Rev. bras. farmacogn. vol.27 número1

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

Original

Article

Comparative

study

of

Passiflora

taxa

leaves:

II.

A

chromatographic

profile

Luma

Wosch

_,

_Kely

_Cristina

_dos

_Santos

_,

_Daniela

_Cristina

_Imig

_,

_Cid

_Aimbiré

_M.

_Santos

b_{DepartamentodeBotânica,UniversidadeFederaldoParaná,Curitiba,PR,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2February2016 Accepted28June2016 Availableonline28July2016

Keywords: Passiflora Passionfruit Qualitycontrol

Thin-layerchromatography

High-performanceliquidchromatography FlavonoidC-glycosides

a

b

s

t

r

a

c

t

Popularlyknownaspassionfruit,somespeciesofthegenusPassifloraarewidelyusedinfolkmedicines, suchassedativesandtranquilizersinmanycountries.Althoughtheseplantsareemployedforthesame purposes,researchwithdifferentspeciesofPassiflorahasindicatedtheirheterogeneouschemical com-positions.Sincedifferentchemicalcompositionscanresultinvaryingdegreesoftherapeuticefficiency, qualitycontrolbasedonthechemicalconstituentsofeachspeciesisessential.Tothatend,theaimofthis studywastocomparepharmacognosticallyspeciesofPassiflorainordertoestablishachromatographic profileforthequalitycontrolofdrugsinherbalmedicinescontainingpassionfruit.Thestudywas con-ductedbycollectingsamplesofleavesfromtwelvePassiflorataxa(i.e.,tenspeciesandtwoformsofP.

edulis)–P.actinia,P.alata,P.amethystina,P.capsularis,P.cincinnata,P.edulisf.flavicarpa,P.edulisf.edulis,

P.incarnata,P.morifolia,P.urnifolia,P.coccinea,andP.setacea–fromdifferentlocationsandobtainingtheir

chromatographicprofilesviathin-layerchromatographyandhigh-performanceliquidchromatography. BothmethodsusedtheflavonoidC-glycosidesisoorientin,orientin,vitexin,andisovitexinasreference compoundsandcouldultimatelyestablishspecificprofilesforeachspecies.Thechromatographic anal-ysesdiscussedherecanbeusedtoassistindeterminingthequalityandauthenticityofherbaldrugs derivedfromPassifloraspecies.

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

Introduction

BelongingtoPassifloraceaefamily,thegenusPassiflora encom-passesnearly600species(UlmerandMacDougal,2004).Popularly knownaspassionfruit,invariouscountriessomeofthesespecies arewidelyusedinfolkmedicines,typicallyassedativesand tran-quilizers (Conrado et al.,2003). AlthoughPassiflora species are employedfor the samepurposes, investigations have indicated their heterogeneous chemical compositions (Wohlmuth et al., 2010;Lietal.,2011;Zucolottoetal.,2012).Sincedifferentchemical compositionscan result in varying degrees of therapeutic effi-ciency,itiscriticaltoidentifyanddifferentiatespeciesofthegenus. To achieve such identification and differentiation, pharma-cobotanicaltools and chromatographic analysis can offergreat insights,particularlyinregardtoPassifloraspecies(Woschetal., 2015).However,withthesemeans,onceoriginalplantmaterial is processed into a powder or when contaminants are present withsimilarmorphoanatomiccharacteristics,whichoftenoccurs

∗ Correspondingauthor.

E-mail:cid@ufpr.br(C.A.M.Santos).

accidentally (Veiga et al., 2005), pharmacobotanical tools can-notsufficientlyauthenticatespecies(SucherandCarles,2008).In suchcases,itisnecessarytouseanalyticalmethodstoassessthe qualitativeandquantitativecompositionofeachplant’schemical constituents.

Tothatend,chromatographicanalysiscancontributeto devel-oping chemical profiles that aid in distinguishing species. The significance of applying chromatographic analysis also extends tosecondary metabolites, which representa chemical interface betweenaplantanditssurroundingenvironmentandwhose syn-thesisisoftenaffectedbyenvironmentalconditions.Assuch,both seasonaland dailyintraplant,intraspecies,and interspecies dif-ferences can affect thetotal content or relative proportions of secondaryplantmetabolites,ifnotboth.Eveninthepresenceof ageneticcontrol,chemicalconstituentscanbeexpressedin differ-entways owingtotheinteractionofbiochemical,physiological, ecological,and evolutionaryprocesses,which areconsideredto largely compromise anyconstancy in theconcentrationof sec-ondarymetabolites(Gobbo-NetoandLopes,2007).

