Rev. bras. farmacogn. vol.25 número4

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RevistaBrasileiradeFarmacognosia25(2015)328–343

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

Comparative

study

of

Passiflora

taxa

leaves:

I. A

morpho-anatomic

profile

Luma

Wosch

a

_,

_Daniela

_Cristina

_Imig

b

_,

_Armando

_Carlos

_Cervi

b,1

,

Bárbara

Baêsso

Moura

b

,

Jane

Manfron

Budel

c

_,

_Cid

_Aimbiré

_de

_Moraes

_Santos

a,∗

a_Programa_de_{Pós-graduac¸}_ão_em_Ciências_{Farmacêuticas,}_Laboratório_de_{Farmacognosia,}_Universidade_Federal_do_Paraná,_Curitiba,_PR,_Brazil

b_Departamento_de_Botânica,_Universidade_Federal_do_Paraná,_Curitiba,_PR,_Brazil

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

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14May2015 Accepted26June2015 Availableonline14July2015

Keywords: Passiflora

Morpho-anatomy Passionfruit

Pharmacobotanicalstudy Qualitycontrol

a

b

s

t

r

a

c

t

Determiningtheauthenticityandqualityofplantrawmaterialsused intheformulationofherbal medicines,teasandcosmeticsisessentialtoensuretheirsafetyandefficacyforclinicaluse.Some

Pas-sifloraspeciesareofficiallyrecognizedinthepharmaceuticalcompendiaofvariouscountriesandhave

therapeuticuses,particularlyassedativesandanxiolytics.However,thelargenumberofPassifloraspecies, coupledwiththefactthatmostspeciesarepopularlyknownaspassionfruit,increasesthe misidenti-ficationproblem.Thepurposeofthisstudyistomakeapharmacognosticcomparisonbetweenvarious

Passifloraspeciestoestablishamorpho-anatomicalprofilethatcouldcontributetothequalitycontrol

ofherbaldrugproductsthatcontainpassionfruit.Thiswasconductedbycollectingsamplesofleaves fromtwelvePassiflorataxa(tenspeciesandtwoformsofP.edulis):P.actinia,P.alata,P.amethystina,

P.capsularis,P.cincinnata,P.edulisf.flavicarpa,P.edulisf.edulis,P.incarnata,P.morifolia,P.urnifolia,P.

coccineaandP.setacea,fromdifferentlocationsandtheirmorpho-anatomicalfeatureswereanalyzed

usingopticalmicroscopyandscanningelectronmicroscopy.Microscopicanalysisallowedtoindicatea setofcharactersthatcanhelptodifferentiatespecies.Theseincludemidribandpetioleshape,midriband petiolevascularpattern,mediumveinshape,presenceoftrichomes,presenceofbladeepidermalpapillae andsclerenchymaticcellsadjoiningthevascularbundles.Thesecharacterscouldbeusedtoassistinthe determinationofherbaldrugqualityandauthenticityderivedfromaspeciesofPassiflora.

Introduction

Theoriginofthetermpharmacognosy(pharmakon:drugand

gnosis: knowledge) clarifies the discipline of knowledge about drugs.AccordingtotheWorldHealthOrganization(WHO,2000),a drugisamedicinalplant,orpartsthereof,aftercollectionprocesses, stabilizationanddrying,initsfullform,fragmentedorpowdered. Consideringthatmedicinalplantsarechemicallyand microbiologi-callystable,theyareacquiredbyphytotherapicindustriesasherbal drugs,whichmaketheirauthenticationdifficult.

TheuseofPassifloraspeciesasmedicinalplantsbeganinthe sev-enteenthcenturyinEuropeduetotheirsedativeproperty(Hoehne, 1939).Currently,Passifloraspeciesarewidelyusedinfolkmedicine inmanycountries, largelyassedativesandanxiolytics(Conrado etal.,2003).

∗ _{Corresponding}_author.

E-mail:cid@ufpr.br(C.A.M.Santos).

1_In_memoriam.

P.incarnataL.isincludedintheofficialpharmaceuticalcodes of several countries (Gosmann et al., 2011) and is the most widelystudiedinthisaspect.Consequently,inBrazil,mostherbal medicineswerepreparedfromthisspecies.However,P.incarnata

isnativetotheNorthAmericaandcannotadapttotheBrazilian climate.Forthisreason,theBrazilianPharmacopoeia5th _edition (Farmacopeia,2010)chosetomakeP.alataCurtisandP.edulisSims. theconstituentsofherbalmedicines,withoneofeitherspeciesin itsconstitution.

Thesedata,coupledwiththelargenumberofPassifloraspecies (approximately600)andthesamecommonname(passionfruit) giventoseveralspecies,haveincreasedtheprobabilityofmistakes inspeciesidentification,orevenintheadulterationofherbaldrugs. Inordertominimizetheseproblems,pharmacobotanicalanalysis canbeused.Thistechniqueisusedtoidentifymorphologicaland anatomicalcharactersfordifferentiationbetweensimilarspecies. Freitas(1985)employedpharmacobotanicalanalysesona compar-ativestudyoffourPassifloraspecies:P.alata,P.edulis,P.incarnata

andP.quadrangularis,andidentifiedsomedistinguishingfeatures suchasthepresence,locationandtypeoftrichomes.

http://dx.doi.org/10.1016/j.bjp.2015.06.004

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Consideringthemorphologicalsimilarities betweendifferent

Passifloraspecies,theaimofthisresearchwastoexamine, morpho-anatomically,theleavesfromtwelvePassiflorataxa.Therevealed pharmacobotanicalfeaturescanbeusedtoidentifythesemedicinal plants,aswellasdifferentiatebetweenPassifloraspecies, contribut-ingtothequalitycontrol ofherbalproductscontainingpassion fruit.

