w ww . e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Leaf
histochemistry
analysis
of
four
medicinal
species
from
Cerrado
Vinícius
C.
Kuster,
Fernando
H.A.
Vale
∗DepartamentodeBotânica,InstitutodeCiênciasBiológicas,UniversidadeFederaldeMinasGerais,BeloHorizonte,MG,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received24March2016
Accepted9May2016
Availableonline30July2016
Keywords:
Chemicalcompounds
Idioblasts
Secondarymetabolism
SerradoCipó
a
b
s
t
r
a
c
t
Chemicalcomponentsactinplantdefenseandprotection,butmanyofthemareextractedandused medicinally.ForCerrado,activechemicalcomponentsareusedinthetreatmentofdiseases,which strengthensthenecessityforpharmacologicalstudiesofplantsofthatenvironment.Theobjectivewas toevaluatethehistochemistryoftheleafbladeofByrsonimaverbascifolia(L.)DC.,Malpighiaceae, Cam-pomanesiaadamantium(Cambess.)O.Berg,Myrtaceae,RoupalamontanaAubl.,Proteaceae,andSolanum lycocarpumA.St.-Hil.,Solanaceae,speciesthathavebeenreportedasproducersofsecondarymetabolites forpharmacologicaluse.The3rdnodeleaves(median,intercostalandmarginregions)werecollected, fixed,includedinParaplast®or2-hydroxyethylmethacrylate,sectionedinmicrotome,stainedand photo-graphedonmicroscope.Thisanalysisaimedtofindleafregionswhichproducedchemicalcompounds. Forhistochemicaltests,intercostalareaswereselectedfrommedianregionleafofthe3rdnode.Samples freshandnewlycollectedandfixedandembeddedinParaplast®wereused.Testswereconductedfor lipids,terpenoids,phenoliccompounds,alkaloids,sugarsandproteins.Alkaloidswereobservedonlyin R.montana,aswellastheresultsforphenoliccompounds.FlavonoidsarepresentinB.verbascifoliaandR. montana.ThelipidcompositionwasshowedforthechemicalcompoundsofB.verbascifoliaandC. adaman-tium,whichprovedtobepartoftheessentialoilsorresinsoilsinC.adamantiumidioblasts.Thechemical compoundsofB.verbascifolia,C.adamantiumandR.montanaarepresentmainlyinidioblastsamongthe parenchymaandepidermalcells.C.adamantiumhassecretorycavities,butonlywithlipidcontent.The identificationofchemicalcompoundshasnotbeenpossibleinmatureleavesofS.lycocarpum.
©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Plantsecondarymetabolismproducesproductsthatcanaidin thedefenseandprotection,besidestheattractionofpollinators (Evert,2006).Thosesubstanceshavebeenextractedandutilized formedicineproduction,vaccinesand otherforms oftreatment (Barbosa-Filhoetal.,2008;Souzaetal.,2008).Inthiscontext,the anatomic studyof pharmacologicuse plants maycontributeto qualityassuranceand correctidentification(Mauroetal.,2008; Carpanoetal.,2009;Gomesetal.,2009).Beyondthat,itallowsto elucidatetheaspectsreferringtosecretingstructuresand conse-quentlytostorageandsecretionofsecondarymetabolites,which couldleadtothecorrectlocalizationandextractionofmedicinal chemicals.
Secreting structures arefrequently reported tothedifferent organs of Cerrado plants (Castro et al., 1997; Rodrigues et al., 2011;BoudourisandQueenborough,2013),thisbeingBiomerich
∗ Correspondingauthor.
E-mail:fvale@icb.ifmg.br(F.H.Vale).
inmedicinaluseplants(Silvaetal.,2010).Leaves,xylopodiaand barksarequotedasproducersofactivepharmacologicalsubstances (Silvaetal.,2010).AccordingtoRibeiroandWalter(1998)the Cer-radoisthesecondlargestBiomeinBrazil,occupyingaround23%of nationalterritory(OliveiraandMarquis,2002).Itisconsideredthe floristicallyrichestSavannahintheworldwithelevatedendemism, beingoneoftheprioritizedBrazilianareastoconservation(Myers etal.,2000).