Sinceenvironmentalfactorsaffectthefinalcontentofsecondary metabolitesinmedicinalplants,theycanalsosignificantlyaffect thequalityandtherapeuticvalueofherbalpreparations.Toobtain

http://dx.doi.org/10.1016/j.bjp.2016.06.007

qualitativeandquantitativedifferencesamongspeciesare excel-lentchemicalmarkers(Qiminetal.,1991;BokstallerandSchmidt, 1997).Thus,theobjectiveofourstudywastoanalyzetwelve Pas-sifloraspeciesbyusingTLCandHPLC,therebyenablingeffective qualitycontrolforitsleaves,allinordertocontributetothequality ofherbalproductscontainingpassionfruit.

Thin-layerchromatography

Toextract,1mlof60%ethanolwasaddedto200mgofeach sample.Themixtureswerevortexingfor10s,andthesampleswere extractedfor10minwithultrasoundequipment.Afterdecanting,

1.1 1.2 1.3 2.1 2.2 2.3 2.4 2.5 3.1 3.2 Isovitexin

Fig.1.Thin-layerchromatographyprofileofSamples1.1–1.3(Passifloraactinia),Samples2.1–2.5(P.alata),andSamples3.1–3.2(P.amethystina)usingEluentSystem1,with isovitexinasthestandard.

3.3

4.1

4.2

4.3

4.4

5

6.1

6.2

6.3

6.4

Vitexin

7.1 7.2 8.1 8.2 8.3 9.1 9.2 9.3 10.1 10.2 Isoorientin

Fig.3.Thin-layerchromatographyprofileofSamples7.1–7.2(Passifloraedulisf.edulis),Samples8.1–8.3(P.incarnata),Samples9.1–9.3(P.morifolia),andSamples10.1–10.2 (P.urnifolia)usingEluentSystem1,withisoorientinasthestandard.

about2␮lofthesupernatantobtainedwasappliedto

chromato-platesintheformofbands.Silicagel60aluminumplateswere used at 20cm×20cm, without any fluorescence indicator for

high-performancethin layer chromatography(HPTLC) (Merck®_, Darmstadt,Germany,art.5547).

Thesystemwaskeptclosedduringanalysis.Activatedplates wereheldinanovenat90◦_{Cfor90min,andthetemperatureand} humiditywerekeptconstantat20◦_{Cand50%,respectively.The} distancetraveledbytheeluentwasstandardizedto9cm.Two dif-ferenteluentsystems(ES)wereused:oneofethylacetate,acetone, aceticacid,andwaterinaratioof6:2:1:1(Gosmannetal.,2011) andtheother–anadjustmentofthemobilephaseproposedby WagnerandBladt(1996)–ofethylacetate,aceticacid,formicacid, andwaterinaratioof10:1.1:1.1:2.5.

Afterthecompleteevaporationofthesolvents,theplateswere revealedwithdiphenylboriloxietilamine1%inMeOHandlaterwith 5%polyethyleneglycol4000in EtOH(WagnerandBladt,1996). ObservationsweremadeunderUV365nm.

High-performanceliquidchromatography

Toextract,8mlof60%ethanolwasaddedto200mgofthe sam-ple.Themixturewasvortexingfor15s,andthesamplesextracted for30mininultrasoundequipment.Theextractwasfiltered,and thevolume wascompletedto10ml with60% ethanol.Samples werefilteredthroughaMillex®

LCRwithmembranefilterPTFE of0.45␮m,packedinamberglassbottlesat4◦_{Cuntilthetimeof} analysis,andatthattime,injectedat20mg/ml.

Adaptedfromthe methodproposedbyMuller et al.(2005), ourmethodofdeterminingflavonoidisovitexininPassifloraactinia

extractswasdevelopedandvalidatedbyourresearchgroup.Later, themobilephaseconsistedofagradientof0.5%aceticacidinMilliQ water(A),methanol(B),andacetonitrile(C)at0minwith75%(A), 15%(B)and10%(C),at25minwith62%(A),20%(B)and18%(C), andat30minwith75%(A),15%(B)and10%(C).Theflowratewas 1ml/minforarunningtimeof30minwithadetectionwavelength of340nm.

AnalyseswereperformedwithVarianProStarGradient equip-ment,aProStar230pump,andaphotodiodearraydetector335 (ColumnKromasil®_100,5

␮mC-18[250mm×4.6mmin

diame-ter]).

10.3 11 12 Orientin

Fig.4. Thin-layerchromatographyprofileofSamples10.3(Passifloraurnifolia), Sam-ple11(P.coccinea),andSample12(P.setacea)usingEluentSystem1,withorientin asthestandard.

