Rev. bras. farmacogn. vol.25 número5

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w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Comparative

central

effects

of

the

aqueous

leaf

extract

of

two

populations

of

Passiflora

edulis

Adriana

S.F.S.J.

Ayres

a

_,

_Luana

_L.S.

_de

_Araújo

a

_,

_Thaciane

_C.

_Soares

b

_,

_Geison

_M.

_Costa

d

_,

Flávio

H. Reginatto

c

_,

_Freddy

_A.

_Ramos

d

_,

_Leonardo

_Castellanos

d

_,

_Eloir

_P.

_Schenkel

c

_,

Vanessa

P. Soares-Rachetti

a

_,

_Silvana

_M.

_Zucolotto

b

_,

_Elaine

_C.

_Gavioli

a,∗

a_Laboratorio_de_Farmacologia_{Comportamental,}_Departamento_de_Biofisica_e_{Farmacologia,}_Universidade_Federal_do_Rio_Grande_do_Norte,_Natal,_RN,_Brazil b_Laboratório_de_{Farmacognosia,}_Departamento_de_Farmacia,_Universidade_Federal_do_Rio_Grande_do_Norte,_Natal,_RN,_Brazil

c_Programa_de_{Pós-graduac¸}_ão_em_Farmácia,_Universidade_Federal_de_Santa_Catarina,_{Florianópolis,}_SC,_Brazil d_Departamento_de_Química,_Facultad_de_Ciencias,_Universidad_Nacional_de_Colombia,_Bogotá,_Colombia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6March2015

Accepted25June2015

Availableonline26July2015

Keywords:

Passifloraedulisedulis Passifloraedulisflavicarpa

Anxiolytic Antidepressant Sedation

Animalmodels

a

b

s

t

r

a

c

t

PassifloraedulisSims,Passifloraceae,hasbeenusedinBraziliantraditionalfolkmedicinetothetreatment

ofanxietyandinsomnia.P.edulisiscommonlyknownforitseconomicinterestsinBrazil.Thisspecies

exhibitssignificantvariabilityinthefruitrindcolor,thentwosubpopulationshasbeendescribed(P.

edulisfo.flavicarpaO.Deg.(PEF);P.edulisfo.edulis(PEE)).Thisstudycomparedphytochemicalprofile

andbiologicalactionsofaqueousleafextractofPEEandPEF.HPLCanalysisshowedmarkeddistinct

chromatogramstotheP.edulisvarieties.However,inbothextractsthemajorcompoundsobserved

wereflavonoidsC-glycosides.BehavioralstudiesshowedthatPEE(300mg/kg,p.o.)andPEF(100and

300mg/kg,p.o.)reducedanxietyintheelevatedplusmazetest.PEE(300and1000mg/kg,p.o.)andPEF

(1000mg/kg,p.o.)alsoinducedantidepressant-likeactionsintheforcedswimmingtest.PEE1000mg/kg

significantlyreduceddistancemoved,thussuggestingsedation.Noalterationsinsleepingtimewere

observedwithPEEandPEFextracts.Inconclusion,despitethesimilaritiesbetweenthebiologicalactions

observedforbothP.edulisvarieties,quitedifferentphytochemicalprofilewashereinreported.Thesedata

suggestthattheanxiolyticandantidepressantactionsarenotduetoaspecificphytochemicalcomponent.

Introduction

ThegenusPassiflora,Passifloraceae,comprisesabout500plant species,mostlydistributedintropicalandsubtropicalregionsofthe world.Manyspeciesofthisgenushavelongbeenusedintraditional folkmedicinesworldwidetothetreatmentofanxiety,insomnia, epilepsy,spasm,andaches(forreviewsee:Dhawanetal.,2004; Guzman-Gutiérrezetal.,2014).PassifloraedulisSimsisavariety commonlyknownforitstastyfruitbutalsousedinBrazilian tra-ditionalfolkmedicine(Bernaccietal.,2008).Asfirstlynoticedby Degener(1932),P.edulisspeciesexhibitsconsiderable morpholog-icalvariability.Thisplantproducestwotypesoffruit:thepurple (Passiflora edulis fo.edulis) andyellow fruit(Passifloraedulis fo. flavicarpaO.Deg.)(Bernaccietal.,2008).Thesedistinctionsgo fur-ther,theP.edulis fo.edulisspecies presentsflowerswithwhite petalsandpurplefilamentsonthelowerhalf,andwhiteonthe

∗ Correspondingauthor.

