w ww . e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Microscopic
and
UV/Vis
spectrophotometric
characterization
of
Cissampelos
pareira
of
Brazil
and
Africa
Niara
Moura
Porto
a,
Yuri
Lima
de
Barros
b,
Ionaldo
J.L.
Diniz
Basílio
b,
Maria
de
Fátima
Agra
a,∗aLaboratóriodeTaxonomiaeFarmacobotânica,ProgramadePós-graduac¸ãoemProdutosNaturaiseSintéticosBioativos,UniversidadeFederaldaParaíba,JoãoPessoa,PB,Brazil
bDepartamentodeCiênciasFarmacêuticas,CentrodeCiênciasdaSaúde,UniversidadeFederaldaParaíba,JoãoPessoa,PB,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received18May2015 Accepted20October2015 Availableonline31December2015
Keywords: Ethnomedicine Leafanatomy Medicinalplant Menispermaceae Morphoanatomy Pharmacobotany
a
b
s
t
r
a
c
t
CissampelospareiraL.,belongingtoMenispermaceaefamily,hasworldwidedistribution,occurringin tropicalandsubtropicalregionsoftheAmericas,AfricaandAsia.Itisthemostpopularspeciesof Cissam-pelos,knownforitsmedicinalusesofleavesandroots.Thestudyaimstofinddistinctiveleafanatomical characters,andalsodemonstratetheimportanceofspectraldatatoidentifyC.pareirasamples,inorderto contributetoitstaxonomyandqualitycontrolofitsdrugs.Anatomicalleafanalyseswereperformedby opticalandscanningelectronmicroscopy.Thespectralprofilewasobtainedfrommethanolicextractsof C.pareirasamplesfromBrazilandAfrica,withapplicationofUV–visspectrophotometrydata,whichwere analyzedbyprincipalcomponentanalysis(PCA).Someanatomicalcharacterssuchasleafepidermalcells walls,stomata,trichomes,mesophyll,featuresofmidribandpetiole,andthespectralprofilewithinthe wavelengthrangingbetween770and240nm(eightbands)differsbetweenBrazilianandAfrican sam-ples.TheresultsrepresentanadditionalsupporttothetaxonomyofC.pareira,andthequalitycontrolof theirleafdrugs,mainlyinrelationtomisidentifiedsamples.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Cissampelos pareira L., belonging toMenispermaceae family, hasworldwidedistribution,occurringintropicalandsubtropical regionsoftheAmericas,Africaand Asia(Ortiz, 2001).In Brazil, itisencounteredindifferenttypesofvegetation,fromCaatinga, Atlantic Forest and Amazon forest (Braga, 2015).According to
Schmelzerand Gurib-Fakim(2008),inAfricathisspeciesoccurs
insubtropicalforest,savannah,deciduousshrubs,oftenpersisting inclearedlandandplantations,alsoinsecondaryvegetationand nearrockoutcrops.
ItisthemostpopularspeciesofCissampelosnotonlyforitswide distribution,but mainlybecauseitsleavesandrootsarewidely usedasmedicinal.AccordingtoNapralert(2013),C.pareirahas morethaneightyfolknames.InBrazil,itisknownas“parreira”, “abuta”,and“parreira-brava”(LewisandElvin-Lewis,1977;Rury, 1983);inAfrica,it iscalledinfolkmedicineas“chegonde”and “karigi-munana”(Hedbergetal.,1983;Rukungaetal.,2009);andin India,itisknownas“ambastha”,“patha”and“laghupatha”(Vaidya,
1988).
∗ Correspondingauthor.
E-mail:[email protected](M.deFátimaAgra).
Inmanyethnobotanicalreports,theleavesofC.pareiraare rec-ognizedasanaturalmedicineforvariouspurposes.Theleafjuice isusedasantiseptic,anthelmintic,insecticidalandparasiticidal, andagainstdermatitis(SinghandAli,1992),asthmas(Singhand
Maheshwari,1994),genitourinarydisorders(SanchezMedinaetal.,
2001),diarrhea,dysenteriesandgastrointestinaldisorders(Kumar
etal.,2006;Kambleetal.,2008),antifertility(Gangulyetal.,2007;
Priyaetal.,2012),andantidiabetic(Yadavetal.,2013).Thetopical
useofleavesisindicatedtotreathemorrhagesfromcuts,burnsand wounds(RamasubramaniarajaandBabu,2010;Shuklaetal.,2012), andalsototreatabscesses(Abbasietal.,2010;Haqueetal.,2011).In addition,inIndia,theleavesarealsousedascattlefeedtoincrease milk production, and also in somefood systems asthickeners, gellingagents, texturemodifiersandstabilizers (Vardhanabhuti
andIkeda,2006;Priyaetal.,2012),interalia.
The leaves of C. pareira have been reported to be a rich sourceofisoquinolineandbisbenzylisoquinolinealkaloids(Shukla et al., 2012), such asberberine (Kupchan et al., 1960a), curine
(Chowdhury,1972), hayatine (Sharma, 1987)and magnoflorine
(Ahmadetal.,1992).Inaddition,havealsobeenisolated
essen-tialoil(Kupchanetal.,1960b),flavonoids(Ramirezetal.,2003;
Amreshetal.,2007a),polysaccharides(VardhanabhutiandIkeda,
2006),andpectin(Singthongetal.,2004;Arkarapanthuetal.,2005) havealsobeenisolated.
http://dx.doi.org/10.1016/j.bjp.2015.10.006
Biologicaland pharmacologicalactivitiesofleavesand aerial parts (leaves and branches) of C. pareira were demonstrated in severalstudies. The cissampeloflavone, isolated from leaves, showedactivityagainstTrypanosomacruziandT.bruceirhodesiense
(Ramirezetal.,2003).Theplantextractexhibitedantifungal
activ-ityagainstAspergillusnigerandSaccharomycescerevisiae(Kumar etal.,2006).Theethanolextractoftheaerialpartsshowed anti-inflammatoryandanalgesicactivities(Amreshetal.,2007b).The contraceptiveandcytotoxiceffects weredemonstrated byPriya
et al. (2012) and Ganguly et al. (2007), respectively. The
anti-diabeticactivitywasconfirmedbyJannuetal.(2011)andYadav
etal.(2013).Inaddition,apreliminarystudycarriedoutbyThakur
andRana(2013)confirmedtheanxiolyticeffectofC.pareiraleaves.
AccordingtoRhodes(1975)andHootetal.(2009),C.pareira
hasproblemsinitsinterspecificdelimitationwithimpreciselimits, mainlycausedbyitswidedistributionandgreatplasticityoftheir vegetativeforms.Ontheotherhand,theleafanatomicalstudies haveshowntobeanadditionallsupporttotheplanttaxonomy,as alreadydoneinSolanum(Nurit-Silvaetal.,2007;Nurit-Silvaand
Agra,2011;Sampaioetal.,2014),andalsototheMenispermaceae
family,includingCissampelosbyDeWetetal.(2002),Portoetal.
(2008,2011,2012),forexample.
Thespectroscopicchemicaltechniqueshaveemergedand con-tributedasanadditionaltooltocontributetoplanttaxonomy,and alsoasasupporttothequalitycontrolofherbaldrugs,allowing informationtobeobtainedwithouttheneedforprevious isola-tionofchemicalconstituents,asdemonstratedbeforeforBaccharis
(Lonnietal.,2005)andSolanum(Basílioetal.,2012).
