Rev. bras. farmacogn. vol.26 número4

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

Original

Article

Chemical

profiles

of

traditional

preparations

of

four

South

American

Passiflora

species

by

chromatographic

and

capillary

electrophoretic

techniques

Geison

Modesti

Costa

_,

_Andressa

_Córneo

_Gazola

_,

_Silvana

_Maria

_Zucolotto

_,

Leonardo

Castellanos

_,

_Freddy

_Alejandro

_Ramos

_,

_Flávio

_Henrique

_Reginatto

_,

_Eloir

_Paulo

_Schenkel

a_{ProgramadePós-graduac¸ãoemFarmácia,UniversidadeFederaldeSantaCatarina,Florianópolis,SC,Brazil}

b_{DepartamentodeQuímica,UniversidadNacionaldeColombia,Bogotá,Colombia}

c_{DepartamentodeFarmácia,CentrodeCiênciasdaSaúde,UniversidadeFederaldoRioGrandedoNorte,Natal,RN,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received17November2015 Accepted23February2016 Availableonline13April2016

Keywords: Passiflora

C-glycosylflavonoids Alkaloids

HPLC UPLC CE

a

b

s

t

r

a

c

t

SeveralspeciesofthegenusPassifloraaredistributedalloverSouthAmerica,andmanyofthesespecies areusedinpopularmedicine,mainlyassedativesandtranquilizers.Thisstudyanalyzesthechemical profileofextractsoffourPassifloraspeciesusedinfolkmedicine,focusingontheflavonoids,alkaloids andsaponins.Weemployedsimpleandfastfingerprintanalysismethodsbyhighperformanceliquid chromatography,ultraperformanceliquidchromatographyandcapillaryelectrophoresistechniques. TheanalysisledtothedetectionandidentificationofC-glycosylflavonoidsinalltheplantextracts,these beingthemainconstituentsinP.tripartitavar.mollissimaandP.bogotensis.Saponinswereobserved onlyinP.alataandP.quadrangularis,whileharmanealkaloidswerenotdetectedinanyoftheanalyzed extractsinconcentrationshigherthan0.0187ppm,thedetectionlimitdeterminedfortheUPLCmethod. ©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

ThegenusPassifloraisthelargestandmostimportantgenus

ofthefamilyPassifloraceae,comprisingabout500species(Lewis andElvin-Lewis, 1977).InNorth Americaand Europe,themain species,P.incarnata,ispopularlyknownaspassionfruitor passion-flower,whileinSouthAmerica,severalothersspeciesofPassiflora

are widely distributed and known by distinct names, such as

‘maracujá’, ‘curuba’, or ‘badea’, among others. (Arbelaez, 1996; Morsetal.,2000)Manyofthesespecies(P.edulisvar.edulis,P.edulis var.flavicarpa,P.tripartitavar.mollissimaandothers)arecultivated fortheirediblefruitsorforthepreparationofjuices.Infusionsof theirleavesarealsousedinpopularmedicineinmanycountries, asasedativeortranquillizer(PioCorrêa,1978;Arbelaez,1996).

Different countries in South America have registered

phar-maceuticalpreparationsthat usePassiflora speciesastheactive component.In Colombia,for example,theleavesofP.tripartita

var. mollissima are accepted as sedative and hypnotic

compo-nentinphytopharmaceuticalpreparations(Invima,2006).InBrazil,

∗ _{Correspondingauthor.}

E-mail:gmcosta@unal.edu.co(G.M.Costa).

P.alataandP.edulisareincludedinthemostrecentversionofthe BrazilianPharmacopeia(FarmacopeiaBrasileira,2010).

Regardingtheirchemical composition, thecompoundsmore

frequently reported for the genus are flavonoids, especially C

-glycosylflavonoids, which are usually described as the main

components(Ulubelenetal.,1982;Lietal.,2011;Zucolottoetal.,

2012).Thesecompoundshaverecentlybeenassociatedwith

sev-eralpharmacologicaleffectsobservedfordistinctPassifloraspecies (Coletaetal.,2006;Santosetal.,2006;Senaetal.,2009;Zucolotto etal.,2009;Gazolaetal.,2015).Harmanealkaloidsarealso fre-quentlyassociatedwithPassifloraspecies,especially P.incarnata (LutomskiandMalek,1975;Lutomskietal.,1975).Additionally,

several saponins have been described for this genus, although

theiroccurrenceisrestrictedtocertainspecies(Orsinietal.,1985; Reginattoetal.,2001;Doyamaetal.,2005).

