RevistaBrasileiradeFarmacognosia26(2016)147–160
w ww . e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Structure
and
histochemistry
of
medicinal
species
of
Solanum
Laudineia
J.
Matias
a,
Maria
O.
Mercadante-Simões
a,∗,
Vanessa
A.
Royo
b,
Leonardo
M.
Ribeiro
c,
Ariadna
C.
Santos
a,
Jaciara
M.S.
Fonseca
baLaboratóriodeAnatomiaVegetal,DepartamentodeBiologiaGeral,UniversidadeEstadualdeMontesClaros,CampusPr.DarcyRibeiro,MontesClaros,MG,Brazil bLaboratóriodeProdutosNaturais,DepartamentodeBiologiaGeral,UniversidadeEstadualdeMontesClaros,CampusPr.DarcyRibeiro,MontesClaros,MG,Brazil cLaboratóriodeMicropropagac¸ão,DepartamentodeBiologiaGeral,UniversidadeEstadualdeMontesClaros,CampusPr.DarcyRibeiro,MontesClaros,MG,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received12June2015 Accepted4November2015 Availableonline11December2015
Keywords:
Environmentalconservation Herbalmedicine
Plantdrug Pharmacognosy Phytochemical Traditionalmedicine
a
b
s
t
r
a
c
t
Studiesonnativemedicinalplantsstrengtheninitiativestopreservetheenvironmentswherethose speciesnaturallyoccur,manyofthemalreadystronglymenacedevenbeforetheirpotentialtohumankind isknown.Rootandstembarks,leaves,andpericarpssamplesofSolanumagrariumSendtn.,S.lycocarpum
A.St.-Hil.,S.palinacanthumDunal,S.paniculatumL.,andS.stipulaceumRoem.&Schult.,speciesthatoccur intheCerrado(Brazililansavanna)wereprocessedaccordingtocommonlightmicroscopytechniques forstructuralanalysis,andhistochemicaltestswereperformedtolocateandidentifyclassesofchemical compounds.Thedistinctivefeaturesidentifiedwerelowconcentrationofcrystalsandintherootand stem,presenceofterpeneresinintheroot,andabsenceofhypodermisintheleaf,inS.agrarium;bright spots(groupofsclereids)intheroot,isobilateralmesophyll,thickenedcellwallswithhemicellulosesand strongaromainthefruit,inS.lycocarpum;highconcentrationofcrystalsandintherootandstem, oval-shapedlimb,presenceofisolatedcrystalsintheexocarp,inS.palinacanthum;strongsclerificationand rayswithgreatheightintherootandstem,inS.paniculatum;andaccumulationofsolubleproteininthe rootandstem,presenceofconspicuousmembranaceousstipules,absenceofspiniformtrichomes,inS. stipulaceum.Thisworkidentifiesdistinctivestructuralfeatures,itsecologicalimportance,anddetermines thedistributionofsecondarycompoundsassociatedwiththemedicinalpropertiesreportedforthese speciesandcontributestotheconservationofthenaturalenvironmentswheretheyoccur.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
The botanical and chemical characterization of medicinal
speciesisimportantfor thevalidation ofitstraditionaluseand forstudiestoobtainingnewproductsandinnovation(Araújoetal., 2010;Nurit-Silvaetal.,2011;WHO,2013).Thestructuralanalysis ofplantsidentifiesdistinctivefeaturesusefulforthedetermination oftheauthenticityofmedicinalplantspeciesandtheidentification oftheplantorganswherethehighestconcentrationsofactive sub-stancesarepresent,especiallywhentheplantsarefragmentedfor useinherbaldrugs(Argyropoulouetal.,2010;Ferreiraetal.,2011; Coelhoet al.,2012).Thesefeatures haveanecologicalfunction,
relatedtotheenvironment where theplantoccurs.The
under-standingofthisfunctionmayhelpoptimizetheircultivationand collection(Adamsetal.,2013;Moreiraetal.,2013;Sampaioetal., 2014).
∗ Correspondingauthor.
E-mail:maria.simoes@unimontes.br(M.O.Mercadante-Simões).
Chemical studies performed using histochemical techniques
allowaquickandinexpensivepreliminaryevaluationofthe medic-inalpotentialoftaxonomicallyclosespeciesinthesearchfornew pharmaceuticals(Coelhoetal.,2012;Demarcoetal.,2013;Araújo etal.,2014;Mercadante-Simõesetal.,2014).Thistechniquecan minimizecostsinthesearchfornewpharmaceuticalsandincrease thesafetyoftraditionalmedicines(Adamsetal.,2013;Santosetal., 2013),howeverhistochemicalstudiesarerareinSolanum(Araújo etal.,2010;Picolietal.,2013).
Solanaceaeiswidelydistributedwith269speciesofthegenus SolanumL.describedinBrazil(Stehmannetal.,2014).Someofthe speciesoccurringintheCerradobiomearetraditionallyusedfor thecontrolofdiabetes,andhavebeenrecognizedfortheir
medic-inalproperties:SolanumagrariumSendtn.hasanti-inflammatory
effects(Agraetal.,2007;Castroetal.,2011),S.lycocarpumA. St.-Hilishypoglycemic(Farinaetal.,2010),S.palinacanthumDunal isusedasantimicrobialandhypoglycemic(Pereiraetal.,2008),S. paniculatumL.hasantioxidantandanticarcinogenicaction(Ribeiro et al., 2007; Endringer et al., 2010), and S. stipulaceum Roem. & Schult. is used as a blood pressure regulator (Ribeiro et al., 2002).
http://dx.doi.org/10.1016/j.bjp.2015.11.002
MostpharmacognosticanalysesinSolanumspecieswere per-formedonleavesofonlyoneortwospecies(Araújoetal.,2010; Nurit-Silvaetal.,2011),andanatomicalstudiesonalarger num-berofspeciesareontaxonomy(Sampaioetal.,2014;Nurit-Silva etal.,2012).Thereforethegoalofthepresentstudywasto iden-tify distinctive structural features onthe root and stem barks, leavesand pericarpsof S.agrarium, S.lycocarpum, S. palinacan-thum,S.paniculatum,andS.stipulaceum,itsecologicalimportance,
determinethe distributionof secondary compounds associated
with the medicinal properties reported for these species and
contributetotheconservationofthenaturalenvironmentswhere theyoccur.