AlthoughtheleavesofC.pareiraarecommonlyusedin tradi-tionalmedicine,andthereisevidenceofmanyactivitiesoftheir compounds,aliterature surveyshoweda lackof studiesofthe
leaf comparativeanatomy, as wellas spectroscopic analysis of UV–visibleoftheleafextracts.Inthisway,thisstudyaimedtofind leafanatomicalcharacters,distinctivetoC.pareira,onsamplesof plantsfromBrazilandAfrica,revealingtheimportanceof anatom-icalstudiescombinedwithspectraldata,wouldbeusefultothe qualitycontrolofitsdrugs,aswellastothetaxonomyofC.pareira.
Materialsandmethods
Plantmaterial
Botanicalexpeditionsandfieldobservationswerecarriedout byN.M.Porto,inareasofAtlanticForestandRainForest,for sam-plecollectionofMenispermaceae,includingleavesofCissampelos pareiraL.inthefollowingBrazilianStates:Alagoas,ParáMaranhão, Paraíba,PernambucoandSergipe(Table1).Foreachindividual,an averageofthreeleafsamplesweretakenfromthesecondtothe fifthnodesoftheleafbladesandtheproximal,mediananddistal portions,andpetiolewerefixedinFAA(50%)for24h(Johansen, 1940),and preservedin ethanol 70GL.Theotherpartof fertile materialwaspressedanddriedforherbaria,accordingtoBridson
andForman(1999).Thevoucherspecimensweredepositedatthe
HerbariumProf.LauroPiresXavier(JPB),oftheUniversidade Fed-eraldaParaíba.
In addition, leaf samples from herbariumspecimens identi-fiedasC.pareirawerealsoanalyzedfromthefollowingherbaria, acronyms by Thiers (2015): Herbarium of Centro de Pesquisas do Cacau (CEPEC), Herbarium Prof. Jayme Coelho de Morais (EAN),HerbariumofEmbrapaAmazôniaOriental(IAN),Herbário Prof. Lauro Pires Xavier(JPB), Herbário MuseuParaense Emílio Goeldi(MG),HerbariumJardimBotânicodoRiodeJaneiro(RB),
Table1
SelectedvoucherspecimensofCissampelospareiraandspeciesofoutgroup.
Species Specimencode Country,StateandMunicipality Voucherspecimen Herbarium
Anomospermumchloranthum AC Brazil,Pará,Santarém MSilva2619 MG
Anomospermumsteyermarkii AS Brazil,Roraima,Uaicá GTPrances/n MG
Cissampelosandromorpha
CA1 Brazil,Paraíba,Conde NMPorto30 RB
CA2 Brazil,Pará,Belém NMPorto45 JPB
CA3 Brazil,Pernambuco,Catende NMPorto07 JPB
CA4 Brazil,EspíritoSanto,SantaTeresa WPizziolo329 RB
Cissampelospareira
CP1 Brazil,Rondônia,PortoVelho JASilva39 IAN
CP2 Brazil,Pará,MonteAlegre RLFróes30443 IAN
CP3 Brazil,DistritoFederal,Brasília HSIrwins/n IAN
CP4 Brazil,SantaCatarina,Ipumirim ALGasper2020 RB
CP5 Brazil,Goiás,Pirenópolis HSIrwins/n RB
CP6 Brazil,MatoGrossodoSul,Corumbá AC.Cervi3276 RB
CP7 Africa,Ethiopia,Ghion JWAsh655 MO
CP8 Africa,Tanzania,Tangadistrict HFaulkner5631 MO
CP9 Africa,Uganda,Kyadondo PKRwaburindore205 MO
CPa Brazil,Bahia,Filadélfia AMGiulietti1886 CEPEC
CPb Brazil,MatoGrossodoSul,Corumbá APott3158 RB
CPc Brazil,Bahia,Coribe MMLopes1374 CEPEC
CPd Brazil,Paraíba,Maturéia MFAgra5061 JPB
CPe Brazil,SantaCatarina,CapãoAlto MVerdi1156 RB
Cissampelossympodialis
CS1 Brazil,Paraíba,JoãoPessoa MFAgra7133 JPB
CS2 Brazil,Ceará,Fortaleza Celismars/n JPB
CS3 Brazil,Bahia,Juazeiro Zehntren211 RB
Cissampelostropaeolifolia
CT1 Brazil,Sergipe,Capela NMPorto19 JPB
CT2 Brazil,Alagoas,Coruripe NMPorto47 JPB
CT3 Brazil,Maranhão,Ribeirãozinho NMPorto48 JPB
CT4 Brazil,Pará,Conceic¸ãodoAraguaia TPlowman8755 IAN
Hyperbaenadomingensis HD Brazil,Pernambuco,Sirinhaém MOliveira1553 UFP
Orthomenehirsuta OH Brazil,Amazonas,SãoGabriel GABlack48-2473 IAN
Orthomeneschomburgkii OS Brazil,Pernambuco,Igarassu BSAmorim1668 UFP
HerbariumProf.GeraldoMariz(UFP)oftheUniversidadeFederal
dePernambuco(UFPE),andHerbariumofMissouriBotanical
Gar-den(MO).Alistofvoucherspecimensusedinthisstudyisgivenin
Table1.
Anatomicalandhistochemicalanalysis
Theplantmaterialwasdividedintwoportions,onefor anal-ysisby optical microscopy and theother by scanning electron microscopy(SEM).Transversesectionswereperformedonleaves ofC.pareirabyfreehandusingcommercialrazorblades. Subse-quently,thesectionswereclearedbysodiumhypochlorite(20%) until complete clarification,neutralized with acetic acid(0.2%), washedindistilledwater,andstainedwithasolutionofAstrablue and Safranin, modifiedby Bukatsch(1972). Leafepidermiswas separatedfromthemesophyllbydissociationinJeffreysolution
(Johansen,1940),andthenstainedwithSafraninwith1%solution
in50%alcohol,accordingtoFranklin(1945).
Foralkaloid detection,transversesectionsweretreated with Dittman’sandWagnerreagents(FurrandMahlberg,1981).Allleaf sectionsweremountedinglycerinatedgelatin(50%).The obser-vationsandmicrophotographswereperformedbya photomicro-scope(LeicaDM750),withimageprocessingsoftware(Qwin Sys-tem)coupledtoavideocamera(LeicaICC50HD)forimagecapture.
Scanningelectronmicroscopy(SEM)
Scanningelectronmicroscopy(SEM)ofleafepidermiswas per-formedindrymaterialtooptimizetheobservationofwaxesand epidermal appendages.Leaffragments of1cm2 werefixedin a
solutionof4%glutaraldehydein0.1Mpotassiumphosphatebuffer (pH7.0)for24h,at48◦C,thenwashedin0.1Msodium
cacody-latebuffer(pH7.0),followedbypostfixationin1%OsO4,in0.1M
Na-cacodylatebuffer(pH7.0)for1h,atroomtemperature. Subse-quently,thefragmentsweredehydratedinacrescentethylicseries,
and dried atthecritical point, placed onaluminum stubs with double-sidedtape,air-driedand,finally,coatedwithgold.Finally, photomicrographsand microscopicanalysiswereperformedby scanningelectronmicroscopy(JEOLJSM-5600),onleafepidermis, atanacceleratingvoltageof15KV.Micromorphological character-izationwascomplementedbytheanalysisofepicuticularwaxesof theleafepidermisthatwereclassifiedhereaccordingtoBarthlott
etal.(1998).