Spectralanalysis

SpectralanalyseswereperformedusingtheStar Chromatog-raphy Workstation for data acquisition within Varian ProStar GradientHPLC.

Standards

1.1 1.2 1.3 2.1 2.2 2.3 2.4 2.5 3.1 3.2 Isovitexin

Fig.5. Thin-layerchromatographyprofileofSamples1.1–1.3(Passifloraactinia),Sample2.1–2.5(P.alata),andSample3.1–3.2(P.amethystina)usingEluentSystem2,with isovitexinasthestandard.

Results

Thin-layerchromatography

TheresultsobtainedwithES1appearinBox1andFigs.1–4, whereasthoseobtainedwithES2appearinBox2andFigs.5–8. BothES1andES2allowedtheobservationofdifferencesamong samples.WithES2,however,sincethebandsbecamemoredefined, itwaspossibletodisplaymorebands,whichfacilitatedthe com-parisonamongspeciesprofiles(Figs.5–8).

ThethreespecimensanalyzedforthespeciesP.actinia(Samples 1.1–1.3) showed similarchromatographic profiles. An apparent quantitativedifferencewasobservedinthesecondbandof Sam-ple 1.1 (ES 1), which presented a more intense yellow color (Figs.1and5).AccordingtoRfvaluesandcolor,thisbandseemed tocorrespondtothestandardisoorientin.

ThesamplesanalyzedforP.alata,bycontrast,revealed qualita-tivedifferences(Figs.1and5).However,thosesamplescouldbe dividedintotwogroupsbasedonthesimilarityoftheirprofiles: ontheonehand,Samples2.1and2.4,andontheother,Samples 2.2,2.3,and2.5.Thechiefdifferencebetweenthegroupsisthe

presenceofayellowspot(ES1,Rf0.22)oroftwobands(ES2,Rf 0.39and0.33)inSamples2.1and2.4,respectively,thatwereabsent intheothers.

SamplesofP.amethystina(3.1,3.2,and3.3)wereverysimilar (Figs.1,2and5,6).

Bypresentingbandswithhardlyanydetectablestaining inten-sity,Sample4.4ofP.capsularisstandsoutfromtheotherthreeof thespecies,whichweresimilar(Figs.2and6).

ThesinglesampleofP.cincinnata studiedshowednumerous bandsonbothES1andES2(Figs.2and6).

WhereasthefirstsampleofP.edulisf.flavicarpa(6.1)differed entirelyfromtheothersbyshowingfourbandsonES1,allwith aRf(0.93,0.81, 0.70,0.59)greaterthanthelargestoftheother three samples(Samples 6.2–6.4), the others werequite similar (Figs.2and6).MostbandsinSamples6.2–6.4werenotpresent, oratleastindistinguishablefromSample6.1(Fig.2).

ThetwospecimensanalyzedforP.edulisf.edulisshowedstarkly differentprofiles.WhereasbandsofSample7.1presenteda yellow-to-greenbandconcentratedintheupperregionoftheplateand with higher R_f values, bands with yellow coloring from Sam-ple7.2 had lowerRf values (Figs.3 and 7).Moreover,asFig.3

3.3 4.1 4.2 4.3 4.4 5 6.1 6.2 6.3 6.4 Vitexin

7.1 7.2 8.1 8.2 8.3 9.1 9.2 9.3 10.1 10.2 Isoorientin

Fig.7.Thin-layerchromatographyprofileofSamples7.1–7.2(Passifloraedulisf.edulis),Samples8.1–8.3(P.incarnata),Samples9.1–9.3(P.morifolia),andSamples10.1–10.2 (P.urnifolia)usingEluentSystem2,withisoorientinasthestandard.

10.3

11

12

Orientin

Fig.8.Thin-layerchromatographyprofileofSample10.3(Passifloraurnifolia), Sam-ple11(P.coccinea),andSample12(P.setacea)usingEluentSystem2,withorientin asthestandard.

shows,with ES1 Sample7.2 revealed two bands notfound in any other sample analyzed (green and red, Rf 0.86 and 0.77, respectively).

SimilarprofileswerefoundforSamples8.1,8.2and8.3ofP. incarnata(Figs. 3and 7).A lowerintensityofstainingof bands appeared in Sample 8.3,by contrast, thus suggestingthat they havealowerconcentrationofsubstancesviewableinthin-layer chromatography methods. No significant differences were observedbetween theprofilesof P.morifolia (Samples 9.1–9.3) andP.urnifolia(Samples10.1–10.3),asFigs.3,4,7,8illustrate.