Materialsandmethods
Plantmaterial
Fragmentsofrootandstem(apicesandbark,consideringbark thetissuesexternaltothevascularcambium,sensuFahn,1990), leaf,pericarp,androotandstemapexeswerecollectedfromthree
L.J.Matiasetal./RevistaBrasileiradeFarmacognosia26(2016)147–160 149
Fig.2. StructuralandhistochemicalfeaturesofSolanumlycocarpumA.St.-Hil.(A–G,I–L,N,O)Crosssections.(H,M)Radiallongitudinalsection.(A)Rootprimarystructure showingsettlementofphellogenatthecortex(arrow).(B–I)Stem.(B)Primarystructurewithhighdensityoftectortrichomes.(C)Bicollateralbundle.(D)Settlementof phellogenatthecortex(arrow).(EandF)Corticalcellswithplastidspresentingterpenoids.(E)PinkwithNileblue.(F)BluewithNADI.(G)Sievetubeelementsoflarge diameter.(H)Obliquecompoundsieveplates.(I)Accumulationofcrystalsandintheraysandaxialparenchyma.(J–L)leaf.(J)Isobilateralmesophyllpresentingcrystalsand.(K andL)Palisadeparenchyma.(K)LipidsstainedpinkwithNileblue.(L)AlkaloidsstainedyellowwithEllramreagent.(M–O)Pericarp.(M)Mesocarpcellspresentingcellulose wallswithpronouncedthickness.(N)Lowpectinconcentrationinmesocarpcellwalls.(O)ExocarpandmesocarpwithalkaloidsstainedbrownwithDragendorff.(ap)axial parenchyma,(ca)cambium,(cc)companioncell,(cl)collenchyma,(co)cortex,(cs)crystalsand,(ec)epidermiscell,(ex)exocarp,(me)mesocarp,(oc)obliquecompound plate,(pc)pectin,(ph)phloem,(pi)pith,(pl)plastids,(pp)palisadeparenchyma,(pr)protoplast,(ra)ray,(si)sieveplate,(sp)spongyparenchyma,s(t)sievetubeelement, (su)suber,(tr)tectortrichome,(va)vascularbundle,(xy)xylem,(wa)wall.
individualsofSolanumagrariumSendtn.,S.lycocarpumA.St.-Hil.,S. palinacanthumDunal,S.paniculatumL.,andS.stipulaceumRoem. &Schult., occurringin theBrazilian Cerrado, in the municipal-ityofMontesClaros(16◦50′06.1′′S,43◦55′24.8′′W;16◦30′53.2′′S,
44◦04′28.0′′W).Voucherswereobtainedfromfertilematerialand
depositedattheBHCBHerbariumoftheDepartamentodeBotânica,
do Instituto de Ciências Biológicas, da UniversidadeFederal de
MinasGerais,Brazil(116081,168587-168590;LLGiacomin).
Structuralcharacterization
Morphologicalanalysiswasperformedonallfreshplant
mate-rialusing a binocular stereo-microscope,model 0766ZL (Motic,
Richmond, Canada) according to nomenclature proposed by
Junikka(1994).Forstructuralanalysis,theplantmaterialwas eval-uatedfreshorfixedinKarnovskysolution(Karnovsky,1965)for 12h, dehydratedin a gradedethanol series(Jensen,1962), and cold-embedded(Paivaetal.,2011)inhydroxyethyl-methacrylate
resin (Leica Microsystem Inc., Heidenbeg, Germany). Cross
sec-tionsand paradermicsectionswereobtainedusing a LPCtable
microtome(RolembergandBhering,BeloHorizonte,Brazil).Cross
sections and longitudinal sections, 5m thick, were obtained
using a rotary microtome (Atago, Tokyo, Japan). The sections
Fig.3.StructuralandhistochemicalfeaturesofSolanumpalinacanthumDunal.(A,D–I,K–S)Crosssections.(B–C,J)(tangentiallongitudinalsections.(A–E)Rootbark.(A) CortexwithalkaloidsstainedbrownwithDragendorff.(B)Axialparenchymawithcrystalidioblast.(C)Widerays.(D)Starchreserveinradialcell.(E)Formationofconducing elementsatthecambium.(F–J)Stembark.(F)Establishmentofphellogenattheepidermis.(G)PhellodermpresentingflavonoidsstainedredwithDMACA.(H)Crystalsand atthephloem.(IandJ)Formationoflysigenousductcontainingcrystalsand.(K–R)Leaf.(K–M)Shortglandulartrichome.(K)MucilagestainedredwithXP.(L)Lipidsstained bluewithNileblue.(M)Phenolstainedbrownwithironchloride.(N–P)Medianvein.(N)Mucilagestainedblackwithtannicacid.(O)Phenolsstainedbrownwithiron chloride.(P)AlkaloidsstainedyellowwithEllram.(Q-R)Palisadeparenchyma.(Q)FlavonoidsstainedredwithDMACA.(R)Alkaloidsstainedyellow-brownwithDittmar.(S) Exocarpwithundulatedcuticleandsinglecrystals.(ap)axialparenchyma,(ca)cambium,(cc)companioncell,(co)cortex,(cr)crystals,(cs)crystalsand,(ec)epidermiscell, (he)head,l(y)lysigenousduct,(ms)mesophyll,(nu)nucleus,p(a)parenchyma,(pe)periderm,(ph)phloem,(pl)phelloderm,(pp)palisadeparenchyma,(ra)ray,(si)sieve plate,(sg)starchgrains,(st)sievetubeelement,(su)suber,(pd)peduncle,(uc)undulatedcuticle,(va)vascularbundle,(wi)width,(wl)cellwalllysis,(xy)xylem.
