Garibotti Lusa , Maique Weber Biavatti Richetti Amanda Ellen , Everton , Josiane de Athayde Wolff ,Makeli “Arnicas” from Brazil: comparative analysis among ten species

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RevistaBrasileiradeFarmacognosia29(2019)401–424

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

Original Article

“Arnicas” from Brazil: comparative analysis among ten species

Amanda Ellen de Athayde

^a

, Everton Richetti

^b

, Josiane Wolff

^b

, Makeli Garibotti Lusa

^b

, Maique Weber Biavatti

^a,∗

aDepartamentodeCiênciasFarmacêuticas,CentrodeCiênciasdaSaúde,UniversidadeFederaldeSantaCatarina,Florianópolis,SC,Brazil

bDepartamentodeBotânica,CentrodeCiênciasBiológicas,UniversidadeFederaldeSantaCatarina,Florianópolis,SC,Brazil

a r t i c l e i n f o

Articlehistory:

Received26October2018 Accepted6February2019 Availableonline23March2019

Keywords:

Arnica Morphoanatomy Phytochemicalreview Qualitycontrol Asteraceae Ethnopharmacology

a b s t r a c t

The“arnicas”foundinBrazilareexamplesofdifferentspeciesofthefamilyAsteraceaeusedinpopular medicineforitsattributedanti-inﬂammatoryaction.Amongthespeciesknownandusedas“arnica”

weselected:CaleaunifloraLess.,Chaptalianutans(L.)Polák,LychnophoraericoidesMart.,Lychnophora pinasterMart.,LychnophorasalicifoliaMart.,LychnophoradiamantinanaCoile&S.B.Jones,Porophyllum ruderale(Jacq.)Cass.,Pseudobrickelliabrasiliensis(Spreng.)R.M.King&H.Rob.,Sphagneticolatrilobata(L.) Pruski,andSolidagochilensisMeyen,duetotheirextensiveuse.Thisresearchprovidesnewinformation onleafmorphologyandanatomyandonchemistryofthemajormetabolitesfoundinthesespecies throughhistochemicaltestsandphytochemicalreview.Theresultsrevealedanatomicalcharactersforthe differentiationandqualitycontrolofthevegetaldrugs,beingthese:distinctiveepidermalattachments, epidermiscells,parenchymalcellsofthemesophyll,vascularbundles,midveinpatternsandsecretory structuresofexudationofsecondarymetabolites.Thereviewofchemicalprofilesshoweddifferencesin thechemicalcompositionofthespecies,asdifferentskeletonsofsesquiterpenelactonesinthespecies evaluatedinadditiontootherchemicalclassessuchasterpenes,flavonoids,chromenesandphenolicacids derivate.Basedontheresultsobtainedinthisworkitisimportanttoemphasizethattheinformation aboutthetenspeciesofarnicageneratesubsidiesfordifferentiationandidentificationofcharacteristic markersandforthediagnosisofthespeciesanditcanbeappliedinthe“arnicas”qualitycontrol.

Introduction

Brazilhasahugeplantdiversityandwidesociodiversity.Forthis reason,thecountrypresentsgreatpotentialforthedevelopmentof phytotherapyandcorrelatedsciences.Thepopularuseofmedicinal plantsinBrazilisaprocessofproductionandreproductionofvaried knowledgeandpracticesoriginatedindifferentcultures,resulted fromthesocialinteractionamongseveralgroupsthatcomposes, inthiscase,theBrazilianculture(European,Africanandindige- nouscommunities,amongmanyotherculturalgroups)(Salesetal., 2015).

TheEuropeanimmigrantsthatarrivedtoworkmainlyincof- feeplantationinBrazil(19–20thcenturies)couldnotﬁndherethe Europeanmedicinalplants,suchastheverywellknownArnica montana, used as topical anti-inﬂammatory. To meet the need forthisspecies,immigrantsbegantolookforotherspeciesthat could provide the same therapeutic effects; this selection was anecdotallybasedonmorphologicalandsensorialcharactersthat

∗ Correspondingauthor.

E-mail:maique.biavatti@ufsc.br(M.W.Biavatti).

couldresemblethetruearnica(A.montana).Overtheyears,the combination of the European heritage with the miscegenation withotherethnicandculturegroups,ledtoaselectionofseveral plants named popularly as “arnica”,arising from some resem- blance totheA.montana (Semir etal., 2011).Similarprocesses occurredalsoinothercountries,forexampleinMexicoiscom- montheuseofHeterothecainuloidesCass.,Asteraceae,knownas

“arnica-mexicana”(Rodríguez-Chávezetal.,2017).Accordingto Obónet al.(2012),in theIberic peninsulaand BalearicIslands, about32speciesareknownas“arnica”belongtosixbotanicalfam- ilies.Atotalof24speciesareAsteraceae,belongingtoeighttribes:

Anthemideae Cass.(Achillea ageratumL.); Astereae Cass. (Conyza bonariensis (L.) Cronquist); Cardueae Cass. (Centaurea granaten- sisBoiss.ex DC.);CichorieaeLam. &DC. (AndryalaintegrifoliaL., A. ragusinaL.,Crepispaludosa (L.)Moench, C.vesicaria L.subsp.

taraxacifolia(Thuill.)Thell.,andHieraciumsp.);DoroniceaePanero (DoronicumcarpetanumBoiss.&Reut.exWillk.&Lange,D.gran- diﬂorumLam.andD.pardalianchesL.);InulaeaeCass.(Chiliadenus glutinosus(L.)Fourr.,Dittrichiaviscosa(L.)Greuter,Inulabritannica L.,I.helenioidesDC.,I.helveticaGrauer,I.montanaL.,I.salicinaL., Pallensisspinosa(L.)Cass.,Pulicariaodora(L.)Rchb.,andP.palu- dosaLink.);MadieaeJeps.(A.montanaL.).SenecioneaeCass.(Senecio

https://doi.org/10.1016/j.bjp.2019.02.006

creativecommons.org/licenses/by-nc-nd/4.0/).

