Rev. bras. farmacogn. vol.27 número2

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

Original

Article

Morpho-anatomy

and

chemical

profile

of

native

species

used

as

substitute

of

quina

(Cinchona

spp.)

in

Brazilian

traditional

medicine.

Part

II:

Remijia

ferruginea

Nádia

S.

Somavilla

,

Gustavo

P.

Cosenza

,

Christopher

W.

Fagg

,

Maria

G.L.

Brandão

a_{LaboratóriodeAnatomiaVegetal,UniversidadedeBrasília,Brasília,DF,Brazil}

b_{DepartamentodeBotânica,UniversidadeFederaldeJuizdeFora,CampusUniversitário,JuizdeFora,MG,Brazil} c_{LaboratóriodeFarmacognosia,FaculdadedeFarmácia,UniversidadeFederaldeMinasGerais,BeloHorizonte,MG,Brazil}

d_{CentroEspecializadoemPlantasAromáticas,MedicinaiseTóxicas,MuseudeHistóriaNaturaleJardimBotânico,UniversidadeFederaldeMinasGerais,BeloHorizonte,MG,Brazil} e_{DepartamentodeBotânica,FaculdadedeCeilândia,UniversidadedeBrasília,Brasília,DF,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29May2016 Accepted5September2016 Availableonline26October2016

Keywords:

Stembarkanatomy Qualitycontrol Quininederivatives

a

b

s

t

r

a

c

t

ThisresearchispartofalargerstudyoftheBrazilianspeciesthatarecommonlyreferredtoas“quinas” andusedassubstituteofCinchonaspecies.Inthisstudy,wehaveperformedthebotanical characteriza-tionofthestembarkofRemijiaferruginea(A.St.-Hil.)DC.,Rubiaceae,bymorphologicalandanatomical description,andtheanalysisofitschemicalprofile.Stembarkisthinandhasthecolorandthetextureof itsexternalandinternalsurfacesasdiagnosticfeatures.Typesandsizesofsclerifiedcellsinthecortical parenchymaandinthesecondaryphloemareimportantfeaturesforanalysisofthetransversalsections andinthemacerate.Alkaloids,flavonoidsandchlorogenicacidweredetectedinthechemicalanalysis forTLC.Thesestandardreferencescanbeusedinthequalitycontrolofthebarkofquinas.

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Quina(orchina)isthetraditionalnameattributedtoCinchona calisayaWedd.andC.succirubraPav.exKlotzsch,Rubiaceae,species nativefromPeruthatproducestheantimalarialquinine(Kauretal.,

2009;Dondorpetal.,2009).InBrazil,speciesfromdifferent

botan-icalfamiliesareusedforcenturiesassubstituteofthesetruequina. Theyhaveitsnamelinkedtothebittertasteofitsstembarkandthe medicinaluseasfebrifuge(Cosenzaetal.,2013).Recently,weare focusingintoperformermorphological,anatomicalandchemical profilethatcanbeusefulinthequalitycontrolofquinabarks.In thepartIofthesework,wehavestudiedthebarksofPolyouratea hexasperma(A.St.-Hil.)Tiegh.(sin.Ourateahexasperma(A.St.-Hil.) Baill.)(Somavillaetal.,2013).

StembarksofRemijiaferruginea(A.St.-Hil.)DC.,Rubiaceae,are known as “quina-da-serra”, “quina-de-remijo”, “quina-mineira”

(Corrêa,1984;Botsaris, 2007;Saint-Hilaire,2014).Thisshrubby

species(Fig.1AandB)isendemicofBrazil,occurringmainlyin rockyoutcrops(INCT,2014).Thegeographicdistribution includ-ingthestatesofMinasGerais,MatoGrosso,MatoGrossodoSul,

∗ Correspondingauthor.

E-mail:nadia.somavilla@ufjf.edu.br(N.S.Somavilla).