Histochemicaltests
Histochemical tests were performed on sections obtained
from fresh material as described above using the following
reagents: Lugol(Johansen,1940)for detection ofstarch, tannic acid(Pizzolato and Lillie, 1973) and ruthenium red (Johansen,
1940) for acid polysaccharides, xylidine Ponceau (XP) (Vidal,
1970) and Coomassie blue (Fisher, 1968) for protein, Sudan
red IV and Sudan black B (Pearse, 1980) for total lipophilic
compounds, Nile blue (Cain, 1947) for acid and neutral lipids,
␣-naphthol and dimethylparaphenylenediamine hydrochloride
(NADI)(DavidandCarde,1964)foressentialoilsandoleoresins, Ell-ram,Dittmar(FurrandMalhberg,1981),andDragendorffreagents (SvendsenandVerpoorte,1983)foralkaloids,potassium dichro-mate(Gabe,1968)andironchloride(Johansen,1940)forphenols; vanillin chloride (Mace and Howell, 1974) for tannins; and p
-dimethylaminocinnamaldehyde(DMACA)(Feuchtetal.,1986)for
flavonoids.Controltestswereperformedsimultaneouslyaccording totherecommendationsoftherespectiveauthors.
PhotographicdocumentationwasperformedusinganAxioCam
MRccameracoupledtoanAxioVisionLEphotomicroscope(Zeiss,
Oberkochen,Germany)andanA620digitalcamera(Canon,Tokyo,
Japan)coupledtoaEclipseE-200lightmicroscope(Nikon,Tokyo, Japan).
Results
Structuralandhistochemicalcharactersreportedforthefour organsofthefivespeciesofSolanumarepresentedbyspecies,in Figs.1–5,andbyorgansinTables1–8.Thedatapresentedbyspecies
cancontributetotheelaborationofmonographs,andpresented
bytheorgansaretocontributetothequalitycontrolofthedrug obtainedfromthespecies.
S. agrarium presented root bark with a corrugated internal texture,resinaccumulationatthesuber,voluminousintercellular
spaces, low lignification, and considerable starch and protein
L.J.Matiasetal./RevistaBrasileiradeFarmacognosia26(2016)147–160 151
Fig.4. StructuralandhistochemicalfeaturesofSolanumpaniculatumL.(A,G–I)Longitudinalradialsections.(B,C,E,F,L–R)Crosssections.(D,J–K)Tangentiallongitudinal sections.(A–D)Rootbark.(A)Sclerifiedcortexwithvoluminousintercellularspaces(asterisk).(B)Cortexwithphenolsstainedbrownwithdichromate.(C)Phloemwith higherproportionofconducingelements(rectangle)thantheaxialparenchyma(circle)andcloserays(dottedline).(D)Highradialheight.(E–H)stembark.(E)Sievetube elementsatthepith.(F)CortexwithflavonoidsstainedredwithDMACA.(G)Pectinprojectionsincorticalcells.(H)Sclereidswithobservablepit.(I–K)sievetubeelements. (I)Obliqueplates.(J)Compoundplates.(K)Small-diametersieveareas.(L–R)leaf.(L–M)Glandulartrichome.(L)AlkaloidsstainedbrownwithDittmar.(M)Mucilagestained redwithXP.(N)Dorsiventralmesophyll.(O–Q)palisadeparenchyma,omucilagestainedblackwithtannicacid.(P)Phenolsstainedbrownwithdichromate.(Q)Flavonoids stainedredwithDMACA.(R)Lignifiedhypodermisattheabaxialsurfaceofthemedianvein.(cl)collenchyma,(co)cortex,(cs)crystalsand,(ec)epidermiscell,(ep)external phloem,(ip)internalphloem,(he)head,(hi)height,(hy)hypodermis,(oc)obliquecompoundplate,(op)obliqueplate,(pc)pectin,(pe)periderm,(ph)phloem,(pi)medulla, (pp)palisadeparenchyma,(pt)pit,(ra)ray,(sa)sieveareas,(sc)sclerification,(sp)spongyparenchyma,(st)sievetubeelement,(xy)xylem.
bitter taste and low sclerification; the cortex had voluminous
intercellularspaces,highstarchaccumulation,andasmall quan-tityofcrystalsand;thevolumeoccupiedbytheaxialparenchyma wassignificantlylargerthanthatofthesievetubeelementsatthe phloem(Tables3and4).Leavespresentedamedianveincomposed ofpolyhedricepidermiscells,nohypodermis,andaccumulationof terpenoidsinthesecretorytrichomes;thelower-calibervascular bundlesatthepetiolewerecollateralandnotsurroundedby per-icyclicfibers(Fig.1E–HandTable5).Theexocarpwaspurple,and alkaloidsandterpenoidsaccumulatedatthemesocarp(Fig.1I–L andTables7and8).
InS.lycocarpum,theestablishmentofphellogenintherootat theperipheralcorticalcells,andthesecondaryphloempresented
acompact arrangementofaxial parenchyma,homogeneousray
height,andsievetubeelementswithdiminutecaliberandpores
(Fig.2AandTables1and2).Thesteminprimarystructurewas
pilous;thestembarkpresentedlenticelsandcuboid-shapedsuber cells;thecortexaccumulatedterpenoids;andthephloemhad thin-walled sievetube elements(Fig.2B–Iand Tables 3 and4).The mesophyllwasisobilateral.Themedianveinregionpresented epi-dermalcellswithstraightanticlinalwalls,accumulationofcrystal sand,andhighsclerificationindex(Fig.2J–LandTable5).Thefruit hadastrongaroma,theexocarpexhibitedthickcollenchyma,and thecellsofthemesocarphadthickwallswithlowconcentrationof pectinbuthighconcentrationofmucilage,protein,andstarch,and analkaloid-richprotoplast(Fig.1M–OandTables7and8).
Table1
StructuralfeaturesofrootofSolanumagrariumSendtn.,SolanumlycocarpumA.St.-Hil.,SolanumpalinacanthumDunal,SolanumpaniculatumL.,andSolanumstipulaceum Roem.&Schult.