E-mail:ecg@pq.cnpq.br(E.C.Gavioli).

tophalfandbrownish-purplefruit,withalengthof6–7cm.The flowersofP.edulisfo.flavicarpaexhibithighlypurplecoronaatthe baseandlargeryellowfruits(i.e.,6–12cmlong)(Bernaccietal., 2008).Indeed,theirgeneticconstitutionswerefoundhavinglow similarity(Aukaretal.,2002),andtheirvolatilecompoundswere significantlydifferent(Pontesetal.,2009).Despitethese dissimi-larities,P.edulisfo.flavicarpaisusuallyconsideredjustavarietyof theP.edulisspecies,thustheinfraspecifictaxonomyofP.edulisis contradictoryandworthfurtherdiscussion(forreviewseeBernacci etal.,2008).

In2010,theofficialmonographofP.edulisspecies(no descrip-tionofthevariety)wasincludedintheBrazilianPharmacopoeia (2010)aswellastheofficialmonographofP.alataCurtis.An impor-tantfeatureofPassifloraspeciesisthat,duetothefolkmedicinal uses,P.edulis,P.alataandP.incarnatawereincludedintheNational ListofMedicinalPlantsofInteresttoBrazilianPublicHealth Sys-tem(MinistériodaSaúdedo Brasil,2009).Thislistincludes71 species ofmedicinalplants withpotentialtogenerate pharma-ceuticalproductsofinteresttoBrazilianpublichealth(Ministério daSaúdedo Brasil,2009).Regardingtherelevance ofPassiflora

http://dx.doi.org/10.1016/j.bjp.2015.06.007

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speciesas food,Brazilis considered thetop worldproducer of yellowfruits(P.edulisfo.flavicarpa).Theseobservationsreinforce thevalueof pharmacologically validating thetraditional useof Passifloraspeciesabundantlycultivatedand medicinallyusedin Brazil.

Invivostudieshavelongbeenreportingthebiologicalactions ofP.edulisSimsinrodents(Petryetal.,2001;Coletaetal.,2006; Reginattoetal.,2006).However,noformaldistinctionbetweenP. edulisvarietieshasbeenregistered.Anexceptionwasfoundina studypublishedin2011byaChinesegroup,whichcomparedthe chemicalconstitutionandbiologicaleffectsoftheethanolextract oftwopopulationsofP.edulis(‘edulis’and‘flavicarpa’)cultivated inChina(Lietal.,2011).In2010,thesameChineseresearchgroup hasreportedthebehavioraleffectsofethanolextractoftheaerial partofP.edulisfo.flavicarpaanditsfractionsinmice(Dengetal., 2010). Considering theimportance of validating the traditional folkusesofP.edulisvarieties,whichhashugeeconomicrelevance inBrazil,thepresentstudyaimedtocomparethephytochemical profileandthecentraleffectsoftheaqueousleafextractofthe twopopulationsofP.edulis(‘edulis’and‘flavicarpa’)cultivatedin SouthAmerica.