BahiaandEspíritoSanto(DelpreteandCortés,2006;Zappi,2014). Thesebarkswerewidelyusedin19thcenturytotreatfeversand malaria(Cosenzaetal.,2013).Lindley(1838),forexample,citedthis speciesinhisbookFloraMedicaasoneofthemostimportantplant usedintheworldand,despitehavinganinferiorefficacycouldbe consideredasasubstitutePeruvianquina.Dueitsusealsoin con-ventionalmedicine,monographsforthebarksofR.ferrugineawere includedinthefirsteditionoftheBrazilianPharmacopeia(Silva,

1926;Brandãoetal.,2009).Extractsfromthebarksareingredient

ofthetraditionalformulaIerobina®_{,usedtotreatdyspepsia(Botion}

etal.,2005).Ontheotherside,inourrecentstudy,inwhichwe iden-tifiedbarksofquinasoldinpopularmarketbyDNAbarcode,we observeadeclineinusebarksfromR.ferrugineaasquina(Palhares

etal.,2014).

Study shown that high doses of thebark extracts of R. fer-rugineainduced reduction ofthe parasitaemiaand mortalityin mice infected by Plasmodium berghei, and indicating moderate antimalaricalactivity(Andrade-Netoetal.,2003)althoughinthis alkaloids-producingspecieshasnotbeendetectedthepresenceof quinine.

Theaimofourworkistodescribethebotanicalfeaturesandto analyzethechromatographicprofileofstembarkoftheR. ferrug-ineainordertoprovidesupportintheidentification,analysesand standardizationofthisrawmaterial.

http://dx.doi.org/10.1016/j.bjp.2016.09.005

Fig.1. Remijiaferruginea(A.St.Hil.)DC.(A)Shrubbyhabitonrockysoilofrockoutcrop.(B)Detailofinflorescenceandleaves.(C)Generalexternalviewofstembarkwith highlightingforpresenceoflichens(arrows).(D)Samplesofstembarkshownexternal(left)andinternal(right)aspect.Bars:1cm.

Materialsandmethods

Plantmaterial

The samples of the stem bark of Remijia ferruginea (A.

St.-Hil.) DC., Rubiaceae, for analysis were collected in São Gonc¸alo do Rio das Pedras,Serro, Minas Gerais (S 18◦₂₅′₄₁′′_W 043◦₃₀′₀₃′′_{) and registered as DAT-134 in the DATAPLAMT}

(http://www.dataplamt.org.br).

Morphological,anatomicalandhistochemicalanalysis

The samples were described as to external and internal

aspectssuchascoloring,textureandtestsorganoleptic.For pur-poses anatomical characterization part of these samples were fixedinsolutionofformaldehyde–aceticacid–ethanol70(1:1:18,

Johansen,1940),rinsedindistilledwaterandstoredinethanol70.

After,thesesamplesweresectionedinmicrotometypeRanvierand stainedwithastrablueandfuchsindyes(KrausandArduim,1997) andmountedonslideswithvernizvitralincolor500®_(Paivaetal.,

2006).Freshsamplesweresubmittedtohistochemicaltests:ferric

chloride(Johansen,1940)andpotassiumdicromate(Gabe,1968)to detectphenolicscompounds,vanillinhydrochloricacidfortannins

(Gardner,1975),acidphloroglucinforlignin(Sass,1951),solution

oflugolforstarch(KrausandArduim,1997),SudanIII(Sass,1951) andSudanIV(Gerlach,1984)forlipids.Partofsamplewas submit-tedtomacerationprocessfordissociationandtissuecomponents analysis.Forthat,thesampleswereplacedinFranklinsolutionand maintainedinakiln(60◦_{C)for72h(KrausandArduim,1997).After} thisprocess,themaceratewaswashedwithdistilledwaterto com-pleteremovalofFranklin solutionandkeptin50%ethanol. For stainingwasemployedethanolicsafranin1%.Theslidesobtained fromthesepreparationswereanalyzedanddescribedbyOlympus CX31opticalmicroscopeandphotographedwithadigitalcamera OlympusC-7070,withwidezoom.Thebotanicaldescription fol-lowedtherecommendationsofJunikka(1994)andRichteretal.

(1996).