Features Solanumspecies
ag ly pl pn st
Morphology
Bittertaste 1 1 2 1 2
Externalsurface
Texture wr ro wr ro ro
Color br br br br br
Internalsurface
Corrugatedtexture 2 1 1 1 1
Peridermcrosssection
Color br br br br br
Thickness 1 2 1 2 2
Cortex/phloem
Color ye ye ye ye ye
Brightspots 1 2 1 1 1
Anatomy
Primarystructure Epidermis
Cellshape tb tb tb tb tb
Phellogenestablishment el el el el el
Cortex
Intercellularspace 2 1 1 1 1
Cellshape tb tb tb tb tb
Crystalsand 0 1 2 0 0
Endophyticfungi 2 1 2 1 2
Vascularsystem di di di di di
Secondarystructure Periderm
Suber
Cellshape tb tb tb cu tb
Cellwallthickening 1 1 1 1 1
Sclerification 0 0 0 2 0
Resin 2 1 1 0 0
Phelloderm
Thickness 1 1 1 1 1
Crystalsand 0 0 1 1 1
Sclereids 0 0 0 1 1
Cortex
Thickness 2 2 2 2 1
Intercellularspaces 2 1 1 1 1
Crystalsand 1 1 2 1 1
Sclereids 0 1 1 2 1
Phloem
Primaryphloemfibers 1 0 1 0 1
Pseudocortex 1 0 1 1 2
Sclerification 0 2 1 1 1
Sievetubeelements
Quantity 1 1 1 2 1
Wallthickening 2 1 2 2 2
Caliber 2 1 2 2 2
Length 1 1 1 1 1
Distribution rb rb rb rb rb
Plates oc oc oc oc oc
Sievediameter 2 1 2 2 2
Sieveareas
Quantity 1 1 1 1 2
Diameter 2 1 2 2 2
Axialparenchyma
Volume 2 2 2 1 2
Caliber 2 2 2 1 2
Crystalsand 1 1 2 2 2
Ray
Width 1–2 1–2 1–8 1–3 1–3
Height 6–20 12–14 1–11 10–25 1–12
Crystalsand 1 1 1 2 1
(ag)S.agrarium,(br)brown,(cu)cuboids,(di)diarch,(el)externallayers(epidermis/cortex),(ly)S.lycocarpum,(oc)obliquecompound,(pl)S.palinacanthum,(rb)radial bands,(ro)rough,(pn)S.paniculatum,(st)S.stipulaceum,(tb)tabular,(wr)wrinkled,(ye)yellow,(0)absent,(1)moderate,(2)pronounced.
Themesophyllaccumulatedtannins,hadacidpolysaccharide-rich cellwalls,and showedannularcollenchymaatthemedianvein cortex (Fig. 3K–R and Tables 5 and 6). Its exocarp wasyellow andhadsinglecrystalsattheepidermis;itsmesocarphadalight
white color, voluminous intercellular spaces, and alkaloid-rich
cells(Fig.1SandTables7and8).
L.J.Matiasetal./RevistaBrasileiradeFarmacognosia26(2016)147–160 153
Fig.5.StructuralandhistochemicalfeaturesofSolanumstipulaceumRoem.&Schult.(A–G,J–M)Crosssections.(HandI)Tangentiallongitudinalsections.(A–E)rootbark.(A) Tangentialexpansion(dottedline)ofrayformingthepseudocortex.(B–E)Proteinaccumulationatthephloemparenchyma.(B,D)StainedredwithXP.(C,E)Stainedbluewith Coomassieblue.(C)Controltest.(F–J)Stembark.(F)LipidsattheexternalcortexstainedblackwithSudanblackB.(G)Evidentannualrings.(H)Obliquecompoundplates withseveralsieveareas.(I)Lateralsieveareaswithlargediameter.(J)Axialparenchymapresentingcellswithlargecaliber.(K–M)Leaf.(K)Petiolewithtectortrichomes. (L–M)terpenoidsatthevein.(L)PinkandbluewithNileblue.(M)BluewithNADI.(ap)axialparenchyma,(cc)companioncell,(co)cortex,(cs)crystalsand,(fp)functional phloem,(np)non-conductingphloem,(oc)obliquecompoundplates,(pa)parenchyma,(pe)periderm,(ra)ray,(sa)sieveareas,(sg)starchgrains,(st)sievetubeelement, (tr)tectortrichome,(va)vascularbundle.
Table2
HistochemicalfeaturesofrootofSolanumagrariumSendtn.,SolanumlycocarpumA.St.-Hil.,SolanumpalinacanthumDunal,SolanumpaniculatumL.,andSolanumstipulaceum Roem.&Schult.
Test Suber Feloderm/cortex Phloem ag ly pl pn st ag ly pl pn st ag ly pl pn st
Primarymetabolites
Starch Lugol 0 0 0 0 0 2 1 1 1 1 2 1 1 1 1 Acidicpolysaccharides Tannicacid 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 Rhuteniumred 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Proteins XP 0 0 0 0 0 1 1 1 1 2 1 1 1 1 2
Coomassieblue 0 0 0 0 0 1 1 1 1 2 1 1 1 1 2 Generallipid SudanVI 1 1 1 1 1 1 1 0 1 0 1 1 1 1 0 Sudanblack 1 1 1 1 1 1 1 0 1 0 1 1 1 1 0 Neutrallipids Nileblue 1 1 1 1 1 1 1 0 0 0 1 0 1 0 0 Acidslipids 1 1 1 1 1 1 1 0 1 0 1 1 1 1 0
Secondarymetabolites
Table3
StructuralfeaturesofstemofSolanumagrariumSendtn.,SolanumlycocarpumA.St.-Hil.,SolanumpalinacanthumDunal,SolanumpaniculatumL.,andSolanumstipulaceum
Roem.&Schult.