Malphigiaceae,Myrtaceae,ProteaceaeandSolanaceaearewell representedontheCerrado(Rizzini,1971;Pereira-Silvaetal.,2004) that alsocontainplantspecies provided withactivesubstances usefultodiseasestreatment.Byrsonima(Malpighiaceae)presents specieswithantimalarialactivity(Milliken,1997).Byrsonima cras-sifolia(L.)KunthandByrsonimaverbascifolia(L.)DC.,andareused forantifeverindifferentcountriesinLatinAmerica(Rutter,1990; Garcia-Barriga,1992).TheleavesofspeciesofCampomanesia (Myr-taceae)areusefulfortreatmentofdiarrheaandbladderissues(Piva, 2002).Besidestheethylacetateextractoffruitsof Campomane-siaadamantium(Cambess.)O.Berghasshownaninhibitoryeffect againstMycobacteriumtuberculosis,a pathogenicbacteriumthat causesmostcasesoftuberculosis(Pavanetal.,2009).Theleavesof theRoupalamontanaAubl.(Proteaceae)areutilizedasantipyretics
http://dx.doi.org/10.1016/j.bjp.2016.05.015
0102-695X/©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
andantisepticsfortreatmentofwoundsandulcers(Butleretal., 2000).Solanaceaeisasourceofalkaloidsprovidedwith pharma-cologicalactions,especiallySolanumcrinitumLam.,S.lycocarpum A.St.-Hil.andS.gomphodesDunal,forbeingusedinthetreatment ofdiabetes(Araújoetal.,2010b).
Basedontheexposure,thisstudyaimedtoevaluatethe histo-chemistryoftheleavesofB.verbascifolia(L.)DC.,Malpighiaceae,C. adamantium(Cambess.)O.Berg,Myrtaceae,R.montanaAubl., Pro-teaceae,andS.lycocarpumA.St.-Hil.,Solanaceae,speciesreported asproducersofsecondarymetabolitesofpharmacological activ-ities. Also, it intends to describe the secretory structures that producesuch compounds,and identifythecells and tissues in whichtheyarestored.
Materialsandmethods
Studiedspeciesanddepositattheherbarium
Byrsonimaverbascifolia(L.)DC.,Malpighiaceae,Campomanesia adamantium(Cambess.)O.Berg,Myrtaceae,RoupalamontanaAubl., Proteaceae,andSolanumlycocarpumA.St.-Hil.,Solanaceae,were collectedatthe“Camposujo”(shrubSavannah)ofCerradowithin SerradoCipó,MinasGerais,Brazil(19◦22′01′’Sand43◦37′10′’W).
VouchersweredepositedattheHerbariumofUniversidadeFederal deMinasGerais(BHCB),undertheregistrationnumbers:161584, 161585,161586and167052.
Lightmicroscopy
Leaves(n=5)ofthe3rdnodeoffiveindividualswerecollected,
fixedinFAA(formalin,aceticacid,50%ethanol,1:1:18v/v/v)and storedinethanol70%(Johansen,1940).
InclusionsinParaplast®
(KrausandArduin,1997)and/orin 2-hydroxyethylmethacrylate (Leica Instruments,historesin)were donewithfragmentsofthemedianregion.Then,crosssectionsof 5–10mwereobtainedusingarotarymicrotome(Leica®
Biocut Jung,USA).Thematerialpreparedinhistoresinwasstainedwith 0.05%toluidineblue–pH4.7(O’Brianetal.,1964)andParaplast®
with0.5%safraninandastrablue,2:8(KrausandArduin,1997).All leavesweremountedinEntellan®(KrausandArduin,1997)and
photographedwithuseofalightmicroscope(PrimoStarZeiss®)
coupledwithdigitalcamera(CanonA650).
Histochemicaltests
Transversesectionsofthemedianareaoftheintercostalregion wereobtainedfromfreshandrecentlycollectedleavesfromthe3rd
node(n=5),usingtablemicrotome(RolembergandBheringTrade, modelLPC).ThetestedmetabolitesclassesarelistedinBox1.Fresh selections,unfixedandunstained,wereutilizedasnegative con-trol.Thepositivecontrolwasconductedasrecommendedbythe respectiveauthorsofhistochemicaltests.Thetestswererepeated onmaterialincludedinParaplast®inordertoobtainthinner
sec-tionsandthusimprovethevisualizationoftheresults.Inbothcases, theslidesweremountedinthereagentitselforjellyglycerin.The sectionswerephotographedunderalightmicroscope(PrimoStar Zeiss®)coupledwithdigitalcamera(CanonA650).
