Rev. bras. farmacogn. vol.26 número1

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Original

Article

Ovary

and

fruit

morphology

and

anatomy

of

Amphilophium

crucigerum

Rosana

Casoti

a

_,

_Melânia

_Palermo

_Manfron

a,∗

,

João

Marcelo

Santos

de

Oliveira

b a_Laboratório_de_{Farmacognosia,}_Universidade_Federal_de_Santa_Maria,_Santa_Maria,_RS,_Brazil

b_Laboratório_de_Botânica_Estrutural,_Universidade_Federal_de_Santa_Maria,_Santa_Maria,_RS,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2April2015 Accepted6August2015

Availableonline28September2015

Keywords:

Crystals Histochemical Morphoanatomy Ovary Seed Trichomes

a

b

s

t

r

a

c

t

Amphilophiumcrucigerum(L.)L.G.Lohmann,knownas“pente-de-macaco”isaspeciesofBignoniaceae nativetoBrazil,andwhoseseedsareusedinfolkmedicine.Thisstudyaimedtodescribe morphoanatomi-calfeaturesofthisspeciesoffruittoaidinitscorrectidentificationandpharmacognosticanalysis.Samples ofovary,pericarpandseedwerefixedwith3%glutaraldehyde,sectionedonarotarymicrotomeand ana-lyzedbystereomicroscope.Theresultsareshowninthreeparts:(1)Theovarypresentspeltatetrichomes, longnon-glandulartrichomesandemergencesintheepidermis;itis2-carpellateandunilocularwithtwo intrudingparietalplacenta;ovulesarenumerousontheplacenta;itpresentsalargequantityofcrystals. (2)Thepericarpiswoody,denselyechinateandellipticshape;itpresentsa2-valvedcapsuleandis septi-cidal;itpresentsemergences,stomata,lenticels,crystalsandalargequantityofclusteredstonescells.(3) Seedsarealate,exalbuminateandexotestal;thereisalargeamountofcrystalsintheexotestalregion;it presentsanendotheliumandremnantendosperm.Histochemicaltestsshowedthepresenceoflipophilic substances,polysaccharides,phenolicsubstances,alkaloidsandasmallquantityofstarch.These phar-macobotanicalfeaturesdescribedforA.crucigerumareessentialforthepharmacognosticanalysisofthe drugplant.

Introduction

The Bignoniaceae family is composed of approximately 406

species,predominantlyneotropical(Lohmann,2015).Onlythree

tribesofthisfamilyoccurinBrazil:Tecomeae,Crescentieaeand

Bignonieae (Sandwith and Hunt,1974; Von Poseret al., 2000).

Amphilophiumcrucigerum(L.)L.G.Lohmannbelongstothe

Bignon-ieaetribe,whosegenusiscomposedof28lianaspecies(Lohmann,

2015). A. crucigerum presents synonymy with Pithecoctenium echinatumandPithecocteniumcrucigerumamongseveralother

syn-onyms(Lohmann,2015).

Thisspeciesfrequentlygrowsonforestclearingsandonthe

bor-dersofhighways.ItbloomsfromOctobertoDecember.InBrazil,A.

crucigerumispopularlyknownas“pente-de-macaco”andis

cul-tivatedasornamental(Sandwith andHunt,1974).Itsfruitsare

usedinfolkmedicinetotreatneuralgia(Bye,1979),inflammations,

skininfectionsandheadachesandasacalmingagent(Francoand

Fontana,2005).

The Bignoniaceae species typically present iridoids,

alka-loids, flavones, naphthaquinones, anthraquinones, tannins, and

∗ Correspondingauthor.

E-mail:[email protected](M.P.Manfron).

anthocyanins(Fischeretal.,2004).Iridoidglycosideswereisolated

from stems of A. crucigerum, showing an antioxidant potential

againstDPPH,bybioautography,andagainstacetylcholinesterase

inhibitors(Martinetal.,2007).