Features Solanumspecies
ag ly pl pn st
Morphology
Bittertaste 2 1 1 1 1
Externalsurface
Color db br gr gr gr
Trichomes 2 0 2 2 2
Textura sm ro wr wr sm
Lenticels 1 0 2 2 2
Thorns 1 1 1 1 0
Internalsurface
Corrugatedtexture 1 1 1 1 2
Peridermcrosssection
Color br br br br br
Thickness 1 1 1 1 1
Anatomy
Primarystructure Epidermis
Cellshape cu cu cu cu cu
Tectortrichomes
Quantity 1 2 0 0 1
Ramification 0 1 0 0 1
Glandulartrichomes
Quantity 0 0 1 0 0
Head 0 0 mc 0 0
Peduncle 0 0 sh 0 0
Cortex/phloem
Anelarcollenchyma 1 1 1 1 1
Corticalparenchyma
Thickness 2 2 2 1 2
Intercellularspaces 0 0 0 2 0
Cellshape ov gl gl ov gl
Crystalsand 0 1 1 2 2
Vascularsystem bi bi bi bi bi
Pericyclicfibers 0 0 0 0 2
Crystalsand 0 0 0 2 2
Vascularbundle bi bi bi bi bi
Medullaryparenchyma
Sclereids 0 0 0 0 2
Crystalsand 0 1 1 0 2
Secondarystructure Periderm
Suber
Cellshape ta cu ta ta ta
Cellwallthickening 1 1 1 1 1
Resin 1 1 0 0 0
Phellogeninstallation ep co ep ep co
Phelloderm
Thickness 1 1 1 1 1
Crystalsand 0 0 0 0 1
Sclereids 0 0 0 0 1
Cortex
Thickness 1 2 1 1 1
Colenchyma 1 1 1 0 1
Parenchyma
Intercellularspaces 2 1 1 1 1
Crystalsand 1 1 2 1 1
Sclereids 0 1 1 2 1
Phloem
Sievetubeelements
Quantity 1 2 2 2 2
Wallthickening 2 1 2 2 2
Caliber 1 2 1 2 1
Length 1 1 1 1 1
Distribution rb rb rb rb rb
Sieveplate oc oc oc oc oc
Sieveareas
Quantity 1 1 1 1 2
Diameter 1 1 1 1 1
L.J.Matiasetal./RevistaBrasileiradeFarmacognosia26(2016)147–160 155
Table3(Continued)
Features Solanumspecies
ag ly pl pn st
Axialparenchyma
Volume 2 1 1 1 1
Caliber 2 2 2 2 2
Crystalsand 1 1 2 1 2
Ray
Width 1 1–2 1–2 1–2 1–2
Height 10–15 14–20 3–7 5–30 6–16
Crystalsand 1 1 0 1 0
Sclerenchyma 0 2 1 2 2
(ag)S.agrarium,(bi)bicolateral,(br)brown,(bs)blackspots,(co)cortex,(cu)cuboid,(db)darkbrown,(ep)epidermis,(gl)globoid,(gr)green,(ly)S.lycocarpum,(mc) multicellular,(oc)obliquecompound,(ov)ovoid,(pl)S.palinacanthum,(pn)S.paniculatum,(rb)radialbands,(ro)rough(sh)short,(sm)smooth,(ta)tabular,(st)S.stipulaceum, (wr)wrinkled,(0)absent,(1)moderate,(2)pronounced.
cortexoftherootbark;thephloempresentednumeroussievetube elementsandrayswithlargeheight(Fig.4A–DandTables1and2).
Thestembarkpresentedalkaloidaccumulationatthecortexand
voluminoussclereids;thebicollateralvascularbundlespresented
internal phloempenetrating deeply into the medulla,and rays
of large heightat thephloem (Fig.4E–K and Tables 3 and 4).
Heterophylly was pronounced; the mesophyll had voluminous
intercellularspaces,andtemporarycellularaccumulationofstarch andflavonoids(Fig.4L–RandTables5and6).Accumulationof sol-ublesugarswasobservedinthefruit,whichconferredasweettaste tothepericarp(Tables7and8).
S.stipulaceumisthemostdistantlyspeciestotheother
stud-iedspecies.Therootbarkdevelopedavoluminouspseudocortex
withevenlyspacedrays,itwasrich insolubleproteinreserves, andwasorientedadjacenttothephloemaxialelements.Theaxial parenchymawasvoluminous(Fig.5A–EandTables1and2).The
stembarkhadnospiniformtrichomes,andtheinternalsurface
wascorrugated.Thephloempresentedaccumulationofterpenoids, well-definedannualrings,andsievetubeelementswithplates pre-sentingnumeroussieveareas(Fig.5F–JandTables3and4).The leavespresentedlargemembranaceousstipulesthatwere distinc-tivemorphologicalfeatureseasilyobservable,andaccumulationof terpenoids(Fig.5K–MandTables5and6).Thefruitwasnotable fortheaccumulationoftannins(Tables7and8).
ThedatapresentedinFigs.1–5andTables1–8showstructural charactersusefulforidentificationandadaptationofthestudied
speciestotheenvironmentwheretheyoccur,andchemical
com-pounds relatedto theirtherapeuticuse:(a)in S.agrarium, low
concentrationofcrystalsandintherootandstem,thepresence ofterpeneresinintheroot,andabsenceofhypodermisintheleaf; (b)inS.lycocarpum,brightspots(groupofsclereids)intheroot, iso-bilateralmesophyll,thickenedcellwallswithhemicellulosesand strongaromainthefruit;(c)inS.palinacanthum,highconcentration ofcrystalsandintherootandstem,oval-shapedlimb,and pres-enceofisolatedcrystalsintheexocarp;(d)inS.paniculatum,strong sclerificationintherootandstem,andrayswithgreatheight;and (e)inS.stipulaceum,accumulationofsolubleproteinintheroot
andstem,presenceofconspicuousmembranaceousstipules,and
absenceofspiniformtrichomes.