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Thus, this study will provide information about the microanatomical characterization of the most usual “arnicas”

inBrazil,deﬁninganatomicalmarkersandcomparingtheirphyto- chemicalcompositionbasedonpublishedpaperstodifferentiate these species, namely: Calea uniﬂora Less. (arnica-da-praia), Chaptalia nutans(L.) Polák (arnica-língua-de-vaca), Lychnophora ericoidesMart.(arnica-da-serra),LychnophoradiamantinanaCoile&

S.B.Jones(arnica-da-serra),Lychnophorapinaster(arnica-da-serra) Mart. and Lychnophora salicifolia Mart. (arnica-falsa, arnica- do-cerrado), Porophyllum ruderale (Jacq.) Cass. (arnica-paulista, arnica-da-praia), Pseudobrickellia brasiliensis (Spreng.) R.M.King

& H.Rob. (arnica-do-campo, arnica-do-mato), Solidago chilensis Meyen (arnica-brasileira, arnica-do-campo) and Sphagneticola trilobata(L.)Pruski(arnica-do-mato).

ThespeciesL.ericoidesandL.pinasterareverysimilarspecies andsometimestheseparationbetweenthemisdifﬁcult.Although thespeciesareconsideredbysomeauthorsassynonyms,inthis workweareconsideringasdifferentspecies.Thecharacteristics usedtoidentifyL.ericoides andL.pinasterare described inthe SupplementaryMaterial.

SpeciesofSenecio(“pseudo-arnica”),Asteraceae,arealsousedas arnicabutwerenotincludedinthisworkbecauseitsmedicinaluse shouldbediscouragedduetothepresenceofthetoxicpyrrolizidine alkaloidsinthisgenus(Sandinietal.,2013).

Materialandmethods Plantmaterialusedinthisstudy

Theplantmaterial analyzed wascollectedin SantaCatarina State(SC)andMinasGeraisState(MG),Brazil.Thesampleswere obtainedinﬁeldorfromexsiccatesdepositedintheFLORHerbar- iumoftheFederalUniversityofSantaCatarinaandfromherborized materialprovidedbyBenoitLoeuille.A.montanawasobtainedof commercialsample inBarcelona,Spain.Wheneverpossible,the leavesusedinthisstudywerecollectedfromtriplicateofthree differentpopulations.Vouchernumberofthespeciescanbefound inTable1.

Anatomicalanalysis

Formorphoanatomicalanalysis,leavesfromthreedistinctpop- ulations (n=3) of species collectedin ﬁeld were ﬁxed in FAA solution(70%ethanol,40%formalin,glacialaceticacid)undervac- uumconditionstoremoveairfromthetissues(Johansen,1940).

Next,thematerialsweredehydratedinagrowingseriesofethanol (30%,50%,70%)andstoredin70%ethanol.Theherborizedmaterial washydratedusingaSmithandSmith(1942)modiﬁedmethod (forthis processwasaddedonedropofdetergent).Afterwards, themedianregionoftheleaf(mediumthird)wasselectedandthe sampleswereinﬁltratedinPEG1500.Thesampleswerethencross-

c CommercialsampleidentiﬁedasdescribedintheBrazilianPharmacopoeia5th edition(FarmacopeiaBrasileira,2010).

sectionedwithavariationbetween15␮mand35␮mthickness using a rotary microtome, model RM 2125 RT Leica Microsys- tem,Nussoch, Germany.Topreparethe permanenthistological slides,thecross-sectionswerestainedwith0.05%ToluidineBlue incitrate-phosphatebuffer,pH4.5(Sakai,1973)andafﬁxedtothe slidesusingsyntheticCanadabalm.Forvisualizationoftheepider- misinfrontalview,dissociationoftheepidermiswasperformed (Johansen,1940).

Histochemicalreactions

Histochemicalreactionswerechosen based onthechemical classesof higher occurrencein theAsteraceaefamily andwere performed on material embedded in PEG 1500. The following chemicalreactionswerecarriedout:SudanIV(Jensen,1962)for lipophiliccompounds;sulphuricacid(GeissmanandGrifﬁn,1971) forsesquiterpenelactones;ferricchloride3%(Johansen,1940)for phenoliccompounds;andRutheniumRedforpecticcompounds.

Thesectionswereexaminedimmediatelyaftereachreaction.Con- trolsectionswereperformedsimultaneoustothehistochemical tests,inaccordancewithstandardprocedure.

Imagescapturing

Allthehistologicalslides,includingthatofhistochemicalreac- tions, were observed of the Leica^® Microscope Model DM2500 coupledtotheLeica^® ModelDFC29videocamera.Tocapturethe imageswithsulphuricacid wasusedan Olympus^® microscope (modelCX21FS1)byattachinga digitalcamera(OlympusT110) toeyepiece.Toverifythecrystals,thehistologicalslideswereana- lyzedunderpolarizedlightusingtheOlympusBX50microscope coupledtotheOlympusDP73camera,throughwhichtheimages werecaptured.

Scanningelectronmicroscopy(SEM)

Toperformthisprocedure,theplantswereﬁxedusingFAAsolu- tionandincubatedforaperiodof24h(Johansen,1940).Afterthat, thesamplesweredehydratedinagrowingseriesofethanol(30%, 50%,70%,80%,90%and100%)wasperformed20mineach.Sub- sequently,thematerialwasdriedusingtheCO₂-criticalmethod and,ﬁnally,thesamplesweremetalizedwithgoldforanalysisby SEM(Fernandesetal.,2016)andanalyzedbySEMwiththeJEOL JSM-6390LVScanningElectronMicroscope.