Despite the pharmacological potential attributed to A.

cru-cigerum,therearenoreportstodatedescribingdiagnosticfeatures

fordiscriminatingthisspecies.Delimitingthegenericlevelofthe

BignonieaetribehasalwaysbeenaproblemaccordingtoLohmann

(2006),duetothelackofdiagnosticfeaturesandbecauseof

over-lappingpatternsofmorphologicalvariationwhichmakeitdifficult

toidentify.Thepresentstudyaimstocharacterizethe

morpho-logical and anatomical featuresof theovary,pericarp andseed

ofA.crucigerum,describingusefulstructuralfeaturesinorderto

improve its descriptionand identification,as wellto present a

histochemicalanalysis.Thesefeaturesareessentialforthe

phar-macognosticanalysisofthedrugplant.

Materialandmethods

Plantmaterial

Amphilophiumcrucigerum(L.)L.G.Lohmann,Bignoniaceae,was

obtainedfromtheSouthernregionofBrazil,at29◦₄₁′₀₂′′_S_and

53◦₄₈′₂₅′′_W._The_flowers_and_mature_fruits,_from_seven_individuals,

http://dx.doi.org/10.1016/j.bjp.2015.08.006

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16 R.Casotietal./RevistaBrasileiradeFarmacognosia26(2016)15–22

Fig.1.DissectedflowerofAmphilophiumcrucigerum.(A)Generalaspectofthegynoecium;(B)ovaryshowingtrichomes(25×);and(C)cross-sectionofovary(35×).

werecollectedfromNovembertoMarch.Thecollectedmaterial

wasidentifiedby GilbertoDolejal Zanettiandthevoucher was

registeredundernumber 12872 SMDBattheherbarium ofthe

BiologyDepartmentattheFederalUniversityofSantaMaria.

Morphologicalandanatomicalcharacterization

AfterdissectionwithstereomicroscopeSZH10(Olympus®

)and

M80(Leica®_),_the_samples_were_fixed_in_3%_{glutaraldehyde}_in_0.1_M

sodiumphosphatebuffer,pH7.2(Gabriel,1982)andsubmittedto

vacuumfor6hforimprovedinfiltration.Subsequently,thesamples

werewashedin0.1Msodiumphosphatebuffer,pH7.2(Gabriel,

1982),andthenindistilledwater.Tween20wasutilizedduring

24hforextractionofepicuticularwaxes.Subsequently,the

sam-plesunderwentdehydrationinanethylalcoholseries,followedby

solutionsofchloroformandpureethanol(1:3,1:1,3:1,1:1,1:3),

andfinallyofpureethanol.Thesampleswerepre-infiltratedina

2-hydroxyethylmethacrylate(HEMA)andpureethanolsolution(1:1)

during12h,followedinpureHEMAandembeddinginthesame

resin,inaTeflonholderuntilreachingpolymerization(Gerritsand

Smid,1983).Sectionsof5␮mthicknessweremadeusingaRM2245

rotarymicrotome(Leica®_)._Toluidine_blue_O_in_0.05%_sodium

ben-zoatebuffer,pH4.4wasusedforstaining(FederandO’Brien,1968).

PermanentslidesweredepositedinthecollectionattheStructural

BotanyLaboratoryoftheBiologyDepartment,UFSM.Observations

andphotomicrographsinbrightfieldandpolarizedlightwere

per-formedusingaDM2000microscope(Leica®

)withaDFC295image

capturesystem(Leica®_).

Histochemicalanalysis

Handsectionsofseedswerepreparedforhistochemicaltestsfor

differentpurposes:Lugol’ssolutionforstarch(Jensen,1962);Sudan

IIItodetectlipophilicsubstances(Brundrettetal.,1991);

Dragen-dorfftodetectalkaloids(FurrandMahlberg,1981);PAS(periodic

acid-Schiff)todetecttotalpolysaccharides(Maia,1979)and

tolui-dineblueforlignins,pectinsandphenoliccompounds(O’Brienand

McCully,1981).

Resultsanddiscussion

Gynoeciummorphology

Thestigmais2-lobed(Fig.1A).Theyellowishanddenselyhairy

ovaryissuperiorandcylindricalinthetransversalsection(Fig.1B

andC).Thetrichomes,situatedintheovary,reflectedlightwhen

observedunderthemagnifyingglass(Fig.1BandC).