Materialandmethods

Botanicalmaterials

Plantmaterial(32samplesfromtwelvetaxa)wascollectedfrom differentareasofBrazil,specificallyfromtheSouth(Paraná,Santa CatarinaandRioGrandedoSul),DistritoFederalandRioGrande doNorte,in2013and2014.Theplantmaterialwasusedtomake voucherspecimensthatwereidentifiedbythetaxonomistDaniela CristinaImig,andarepresentativesamplewasstoredinthe Herbar-iumlistedinBox1.

Morpho-anatomicalstudy

Passiflorataxaplantmaterialwascollected15cmfromtheapex oftheplant.TheleaveswerefixedinFAA70solution(Johansen, 1940)andstoredin70%ethanol(BerlynandMiksche,1976).The remainingleaveswere driedat 50◦_C _and_ground _in _an

analyt-ical mill (Quimis® _G298A21). _The _dried, _ground _samples _were

standardizedbysizeto≤300␮m.Theobtainedpowderwas

ana-lyzedbylightmicroscopy.

For permanent slides, samples were taken fromthe middle regionof thethird leaffromthe apex.Technical embeddingin historesin(LeicaMicrosystems®₎_was_used_by_employing_the

pre-viouslydeterminedmaterials.Thematerialwastrans-sectionedin thetransverseplaneinarotarymicrotome,whichproduced5␮m sections.Toluidinebluewasusedforstaining(FederandO’Brien, 1968).

Forthefrontalviewoftheepidermis,theleavesweresubjected toclearingtechnique(KrausandArduin,1997).Thesampleswere stainedwithsafraninandtheslidesweresetwithglycerinated gela-tine(Kaiser,1880).Semi-permanentslideswerealsopreparedfor drugcharacterizationinpowderusingchloralhydrate.

For theanalysis of scanningelectron microscopy (SEM), the sampleswere fixedin FAA70, dehydratedin a gradedethanol seriesandCO2criticalpointdryingapparatus(Bal-TecCPD-030), coatedwithgold(BalzersSCD-030)andexaminedusingaJeol JSM-6360LVmicroscope.ThisprocedurewasperformedattheElectron MicroscopyCenterattheFederalUniversityofParaná(UFPR).For themorpho-anatomicaldescription,wefollowedtheprocedureof Radfordetal.(1974).

Results

Morphologicalanalysis

Morphological analysis of Passiflora leaves shows important characteristics for differentiatingbetween the taxa (Fig. 1 and

Box1

Datacollection,vouchersandHerbariumofdepositedPassifloraspecies.

Sample Species Localityofcollection Collectiondate Voucher Herbarium

1.1

P.actiniaHook.

C.Politécnico,UFPR,Curitiba-PR 30/08/2013 L.Wosch01 HACC,UPCB 1.2 C.Politécnico,UFPR,Curitiba-PR 06/05/2014 D.C.Imig332 HACC,MBM,UPCB 1.3 C.Botânico,UFPR,Curitiba-PR 06/05/2014 L.Wosch03 HACC,UPCB

2.1

P.alataCurtis

Planaltina-DF 15/08/2013 D.C.Imig52 UPCB,MBM

2.2 Ipira-SC 05/04/2014 D.C.Imig336 HACC,UPCB

2.3 Piratuba-SC 05/04/2014 D.C.Imig347 HACC,MBM

2.4 FozdoIguac¸u-PR 24/04/2014 C.L.Reichert04 HACC

2.5 LargodaOrdem,Curitiba-PR 07/05/2014 L.Wosch04 HACC

3.1

P.amethystinaJ.C.Mikan

Ipira-SC 05/04/2014 D.C.Imig334 HACC,MBM

3.2 Piratuba-SC 05/04/2014 D.C.Imig348 HACC,MBM

3.3 Ipira-SC 05/04/2014 D.C.Imig335 HACC,MBM

4.1

P.capsularisL.

Ipira-SC 05/04/2014 D.C.Imig337 HACC,MBM

4.4 Serradomar–PR 29/05/2014 D.C.Imigetal.,s.n. HACC

5 P.cincinnataMast. Natal-RN 07/2014 L.Wosch05 HACC,MBM

6.1

P.edulisf.flavicarpaO.Deg.

C.Politécnico,UFPR,Curitiba-PR 30/08/2013 L.Wosch02 HACC,UPCB

7.1

P.edulisSimsf.edulis Serradomar–PR 29/05/2014 M.E.Engelsetal.,2490 MBM

7.2 Capinzal–SC 21/07/2014 D.C.Imig346 HACC,MBM

8.1

P.incarnataL. VeraCruzdoOeste-PR 25/04/2014 C.L.Reichert06 HACC

8.2 VeraCruzdoOeste-PR 02/06/2014 C.L.Reichert06 HACC

9.1

P.morifoliaMast.

Ipira-SC 04/04/2014 D.C.Imig338 HACC,UPCB,MBM

9.2 Peritiba-SC 04/04/2014 D.C.Imig339 HACC,UPCB

9.3 Ipira-SC 04/04/2014 D.C.Imig340 HACC,UPCB,MBM

10.1

P.urnifoliaRusby

Irani-SC 04/04/2014 D.C.Imig343 HACC,UPCB

10.2 Concórdia-SC 04/04/2014 D.C.Imig342 HACC,MBM

10.3 Peritiba-SC 04/04/2014 D.C.Imig341 HACC,MBM

11 P.coccineaAubl. Planaltina-DF 15/08/2013 D.C.Imigetal.293 MBM

12 P.setaceaDC. Planaltina-DF 15/08/2013 D.C.Imigetal.284 HACC,UPCB,MBM

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330 L.Woschetal./RevistaBrasileiradeFarmacognosia25(2015)328–343

Fig.1. Passifloraspp.–Morphologicalaspectofleaves–Adaxialside.a.P.actinia;b.P.alata;c.P.amethystina;d.P.capsularis;e.P.cincinnata;f.P.edulisf.flavicarpa;g.P.

edulisf.edulis;h.P.incarnata;i.P.morifolia;j.P.urnifolia;k.P.coccinea;l.P.setacea.Ps.:theimageswereobtainedafterfixationwithFAA.Bars=2cm.