ChromatographicprofileforphenolicandalkaloidsbyTLC

Preparationoffractionsenrichedinphenolicsubstances

co

ph

xy su

pr

vc

D

E

F

G

H

I

J

B

iph oph

pdr

A

pdr

C

Fig.2.StembarkofRemijiaferruginea(A.St.Hil.)DC.(A)Generalaspectoftransversalsection.Arrowindicatesdivisionandexpansionofparenchymacells.(B)Magnification oftheperiderm(bracket)withdestacforphellogen(arrow).(C)Magnificationofsecondaryphloemregionhighlightingthesclerifiedcells(arrow)andcollapsedcellsofouter phloem(oph).(D)Histochemicaltestwithphloroglucinacidshownthesclerifiedcells:corticalcells(arrowhead)andphloemcells(arrow).(EandF)Elongatedsclerifiedcells ofphloem.(E)Fibers.(F)Fiber-sclereids.(GandH)Corticalsclerifiedcells(stonecells).(I)Phloemlongitudinalsectionshownsieveplates(arrows).(J)Transversalsection showncells(arrow)withphenolicscompoundsbypotassiumdichromatehistochemicaltest.Legend:co,cortex;iph,innersecondaryphloem;oph,outersecondaryphloem; pdr,phloemdilatedray;ph,secondaryphloem;phe,phellem;pr,phloemray.Bars:200␮m(A,D,J);100␮m(BandC,I);50␮m(E,GandH);20␮m(F).

filteredthroughfilterpaperand concentratedanddriedusinga rotaryevaporator(crudeextract).Theresultingresiduewasdiluted with50mlwaterandextracted3timeswith30mlethylacetate and30mlofbutanol.Thefractionswereconcentratedtodryness andtheaqueouslyophilized.Eachdriedextractandfractionwere dissolvedin1mlmethanolforsubsequentanalysis.

Preparationofalkaloidfraction

Briefly,3gofdriedR.ferrugineabarkwasextractedunderreflux conditionswith20ml0.1MHClfor30min.Thesolutionwas alka-lizedwithNH4OHtopH9andextractedthreetimeswith20ml ethylether.Theorganicphasewasconcentratedtodryness,and

theresultingresiduewasdissolvedin2mlmethanolforsubsequent analysis.

Chromatographicanalysis

TLC was performed on silica gel plates (Macherey-Nagel

Alugram®_{Xtra SILG UV 254) using solvents and reagents}

reference standards. For the detection of alkaloids, a toluene, methanol, and diethylamine (80:10:10) mixture was used, fol-lowedbysprayingwithDraggendorfreagent.Samplesofquinine hydrochloride(Sigma,BCB3224V),quinidine(Sigma,BCBF1345V), cinchonine(Aldrich,STBB1223),andcinchonidine(Sigma-Aldrich, BCBD9930V)wereusedasreferencestandards.

Resultsanddiscussion

ThestembarkofR.ferruginea measurebetween1and2mm thick,theoutersurfaceisslightlystriated,whitishtolightbrown color and may have grayish color due the presence of lichens (Fig. 1C); inner surface is light browncolor,smooth and slight glossytexture(Fig.1D).Thebarkdoesnothavearemarkablesmell andthetasteisbitterand astringent.In transversalsectionthe stembarkismadeupofperiderm,cortexandsecondaryphloem (Fig.2A).Thereisa oneperiderm and thephellem madeupof 4–12cellslayersandouterlayersdetachfromsurface,the phel-lodermshow1–2cellslayersandthephellogenisinconspicuous (Fig.2B).Mostparenchymacellsofthecortexhavetangential divi-sionandexpansionlinewiththeimprovedincircumferenceofthe stem(Fig.2A,arrow).Somecorticalcellsshowtangential expan-sionand lignificationofitscells walland aremoreclusteredin theperipheryof cortex(Fig.2A and D,arrowhead).In the sec-ondaryphloemispossibletodifferentiatebetweenaninnermost andyoungernon-collapsedphloemwheresclerifiedcellsoccurin reducednumberorlackfromanouterand oldermostcollapsed phloemwithnumerousclusteredsclerifiedcells(Fig.2C andD). Thecellsofrayundergotangentialdivisionandexpansiontoward theperipherygivingrisetodilatedraysandtheoutercellsofdilated rayanastomose withcorticalparenchyma cells (Fig.2Aand C). Sclerifiedcellsofphloemaredifferentfromsclerifiedcellsofthe parenchymacortical.Inthelongitudinalsectionandinthe mac-erateitispossibleidentifythesclerifiedcellsofphloemasfiber orfiber-sclereids(Fig.2EandF)withlengthrangingfrom396.85 to1547.73␮m(832.64±320.68␮m, mean±standard deviation) and widthranging from 26.61to 98.04␮m(47.73±19.13␮m). Thesecellsareaxiallyelongatedandexhibitnon-lamelarcellwall withsimplepitsandintrusivegrowth.Duringtheintrusivegrowth thecellcanformlateralprojectionsandshowndifferentformats (Fig. 2F).Sclerified cells of thecorticalparenchyma are usually shortersclereids(stonecells)withbranchedpitsandlamellarwalls (Fig.2GandH),aretangentially elongatedand canshow intru-sivegrowth(Fig.2G).Lengthofthesecellsvariesfrom180.28to 535.59␮m(321.57±110.59␮m)anditswidthrangingfrom44.21 to123.11␮m(81.0±22.84␮m).Inthephloemthesievedplateare simpleandstraightorslightlyoblique(Fig.2I).