Aspartofourongoingstudieswithspeciesofthegenus Pas-siflora, we evaluate, in this study, the variability of metabolite presentsintheaqueousextractsoffourSouthAmericanPassiflora species:P.alata, P.quadrangularis,P.bogotensisandP.tripartita var.mollissima,focusingspecificallyontheirC-glycosylflavonoid

and alkaloid composition. The presence of saponins in these

species wasalsoevaluated.Chemicalprofileswere obtainedby

different analytical methods, such as high performance liquid

http://dx.doi.org/10.1016/j.bjp.2016.02.005

Table1

Passifloraspecies,withtheirrespectivelocalcommonname,placeofcollectionandidentification.

Species Localcommonname Placeofcollection/timeofyear Identification

P.quadrangularisLinn. Badea,

Maracujá-gigante

Rivera,Huila–Colombia (2◦₅₉′₅₅′′_,−75◦₁₈′₁₆′′_)/July2011

Prof.CarlosAlbertoParra/Herbariumofthe UniversidadNacionaldeColombia(COL 572634)

P.alataCurtis. Maracujá-doce Urussanga,SantaCatarina–Brazil (−28◦

32′ 9′′

,−49◦ 18′

59′′

)/January2011

Mrs.AdemarBrancher(EPAGRI/Urussanga-SC). HerbariumoftheUniversidadeFederalde SantaCatarina(FLOR37823)

P.tripartitavar. mollissima Holm-Nielsen& MüllerJörgensen

Curuba-de-Castilla SantaSofia,Boyacá–Colombia (05◦₄₃′₀₁′′_,

−73◦₃₆′₂₀′′_)/June2011

Prof.CarlosAlbertoParra/Herbariumofthe UniversidadNacionaldeColombia(COL 564522)

P.bogotensisBenth – Nemocón,Cundinamarca–Colombia

(4◦ 35′

60′′ ,−4◦

4′ 60′′

)/May2011

Prof.CarlosAlbertoParra/Herbariumofthe UniversidadNacionaldeColombia(COL 564523)

chromatography(HPLC),ultraperformanceliquidchromatography

(UPLC)andcapillaryelectrophoresis(CE).

Materialsandmethods

Chemicals

Acetonitrile,methanolandformicacid(HPLC-grade)were

pro-videdbyTedia®_{(RiodeJaneiro,Brazil).Waterwaspurifiedwith}

a Milli-Q system(Millipore®_{, Bedford, USA). For theCE}

analy-sis,stock solutionswerepreparedfromtheelectrolytessodium

tetraborate(STB)andammoniumacetate(AcNH4)at100mmol/l.

Sodiumhydroxidesolution(NaOH)at1and0.1mol/lwerealso

pre-pared.Allthesaltsusedwereofanalyticalreagentgrade,andwere

providedbySigma-Aldrich®_{(St.Louis,USA).Allthesolventsand}

solutionswerefilteredthrougha0.22␮mmembranebeforeuse.

Thereferencestandardsorientin,isoorientin,vitexin,isovitexin,

vitexin-2′′_{-O-rhamnoside,harmol,harmaneandharmine(allwith}

purity≥96%)werepurchasedfromSigma-Aldrich®_{.Swertisinwas}

previouslyobtainedfromWilbrandeaebracteataroots,and

iden-tifiedbyNMRspectraldata(Santosetal.,1996).Thecompound

4′_{-methoxyluteolin-8-C-6}′′_{-acetylglucopyranosidewaspreviously}

isolatedfromP.tripartitavar.mollissimaleavesandprovidedby Prof.Dr.FreddyRamos(Ramosetal.,2010).Quadrangulosidewas previouslyisolatedfromPassifloraalataleaves(Costaetal.,2013).

Plantmaterialandpreparationofextractsandsamples

Leavesof adultindividuals of species of Passiflora were col-lectedfrom different regions of Brazil and Colombia (Table1). Leavesofthedifferentspecieswereair-driedseparatelyat40◦_C,

powdered,and extracted byinfusionwithboilingwater (95◦_C,

plant:solvent1:10,w/v)for10min.Theaqueousextractwasthen filtered,frozenandlyophilized.ThesamplesforHPLC,UPLCandCE analysiswerepreparedbydissolvingthelyophilizedcrude aque-ousextractsorreferencestandardsinmethanol:water(1:1,v/v)

andfilteringthrougha0.22␮mmembranebeforeinjection.The

concentrationofthesampleextractswas1000␮g/mlandforthe

referencestandards,theconcentrationwas100␮g/ml.