Lastly,markeddifferenceswereobservableamongspecimens ofP.coccinea(11)andP.setacea(12).Intermsofthebandwith lowerRfvalues,twowereorangeandonewasorange-to-bluefor

P.coccinea,andtwowereyellowforP.setaceainES1(Fig.4).

High-performanceliquidchromatography

Comparisonofprofilesofeachspecies

MostspecimensofeachspeciesanalyzedbyHPLCcoupledtoa diodearraydetectorshowedsimilarqualitativeprofilesandwere thereforesuperimposable(Figs.9and10).HPLCallowsthe detec-tionofqualitativedifferencesalreadyidentifiedintheflavonoid profileofsomesampleswhencomparedtootherspecimensofthe samespeciesobservedwithTLC.Suchdifferenceswereobserved inP.alata,withsamplesdividedintotwogroupsaccordingtothe similarityoftheirprofiles(Samples2.1and2.4andSamples2.2, 2.3,and2.5),aswellasinSample6.1ofP.edulisf.flavicarpa(Fig.9) andtwosamplesofP.edulisf.edulis(Fig.10).

Withallsamplesprocessedinthesamemannerandinjectedin thesameamountsandconcentrations,HPLCallowsaquantitative comparativeanalysisofsampleswithgreaterreliabilitythanthat achievedwithTLC.

Sample 4.4 of P. capsularis presented the most significant quantitativevariation(Fig.9).Overlappingofthechromatograms obtainedforthisspeciesallowedthevisualizationofpeaksin4.4at thesameretentiontimesofthosefoundinothersamples(Fig.11). Givenitslowersensitivity,TLCdidnotpermitsuchvisualization.

Sample8.3ofP.incarnatashowedpronouncedquantitative dif-ferencescompared toSamples8.1and 8.2,chieflybydisplaying lessintensepeaks(Fig.10).Othervariationsobservedwereless significant.

Comparisonofspeciesbycoinjectionwithstandard

Coinjections with standards—that is, at least one flavonoid glycoside—allowedacorrelationofsomepeaksineachprofile,as summarizedinBox3.

Peakswerecoloredaccordingwiththeflavonoidsidentifiedby coinjection:blueforisovitexin,pinkforvitexin,greenfor isoori-entin,andpurplefororientin(Figs.12and13).Partiallycolored peakswerepartiallyco-elutedwithstandards,whereasbicolored peakswereco-elutedwithtwostandards.

Spectralanalysis

50 0

150

50 0 400

200

0 150 100

50

0

5 6.4 6.3 6.2 6.1 5 4.4 4.3 4.2 4.1

10 15 20 25 min

P. capsularis

P. cincinnata

P. edulis f. flavicarpa

Fig.9.Overlayofthechromatogramsobtainedbyhigh-performanceliquid chro-matographyforsamplesofPassifloraactinia,P.alata,P.amethystina,P.capsularis, andP.cincinnatacomparedtoP.incarnataandP.edulisf.flavicarpa.

pureness.Regardingpeaksco-elutedwithisoorientinandorientin, purityvariedaccordingtothechromatogramanalyzed.

ForP.actinia,Peak3 relatedtoisoorientinand,though sym-metrical, exhibited a shift in spectral profile from 276/348 to 278/341nm at its right end. Peak 3, of P. capsularis, was also

600 0 500 1000 0

300

0

300

100 0

5 12 11 10.3 10.1 10.2

10 15 20 25 min

P. setacea P. urnifolia

P. cocciea

Fig.10.Overlayofthechromatogramsobtainedbyhigh-performanceliquid chro-matographyforsamplesofPassifloraedulisf.edulis,P.incarnata,P.morifolia,P. urnifolia,P.coccinea,andP.setacea.

comprisedoftwoseparatespectra,meaningthatitdidnot cor-respondonlytoorientin.

Peak2fromSample2.1ofP.alatashowedaverysimilar spec-trumtoPeak3(275/343nmforPeak2and275/345nmforPeak3), thussuggestingthatitcouldbeasubstancewithastructuresimilar tothatofvitexin.

mAU

150

50 0 20

10

0

5 10

Sample 4.4 Sample 4.2

15 20 25 min

L.

Wosch

et

al.

/

Revista

Brasileira

de

Farmacognosia

27

(2017)

40–49

Box1

Summaryofresultsobtainedwiththin-layerchromatographyusingEluentSystem1fortwelvePassifloraspecies.