Discussion
Structural dataon medicinalplant aresecure characters for
speciesidentificationandqualitycontrolofplantdrugandreveal theirrelationshipwiththeenvironment.Roots,stems,leavesand fruits,dehydratedandfragmentedarewidelyusedasherbaldrugs. Theymaybesubstitutedforeachotherresultinginfraudinthe preparationofherbalmedicines(Adamsetal.,2013).
Thephellogenestablishmentattherootandstem constitute
animportantdistinctivefeaturefortheseorgans.Attheroots,the formationofperidermalwaysstartsatthemoreperipheral corti-callayers(MetcalfeandChalk,1957).Thevascularbundlesinall studiedspeciesinthisworkwerebicollateral,thatisa
character-isticofSolanaceaeandtheoccurrenceofintramedullaryphloem
bundles,situated deeply inthepith,inS.paniculatum hasbeen reportedforspeciesofothergeneraofSolanaceae(Metcalfeand
Table4
HistochemicalfeaturesofstemSolanumagrariumSendtn.,SolanumlycocarpumA.St.-Hil.,SolanumpalinacanthumDunal,SolanumpaniculatumL.,andSolanumstipulaceum Roem.&Schult.
Test Suber Feloderm/cortex Phloem ag ly pl pn st ag ly pl pn st ag ly pl pn st
Primarymetabolites
Starch Lugol 0 0 0 0 0 2 1 1 1 1 2 1 1 1 1 Acidicpolysaccharides Tannicacid 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 Rutheniumred 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Proteins XP 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1
Coomassieblue 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 Generallipid SudanVI 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sudanblack 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Neutrallipids Nileblue 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Acidslipids 1 1 1 1 0 0 0 0 1 0 0 0 0 1 0
Secondarymetabolites
Table5
LeafstructuralfeaturesofSolanumagrariumSendtn.,SolanumlycocarpumA.St.-Hil.,SolanumpalinacanthumDunal,SolanumpaniculatumL.,andSolanumstipulaceumRoem. &Schult.
Features Solanumspecies
ag ly pl pn st
Morphology
Phyllotaxis at at at at at
Stipule 0 0 0 0 1
Heterophylly 1 1 1 2 1
Petiole 1 1 1 1 1
Limb si si si si si
Shape el el ov el el
Margin pi pi pi wh wh
Apex ac ac ac am am
Base ol ol cd ol cn
Consistency hb hb hb hb hb
Surface sm sm sm sm sm
Pilosity 1 2 1 2 2
Nervation pn pn pn pn pn
Anatomy
Interveinregion Trichomes
Spiniform 2 1 2 1 0
Tectors
Quantity 1 2 1 2 2
Ramification 0 1 0 1 1
Glandular Shorterect
Quantity 0 0 2 2 2
Head 0 0 mu mu mu
Longerect
Quantity 1 0 1 0 0
Head un 0 un 0 0
Bent
Quantity 2 0 2 1 2
Head mu 0 un mu mu
Epidermis
Cellshape t t t t t
Undulationofanticlinalwalls 1 1 0 2 0
Mesophyll dv is dv dv dv
Crystalsand 0 1 1 1 1
Palisadeparenchyma
Numberoflayers 1 1 1 1 1
Celllength he he ho ho ho
Spongyparenchyma
Intercellularspaces 1 1 1 2 1
Vascularbundle bi bi bi bi bi
Medianvein Epidermis
Cellshape po cb cb cb cb
Undulationofanticlinalwalls 2 0 1 2 1
Hypodermis 0 1 1 1 1
Collenchyma 1 2 2 2 2
Type an an al an an
Crystalsand 0 1 1 1 0
Sclereids 0 1 0 0 0
Parenchyma
Crystalsand 0 2 1 1 1
Sclereids 0 1 0 0 0
Vascularbundle bi bi bi bi bi
Pericyclicfibers 2 2 1 0 0
Crystalsand 0 1 1 1 1
Petiole Epidermis
Cellshape cb cb cb cb cb
Undulationofanticlinalwalls 1 0 1 0 0
Collenchyma 1 2 2 2 2
Wallthickening an an al an an
Parenchyma
Crystalsand 1 2 2 2 1
Intercellularspaces 1 2 2 1 1
Sclereids 0 1 0 0 0
Vascularsystemshape hm hm hm hm hm
Pericyclicfibers 0 2 0 0 0
Crystalsand 0 1 1 1 1
Lowercaliberbundles co bi bi bi bi
L.J.Matiasetal./RevistaBrasileiradeFarmacognosia26(2016)147–160 157
Table6
HistochemicalleaffeaturesofSolanumagrariumSendtn.,SolanumlycocarpumA.St.-Hil.,SolanumpalinacanthumDunal,SolanumpaniculatumL.,andSolanumstipulaceum
Roem.&Schult.
Test Interveinregion Medianvein
ag ly pl pn st ag ly pl pn st
Primarymetabolites
Starch Lugol 0 0 1 1 0 0 0 1 1 0
Acidicpolysaccharides Tannicacid 1 1 1 1 1 1 1 1 1 1
Rutheniumred 1 1 1 1 1 1 1 1 1 1
Proteins XP 0 1 0 0 1 0 0 1 1 1
Coomassieblue 0 1 0 0 1 0 1 1 1 1 Generallipid SudanVI 1 1 1 1 1 1 1 1 1 1
Sudanblack 1 1 1 1 1 1 1 1 1 1
Neutrallipids Nileblue 1 1 1 1 1 1 1 1 1 1
Acidslipids 1 1 1 1 1 1 1 1 1 1
Secondarymetabolites
Terpenoids NADI 1 1 1 1 1 1 1 1 1 1
Alcaloids Ellram 2 2 2 2 2 1 1 1 1 1
Dittmar 2 2 2 2 2 1 1 1 1 1
Phenoliccompounds Dichromate 2 2 2 2 2 1 1 1 1 1
Tannins Vanillin 2 2 2 2 2 1 1 1 1 1
Flavonoids DMACA 2 2 2 2 2 1 1 1 1 1
(ag)S.agrarium,(ly)S.lycocarpum,(pl)S.palinacanthum,(pn)S.paniculatum,(st)S.stipulaceum,(0)absent,(1)moderate,(2)pronounced.