Materialsandmethods

Chemicalandreagents

AllsolventsusedwereHPLCgradeandMilliQwater,filtered in 0.45␮m membranes (MILEX®) and degassed by ultrasound bath.Thereferencestandardsusedwerevitexin-2′′_-_O_-rhaminoside (Fluka,98.2%),isoorientin(Sigma,>98%), orientin(Sigma,>97%), isovitexin(Fluka,>95%), vitexin(Fluka, >95%),and luteolin-7-O -glucoside(Fluka, >98%).The compound6,8-di-C-glycosylchrysin wasisolatedfromLychnophoraericoidesleavesandwasprovidedby Dr.NorbertoPeporineLopes.Spinosinandswertisinwereisolated fromWilbrandea ebracteataroots (Santos etal., 1996). Vicenin-2wasisolatedfromP.edulisfo. flavicarpaleavesand identified byNMRspectraldata(1_H_and _2D-NMR_{techniques-COSY,} _HSQC andHMBC)andthepuritywasconfirmedbyHPLC/DAD(Zucolotto etal.,2009).Thereferencestandardswereanalyzedat50␮g/ml concentration.Thesolutionswerefilteredin0.45␮mmembranes (MILEX®₎_and_a₂₀

␮laliquotofthefiltratewasinjectedforHPLC analysis.

Plantmaterial

Theleavesof Passifloraedulisfo.flavicarpa O.Deg.were col-lectedinAntônioCarlos,SantaCatarina,Brazilandidentifiedby thebotanistProf.Dr.DanielFalkenberg(DepartmentofBotanyof theFederalUniversityofSantaCatarina,Florianópolis,SC,Brazil). AvoucherspecimenwasdepositedintheHerbariumatthesame university(FLOR33886).TheleavesofP.edulisSimsfo.eduliswere collectedin SantaSofia,Boyacá,Colombiaand identifiedbythe botanistLuisCarlosJimenez(InstitutoNacionaldeCiencias, Univer-sidadNacionaldeColombia).Avoucherspecimenwasdepositedin theherbariumatthesameuniversity(COL530661).

Preparationofextracts

The leaves of P. edulis fo. edulis and P. edulis fo. flavicarpa wereair-driedat40◦_C,_powdered_and_extracted_using_hot_water (90◦_C) _by _infusion_(plant: _solvent,_1:10, _w/v) _for ₁₀_min._After that,theextractswerefilteredandfreeze-dried.ForHPLCanalysis, eachextractwaspreviouslyfilteredthrougha0.45␮mmembrane (MILEX®_)._The_extracts_were_solubilized_in_{methanol:water}_(1:1,

v/v)at7.5mg/mland20␮lwasusedforHPLCanalysis.

Phytochemicalanalysis

HPLC analysis was performed in a Varian® _{chromatograph}

pumpProStar240,ProStar410autoinjector,coupledtoProStar 335 DAD detector and Phenomenex-Luna 5␮m C18 (2) 100A (250mm×4.6mm)(column).Themobilephasewas(A) acetoni-trileand(B)aceticacid0.3%,usingthefollowinggradient0–10min 10:90%(A) in (B),10–40min 20:80%(A) in (B),and 40–90min 20:80%(A)in(B)ataflowrateof1ml/minandUV345nm.Peaks foundin thechromatogramswere identified bycomparingthe retentiontime(RT)andUVspectraofthereferencestandardsand byco-injectionofreferencestandardsplusextractstocomparethe increaseofpeakarea.

Animals

Male Swiss mice (weighing 25–40g) from our breeding colony were used. Animals were housed in plastic cages (41cm×34cm×16cm),12percage,undera12-hlight/darkcycle (lightson6a.m.) atroom temperatureof 23±1◦_C, _with_water andfoodadlibitum.Allexperimentalprocedureswereconducted between2 and 5p.m.and were performedin accordance with theBrazilianlawn◦_11.794/2008_for_experimental_use_of_animals. ThisstudywasapprovedbylocalEthicCommitteeforAnimalUse (LicenseN◦_032/2010).

Drugsandtreatments

Diazepam(CristáliaProd.Quím.FarmacêuticosLtda.,SãoPaulo, Brazil), an anxiolyticdrug, was solubilized in tween 80 (0.5%) andsaline,anditwasinjectedintraperitoneally(i.p.)at1mg/kg (10ml/kg),30minbeforetesting.Nortriptyline(Novartis Biociên-ciasS.A.,SãoPaulo,Brazil),anantidepressantdrug,wassolubilized insalineandi.p.administeredat30mg/kg(10ml/kg),30minprior theforcedswimmingtest.Salinewasusedascontrol.