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A.E.Athaydeetal./RevistaBrasileiradeFarmacognosia29(2019)401–424 403 Phytochemicalreview

Phytochemical investigations of “arnicas” species was per- formedusingtheReaxys,Sciﬁnder,Pubmed,Sciencedirect,Google Scholar and Tropicosdatabases. The dataobtainedwas plotted incomparativetable,correlatingthe“arnica”specieswiththeir respectivemetabolitesaccordingtotheliterature.Phytochemical surveywascarriedoutfromMarch2017toJuly2018.Therewas nominimumdateforinclusionofthedata,soalldatawasincluded untiltheyear2018.

Results

Micromorphologicalcharacterization

Allthesamplesofleavesofthespeciesanalyzedareillustrated inFigs.1(A-J),2(A-F),3(A-I),4(A-J),5(A-G),6(A-G),7(A-H),8(A- H), 9(A-J), 10(A-J), 11(A-G) and the aim characteristics are summarized in Figs. 12–14. The complete morphoanatomical descriptionofthesesamplesfollowsbelow.

Middlevein

Initiallythemiddlevein(MV),incrosssection,wasevaluated.

ThespeciesC.uniﬂora,C.nutansandS.chilensispresentedtheMV withabiconvexform,withtheabaxial regionmoreprominent (Fig.1A,B,J).ThespeciesL.diamantinana,L.ericoides,L.pinaster and L.salicifolia presented adaxial surface withcentral depres- sionandabaxialfacewithroundedprojection(Fig.1C–F).ForP.

brasiliensisitwasobservedalittledeﬁnedMV,withoutgreatdiffer- entiationofthestrataofparenchyma,andisnotpossibletodiscuss clearlythemformat(Fig.1H).ThespeciesP.ruderaleandS.trilobata

presented,respectively,aconvexandconcave-convexmiddlevein form(Fig.1G,I).

Theepidermalcells,forallspecies,presentuniseriateformand coveredbycuticle.AdjacenttotheepidermisoftheC.unifloraand C.nutanswasobservedanangularcollenchymaturnedtoadax- ialface (Fig.1A and B).For theS. trilobata andS. chilensis was foundangularcollenchymainbothsurfaces(Fig.1I–J).Lychnophora speciespresentedalotofcollenchymacellsinbothsurfaces,being themlamellarcollenchymatoL.ericoidesandsclerehydeifiedcells orfibersassociatedwiththephloem(Fig.1C–F).P.ruderale,showed astratumofangularcollenchymainbothfaceswiththeextension ofthesheathofthecollenchymabundle(Fig.1G).

Internalsecretorystructureswereobservedclosetothevascu- larbundles,dispersedintheparenchymaoftheMV.ThetaxaC.

uniﬂoraandS.trilobatapresentedtwosecretorycavitiesinserted intheparenchymafacingtheabaxialsurface,closetothephloem, andasecretorycavityjustabovethexylem,turnedtoadaxialsur- face(Fig.1AandI).S.chilensispresentedasecretorycavityturned toabaxialface,insertedbelowthephloemoftheMVoftheleaf (Fig.1J).S.trilobatapresentedthreesecretorycavities,twoturned toabaxialface,belowthephloemandonefacingtheadaxialsur- face,abovethexylem;theotherspeciesdidnotpresentcavitiesin theMVregion(Fig.1I).

Thevascularbundlesintransversalsectionpresentinthemiddle vein(MV)ofC.uniﬂoraisorganizedinopenarchform(Fig.1Aand E,respectively).C.nutanspresentsarelativelycilindricvascular bundle(Fig.1B).ThespeciesofLychnophorapresentedthreefree bundleswithcilindricform(Fig.1C,D,F).P.ruderalepresentssmall andstraightvascularpatternwithextensionofthebundlesheath (Fig.1G).P.brasiliensisdoesnotpresentspeciﬁcdelimitations,with vascularbundlesdistributedthroughoutthemesophyll(Fig.1H).

S.chilensispresentedacordformatvascularsystemand,thetaxon

Fig.1. Middlevein(MV)incross-sectionsofleavesfullyexpandedof“arnicas”.A–J.Lightmicroscopy.A.Caleauniﬂora;B.Chaptalianutans;C.Lychnophoradiamantinana;D.

Lychnophoraericoides;E.Lychnophorapinaster;F.Lychnophorasalicifolia;G.Porophyllumruderale;H.Pseudobrickelliabrasiliensis;I.Sphagneticolatrilobata;J.Solidagochilensis.

A.Stainingwithrutheniumred;B–D,F–J.Stainingwithtoluidineblue;E.StainingwithSudan.Abbreviations:COL,collenchyma;XI,xylem;FLO,phloem;PAR,regular parenchyma;ESC,sclerehydes;CV,internalsecretorycavity.A,J,I.presenceofCV;E.presenceofESC.C–F.possiblesclerenchymaﬁbersbelowthephloem.Findings:A, B,J.MVwithbiconvexform;C,F.adaxialfacewithcentraldepressionandabaxialfacewithroundedprojectiontotheMVform;H.undeﬁnedMV;G.convexMVform;

I.concave-convexMVform;A–J.uniseriateepidermalcells,coveredbycuticle;A.openarchvascularbundlesform;B.cordformatvascularbundlesform;C–F.threefree bundleswithcilindricform;G.smallandstraightvascularpatternwithextensionofthebundlesheath;H.undeﬁnedvascularbundlesform;I.cordformatvascularbundles form;J.conicalandsmalldimensionsvascularbundlesform.

