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

Pharmacobotanical

study

of

Baccharis

pentaptera

Jane

Manfron

Budel

a

_,

_Josiane

_Padilha

_de

_Paula

a

_,

_Vera

_Lucia

_Pereira

_dos

_Santos

b

_,

Celia

Regina

Cavichiolo

Franco

c

_,

_Paulo

_Vitor

_Farago

a,∗

,

Márcia

do

Rocio

Duarte

d

a_Departamento_de_Ciências_{Farmacêuticas,}_Universidade_Estadual_de_Ponta_Grossa,_Ponta_Grossa,_PR,_Brazil b_Departamento_de_Engenharia,_Saúde_e_Meio_Ambiente,_Centro_{Universitário}_{Internacional,}_Curitiba,_PR,_Brazil c_Departamento_de_Biologia_{Celular,Universidade}_Federal_do_Paraná,_Curitiba,_PR,_Brazil

d_Departamento_de_Farmácia,_Universidade_Federal_do_Paraná,_Curitiba,_PR,_Brazil

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r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16May2015 Accepted7July2015 Availableonline29July2015

Keywords:

Compositae Herbaldrugs Medicinalplant Morpho-anatomy

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b

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BaccharisL.sect.Caulopterae,Asteraceae,comprisesthirtyspeciesinBrazilthatshowstemsrepresented bycladodes,whichareverysimilarinmorphology.Thesespeciesarepopularlyknownas“carqueja” inBrazilandArgentinaandareusedinpopularmedicineasdiureticandstomachic.Theaimofthis workwastoexaminethemorpho-anatomicalcharactersofcladodesofBaccharispentaptera(Less.)DC. fordiagnosispurposes.Theplantmaterialwaspreparedbylightandscanningelectronmicroscopy.B. pentapterashowsoppositeandspreadwingsinthetwo-wingedcladodeaxisandirregulararrangement inthethree-wingedcladode.Thewingshaveauniseriateepidermiswithpalisadeparenchymanextto bothsidesofepidermis.Thespongyparenchymacrossedbyminorcollateralvascularbundlesisobserved inthecentralregionofwings.Theglandulartrichomesarecapitateandbiseriateandthenon-glandular trichomesareuniseriateandflagelliformwith2–3cellsthatextendfromthebase.Incaulinaraxis, thereareuniseriateepidermis,chlorenchymaalternatingwithangularcollenchymaandperivascular fibercapsadjoiningthephloemwhichisoutsidethexylem.Prismaticandstyloidcrystalsareverified intheperimedullaryzone.ThesecombinedcharacterscanassistthediagnosisofBaccharisspeciessect. Caulopterae.

Introduction

BaccharisL.sect.CaulopteraeincludesOrganensis,Trimeraand

Genistelloidesgroups(Heidenetal.,2009).Thissectionoccursin SouthernAmericaandcomprisesthirtyspeciesinBrazilthatshow stemsrepresentedbycladodes(Heidenetal.,2009)whichare simi-larinmorphology(Budeletal.,2008).Inthatsense,agreatefforthas beendevotedtoexaminetheanatomicalcharactersofaerial veg-etativeorgansfromBaccharisfordiagnosispurposes.Inaddition, thesespeciesareusedinpopularmedicineasdiureticand stom-achicandarepopularlyknownas“carqueja”inBrazilandArgentina

(Budeletal.,2008).

Baccharis pentaptera (Less.) DC., Asteraceae, is a shrub that inhabits exclusively Southern America. The scientific names

BaccharisstenocephalaBakerandBaccharisfastigiataBakerare con-sideredsynonymsofB.pentapteraduetoseveralupdatesprovided bytaxonomists(Schneideretal.,2009).InspiteofB.pentaptera

is not officially accepted in national pharmacopoeias, Baccharis

∗ Correspondingauthor.

E-mail:pvfarago@uepg.br(P.V.Farago).

trimera(Less.)DC.,aquitesimilartaxonthatiswidelysoldin phar-macies,herbalandstreetshops,isclaimedtobethemostpopular andinvestigatedspeciesof“carquejas”andisincludedin Farma-copeiaBrasileiraV(2010).