Aqueousextracts of Passiflora weresolubilized in tap water andadministered orally(p.o.), byusinga syringecoupledtoan oral cannula (0.1cm×4cm), in a volume of 10ml/kg, 60min beforetesting.Tapwaterwasusedascontrol.Thiopentalsodium 50mg/kg (Cristália Prod. Quím. FarmacêuticosLtda., São Paulo, Brazil),abarbituricacid,wasinjectedi.p.60minpriorthePassiflora extracts.

Behavioraltests

Elevatedplus-maze(EPM)test

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Openfieldtest

Spontaneouslocomotoractivityofmicewasmeasuredusingthe openfieldtest.Theapparatus,madeofwoodcoveredwith imper-meableformica,hadablackfloorof40cm×40cmandblackwalls of40cmhigh.Thetestroomhadacontrolledillumination (dimly-lightcondition).Eachmousewasplacedinthecenteroftheopen fieldandthedistancetraveledevery5minwereregistered dur-ing30minthroughautomaticobservation(Anymaze,StoeltingCo., WoodDale,IL,USA).Afterthebehavioralevaluationofeachmouse, thearenawascleanedwith10%ethanolsolution.

Forcedswimmingtest

ThistestwasperformedaccordingtoPorsoltetal.(1977).Mouse wasindividuallyforced toswim ina transparentglasscylinder (24cminheight,18cmindiameter)filledwith18cmofwaterat 23±1◦_C._The_time_spent_immobility_(in_s)_was_measured_during thelast4minofasingle6-mintestsession.Micewereconsidered immobilewhentheymadenofurtherattemptstoescapeexcept themovementsnecessarytokeeptheirheadsabovethewater.

Thiopental-inducedsleepingtimetest

Thiopentalwasusedtoinducesleepaspreviouslydescribedby Vogel andVogel (1997).Thiopental50mg/kg wasadministered i.p. 60minafter administrationof Passiflora extracts.The inter-val time betweeninjection of thiopental and time that animal lossrightingreflexwasrecorded(ins)aslatencytime.The inter-val,inseconds,betweenlossandrecoveryofrightingreflexwas

registeredassleepingduration,anditwasusedasindexofhypnotic effect.

Statisticalanalysis

Thedatahereinpresentedwerereportedasmean±SEM.All datawereanalyzedusingGraphPadInStat3.06(GraphPadSoftware Inc.,SanDiego,USA).AllresultswereinitiallysubmittedtoLevene’s testforhomogeneityofvarianceandtoKolmogorov–Sminorv’stest fornormality. Comparisonsbetweentreatedandcontrolgroups wereperformedusingone-wayANOVAfollowedbyDunnett’stest orStudent’st-test,asdetailedin thefigurelegends.Differences wereconsideredsignificantwhenp<0.05.

Resultsanddiscussion

Phytochemicalanalysis

TheaqueousextractsfromP.edulisfo.edulisandP.edulisfo. flavi-carpawereanalyzedbyHPLC/PAD.Thechemicalprofileshowedin chromatogramsismarkedlydifferenttotheP.edulisvarieties.P. edulisfo.edulispresentspeaksbetween30and80min,themajor peakswereobservedinRT=32.4and58min(Fig.1A).Incontrast,P. edulisfo.flavicarpashowspeaksbetween20and50minandmajor peakswithRT=29.3and36min(Fig.1B).Accordingtothe max-imaabsorptionfoundintheUVspectra,themainpeaksobserved in thechromatograms of both extracts seem to be flavonoids.