Species Sample(s) Numberofmajorbands Color Rfa Standardsassigned

P.actinia 1.1,1.2and1.3 6 Yel-Gn;yellow;yellow;BBe;yellow;Be 0.57;0.49;0.35;0.32;0.28;0.04 Isoorientin,isovitexin

P.alata 2.1_2.2,and_2.3and2.4_{2.5 5}5 _Yel-Gn;Yel-Gn;yellow;_Be;yellowGn;_Gn;Yel-Gn;_Yel Yel _0.57;0.57;_0.51;0.49;_0.49;0.41;_0.41;0.31;_0.310.22 _Isoorientin,Isoorientin,isovitexin_isovitexin

P.amethystina 3.1,3.2and3.3 5 Yel-Gn;Yel;BBe;Be;BGn 0.57;0.49;0.32;0.19;0.10 Isoorientin,isovitexin

P.capsularis 4.1,4.2and4.3 3 Yel;BBe;Yel 0.49;0.33;0.15 Isoorientin

P.cincinnata 5 6 Yel-Gn;Yel;Yel-Gn;Oe;Yel-Gn;Oe 0.57;0.49;0.44;0.33;0.26;0.16 isoorientin,isovitexin

P.edulisf. flavicarpa

6.1 7 Oe;Be;Yel-Gn;Oe;Yel;Oe;Oe 0.93;0.81;0.70;0.59;0.43;0.31;0.15 Isoorientin,isovitexin

6.2,6.3and6.4 7 Yel-Gn;Yel;BBe;Yel-Gn;Bn;Yel-Gn;Oe 0.57;0.49;0.33;0.26;0.22;0.19;0.15 Isoorientin,isovitexin P.edulisf.

edulis

7.1 8 Gn;Bn;Gn;Gn;Oe;Gn;Gn;BBe 0.78;0.75;0.70;0.62;0.52;0.49;0.45;0.34 Vitexin

7.2 8 Gn;Yel;Yel-Gn;Yel-Gn;BBe;Yel-Gn;Yel-Gn;Oe 0.86;0.77;0.58;0.47;0.34;0.27;0.19;0.14 Isovitexin

P.incarnata 8.1_8.3and8.2 ₅6 _Yel-Gn;Yel-Gn;Yel-Gn;_Yel-Gn;Yel;_Yel;BBe;_Yel-Gn;Yel-Gn;_Yel Yel 0.69;_0.69;0.57;_0.57;0.50;0.50;_0.27;0.33;_0.210.27;0.20 _Isoorientin,Isoorientin,_vitexin,vitexin,isovitexin_isovitexin

P.morifolia 9.1,9.2and9.3 9 Yel;Yel;Yel;Yel;Yel-Gn;BBe;Yel-Gn;Oe;BGn 0.80;0.68;0.58;0.50;0.44;0.33;0.27;0.16 Isoorientin,vitexin,isovitexin

P.urnifolia 10.1,10.2and10.3 7 Gn;Yel-Gn;Yel;BBe;Yel;Yel;Oe 0.71;0.57;0.50;0.33;0.27;0.19;0.16 Isoorientin,isovitexin

P.cocinea 11 6 Yel;Yel;BBe;Oe;Be;Oe 0.59;0.51;0.35;0.33;0.26;0.17 Isoorientin,isovitexin

P.setacea 12 6 Yel;Yel;Yel;BBe;Yel;Yel 0.59;0.51;0.38;0.35;0.30;0.20 Isoorientin,isovitexin

Standards

Isovitexin 1 Yel-Gn 0.57

Vitexin 1 Yel-Gn 0.69

Isoorientin 1 Yel 0.50

Orientin 1 Yel 0.60

Yel,yellow;Yel-Gn,greenishyellow;Oe,orange;Be,blue;BBe,brightblue;Gn,green;BGn,brightgreen;Bn,brown. a_{Dataarepresentedinorderofappearance,fromtoptobottom,onthechromatographicplate.}

Box2

Summaryofresultsobtainedwiththin-layerchromatographyusingEluentSystem2fortwelvePassifloraspecies.

Species Sample(s) Numberofmajorbands Color Rfa Standardsassigned

P.actinia 1.1,1.2and1.3 7 Yel-Gn;Be;Yel;Red;Yel-Gn;Yel-Gn;Be 0.66;0.62;0.59;0.47;0.42;0.36;0.04 Isoorientin,isovitexin

P.alata 2.1_2.2,and_2.3and2.4_2.5 7_{5 Yel-Gn;}Yel-Gn;Be;_Be;Yel;Yel;_Gn;Gn;_Yel;Yel;Gn;Yel _0.66;0.66;_0.62;0.62;_0.59;0.59;_0.50;0.50;_0.420.42;0.39;0.33 _Isoorientin,Isoorientin,isovitexin_isovitexin