Chalk,1957).Awidevariationofsievetubeelementsandpores diameterswasobserved,andindicatesthatthesefeaturescanbe reliably used todistinguishspecies of this genus.A wide vari-ation of the characteristicsof theseconducting cells, including theinclinationandnumberofsieveareas,hasbeenreportedfor thegenusSolanum(Chavanetal.,2000).Speciespresentingsieve tubesoflargediameterandespeciallywideporescanbenefitfrom
higher nutrient flow rates into developing organs (Mullendore
etal.,2010).
Thefoliarfeaturespresentedinthisstudyareusefulfor diag-nosticpurposes,especiallytheepidermalfeatureswhicharewell preservedintherawmaterial.Thetypesoftrichomesandstomata, andoutlineofcellwallsarewelldescribedonSolanum(Araújoetal., 2010;Nurit-Silvaetal.,2012;Sampaioetal.,2014).Theleavesare widelyusedasdrugs,andaredesirabletoreplacestemandroot
barksperleaves,becausetheharvestofbarksisdamagingforthe individual.
Thefruitsofallthestudiedspecies wereberrieswhich isin accordancewithpreviousreports forSolanumspecies(Feliciano andSalimena,2011),andthepresenceofcalciumoxalatecrystals, andvoluminousintercellularspaceswererelatedbyChiariniand Barboza(2009).Verythickwalls,richinacidsandbasic polysac-charides,asobservedforS.lycocarpummesocarp,constitutewater andnutrientreservemechanisms(Nevesetal.,2013).
AnalyzingthedataobtainedforS.stipulaceum,somefeatures inparticular,suchastheabsenceofspiniformtrichomes,presence of stipules,sclerificationof thevascular systemand medullaof
thestemprimarystructure,andpronouncedaccumulationof
ter-penoidsinalmostallorgans,indicateitslargerstructuraldifference fromtheremainingstudiedspecies. Infact,accordingtorecent
Table7
StructuralfeaturesofpericarpsofSolanumagrariumSendtn.,SolanumlycocarpumA.St.-Hil.,SolanumpalinacanthumDunal,SolanumpaniculatumL.,andSolanumstipulaceum Roem.&Schult.
Features Solanumspecies
ag ly pl pn st
Morphology
Typeoffruit be be be be be
Aroma 0 2 0 0 0
Exocarp
Color pu gr yl gr gr
Consistency 1 2 2 1 2
Mesocarp
Color yl yl wh gr gr
Thickness 1 2 1 1 1
Anatomy
Exocarp Cuticle
Undulation 2 1 2 1 1
Thickness 1 2 1 1 2
Epidermis
Cellshape po ab ab ab po
Singlecrystals 0 0 1 0 0
Collenchyma 1 2 1 0 1
Mesocarp
Cellshape gl po wh wh t
Wallthickening 1 2 1 1 2
Intercellularspaces 1 1 2 1 1
Table8
HistochemicalfeaturesofpericarpsofSolanumagrariumSendtn.,SolanumlycocarpumA.St.-Hil.,SolanumpalinacanthumDunal,SolanumpaniculatumL.,andSolanum stipulaceumRoem.&Schult.
Test Exocarp Mesocarp
ag ly pl pn st ag ly pl pn st
Primarymetabolites
Starch Lugol 1 0 0 0 1 1 1 1 1 1
Acidicpolysaccharides Tannicacid 1 1 1 1 1 1 1 1 1 1
Rhuteniumred 1 1 1 1 1 1 1 1 1 1
Proteins XP 1 1 1 1 1 1 1 1 1 1
Coomassieblue 1 1 1 1 1 1 1 1 1 1 Generallipids SudanVI 1 1 1 1 1 1 1 1 1 1
Sudanblack 1 1 1 1 1 1 1 1 1 1
Neutrallipids Nileblue 1 1 1 1 1 1 0 1 1 1
Acidslipids 0 1 1 0 1 0 1 0 0 1
Secondarymetabolites
Terpenoids NADI 1 1 1 1 1 1 1 1 1 1
Alcaloids Ellram 1 1 1 1 1 1 1 1 1 1
Dittmar 1 1 1 1 1 1 1 1 1 1
Phenoliccompounds Dichromate 1 1 1 1 1 1 1 1 1 1 Ferricchloride 1 1 1 1 1 1 1 1 1 1
Tanniss Vanillin 1 1 1 1 1 1 1 1 1 1
Flavonoids DMACA 1 1 1 1 1 1 1 1 1 1
(ag)S.agrarium,(ly)S.lycocarpum,(pl)S.palinacanthum,(pn)S.paniculatum,(st)S.stipulaceum,(0)absent,(1)moderate,(2)pronounced.
taxonomyrevisions,S.agrarium,S.lycocarpum,S.palinacanthum, andS.paniculatumareincludedinthesubgenusLeptostemonum (Dunal)Bitter(Levinetal.,2006),whereasS.stipulaceumisincluded inthesubgenusBrevantherumSeithe(Roe,1972;Bohs,2005).
Someanatomical features can revealthe species adaptation
mechanismsforthesehabitats.Aspectsofleafanatomyare
infor-mativeof theenvironment where theindividuals live(Rossatto
andKolbm,2012).Thepresenceoftectorandglandulartrichomes withavarietyofshapeshasbeenreportedforSolanum(Nurit-Silva etal.,2011).Ahighdensityofbothtypesoftrichomesindicated adaptationtohighlight,lowwateravailability,andhighherbivory rate,typicaloftheCerrado.Thesameoccurredforthe hypoder-misobservedinallstudiedspecies,whichconstitutesanadditional
xeromorphicfeature (Rossattoand Kolbm, 2012).The presence
of isobilateral mesophyll in S. lycocarpum has been previously
reportedforSolanumspeciesgrowinginhabitatswithhighlight butisnotinaccordancewiththedorsiventralmesophyllusually observedforthisfamily(MetcalfeandChalk,1957).Strong scleri-ficationwasobservedinS.lycocarpum.Sclerophyllyresultsinthe increaseofmechanicalresistance,asanadaptationto environmen-talstresses.Highlevelsofsclerophyllycanbeinducedbylowwater availability,highlightintensity,andlowconcentrationof macronu-trients,whicharecommonconditionsintheCerrado(Costaetal., 2012;RossattoandKolbm,2012).
