., 2011).However,othertypesofstomatahavealsobeenreported, suchascyclocytic(Budeletal.,2013),staurocytic,tetracytic(Freire etal.,2007)andactinocytic(Pereiraetal.,2014).

In the present study, B. brevifolia shows hexacytic (Fig. 1E) andcyclocyticstomata(Fig.1I),B.microdontahassignsof stauro-tetracyticandtetracyticstomata(Fig.1F),B.pauciflosculosashows tetracytic (Fig. 1G) and anomocytic stomata (Fig. 1K), and B. trilobata has stauro-tetracytic (Fig. 1H and L) and tetracytic stomata.

Asingle-layerepidermisinacross-sectioncovered bya thin cuticle, biseriate glandulartrichome, flagelliform non-glandular trichome,isobilateral mesophyll,minorcollateral vascular bun-dlessurroundedbyanendodermis,andsecretoryducts,havebeen extensivelydescribedinBaccharis(Molaresetal.,2009;Budeletal., 2012,2015;Barretoetal.,2015;Bobeketal.,2015)andwerefound inallthestudiedspecies(Figs.1M,PandFigs.2A–D).

The midrib shape is a significant feature for differentiating species(Oliveiraetal.,2011;Gogoszetal.,2012; Barretoetal., 2015;Woschetal.,2015).Inthepresentstudythisinformation wasalsousedtodifferentiateBaccharisspecies.B.brevifoliahasa flat-convexshapeinthemidrib(Fig.2A),whereasB.microdontahas aconcave–convexshape(Fig.2B),B.pauciflosculosahasabiconvex shape(Fig.2C)andinthecaseofB.trilobatathemidribisalmost flatonbothsides(Fig.2D).

Inthecase ofallthestudiedspecies,beneath theuniseriate epidermisthereare 2–5layers ofangular collenchymaonboth sidesandthereisasinglecollateralvascularbundleintheground parenchymatissue(Figs.3A–D).

Inthisstudy,onlytheB.microdontaleafhasapetiole(Fig.4A), whichwhenviewedincross-section,isconcave-convexwiththree slightprojectionsontheabaxialsurface,thecentralpartbeingmore prominent.Theepidermalcoatinghasthesamecharacteristicsas thoseobservedintheleaf.Thecollenchymaisanangulartypeand

occursincontinuousstripsof1–2setsofcells.Therearethreefree collateralvascularbundlessurroundedbytheendodermisandin openarcorganization(Fig.4A)withsecretoryductsinanexternal phloemposition(Fig.4B).

Intransection,thestemofB.brevifolia(Fig.5A)andB. pauci-flosculosa(Fig.5C)haveanirregularshapewithfiveconspicuous ribs.B.microdonta(Fig.5B)andB.trilobata(Fig.5D)haveanalmost hexagonalshapewithfourconspicuousribs.

Inthisstudy,thestemepidermisisverysimilartotheleaf epi-dermisforthesetaxa,sinceitpossessesstomata,astriatedcuticle, andnon-glandularorglandulartrichomes(Fig.6AandD)which can either appear isolated or in clusters. Angular collenchyma alternateswithchlorenchymabeneaththeepidermisoccursinall studiedspecies(Fig.6B–D)althoughacontinuousstratumof col-lenchymacouldbeobserved(Fig.6AandB).Thischaracteristichas beenreportedforotherBaccharisspecies,suchasB.singularis(Vell.) G.M.Barroso(Souzaetal.,2011).Onetofivelayersofangular col-lenchymacanbefoundforthefourstudiedspecies,particularlyin theribs.

Theendodermisboundthecortexinternallywithvisible Cas-parianstrips.Secretoryductsandperivascularfibercapsarefound nexttothephloem.Fiberisobservedinthephloem.Thexylem tracheary elements are arranged in rows in an orderly man-ner and separated by parenchyma cells and fibers (Fig.6A–D). Thepithconsistsofisodiametricthin-walledparenchymaticcells (Fig.5A–D).