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Fig.2. Viewofstomataandepidermalcellsinabaxialsurfaceofleavesfullyexpandedof“arnicas”.A–F.Lightmicroscopy;A.Caleauniﬂora;B.Chaptalianutans;C.Porophyllum ruderale;D.Pseudobrickelliabrasiliensis;E.Sphagneticolatrilobata;F.Solidagochilensis;A–B.Stainingwithtoluidineblue.C–F.Stainingwithsafranine.Findings:A–F.anomocytic characteristicsbystomata.

Fig.3.Viewofstomatainabaxialsurfaceofleavesfullyexpandedof“arnicas”bySEM.A.Caleauniﬂora;B.Chaptalianutans;C.Lychnophoraericoides;D.Lychnophorapinaster;

E.Lychnophorasalicifolia;F.Porophyllumruderale;G.Pseudobrickelliabrasiliensis;H.Sphagneticolatrilobata;I.Solidagochilensis.Findings:A,B,H.regularlevelofstomatain relationtotheepidermis.C,F–G.belowoftheepidermislevel.D,E,I.abovetheepidermislevel.

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A.E.Athaydeetal./RevistaBrasileiradeFarmacognosia29(2019)401–424 405

Fig.4. Epidermiscells(adaxialvs.abaxialsurface)ofleavesfullyexpandedof“arnicas”bySEM.Adaxialface:A.Caleauniﬂora;B.Chaptalianutans;C.Lychnophoradiamantinana;

D.Lychnophoraericoides;E.Lychnophorapinaster;F.Lychnophorasalicifolia;G.Porophyllumruderale;H.Pseudobrickelliabrasiliensis;I.Sphagneticolatrilobata;J.Solidagochilensis.

Abaxialface:A.Caleauniﬂora;B.Chaptalianutans;C.Lychnophoradiamantinana;D.Lychnophoraericoides;E.Lychnophorapinaster;F.Lychnophorasalicifolia;G.Porophyllum ruderale;H.Pseudobrickelliabrasiliensis;I.Sphagneticolatrilobata;J.Solidagochilensis.Findings:A–JandA–J.uniformepidermisoftheleafsurface(bothfaces);A,B,Iand A,B,I.sinuouswallsoftheepidermalcells;G,H,JandG,H,J.straightwalloftheepidermalcells;C–FandC–F.straightwalloftheepidermalcellsintheadaxialface (C–F)beingmoreroundedandtheabaxialcells(C–F)moreelongated.

hypostomaticleafpatternwasobservedtotheC.nutans,L.diamantinana,L.ericoides,L.pinasterandL.salicifolia(Fig.4B–F).

Thesinuosityoftheepidermalcellsshowedsigniﬁcantdiffer- enceforeachspecies.The“arnicas”presentedtheleafsurface(both faces)withuniformepidermis,whose cellsinfrontalviewpre- sented sinuous wallsto thetaxons C. uniﬂora,C. nutansand S.

trilobata(Fig.4A–B,I–I).StraightwallwasobservedforP.ruderale (longandstraightcellsinadaxialface),P.brasiliensis(bothsides) andS.chilensis(bothsides)(Fig.4G–H,J–J).FortheLychnophora species,theformofthecellswasidentiﬁedasstraight,withthe cellsintheadaxialfacebeingmoreroundedandtheabaxialcells moreelongated(Fig.4C–FandC–F).

Thepresenceofglandular(GT)and non-glandulartrichomes (NGT)wereveriﬁedtotheC.uniﬂora,C.nutans(NGTonly),L.ericoides,L.diamantinana,LpinasterandL.salicifolia,S.chilensisand

S.trilobata(Figs.5A–G,6A–G, 6A–Hand 7A–H).ThespeciesP.

ruderaleandP.brasiliensispresentedglabrousleaf.InC.uniﬂora, theGTareinsertedinsmalldepressionintheepidermisandcan bemulticellularandbicellularorcapitatewithunicellularpedi- celandgloboidhead(Figs.5Aand6A).TheLychnophoraspecies evaluatedshowedGTconcentratedontheabaxialfaceinclosedin crypts(Figs.5B–Eand6B–E).S.chilensispresentedmulticellulartri- chomes,plurisseriateand/orcapitatewithunicellularpedicelwith elongatedterminalcell(Figs.5F–F,6G).S.trilobatapresentedbis- seriedGTindepressionand/ormulticellularunisseriateGTwith elongatedterminalcell(Figs.5G,6F).

Multicellular (5–8 cells) and unisseriate NGT were observed in the species C. uniﬂora, S. chilensis and S. trilobata (Figs. 7A, G–H and 8A, G–H). C. nutans presented long and numeroustrichomes(Figs.7Band8B).TheLychnophoraspecies

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Fig.5.Glandulartrichome(GT)incross-sectionsofleavesfullyexpandedof“arnicas”.Lightmicroscopy:A.Caleauniﬂora;B.Lychnophoradiamantinana;C.Lychnophora ericoides;D.Lychnophorapinaster;E.Lychnophorasalicifolia;F,F,F.Solidagochilensis;G,G.Sphagneticolatrilobata;A,F,F,GandG.stainingwithsafranine;F.stainingwith toluidineblue;B,C,D,E.uncolored.Findings:A.GTinsertedinsmalldepressionandmulticellularandbicellularorcapitedwithunicellularpedicelandgloboidhead.B–D.GT inclosedincrypts;F.GTmulticellularplurisseriateand/orcapitatewithunicellularpedicelwithelongatedterminalcell;G.GTbisseriedindepressionand/ormulticellular unisseriatewithelongatedterminalcell.