HPLCanalysis

TheHPLCanalyseswerecarriedoutinaPerkinElmer®_Series200

system,equippedwithDiodeArrayDetection(DAD),quaternary

pump,on-linedegasserandautosampler.Thedatawereprocessed

usingthesoftwareChromera®_{Workstation.Thechromatographic}

analysesfor all sampleswere performed at room temperature

(21±2◦_{C),withaninjectionvolumeof20}

␮l.TheDADspectrawere

acquiredattherangeof190–450nm.Thepeaksin thesamples

werecharacterizedbycomparingtheretentiontime,UVspectra

andco-injectionwiththereferencestandards.Vertical®_VertSep

C18column(250mm×4.6mmi.d.;5␮m)wasusedasstationary

phase.Intheanalysisofflavonoids,agradientsystemof acetoni-trile[solventA]andformicacid0.5%[solventB]wasusedasmobile phase,inasinglestep:15–35%A(0–20min).Theflowratewaskept

constantat1.2ml/minandthechromatogramswererecordedat

340nm.Foralkaloidanalysis,themobilephaseusedwascomposed

ofanaqueousbufferofsodiumphosphatedibasic(50mmol/l,pH

8.0)[A],methanol[B]andacetonitrile[C]atisocraticconditionsof 56%A:12%B:32%C(0–20min).Theflowratewaskeptat1ml/min

andtheUVdetectionat245nm.Thechromatographicconditions

fortheanalysisofsaponinswerepreviouslydescribedbyourgroup (Costaetal.,2013).

UPLCanalysis

AnUPLCWatersAcquity® _{HClasssystemwithDADdetector,}

quaternarypump,on-linedegasserandautosamplerwasusedfor

theseanalyses.Thechromatographicparameterswereconverted

fromHPLCtoUPLCusingthesoftwareEmpower®

.Separationsof bothflavonoidsandalkaloidswerecarriedoutinaPerkinElmer®

BHE C18 column (100mm×2.9mm i.d.; 1.8␮m). The analyses

werealsoperformedatroomtemperature(21±2◦_C),withDAD

spectra acquired at the range of 190–450nm. Flavonoid

anal-ysis used a two-steps gradient of acetonitrile [solvent A] and

formic acid 0.5% [solvent B]: 15–35%A (0–8min), followed by

35%A(8–10min).Theflowratewaskeptconstantat0.25ml/min.

Theinjectionvolumewas3␮l.Thechromatogramwasrecorded

at 340nm. For the alkaloids, the same mobile phase and

iso-cratic system as the HPLC was used, withan analysis time of

7min. The flow rate was determinate as 0.2ml/min, with an

injectionvolume of 2␮l. The UV detection was determined at

245nm.

CEanalysis

TheanalyseswereperformedonanAgilent7100capillary

elec-trophoresisinstrumentequippedwithDADdetector,temperature

controldevice,andautosampler.Foralltheexperiments,a

fused-silicacapillary (Agilent,modelG1600-61232) of60.5cm (52cm

effectivelength),with50␮minnerdiameterandexpanded

detec-tionwindowwasused.Thedatawereprocessedusingthesoftware Agilent ChemStation®_{. For the first use, the capillary was}

pre-treatedwithapressureflushwith1mol/lNaOHsolution(30min). Eachday,thecapillarywasconditionedwithNaOH1mol/l(5min),

waiting time (1min), Milli-Q water (5min) and running buffer

(5min).Inbetweenruns,thecapillarywasflushedwithrunning

buffer(2min). The DAD spectrawere acquiredat the range of

200–500nm.Amethodfortheanalysisofflavonoidswas

1

500 mAU

0

45

0

120

60

0

0 200

100

50

0

0 10 20

8

P. tripartita P. bogotensis P. quadrangularis P. alata

7 5 2 1

min 100

25

10

3

2 6

5

1

6 4

∗C

∗_B

∗_A

∗D

∗_E

∗F

∗G 20 min

mAU

2 3,4567 8 _{with20%ofMeOHasrunningbuffer.Thesampleswereintroduced}

tothesystembyhydrodynamicinjection(50mbar/10s).All separa-tionswereperformedatavoltageof+25kV,constanttemperature of30◦_{C,anddirectdetectionat390nm.Apigeninwasusedas}