Results
Therewasnooccurrence ofexternal secretorystructureson theleaflaminainanyspeciesstudied,butonlyinternalsecretory structuresoccurredinthem(classificationofEvert,2006).These structuresoccurinthemidrib(Fig.1A,DandG),theintercostal region(Fig.1B,E,H)andthemargin(Fig.1C,FandI),particularly
Box1:Metabolitegroups,reagentsandauthorsofthe methodologiesusedinhistochemicaltests.
Metabolite groups
Reagent References
Lipids
Total SudanredB Brundrettetal.
(1991)
Terpenoids
Essentialoils andresin-oils
Nadireagent DavidandCarde
(1964)
Steroids Antimonytrichloride Hardmanand
Sofowora(1972), Maceetal.(1974)
Phenoliccompounds
General FerricchlorideIII Johansen(1940)
Tannins Vanillin–hydrochloricacid MaceandHowell
(1974)
Lignin Phloroglucinol Johansen(1940)
Flavonoids DMACA-
p-Dimethylaminocinnamaldehyde
FeuchtandSchmid (1983)
Alkaloids
General Dragendorff FurrandMahlberg
(1981)
Polysaccharides
General PAS-Periodicacid–Schiff’s reagent
Mcmanus(1948)
Starch Lugol Jensen(1962)
Pectins Rutheniumred Johansen(1940)
Mucilage Tannicacid/FerricchlorideIII PizzolatoandLillie (1973)
Proteins
Total Bromophenolblue Maziaetal.(1953)
inidioblasts(Fig.1A–I),inmostspecies.Inthesecells,thechemical compoundsoccupyuniformlyallvacuole,ingeneral(Fig.1Aand I).Theidioblastspresentprimarywallswhenintheepidermisand parenchyma(Fig.1A–F),orsecondary,whenattachedtothefibers (Fig.1G–I).
Thesizeandshapevaryaccordingtothetissuetowhichitis associated,inmostcasesbeingsimilartotheneighboringcellsof thesamemeristematicorigin(Fig.1A–I).Insuchcases,recognition isonlypossiblebythepresenceofchemicalcompounds.
Byrsonimaverbascifolia
Inmidrib,idioblastscontainingchemicalcompoundsareshown betweenxylemandphloemcells,andinparenchymaoriginated fromthegroundmeristem(Fig.1A).Intheintercostalregionand intheedgeoftheleaf,thecompoundsarefoundinepidermalcells onbothsides,inthemesophyllandinthesmallerdiameterbundles (Fig.1BandC).Inthenegativecontrolitwasnotpossibletoidentify chemicalcompounds(Fig.2A).
Thehistochemicalanalysisshowedlipidsinallidioblastswith content(Fig.2B;Box2)andconcentratedflavonoidsinthecellsof thespongyandpalisadeparenchyma(Fig.2C;Box2).
Campomanesiaadamantium
Midrib
B
. v
erbascif
olia
C
. adamantium
R.
montana
S
. lycocar
pum
Intercostal
Xy
Xy
Fi
Fi
PP
PP
PP SP
SP
Cu
Cr Sc
Xy
Xy Ph
Ph
PP
SP
BSE SC
Ph
Ep
Ep
PP
SP Ph
A
B
C
F
E
D
G
H
I
J
K
L
Margin
Fig.1.CrosssectionsofByrsonimaverbascifolia(A–C),Campomanesiaadamantium(D–F),Roupalamontana(G–I)andSolanumlycocarpum(J–L).(A,D,G,J)Midrib;(B,E,H,
K)Intercostalregion;(C,F,I,L)Margin.Abbreviations:Xy,xylem;Ph,phloem;Fi,fiber;PP,palisadeparenchyma;SP,spongyparenchyma;BSE,bundlesheathextension;SC,
secretorycavity;Cr,crystal;Sc,sclereid;Cu,cuticle;Ep,epidermis.Arrowsindicateidioblastswithchemicalcompounds.Bars=50m.
Inthesectionwithoutchemicaltreatment(negativecontrol)it wasnotpossibletovisualizeanychemicalcompoundsof poten-tiallypharmacologicaluse(Fig.2D).Fromthehistochemicaltestsit wasnotedthatessentialoilsoroilresins(Fig.2E,Box2)andlipids (Fig.2F;Box2)arewidelydistributedintheleafblade.Inthe secre-torycavitiespositiveresultsforgenerallipidswerefound(Fig.2G;
Box2).