Box 2). The leaves are simple and the blade shape ranges from entire to 2-, 3-, 5-lobed or 5-partite. The leaf margin characteristics may be serrate or crenulate. The apex ranges fromacute, obtuse, to acuminate. The bases are rounded, cor-date,subcordateor attenuate.Thedimensionsof theleafblade and petiole vary significantly between the taxa. The presence and number of glands on the petiole also vary consider-ably.

Anatomicalanalysis

Epidermis

Thesurfaceviewofthebladeshowsdifferentepidermalcell anticlinalwallsasobservedinBox3andinFigs.2and3forthe adaxialandabaxialside,respectively.

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Box2

MorphologicalcharacteristicsofPassifloraspeciesleaves.

P.actinia P.alata P.amethystina P.capsularis P.cincinnata P.edulisf.flavicarpa

Leafblade

Shape Ovate Ovatetoelliptical 3-Oblonglobs 2–3Ovatelobs 5-Lobedto 5-partite

3-Oblonglobs

Size

Width(cm) 4–7 7–15 6–10 6–8.5 9 11–22.5

Length(cm) 5–9 9.5–17.5 5–8 7–9.5 6–7 9–17

Apex Obtusetoacute Acuminate Obtuse Acute Acutetoobtuse Acutetoacuminate Base Rounded Roundedto

subcordate Cordateto subcordate Cordateto subcordate Truncate Subcordate

Margin Entire Entiretosparsely glandularserrate

Entirewithglands inthesinus

Entire Glandularserrate Serrate

Color Lightgreenand glossyonboth sides

Lightgreenonboth sides

Darkgreen, glaucousonabaxial side

Darkgreenonboth sides,slightly opaqueonabaxial side

Darkgreenonboth sides,glossyonthe adaxialsideand opaqueonabaxial side

Colorafterdrying Cleareronabaxial side

Slightlycleareron abaxialside

Darkened,clearer onabaxialside

Clearerandgrayon abaxialside

Clearerand uniformonabaxial side

Clearerandopaque onabaxialside

Venation Pinnate Pinnate Palmate Palmate Palmate Palmate

Petiole

Size(cm) 2.5–3.5 3.5–5 2–4.5 2.5–4 3 2–6

Glands

Frequency ∼6 2–4 5–7 Absent 2 2

Location Scattered Abovethemedian region

Scattered,nearthe leafblade

– About1cmfrom thebase

Nexttotheleaf blade

P.edulisf.edulis P.incarnata P.morifolia P.urnifolia P.coccinea P.setacea

Leafblade

Shape 3-Oblongto lanceolatelobs

3-Oblongto lanceolatelobs

3-Lobedattheapex 2-Lanceolatelobs Oblongtoelliptical 3-Oblonglobs

Size

Width(cm) 4.5–6 4.5–6 5.5–10.5 5.5–6 4–5 9.5

Length(cm) 4–6 4–6 5–9.5 7–8 11 10.5

Apex Acuminate Acutetoacuminate Acutetoobtuse Acutetoobtuse Acute Acuminatetoacute Base Subcordateto

attenuate

Cordateto subcordate

Cordate Attenuate Roundedto subcordate

Cordateto subcordate Margin Serrate Serrate Serratetocrenulate Entire Serrate Serrate Color Darkgreenonboth

sides,glossyon adaxialsideand opaqueonabaxial side

Greenonboth sidesandglaucous onabaxialside

Lightgreenand opaqueonboth sides

Green,sometimes variegatedand opaqueonabaxial side

Greenonboth sides

Greentolight greenonbothsides andopaqueon abaxialside

Colorafterdrying Cleareronabaxial face

Clearerinthe abaxialface

Similaronboth sides

Clearerinthe abaxialface

Venation Palmate Palmate Palmate Palmate Pinnate Palmate

Petiole

Size(cm) 5–6 1.5 2–4 2–2.5 2 4–5

Glands

Frequency 2 2 2 Absent Absent 4

Location Nexttotheblade Nexttotheblade Atthemedian region

– – Atthemedian

region

Box3

Epidermalcellanticlinalwallsonadaxial(AD)andabaxial(AB)sidesofPassifloraspecies.

Epidermalcell anticlinalwalls P. actinia P. alata P. amethystina P. capsularis P. cincinnata

P.edulisf.

flavicarpa

P.edulisf.edulis P.

incarnata P. morifolia P. urnifolia P. coccinea P. setacea 7.1 7.2 Straightto slightlywavy

AD AD AD AD AD

Slightlywavy towavy

AB AB AD AB AD

Wavyto slightly sinuous AD AB Slightly sinuousto sinuous

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Fig.2.Passifloraspp.–Viewoftheleafsurface(epidermis)–Adaxialside.a.P.actinia;b.P.alata;c.P.amethystina;d.P.capsularis;e.P.cincinnata;f.P.edulisf.flavicarpa;g1.