Arelativelywell-developedring-shapednectaryispresentat

thebaseoftheovary(Fig.1A),wheretherearestomataand

tri-chomessimilartothosefoundintheovary.Thetypeofnectaryand

thepresenceoftrichomesinthenectaryareconsideredinvariable

features,makingthemimportantinthedeterminationofspeciesof

differentgenera,includingAmphilophium(Rivera,2000).Someof

thefeaturesdescribedabovewerealsodescribedforgenus

deter-minationbySandwithandHunt(1974),Galetto(1995),Nogueira

etal.(2013)andLohmannandTaylor(2014).

Ovaryanatomy

Theovaryis2-carpellate,unilocularandplurispermic,withtwo

intrudingparietalplacenta(Fig.2AandB),similartothatdescribed

byFischeretal.(2004).Theovulesareanatropous(Fig.2C).

Theouterepidermisiscomposedofonecelllayer,andpresents

alargequantityofnon-glandulartrichomes(Fig.3AandD).These

trichomesaremulticellular,longandnon-branched.Studiescarried

outbyNogueiraetal.(2013),inleavesofA.crucigerum,showedthe

presenceofbranchednon-glandulartrichomes.Inthenectarywas

observedstomata(Fig.3E).

Intheovary,emergenceswerealsoobserved,besidesa

rela-tivelyfewernumberofglandulartrichomesatdifferentstagesof

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Fig.3.LongitudinalsectionoftheovaryofAmphilophiumcrucigerum.(A)Generalaspectofovary;(B)outerepidermis;(C)trichomeandemergence;(D)outerepidermis withcrystals;and(E)stomata.cr,crystals;em,emergence;ep,epidermiscells;gt,glandulartrichomes;mp,mesophyll;ne,nectary;ov,ovule;st,stomata;tr,trichomes.

development(Fig.3BandD).Theemergencespossesswidebases

and the subepidermal cells are only partof thebase, possibly

derivedfromoneortwosubepidermalcells(Fig.3C).Alarge

por-tionofthisstructureismadeofcellsderivedfromtheepidermis.

Thearrangementandsizeofthesecellsareirregular.Inaddition,

theemergencespresentasharpapexdefinedbyasinglecell.

Theglandulartrichomeisapeltatemorphotype,composedof

sixcellslocatedontheheadofthetrichomeandasinglecellon

thestalk(Fig.3B).AccordingtoNogueiraetal.(2013),thepresence

ofpeltatetrichomesisverycommoninspeciesoftheBignonieae

tribe.Thepresenceoftrichomesisoftenassociatedwithdesiccation

and/orprotectionagainstherbivores(Wagneretal.,2004).

The mesophyll is composed of parenchymatous tissue

(Figs. 2A and 3A). The marginal vascular bundles bifurcate

radiallywithhalfofthebundlefacingtowardacentralposition

neartheplacentae.Thus,theovaryhastwoventrallargevascular

bundlesandtwolargedorsalvascularbundlesonthecarpelwall,

Fig.4.LongitudinalsectionoftheovaryofAmphilophiumcrucigerum.(A)Detailof parenchymaticandvasculartissueswithcrystalaccumulation(brightfield)and(B) crystalsintheparenchymatictissueandinvascularbundlesafterpolarizedlight. cr,crystals;vb,vascularbundle.

besidesmanysmalllateralvascularbundlesfoundontheovary

wall(Figs.2Aand3A).

Numerouscrystalliferouscellsoccurinthemesophyll(Fig.2B).

There is also a large accumulation of calcium oxalate crystals

on the vascular tissue, which apparently obliterate some

ves-selelements,andtherefore,withoutanidentifiableformortype

(Figs.2Band4AandB).Theinnerepidermispresentsisodiametric

cellsthatdevelopvacuoles.Theepidermis,whichcoversthe

pla-centa,presentscellswithdensecytoplasmthataresmallerthan

thelocularcells (Fig.2B). Theovulesareanatropous,unitegmic

and tenuinucellate. They are located on the placenta and are

numerous (Fig. 3A). Part of the results is in agreement with

thoseusuallydescribedforBignoniaceaeandBignonieae(Corner,

1976;Armstrong,1985;Fischeretal.,2004,LohmannandTaylor,

2014).