Roupalamontana
Inmidrib,thechemicalcompoundsarepresentinsubepidermal parenchyma cells, in thefibers that surroundthevascular sys-temandintheparenchymaandfibercellsofxylemandphloem (Fig.1G).In theintercostalregion,thecompoundsoccurinthe epidermisof bothleaf surfaces,parenchyma cellsin the meso-phyll andin theminor vascular bundles (Fig.1H).In theedge, thecompoundsareassociatedwithfibersandparenchymacells (Fig.1I).
Chemicalcompoundswerenotvisualizedinnegativecontrol (Fig.2H).Positiveresultswerefoundforphenoliccompoundsin theepidermalcellsofadaxialsurface(Fig.2I,Box2).Flavonoids (Fig.2J,Box2)andalkaloids(Fig.2J,Box2)werepresentinthe mesophyllcells.
Solanumlycocarpum
Noevidenceofchemicalcompoundswasfound(Fig.1J,K,L)and allthehistochemicaltestshadnegativeresults(Box2).
Discussion
Negative control Lipids
Lipids Lipids
Flavonoids
Negative control
B
. v
erbascif
olia
C
. adamantium
R.
montana
Negative control Phenolic compounds Flavonoids Alkaloids
Essential oils Ep
SP
A
B
C
D
E
F
G
H
I
J
K
SP
Ep
SP
SP BSE
SP SP
PP PP
PP
PP
PP
PP
SP
PP
SP
SC
PP
Fig.2. Crosssectionswithpositiveresultsforhistochemicaltests.(A–C)Byrsonimaverbascifolia;(D–G)Campomanesiaadamantium;(H–K)Roupalamontana.(A,D,H)
Negativecontrol;(B,F,G)lipids;(C,J)flavonoids;(E)essentialoils;(I)phenoliccompounds;(K)alkaloids.Abbreviations:Ep,epidermis;PP,palisadeparenchyma;SP,spongy
parenchyma;BSE,bundlesheathextension;SC,secretorycavity.Arrowsindicateidioblastswithchemicalcompounds.Bars=50m.
(Fahn,1979).Thosedifferentcellspresentalargedistributionin theleaf,fromthemidribuntiltheedge,exceptforS.lycocarpum, thatcontrarytoexpectationsdidnotshowpositiveresultsfromthe histochemicalanalysisadopted.
Rinaldoetal.(2010)studiedsixspeciesofBysonimaandfound thepresenceoftwoflavonoids:catechinandepicatechin, includ-ingforBysonimaverbascifolia.Thesecompoundspresentantitumor andantioxidantactivityandmayactinthetreatmentofgastric ulcers, inflammation, skininfections and fever (Heinrich et al., 1992;Aguiaretal.,2005).Theresultsindicatethattheproduction
siteand/oraccumulationofflavonoidsinB.verbascifoliaare associ-atedwithparenchymacells,especiallyinthepalisadeandspongy parenchyma. Parenchyma mesophyll cells containing chemical compoundshavebeenreportedinsixspeciesofByrsonimafrom BrazilianCerrado(Araújoetal.,2010a),butwithouthistochemical characterization.
InMyrtaceae,secretory cavitiesproducersoflipophilic com-pounds are widely foundin species (Solereder, 1908; Metcalfe andChalk,1950),asobservedforCampomanesiaadamantium.The wideproduction ofessential oilsoroilresinsinC. adamantium
Box2:Positiveresultsofhistochemicaltestsforthesecretorystructuresontheleafbladeofthefourspeciesstudied. Abbreviations:(+)=positiveresult.