inter-nalstandard(I.S.)inordertoalignthemigrationtime.Thealkaloid

analysiswasperformedbasedonthemethodpreviouslydescribed

byUngeretal.(1997).Briefly,AcNH4(100mmol/l;pH4.0,adjusted

withaceticacid),with50%ACNwasusedasrunningbuffer. Hydro-dynamicinjection(50mbar/5s)wasused,withseparationvoltage of+15kV,constanttemperatureof15◦_{C,anddetectionat245nm.}

Experimentaldeterminationofthesensitivityofthemethodsfor alkaloidanalysisbyUPLCandCE

Thelimitsofquantification(LOQ)anddetection(LOD)forthe analyticalmethodsusedintheanalysisofalkaloidswere

experi-mentallydeterminedusingstandardsolutionsfromtheharmane

alkaloid,preparedindifferentconcentrationsinthematrix (sam-ples),intherangeof50–0.0187␮g/ml.Allsolutionswereprepared

andanalyzedintriplicatebybothtechniques.Linearitywas deter-minedusingnine-pointandfive-pointregressioncurves,forUPLC andCE,respectively.LOQwasdefinedbysignal:noiseratioof10:1 andalsobyrelativestandarddeviation(RSD>5%).LODwasdefined byasignal:noiseratioof3:1(ICH,2005).

Resultsanddiscussion

Plant extracts are often complex mixtures whose

therapeu-tic effect cannot always attributedto a single component, and

sometimes,thecomponentsresponsibleforaparticulareffectare

notknown.Asnotallthecomponentshavereferencestandards

foridentificationandquantitation,somequalitycontrolanalyses

maybeperformedbyafingerprintanalysis,inwhichthe

exper-imental datafromthechemical analysisof differentextractsis comparedwithoutaccuratequantification.Thismethodisaccepted bytheWorldHealthOrganizationforthequalitycontrolofherbal medicines(WHO,1991).

C-glycosylflavonoidanalysis

Forthedevelopmentofthechromatographicmethods,seven

authenticsamplesofC-glycosylflavonoidswereusedasreference

compounds. After evaluating several parameters, including the

distinctchromatographicsystemsdescribedinliterature,the

ini-tialconditionsusedweredeterminedasthosethatallowedgood

resolutionbetweenallreferencecompounds,andenabledthe dif-ferentiationoftheextractsanalyzed,accordingtotheflavonoid profile.Theanalyticalparameterswerethenoptimized,basedon

visualizationofthemaximumnumberofpeaks,theirresolution

index,thetimerequiredfortheanalysis,andthesimplicityofthe method.Theflavonoidfingerprintsobtainedforeachspeciesinthe finalselectedmethodarepresentedinFig.1,andthepeak assign-mentsforeachextractaredescribedinTable2.

Amongthefouranalyzed species,P.alataand P.

quadrangu-larispresentedtheleastcomplex flavonoidprofile.It isnotable

Fig.1. HPLCchromatogramsofflavonoidsstandards(upperchromatogram; nor-malizedview)andaqueousextractsoftheleavesofPassifloraspecies.1:isoorientin, 2:orientin,3:vitexin-2′′_{-O-rhamnoside,4:vitexin-2}′′_{-O-xyloside,5:vitexin,6:}

isovitexin,7:swertisin,and8:4′_{-methoxyluteolin-8-C-6}′′_{-acetylglucopyranoside.}

*A:orientin-2′′_{-O-glucoside,*B:orientin-2}′′_{-O-xyloside,*C:vitexin-2}′′_-O-glucoside

(identifiedbyGazola,2014),*D:isoorientin-2′′

-O-rhamnoside,*E:isovitexin-2′′

-O-rhamnoside, *F: luteolin-6-C-˛-l-rhamnopyranosyl-(1→2)-(6′′_-O

-acetyl)-ˇ-d

-glucopiranoside,and*G:apigenin-6-C-˛-l-rhamnopyranosyl-(1→2)-(6′′_-O

Table2

PeakassignmentsoftheaqueousextractsbyHPLC–DAD.