Chemicalanalysis
Samplesofdriedleavesof C.pareira(fromBrazilandAfrica) andofninespeciesusedasanoutgroupofMenispermaceae fam-ilywereinvestigated:CissampelosandromorphaDC.,Cissampelos sympodialisEichl., Cissampelostropaeolifolia DC.,Anomospermum chloranthumDiels,AnomospermumsteyermarkiiKrukoff&Barneby,
Hyperbaenadomingensis(DC.)Benth.,Orthomenehirsuta(Krukoff &Moldenke)Barneby&Krukoff,Orthomeneschomburgkii(Miers) Barneby&Krukoff,SciadoteniabrachypodaDiels.AllUV spectro-photometricanalysiswereperformedusingamodifiedversionofa previouslypublishedmethod(Basílioetal.,2012).Briefly,methanol extractsofdriedleaveswerepreparedbyultrasoundextractionfor 20minatroomtemperature,andthenfilteredthroughmembranes withaporesizeof0.45mm,modifiedfromBasílioetal.(2012).
TheUV/Visspectrawererecordedwithaspectrophotometer (UV-1650PC,Shimadzu,Kyoto,Japan).Forallabsorbance measure-mentsQuartzcells(1cm)wereused.Thespectrawererecorded intriplicatefrom770to200nm.Thestandardizedprocedurewas repeatedforallspeciesandthedatawasautomaticallyreducedto anASCIIfile.
The spectra were normalized by setting the absorbance at 770nmequaltozeroandsubsequentlymeancentered.Thedata matrixwasprocessedbyFITOPACv.2.1.2software.Finally,a prin-cipalcomponentanalysis(PCA)wasperformedusingthemethods
variance-covariancebetweenthegroupswithPastsoftware,
ver-sion2.15(Hammeretal.,2001).
Resultsanddiscussion
Anatomicstudy
TheleafepidermisofC.pareira,infrontview,presentedcells withcurvetowavedanticlinalwallsontheglabrescentadaxial surface(Fig.1AandC),andwavedandhairyontheabaxial
sur-face(Table2,Fig.1BandD).Intransversesection,theepidermisof
C.pareirawasuniseriatewithtabularcells,andathinandsmooth cuticle(Fig.3A).MetcalfeandChalk(1950),Portoetal.(2011)and
Sudhakaran(2012)havereportedthispatternofcellwallstoC.
pareira.ItwasalsorecordedinotherspeciesofCissampelos(Hong
etal.,2001;DeWetetal.,2002;Portoetal.,2011),andothergenera
ofMenispermaceaeasCocculusandStephania(MetcalfeandChalk, 1950).OnlytwosamplesfromBrazil(CP6andCPb),andtwofrom Africa(CP8andCP9)showedthickenedcuticle(Figs.4Eand5C,E) thatdifferedfromtheC.pareirapattern(Table2).ToWilkinson
(1979), in generalthe presence and thickness of the cuticle is
determinedbyenvironmentalfactoranddoesnothavetaxonomic importance.
EightspecimensidentifiedasC.pareiradisplayedtheleaf epi-dermispatterndifferentfromthoseknownforthisspecies.Two samplesfromBrazil(CP6andCPb)showedstraightanticlinalwalls onthe adaxial, and curve onthe abaxial (Table 2, Fig. 4C and D).AccordingtoStace(1965),thefeaturesofanticlinalwallsare amesomorphiccharacter,andenvironmentalconditionssuchas humidityplayasignificantroleindeterminingthepatternof anti-clinalcellwalls.These featuresalsohavebeenreportedtovary inresponsetochangesinlightregimesbyRôc¸asetal.(2001)and
Mantuanoetal.(2006).
Four samples from Brazil, CP3, CP5, CPa and CPc (Table. 2,
Fig.4G), and three fromAfrica (CP7, CP8and CP9) showed an epidermalpapillae(Table.2,Fig.5A).Thepresence,numberand dis-tributionofpapillaeconstituteanimportantfeaturethathasbeen usedtodefineboundariesatspecificandgenericlevelinmany tax-onomicgroups(Hongetal.,2001).AccordingtoBoneetal.(1985), thecellwallsoftheleafepidermiswithconvexcurvaturewouldbe advantageoustoincreasetheenergycaptureefficiency,whichis importantforplantsthatmustsurviveatextremelylowlight lev-els.Furthermore,papillaeintheleafepidermisminimizethearea ofcontactcausingaverylowadhesionofwateronleaf(Ensikat
etal.,2011).
Epicuticularwaxesastubulesandclustersoftubuleswere con-firmedinSEM,in bothsurfacesofC.pareira(Fig.2CandD), as previouslyreferredbyPortoetal.(2011).Theepicuticularwaxes alsohavetaxonomicvalueinthecharacterizationoftheleaf epider-mis,accordingtoBarthlottetal.(1998).Moreover,thepresenceof waxtubulesinleafepidermiscouldberelatedtothehigherwater repellency(Ensikatetal.,2011).
Theleaveswerehypostomaticwithanomocyticandanisocytic stomata occurring simultaneously, both at the epidermal level (Fig.3A),however,theanomocytictypewaspredominant(Fig.1D), which corroborate with thepattern previously described to C. pareirabyDeWetetal.(2002)andPortoetal.(2011).Simple bi-celledtomany-celledtrichomeswerepresentonbothsurfacesof theleafepidermis(Figs.1A,Band2A,B),whicharecommonon theleafepidermisofsomeMenispermaceaespecies,accordingto
Wilkinson(1978).
Dorsiventralleaveswithadaxialmesophyllandasinglelayer ofpalisadewasthepatternforC.pareira(Table2,Fig.3A). How-ever,bisseriatepalisadewasobservedinfivesamples,beingtwo
fromBrazil(CP6andCPb),andthreefromAfrica(CP7,CP8,CP9) Table
Fig.2.Cissampelospareira(RLFróes30443),leafepidermisinfrontviewbySEM:(A)adaxialsurfacewithlongfingerhairs;(B)detailofthewrinkledepidermisandtrichomes ontheabaxialsurface;(C)detailoftheabaxialepidermiswithepicuticularwaxes,astubule;(D)stomaindetailandepicuticularwaxesontheabaxialsurface(Legends:epw, epicuticularwax;st,stomata;tri,trichome).
(Table 2, Fig. 5C) According to Esau (1972), Levitt (1980) and
Rozemaetal.(1997),palisadeandspongyparenchymaaretissues
knowntorevealresponsesrelatedtolightandsoilwater varia-tions.Allsamplesshowed3–5-layeredspongyparenchymawith largeintercellularspaces(Fig.3A),andseveralcollateralvascular bundlesdistributedthroughoutthemesophyll.
Theleafmarginwasslightlycurvedtowardtheabaxialsurface withamany-layeredcollenchymaandasinglevascularbundle.The dorsiventralorganizationoftheleafmesophyllischaracteristicto
Cissampelos,accordingtoMetcalfeandChalk(1979)andDeWet
etal.(2002),anddoesnotconstituteanexclusivecharacterofC.
pareira.