70

60

50

40

30

20

10

0

70 80

Absorbance Units (mA

U)

5

0 10 20

OH

OH HO

mAU

mAU rham-O-glc

glc

glc glc

glc

glc glc

HO

HO O

O O

O

O O OH

OH

OH O

O O O

O O

O

(2)

(4)

(5)

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1

2

3

5

6

7

4

1

3

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7 (3)

A

B

25

15 30 35 40 45 50 55 60 65 70 75 80 85 90

0 5 10 15 20 25 30 40 45 50 55 60

Time (min)

65 70 75 80 85 90

35 60

50

40

30

20

10

0

Fig.1.HPLCchromatogramsofaqueousextractsofPassifloraedulisfo.edulis(A)andPassifloraedulisfo.flavicarpa(B)leaves7.5mg/ml.UV=345nm.(A)1,4,5and

7=luteolinderivatives,2=vitexin-2′′_-_O_-rhamnoside,₃₌_luteolina-7-_O_-glucoside,₆₌_apigenin_derivative._(B)₁_and₆₌_luteolin_derivatives,₂₌_vicenin-2,3₌_apigenin

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Table1

FlavonoidsidentifiedinPassifloraedulisfo.edulisandP.edulisfo.flavicarpa.

Flavonoid Isoorientin Orientin Vitexin Isovitexin Vitexin-2′′_-_O

-rhamnoside

Luteolin-7-O -glucoside

Vicenin-2 Spinosin 6,8-di-C-glycosyl chrysin

Swertisin

P.edulisfo.flavicarpa + − − ++ − − ++ − ++ −

P.edulisfo.edulis − − − − + + − − − −

Notes:+,Compoundidentified;++majorcompound;−compoundnotidentified.

Co-injection of extracts plus reference standards showed an

increasein thepeakareaofsomeflavonoids assummarized in

Table1.

RegardingtheP.edulisfo.edulisextract,onlytwominoritypeaks were identified: vitexin-2′′_-_O_-rhamnoside _(peak _2, _RT₌₃₆_min) and luteolin-7-O-glucoside (peak 3, RT=42min) (Fig. 1A). In general, peaks showed the typical UV absorption of luteolin derivatives:peak1(max=261,349nm),4(max=264,348nm),5 (max=264,348nm),and 7(max=297,348nm);exceptfor peak 6 (max=267,342nm) that presented UV absorption similar to flavonoidapigenin (Fig. 1A) (Mabry et al., 1970).As regardsP. edulisfo.flavicarpaextract,vicenin-2(peak2,RT=28.2min), isoori-entin (peak 4, RT=30.2min), 6,8-di-C-glycosylchrysin (peak 5, RT=35.5min)andisovitexin(peak7,RT=38.5min)wereidentified asthemajorpeaks(Fig.1B).Thepeaks1(max=266,347nm)and6 (max=266,345nm)displayedanUVabsorptionsimilartoluteolin derivatives,whilepeak3(max=268,339nm)wascharacterizedas anapigeninderivative(Fig.1B)(Mabryetal.,1970).

Briefly,thechromatogramsofP.edulisfo.flavicarpawerequite differentfromthoseofP.edulisfo.edulis.Sixmajorpeaks(between 55and80min)observedintheP.edulis fo.edulishadnotbeen detectedin P.edulis fo. flavicarpa. Theseobservations reinforce theviewthatthesetwoformsofP.edulisaredifferentfromeach other.Zucolottoandcolleagues(2011) havepreviously demon-stratedthepresenceofC-glycosyl flavonoidsinSouthAmerican Passiflora species, includingthe P.edulis fo. edulisand P.edulis fo.flavicarpa.ConcerningtheP.edulisfo.flavicarpa,inthisstudy, vicenin-2,isoorientin, isovitexin, orientin, vitexin, spinosin and 6,8-di-C-glycosylchrysin were identified, while to the P. edulis fo.edulisextractnoreferencestandardsusedinthis workwere identified(seeTable1).Previously,Lietal.(2011)showedthe pres-enceofthefollowingflavonoidsinP.edulisfo.flavicarpaethanol extract: lucenin-2, vicenin-2, isoorientin, isovitexin, luteolin-6-C-chinovoside,andluteolin-6-C-fucoside.Inagreementwithour data,Lietal.(2011)alsodemonstratedthatnoneoftheseflavonoids

weredetectedinP.edulisfo.edulis.Theseobservationsreinforce theviewthatthesetwovarietiesofP.edulisaredifferentfromeach other.However,distinctlocaland collectiontimesoftheherein studiedP.edulisvarietiesshouldbetakenintoaccounttoexplain differencesinthephytochemicalprofile.

