Rev. bras. farmacogn. vol.26 número4

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

Original

Article

Foliar

anatomy

of

Viola

maculata

growing

in

Parque

Nacional

Los

Alerces,

Chubut,

Patagonia,

Argentina

Chana

Pilberg

a

_,

_Maria

_V.

_Ricco

a

_,

_Maria

_A.

_Alvarez

a,b,∗

a_{Farmacobotánica}_y_{Farmacognosia,}_Farmacia_y_Bioquímica,_Facultad_de_Ciencias_de_la_Salud,_Universidad_Maimónides,_Ciudad_de_Buenos_Aires,_Argentina b_Consejo_Nacional_de_{Investigaciones}_{Cienti´ficas}_y_Tecnolo´_gicas,_Ciudad_de_Buenos_Aires,_Argentina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6October2015 Accepted17March2016 Availableonline16April2016

Keywords: Violamaculata

Argentina Medicinalplant Traditionalmedicine Leaf

a

b

s

t

r

a

c

t

ViolamaculataCav.,Violaceae,growsintheunderwoodalongtheLosAndesmountainsystemfrom NeuquéntoTierradelFuegoandIslasMalvinas.Itisalsoknownasvioletaamarillaoraspilundeu,andis usedinpopularmedicinebytheMapuchesasstimulant,indermatology,andasalavageineyediseases. Theaimofthisworkistoexaminethemorpho-anatomicaltraitsoftheleavesandpetioleofV.maculata

(collectedatParqueNacionalLosAlerces,Patagonia,Argentina).Crosssectionsandscanningelectron microscopyofleavesbladeandpetioleshowedasimpleorganizationwithsimpleunicellulartrichomes andcellscontainingtannins,andcrystalsofcalciumoxalatemainlyinthespongyparenchyma.AsV. maculataisusedintraditionalmedicine,thespecificationoftheanatomicalcharactersisrelevantto describethespecies.

Introduction

ViolaisthelargestgenusoftheViolaceaefamilymostly consti-tutedbyshrubsorherbs.Someauthors,basedinaphylogeny-based revisionofthegenus,havesuggestedtheworldwideexistenceof twosubgeneraandsixteensections(Yousefietal.,2012;Mehrvarz andMarcussen,2013).Numerousanatomical studieshavebeen publishedonthegenusViola(Metcalfeand Chalk,1950; Rubin and Paolillo, 1978;Akarsu,1989; Dinc¸ and Yıldırımlı,2007).In Argentina,there arenineteenspecies ofViolaceae,two ofthem exoticandnaturalized(MehrvarzandMarcussen,2013).Among themViolamaculataCav.,commonlyknownaspileundeuor vio-letaamarillaororejaderatón,growsintheunderwoodalongthe LosAndesmountainsystemfromNeuquéntoTierradelFuegoand IslasMalvinas.CusatoandRossow(1991)havecitedV.maculata

aspartof thenative florafrom ParqueNacional Lago Puelo.V. maculataisa perennialherb upto3–25cm,withrootstockand stolons,pubescentorhairless.Leavesareupto6–65×3–55mm, ovate or lancelolate, pubescent or hairless, crenated. Petioles are 0.5–20cm long, in general pubescent. Stipules are serrate, 2–12mm.Peduncleis2–25cmlong,pubescentorhairless.Flowers arenotfragrant.Sepalsareovate,narrowlytriangularor lanceo-late,2–8mm,pubescent,exceptionallyhairless.Petalsareyellow, occasionallywithreddishorbrownish lines,thelowestpetalis

∗ _{Corresponding}_author.

E-mail:alvarez.mariaalejandra@maimonides.edu(M.A.Alvarez).

Fig.1. Clarifiedleaf,surfaceview:A:centralbundle;B:leafmargin;C:lateral bun-dle;D:petiole.

http://dx.doi.org/10.1016/j.bjp.2016.03.008

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Fig.2.(A)CrosssectionofViolamaculataCav.leaf,centralbundlewithxylem,phloemandaparenchymaticbundlesheathsurroundingthem.(B)Crosssectionofleafmargin, detailshowingcalciumoxalatecrystals(1),parenchymawithchloroplasts(2),andcellswithtannins(3).