P.edulisf.edulissample7.1;g2.P.edulisf.edulissample7.2;h.P.incarnata;i.P.morifolia;j.P.urnifolia;k.P.coccinea;l.P.setacea.st:stomata.Bars=20␮m.

observedontheadaxialside(Figs.2band2h).Allothertaxahave hypostomaticleaves(Figs.2and3).Thestomataarelocatedatthe samelevelofotherepidermalcellsinP.actinia,P.alata,P. cincin-nataandP.coccinea;atthesamelevelorinaslightdepressioninP. amethystina,P.morifoliaandP.urnifolia;atthesamelevelorslightly aboveinP.capsularis,P.edulisf.flavicarpa,P.edulisf.edulisandP. setacea,andslightlyaboveinP.incarnata.

P. actinia shows anomocytic stomata (Fig. 3a). P. alata, P. amethystina,P.capsularis,P.cincinnata,P.edulisf.flavicarpa,P.edulis

f.edulisandontheabaxialsideofP.incarnatashowsanomocytic

stomata(Fig.3h),anisocyticstomata(Fig.3c)andparacyticstomata (Fig.3f).Anisocyticstomatacanbeobservedontheadaxialsideof

P.incarnata.AlthoughP.morifoliaandP.urnifoliashowanomocytic stomata(Fig.3i,j),afewanisocyticstomatacanalsobeobserved.In

P.coccinea,anomocytic(Fig.3k),anisocyticandparacyticstomata areobservedbutanisocyticstomataarerare.InP.setacea(Fig.3l), allthreetypesareobservedbutparacyticstomataareuncommon.

P.actinia(Fig.4a, b), P.alata(Fig.4c,d) and P.amethystina

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Fig.3.Passifloraspp.–Viewoftheleafsurface(epidermis)–Abaxialside.a.P.actinia;b.P.alata;c.P.amethystina;d.P.capsularis;e.P.cincinnata;f.P.edulisf.flavicarpa;

g1.P.edulisf.edulissample7.1;g2.P.edulisf.edulissample7.2;h.P.incarnata;i.P.morifolia;j.P.urnifolia;k.P.coccinea;l.P.setacea.st:stomata;arrowsindicatepapillae.

Bars=20␮m.

non-glandulartrichomes(Fig.5b)onlyontheadaxialside.P. capsu-laris(Fig.5a),P.edulisf.flavicarpa(Fig.5c),P.edulisf.edulis(Fig.5d, e),P.incarnata(Fig.5f),P.morifolia(Fig.5g),P.urnifolia(Fig.5h),P. coccinea(Fig.5i)andP.setacea(Figs.4g,hand5j)arepubescenton bothsides.

P.capsularis presents two types of non-glandular trichomes distributedthroughouttheleafblade;however,therearemore tri-chomesontheabaxialside.Thefirsttypeisconical,straight(Fig.5a, rightside)andcommonlybicellular,butit canbeunicellularor

multicellular;thebasecell iswider and showsa regionin half sphere;thecuticleisstriate.Thesecondtypeiscylindrical(Fig.5a, left side) and unicellular; thecell wall is thin and the apex is rounded;thewidthisconstantandcoveredbyastriatecuticle; theyaresmallerthantheothertrichomepreviouslydescribed.

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Fig.4.Passifloraspp.Cross-sectionsofmidribandpetioleofglabrousspeciesP.actinia,a–b;P.alata,c–d;P.amethystina,e–f;anddenselypubescentP.setacea,g–h.Bars

(␮m)=100(a),200(b,c,e–g),500(d,h).

InP.edulisf.flavicarpa,afewnon-glandulartrichomescanbe seenonboth sidesofthemidrib.Theyareconical and straight (Fig.5c),shortormediuminlengthandthecuticleisstriate.

P.edulisf.edulis(sample7.1)hascylindrical(Fig.5d)and unicel-lularorbicellularnon-glandulartrichomeswithaacuteapex.These trichomesareencounteredonbothsides,particularlyonlyonthe largerveinontheadaxialsideandmoreuniformlyscatteredon theabaxialsurface.Insample7.2,conicalnon-glandulartrichomes (Fig.5e)wereencountered,positionedontheveinsonbothsides.

P.incarnatapresentscylindrical,slightlycurved,non-glandular trichomes(Fig.5f)onbothleafsides.Ontheadaxialfacetheyare unicellularorbicellular,wideandwithathickcellwall.Theyhave differentlengthsandataperingapicalcell.Similartrichomescanbe observedontheabaxialside,buttheyaremulticellularandlonger. Onboth leafsides,thesetrichomesareobservedmainlyonthe largervein.

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Fig.5. Passifloraspp.–Non-glandulartrichomes.a.P.capsularis;b.P.cincinnata;c.P.edulisf.flavicarpa;d.P.edulisf.edulissample7.1;e.P.edulisf.edulissample7.2;f.P.

incarnata;g.P.morifolia;h.P.urnifolia;i.P.coccinea;j.P.setacea.t1:trichometype1;t2:trichometype2;arrowindicatecellsorganizedinarosette.Bars(␮m)=20(c,h),

50(a,b,d,e,g,i),100(f),200(j).

uncinated(Fig.5g).It hasa thickcellwalland a striatecuticle. Thesecondtypeisunicellularandcylindricalwitharoundedapex (Fig.5g).Ithasathincellwallandastriatecuticle.Theuncinated, non-glandulartrichomeislongerthanthecylindricaltrichomeand ontheabaxialside,thecylindricaltypeislesscommon.

P. urnifolia presents cylindrical, non-glandular trichomes (Fig.5h)witha roundedapex,thickcellwalland astriate cuti-cle.Theyaredistributedthroughouttheleafbladealthoughmore frequentonthelargervein.

P.coccineapresentscylindrical(Fig.5i)andunicellular, bicellu-larormulticellularnon-glandulartrichomes.Theyshowatapering apex,widecellwallandthebasecellhasaregioninhalfsphere. Thesetrichomesaredistributeduniformlyontheleafblade,more frequentlyontheabaxialside.