Flavonoids R1 R2 R3 R4 R5 Rta(min) UVmax(nm) Species

Isoorientin(1) glu H H OH OH 9.2 258,268sh,348 P:bogotensis;

P.tripartitavar.

mollissima

Orientin(2) H H glu OH OH 9.8 258,268sh,348 P.alata;

P.tripartitavar.

mollissima

Vitexin-2′′_{-O-rhamnoside(3) H H glu-O-rham H OH 10.5 269,335 P.alata}

Vitexin-2′′_{-O-xiloside(4) H H glu-O-xil H OH 10.8 269,335 P.quadrangularis}

Vitexin(5) H H glu H OH 11.4 269,338 P.quadrangularis;

P.tripartitavar.

mollissima

Isovitexin(6) glu H H H OH 11.7 269,338 P.alata;

P:bogotensis

Swertisin(7) glu OCH3 H H OH 12.3 270,334 P.tripartitavar.

mollissima

4′_{-Methoxyluteolin-8-C-6}′′

-acetylglucopyranoside (8)

H H glu-O-acetyl OH OCH3 17.8 269,295,346 P.tripartitavar.

mollissima

glu,glucose;rham,rhamnose;xil,xiloside.

a_{PeaknumbersinFig.1.}

thatthemajorflavonoidsofbothspecieshave almostthesame

retentiontimes.Thisobservationhaspreviouslybeendescribed

byourresearchgroup,thetwodistinctmajorcompoundsbeing

identifiedasvitexin-2′′_{-O-rhamnoside(3)(P.alata)andvitexin-2}′′

-O-xyloside(4)(P.quadrangularis),proposedaschemicalmarkers for these species (Costa et al., 2013).Additionally, orientin (2) and isovitexin (6)were identified in P.alata, while vitexin (5) wasobservedonlyinP.quadrangularis.OtherC-glycosylflavonoids fromP.quadrangularis,whichhave beenisolatedand identified (Gazola,2014),werealsoassigned(Fig.1,peaks*A–*C).Someof thesecompounds(orientin-2′′_{-O-xyloside,vitexin-2}′′_{-O-glucoside,}

vitexin-2′′_{-O-xyloside)havealsobeendescribedbySakalemand}

co-workers(2012).

Ahigheraccumulationofflavonoidswasobservedintheextract ofP.bogotensis,althoughonlytwoflavonoidscouldbeidentified byspikingexperimentswithcommercialstandards(isoorientin(1) andisovitexin(6)).Recently,workontheflavonoidcompositionof P.bogotensisleaveshasreportedthepresenceofthesecompounds, togetherwithotherC-glycosylflavonoids,indicatedinFig.1(peaks *D–*G)(Costaetal.,2015).

Among the extracts analyzed, P. tripartita var mollissima

displayed the most complex flavonoid profile. Isoorientin,

orientin, vitexin, swertisin and 4′_{-methoxyluteolin-8-C-6}′′

-acetylglucopyranoside could be identified by co-injection and

UVspectra.Inapreviouswork,thesameauthorshaveevaluated

the C-glycosylflavonoid profile of P. tripartita var. mollissima leavesandpericarpbya distinctHPLCmethod(Zucolottoetal., 2012).Althoughbotharesuitablefortheirpurposes,themethod

described herein allows the great diversity of flavonoids tobe

betterobserved,withananalysistimeofonly20min.Simirgiotis andco-workers (2013)have studiedthe peeland fruit juice of thisspecies,whichalthoughdifferentpartsoftheplant,presented severalflavonoidsthatwerestructurallyrelatedtothoseobserved inourwork.

TheparametersdevelopedfortheHPLCanalysisofthe

refer-encecompounds(authenticsamplesofC-glycosylflavonoids)and

theextracts(Fig.1),weresubsequentlyusedintheUPLCanalysis, yieldingsimilarresults(Fig.2).AlthoughtheHPLCanalysisallowed rapidanalysiswithanadequateresolutionbetweenpeaks,

compar-atively,theanalysistimebyUPLCwasreducedby50%(10min),

withvirtuallyunchangedfingerprints.AsfortheHPLC analysis, theUPLCanalysisalsoenabledustodifferentiatethefourextracts, especiallyinthedifficultdistinctionofthemayorflavonoidsfrom P.alataandP.quadrangularis.