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Fig.6. SeedsofAmphilophiumcrucigerum.(A)Generalaspectofseed:dorsalface;(B)generalaspectofseed:ventralface;(C)embryocoveredcoriaceouswrapper:raphe; (D)embryocoveredcoriaceouswrapper:anti-raphe;and(E)wingfrontview.

Fruitmorphology

Pericarp

Thefruitiselliptic,woodyanddenselyechinatewith

approx-imately12cminlength,6cminwidthand2cminthickness,in

themedianregion.Itiscomposedofa2-valvedcapsuleslightly

flattenedwithathickpericarpandaseminiferouscolumn(Fig.5A

andC).

Thefruitsaresepticidalcapsules,whichisoneofthekey

identi-fyingfeaturesfortheBignonieaetribe(Gentry,1980;Barrosoetal.,

1999;Fischeretal.,2004).Thepericarpsarebiconvexwithadorsal

vascularbundle.Thefruitisgreenish-browntorust-colored,when

dry.

AmongthemainfeaturesofA.crucigerumfruit,thereare

echi-nate(horn-like)projectionsoftheexocarp,whichisconsideredas

morphologicalsynapomorphiesinthisgenus(Pool,2007;Lohmann

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Fig.8.LongitudinalsectionsthroughthelateralplaneoftheseedsofAmphilophiumcrucigerum.(A)Generalaspect;(B)testa;(C)endospermandendothelium;(D)detail ofthecotyledonnode;(E)hypocotylradicleaxis;and(F)calazalregion.cn,cotyledonnode;co,cotyledon;de,cotyledondorsalepidermis;eb,embryo;ed,endosperm;eh, hypocotylradicleaxis;et,endothelium;ex,exotesta;mt,mesotesta;t,testa.

andTaylor,2014).AccordingtoGentry(1973),theseechinate

pro-jectionsinthevalvesmayserveasprotectionagainstpredatorsto

allowtheripeningoffruitsandseeds.

Seedmorphologyandclassificationofthematureembryo

A.crucigerum presentswingedseedsalong thelengthofthe

fruitandoccupyingtheentirelocule(Fig.5C).Theseedspresent

abrownish-goldcolorandanevidentseminalnucleus(Fig.6A–D).

Theyhavebrighttranslucentwingsandpapyraceousconsistency

withapproximately5cminwidthand2.5cminheight.The

papil-lateseedcoatisanothersynapomorphyofthegenus(Lohmannand

Taylor,2014).Thewingsconsistoflateralexpansionofthe

tegu-mentandchalazalregion.Thenumberofcelllayersreducestoward

theedgeoftheseminalnucleusandofthewing,whereonlyone

celllayeroccurs(Fig.6E).

According to Fischer et al. (2004), in Pithecoctenium,

Anemopaegma and Jacaranda, the seminal body is enveloped

byalargewing.Thesamecanbeobservedforothertaxafrom

this family,suchasTabebuiaochracea (Sampaio etal.,2007), T.

chrysotrica(Souzaetal.,2005)andMacfadyenaunguis-cati(Souza

etal.,2008),wherethechalazalportionoftheseedexpandsand

becomespartofthewing,exceptinT.caraiba(FerreiraandCunha,

2000).However,insomeBignoniaceaethereisanalmostvestigial

chalazalexpansionof thewing,asin Tecomastans(Renòetal.,

2007).

Fruitanatomyandhistology

Pericarp

Thepericarpiscomposedofthreedifferentportions(Fig.7A).

Theouterportioncomprisestheexocarp.Belowexocarpoccursthe

mesocarp,dividedintothreeregions.Theinnerportioncomprises

theendocarp.

Theexocarpiscomposedofonetothreecelllayers.Lenticels

andemergencesarepresent(Fig.7B–D).Theouterpericlinalwall

ofthecellsisthickandlignified(Fig.7D).Stomatalguardcellsoccur

slightlyabovethesurfaceoftheexocarp(Fig.7D).