Metabolitegroups Secretorystructures Species
B.verbascifolia C.adamantium R.montana S.lycocarpum
Alkaloids General Idioblast +
Phenoliccompounds General Idioblast +
Flavonoids Idioblast + +
Lipids Total Idioblast + +
Secretorycavity +
leavescorroborates the studies of chemical characterization of other species of Campomanesia. Essential oils are present in C. guazumifolia(Cambess.)O.Berg,C.xanthocarpa(Mart.)O.BergandC. rhombeaO.Berg,beingrichinsesquiterpenes,whileC.aureaO.Berg featurespredominanceofmonoterpenes(Limbergeretal.,2001). ForC.adamantium,essentialoilsoftheleaveshavealowincome, withpresenceofmono andsesquiterpenes(Valliloetal.,2006; Coutinhoetal.,2008),howeverwiththewealthofflavanonesand chalcones(Coutinhoetal.,2008).Despitetheseresults,thetest performedtoflavonoids(DMACA)hasnotdetectedsucha com-poundintheevaluatedleafareas.Coutinhoetal.(2010)reported thattheflavonoidscontentinC.adamantiumleavesisinfluencedby seasonalvariation,whichmayexplainthefactthatthiscompound wasnotfoundintheanalysis,especiallyconsideringthatthe envi-ronmentatthelocationofcollectionshowstwodistinctseasons (KlinkandMachado,2005).
The Proteaceae chemical profile is composed mainly by flavonoids,saponins,polyphenolsglycosides,coumarinsand alka-loids(Butleretal.,2000).Theflavonoidsisolatedfromtheaerial partofR.montanahavebeenusedtoreducethemotoractivityof adultSchistosomamansoni(Nevesetal.,2015).Thesecompounds were identified in the mesophyll cells in the spongy and pal-isadeparenchyma.Furthermore,thealkaloids,typicalofProteaceae (Butleretal.,2000),werealsofoundinleavesofR.montana,inthe sameregionwheretheflavonoidsarepresent.
InS.lycocarpum,thepharmacobotanyworkusuallyshowsthe mainuseofthefruitforthetreatmentofdiseasessuchasasthma, flu,colds,andalsoasatonic(RodriguesandCarvalho,2001).The leavesareused asemollient and antirheumatic(Rodrigues and Carvalho, 2001).Despite the use, noevidence of production of potentiallyactivechemicalcompoundsingreenandmatureleaves wasfound.HistochemicalstudiesforS.lycocarpumwereconducted byAraújoetal.(2010b),butonlyelucidatedthechemical char-acteristicsofstructural components.Aireset al.(2005)showed allelopathiceffectinsenescentleavesofthisspecies,which indi-catespossibleproductionofchemical compoundsinthatorgan. Inyoungleavesglandulartrichomeswerereported(Elias etal., 2003),whichdonotremaininthematureleaf(Araújoetal.,2010b). Chemicalanalysisuncoveredthepresenceoftannins,flavonoids, steroidsandtriterpenes,coumarinsandsaponins,forS.lycocarpum leaves(Gallonetal.,2015),beingassignedantibacterial,antifungal andantiviralfortannins(Carvalhoetal.,2007).Thus,itispossible thatthechemicalcompoundsfromtheleavesofS.lycocarpumare producedonlyinyoungleavesorinsenescence,orwhichare unde-tectablebyhistochemicaltestsinspecificmonthsoftheyeardue tolowproductivity.
Insummary, throughtheadoptedhistochemicaltestsitwas possibletoidentifyintissuelevel,theregionofsynthesisand/or storageofmetabolitesofpharmacologicaluseintheleavesofB. ver-bascifolia,C.adamantiumandR.montana.Thesamewasnotpossible tothematureleavesofS.lycocarpum.Thepotentiallyactive com-poundswereconcentratedonidioblastspresentprimarilyamong theparenchymaandepidermalcells,whicharedifferentiatedfrom theothersonlybythepresenceofchemicalcompounds.
Author’scontributions
VCK(PhDstudent),partfromhisthesis,participatedinallstages ofthework;FHAVsupervisedthedoctoralstudentVCKinallstages ofthework.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
References
Aguiar,R.M.,David,J.P.,David,J.M.,2005.Unusualnaphthoquinones,catechinand
triterpenefromByrsonimamicrophylla.Phytochemistry66,2388–2392.
Aires,S.S.,Ferreira,A.G.,Borghetti,F.,2005.Efeitoalelopáticodefolhasefrutosde
SolanumlycocarpumA.St.-Hil.(Solanaceae)nagerminac¸ãoecrescimentode SesamunindicumL.(Pedaliaceae)emsolosobtrêstemperaturas.ActaBot.Bras. 19,339–344.
Araújo,J.S.,Azevedo,A.A.,Silva,L.C.,Meira,R.M.S.A.,2010a.Leafanatomyasan
additionaltaxonomytoolfor16speciesofMalpighiaceaefoundintheCerrado area(Brazil).PlantSyst.Evol.286,117–131.