Fig.6. Glandulartrichome(GT)ofleavesfullyexpandedof“arnicas”bySEM.A.Caleauniﬂora;B.Lychnophoradiamantinana;C.Lychnophoraericoides;D.Lychnophora pinaster;E.Lychnophorasalicifolia;F.Sphagneticolatrilobata;G.Solidagochilensis.Abbreviations:EP,epidermis;ST,stoma;GT,glandulartrichome;NGT,nonglandular trichome.Findings:A.GTinsertedinsmalldepressionandmulticellularandbicellularorcapitedwithunicellularpedicelandgloboidhead;B–D.GTinclosedincrypts;F.GT multicellularplurisseriateand/orcapitatewithunicellularpedicelwithelongatedterminalcell;G.GTbisseriedindepressionand/ormulticellularunisseriatewithelongated terminalcell.

Fig.7. Nonglandulartrichome(NGT)incrosssectionofleavesfullyexpandedof“arnicas”.A.Caleauniﬂora;B,B.Chaptalianutans;C.Lychnophoradiamantinana;D.

Lychnophoraericoides;E.Lychnophorapinaster;F.Lychnophorasalicifolia;G,G.Solidagochilensis;H.Sphagneticolatrilobata;Findings:A,G,G,H.stainingwithsafranine;B,C, D,E,F.uncolored;A,G,G,H.multicellular(5–8cells)andunisseriateNGT;B.longandnumerousNGT;C,F.starredtrichomes(3–5arms);G,G.elongatedTNG,withslightly enlargedbaseandslimterminalcell;H.NGTwiththebasalcellofverrucouscuticle.

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Fig.8.Non-glandulartrichome(NGT)ofleavesfullyexpandedof“arnicas”bySEM.A,A.Caleauniﬂora;B,B.Chaptalianutans;C,C.Lychnophoradiamantinana;D.Lychnophora ericoides;E.Lychnophorapinaster;F.Lychnophorasalicifolia;G.Solidagochilensis;H,H,H.Sphagneticolatrilobata;Abbreviations:EP,epidermis;ST,stoma;GT,glandular trichome;NGT,nonglandulartrichome.Findings:A,G–H.multicellular(5–8cells)andunisseriateNGT;B.longandnumerousNGT;C–F.starredtrichomes(3–5arms);G.

elongatedTNG,withslightlyenlargedbaseandslimterminalcell;H.NGTwiththebasalcellofverrucouscuticle.

Fig.9. Intermediateregionofmesophyll(IRM)incrosssectionofleavesfullyexpandedof“arnicas”.A.Caleauniﬂora;B.Chaptalianutans;C.Lychnophoradiamantinana.D.

Lychnophoraericoides;E.Lychnophorapinaster;F.Lychnophorasalicifolia;G.Porophyllumruderale;H.Pseudobrickelliabrasiliensis;I.Solidagochilensis;J.Sphagneticolatrilobata.

Abbreviations:CUT,cuticule;PP,palisadeparenchyma;LP,lacunarparenchyma;AP,aquiferousparenchyma;PR,regularparenchyma;VB,vascularbundleblond;ESC, sclerehyde;EP,epidermis;GT,glandulartrichome;NGT,non-glandulartrichome;IC,internalcavity.Findings:A–F.stainingwithrutheniumred;G,J.stainingwithtoluidine blue;A,B,G,J.dorsiventralparenchyma;C–F.palisade(adaxial),spongy(abaxial)andaquiferousparenchymastrataclosetothevascularbundleswithextensionofthe bundlesheath;H.homogenousmesophyll;I.isobilateralmesophyllandaquiferousparenchyma(interconnectingthevascularbundles);A–J.collateraltypeofthebundles.

presentedstarredtrichomes(3–5arms)varyinginsizeandwidth, withcellulosicthickeningandconcentratedontheabaxialfaceof theleafbetweencrypts(Figs.7C–Fand8C–F).S.chilensisshowed elongatedTNG,withslightlyenlargedbaseandslimterminalcell (Figs.7G–Gand8G–G).S.trilobatapresentedtrichomeswiththe basalcellofverrucouscuticle(Figs.7Hand8H–H).

Theintermediateregionofthemesophyll(IR)wasdorsiven- tralparenchymaforthespeciesC.uniﬂora,C.nutans,P.ruderale andS.trilobata(A,B,G,J).TheLychnophoraspeciespresentedpal- isade(adaxial), spongy(abaxial)and aquiferparenchyma strata close to the vascular bundles with extension of the bundle sheath,excepttoL.pinaster(Fig.9C–F).P.brasiliensispresented

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Fig.10. Leafmarginincrosssectionofleavesfullyexpandedof“arnicas”.A.Caleauniﬂora;B.Chaptalianutans;C,C.Lychnophoradiamantinana;D.Lychnophoraericoides;

E.Lychnophorapinaster;F.Lychnophorasalicifolia;G,G.Porophyllumruderale;H.Pseudobrickelliabrasiliensis;I.Solidagochilensis;J.Sphagneticolatrilobata;A,B,F.staining withferricchloride;C–EandG–I.stainingwithrutheniumred;J.toluidineblue.Findings:A–F,J.roundedandflexedsheetedgefortheabaxialface;C–F.flexionwithafinal marginnarrowing.I.discreteflexionofthemarginwithepidermalprojectionstotheoutsideoftheleaf;G–H.straightmargin,withoutflexionorlittleflexure.G,G.large secretorycavityonthemarginofleaf.

Fig.11.AnalysesofleavesfullyexpandedofArnicamontanabySEM.A.adaxialface;B.abaxialsurface;C.stomata;D.baseoftheNGT;E.peduncleofNGT;F.apexofNGT.