B.pentapteraisusedinfolkmedicinesimilarlytoother “car-quejas”(BarrosoandBueno,2002).However,ithasbeenusually mistaken for B. crispa Spreng., B. microcephala (Less.) DC., B. opuntioides Mart.ex Baker, B. trimera (Less.)DC., and B. usterii

Heering (Barroso and Bueno, 2002; Alonso and Desmarchelier,

2006;Rodrigues et al.,2008).Moreover,few studies were

per-formedinordertoinvestigatechemicaldifferencesamongspecies from Caulopterae section. Regarding B. pentaptera, Bona et al.

(2002) showed that its essential oil yielded 1.8% and

Simões-Piresetal.(2005)identified␤-pinene(41.3%)asitsmainvolatile

compound.

Consideringthatmedicinalplantsarewidelysoldasfragments orpowders,morpho-anatomical descriptionisthefirst parame-terthatcanbeusedfordiagnosis.Inthiscontext,morphological andanatomicalcharacterizationcanbeasimpleandfasttoolfor identificationofherbaldrugs.Thus,thegoalofthispaperwasto dis-cussthesefeaturesforcladodesofB.pentapterainordertoprovide furtherdataaboutCaulopteraesection.

http://dx.doi.org/10.1016/j.bjp.2015.07.007

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Fig.1. Baccharispentaptera(Less.)DC.,Asteraceae.(A)Generalaspect;(B)faceviewofepidermalcells,stoma(st)andstriatedcuticle(cu)bySEM;(C)surfaceviewof epidermisshowingflagelliformnon-glandulartrichome(ng)(SEM).Bar=0.5cm(A).

Materialsandmethods

Plantmaterial

Baccharispentaptera(Less.)DC.,Asteraceae,wascollectedfrom specimensof sunnyareasatFazendaSão MaximianoinGuaíba (coordinates30◦₁₀′_S_and₅₁◦₂₀′_W,_and₂₇_m_altitude),_Serra_do

Sudoeste, Rio Grande do Sul, Brazil during December 2003. A voucherwasidentifiedbytaxonomistsandregisteredunderthe numberICN128450intheherbariumattheInstituteofBiosciences, FederalUniversityofRioGrandedoSul.

Morpho-anatomicalassays

Cladode fragments were collected 5–15cm from apex. At least five specimens were fixed in FAA 70 (Johansen, 1940), which wasreplaced by70% ethanol (v/v)(Berlyn andMiksche, 1976).Theplantmaterialwassectionedbyhandordehydrated, embeddedinglycolmethacrylate(Leicahistoresin)andsectioned usingtheLeicaRM-2145microtome.Cross-sectionsand longitu-dinal sectionswere stained withtoluidine blue (O’Brien et al., 1964) or astra blue and basic fuchsine combination (Roeser,

1972).

Thefollowingstandard solutionswereusedfor microchemi-caltests:hydrochloricphloroglucintoreveallignin(Sass,1951), SudanIIIforlipophiliccompounds(Foster,1949),ferricchloride forphenolicsubstances(Johansen,1940),sulphuricacidfor cal-ciumoxalatecrystals(Oliveiraetal.,2005)andiodine-iodidefor starch(BerlynandMiksche,1976).Photomicrographsweretaken withtheOlympusBX-40lightmicroscopeequippedwithadigital camera.

Fortheanalysisbyscanningelectronmicroscopy(SEM)(Souza, 1998),thefixedmaterialwasdehydratedusingincreasingamounts ofethanolandtheCO2criticalpointapparatus(BalzersCPD-030)

andcoatedwithgold(BalzersSputteringSCD-030).Electron micro-graphsweretakenwiththeJeolJSM-6360LVscanningmicroscope.

Results

ThestemofB.pentaptera(Less.)DC.(Fig.1A)usuallygrowstoa heightof0.5–1mandshowsthreewings(Fig.2C)inthevegetative axisandtwowingsinthereproductivebranches(Fig.2B).Theaxis isrigidandthewingsarediscontinuous.Thewingsareopposite andspreadintheaxisofthetwo-wingedcladode(Fig.2B);they areirregularlyarrangedinthecladodewiththreewings(Fig.2C).