P.amethystina 3.1,3.2and3.3 9 Be;Yel-Gn;Be;Yel;Yel;Yel-Gn;BGn;BGn;Be 0.71;0.66;0.62;0.59;0.50;0.42;0.34;0.22;0.07 Isoorientin,isovitexin

P.capsularis 4.1,4.2and4.3 7 BBe;Yel;Be;Yel;Yel;Yel;Oe 0.76;0.70;0.65;0.61;0.35;0.31;0.24 Isoorientin,orientin

P.cincinnata 5 10 Yel;Be;Yel;Yel;Yel;Be;Yel;Oe;Yel;Yel 0.68;0.65;0.61;0.52;0.45;0.36;0.33;0.28;0.21;0.13 Isoorientin,isovitexin P.edulisf.

flavicarpa

6.1 8 Oe;Yel;Yel;Yel;Yel;Yel;Oe;Yel 0.98;0.91;0.86;0.78;0.71;0.62;0.50;0.43 Isoorientin,isovitexin

6.2,6.3and6.4 7 Yel;Be;Yel;Yel;Bn;Yel;Oe 0.68;0.65;0.60;0.34;0.32;0.29;0.23 Isoorientin,isovitexin

P.edulisf. edulis

7.1 11 Oe;Bn;Yel;Yel;Yel;Bn;Yel-Gn;Bn;Yel;Oe;Be 0.98;0.80;0.75;0.68;0.61;0.58;0.55;0.52;0.48;0.41;0.33 Vitexin,orientin 7.2 10 Gn;Red;Be;Yel-Gn;Gn;Oe;Yel;Yel;Yel;Yel 0.90;0.84;0.62;0.58;0.53;0.44;0.41;0.33;0.28;0.22 –

P.incarnata 8.1and8.2 7 Yel;Yel;Be;Yel;Yel;Yel;Yel 0.77;0.67;0.62;0.59;0.37;0.33;0.27 Isoorientin,vitexin,isovitexin

8.3 6 Yel;Yel;Yel;Yel;Yel;Yel 0.77;0.67;0.59;0.37;0.33;0.27

P.morifolia 9.1,9.2and9.3 10 Yel;Yel;Yel;Be;Yel;Yel-Gn;Oe;BBe;Yel;Oe 0.82;0.75;0.67;0.63;0.59;0.50;0.41;0.36;0.32;0.26 Isoorientin,vitexin,isovitexin

P.urnifolia 10.1,10.2and10.3 6 Yel-Gn;Yel-Gn;Yel;Yel;Yel;Yel 0.77;0.65;0.58;0.34;0.28;0.23 Isoorientin,isovitexin

P.cocinea 11 7 BBe;Yel-Gn;Yel;Be;Oe;Yel;Oe 0.96;0.64;0.57;0.48;0.43;0.39;0.28 Isoorientin,isovitexin

P.setacea 12 8 BBe;Yel-Gn;BBe;Yel;BBe;Yel-Gn;Yel;Yel 0.96;0.65;0.62;0.58;0.48;0.42;0.37;0.30 Isoorientin,isovitexin

Standards

Isovitexin Yel,Yel-Gn 0.66

Vitexin Yel-Gn 0.78

Isoorientin Yel 0.58

Orientin Yel 0.68

Roseflora Isoorientin,isovitexin

P. actinia

P. alata 2.1

P. alata 2.2

P. amethystina

P. capsularis

P. cincinnata

P. edulis f. flavicarpa 6.1

P. edulis f. flavicarpa 6.2 mAU 250 3 3 3 3 3 3 3 4 4 4 4 4 4 4 5 5 5 5 5 5 5

5 10 15 20 25 min

6 6 6 7 9 8 8 7 6 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 100 0 100 50 0 150 50 0 250 100 0 150 75 0 250 100 0 50 0 150 75 0

Fig. 12.Comparison of different chromatographicprofiles obtained by high-performanceliquidchromatography for the speciesPassiflora actinia, P.alata (Samples2.1and2.2),P.amethystina,P.capsularis,P.cincinnata,andP.edulisf.

flavi-carpa(Samples6.2and6.1).Peaksareidentifiedbycoinjectionwithstandard:green

forisoorientin,purplefororientin,pinkforvitexin,andblueforisovitexin.

P. urnifolia P. coccinea P. setacea 3 3 3 3 4 4 4 5 5 5 6 6 7

5 10 15 20 25 min

6 7 8

1 1 1 1 2 2 2 0 300 600 0 300 700 0 150 500 200 0

Fig. 13.Comparison of different chromatographic profilesobtained by high-performanceliquidchromatographyforthespeciesPassifloraedulisf.edulis(7.2),

P.incarnata,P.morifolia,P.urnifolia,P.coccinea,andP.setacea.Peaksidentifiedby

coinjectionwithstandard:greenforisoorientin,purplefororientin,pinkforvitexin, andblueforisovitexin.