The outer mesocarp is formed during fruit differentiation

throughtheproliferationofoutertissue,resultinginhorn-like

pro-jections(Fig.7A).Theoutermesocarppresentsparenchymatous

cellsandclusteredstonescells(Fig.7B)thatformanalmost

con-tinuouslayer.Atthebaseofthehorn-likeprojections,thereare

layersofsclerifiedcells,demarcatingtheoutermesocarpfromthe

middlemesocarp(Fig.7AandE).Needle-likecrystalsarepresent

ontheoutermesocarpinaradialorganization(Fig.7F).

The middle mesocarpis composed of vascular bundles, and

parenchymatous tissue withthin sclerified walls (Fig. 7E).The

innermesocarpispredominantlycomposedoftissuesimilartothat

foundinthemiddlemesocarp,buttherearenovascularbundles

(Fig.7A).Inaddition,theinnermesocarppresentstwo

isodiamet-riccelllayerswiththickwalls.Theendocarpisformedbyasingle

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Fig.9.LongitudinalsectionoftheseedofAmphilophiumcrucigerum.(A)testa;(B) wallthickening,afterpolarizedlight;(C)detailsoftherapheregion(testa);and (D)crystalsontestaandoncotyledon,afterpolarizedlight.cn,cotyledonnode;co, cotyledon;ed,endosperm;eh,hypocotylradicleaxis;ex,exotesta;de,cotyledon dorsalepidermis;et,endothelium;mt,mesotesta.

Seed

TheseedsareexalbuminousinA.crucigerum(Fig.8A),asthey

alsoareinTabebuiaochracea(Sampaioetal.,2007).Thisremnant

endospermisformedbyalayerofparenchymatouscellswithout

anytypeofnutritivereserve(Fig.8BandC).However,intheregion

neartotheraphe,theendospermpresentsthreeorfourcelllayers,

whichgraduallylessenuntilformingasinglecelllayer(Fig.8Eand

F).ThisfeatureistypicalinBignoniaceae,asdescribedbyCorner

(1976),Esau(1985)andJohrietal.(1992).

TheseauthorsreportBignoniaceaeseedsasexalbuminouswith

absent or scarce endosperm, except in some species, such as

Pyrostegiavenusta,whichpresentsanevidentendosperm(Gabrielli

andCastro,1995).

Theembryopresentsjuxtaposed,2-lobed,foliaceouscotyledons

fromtheapextothebase,andayellowish coriaceouswrapper.

Theembryosareonaverage0.8cminlength,0.6cminwidthand

0.1cminthickness(Fig.6CandD).Theembryopresentsa

contin-uousaxialandstraighthypocotyl-radicleaxis,whichisverysmall

incomparisontothecotyledonlobules(Figs.6Dand8A).In

con-trast,T.stanspresentsamoreevidenthypocotyl-radicleaxis(Renò

etal.,2007).Inthissection,embryospresentanoblong-transverse

andlenticularshape(Fig.8A),whicharefeaturesofBignoniaceae

(Barrosoet al.,1999; Ferreiraand Cunha,2000; Sampaioet al.,

2007).

Theseedisexotestal(Fig.8A–C),possessingelongatedcellswith

secondaryradialwallsintheformofbandswiththepresenceof

crystals(Fig.9A–D).Theparietalthickeningoftheexotestalcells

aremoredevelopedintheseminalnucleus,whichhasneverbeen

reportedforthisgenus.

In the mesotestal tissue composed of sclerenchymatous

parenchyma,maturecellsundergocompressionfromthe

expand-ingembryo (Figs. 8C,9A, C, and 10A, B). Themesotesta in the

chalazalregionpresentsagreaternumberofcellsandcell

rem-nants,incontrasttothemicropylarregion,whichpresentsonly

cellremnants(Fig.8AandD–F).

Thehistologicalanalysisrevealedphenoliccompoundsinthe

micropylarregionveryclosetothemesotestalcellscitedabove,

togetherwithcellremnantsfromthevasculartissueinthechalazal

region(Fig.8A,E,andF).Theendotestaisabsentbecauseinthis

familytheinnerepidermisisdestroyedduringthedevelopmentof

thegynophyte.