Inthewings,theepidermalcellsarepolygonal(Fig.1BandC) andhavethickanticlinalwallswithevidentprimarypitsinfrontal view.Anomocyticstomata(Fig.1B)arelocatedatthesamelevelor slightlyabovethesurroundingcells.Incross-section,theepidermis isuniseriate(Fig.2D–F)andcoatedwithathinandslightlystriated cuticle(Figs.1B,Cand2E).Inparticular,cuticleisstriatedaround stomata(Fig.1B).

Glandular (Fig. 2A) and non-glandular trichomes

(Figs. 1C and 2A) are inserted in the same small epidermal

depressionsandareeitherisolatedorinclusters(Fig.2AandD). Theglandulartrichomes(Fig.2A)arecapitateand biseriateand have8cellsatthebase.Thenon-glandulartrichomesareuniseriate andflagelliformwith2–3cellsthatextendfromthebase(Fig.1C). Bothglandularandnon-glandulartrichomesshowcellsatthebase withdensecytoplasmandanevidentnucleusthatwerestained positivelyforlipophilicsubstances.

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15kv X1, 500 B.steno

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Fig.2.Baccharispentaptera(Less.)DC.,Asteraceae.(A)Surfaceviewofepidermalcellsexhibitingbiseriateglandulartrichomes(gt)andnon-glandulartrichomes(ng)in clusterbySEM;(B)cladodeincross-sectionshowingaxis(ax),chlorenchyma(cl),collenchyma(co),endodermis(en),fibers(fi),pith(pi),phloem(ph),secretoryducts(sd), xylem(xy)andtwowings(wi);(C)cladodeincross-sectionindicatingaxis(ax)andthreewings(wi);(D)wingincross-sectionneartothebordershowingepidermis(ep), fibers(fi),palisadeparenchyma(pp),spongyparenchyma(sp),stoma(st),trichomesinclusters(tc)secretoryduct(sd)andvascularbundle(vb);(E)wingincross-section showingcuticle(cu),epidermis(ep),stoma(st)andpalisadeparenchyma(pp);(F)wingandaxisindicatingepidermis(ep),palisadeparenchyma(pp),spongyparenchyma (sp),stoma(st),secretoryduct(sd),vascularbundle(vb)andpith(pi).Bar=20␮m(E),100␮m(B,D,F),200␮m(C).

In stem axis,the epidermis exhibits similar features to the wingsand, below it;thechlorenchymaalternates withangular collenchyma,whichhas2–3rowsintheribs(Figs.2Band3B,F). Secretoryducts,similartothoseviewedinthewings,canbefound nextto endoderm that bounds the internal part of the cortex

(Figs.2Band3B,C,F)anditswallsareimpregnatedwithlipophilic

compounds.

Thevascular cylindershowscambium thatproduces inward xylem and outward phloem (Fig. 3B and C). Perivascular fiber capsareadjoiningtophloemandsomefiberscanalsoappearin phloem(Fig.3B,CandF).Thepithiscomposedbyrelativelylarge parenchymatouscells withthinwalls(Fig.3B–D).Prismaticand styloidcrystals(Fig.3DandE)ofcalciumoxalateoccurinthe per-imedullaryregion.

Discussion

Baccharis L.sect. Caulopterae comprises shrubs withwinged stems (Heiden et al., 2009) that can be either continuous or discontinuous and form different-sized segments (Barroso and

Bueno,2002).Inthissection,thereareseveralspecieswhichshow

three-winged vegetative and reproductive branches, suchas B. apicifoliosaA.A.Schneid.&Boldrini,B.crispaSpreng.,B. flexuosir-amosaA.A.Schneid.,B.glazioviiBaker,B.jochenianaG.Heiden& Macias,B.junciformisDC.,B.microcephala(Less.)DC.,B.milleflora

(Less.)DC.,B.myriocephalaBakerandB.penningtoniiHeering.The speciesB.articulata(Lam.)Pers.presentstwo/three-winged veg-etativebranchesandtwo-wingedreproductivecladodes(Heiden etal.,2009).Inthisstudy,B.pentaptera(Less.)DC.showedthree wingsinthevegetativeaxesandtwowingsinthereproductive branches.