Themidribwasbiconvex,intransversesection,more promi-nent and rounded to theabaxial surface witha sub-epidermal 3–5-layered lacunarcollenchyma,followed by thefundamental parenchyma.Thevascularsystemhasasinglevascularbundlewith asclerenchymatousringatthemiddleportion,and1–2smaller vas-cularbundlesatthebaseandapex(Table2,Fig.3B).Fivesamples, threefromAfrica(CP7,CP8andCP9)andtwofromBrazil(CP6and CPb)showedmidribwithplane-convexshape,differingfromthe morecommonbiconvexpatterncharacteristicofC.pareira(Table2,
Figs.4Dand5D).Inourstudies,themorphologyofthemidribin
CissampelosandothergeneraofMenispermaceae(inprep.), consti-tutesanimportantfeaturetoseparatetaxaatspecificlevel,which isalsocorroboratedbystudiesofDeWetetal.(2002).
Intransversesection,thepetioleofC.pareirashowedcircular contourwithuniseriateepidermisandsimplelongtrichomes;the cortexundertheepidermishascontinuouscollenchymasimilarto themidrib;thevascularsystemisformedby6–8collateralvascular bundles(Fig.3C–E),whicharesurroundedbyasclerenchymatic ringatthemiddleportion(Table2,Fig.3D).Sixsamples(CP3,CP5, CP6,CPa,CPbandCPc)showed9–12collateralvascularbundles
(Table2,Fig.4E),thusdifferingfromthepatternobservedforC.
pareiraandthereforesuggestingthesesamplesbelongtodifferent taxa.
According to Sinnot (1914), Metcalfe and Chalk (1950) and
Howard(1979),thecharacteristicsoftheangiospermshavegreat
taxonomicsignificancebecausetheenvironmentdoesnot influ-ence it. Moreover, the petiole vascularization is diagnostic to distinguishsomegeneraandspeciesandalsohastaxonomic impor-tance (Wilkinson, 1978, 1986, 1989). On the other hand, the sclerenchymaticsheathwasalsoreferredtoC.sympodialisbyPorto
etal.(2008),andtootherSouthAfricanCissampelosspeciesbyDe
Wetetal.(2002).Itiscommoninvegetativeorgansof
Menisper-maceae,especiallyinlianas(Carlquist,1996).
Idioblasts wereobserved in thevascular tissue ofthe corti-calparenchymaofthemidribandpetioleandshowedapositive reactiontoalkaloidsinallsamplesofC.pareira(Table2,Fig.3F). Differently, thesamplesCP3, CP5,CP6,CP8, CP9,CPa, CPband CP5 showed no positive reaction to alkaloids (Fig. 5G and H). Theseresultscorroborate withexistingchemical studiesof Cis-sampelos(Semwaletal.,2014),aswellasconfirmthelocalization of the production and/or accumulation of these compounds in
theleaf(Chowdhury,1972;Cavalcantietal.,2014).Accordingto
Menachery(1996)andBarbosa-Filhoetal.(1997),theoccurrence
ofalkaloidscanbeachemotaxonomiccharacterof Menisperma-ceae.
Secretorycavitieswereobservedattheleafapexoftwosamples fromAfrica(CP8andCP9),andalsointhemidribandpetioleofeight samples(Table2,Figs.4Hand5E,F),threefromAfrica(CP7,CP8and CP9),andsixfromBrazil(CP3,CP5,CP6,CPa,CPbandCPc).Although thischaracterhasbeenreportedforsomespeciesofCissampelos
andothergeneraofMenispermaceae(MetcalfeandChalk,1950,
1979;Wilkinson,1989;DeWetetal.,2002),itwasnever
Fig.3. Cissampelospareira(ALGasper2020)intransversesections:(A)dorsiventralmesophyll;(B)midribwithcollateralvascularbundle;(C)vascularbundlesofpetiolein thebasalportion;(D)vascularbundlesofpetioleinthemedianportionwithasclerenchymatousring;(E)detailofcollateralvascularbundle;(F)stainedidioblastsshowing positivereactionforalkaloids(Legends:col,collenchyma;ef,exchangefascicular;ep,epidermis;fc,fascicularcambium;fp,fundamentalparenchyma;id,idioblast;per, pericycle;ph,phloem;pp,palisadeparenchyma;pt,pith;rsc,sclerenchymatousring;scl,sclerenchyma;sp,spongyparenchyma;st,stomata;tri,trichome;vb,vascular bundle;xyl,xylem).
toFahn(1988),secretorycavitiesaredistinctiveforangiosperms
andhaveimportanttaxonomicvalue.
Theresultsoftheleafanatomyofthirteensamplesanalyzed identifiedasC.pareira,revealedthatonlyfiveofthesesamples(CP1, CP2,CP4,CPdandCPe),allfromBrazil,showedasetofcharacters thatmatchthepatterndescribedforC.pareirabyPortoetal.(2011)
andSudhakaran(2012),whicharesummarizedhere:hypostomatic
leafblades;anomocyticandanisocyticstomatawithpredominance ofthefirsttypeattheepidermislevel;epidermalanticlinalwalls curvesontheadaxialsurface,and wavyontheabaxialsurface; epidermiswiththincuticleandsimpleuniseriatetrichomes; dor-siventralmesophyllwithuniseriatepalisadeparenchyma,withfew andrarestyloidcristals(Table2,Fig.4H);biconvexmidrib; collat-eralvascularsystem;petiolewith6–7vascularbundles;idioblasts positiveforalkaloids;andnosecretorycavities.
Ninesamples(CP3,CP5,CP6,CP7,CP8,CP9,CPa,CPbandCPc) weremistakenlyidentifiedasC.pareira,astheyhaveasetof char-actersdifferentofthepatternofC.pareira.Thesesetofdifferent
charactersdisplayedbythesesamplesallowtoseparatetheminto threegroups,whichcouldbelongtodistinctandunidentifiedtaxa
(Table2):Cissampelossp1,CP3,CP5,CPaandCPc(Fig.4AandB);
Cissampelossp2(Fig.4C–H),CP6andCPb;andCissampelossp3,CP7, CP8andCP9(Fig.5A–H).
Cissampelos sp1and Cissampelos sp3have secretory cavities, biconvexmidrib,and epidermalpapillae ontheabaxialsurface. However,theypresentdistinctivecharacters:Cissampelossp1has uniseriatepalisadeparenchyma,and thepetiolewith9–12 vas-cular bundles (Table 2); while in Cissampelos sp3 the palisade parenchymaisbiseriateandthepetiolehas6–8vascularbundles.
On the otherhand, samples of Cissampelos sp2 differsfrom
Fig.4.(A,B)Cissampelossp1(HSIrwins/n–IAN),leafepidermisinfrontview:(A)Leafepidermiswithstraightanticlinalcellswallsontheadaxialsurface;(B)Leafepidermis withcurveanticlinalcellswallsontheabaxialsurface;(C–H)Cissampelossp2(ACCervi3276),transversesections:(C)leafmarginsomewatrounded;(D)Planeconvex midribwithcollateralvascularbundle;(E)Petioleof12collateralvascularbundlesinthebasalportion;(F)detailofparenchymawithstyloidscristals,(G)detailofepidermal papillaeontheabaxialsurface;(H)detailofmesophyllwithsecretorycavities;(Legends:cr,crystals;ep,epidermis;pap,papillae;pp,palisadeparenchyma;pt,pith;sc, secretorycavity;scl,sclerenchyma;sct,scaroftrichome;sp,spongyparenchyma;st,stomata;tri,trichome;vb,vascularbundle).