Histochemical tests were positive for phenolic compounds insideofthephellemcells andparenchyma cellslocatedinthe ray and axial system of the phloem as well as at the cortex (Fig.2J).Probablythisdepositionreferstobrown-yellowish sub-stancementionedinthedescriptionofthisspeciesbytheBrazilian Pharmacopeia(Silva,1926).Starchwasidentifiedinsideofcortical andphloemraysparenchymacells.Ligninoccurinthecellwallof sclerifiedcells(Fig.2D).

Theanalysis performedto phenolic compounds showedthe

presenceofrutininRfof0.5andchlorogenicacidinRfof0.6in thefractionsenrichedthesesubstances,sprayingNP/PEGreagent followedbyUV365nm(Fig.3).Thechromatographicprofilefor alkaloidsinTLC,obtainedforapurifiedfractionshowedthe pres-enceofbandsinthesameRfofcinchonine(0.65)(Fig.4).Cinchona alkaloidsoccurmostnotably ingenusCinchonaand therelated generaRemijiaandLadenbergiaandcinchonineisoneofthefour principal alkaloids of a total of 35 cinchona alkaloids known

(HofheinzandMerkli,1984).Ruiz-Mesiaetal.(2005)andArana

1 2 3 4 5 6 7 8

− Start − Front − Rf=0.85

− Rf=0.50 − Rf=0.60

Fig.3. TLCforidentificationofphenolicssubstancesinRemijiaferrugineabarks.

5 4 3

2 1

− Start − Front

− Rf=0.60

− R_f=0.70

Fig.4. TLCforidentificationofalkaloidsfromRemijiaferruginea.

etal.(2011)observedthepresenceofalkaloidsofthequininetype

inRemijiaperuvianahighlighttherichnessofthesealkaloidsgroup inthegenusRemijia.Díazetal.(2004)detectedbioactiveCinchona

alkaloidsintheleavesofR.peruvianaindicatingtheimportanceof researchesinvolvingplantorganswhichobtainingtheraw mate-rialismoreprofitableandlessharmfultotheconservationofthe species.

Bathysacuspidate(A.St.Hil.)Hook.f.exK.SchumisaBrazilian floraRubiaceaealsoknownasquinaanditsstembarkhadbitter tasteandthefolkmedicineusethisspecieforthemalariatreatment

(Corrêa,1984;Botsaris,2007).Inthephytochemicalprospection

byTLCinthestembarkwerefoundterpen,flavonoids,tannins, cumarinsandalkaloids(Coelhoetal.,2012;Gontijoetal.,2012). Anatomicaldescriptionandhistochemicaltestsalsoshown resem-blance toR. ferruginea andmaycausemisidentification (Coelho etal.,2012).Howeversomemorphologicalandanatomical charac-teristicsenablesusdifferentiatestructurallythestembarkofboth speciestakeintoaccountthecolornotreddishofinnersurface, theabsenceofcrystalsinthesecondaryphloemandcortexcells, themostelongatedsclereidsinthecortexandonlyoneperiderm (lackofrhytidome)inR.ferruginea.InrelationtoCinchonaspp.stem

bark(GilgandBrandt,1926;Costa,1982;BritishPharmacopoeia,

thefibersformatandthethicknessandcolorofthebarkarethe principaldiagnosticcharacteristics.