TheepidermalcharacteristicsstablishedforB.pentapteraare observedinmostof“carquejas”(Cortadietal.,1999;Budeletal.,

2003,2004;BudelandDuarte,2010;Pereiraetal.,2014;Jasinski

etal., 2014), includingthe anatomicalpattern of anticlinal epi-dermalcellswalls.Anomocyticandanisocyticstomataareusually describedforseveralspeciesofBaccharis(Souzaetal.,2011,2013;

Budeletal., 2013;Jasinski etal.,2014).However,Pereira etal.

(2014)verifiedactinocyticandanomocyticstomataforB.milleflora

DC.Actinocyticstomatawerealsofoundonlyinthenon-wingedB. confertaKunthandB.boliviensis(Weed)Cabrera(Freireetal.,2007).

Freireetal.(2007)reportedthatthestomataltypeandepidermal

cellwallscanbeusedtodifferentiateBaccharisspecieswhenthey havesimilartrichomes.

Biseriatecapitateglandulartrichomesaretypicallyregistered for“carquejas”.Similartrichomeswerereportedinmanyspecies ofBaccharis,suchasB.coridifoliaDC. (Budeland Duarte,2007),

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Figure3. Baccharispentaptera(Less.)DC.,Asteraceae–Cladodeincross-section.(A)Detailofwing,showingaminorvascularbundle(vb),endodermis(en),fibers(fi),palisade parenchyma(pp),spongyparenchyma(sp)andsecretoryduct(sd).(B)Axisindirectiontocollenchyma(co)exhibitingepidermis(ep),endodermis(en),fibers(fi),secretory duct(sd),phloem(ph),xylem(xy)andpith(pi).(C)Axisindirectiontochlorenchyma(cl)indicatingcambium(ca),endodermis(en),fibers(fi),secretoryduct(sd),phloem (ph),xylem(xy)andpith(pi).(D)Prismatic(pcr)andstiloid(scr)crystalsofcalciumoxalateinthepith(pi).(E)Prismaticandstyloidcrystalsofcalciumoxalateinpith(SEM). (F)Axisshowingfibers(fi)inphloem(ph),cuticle(cu),collenchyma(co),secretoryduct(sd),endodermis(en)andepidermis(ep).Bar=20␮m(A–E).

B.notosergilaGriseb.,B.pedunculata(Mill.)Cabrera(Heidenetal., 2009),B.teindalensisKunth,B.tolaPhil.(Freireetal.,2007),and

B.usteriiHeering(BudelandDuarte,2010).Moreover,other gen-eraofAsteraceaehavetheseglandulartrichomes,mainlyGochnatia

(Youssef etal.,2013), Calea(Budelet al.,2006; Camilottietal.,

2014)andMikania(Gasparettoetal.,2010;Amorinetal.,2014;

Araújo et al., 2015).Furthermore, other types of glandular

tri-chomeswerealsodescribedfor“carquejas”asuniseriatecapitate glandulartrichomesinB.sagittalis(Less.)DC.andB.triangularis

Hauman(Petenattiet al.,2007)and non-capitate glandular tri-chomesinB.usterii(BudelandDuarte,2010).

Thenon-glandulartrichomesobservedinthisstudywerefound for both winged and non-winged species, suchas B. coridifolia

(BudelandDuarte,2007),B.anomalaDC.(BudelandDuarte,2008a),

B. singularis (Vell.) G.M. Barroso (Souzaet al., 2011), B. spicata

(Oliveiraetal.,2011),B.cognataDC.(Budeletal.,2013),B.glaziovii

(Jasinski et al., 2014) and B. milleflora (Pereira et al., 2014). In

addition,simple and flagelliform non-glandulartrichomes with uniseriateisodiametric cells at thebaseweredescribed forthe non-wingedB.uncinellaDC.(BudelandDuarte,2008b).

Asusual,thetrichomesmorphologywasusedasataxonomic standardfortheirclassificationinthisinvestigation.However,the non-glandulartrichomesofB.pentapterademonstratedbasalcells stainedforlipophiliccompounds.Hence,thesetrichomescanbe

alsoestablishedasamixedtypeowingtotheirfunctional proper-tiesforprotectionandsecretion.