Cissampelossp2issimilartothatshowedbyCissampelossp1(9–12), butdifferentfromCissampelossp3,with6–8vascularbundles.
Theevidencesfoundinthisworksuggestthatninesampleswere mistakenlyidentifiedasC.pareira,andprobablybelongtothree
Fig.5. Cissampelossp3,transversesectionsofleafblades.(A)(JWAsh655):detailofepidermalpapillae;(B–D)(PKRwaburindore205):(B)detailofastomaontheabaxial surface;(C)dorsiventralmesophyllwithbisseriatepalisade;(D)plane-convexmidribwithcollateralvascularbundle;(E)(HFaulkner5631):detailofmesophyllwithsecretory cavities;(F,H)(PKRwaburindore205):(F)Palisadewithsecretorycavities;(G,H)stainedsecretorycavitiesshowingpositivereactionforalkaloids(Legends:ct,cuticle;ep, epidermis;pap:papillae;pp,palisadeparenchyma;sc,secretorycavity;sp,spongyparenchyma;st,stomata;tri,trichome;vb,vascularbundle).
Spectroscopyanalysisandprincipalcomponentanalysis(PCA)
Thespectralprofileshowedsaturatedabsorbanceinthe charac-teristicregionofconjugatedenones(240–190nm)whiletheregion above550nmshowednorelevantabsorbance,onlybandsof pos-sibleinterferents,relatedtochlorophylland/orothersubstances
(Shipmanetal.,1976).Theseresultsledustoselectthespectral
rangefrom550to250nm(600variables)forchemometricstudies (Fig.6).
Thecharacterization ofspectrawascarried outby the anal-ysisofmethanolextractsofspecimens.Fourdistinctabsorption bandswereobserved(Table3):BandI(max500–504.5nm);Band
II(max391.5–468nm);BandIII(max312.5–368.5nm);BandIV
(max223–291nm).
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
250 300 350 400
Wavelength (nm)
Arbitrary units
450 500 550
CA4
CP1 CP3 CP5 CP7 CP9 CA2
CA3 HD AE CA1 AC SB CP8 OH
OS CP6 CT3 CT2 CP4 CT1 CS3 CP2 CS2 CS1
Fig.6. Cissampelosspecies:UV–visspectraofinthespectralregionselectedforchemometricanalysis(550–250nm).ForthenumericalcodeofthespeciesseeTable1.
significantlydifferentabsorptionvaluesatmax 466.5nm,when
comparedwithotherspecies.
The intraspecific analysis of samples of C. pareira revealed shiftsinthewavelengthofmaximumabsorptioninthebandIV between Brazilian (max at 261.5–272.0nm) and African (max
at 282.0–284.0nm) specimens, except for the sample CP9 that showednoband.ThespectralanalysisofC.pareirasamplesfrom Brazilrevealedabsorptionmaximaat261.5and272nm,whichare characteristicoftheC Nchromophore(Nagarajan etal.,2011).
Basedonthedataavailable,suchabsorbancecanberelatedtothe presenceoftropoisoquinolinealkaloids,whichwerealready iso-latedfromC.pareira(Moritaetal.,1993),andisalsocommonin otherspeciesofCissampelos(Menachery,1996).
Thespectraldataoftheoutgroupsamples,A.chloranthum,A. steyermarkii,H.domingensis,O.hirsuta,O.schomburgkii,S. brachy-podaweredifferent,asexpected,fromC.pareiraandotherspecies ofthegenus,especiallyC.andromorpha,C.sympodialisandC. tropae-olifoliainthePC1factor(Fig.7A).
Table3
Maximumabsorptionvalues(max)forCissampelosspeciesandout-group.
Species(Country) Collector(Herbarium) Code max(nm)a
BandIV BandIII BandII BandI
Anomospermumchloranthum MSilva2619(MG) AC 271.5 319.5 – 501.5
Anomospermumsteyermarkii GTPrances/n(MG42173) AS – – 405.5 501.5
Cissampelosandromorpha
NMPorto30(JPB) CA1 270.5and279.5 323and366.5 409.5 –
NMPorto45(JPB) CA2 269 – 409 503
NMPorto07(JPB) CA3 270 320.5 406.5 –
WPizziolo329(RB) CA4 – – 408 504.5
Cissampelospareira(Brazil)
JASilva39(IAN) CP1 267.5 – 407 504.5
RLFróes30443(IAN) CP2 270 334 407.5 504.5
HSIrwins/n(IAN129416) CP3 269.5 320.5 405.5 504.5
ALGasper2020(RB) CP4 272 312.5 408 504.5
HSIrwins/n(RB144809) CP5 261.5 – 407.5 504.5
APott3158(RB) CP6 271.5 319.5 404.5 503.5
Cissampelospareira(Africa)
JWAsh655(MO) CP7 283 336.5 – –
HFaulkner5631(MO) CP8 284 – 407 –
PKRwaburindore205(MO) CP9 – 335.5 – –
Cissampelossympodialis
Agra7133(JPB) CS1 270 318.5and361.5 407 503.5
Celismars/n(JPB) CS2 270 326 403.5 503.5
Zehntren211(RB) CS3 269.5 317.5 406 504
Cissampelostropaeolifolia
NMPorto19(JPB) CT1 257 326 410and468 –
NMPorto47(JPB) CT2 – 320.5and368.5 409.5and466.5 –
TPlowman8755(IAN) CT4 278and282.5 – – –
Hyperbaenadomingensis MOliveira1553(UFP) HD 273 318 – 504.5
Orthomenehirsuta GABlack48-2473(IAN) OH – – 406.5 –
Orthomeneschomburgkii BSAmorim1668(UFP) OS 276 318.5 – 503
Sciadoteniabrachypoda NTSilva4146(IAN) SB 269.5 328 403 503
40
32
24
–24 –24
–36 –48
16
16 –16 8
8
4
–4
–8
–12
–16
–20
Component 1
Component 3
–8
–12 12
12
12 –12
–24 –36
B
–48 24 36 48 60
24
Component 1
A
Component 2 36 48 60
C. pareira (Brazil)
Anomospermum chloranthum
Anomospermum steyermarki Orthomene hirsuta e Orthomene schomburgkii C. pareira (Africa)
C. andromorpha
C. sympodialis
C. tropaeolifolia
Sciadotenia brachypoda
Hyperbaena domingensis
Fig.7.Cissampelosspecies(A)PC1×PC2;(B)PC1×PC3:PCAspectralprofile(770–250nm).