Araújo,N.D.,Coelho,V.P.M.,Agra,M.F.,2010b.Estudofarmacobotânico
compara-tivodefolhasdeSolanumcrinitumLam.,SolanumgomphodesDunaleSolanum lycocarpumA.St.-Hil.,Solanaceae.Rev.Bras.Farmacog.20,666–674.
Barbosa-Filho, J.M., Alencar, A.A., Nunes, X.P., Tomaz, A.C.A., Sena-Filho, J.G.,
Athayde-Filho,P.F.,Silva,M.S.,Souza,M.F.V.,D.A-Cunha,E.V.L.,2008.Sourcesof
alpha-,beta-,gama-,delta-andepsilon-carotenes:atwentiethcenturyreview. Rev.Bras.Farmacogn.18,135–154.
Boudouris, J., Queenborough, S.A., 2013. Diversity and distribution of
extra-floral nectaries in the cerrado savanna vegetation of Brazil. Peer J.,
http://dx.doi.org/10.7717/peerj.219.
Brundrett, M.C., Kendrick, B., Peterson, C.A., 1991. Efficient lipid staining in
plantmaterialwith SudanRed 7BorFluoral Yellow088 in polyethylene glycol–glycerol.Biotech.Histochem.66,111–116.
Butler,M.S.,Katavic,P.L.,Davis,R.A.,Forster,P.I.,Guymer,G.P.,Quinn,R.J.,2000.
10-Hydroxydarlingine,anewtropanealkaloidfromtheAustralianproteaceous planttriuniaerythrocarpa.J.Nat.Prod.63,688–689.
Carpano,S.M.,Castro,M.T.,Spegazzini,E.D.,2009.Caracterizaciónmorfoanatómica
comparativaentreAloevera(L.)Burm.F.,AloearborescensMill.,Aloesaponaria Haw.yAloeciliarisHaw.(Aloeaceae).Rev.Bras.Farmacogn.19,269–275.
Carvalho,J.C.T.,Gosmann,G.,Schenkel,E.P.,2007.Compostosfenólicossimples
eheterosídicos.In:Simões,C.M.O.,Schenkel,E.P.,Gosmann,G.,Mello,J.C.P., Mentz,L.A.,Petrovick,P.R.(Eds.),Farmacognosia:daplantaaomedicamento. UFRGS,PortoAlegre,pp.519–535.
Castro,M.M.,Leitão-Filho,H.F.,Monteiro,W.R.,1997.Utilizac¸ãodeestruturas
secre-torasnaidentificac¸ãodosgênerosdeAsteraceaedeumavegetac¸ãodecerrado. Rev.Bras.Bot.20,163–174.
Coutinho,I.D.,Kataoka,V.M.F.,Honda,N.K.,Coelho,R.G.,Vieira,M.C.,Cardoso,C.A.L.,
2010.Influênciadavariac¸ãosazonalnosteoresdeflavonoideseatividade
antiox-idantedasfolhasdeCampomanesiaadamantium(Cambess.)O.Berg,Myrtaceae. Rev.Bras.Farmacogn.20,322–327.
Coutinho,I.D.,Poppi,N.R.,Cardoso,C.L.,2008.Identificationofthevolatile
com-poundsofleavesandflowersinGuavira(CampomanesiaadamantiumO.Berg). J.Essen.OilRes.20,405–407.
David,R.,Carde,J.P.,1964.Colorationdifférentielledêsinclusionslipidiqueet
ter-peniquesdêspseudophyllesduPinmaritimeaumoyendureactifNadi.C.R. Acad.Sci.258,1338–1340.
Elias,S.M.R.,Assis,R.M.,Stacciarini-Seraphin,E.,Rezende,M.H.,2003.Anatomia
foliaremplantasjovensdeSolanumlycocarpumA.St.-Hil.(Solanaceae).Rev.Bras. Bot.26,169–174.
Evert,R.F.,2006.Esau’sPlantAnatomy:Meristems,Cells,andTissuesofthePlant
Body:TheirStructure,Function,andDevelopment.JohnWiley&Sons,Hoboken.
Fahn,A.,1979.SecretoryTissuesinPlants.AcademicPress,London.