P.setaceapresentsuniserate,filiform,non-glandulartrichomes. Theyarebicellular, formedby a basecellwitha regioninhalf sphere,ormulticellular,formedbytenormorecells.Ontheadaxial side,thesetrichomesareonlydispersedonthelargervein.Onthe

abaxialside,theyaremorehomogeneouslydistributedontheleaf blade(Fig.5j).

The adjacentepidermal cells of thetrichomesmaybe simi-lartoothercellsorgroupedinarosette.Theyvaryaccordingto taxa,trichometypeorbladeside.InP.capsularis,thebasalportion ofthetrichomeissurroundedbycellsorganizedinarosetteor arrangedslightly abovetheotherepidermal cells(Fig.5a).In P. incarnata, P.morifolia, P. urnifolia(Fig. 5h) and P. coccinea, the basal portionof the trichomeis surrounded bycells organized in a rosette onboth sides.In P.setacea,the adjacent cells are arrangedslightlyabovetheotherepidermalcellssurroundingthe trichomesontheadaxialside.InP.edulisf.edulis(sample7.1),the adjacentepidermalcellsofthetrichomesaresimilartotheother cells.

Crosssectionofleafblade

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Fig.6.Passifloraspp.–Leafbladeincross-sectionatmediumvein.a.P.actinia;b.P.alata;c.P.amethystina;d.P.capsularis;e.P.cincinnata;f.P.edulisf.flavicarpa;g.P.edulisf.

edulis;h.P.incarnata;i.P.morifolia;j.P.urnifolia;k.P.coccinea;l.P.setacea.ep:epidermis;pp:palisadeparenchyma;sp:spongyparenchyma;st:stomata;ps:parenchymatic

sheath;ec:sclerenchyma;dr:druse;pa:papillae.Bars(␮m)=50(a,c,d–l),100(b).

distributedontheabaxialside(Fig.6a)andP.coccineaandP.setacea

showsomepapillaeonaminorveinontheabaxialside(Fig.6kand l).

The mesophyll is dorsiventral and a stratum of palisade parenchyma appears in all taxa (Fig. 6). The layers of spongy parenchymavarybyspecies;3–6forP.amethystina(Fig.6c),3–4 forP.capsularis(Fig.6d),3–5forP.morifolia(Fig.6i)andP.setacea

(Fig.6l),and7–10forP.alata(Fig.6b),forexample.Thesamples ofP.capsularisdifferedinleafbladethickness.Thesample4.4 pre-sentedpalisadeparenchymanarrowerthantherestofthesamples ofthisspecies(Fig.7).

Smallandmediumcollateralvascularbundlescanbedetected immersedinthemesophyllandsurroundedbytheparenchymatic sheath (Fig. 6). P.actinia, P. alata, P.amethystina, P.capsularis,

P. cincinnata, P. edulis f. flavicarpa, P. edulis f. edulis, P. incar-nata,P.urnifoliaandP.setaceashowdrusesintheparenchymatic sheath,however,P.coccineapresentsdrusesonlyinthepalisade parenchyma.NodrusesappearedintheleafbladeofP.morifolia.

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Box4

Midribshapeincross-sectionofPassifloraspecies.

Species Midribshape

P.actinia Biconvexwithprominentandroundedconvexityontheabaxialside.

P.alata Biconvexwithslightconvexityontheadaxialsideandprominentandangularconvexityontheabaxialside.

P.amethystina Biconvexwithprominentandroundedconvexityontheabaxialside.

P.capsularis Biconvexwithprominentandtruncateconvexityontheabaxialside,irregularprojectionsonbothsides.

P.cincinnata Biconvex,withanacuteprojectiontotheadaxialsideandprominentandroundedconvexityontheabaxialside.

P.edulisf.flavicarpa Biconvexwithcylindricalprojectionontheadaxialsideandprominentandroundedconvexityontheabaxialside,forminganovateshape.

P.edulisf.edulis Biconvexwithcylindricalprojectionontheadaxialsideandprominentandroundedconvexityontheabaxialside,ovateinoutline.

P.incarnata Biconvexwithacuteprojectionontheadaxialsideandprominentandtruncateconvexityontheabaxialside,triangularinoutline.

P.morifolia Biconvexwithacuteprojectionontheadaxialsideandprominentconvexitywithsmallprojectionsontheabaxialside.

P.urnifolia Flat-convexshape,withaminorprojectionontheadaxialsideandprominentconvexityontheabaxialside.

P.coccinea Biconvexwithprominentandroundedconvexityontheabaxialside.

P.setacea Biconvexwithacuteprojectionontheadaxialsideandprominentandroundedconvexityontheabaxialside,withsmallprojections.

Fig.7.Passifloracapsularis.Leafbladeincross-section-Thicknessdifferencesofthe

leafblade.a.sample4.2(similarasallothersamplesofthisspecies);b.sample4.4. ep:epidermis;pp:palisadeparenchyma;sp:spongyparenchyma;dr:druse;vb: vascularbundle.Bars=50␮m.

Box5

MidribvascularpatternsofPassifloraspecies.

Species Midribvascularpattern

P.actinia Threefreetracesinaflatarcandonedorsaltrace.

P.alata Oneflatarcwithonedorsaltrace.

P.amethystina Threefreetracesinaflatarcandonedorsaltrace,

organizinginacentralring.

P.capsularis Oneflatarcwithoneminordorsaltrace,organizingin

acentralringwithamedullarregion.

P.cincinnata Threefreetracesinflatarc,onedorsalandonelateral

trace.

P.edulisf.flavicarpa Fourtracesinacentralring.

P.edulisf.edulis Fourtracesinacentralring.

P.incarnata Threefreetracesinaflatarcandonedorsaltrace.