S.agrariumistheonlyannualspeciesofthefivestudiedspecies. Thepresenceoflooseparenchymaattherootsmayberelatedtoits shortlifecycle,whichoccursduringtheshortrainfallperiodthat occursintheCerrado,whenthesoilpresentshighwatersaturation.
Voluminousintercellular spaces favor aerationin organs
devel-oping inwater saturated soils(Somavillaand Graciano-Ribeiro,
2012).Decreased intercellularspaces minimizeexcessive water
loss,whichisrelevantinspeciesgrowingunderthepredominantly dryCerradoclimateconditions(Milaneze-Gutierreetal.,2003).The investmentinstarchandproteinreservesattherootandstemmay beassociatedwiththefactthatthisspeciesisannual,withtheneed forfastmetabolismduetotheemissionofalargenumberofsprouts inashortperiodoftimeduringtherainyseason(Nurit-Silvaetal., 2011).
Thepresenceofcrystalsandisaunifyingfeatureofthegenus Solanum(MetcalfeandChalk,1957),remainingidentifiableeven incalcinedmaterials(FurrandMalhberg,1981).Calciumoxalate crystalsaresynthesizedfromendogenousoxalicacidandcalcium originatingfromtheenvironment(FranceschiandNakata,2005).
In additiontoaccumulatingatthe vacuoles,theycanbe found
attheapoplast,inductsresultingfromlysisofcrystalidioblasts (Liangetal.,2006).Themainfunctionoftheirformation, espe-ciallyindevelopingtissues,seemstobetheregulationofcalcium concentration,whichisneededfortheexpansionofcells under-goingdivision(FranceschiandNakata,2005;PaivaandMachado, 2005).Theycanalsobeinvolvedinprotectionagainstherbivores
and aluminumdetoxification (Nakata,2003),which are
charac-teristicoftheCerradobiome.The developmentof crystalducts
frombreakingidioblasts,whichhavecellwallscomposedmainly
ofpectin,inrecentlyformedphloemcellsclosetothecambium
layer,wasobservedinthepresentstudywasnotrelatedbeforefor thegenus.
Themedicinal properties mentioned for thespecies maybe
associatedwiththedistributionofsecondary compoundsinthe
different organs. Histochemical techniques are fast and cheap
methods thatcan beusedto identifybioactiveclasses of
com-pounds in tissues, and cell compartments (Coelho et al., 2012;
Demarco et al., 2013; Bedetti et al., 2014). The location of
compounds of interest facilitates studies of domestication or
obtainmentofactiveprinciplesthroughbiotechnologybecauseit allowstheidentificationoftargetcompartmentsforplant
improve-ment. The interpretation of histochemical results allows us to
compare organs, species, or materials originating from
differ-entenvironmentsorseasons (Coelho etal.,2012; Adamsetal., 2013).Previoushistochemicalscreeningcanminimizethesearch for interestingcompounds in relatedplant species,resulting in decreasedcostsfor pharmaceuticalresearch(Saslis-Lagoudakisa etal.,2012).
L.J.Matiasetal./RevistaBrasileiradeFarmacognosia26(2016)147–160 159
acidinhibitsmaltaseactivityinS.palinacanthum,decreasingthe releaseofglucoseintotheblood(Pereiraetal.,2008;Kumaretal., 2011).Thisindicatesa possibleuseof thisspecies forthe con-trolofdiabetes,whichhasnotbeentraditionallyexplored.Rutin, aflavonoidwithanti-microbialaction,hasbeenisolatedfromits leaves(Pereiraetal.,2008).TheaccumulationofflavonoidsinS. paniculatumseemstoberelatedtoitsantioxidantaction(Ribeiro etal.,2007).ThehypotensiveactionofS.stipulaceummayberelated tothepresenceofterpenoids,saponins,andalkaloids(Ribeiroetal., 2002).
Conclusions
Theidentificationofstructuraldiagnosticfeaturesandclasses
of chemical compounds withknownbiological activity present
indifferentorgans ofthestudiedspecies increasesthe reliabil-ityofpreparationsofpharmaceuticalformulations,andrevealsa vastareaofresearchinthechemicalcharacterizationofmolecules relatedtotheirtraditionalmedicinaluse.Studiesrelatingthe
struc-turalfeatureswiththeenvironmentalpressureonthemedicinal
species,andstudiestotestthepopularlybelieved propertiesof nativemedicinalplantsstrengtheninitiativestopreservethe
envi-ronments where those species occur naturally, many of them
alreadystronglymenacedevenbeforetheirpotentialtohumankind
isknown.
Authors’contributions
LJM,ACS,andJMSFcontributedincollectingtheplantsamples, runningthelaboratorywork,andanalysisofthedata.MOMS,VAR, andLMRdesignedthestudyandsupervisedthelaboratorywork.All theauthorshavecontributedindraftedthepaper,criticalreading, writingofthemanuscript,andapprovedthesubmission.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare
thatnoexperimentswereperformedonhumansoranimalsfor
thisstudy.
Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.
Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
The authors wish to thank the FAPEMIG for the Research
and Technological Development Incentive Scholarship to M.O.
Mercadante-Simões(CRA-BIPID-00152-12)andL.M.Ribeiro
(CRA-BIPIT-00137-11);theCNPqandFINEPfortheIndustrialTechnology
DevelopmentscholarshiptoL.J.Matias (DTI-III-381914/2012-7);
theResearchPro-RectoryoftheStateUniversityofMontesClaros fortheScientificInitiationScholarshiptoAriadnaConceic¸ãodos
Santos; and Waldimar Ferreira Ruas for collecting the plant
material.
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