Peak 7, of P. amethystina, in being unidentified, presents a spectrumwithmaximumabsorptionat277/343nm,whichwas vir-tuallyidenticaltothatofPeaks5(277/343nm)and6(276/343nm), therebycorrespondingtovitexinandisovitexin,respectively.

Peaks1–4ofP.capsulariswereimpure.Peak1probably rep-resented a flavonoid, for its spectrum presented a maximum absorptionatonly335nm.ThesameoccurredforPeak4,which presentedamaximumabsorptionat318and336nm.Peak3was contaminatedwithasubstance,whichcausedittoshowaspectrum withamaximumat320nm.

TheprofilepresentedforPeak3bySample6.2 ofP.edulisf. flavicarpashowsamaximumabsorptionatonly279nm,whichis inconsistentwiththatofflavonoids.

Only Peak 4 of P. edulis f. edulis (Sample 7.1) presented a spectrumunliketheothers,withamaximumat276nm.Peak2, althoughasymmetrical,showedthesamespectra,withamaximum absorptionat274/346nm.

For Sample 7.2, Peaks 4 and 7 showed only an absorp-tion maximum in their spectra.Peak 3 of this sample showed a spectrum with absorptions at 280/339nm, which were rela-tivelydistantfrom275/355nm—thatis,thepatternpresentedfor isoorientin—probablycorrespondingtoaflavonoidwitharetention timeclosertothatofisoorientin.

Allotherpeaksnotquotedexhibitedtwoabsorptionmaximaat wavelengthssimilartothosepresentedbythestandards.

Discussion

Chromatographicanalysis

Asamethodinthequalitycontrolofdrugmaterials,TLCwas chosen for beingthesimplest and mosteconomical chromato-graphictechnique for rapidseparation and visualidentification (Lopes,2006).BothES1andES2allowedustodifferentiatespecies bypresenting a profileforeach. ES2 allowedthevisualization ofmorebands,whichgreatlyfacilitatedcomparison.Atthesame time,allvariationsdetectedbyHPLCwerealsoobservedwithTLC. Similarly,thetwoeluentsystemsallowedthecorrelationofsome bandswithpatternsused(Boxes1and2),whichwasalsopossible withHPLC(Box3).

For thesereasons, thetwo methods developed canbe used forthe qualitycontrol of drugsand extractsof leavesof Passi-floraspecies.Suchmethodsallowthedetectionofqualitativeand quantitativevariablesamongsamplesofthesamespeciesorform. Regardingqualitativevariations,samplesofP.alatadifferedgreatly andcouldbeplacedintwogroups,whosechiefdifferencewasthe presenceofanadditionalpeak,asinSamples2.1and2.4.Similar profilestothetwoprofilesfoundinourstudywerealsoreported byMulleretal.(2005)andMadoglio(2011),thelatterofwhom showedthatthepeakco-elutedwithstandardvitexinwas vitexin-2-O-rhamnoside.

ThespeciesP.alatapresentedavariabilityalreadywell docu-mentedintheliterature.Melettietal.(2003)foundavariationin itsmorphologicalandagronomiccharacters,whereasBellonetal. (2009)detecteditsgeneticvariability,whichismorepronounced inwildaccessionsdue tothewide geographical distributionof thespecies.Suchwell-documentedgeneticvariabilitycouldrelate tothediversityinthechemicalconstitutionofdifferentP.alata

species,andsincedifferentchemicalconstitutionscausedifferent therapeuticefficiencies,thestandardizationoftheplantextractand productdrugsisnecessarybasedontheirchemicalconstitution. Thisbecameevidentinourstudybyobservingthechromatograms ofthetwoformsofP.edulis(Figs.3,7and10)thatshowed signifi-cantchemicaldifferences,aswellasinthesamples.

Zucolottoetal.(2012)studiedthetwoformsofP.edulisand obtaineda chromatogram for P.edulis f. flavicarpa that closely resembledthatforourSamples6.2–6.4thanforourSample6.1. Theirchromatogramfor P.edulisf.eduliswasalsoclosertoour Sample7.1.Onthispoint,theauthorssuggestedthattheresultsfor thetwoformsofP.eduliswereduetoawiderangeofinter-and intrachemicalcompositionalforms.