Fig.10.Longitudinalsectionthroughtheanteroposteriorplaneoftheseedof

Amphilophiumcrucigerum.(A)Generalaspect;(B)distalregion(seedwing);(C) cotyledon;(D)endospermandendothelium.cn,cotyledonnode;co,cotyledon;ed, endosperm;eh,hypocotylradicleaxis;ex,exotesta;de,cotyledondorsalepidermis; et,endothelium;mt,mesotesta;ve,cotyledonventralepidermis.

The endothelium presents membranous layer of cells that

envelop the entire embryo (Fig. 10A). This structure presents

phenoliccompoundsthatacquireayellowishcolor,eveninthe

presenceoftoluidineblue(Fig.8E).

Thepresenceofanendotheliumandscarceendosperm(ab

ini-tio)iscommoninspecies ofBignoniaceae,whichareunitegmic

andtenuinucellate(Souzaetal.,2005;LohmannandTaylor,2014).

Pyrostegia venusta presents endosperm with two to six layers

(GabrielliandCastro,1995).

Inthelongitudinalsection,theepidermalcellsofthecotyledons

areslightlytangentiallyelongated,inboththedorsalandventral

region(Fig.10C).Themesophyllisparenchymatouswith

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Fig.11.HistochemicalanalysisofembryoofAmphilophiumcrucigerum.(A)Dragendorff(alkaloids);(B)PAS(polysaccharides);(C)SudanIII(oils);and(D)Lugol(starch).

oils,aswellasascarcepresenceofstarch.PAS-positive

polysac-charideswereidentified onthecellwallsand inthecytoplasm

(Fig.11A–D).

InA.crucigerum,themembranouswrapperlocatedbetweenthe

embryoandthetestaissimilartothestructurefoundin

Bignoni-aceae,acommonfeatureinthefamily(GabrielliandCastro,1995;

Costa,2003;Souzaetal.,2005;Renòetal.,2007).This

membra-nouswrapperiscomposedofcellscontainingphenoliccompounds,

whichplayaroleinprotectingagainstpredatorsand

microorgan-isms,andincreasethehardnessofthetegumentandgivecolorto

theseed(BeltratiandPaoli,2006).

Understandingthe maingroups of secondary metabolitesin

medicinalplantsis importantas astarting point forstudies on

biologicalactivityorfortheisolationandidentificationof

bioac-tivecompounds,especiallythoseusedinfolkmedicine.Phenolic

compoundsarelinkedtoantioxidant,anti-inflammatoryand

anal-gesicactivities,amongmanyotherbiologicaleffects(DeOliveira

etal.,2012;Figueiredo-Rinheletal.,2013).Alkaloidsare

associ-atedtoagreaternumberofactivities,includingantimicrobialand

analgesicactivities(Huetal.,2013).Thus,thepresenceofthese

typesofcompoundscorroborateswiththeuseofthisspeciesin

folkmedicine.

Structuraldetailsofcellsarerelevantwhenobservingtornor

groundmaterial,bothforpharmacognosticcontrolandforquick

identification of species, as reported by Oliveira et al. (2003),

Verdametal.(2012),Jasinskietal.(2014),andSantosetal.(2015).

SeedsofA.crucigerumpresentmorphologicalandanatomical

fea-turescommontothefamily.In addition,specificfeatures allow

theidentificationandreliableanalysisofthespecies,including:

presenceoftrichomesintheovaryandpartofthenectary;large

quantityofcrystalsonfloraltissue;exotestalcellswithsecondary

radialwallswiththepresenceofcrystalsonseedwingsandthe

presenceofaremnantendospermintheseed;andapositivetest

foralkaloidsintheembryo.

Authors’contributions

RCcontributedtothecollectionofplantsamples,preparation

ofherbarium,runningthelaboratorywork,analysisofthedata

anddraftingthepaper.MPMandJMSOdesignedthestudy,

super-visedthelaboratoryworkandcontributed tocriticalreading of

themanuscript.Alltheauthorshavereadthefinalmanuscriptand

approvedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthanktheFederalUniversityofSantaMariaand

CAPES(Coordenac¸ãodeAperfeic¸oamentodePessoaldeNível

Supe-rior)forfinancialsupport.

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