P.morifolia Oneflatarcwithonedorsaltrace,organizingina

centralringwithasmallmedullarregion.

P.urnifolia Medullatedcylinder.

P.coccinea Fivefreetracesinaflatarcandonedorsaltrace.

P.setacea Fourfreetracesinring.

P.urnifoliashowedsclerenchymaticcellsatdifferentstagesof lig-nificationlocatedinthecenterofthevascularbundle(Fig.6j).No sclerenchymaappearedintherestofthetaxa.

Insometaxa,themediumveinmakesprojectionsor depres-sionsonbothsidesoftheleafblades.Aconvexityisobservedon theabaxialsideofP.capsularis(Fig.6d),P.cincinnata(Fig.6e),P. incarnata(Fig.6h),P.morifolia(Fig.6i),P.urnifolia(Fig.6j),P. coc-cinea(Fig.6k),andonbothsidesofP.edulisf.edulis(Fig.6g).Only

P.setaceapresentsaconvexityontheabaxialsideandaconcavity ontheadaxialsideonthemediumvein(Fig.6l).

Crosssectionofmidrib

Inthecross-section,themidribshapeshowsadifferentstandard forPassiflorataxaasobservedinFig.8andsummarizedinBox4.

Box6

PetioleshapeofPassifloraspecies.

Species Shape Description

P.actinia Flat-convexto

concave-convex

Flatorslightlyconcaveontheadaxialsideandroundedconvexityontheabaxialside.

P.alata Concave-convex Adaxialsidewithtworibsattheendandacuteandprominentontheabaxialside.

P.amethystina Flat-convex Almostcircular,buttruncateontheadaxialside.

P.capsularis Flat-convex Almostcircular,buttruncateontheadaxialside.

P.cincinnata Concave-convex Adaxialsidewithtwosmallribsattheendandacuteandprominentontheabaxialside,pentagonalinoutline.

P.edulisf.flavicarpa Concave-convex Adaxialsidewithtwoconspicuousribsattheendandprominentconvexitysharperinthecentralregionontheabaxial

side.

P.edulisf.edulis Concave-convex Adaxialsidewithtwoconspicuousribsattheendandprominentconvexitysharperinthecentralregionontheabaxial

surface.

P.incarnata Concave-convex Adaxialsidewithtwoconspicuousribsattheendandroundedandprominentconvexityontheabaxialsurface.

P.morifolia Flat-convex Almostcircular,buttruncateontheadaxialside.

P.urnifolia Flat-convexto

concave-convex

Flatorslightlyconcaveontheadaxialsideandroundedconvexityontheabaxialside.

P.coccinea Concave-convex Adaxialsidewithtwoconspicuousribsattheendandroundedandprominentconvexityontheabaxialside.

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Fig.8.Midribincross-section.a.P.actinia;b.P.alata;c.P.amethystina;d.P.capsularis;e.P.cincinnata;f.P.edulisf.flavicarpa;g.P.edulisf.edulis;h.P.incarnata;i.P.morifolia;

j.P.urnifolia;k.P.coccinea;l.P.setacea.ep:epidermis;co:chollenchyma;ec:sclerenchyma;tr:non-glandulartrichome.Bars(␮m)=200(a,c,d,e,h,i,j),500(b,f,g,k,l).

Beneaththeepidermisoftheadaxialside,thenumberofangular collenchymastratavaryfrom7to8inP.morifolia(Fig.8i),7to9in

P.cincinnata(Fig.8e)andP.edulisf.flavicarpa(Fig.8f),7to14inP. edulisf.edulis(Fig.8g)andP.incarnata(Fig.8h).Ontheabaxialside, 2–5angularcollenchymastrataarepresentinallthetaxa.Druses aredispersedinthegroundparenchymainalltaxa(Fig.8).

Sclerenchymatictissueisrepresentedbycellsorcaps. Perivas-cularcapsadjointhephloemandfreecellsnearthexylem inP. actinia(Fig.8a).P.alatashowssclerenchymaticcellsadjoiningthe phloem(Fig.8b).P.urnifoliashowsperivascularcapsadjoiningthe

phloem(Fig.8j).P.coccineashowsperivascularcapsadjacenttothe phloemofthedorsalsheathbundle(Fig.8k).

Thevascularsystemisrepresentedbycollateralvascular bun-dles;midribvascularpatternsaresummarizedinBox5.

Crosssectionofpetiole

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Fig.9.Petioleincross-section.a.P.actinia;b.P.alata;c.P.amethystina;d.P.capsularis;e.P.cincinnata;f.P.edulisf.flavicarpa;g.P.edulisf.edulis;h.P.incarnata;i.P.morifolia;

j.P.urnifolia;k.P.coccinea;l.P.setacea.ep:epidermis;co:chollenchyma;ph:phloem;xy:xylem;tr:trichome.Bars(␮m)=200(a,c),500(b,d–l).

Theepidermisshowsthesamecharacteristicsasdescribedfor theleafblade.Trichomescanbeobservedinalltaxa,exceptinP. actinia,P.alataandP.amethystina.

Thevascularsystemisrepresentedbycollateralvascular bun-dles and the petiole vascular patterns are summarized in Box 7. Beneaththeepidermis, 2–4 continuousangular collenchyma strataanddrusesarepresentscatteredinthegroundparenchyma (Fig.9).

ThemainanatomicalcharacteristicsaresummarizedinBox8.

Microscopicanalysisofpowder

Themicroscopicanalysisofpowderidentifiedsome character-isticspreviouslydescribedintheanatomicalanalyses(Fig.10).

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Box7

PetiolevascularpatternsofPassifloraspecies.

Species Petiolevascularpattern

P.actinia Fivefreetracesinaflatarc,alargerdorsaltraceandtwolateralribtraces.