Authors’contribution

NSScontributedinanatomyandhistochemicalstudies.MGLBis thecoordinatoroftheresearchandGPChasdonethe chromato-graphic analyses.CWF contributed in collectingplant material, identificationandherbariumconfection.Alltheauthorshaveread thefinalmanuscriptandapprovedthesubmission.

Conflictofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

The authors are grateful to CNPq for financial support

(563311/2010-0,563563-2010-9)andfellowships (150523/2011-4and150453/2012).SpecialthankstoPhDAugustoCésarFranco ofthePhysiologyLaboratoryofBotanicalDepartament/University ofBrasíliaforuseofphotomicroscopyduringthePostdoctoralof firstauthorinthisinstitution.

References

Andrade-Neto,V.F.,Brandão,M.G.L.,Stehmann,J.R.,Oliveira,L.A.,Krettli,A.U.,2003. AntimalarialactivityofCinchona-likeplantsusedtotreatfeverandmalariain Brazil.J.Ethnopharmacol.87,253–256.

Arana,G.V.,Mesía,L.R.,Mesía,W.R.,Reina,M.,2011.Estudioquímicodelacorteza deRemijiaperuviana“Cascarilla”.Cienc.Amaz.1,41–49.

Botion,L.M.,Ferreira,A.V.M.,Côrtes,S.F.C.,Lemos,V.S.,Braga,F.C.,2005.Effectsof theBrazilianphytopharmaceuticalproductIerobina®_{onlipidmetabolismand}

intestinaltonus.J.Ethnopharmacol.102,137–142.

Botsaris,A.S.,2007.PlantsusedtraditionallytotreatmalariainBrazil:thearchives ofFloraMedicinal.J.Ethnobiol.Ethnomed.3,18–21.

Brandão,M.G.L.,Cosenza,G.P.,Grael,F.F.,NettoJunior,N.L.,Monte-Mór,R.L.M.,2009. TraditionalusesofAmericanplantspeciesfromthe1steditionofBrazilian OfficialPharmacopoeia.Rev.Bras.Farmacogn.19,478–487.

BritishPharmacopoeia,2009.HerMajesty’sStationaryOffice,London.

Coelho,V.P.M.,Leite,J.P.V.,Nunes,L.G.,Ventrella,M.C.,2012.Anatomy, histoche-mistryandphytochemicalprofileofleafandstembarkofBathysacuspidata (Rubiaceae).Austral.J.Bot.60,49–60.

Corrêa,M.P.,1984.DicionáriodeplantasúteisdoBrasiledasexóticascultivadas, vol.6.RiodeJaneiro,DiGiorgio.

Cosenza,G.P.,Somavilla,N.S.,Fagg,C.W.,Brandão,M.G.L.,2013.Bitterplantsused assubstitutiveofCinchonaspp.(quina)inBraziliantraditionalmedicine.J. Ethnopharmacol.149,790–796.

Costa,A.F.,1982.Farmacognosia,vol.3.CalousteGulbenkian,Lisboa.

Delprete,P.G.,Cortés,B.R.,2006.AsynopsisoftheRubiaceaeofthestatesofMato GrossoandMatoGrossodoSul,Brazil,withakeytogenera,andapreliminary specieslist.Rev.Biol.Neotrop.3,13–96.

Díaz,J.G.,Sazatornil,J.G.,Rodríguez,M.L.,Mesía,L.R.,Arana,G.V.,2004.Fivenew alkaloidsfromtheleavesofRemijiaperuviana.J.Nat.Prod.67,1667–1671. Dondorp,A.M.,Nosten,F.,Yi,P.,Das,D.,Phyo,A.P.,Tarning,J.,Lwin,K.M.,Ariey,F.,

Hanpithakpong,W.,Lee,S.J.,Ringwald,P.,Silamut,K.,Imwong,M.,Chotivanich, K.,Lim,P.,Herdman,T.,An,S.S.,Yeung,S.,Singhasivanon,P.,Day,N.P.J., Linde-gardh,N.,Socheat,D.,White,N.J.,2009.ArtemisininresistanceinPlasmodium falciparummalaria.N.Engl.J.Med.361,455–467.