Feucht,W.,Schmid,P.P.S.,1983.Selektiverhistochemischernachweisvonflavanen
(catechinen)mitp-dimethylamino-zimtaldehydinsprosseneiniger obstge-holzi.Gartenbauwissenschaft48,119–124.
Furr,M.,Mahlberg,P.G.,1981.Histochemicalanalysesoflaticifersandglandular
trichomesinCannabissativa.L.Nat.Prod.44,152–158.
Gallon, M.E., Barros, B.S.P., Silva, M.A., Dias, S.H.M., Alves-da-Silva, G., 2015.
Determinac¸ãodosparâmetrosanatômicos,físico-químicoefitoquímicosdas folhasdeSolanumlycocarpumA.St.-Hill.Rev.Bras.PlantasMed.17,937–944.
Garcia-Barriga,H.,1992.FloraMedicinaldeColombia.TercerMundo,Bogotfi.
Gomes,R.S.D.L.,Oliveira,V.C.,Jácome,R.L.R.P.,Pinto,J.E.B.P.,Lameira,O.A.,Barros,
A.M.D.,2009.Estudomorfoanatômicocomparativoentreapoaia(Psychotria
ipecacuanha(Brot.)Stokes–Rubiaceae)obtidadaregiãoAmazônica(habitat original)eprovenientedeprocessobiotecnológicosubmetidaadiferentes trata-mentosdeinterceptac¸ãodaradiac¸ãosolar.Rev.Bras.Farmacogn.19,276–283.
Hardman, R.,Sofowora,E.A., 1972. Antimonytricholoride astestreagentsfor
steroids,especiallydiosgeninandyamogenin,inplanttissues.StainTechnol. 47,205–208.
Heinrich,M.,Rimpler,H.,AntionioBarrera,N.,1992.Indigenousphytotherapyof
gastrointestinaldisordersinalowlandMixecommunity(Oaxaca,Mexico): ethnopharmacologicevaluation.J.Ethnopharmacol.36,63–80.
Jensen,W.A.,1962.BotanicalHistochemistry:PrinciplesandPractice.W.H.Freeman
andCo,SanFrancisco.
Johansen,D.A.,1940.PlantMicrotechnique.McGraw-Hill,NewYork.
Klink,C.A.,Machado,R.B.,2005.ConservationoftheBrazilianCerrado.Conserv.Biol.
19,707–713.
Kraus,J.,Arduin,M.,1997.ManualBásicodeMétodosemMorfologiaVegetal.EDUR,
Seropédica.
Limberger,R.P.,Apel,M.A.,Sobral,M.,Moreno,P.R.H.,Henriques,A.T.,Menut,C.,
2001.AromaticplantfromBrazil:chemicalcompositionofessentialoilsfrom
someCampomanesiaspecies(Myrtaceae).J.Essent.OilRes.13,113–115.
Mace,M.E.,Bell,A.A.,Stipanovic,R.D.,1974.Histochemistryandisolationof
Mace,M.E.,Howell,C.R.,1974.Histologicalandhistochemicalusesofperiodicacid. StainTechnol.23,99–108.
Mauro,C.,Silva,C.P.,Missima,J.,Ohnuki,T.,Rinaldi,R.B.,Frota,M.,2008.Estudo
anatômicocomparadodeórgãosvegetativosdeboldomiúdo,Plectranthus orna-tusCodd.emalvaric¸o,Plectranthusamboinicus(Lour.)Spreng.–Lamiaceae.Rev. Bras.Farmacogn.18,608–613.
Mazia,D.,Brewer,P.A.,Alfert,M.,1953.Thecytochemistrystainingand
measure-mentofproteinwithmercuricbromophenolblue.Biol.Bull.104,57–67.
Mcmanus,J.F.A.,1948.Histologicalandhistochemicaluseofperiodicacid.Stain
Technol.23,99–108.
Metcalfe,C.R.,Chalk,L.,1950.AnatomyoftheDicotyledons.ClarendonPress,Oxford.
Milliken,W.,1997.Traditionalanti-malarialmedicineinRoraima.Brazil.Econ.Bot.
51,212–237.
Myers,N.,Mittermeier,R.A.,Mittermeier,C.G.,Fonseca,G.A.B.,JenniferKent,J.,2000.
Biodiversityhotspotsforconservationpriorities.Nature403,853–858.