P.alata Eightfreetracesformingacentralringwithamedullartraceandtwolateralribtraces.

P.amethystina Fivefreetracesinaflatarc,alargerdorsaltraceandtwolateralribtraces.

P.capsularis Threefreetracesinaflatarc,alargerdorsaltraceandtwolateralribtraces.

P.cincinnata Sevenfreetracesinaflatarc,alargerdorsaltraceandtworibtraces.

P.edulisf.flavicarpa Sevenfreetracesinaflatarc,adorsaltraceandtworibtraces.

P.edulisf.edulis Ninefreetracesinaflatarcandoneortwodorsaltracesandtworibtraces.

P.incarnata Fiveu-shapedfreebundlesandadorsaltrace.

P.morifolia Threefreetracesinaflatarc,adorsaltraceandtwolateralribtraces.

P.urnifolia Threefreetracesinaflatarc,adorsaltraceandtwolateralribtraces.

P.coccinea Sixu-shapedfreebundles,adorsaltraceandtworibtraces.

P.setacea Sixfreetracesformingacentralringwithtwoorthreetracesinthemiddleandtwolateralribtraces.

Fig.10. Microscopicanalysisofpowder.a.P.actinia,papillaeinfrontalviewofepidermisabaxialside;b.P.amethystina,sinuousanticlinalepidermalcellwall;c.P.edulis

f.edulis,epidermisandangularchollenchyma;d.P.morifolia,uncinatenon-glandulartrichome;e.P.morifolia,cylindricalnon-glandulartrichomewithroundedapex;f.P.

urnifolia,cylindricalnon-glandulartrichomewithroundedapex;g.P.setacea,slightlywavyanticlinalepidermalcellwall;h.P.setacea,filiformnon-glandulartrichomeon

thevein;i.P.incarnata,cylindricalnon-glandulartrichomewithacuteapex.ep:epidermis;st:stomata;co:chollenchyma;tr:non-glandulartrichome;arrow:papillae.Bars (␮m)=20(a,b,e),50(c),100(d,f,g,i),200(h).

Innon-glabrous taxa, non-glandulartrichomesareobserved, exceptforP.edulisf.flavicarpathathasfewtrichomes.Inaddition,

P.actiniashownotonlystomatabutalsonumerouspapillaeonthe abaxialside(Fig.10a).

Discussion

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L. Wosch et al. / Revista Brasileira de Farmacognosia 25 (2015) 328–343 341

AnatomicalfeaturesofdifferentiationbetweenPassifloraspecies.

P.actinia P.alata P.amethystina P.capsularis P.cincinnata P.edulisf.flavicarpa

Epidermalcellanticlinalwalls

Adaxial Wavytoslightlysinuous Straighttoslightlywavy Slightlysinuoustosinuous Slightlysinuoustosinuous Straighttoslightlywavy Slightlywavytowavy Abaxial Slightlysinuoustosinuous Slightlywavytowavy Slightlysinuoustosinuous Slightlysinuoustosinuous Slightlywavytowavy Slightlysinuoustosinuous

Non-glandulartrichomes

Adaxial Glabrous Glabrous Glabrous (1)Conical(2)cylindrical (roundedapex)

Glabrous

Abaxial Conical Conical

Stomatalocation Hypo Amphi Hypo Hypo Hypo Hypo

Presenceofpapillae Internervalregionon

abaxialface

– – – – –

Sclerenchymaticcells Capsadjointophloemand

xylem

Cellsadjointophloem – – – –

Mediumveinshape Flat/flat Flat/flat Flat/flat Flat/convex Flat/slightlyconvex Flat/flat

Midribshape

Midribvascularization

pattern

3Freetracesinflatarcand 1dorsaltrace

1Flatarcwith1dorsal trace

3Freetracesinflatarcand 1dorsaltrace,organizing inacentralring

1Flatarcwithoneminor dorsaltrace,organizingina centralringwitha medullarregion

3Freetracesinflatarc,1 dorsaland1lateraltrace

4Tracesinacentralring

Petioleshape Flat/convexto

concave-convex

Concave/convex Flat/convex Flat/convex Concave/convex Concave/convex

Petiolevascularization

pattern

5Freetracesinflatarc,a largerdorsaltraceand2 lateralribtraces

8Freetracesforminga centralringwitha medullartraceand2 lateralribtraces

7Freetracesinflatarc,a largerdorsaltraceand2rib traces

7Freetracesinflatarc,a dorsaltraceand2ribtraces

P.edulisf.edulis P.incarnata P.morifolia P.urnifolia P.coccinea P.setacea

7.1 7.2

Epidermalcellanticlinalwalls

Adaxial Straighttoslightlywavy Slightlysinuousto sinuous

Slightlysinuousto sinuous

Slightlysinuoustosinuous Slightlywavytowavy Straighttoslightly wavy

Straighttoslightlywavy

Abaxial Slightlywavytowavy Slightlysinuousto sinuous

Slightlysinuoustosinuous Slightlysinuousto sinuous

Wavytoslightly sinuous

Non-glandulartrichomes

Adaxial Cylindrical(pointed apex)

Conical Cylindrical(pointed apex)

(1)Uncinate(2)cylindrical (roundedapex) Cylindrical(rounded apex) Cylindrical(pointed apex) Filiform Abaxial

Stomatalocation Hypo Amphi Hypo Hypo Hypo Hypo

Presenceofpapillae – – – – Mediumvenation Mediumvenation

Sclerenchymaticcells – – – Cellsinthecenterof

thevascularbundle

Cellsadjoinedto phloem

–

Mediumveinshape Slightlyconvex/slightlyconvex Flat/slightlyconvex Flat/slightlyconvex Flat/slightlyconvex Flat/slightlyconvex Concave/convex