In addition to the analysis by chromatographic methods,

theflavonoid composition wasalso analyzedby capillary

elec-trophoretic(CE).Theliteraturemostlyreportstheuseofborate buffersasrunningelectrolytesintheanalysisofflavonoidsbyCE (Molnár-PerlandFüzfai,2005;Marchartetal.,2003;Rijkeetal., 2006).Consideringthatthepreviouslychromatographicanalyses haverevealedahighercomplexityofflavonoidsinP.tripartitavar. mollissima,theCEmethodwasinitiallydevelopedforthisextract.

Someparameterswereevaluatedforthechangeinelectrosmotic

flow,suchastheelectrolyteconcentration,voltage,capillary tem-peratureandinjectionvolume.Theconditionsthatprovidethebest separationforP.tripartitavar.mollissima(see‘Materialsand meth-ods’section)wereappliedtotheotherextracts(Fig.3),enablingto distinguishtheflavonoidfingerprintsforeachspecies.

Comparedwiththechromatographictechniques,theprofiles

obtainedbyCEshowedalsoashorttimeanalysis,goodpeak reso-lutionandsymmetry.Qualitatively,anothersubstantialdifference

betweenthechromatographicandelectrophoreticmethodsisthe

detectionwavelength.InHPLC,flavonoidsareusuallydetectedin

therangeof330–350nm,whichisthebandofmaximum

absorp-tionfor these compounds,providing selectivitytothe method.

mAU 1200

500

0

0 5 10

min P. tripartita P. bogotensis P. quadrangularis P. alata

Fig.2.UPLCchromatogramofflavonoidsfromaqueousextractsoftheleavesofPassifloraspecies.Fordetailsofthechromatographicmethod,seethe‘Experimental’section.

mAU

30

15

0

0 4 8 12

min I.S.

P. tripartita P. bogotensis P. quadrangularis P. alata

Fig.3. CEelectropherogramofflavonoidsfromaqueousextractsoftheleavesof

Passifloraspecies.Internalstandard(I.S.):apigenin.Fordetailsofthecapillary elec-trophoreticmethod,seethe‘Materialsandmethods’section.

Alkaloidsanalysis

Somepreviousstudiesreportthepresence of alkaloidsin P.

incarnata and P. edulis (Poethke et al., 1970; Lutomski et al., 1975).Nevertheless,morerecentworks,withmoresensitive

meth-ods,havequestionedthepresenceorlevelsofthesecompounds

(SperoniandMinghetti,1988;Rehwaldetal.,1995;Griceetal., 2001).

Inthealkaloidanalysispresentedinthisstudy,chromatographic

andelectrophoreticmethodsweredevelopedusingthestandards

harmol,harmaneandharmine,compoundsthathavepreviously

beendescribedforP.incarnataandP.edulis(Poethkeetal.,1970;

Lutomskietal.,1975).Qualitatively,thepresenceofthesealkaloids wasnotobservedinthecrudeaqueousextractfromtheleavesof thesespeciesbyHPLC,UPLCorCE(Fig.4).Forthisreason,a

calibra-tioncurveforharmanewasbuiltinUPLCandCE(thetechniques

withthefastestmethods)todeterminethelinearityandsensitivity ofthesemethods.TheresultsarepresentedinTable3.

The quantitative data showed an excellent linear

relation-ship betweenpeak areaand concentration (r2_{>0.999) for both}

techniques.Aboutthesensitivityofthesemethodologies,itwas observedthatUPLCpresentedasensitivity15-foldhigherthenCEin theseconditions,whichcanbemainlyexplainedbythedifference oftheinterparticulesspacesintheUPLCcolumnandtheinternal diameterofthecapillarycolumninCE.

Considering thedetermined limitsfor thesemethods, itcan

bestatedthattheaqueousextractsanalyzedinthisworkdonot

have harmane type alkaloids at levels exceeding 0.0187␮g/ml

(=0.0187ppm). Thisresultis inaccordance withsomeprevious

quantitativeworksbyHPLC,whichalsodidnotdetectalkaloids

in the Passiflora species investigated, at concentrations higher than0.1ppm(Rehwaldetal.,1995).Griceandco-workers(2001) detectedalkaloidsincommercialsamplesofP.incarnataatlevels lowersthan0.018ppm,butusingafluorescencedetectorinstead ofadiodearraydetector.