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10–25×4–10mm,ovate,slightlyordeeplyemarginated,the lat-eraloneshaveclaviformhairs.Spur1–3mm.Ovaryisconicaland hairless.Styleis2mm,geniculateatbase,widerattheapex.Capsule is5–8mmlong.ThesampleswestudiedwerecollectedinParque NacionalLosAlerces,aNationalParkestablishedin1937to pro-tectthenativeforestoflahuanoralerce(FitzroyacupressoidesMol Johnst.).ItislocatedinthewestsectorofChubutprovincewhich isanareaof259,570hectaresintheecoregionBosques Patagóni-cos(PatagoniaForests)(CabreraandWillinik,1980;Rossow,1988; ToledoandKutschker,2012).Theareahasacomplexlacustrine sys-tem(Conticelloetal.,1997),glaciers(e.g.,Torecillas),mountains, andpaths,inhabitedbyrareanimalspeciessuchasthehuemul (HippocmelusbisculusMolina),thepudú(Pudumephistophiles),the palomaaraucana(Patagioenasaraucana),andthegatohui ˜na( Leop-ardus guigna). V. maculata is referred to as used in traditional medicinebytheMapuchepeople,formerlysemi-nomadic horticul-turistsandgatherersinhabitantsoftheNorth-WesternPatagonia (ArgentinaandChile).Theirdescendantsstillpreservetheir ances-tralknowledgeaboutthemedicalusesofsomewildnativeplants,in

spiteoftheundergoinglossesandtransformations(Citarella,1995). Estombaetal.(2006)andConticelloetal.(1997)havegathered partofthatknowledgebyinterviewingsomeMapuche communi-ties,describingtheuseofV.maculata,pilundeuororejaderatónby theChurruhincamapuchecommunityindifferentsituations,e.g., thewholeplantisusedasanenergizerandindermatology,and theinfusionpreparedusingtheleavesisusedasalavageincaseof someeyediseases.Tourkarkissian(1980)alsomentioneditsuseas pectoralanddiaphoretic,andingastrointestinaldiseases(Molares andLadio,2014).Inthisworkwehaveperformedtheanatomical analysisfromcrosssectionsofleavesandpetiolefromV.maculata

collectedinParqueNacionalLosAlerces.

Materialsandmethods

Plantmaterial

LeavesofViolamaculataCav.var.maculata,Violaceae,were col-lectedinParqueNacionalLosAlerces(42◦₄₈′₂₇′′_S,₇₁◦₅₃′₅₆′′_W),

2

A

6

5

C

4

B

1

3

20 µm 2

5

1

2

22.5 µm

3 4

1

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A

50 _µm

B

1

2

3

4

Fig.5.CrosssectionofViolamaculataCav.petiole:(A)safranine-fastgreendye;and(B)centralvascularbundle,1–epidermis,2–hypodermis,3–parenchymawith4-cells containingtannin.

Argentina,andidentifiedbyCusato,numberofdeposit4068BAF. February1997.

Assays

Leafbladesandpetiolesfromtwentyrandomlyselectedplants werefixedinFAA(formalin,glacialaceticacidandethanol80◦_,

1:1:8)forperforminganatomicalstudies.Fivecentralleafletsfrom eachplantwereclarifiedaccordingtoDizeodeStrittmatter(1984). Theparameters analyzed werestomata and hair sizeand den-sity.ThefoliararchitecturewasdescribedfollowingHickey(1979). Free-handcrosssectionsandcutswithaMinotmicrotomefrom leafbladesandpetioleswerestainedwithsafranine-fastgreenand mountedonmicroscopeslidesusingglycerine-gelly.Thefollowing characterswereinvestigated:presenceofcalciumoxalatecrystals, presenceoftannins,andpresenceofhairs.LeaveswerefixedinFAA tobeanalyzedwithScanningElectronicMicroscopy(SEM).Also,a Zeisspolarizedlightmicroscopewasusedfortheobservationof crystals.DrawingsweremadeinaLeizopticmicroscope.Three tofourmeasurementsandobservationreplicatesweremadefrom severalcross-sections.