G.GT.

homogenous mesophyll, without distinction between the cells (Fig.9H).S.chilensispresentedmesophylloftheisobilateraltype withpresenceofaquiferparenchymainthemedialregionofthe mesophylland interconnectingthevascularbundles (Fig.9I). In allspecies, the bundles wereof the collateral type, distributed inthe median regionof themesophyll,being surroundedby a parenchymaticsheath. Inthespecies C.uniﬂoraand S.chilensis, these may be associated with secretory ducts (Fig. 9A, I). The Lychnophorapresentedextensionofthesheathofthebundleand

alsolargerdimensionsoftheregionwherethevascularbundles meet(Fig.9C–F).Thepresenceof crystalidioblastswasveriﬁed intheL.diamantinana(smalldruses).Theoccurrenceofphenolic, lipophilicandpecticcompoundswasalsoveriﬁed(Item4.2).

Leafmargin

In the present study, among the “arnica” specimens, there wasa differenceinleafmargininrelationtoﬂexion anddistri- butionofparenchymacells. ThespeciesC.uniﬂora,C.nutans,L.

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Fig.12. Flowcharttoqualitycontrolofthe“arnicas”evaluatedusingtheleavesfullyexpanded,throughthemiddlevein.Orange,anatomicalparameters;Blue,anatomical deﬁnitionandtarget;Green,specieconﬁrmation.

Fig.13.Flowcharttoqualitycontrolofthe“arnicas”evaluatedusingtheleavesfullyexpanded,throughthestomata.Orange,anatomicalparameters;Blue,anatomical deﬁnitionandtarget;Green,specieconﬁrmation.

ericoides,L.diamantinana,L.pinaster,L.salicifoliaandS.trilobata showedroundedandrevoluteleafbordertowardtheabaxialface (Fig.10A–F,J).FortheLychnophoraspeciesthereismarkedflexion withafinalmarginnarrowing(Fig.10C–F).S.chilensispresented discreteflexionofthemarginandwithepidermalprojectionsto theoutsideoftheleaf(Fig.10I).Ontheotherside,thespeciesP.

ruderaleand P.brasiliensisshowedastraightmargin,withoutor littleﬂexion(Fig.10G–H).Inparticular,thereisalargesecretory cavitypresentonthemarginofP.ruderale(Fig.10G–G).Theorga- nizationofthemesophyllcellsfollowsthesamedistributionofthe

intermediateportion,withexceptionforS.chilensiswithpredom- inanceofaquiferparenchyma(Fig.10I).

A.montana(“thetrueArnica”)

Analyses of the A. montana leaveswere performed only by scanningelectronmicroscopy(Fig.11A–G).Weareobservedthe amphistomatic pattern ofthe leaves, witha greaternumber of stomataontheabaxialfaceoftheleaf(Fig.11AandB).Stomawas lightlyprojectedontotheepidermis(Fig.11C).Thenon-glandular trichomes(4–5cells)showedaverrucousapicalcellandsmooth

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Fig.14.Flowcharttoqualitycontrolofthe“arnicas”evaluatedusingtheleavesfullyexpanded,throughthetrichomes.Orange,anatomicalparameters;Blue,anatomical deﬁnitionandtarget;Green,specieconﬁrmation.

cuticletothebasecells(Fig.11A–B,D–F).Theglandulartrichomes arecapitate(Fig.1G).

Identiﬁcationﬂowchartforthe“arnicas”evaluatedinthisstudy

Tosystematizethemicromorphologicalcharacterizationofthe

“arnicas”evaluatedinthisstudy,aflowchartwascreatedtodiffer- entiatethespecies.Diagnosticanatomicalelementswereidentified andplotted makingpossiblethe quickidentificationof species, intactorcrushed(Figs.12–14).

Histochemicalcharacterization

Thehistochemicaltestsrevealedthatsecondarymetabolitesthe mainchemotaxonomicmarkersofthe“Arnicas”weresecretedand accumulatedinspeciﬁcssecretorystructuressuchasglandulartri- chomes,epidermalidioblasts,secretory tissuessuchaspalisade andspongychlorophyllparenchyma,themiddleveinintheleaves

and in theinternal secretory structures.The ﬁgures showpos- itive reactionsto:lipophilic substances, evidenced bySudan IV (Figs.15and16)inorangetored;sesquiterpenelactonesevidenced bysulphuricacid(Figs.17and18)inredtoorangeandyellowand phenoliccompoundsevidencedbyferricchloride(Figs.19and20) withbrowncolor.

Thehistochemicalreactions,classesofmetabolitesfoundand thesites of secretion in theevaluated species will beshow in Table2.

Chemicalreview

Thephytochemical review of thespecies of “arnica” reveals diversiﬁcationofcompounds.Basedonthedataobtained,ispos- sibleverifythemainclassesofmetabolitesineachofthespecies (Fig.21).Table3compilestheliteraryﬁndingsreferringtothemain substancesdescribedforeachofthespecies.

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Fig.15.Histochemicalreactionforlipophilicsubstancesincross-sectionsofleavesof“arnicas”,highlightedbySudan.Lightmicroscopy.Theﬁgureshowspositivereactions (red)forlipophilicsubstanceswithaccumulationofmetabolitesinglandulartrichomes.A,A.Caleauniﬂora;B,B.Lychnophoradiamantinana;C.Lychnophoraericoides;D.

Lychnophorapinaster;E.Lychnophorasalicifolia;F.Solidagochilensis;G.Sphagneticolatrilobata.