Nevertheless,thenon-detectionofalkaloids,despiteappearing asanegativeresult,hasgreatrelevance.Theaqueousextracts eval-uatedwerepreparedaccordingtotheuseofPassifloraleavesin

folkmedicine.Thus,itwasdemonstratedthatthesecompounds

areabsentinthetraditionalpreparations.However,thesedatado notruleoutthepresenceofharmanealkaloidsinthesespecies,

Table3

Calibrationdataofharmane.

Technique Linearityrange(␮g/ml) Calibrationequationa _{Correlationfactor(r}2_{) LOQ}b_{(␮g/ml) LOD}b_(␮g/ml)

CE 50.0–1.0 y=2.4144x+1.0596 0.9997 0.5 0.25

UPLC 50.0–0.065 y=123720x−6479.9 0.9999 0.0315 0.0187

I

II

III

B

A C

mAU

1200

600

0

0

100

50

0

3 5 7

min

18 min 80

0

0 5 15

BC A

10 0

mAU

25 min 40

P. tripartita P. bogotensis P. quadrangularis P. alata

P. tripartita P. bogotensis P. quadrangularis P. alata

Fig.4. Chromatograms(I:HPLC;II:UPLC)andelectropherogram(III)ofalkaloidsstandards(A:harmol;B:harmane;C:harmine)andofaqueousextractsoftheleavesof

whichmaybepresentinotherorgansoftheplant,oreveninthe leaves,inwhichaspecificalkaloidextractivemethodcouldbeused forthispurpose.

Saponinanalysis

Eventhoughmanysaponinshavebeendescribedforthegenus

Passiflora,theoccurrenceofthesemetabolitesisrestrictedtojust afewspecies.ThepresenceofsaponinsinP.alataandP. quadran-gularishasalreadybeenreportedinpreviousworksofourgroup (Reginattoetal.,2004;Birketal.,2005),ashasacomparative anal-ysisbyHPLCofthesetwospecies(Costaetal.,2013).Theadditional

resultspresentedherein,usingthesamechromatographic

condi-tions,indicatethattheextractsofP.bogotensisandP.tripartitavar.

mollissimaleavesshowednoevidenceofthesecompounds(see

supplementarymaterial).

Inconclusion,fastandsimpleanalyticalmethodsforthe finger-printingofflavonoidsandalkaloidsfromP.alata,P.quadrangularis, P.bogotensisand P.tripartita var. mollissimaextracts were suc-cessfullyestablishedbythreedifferenttechniques,showinggood

resolution and sensitivity. A wide diversity of flavonoids was

observedforthesefourspecies,whilesaponinswereaccumulated onlyinP.alataandP.quadrangularisextracts.Alkaloids,whose pres-enceiscontroversialinpreviouspapers,werenotdetectedbyanyof themethodsused.Theanalyticalmethodsandtechniquesreported hereinaresuitableforqualitycontrolanalysisofthesemetabolites inplantsamples,andwouldbeofgreathelpinfutureworkswith otherPassifloraspecies.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethattheyhave

fol-lowed theprotocolsof theirworkcenter onthe publicationof

patientdata.

Right to privacy and informed consent. The authors have obtainedthewritteninformedconsentofthepatientsorsubjects mentionedinthearticle.Thecorrespondingauthorisinpossession ofthisdocument.

Authors’contributions

GMCperformedtheextraction,analyticalwork,dataanalysis, anddraftingofthepaper.ACGcontributedtothechromatographic analysis.SMZcontributedtotheplantcollectionandextraction.LC andFARcontributedtothecollectionoftheplants,preparationof thevoucherspecimen,supervisionoflaboratorywork,andcritical readingofthemanuscript.FHRandEPSdesignedthestudy, super-visedthelaboratorywork,andcontributedtoacriticalreadingof themanuscript.Alltheauthorshavereadthefinalmanuscriptand

approvedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Theauthorsgreatlyappreciatethefinancialsupportprovided byFondoNacionaldeFinanciamientoparalaCiencia,laTecnología

y la Innovación, FranciscoJosé de Caldas, contract No. 0459 –

2013,RedNacionalparalaBioprospeccióndeFrutas

Tropicales-RIFRUTBIO.TheauthorsE.P.SchenkelandF.H.Reginattothankthe

CNPq-NationalCouncilofScientificandTechnologyDevelopment

(Brazil)forprovidingtheresearchfellowships.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2016.02.005.

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