Midribshape

Midribvascularization

pattern

4Tracesinacentralring 3Freetracesinflatarc and1dorsaltrace

1Flatarcwith1dorsal trace,organizingina centralringwithasmall medullarregion

Medullatedcylinder 5Freetracesinflatarc and1dorsaltrace

4Freetracesinring

Petioleshape Concave/convex Concave/convex Flat/convex Flat/convexto

concave/convex

Concave/convex Flat/convex

Petiolevascularization

pattern

9Freetracesinflatarcand1or2dorsal tracesand2ribtraces

U-shaped5free bundlesandadorsal trace

3Freetracesinflatarca dorsaltraceand2lateral ribtraces

About3freetracesin flatarc,adorsaltrace and2lateralribtraces

U-shaped6free bundles,adorsaltrace and2ribtraces

About6freetraces formingacentralring with2or3tracesinthe middleand2lateralrib traces

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and cosmetics are essential to ensure its security and effi-cacy for clinicaluse. Misidentificationand tampering increases the probability of adverse events, such as the hepatotoxic-ity cases reported by Pittler and Ernst (2003). Several raw materials are intentionally adulterated by other species that are morphologically similar, cheaper in price, or more read-ily available (Ma et al., 2002; Zhao et al., 2006; Zhang et al., 2007).

P.edulishasbeenwidelycultivatedinBrazilandMoraesetal. (1997)reportedthatleavesandstemshavebeenusedasan adul-terantforP.alata,whichwasincludedinthefirstthreeeditions oftheBrazilianPharmacopoeiaandwasofficialinthecountryat thattime.Moreover,inBrazil,severalPassifloraspeciesare pop-ularlyknownas‘maracujá’, whichreinforces theimportanceof identificationanddifferentiatingbetweenthem(BeraldoandKato, 2010).

Morphological characteristicsare very important for correct speciesidentification.However,severalmedicinalspeciesaresold fragmentedorpowdered.Thus,theanatomicalfeaturescangreatly contribute,plustomorphology,byindicatingthecharacteristicsfor botanicalidentification.

In this research, similar morpho-anatomical characteristics were encountered in all Passiflora taxa, such as dorsiventral mesophyll, collateral vascular bundles and the presence of druses.However,thefollowing structuresmightbeemphasized as useful to distinguish species: midrib and petiole shape in cross-section, midrib and petiole vascular patterns, outline of medium veins in cross-section, sclerenchymaticcells adjoining the vascular bundle, presence and type of non-glandular tri-chomes and presence of papillae in the epidermis of the leaf blade.

Theimportanceofstandardizingmedicinalplantcultivationis highlightedbyadifferencefoundbetweenthesamplesofP. capsu-laris.Themesophyllthicknessofsample4.4wasnarrowerthanthe oneofothersamples,duetoenvironmentalinfluences.The sam-ple4.4wascollectedfromanAtlanticForestwhereithadgrown intheshadowof trees.The othersampleswerecollectedfrom anopenspacewithdirectsunexposure.Pires(2008)studiedthe behaviorofsomePassifloraspeciesinrelationtoshadingandfound thatP.morifolia,growinginfullsunwith25%ofshade,showeda thickermesophyllformedbylongerpalisadecellsandbigger inter-cellularspaceswhencomparedwithothersgrowingin50–75%of shade.

Crochemore et al. (2003) studied the diversity of Passiflora

speciesinrelationtomorphologicalcharacteristics.Theyreported a great variability between the analyzed samples of P. edulis

f. edulis in coincidencewith the morpho-anatomical variability found in this study for the same form. Amorim et al. (2014) interpretedthattheintraandinterspecificvariabilitywithinthe

Passifloragenus wasassociated withan ability toadapt to dif-ferent environments. In addition,Viana et al. (2003) suggested thatanotherreasonwastheeasinessofPassifloraspeciessexual reproduction.

Itisimportanttohighlightthevariabilityfoundinthisstudyin

P.edulisf.flavicarpaandP.edulisf.edulis,especiallyregardingtothe presenceandtypeoftrichomes.However,theliteraturediscusses thistopiconlyatthespecieslevelandnotattheformlevel.Besides, thereportsareinconsistents(Pereiraetal.,2009;BeraldoandKato, 2010;Farmacopeia,2010;Barbosaetal.,2013;Leiteetal.,2013; Farias,2014;Cervi,1997).

Inthis study,only P.alataandP.incarnata showed amphis-tomatic leaves. However, they are considered as hypostomatic by Farmacopeia (2010), Pereira et al. (2009), Farias (2014) and WHO (2007). This probably happened because of the low density of stomata present on the adaxial side for both species.

Conclusion

ThemorphologicalcharacteristicsofPassifloraspp.leaves sup-portthedifferentiationofspecies;however,whenusedinherbal drugs,fragmentedorpowdered,anatomicalcharacteristicsprovide additionalusefuldatafordifferentiatingthem.

Authors’contributions

LW(MScstudent) wasresponsibleforcollectingplant mate-rial,identification,confectionofherbarium,runningthelaboratory work,analysisofthedataanddraftingthepaper;DCIcontributed toplantcollecting,identificationandherbariumconfection;BBM contributedinmakingtheslidesandanalysisofthedata;ACC,JMB andCAMSdesignedthestudy,supervisedthelaboratoryworkand readthemanuscriptcritically.Alloftheauthorshavereadthefinal manuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthankVeraL.P.dosSantosandCeliaR.C.Franco fortheirvaluableworkontheelectronicmicroscopyslides prepa-ration; an acknowledgment is alsodue toDr. Fabio G.Faleiro, Embrapa Cerrados, Planaltina-DF for collecting some Passiflora

species.CAMSthanksCNPqfortheresearchschorlarship.

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