Gardner,R.O.,1975.Vanillin–hydrochloricacidasahistochemicaltestfortannin. Biotech.Histochem.50,315–317.

Gabe,M.,1968.Techniqueshistologiques.Masson&Cie,Paris. Gerlach,D.,1984.BotanischeMikrotechnik.Thieme,Stuttgart.

Gilg,E.,Brandt,W.,1926.Farmacognosia:matériafarmacéuticavegetalyanimal. Labor,Barcelona.

Gontijo, D.C.,Nunes, L.G., Souza,C.J.A., Fietto,L.G.,Leite, J.P.V.,2012. Evalua-tionofthegenotoxicpotentialofethanolicextractsofstembarkandleaves of Bathysa cuspidata (A. St.-Hil.) Hook.Rev. Cienc. Farm. Basica Apl. 33, 355–359.

Hofheinz,W.,Merkli,B.,1984.Quinine andquinineanalogues.In:Peters,W., Richards,W.H.G.(Eds.),AntimalarialDrugII:CurrentAntimalarialandNewDrug Developments.Springer-Verlag,Berlin.

INCT,2014.HerbárioVirtualdaFloraedosFungos,http://inct.splink.org.br[accessed 23.11.14].

Johansen,D.A.,1940.PlantMicrotechnique.Macgraw-HillBookCompany,New York.

Junikka,L.,1994.SurveyofEnglishmacroscopybarkterminology.IAWAJ.15,3–45. Kaur,K.,Jain,M.,Kaur,T.,Jain,R.,2009.Antimalarialsfromnature.Bioorg.Med.

Chem.17,3229–3256.

Kraus,J.E.,Arduim,M.,1997.Manualbásicodemétodosemmorfologiavegetal. EDUR,RiodeJaneiro.

Lindley,J.,1838.FloraMedica;abotanicalaccountofallthemoreimportantplants usedinmedicine,indifferentpartsoftheworld.Longman,Orme,Brown, Green-sandLongmans,London,pp.429.

Paiva,J.G.A.,Fank-de-Carvalho,S.M.,Magalhães,M.P.,Graciano-Ribeiro,D.,2006. Vernizvitralincolor500®_{:umaalternativademeiodemontagem}

economica-menteviável.ActaBot.Bras.20,257–264.

Palhares,R.M.,Drummond,M.,Brasil,B.S.A.F.,Krettli,A.U.,Oliveira,G.C.,Brandão, M.G.L.,2014.Molecularidentificationassociatedtochemicalandbiological activitytoevaluatespeciesofBrazilianquina,usedassubstituteofCinchona spp.J.Ethnopharmacol.155,815–822.

Richter, H.G., Mazzoni-Viveiros,S.C., Alves, E.S.,Luchi, A.E., Costa,C.G., 1996. Padronizac¸ãodecritériosparaadescric¸ãoanatômicadacasca:listade carac-terísticaseglossáriodetermos.IF-Sér.Reg.S.Paulo16,1–25.

Ruiz-Mesia,L.,Ruiz-Mesia,L.W.,Reina,M.,Martínez-Dias,R.,Guadanõ,A., Gonzalez-Coloma,A.,2005.BioactivecinchonaalkaloidsfromRemijiaperuviana.J.Agric. FoodChem.53,1921–1926.

Saint-Hilaire,A.,2014.Plantasusuaisdosbrasileiros.FinoTrac¸oEditoraLtda,Belo Horizonte.

Sass,J.E.,1951.BotanicalMicrotechnique.TheIowaStateCollegePress,Ames. Silva,R.A.D.,1926.FarmacopéiadosEstadosUnidosdoBrasil,1a_{.ed.IndústriaGráfica}

Siqueira,São Paulo,http://www.anvisa.gov.br/hotsite/farmacopeiabrasileira/ publicacoes/1edicao.pdf.

Somavilla,N.S.,Cosenza,G.P.,Fagg,C.W.,Brandão,M.G.L.,2013.Morpho-anatomy andchemicalprofileofnativespeciesusedassubstitutesofquina(Cinchona spp.)inBraziliantraditionalmedicine.PartI:Polyourateahexasperma.Rev.Bras. Farmacogn.23,592–599.