Behavioraltests

Elevatedplus-mazetest

BeforestartingthebehavioralevaluationofPassifloraextracts, theeffectsofthestandardanxiolyticdrug,diazepam,wasassessed inourexperimentalconditions.Micei.p.injectedwithdiazepamat 1mg/kgdisplayedasignificantincreaseinthepercentageoftime spentin (vehicle: 12.3±7.0%; diazepam: 42.0±4.5%*; *p<0.05, Student’st-test)andentriesinto(vehicle:19.3±5.0%;diazepam: 51.0±4.5*%;*p<0.05,Student’st-test)openarms.Asshowedin Fig.2,P.edulisfo.edulis300mg/kgsignificantlyincreasedthe per-centageofentriesintoopenarms(Fig.2A;F(3,42)=3.096;p<0.05, ANOVA,Dunnett’stest).Similarly,P.edulisfo.flavicarpa100and 300mg/kgevokedasignificantincreaseinthepercentageofentries intoopenarms(Fig.2D;F(4,51)=4.206;p<0.05,ANOVA,Dunnett’s test).Nosignificanteffectsinthepercentageoftimespentintoopen armsandfrequencyofentriesintoclosedarmswereobservedin micetreatedwiththeextractsofthesetwopopulationsofPassiflora (Fig.2B,C,E,F;p>0.05).

Open-fieldtest

Consideringthecumulativedistancetraveled,atrendtoreduce this behavioral parameter was observed in P. edulis fo. edulis (1000mg/kg)-treated mice (Fig. 3A; F(2,25)=2.965; #p=0.07, ANOVA,Dunnett’stest).TheadministrationofP.edulisfo.edulisat 1000mg/kgsignificantlyreducedthedistancetraveledatthefirst 5minofobservationcomparedtocontrol(Fig.3B;F(2,25)=3.233; *p<0.05, ANOVA,Dunnett’stest).P.edulis fo.flavicarpa didnot

P. edulis fo. edulis

10 20 30 40 50

A

*

0 100 300 1000 mg/kg

0 30 100 300 1000

% Entries into open arms

P. edulis fo. edulis

10 20 30 40 50

B

% Time in open arms

0 5 10 15 20

C

Entries into closed arms

P. edulis fo. flavicarpa

10 20 30 40 50

D

*

_*

mg/kg

0 100 300 1000 mg/kg 0 100 300 1000 mg/kg

0 30 100 300 1000 mg/kg 0 30 100 300 1000 mg/kg

% Entries into open arms

P. edulis fo. flavicarpa

0 10 20 30 40

E

% Time in open arms

0 5 10 15 20

F

Entries into closed arms

Fig.2.EffectsoftheacuteadministrationofP.edulisfo.edulis(100,300and1000mg/kg,p.o.)andP.edulisfo.flavicarpa(30,100,300and1000mg/kg,p.o.)onthepercentage

ofentriesinto(A,D)andtimespentinopenarms(B,E),besidesonthefrequencyofentriesintoenclosedarms(C,F)intheelevatedplusmazetest.Resultsarerepresented

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20 40 60 80

A

&

0 300 1000 mg/kg

Distance traveled (m)

0 5 10 15 20 25 30 35

0 5 10 15

20 Control

300 mg/kg

1000 mg/kg

B

*

Time (min)

20 40 60 80

C

0 300 1000 mg/kg

0 5 10 15 20 25 30 35

0 5 10 15

20 Control

300 mg/kg

1000 mg/kg

D

time (min)