Theprincipalcomponentanalysisofspectraldataallowedthe differentiationof samples of C.pareira of Africanand Brazilian originmainlybasedontheirscorevaluesatPC3.Thespectraof BraziliansamplesCP4andCP6showedproximitywiththesamples oftheAfricangroup,CP7,CP8andCP9(Fig.8).ThesamplesofC. pareiraspecimenswereclosetoC.tropaeolifoliaandC.sympodialis
withpositivescorevaluesinthePC3(Fig.7B).Itisworthnotinga groupofAfrican(CP7,CP8andCP9)andBraziliansamples(CP4and CP6)that,althoughsimilar,hasspreadthroughoutthedispersion diagram(Fig.8).Basedontheresults,ingeneral,thePCAanalysis wasabletodelimitthespeciesofCissampelosanalyzed.
The results showed that application of UV/Vis spectropho-tometryisavalidtechniqueforidentifyingCissampelossamples, confirmingtheobservationsalreadymadeforotherangiosperms groupssuchasAsteraceaebyLonnietal.(2005)andSolanumby
Basílioetal.(2012).
Thechemicaldatacorroborateswiththeanatomical features ofC.pareirasamples,whichdifferentiatesthemfromsamplesof otherspecieswiththefollowingcharacters:biseriateparenchyma, stomataabovetheleveloftheepidermis,presenceofepidermal papillaeaswellassecretorycavitiesinthemesophyll.
Theprincipalcomponentsanalysis(PCA)providedimportant spectral information, which allowed the separation of African-tropicalandNeotropicalsamples.Thesedifferencesmayberelated togeographicaldistribution,consideredbyGriffinandLin(2000)
2.4
–2.4
–3.0
Component 1
Component 2
1.8
–1.8 1.2
–1.2 0.6
+ +
–0.6
–12 12
–24 24
–36 36 48 60
C. pareira (Brazil)
C. pareira (Africa)
Fig.8.CissampelospareiraandrelativesfromBrazilandAfrica(PC1×PC2):PCA spectralprofile(770–250nm).
Conclusions
Charactersofepidermalcells,mesophyll(palisadeparenchyma layers),midribshape,numberofvascularbundlesinpetiole, secre-torycavities, and thechemical resultsproved tobeuseful and distinctivetoseparateC.pareirafromrelatedspecies,providingan additionaltoolforitstaxonomy,andqualitycontroloftheirdrugs composedbyleaves.Furtherchemicalandtaxonomicstudiesare neededtoenabletheidentificationofalltaxa,aswellastoexplain somevariationsinspectralandanatomicaldatatoC.pareiraand itsrelatedspecies.
Authors’contributions
NMP (PhD student) contributed in collecting plant samples and identification,and runningthe laboratorywork (preparing herbariasamples,chemical extraction, obtainingof spectraand plantanatomystudies),analysisofthedataandpreparationofthe paper.YLBcontributedtoUVanalysis,supervisedbyIJLDB.MFA designedthestudywithCisampelospareira,andsupervisedall lab-oratoryandfieldwork,aswellascontributedtocriticalreadingof themanuscript.Alltheauthorshavereadthefinalmanuscriptand approvedthesubmission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
TheauthorsaregratefultoCAPESforfinancialsupport,andCNPq forthescholarshipstoNMPortoandMFAgra.Theauthorsthankto Dr.EduardodeJesusOliveirafortheEnglishrevisionand sugges-tions,andDr.RosaOrtizforhervaluablecommentsandhelpwith thebibliography;toMissouriBotanicalGardenfortheinstitutional support;thecuratorsofherbariaCEPEC,IAN,JPB,MG,RB,UFPand MOfortheirpleasantcooperationduringourvisitsandloanofplant material;andDulceGonc¸alvesforhertechnicalsupport.
References
Abbasi,A.M.,Khan,M.A.,Ahmad,M.,Zafar,M.,Jahan,S.,Sultana,S.,2010. Ethnophar-macologicalapplicationofmedicinalplantstocureskindiseasesandinfolk
cosmeticsamongthetribalcommunitiesofNorth-WestFrontierProvince, Pak-istan.J.Ethnopharmacol.128,322–335.
Ahmad,R.,Malik,M.A.,Zia-ul-Haq,M.,1992.AlkaloidsofCissampelospareira. Fitoter-apia63,282–285.
Amresh,G.,Zeashan,H.,Rao,C.V.,Singh,P.N.,2007a.Prostaglandinmediated anti-inflammatoryandanalgesicactivityofCissampelospareira.ActaPharm.Sci.49, 153–160.
Amresh,G.,Zeashan,H.,Gupta,R.J.,Kant,R.,Rao,C.V.,Singh,P.N.,2007b. Gastro-protectiveeffectsofethanolicextractfromCissampelospareirainexperimental animals.J.Nat.Med.61,323–328.
Arkarapanthu,A., Chavasit,V., Sungpuag, P.,Phuphathanaphong, L.,2005.Gel extractedfromKhruea-ma-noi(CycleabarbataMiers)leaves:chemical com-positionandgelationproperties.J.Sci.FoodAgric.85,1741–1749.
Barbosa-Filho,J.M.,Agra,M.F.,Thomas,G.,1997.Botanical,chemicaland pharma-cologicalinvestigationonCissampelosspeciesfromParaíba(Brazil).Sci.Cult.49, 386–394.
Barthlott,W.,Neinhuis,C.,Cutler,D.,Ditsch,F.,Meusel,I.,Theisen,I.,Wilhelmi,H., 1998.Classificationandterminologyofplantepicuticularwaxes.J.Linn.Soc. 126,237–260.
Basílio,I.J.L.D.,Bhattacharyya,J.,Moura,R.K.P.,Agra,M.F.,2012.Applicationof UV/VISspectrophotometryandmultivariateanalysistocharacterizationofthe speciesofSolanumsect.ErythrotrichumCHILD.Chem.Biodivers.9,1114–1124. Bone,R.E.,Lee,D.W.,Norman,J.M.,1985.Epidermalcellsfunctioningaslensesin
leavesoftropicalrainforestshadeplants.Appl.Opt.24,1408–1412.
Braga,J.M.A.MenispermaceaeinListadeEspéciesdaFloradoBrasil.JardimBotânico doRiodeJaneiro.http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB10031 (accessedJuly2015).
Bridson,D.,Forman,L.,1999.TheHerbariumHandbook,3rded.RoyalBotanic Gar-dens,Kew.
Bukatsch,F.,1972.AzuldeAstraeSafranina.In:Kraus,J.,Arduin,M.(Eds.),Manual básicodemétodosemmorfologiavegetal.Edur,Seropédia,RiodeJaneiro,p.26. Carlquist,S.,1996.WoodandstemanatomyofMenispermaceae.Aliso14,155–170. Cavalcanti,A.C.,Gomes,A.N.P.,Porto,N.M.,Agra,M.F.,Moura,T.F.A.L.,Oliveira,E.J., 2014.PhamacognosticevaluationofCissampelossympodialisEichlleaves.S.Afr. J.Bot.93,70–78.
Chowdhury,A.R.,1972.ChemicalinvestigationsonCissampelospareira.Sci.Cult.38, 358.
DeWet,H.,Tilney,P.M.,VanWyk,B.E.,2002.Vegetativemorphologyandanatomy ofCissampelosinSouthAfrica.S.Afr.J.Bot.68,181–190.
Ensikat,H.J.,Ditsche-Kuru,P.,Neinhuis,C.,Barthlott,W.,2011.Superhydrophobicity inperfection:theoutstandingpropertiesofthelotusleaf.BeilsteinJ. Nanotech-nol.2,152–161.