Similar chlorenchymaorganization was reported for several “carquejas”(SáandNeves,1996;Cortadietal.,1999;Budeletal.,

2003;Rodriguesetal.,2008;BudelandDuarte,2009,2010;Pereira

etal.,2014).ThewingedgearrangementofB.pentapterathat pre-sentedvascularbundlesandsecretoryductswasalsofoundinB. milleflora(Pereiraetal.,2014),B.myriocephala(SáandNeves,1996),

B.trimera(BudelandDuarte,2009),andB.usterii(BudelandDuarte, 2010).However,B.crispashowedonlysecretoryductsonthewing edges,whichwerenotaccompaniedbyvascularbundles(Cortadi

etal.,1999).

SeveralmembersofBaccharisarehighproducersofessential

oils(Simões-P_ires_et_al.,_2005;_Molares_et_al.,_2009;_Budel_et_al.,

2012;Florãoetal.,2012;Lageetal.,2015).Thesevolatileoilsare

usuallyfoundinglandulartrichomesandsecretoryductsof Aster-aceaespecies(Budeletal.,2012;Jasinskietal.,2014)andhavesome well-knownmedicinalpropertiesasanti-inflammatory, antiproto-zoal,schistosomicidal,andantimicrobialactivities(Parreiraetal.,

2010;Florãoetal.,2012;Valarezoetal.,2015).Theremarkable

presenceofthesesecretorystructuresconfirmssomeprevious lit-eraturedatathatdemonstratedayieldof1.8%fortheessentialoilof

B.pentaptera,inwhich␤-pinenewasitsmainvolatilecomponent

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Inspiteoftheirresponsivenesstochangesintheenvironmental calciumconcentration,theoccurrenceofcalciumoxalatecrystals isstronglydependentofageneticcontrol.Inaddition,crystaltype anddistributionmaybeconsideredataxonomiccharacterfor clas-sificationofspecies(FranceschiandNakata,2005).B.pentaptera

showedprismaticandstyloid crystalsof calciumoxalateinthe perimedullaryregion.Thesetypesaretypicallyencounteredinthe perimedullaryregionofcladodesof“carquejas”,suchasB. articu-lata,B.crispa,B.trimera(Cortadietal.,1999),B.myriocephala(Sáand

Neves,1996),B.microcephala(BudelandDuarte,2009),B.glaziovii

(Jasinskietal.,2014),B.sagittalis,andB.triangularis(Petenattietal.,

2007).However,B.milleflorarevealednocrystalintheinvestigation performedbyPereiraetal.(2014).

Asexpected,themorpho-anatomicalcharactersofthecladodes ofB.pentapteracorrespondedwiththoseofthesectionCaulopterae. Thesefeaturesneedtobeevaluatedasawholeforspecies diag-nosis.Howeverthefollowingstructuresshouldbeemphasizedas distinguishingcharacters ofother Caulopteraespecies: opposite andspreadwingsintheaxisofthetwo-wingedcladode,irregular arrangementinthethree-wingedcladode,uniseriate epidermis, palisadeparenchymanexttobothsidesoftheepidermis,spongy parenchymatraversedbyminorcollateralvascularbundlesinthe centralregion,capitateandbiseriateglandulartrichome,uniseriate andflagelliformnon-glandulartrichomewith2–3cellsthatextend fromthebase,caulinaraxiswithchlorenchymaalternatingwith angularcollenchyma,perivascularfibercapsadjoiningthephloem whichisoutsidethexylem,andprismaticandstyloidcrystalsin theperimedullaryzone.

However,thesediagnosticelementsarenotusuallydetectedor preservedforhighlyfragmentedcommercialsamples.Inthatsense, furtheranalysesshouldbeperformedinordertoafinal differenti-ationofthisspeciesfromother“carquejas”,includingquantitative micrograph(Petenetattietal.,2007)andadditionalhistochemical reactionasreportedbyVentrellaandMarinho(2008).

Authors’contributions

JMBdevelopedtheresearchandpreparedthemanuscript.JPP, VLPS,CRCFandPVFcontributedinperformingthescanning elec-tronmicroscopy (SEM)analysis,describing theanatomical data ofthespeciesandprovidingacriticalreadingofthemanuscript. MRDcreatedtheprojectandsupervisedthelaboratorywork.All theauthorshavereadthefinalmanuscriptandapprovedthe sub-mission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

TheauthorsthanktheElectronMicroscopyCenteroftheFederal UniversityofParanáforprovidingSEMimages.

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