Thetype,presenceorabsenceofcrystalscanbeconsideredtobe taxonomiccharacteristics(Meric,2009).Calciumoxalatecrystals areoftenfoundintheperimedullaryregionofthepithinBaccharis (BudelandDuarte,2008b;Souzaetal.,2011;Oliveiraetal.,2011; Jasinskietal.,2014;Barretoetal.,2015;Bobeketal.,2015).

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Fig.6.(A)BaccharisbrevifoliaDC.,(B)BaccharismicrodontaDC.,(C)BaccharispauciflosculosaDC.,(D)BaccharistrilobataA.S.Oliveira&Marchiori.Stemincross-section, showingchlorenchyma(cl),collenchyma(co),cuticle(ct),epidermis(ep),endodermis(en),fibers(fi),glandulartrichome(gt),phloem(ph),secretoryduct(sd),andxylem (xy).Scalebar=50␮m.

(Fig.7D),andB.trilobatahassignsofcrystalsand,square dipyra-mids, elongated square dipyramids, styloids with pointed and squareends(Fig.7EandF),aswellastabularcrystalsinpilesthat looklikeatower(Fig.7G).Thistypehasnotbeenmentionedbefore inthegenus.

Anatomicalanalysisisaninherentprocedurefornearlyall phar-macopeiasandisoneofthemainidentificationtestsrequiredfor theherbalindustry.Althoughtheindividualstructuralelements arerelativelycommonwithinthesametype ofplantparts,the

mannerinwhich theelementsaresetgivesavegetablespecies itscharacteristicfingerprint(Uptonetal.,2011).

Inthatsense,eventhoughmostoftheanatomicalfeaturesofthe leafandstemofBaccharisspp.arequitesimilar,several character-isticsobservedinthisstudysupporttheneedforadifferentiation ofthefourstudiedspecies,asconfirmedinBox1.Theoccurrence andtypeofthestomata,midrib,stemandcrystalsshapes,andthe presenceofthepetiolearerecommendedasgoodmarkersfora diagnosisofthespecies.

Box1:AnatomicalcharacteristicsofBaccharisbrevifolia,B.microdonta,B.pauciflosculosaandB.trilobata.

Anatomicalcharacteristics B.brevifolia B.microdonta B.pauciflosculosa B.trilobata

Occurrenceofstomataintheleaves Amphistomatic Hypostomatic Amphistomatic Amphistomatic Typesofstomata Ciclocyticandhexacytic Stauro-tetracytic

andtetracytic

Anomocyticand tetracytic

Stauro-tetracyticand tetracytic

Shapeofmidribshape Flat-convex Concave-convex Biconvex Flatonbothsides

Presenceofpetiole Absent Present Absent Absent

Shapeofstem Irregularwithsixribs Hexagonalwith fourribs

Irregularwithsix ribs

Hexagonalwithfourribs

Typesofcrystals Crystalsand,styloidswith pointedorsquareendsand squaredipyramids

Rarestyloidswith pointedends

Crystalsandand squaredipyramids

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Fig.7.(A,B)BaccharisbrevifoliaDC.,(C)BaccharismicrodontaDC.,(D)BaccharispauciflosculosaDC.,(E,F,G)BaccharistrilobataA.S.Oliveira&Marchiori.Stemincross-section, showingperimedullaryregionwithcrystals,sand(sa),stiloydswithpointedends(slp),stiloydswithsquareends(sls),squaredipyramids(sd),elongatedsquaredipyramids (esd),andtabularcrystalsinpiles(tb).Scalebar=10␮m(A,B,C,D).

Authors’contributions

VBBcarriedouttheresearch.Theseresultsformapartofher MAcoursework.GHidentifiedtheplantsamplesandperformed thevoucher.VPAandCFOassistedbyconductingtheanatomical tests.JPPandPVFprovidedacriticalreadingofthemanuscript.TN createdtheprojectandwasresponsibleforarrangingthe scholar-ship.JMBcreatedtheprojectandsupervisedthelaboratorywork, aswellasanalyzingthedataandprovidingadraftofthearticle. Alltheauthorshavereadthefinalmanuscriptandapprovedits submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsaregratefultoCAPESforgrantingscholarshipsto VBB.TheauthorswouldalsoliketothanktheCMEoftheFederal UniversityofParanáforthescanningelectronmicrographs.

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