Vianaetal.(2003)detectedgeneticvariabilityamongtheforms ofP.edulis,whichalongwiththeresultsobtainedinthisstudy indi-catetheimportanceofidentifyingtheformofthespecies,aswellas thechemicalcharacterizationandstandardizationofherbaldrugs andextractsofP.edulis.Thesedrugsandextractscouldformpart oftheconstitutionofteas,herbalremedies,orcosmetics, consider-ingthattheconcentrationandcompositionofphenolicsubstances correlatewithbiologicalactivity(Colomeuetal.,2014).

Regardingquantitativevariations,P.capsularissamplesshowed themostpronounceddifference,whichrelatedto morphoanatom-icalvariation.Sample4.4ofP.capsularisshowedpeaksatfarlower intensitiesthanthoseofSamples4.1–4.3(Figs.2,6and9),asclearly showninFig.11,inwhichthequalitativecompositionofSample 4.4greatlyresemblestothecompositionpresentedbytheothers.

Inadditiontopeakswithlowerintensities,themesophyll thick-nessofSample4.4wasnarrowerthanthatofoneothersample duetoenvironmentalinfluences.Sample4.4wascollectedfroman

Atlanticforest,whereithadgrownintheshadowoftrees,whereas theothersampleswerecollectedfromanopenspacewithdirect exposuretothesun.

Inadditiontoreportingthesamemorphoanatomicdifferences forP.capsularis,Tattinietal.(2000)showedthatthespecies Phyl-lyrealatifoliagrowninshadyspacesunderathickforestofPinus pineaorgrownin thefullsunofopendunesdemonstratedthe accumulationofflavonoidsandglandulartrichomesintheleaves. Thoseauthorsreportedthattheconcentrationofflavonoid glyco-sidesshowedasharpincreaseinleavescollectedfromsunnyplaces. Nevertheless,thecompositionofflavonoidsremainedunchanged, asinagreementwithfindingsforP.capsularisinourwork.

Suchdifferencesinconcentrationresultfromplantadaptations todifferentlightintensitiesbyregulatingtheirphysiologicalstates andchangingtheirprimaryand secondarymetabolicpathways. Indoingso,theiranabolicandcatabolicprocessesachieve their maximumfunctionalstatus(Nobel,1991).

P.incarnatasamplesalsoexhibitedquantitativevariations,as inSample8.3,whichshowedmainpeakswithlowerintensities along withUV absorption.Unlikeothers of thespecies,Sample 8.3 wasa dried extract provided by theherbalindustry, while theothertwo sampleswere collectedata cultivationsite. The detectionofproblemsincommercialextractsisnotuncommon, however.Theimportanceofqualitativeandquantitative standard-izationofflavonoidspresentinthedrugsorextractsofPassiflora

species,especiallyinpreparationsofP.incarnata,reliesonthefact thatflavonoidssuchasvitexin,isovitexin,orientin,andisoorientin contributetothedrugactivity(MenghiniandMancini,1988).

Unfortunately,itisnotunusualtodetectproblemsin commer-cialextracts.Silvaetal.(2013)testedtheeffectiveness,regardingto theprotectionoffibroblastsagainsttheeffectsofUVrays,fivegreen tea commercialextracts used to enrich formulations cosmetic, comparedtoafluidextractpreparedaccordingtothe recommenda-tionsoftheBrazilianPharmacopoeia(2010).Theauthorsobserved thattheEGCGcontentwasmuchhigherintheextractprepared accordingtothepharmacopoeia,beingtheonlyonetoshow sig-nificantantioxidantactivity.Itisevident,thus,theneedtoapply amorerigorousqualitycontrolforcommercialextracts,covering thedeterminationnotonlyqualitativebutalsoquantitativeoftheir chemicalmarkers.

EachspeciesofPassifloraanalyzedexhibitedadistinctive chro-matographicprofileofitsleaves’hydroalcoholicextracts,thereby makingitpossibletosuggestacorrelationbetweensomebandsin TLCandsomepeaksinHPLCofC-glycosylatedflavonoidssuchas isoorientin,orientin,vitexin,andisovitexin.Thesecompoundscan thereforebeusedinthequalitycontrolofrawmaterialsas chemi-calmarkersforauthenticatinganddifferentiatingspecieswiththe proposedmethods.

Conclusion

Theevaluationofthemorphologicalandanatomicalfeaturesof drugsderivedfromplantsinthegenusPassiflora,asshown previ-ously(Woschetal.,2015),whenalliedtochromatographicprofiles, cancontributetothediagnosisanddifferentiationofspecies. Vari-ationsfoundwithinthesamespeciesorwithinandbetweenforms underscoretheimportanceofconductingpharmacognostic analy-sisandstandardizingcultureconditionsofPassifloraspecieswhose productsareusedfortherapeuticpurposes.

Authors’contributions

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