Neves,B.J.,Andrade,C.H.,Cravo,P.V.L.,2015.Naturalproductsasleadsin
schisto-somedrugdiscovery.Molecules20,1872–1903.
O’Brian,T.P.,Feder,N.,Mccully,M.E.,1964.Polychromaticstainingofplantcellwalls
bytoluidineblueO.Protoplasma59,368–373.
Oliveira,P.S.,Marquis,R.J.,2002.TheCerradosofBrazil:EcologyandNaturalHistory
ofaNeotropicalSavanna.ColumbiaUniversityPress,NewYork,pp.398.
Pavan,F.R.,Leite,C.Q.F.,Coelho,R.G.,Coutinho,I.D.,Honda,N.K.,Cardoso,C.A.L.,
Vilegas,W.,Leite,S.R.A.,Sato,D.N.,2009.Evaluationofanti-Mycobacterium
tuberculosisactivityofCampomanesiaadamantium(Myrtaceae).Quim.Nova 32,1222–1226.
Pereira-Silva,E.F.L.,Santos,J.E.,Kageyama,P.Y.,Hardt,E.,2004.Florísticae
fitossoci-ologiadosestratosarbustivoearbóreodeumremanescentedecerradãoemuma UnidadedeConservac¸ãodoEstadodeSãoPaulo.Rev.Bras.Bot.27,533–544.
Piva,M.G.,2002.OCaminhodasPlantasMedicinais:EstudoEtnobotânico.Mondrian,
RiodeJaneiro.
Pizzolato,T.D.,Lillie,R.D.,1973.Mayer’stannicacid-ferricchloridestainformucins.
J.Histochem.Cytochem.21,56–64.
Ribeiro,J.F.,Walter,B.M.T.,1998.FitofisionomiasdobiomaCerrado.In:Sano,S.M.,
Almeida,S.P.(Eds.),Cerrado:AmbienteeFlora.Embrapa,Brasília,pp.87–166.
Rinaldo,D.,Batista,J.,Rodrigues,J.,Benfatti,A.C.,Rodrigues,C.M.,Santos,L.C.,Furlan,
M.,Vilegas,W.,2010.Determinationofcatechindiastereomersfromtheleaves
ofByrsonimaspeciesusingchiralHPLC-PAD-CD.Chirality22,726–733.
Rizzini,C.T.,1971.Aspectosecológicosdaregenerac¸ãoemalgumasplantasdo
cer-rado.In:Ferri,M.G.(Ed.),AnaisdoIIISimpósioSobreCerrado.EditoraEdgard Blücher,SãoPaulo,pp.61–64.
Rodrigues,V.E.G.,Carvalho,D.A.,2001.Levantamento etnobotânicode plantas
medicinaisnodomíniodocerrado,regiãodoaltoRioGrande,MinasGerais. Cienc.Agrotec.Lavras25,102–123.
Rodrigues,T.M.,Teixeira,S.P.,Machado,S.R.,2011.Theoleoresinsecretorysystemin
seedlingsandadultplantsofcopaíba(CopaiferalangsdorffiiDesf.,Leguminosae –Caesalpinioideae).Flora206,585–594.
Rutter,R.A.,1990.CatálogodeplantasútilesdelaAmazoniaPeruana.Instituto
LingüísticodeVerano,Lima.
Silva,N.L.A.,Miranda,F.A.A.,Conceic¸ão,G.M.,2010.Triagemfitoquímicade
plan-tasdeCerrado,daáreadeprotec¸ãoambientalmunicipaldoInhamum,Caxias, Maranhão.ScientiaPlena6,1–17.
Solereder,H.,1908. SystematicAnatomyoftheDicotyledons.ClarendonPress,
Oxford.
Souza,F.C.F.,Melo,C.T.V.,Citó,M.C.O.,Félix,F.H.C.,Vaconcelos,S.M.M.,Fonteles,
M.M.F.,Barbosa-Filho,J.M.,Viana,G.S.B.,2008.Plantasmedicinaiseseus
princí-piosbioativos:Umarevisãodabioatividadeepotenciaisbenéficosnosdistúrbios daansiedadeemmodelosanimais.Rev.Bras.Farmacogn.18,642–654.
Vallilo,M.I.,Lamardo,L.C.A.,Garbelotti,M.L.,Oliveira,E.,Moreno,P.R.H.,2006.