Fig.3.EffectsoftheacuteadministrationofP.edulisfo.edulis(300and1000mg/kg,p.o.)andP.edulisfo.flavicarpa(300and1000mg/kg,p.o.)onthecumulativedistance

traveledduring30min(A,C)andontheaccumulateddistancetraveledoverconsecutive5mintime(B,D)intheopenfieldtest.Resultsareexpressedasmean±SEMof8–10

animalspergroup.*p<0.05vs.control;&_p₌_0.07_vs._control,_ANOVA_followed_by_Dunnett’s_test.

affectthedistancetraveledbymiceduringthe30minof obser-vation(Fig.3CandD;p>0.05).

SeveralstudiesinvestigatedtheeffectsofP.edulisinanxiety. However,experimentalresultshave broughtsomecontroversy. Petryetal.(2001)showedthatP.edulisSimsreducedanxietyin rodents,aswellasColetaetal.(2006)andBarbosaetal.(2008). However,Dhawanetal. (2001)showedthat P.edulis Simswas devoidof anxiolyticactivity.These differences couldbedue to the fact that distinct subpopulations of P. edulis were used. In favorof thisview,Lietal. (2011)comparedtheanxietyeffects ethanolleafextractsof twovarietiesof Passifloraedulis(‘edulis’ and ‘flavicarpa’). They showed that P. edulis fo. flavicarpa dis-playedanxiolytic-likeactivityonlyatthehigherdosetested(i.e., 400mg/kg). However, P. edulis fo. edulis wasinactive at lower doses,whileat400mg/kgevokedsedation.Basedonthese obser-vations,Liet al.(2011) suggestedthat thebiological actionsof thesesubpopulationsofP.edulisaredistinct.IncontrastwithLi etal.,weobservedthat bothvarietiesofP.edulisreduces anxi-etybehaviors;beingP.edulisfo.flavicarpamorepotentthanthe variety‘edulis’.Indeed, thehypolocomotor/sedativeeffects of P. edulisfo.edulis,firstlyreportedbyLietal.(2011),wasalsoherein observed.DifferencesobservedbetweenthepresentstudyandLi etal.(2011)canbeexplainedbythedistinctionsintechnicalextract preparations(i.e.,aqueousandethanol,respectively),besidesthe differences in local and collection times of the used P. edulis varieties.

Forcedswimmingtest

Student’st-testrevealedasignificanteffectfortheacute admin-istration of nortriptyline in mice in the forced swimming test. Nortriptylinereducedthetimethatanimalsspentimmobileinthe watercomparedtocontrols(saline:100.16±15.11s;nortriptyline:

49.28±7.31s*;*p<0.05,Student’st-test).AsillustratedinFig.4, theadministration of theaqueous extract of P.edulis fo.edulis 300mg/kg(Fig.4A;F(3,81)=5.028;p<0.05,ANOVA,Dunnett’stest) and P.edulis fo. flavicarpa 1000mg/kg (Fig. 4B; F(2,56)=3.489; p<0.05,ANOVA,Dunnett’stest)significantlyreducedimmobility timeofmiceintheforcedswimmingtest.

Verylittleliteratureinformation supportstheantidepressant actionsof Passifloraspecies. A studysuggestedthat the antide-pressive effects of Hypericum perforatum are potentiatedwhen it iscombinedwithP.incarnata(Fiebichetal., 2011).Recently, forthefirsttimeitwasshowedthataPassifloraextractinduces antidepressant-likeeffects inmice.Anethanolextract ofleaves andstemsofP.edulis(thevarietyofP.eduliswasnotdetailed), 400–500mg/kg,givenorallyduringsevendays,reduced immobil-itytimeinthetailsuspensionandforcedswimmingtests(Wang etal.,2013).Inthesamestudy,cyclopassiflosidesIXandXI, com-pounds observed in largeamountin theethanol extract, were isolated, andthese cycloartanetriterpenoids weresuggested to bemediatingtheseeffects(Wangetal.,2013).Inourstudy,both extractsevokedantidepressantactions.However,P.edulisfo.edulis seemed tobemore potentcompared tothevariety‘flavicarpa’. Thesefindingsarepromisingandtheysupportaninnovative bio-logicalactioninducedbyP.edulisvarieties.