Resultsanddiscussion

Violamaculata leafblade organization is simple (Fig.1), the laminashapeissymmetricshowingacrenateleafmarginwitha chartaceoustextureandanormalpetiolewithacraspedodromous venation(Hickey,1979).Athincuticlelayeronbothsurfaces (adax-ialandabaxial)coverstheleaves.Theupperepidermisconsistsof asinglerowofsquareorrectangularcells,65–85␮mwide(Fig.2). Stomataareparacytic,locatedatthesameleveloftheother epi-dermalcells, witha stomatadensityof 3permm2_._Cells_of_the lowerepidermisarewavy,24–42␮mwide,withanomocytic sto-mataandastomatadensityof80permm2₍_Fig.₃_)._The_analysis_with SEMshowedthatcuticleisthin,foldedoverthenervesandsmooth amongthem.Also,therearesimpleandone-cellhairsonboth epi-dermises.Leafbladesarepolymorphous,egg-shaped,crenated,and pubescentatthemargin.Hairsaresimple,unicellular.Mesophyllis dorsiventral,amphistomatic,palisadeparenchymaare1–2layered belowtheupperepidermis,withcellscontainingtannins.Spongy parenchymacells are 2–3layered(Fig.2)withcells containing tanninsandcalciumoxalatecrystals.Thevascularbundleinthe mid-ribiscollateralandthebundlesheathconsistsofparenchyma cells(Fig.4).Thereissclerenchymatowardtheupperepidermis

andcollenchymatowardthelowerepidermis.Thebundlesheath alsosurroundsthesecondaryvein.

Petiole(Fig.5)iscoveredbyepidermalcellswithathick cuti-cleandfewstomata.A2–3layeredcellscollenchymaissituated undertheepidermis.Parenchymatouscortex consistsof spheri-calcells,10–12layeredwithintercellularspaceandabundantcells withtannin.Thereisalargecentralvascularbundle,collateral with-outabundlesheath.Thexylemturnstowarduppersideofpetiole, andthephloemturnstowardthelowersideofthepetiole.

As it was reported for some other species of Violacea (e.g., V. odorata, V. alba, V. sintenisii, V. tineorum, V. arvensis),

V.maculatapresentscalciumoxalatecrystals,isolatedorless fre-quentlygrouped,betweentheleaves’palisadeandlacunosetissues (Colomboetal.,2007;WatsonandFlores,2009;Toiuetal.,2010). Also,somespecies(e.g.,V.kizildaghensis,V.tineorum,V.ucriana) containbrownmucilaginousbodies inthemesophyll(Colombo etal.,2007;Dinc¸andYıldırımlı,2007;Ba˘gcıetal.,2008).Asfor tri-chomes,someViolaspeciesareglabrous(V.metajapoica,V.tricolor,

V.micranthellaWedd)whileothershavesimpleandunicellular tri-chomes(V.odorata,V.kizildaghensis,V.arvensis,V.hirta,V.elatior, V.alba suspalba)(Sansoetal.,2003,2005;Dinc¸andYıldırımlı, 2007;Ba˘gcıetal.,2008;Toiuetal.,2010;WatsonandFlores,2011; Yousefietal.,2012;MehrvarzandMarcussen,2013;Mareacreetal., 2014).V.kizildaghensis,V.arvensis,V.hirta,V.elatior,V.tineorum, haveanisocytic stomata(Dinc¸and Yıldırımlı,2007;Ba˘gcı etal., 2008;MehrvarzandMarcussen,2013;Mareacreetal.,2014)while

V.maculatastomataareparacyticintheadaxialleafsurfaceand anomocyticintheabaxialleafsurface.Asthespeciesisusedin tra-ditionalmedicinethisstudycancontributetotheknowledgeand characterizationofthespecies.

Authors’contributions

CPcontributed incollecting plantsample and identification, designingthestudyandanalysisofdata,andinwritingthepaper. MVRcontributedinrunningthelaboratoryworkandanalysisof data.MAAcontributedincriticalanalysisofdata,supervisedthe laboratorywork,andinwritingthemanuscript.Alltheauthorshave readthefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

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Acknowledgments

M.A.AlvarezismemberofCONICET.UniversidadMaimo ´nides fundedthiswork.WewishtothankMs.IsabelRilloforheradvice andcarefulrevisionofEnglish.

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