Fig.16.Histochemicalreactionforlipophilicsubstancesincross-sectionsofleavesof“arnicas”,highlightedbySudan.Lightmicroscopy.Theﬁgureshowspositivereactions forlipophilicsubstanceswithaccumulationandsecretionofmetabolitesintheepidermalcellsandothersecretorystructuresofleaves.A,A.Caleauniﬂora;B.Lychnophora diamantinana;C.Lychnophoraericoides;D.Lychnophorapinaster;E.Lychnophorasalicifolia;F.Solidagochilensis;G.Sphagneticolatrilobata.Findings:A,B,C,D,I,F.midvein withaccumulationofmetabolitesinparenchymacells.A,B,B,C,D,D,E,F,G,H,I,J.accumulationofmetabolitesintheepidermisand/orchlorophyllparenchymaofthe mesophyll;A,A,G,I,I.accumulationofmetabolitesintheinternalsecretorystructure.Reactionpatternatthetipofthearrow(red).

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Fig.17.Histochemicalreactionforscreeningofsesquiterpenelactonesincross-sectionsofleavesof“Arnicas”,evidencedbysulphuricacid.Lightmicroscopy.Theﬁgure showspositivereactions(brown/orange)forscreeningofsesquiterpenelactoneswithaccumulationofmetabolitesinglandulartrichomes.A.Caleauniﬂora;B.Lychnophora diamantinana;C.Lychnophoraericoides;D.Lychnophorapinaster;E.Lychnophorasalicifolia;F.Solidagochilensis;G,G.Sphagneticolatrilobata.

Fig.18.Histochemicalreactionforscreeningofsesquiterpenelactonesincross-sectionsofleavesof“arnicas”,evidencedbysulphuricacid.Lightmicroscopy.Theﬁgureshows positivereactionsforscreeningofsesquiterpenelactoneswithaccumulationandsecretionofmetabolitesintheepidermalcellsandothersecretorystructuresofleaves.

A.Caleauniﬂora;B.Lychnophoradiamantinana;C.Lychnophoraericoides;D.Lychnophorapinaster;E.Lychnophorasalicifolia;F.Solidagochilensis;G.Sphagneticolatrilobata.

Findings:B,D,E,F,H.midveinwithaccumulationofmetabolitesinparenchymacells;A,C,F,G,H,I.accumulationofmetabolitesintheepidermisand/orchlorophyll parenchymaofthemesophyll;F,G,H.accumulationofmetabolitesintheinternalsecretorystructure.Reactionpatternatthetipofthearrow(brown/orange).

Discussion

Micromorphologicalcharacterization

Theanatomicaldataobservedinthemiddlevein(MV)forthe speciesC.uniﬂora,C.nutansandS.chilensisareinagreementwith

thosedescribedbyBudeletal.(2006),EmpinottiandDuarte(2006) andSouzaetal.(2017),respectively.TheMVobservedinS.trilo- bataandP.ruderalewasaccordingtoBaccarinetal.(2009)and Milanetal.(2006),respectively.TheleavesoftheLychnophoraare relativelysimilar:theMVofL.salicifolia,L.pinasterandL.diamanti- nanapresentedadaxialfacewithcentraldepressionandabaxial

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Fig.19.Histochemicalreactionforphenolicsubstancesincross-sectionsofleavesof“arnicas”,highlightedbyferricchloride.Lightmicroscopy.Theﬁgureshowspositive reactions(brown)forphenolicsubstanceswithaccumulationofmetabolitesinglandulartrichomes.A.Caleauniﬂora;B.Lychnophoradiamantinana;C.Lychnophoraericoides;

D.Lychnophorapinaster;E.Lychnophorasalicifolia;F.Solidagochilensis;G.Sphagneticolatrilobata.

Table2

Histochemicaltestsandsuggestedmetabolicclassesforleafsecretionsof“Arnicas”species.

Species Histochemicaltests SudanIV Sulphuricacid Rutheniumred Ferricchloride

Metabolicclasses Lipophilicsubstances Sesquiterpenelactones Pecticcompounds Phenoliccompounds

Arnicamontana IR,GT,MDSS Notidentiﬁed IR,GT,MD,EP IR,MS,MD

Caleauniﬂora IR,GT,MDSS IR,GT GT,SS IR,GT,MD,SS

Chaptalianutans IREP IR,MD, IR,SS MS

Sphagneticolatrilobata IR,GT,MD,SSEP IR,GT IR,GT,MD,SS IR,GT,MD,SS

Solidagochilensis IR,GTSS IR,GT,MD,SS IR,GT,SS,NGT IR,GT,MD,SS

Porophyllumruderale IR,MD,SS – IR,MD,SS IR,MD,SS

Pseudobrickelliabrasiliensis MS SS,MS MS MS

Lychnophoraericoides IR,GT,MD GT,MD IR,MD,NGT IR,GT,MD,NGT

Lychnphorasaicifolia IR,GT,MD GT IR,GT,MD,NGT IR,GT,MD,NGT

Lychnophorapinaster IR,GT,MD GT IR,MD,NGT IR,GT,MD,NGT

Lychnophoradiamantinana IR,GT,MD,NGT GT,MD IR,GT,MD,NGT,EP IR,GT,MD,NGT

IR,intermediatemesophyllregion;EP,epidermalcells;ST,stomata;MV,middlevein;GT,glandulartrichome;NGT,non-glandulartrichome;MS,mesophyll;“–”,negative;

SS,secretorystructure.

surfacewithroundedprojection.L.ericoidespresentedontheadax- ialfacediscretecentraldepressionandabaxialsurfacealsowith roundedprojection.ThroughthecharacterizationoftheMVofthe

“arnica”species,itwaspossibletoobtainapreliminarydifferen- tiationamongtheevaluatedspecies,especiallyinrelationtothe formofMV,beinganimportantanatomicalcharacterforquality control.