Esau,K.,1972.AnatomiaVegetal.Omega,Barcelona.
Franklin,G.L.,1945.Preparationofthinsectionsofsyntheticresinsandwood-resin composites,andanewmaceratingmethodforwood.Nature155,51. Fahn,A.,1988.Secretorytissuesinvascularplants.NewPhytol.108,229–257. Furr,M.,Mahlberg,P.G.,1981.Histochemicalanalysesoflaticifersandglandular
trichomesinCannabissativa.J.Nat.Prod.44,153–159.
Ganguly,M.,KrBorthakur,M.,Devi,N.,Mahanta,R.,2007.Antifertilityactivityof themethanolicleafextractofCissampelospareirainfemalealbinomice.J. Ethnopharmacol.111,688–691.
Griffin,W.J.,Lin, G.D.,2000.Chemotaxonomyand geographicaldistributionof tropanealkaloids.Phytochemistry53,623–637.
Hammer,Ø.,Harper,D.A.T.,Ryan,P.D.,2001.PAST:Paleontologicalstatistics soft-warepackageforeducationanddataanalysis.Palaeontol.Electron.4,1–9. Haque,M.A.,Shaha,M.K.,Ahmed,S.U., Akter, R.,Rahman, H.,Chakravotry,S.,
Imran,A.H.M.N.,Islam,M.T.,Das,R.C.,Rahmatullah,M.,2011.Useofinorganic substancesinfolkmedicinalformulations:acasestudyofafolkmedicinal practitionerinTangaildistrict,Bangladesh.Am.EurasianJ.Sustain.Agric.5, 415–423.
Hedberg,I.,Hedbrerg,O.,Madati,P.J.,Mshigeni,K.E.,Mshiu,E.N.,Samuelsson,G., 1983.InventoryofplantsusedintraditionalmedicineinTanzania.II.Plantsof thefamiliesDilleniaceae-Opiliaceae.J.Ethnopharmacol.9,105–127. Hong,Y.P.,Pan,K.Y.,Chen,Z.D.,Lu,A.M.,2001.Charactersofleafepidermisandtheir
systematicsignificanceinMenispermaceae.ActaBot.Sin.43,615–623. Hoot,S.B.,Zautke,H.,Harris,D.J.,Crane,P.R.,Neves,S.S.,2009.Phylogeneticpatterns
inMenispermaceaebasedonmultiplechloroplastsequencedata.Syst.Bot.34, 44–56.
Howard,R.,1979.ThePetiole.In:Metcalfe,C.R.,Chalk,L.(Eds.),Anatomyofthe Dicotyledons,1.ClarendonPress,Oxford,pp.88–96.
Jannu,V.,SaiVishal,D.,RanjithBabu,V.,Harisha,B.,RaviChandraSekharaReddy, D.,2011.Antidiabeticactivityofhydro-alcoholicextractofCissampelospareira Linn.Leavesinstreptozotocininduceddiabeticrats.Int.J.Pharm.Technol.3, 3601–3611.
Johansen,D.A.,1940.PlantMicrotechnique.McGrawHill,NewYork.
Kamble,S.Y.,More,T.N.,Patil,S.R.,Pawar,S.G.,RamBinduraniBodhankar,S.L.,2008. PlantsusedbytribesofNorthwestMaharashtraforthetreatmentof gastroin-testinaldisorders.IndianJ.Tradit.Knowl.7,321–325.
Kumar,V.P.,Chauhan,N.S.,Padh,H.,Rajani,M.,2006.Searchforantibacterialand antifungalagentsfromselectedIndianmedicinalplants.J.Ethnopharmacol.107, 182–188.
Kupchan,S.M.,Yokoyama,N.,Beal,J.L.,1960a.Menispermaceaealkaloids.I.The alka-loidsofCissampelospareiraLinn.andtheoriginofradixpareirabrave.J.Am. Pharm.Assoc.49,727.
Levitt,J.,1980.ResponsesofPlantstoEnvironmentalStresses,vol.2.NewYork, AcademicPress.
Lewis,W.H.,Elvin-Lewis,M.P.F.,1977.MedicalBotany.Wiley-Interscience,New York.
Lonni,A.A.S.G.,Scarminio,I.S.,Silva,L.M.C.,Ferreira,D.T.,2005.Numericaltaxonomic characterizationofBaccharisgenusspeciesbyultravioleta-visible spectropho-tometry.Anal.Sci.21,235–239.
Mantuano,D.G.,Barros,C.F.,Scarano,F.R.,2006.Leafanatomyvariationwithinand betweenthreerestingapopulationsofErythroxylumovalifoliumPeyr. (Erythrox-ylaceae)inSoutheastBrazil.Rev.Bras.Bot.29,209–215.
Menachery,M.D.1996.ThealkaloidsofSouthAmericanMenispermaceae.InS.W. Pelletier(org),Alkaloids:ChemicalandBiologicalPerspectives.NewYork:Ed. Pergamon,p269–302.
Metcalfe,C.R.,Chalk,L.,1950.AnatomyofDicotyledons:Leaves,Stem,andWoods inRelationtoTaxonomywithNotesonEconomicUses,vol.1.ClarendonPress, Oxford,UnitedKingdom,pp.52–62.
Metcalfe,C.R.,Chalk,L.,1979.AnatomyoftheDicotyledons:SystematicAnatomyof LeafandStem,withaBriefHistoryoftheSubject,vol.1.,2nded.ClaredonPress, Oxford/UnitedKingdom,pp.267.
Morita, H., Matsumoto, K., Takeya, K., Itokawa, H., Iitaka, Y., 1993. A novel antileukemic tropoloisoquinoline alkaloid, pareirubrine, from Cissampelos pareira.Chem.Lett.,339–342.
Nagarajan,K.,Chauhan,N.,Mittal,A.,Singh,V.,Bodla,R.B.,Tiwari,R.K.,2011. Phyto-chemicalextraction,optimizationandphysico-chemicalcharacterizationoftwo bioactiveisolatesfromtheleavesandstemofCissampelospareira.DerPharma Chem.3,327–337.
NAPRALERT,NaturalProductsAlert.http://www.napralert.org/(accessedMarch 2013).
Nurit-Silva,K.,Basi´lio,I.J.L.D.,Agra,M.F.,2007.Estudofarmacobotânicocomparativo entreSolanumpaniculatumL.eSolanumrhytidoandrumSendtn.Rev.Bras.Biol. 5,243–245.
Nurit-Silva,K.,Agra,M.F.,2011.LeafepidermalcharactersofSolanumsect. poly-trichum(Solanaceae)astaxonomicevidence.Microsc.Res.Tech.74,1186–1191. Ortiz,R.,2001.Menispermaceae.InStevens,W.D.,UlloaUlloa,C.,Pool,A.,Montiel, O.M.(orgs.),FloradeNicaragua.Missouri:MonographsinSystematicBotany fromtheMissouriBotanicalGarden,p.1432–1442.
Porto,N.M.,Basílio,I.J.L.D.,Agra,M.F.,2008.Pharmacobotanicalstudyoftheleaves ofCissampelossympodialisEichl.,(Menispermaceae).Braz.J.Pharmacogn.18, 102–107.