Inrelationtoepidermalattachments,weobservevariablestruc- turesof great diagnosticvaluefortaxonomy, suchas glandular trichomes(GT)thatareinvolvedinthesecretionofvariousbioac- tivesubstances(Lusaetal.,2016a,b).Inthepresentstudy,different typesofGTweredescribed,beingabletobeusedforqualitycon- trolofthespecies.TheﬁndingsregardingS.chilensisdifferfrom thoseregisteredbySouzaetal.(2017)thatdescribes,throughpara- dermalsections,onlythepresenceofnon-glandulartrichomesin theleavesofthespecie,whileweobservedthepresenceofnon- glandulartrichomesandglandulartrichomesincross-sections.InS.

trilobatatheGTwerepresentintwoforms:bisseriateindepression andamulticellularwithelongatedapex,identiﬁedfortheﬁrsttime.

ForC.uniﬂora,theGTareaccordingtodescriptionmadebyBudel etal.(2006).FortheLychnophoragenus,GTweredetectedonthe

abaxialsurface,betweenthecrypts.Thesedataarecorrelatewith those described by Lusa et al. (2016a,b)for otherLychnophora species.NoglandulartrichomeswasfoundinthespeciesP.rud- eraleandC.nutans,inagreementwithdescribedbyEmpinottiand Duarte(2006)andDuarteetal.(2007),respectively.InP.brasiliensis noGTwasobservedeither.

Someanatomical diagnosticfeatures wereobservedthrough nonglandulartrichomes(NGT).InP.ruderale,aleafglabrouswas observed,withoutthetrichomespreviouslydescribedbyDuarte etal.(2007).TheabsenceofhairswasalsoobservedforP.brasiliensis.InC.nutansnumerousNGTwerefoundpredominantlyonthe abaxialsurface(EmpinottiandDuarte,2006).InrelationtoS.chilensis,S.trilobata and C.uniﬂora,similaranatomical ﬁndings were describedbySouzaetal.(2017),Baccarinetal.(2009)andBudel etal.(2006).

AccordingtoMetcalfeandChalk(1988)anomocyticstomataare characteristicoftheAsteraceaefamily,asweobservedpredomi- nantly.FortheLychnophoraitwasnotpossibletocharacterizethe typeofstomataduetotheinnumerablepresenceofnon-glandular trichomesandalsobythestomata beinginsertedinthecrypts, makingtheirvisualizationevenmoredifﬁcult.

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Fig.20. Histochemicalreactionforphenolicsubstancesincross-sectionsofleavesof“arnicas”,highlightedbyferricchloride.Lightmicroscopy.Theﬁgureshowspositive reactionsforphenolicsubstanceswithaccumulationandsecretionofmetabolitesintheepidermalcellsandothersecretorystructuresofleaves.A.Caleauniﬂora;B.

Lychnophoradiamantinana;C.Lychnophoraericoides;D.Lychnophorapinaster;E.Lychnophorasalicifolia;F.Solidagochilensis;G.Sphagneticolatrilobata.Findings:A,B,C,D,E, F,F,I,J.midveinwithaccumulationofmetabolitesinparenchymacells;A,B,C,D,G,G,H,I,J.accumulationofmetabolitesintheepidermisand/orchlorophyllparenchyma ofthemesophyll;A,G,I,J.accumulationofmetabolitesintheinternalsecretorystructure.Reactionpatternatthetipofthearrow(brown).

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A.E.Athaydeetal./RevistaBrasileiradeFarmacognosia29(2019)401–424415 Table3

Reviewofmaincompoundsdescribedfortheknownspeciesofthe“arnicas”inBrazil.

Compounds A.mon-

tana

S.trilobata

S.

chilensis C.

uniﬂora

P.ruderale

C.

nutans P.

brasiliensis

L.ericoides

L.salicifolia

L.

pinaster L.

diamantinana Flavone

Apigenin x^(F,G)

Apigeninglycosylated x^(F,G)

Chrysin x^(F) x^(K) x^(K) x^(G)

Chrysinglycosyl x^(F,G)

Luteolin x^(k) x^(a) x^(C)

Luteolinglycosil x^(F)

3,4,7-Trihydroxi-ﬂavone-7-O-␤- glucopyranosyl

x^(s)

5,4-Dihydroxy-7-methoxyﬂavone x^(j)

5,3,4-Trihydroxy-7- methoxyﬂavone

x^(j) 5,7-Dihydroxy-4-methoxyﬂavone

(acacetin)

x^(F) 3,7,4-Trihydroxyﬂavanone

(garbanzol)

x^(F)

3-Methyl-galangin x^(F)

Flavanone

Naringenin x^(F,G)

Pinocembrin x^(F,G) x^(K) x^(K) x^(G)

Pinostrobin x^(F,G) x^(K) x^(K) x^(G)

Naringenin x^(F,G)

Pinocembrin x^(F,G) x^(K) x^(K) x^(G)

Pinobanksin x^(F,G) x^(K) x

Flavonol

Afzelechin x^(a)

Galangin x^(F)

Kaempferol x^(N) x^(a)

Resokaempferol x^(F)

Patuletin x^(N) x^(a)

Quercetin x^(N) x^(k) x^(b) x^(s) x^(v) x^(A) x^(C) x^(F,G) x^(J) x^(M)

Quercetinheterozyl x^(N) x^(k) x^(a) x^(s)

Quercetin-7,3,4-trimethylether x^(J)

Quercetol x^(F,G)

Rutin x^(N) x^(v)

Chalcone

Buteinchalcona x^(r)

Butein4-O-glucopyranosyl x^(r)

␣-Hydroxy-butein x^(r)

2,4-Hihydroxy-3-methoxy- chalcone-4-O-␤-glucopyranosyl

x^(s)

2,4,3,4-␣-Pentahydroxychalcone x^(s)

Pinostrobinchalcone x^(F,G)