Porto,N.M.,Figueiredo,R.C.B.Q.,Oliveira,A.F.M.,Agra,M.F.,2011.Leafepidermal characteristicsofCissampelosL.(Menispermaceae)speciesfromNortheastern Brazil.Microsc.Res.Tech.74,370–376.
Porto,N.M.,Araújo,N.D.,Basílio,I.J.L.D.,Agra,M.F.,2012.Analysisofleafepidermal charactersofmedicinalandpoisonousBrazilianMenispermaceae.PlantaMed. 78,1111.
Priya,G.,Saravanan,K.,Renuka,C.,2012.Medicinalplantswithpotentialantifertility activity–areviewofsixteenyearsofherbalmedicineresearch(1994–2010). Int.J.Pharm.Tech.Res.4,481–494.
Ramasubramaniaraja,R.,Babu,N.M.,2010.Antihelminthicstudiesandmedicinal herbs–anoverview.Int.J.Pharm.Sci.Rev.Res.5,39–47.
Ramirez,I.,Carabot,A.,Melendez,P.,Carmona,J.,Jimenez,M.,Patel,A.V.,Crab, T.A.,Blunden,G.,Cary,P.D.,Croft,S.L.,Costa,M.,2003.Cissampeloflavone, a chalcone–flavone dimer from Cissampelos pareira. Phytochemistry 64, 645–647.
Rhodes,D.G.,1975.ArevisionofthegenusCissampelos.Phytologia30,415–485. Rôc¸as,G.,Scarano,F.R.,Barros,C.F.,2001.LeafanatomicalvariationinAlchornea
triplinervia(Spreng.)Müll.Arg.(Euphorbiaceae)underdistinctlightandsoil waterregimes.Bot.J.Linn.Soc.136,1–8.
Rozema, J., Chardonnens, A., Tosserams, M., Hafkenscheid, R., Bruijnzeel, S., 1997.LeafthicknessandUV-Babsorbingpigmentsofplantsinrelationto anelevationalgradientalongtheBlueMountains,Jamaica.PlantEcol.128, 150–159.
Rukunga,G.M.,Gathirwa,J.W.,Omar,S.A.,Muregi,F.W.,Muthaura,C.N.,Kirira,P.G., Mungai,G.M.,Kofi-Tsekpo,W.M.,2009.Anti-plasmodialactivityoftheextracts ofsomeKenyanmedicinalplants.J.Ethnopharmacol.121,282–285. Rury,P.M.,1983.PareiraBrava:19thCenturynotesandcommercialsamplesfrom
E.R.SquibbM.D.Bot.Mus.Leaf.29,27–48.
Sampaio,V.S.,Araújo,N.D.,Agra,M.F.,2014.CharactersofleafepidermisofSolanum oftheBrevantherumCladefromAtlanticForestofNortheasternBrazil.S.Afr.J. Bot.74,108–113.
SanchezMedina,A.,GarciaSosa,K.,MayPat,F.,PenaRodriguez,L.M.,2001. Antioxi-dant,antimicrobialandbeta-glucosidaseinhibitionactivities.Phytomedicine8, 144–151.
Schmelzer, G.H., Gurib-Fakim, A. (Eds.), 2008. Plant Resources of Tropical Africa11(1).MedicinalPlants1.PROTAFoundation/BackhuysPublishers/CTA, Wageningen,Netherlands/Leiden,Netherlands/Wageningen,Netherlands,791 pp.
Semwal,D.K.,Semwal,R.B.,Vermaak,I.,Viljoen,A.,2014.Fromarrowpoisonto herbalmedicine:theethnobotanical,phytochemicalandpharmacological sig-nificanceofCissampelos(Menispermaceae).J.Ethnopharmacol.155,1011–1028. Sinnot,E.W.,1914.Theanatomyofthenodeasanaidintheclassificationof
angiosperms.Am.J.Bot.7,303–322.
Sharma,V.,1987.Biosynthesisofhayatin.IndianJ.Chem.26,589–591.
Shipman,L.L.,Cotton,T.M.,Norris,J.R.,Katz,J.J.,1976.Ananalysisofthevisible absorptionspectrumofchlorophyllamonomer,dimer,andoligomersin solu-tion.J.Am.Chem.Soc.98,8222–8230.
Singh,V.K.,Ali,Z.A.,1992.Acontributiontotheethnopharmacologicalstudyofthe UdaipurforestsofRajasthan,India.Fitoterapia63,136–144.
Singh,K.K.,Maheshwari,J.K.,1994.Traditionalphytotherapyofsomeplantsusedby thetharusoftheNainitalDistrict,UttarPradesh,India.Int.J.Pharmacogn.32, 51–58.
Stace,C.A.,1965.Cuticularstudiesasanaidtoplanttaxonomy.Bull.Br.Mus.Nat. Hist.Bot.4,1–78.
Singthong,J.,Cui,S.W.,Ningsanond,S.,DouglasGoff,H.,2004.Structural character-ization,degreeofesterificationandsomegellingpropertiesofKrueoMaNoy (Cissampelospareira)pectin.Carbohydr.Polym.58,391–400.
Sudhakaran,M.V.,2012.Histo-morphological,fluorescentandpowdermicroscopic characterizationofCissampelospareiraLinn.Phcog.J.4,57–68.
Shukla,P.,Shukla,P.,Gopalakrishna,B.,2012.Investigationofin-vitroanthelmintic activityofCissampelospareiraLinnagainstPheretimaposthuma.Int.J.Pharm.Sci. Res.3,265–267.
Thakur,P.,Rana,A.C.,2013.EffectofCissampelospareiraleavesonanxiety-like Behaviorinexperimentalanimals.J.Tradit.Complement.Med.3,188–193. Thiers,B.(continuouslyupdated)IndexHerbariorum:AGlobalDirectoryofPublic
HerbariaandAssociatedStaff.NewYorkBotanicalGarden’sVirtual Herbar-ium.NewYorkBotanicalGarden.Availablefrom:http://sweetgum.nybg.org/ih/ (accessed29.10.15).
Vaidya,B.G.,1988.NighantuAdarsh,vol.1.ChaukhambhaBhartiAcademy Publica-tions,Varanasi,India,pp.44–45.
Vardhanabhuti,B.,Ikeda,S.,2006.Isolationandcharacterizationofhydrocolloids frommonoi(Cissampelospareira)leaves.FoodHydrocolloids20,885–891. Yadav,K.S.,Yadav,N.P.,Shanker,K.,Thomas,S.C.,Srivastav,S.,Srivastava,S.,Kumar
Rai,V.,Mishra,N.,Sinha,P.,2013.Assessmentofantidiabeticpotentialof Cis-sampelospareiraleafextractinstreptozotocinenicotinamideinduceddiabetic mice.J.Pharm.Res.6,874–878.
Wilkinson,P.,1978.LeafanatomyoftheTribeCoscinieaeHook.f.&Thoms. (Menis-permaceae).KewBull.32,347–360.
Wilkinson,H.P.,1979.Plantsurface.In:Metcalfe,C.R.,Chalk,L.(Eds.),Anatomyof theDicotyledon.ClaredonPress,Oxford,pp.97–162.
Wilkinson,H.P.,1986.LeafanatomyofTinomisciumandFibraurea (Menisperma-ceaetribeFibraureeae)withspecialreferencetolaticifersandastrosclereids. KewBull.41,153–169.