Thiopental-inducedsleepingtimetest

Atthedosestested,thetreatmentwiththeaqueousextractofP. edulisfo.edulisandP.edulisfo.flavicarpadidnotsignificantlyaffect neitherthelatencytosleepnorthesleepingtimedurationinduced bythiopental(Table2).

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50 100 150 200

*

A

0 100 300 1000 mg/kg

Immobility time (s)

P. edulis fo. flavicarpa

50 100 150 200

B

*

0 300 1000 mg/kg

Immobility time (s)

Fig.4.EffectsoftheacuteadministrationofP.edulisfo.edulis(100,300and1000mg/kg,p.o.)(A),andP.edulisfo.flavicarpa(300and1000mg/kg,p.o.)(B)ontheimmobility

timeofmiceintheforcedswimmingtest.Resultsarerepresentedmean±SEMof16–18animalspergroup.*p<0.05,ANOVAfollowedbyDunnett’stest.

Table2

EffectsofacuteadministrationofPassifloraedulisfo.edulis(PEE;300and1000mg/kg,

p.o.)andP.edulisfo.flavicarpa(PEF;300and1000mg/kg,p.o.)onthelatencytosleep

andsleepingtimedurationinducedbythiopental.

Extract Dose Latencytosleep(s) Sleepingtimeduration(s)

Vehicle – 155.2±8.0 403.5±66.9 PEE 300mg/kg 163.3±17.3 599.8±167.2 PEE 1000mg/kg 146.2±27.8 584.2±87.3

Vehicle – 223.1±17.0 647.7±187.1 PEF 300mg/kg 174.7±15.3 513.2±117.1 PEF 1000mg/kg 181.3±14.0 686.0±267.4

Resultsareexpressedasmean±SEMof10–12animalspergroup.

morphine-inducedsleeptimeinmice,andthisextractalsopartially blockedtheamphetamine-induced stimulatedeffects (Doetal., 1983).FewstudiesshowedthatP.edulisaqueousextractsreduce spontaneouslocomotoractivityandprolongsleepinmice(Maluf etal.,1991;Meier,1995).Senaetal.(2009)showedthatthe aque-ousextractfromthepericarpofP.edulisfo.flavicarpapotentiated thehypnoticeffectsofethyletherinmice.Inourstudy,the treat-mentwiththeaqueousextractofP.edulisfo.edulisandP.edulis fo.flavicarpadidnotsignificantlyaffectthiopental-induced sleep-ingtime.Ascommentedabove,apotentialsedativeeffectwasonly detectedfortheaqueousextractofP.edulisfo.edulisinmiceat 1000mg/kg.Possibly,thelowsedativeeffectofourextractscould beduetothepartofplantusedinourstudy(whichwasnotstated inthoseoldreports),methodsofextractpreparationordifferences betweenbioassays;additionally,aspectsrelatedtoseasonal varia-tionandgeographicaloriginofplantsshouldbetakenintoaccount.

Conclusion

Thepresentfindingssupportthatbothextractsshare anxiolytic-andantidepressant-likeactivities.Bycontrast,quitedistinct phy-tochemicalprofilewasreportedfortheaqueousextractofP.edulis varieties.Inboth extractsthemajorcompoundsobservedwere flavonoidsC-glycosides,suggestingthatthesebiologicalactionsare notduetoaspecificglycoside.Apossibleexplanationtothe biolog-icalsimilaritiesbetweenP.edulisfo.edulisandP.edulisfo.flavicarpa couldbeonthefactthatflavonoidsC-glycosidesorothersimilar productofthemetabolismofP.edulisvarietiesarepromotingthese sharedbiologicalactions.Finally,bothvarietiesofP.eduliscouldbe usedasaremedyforanxietyanddepression.

Authors’contributions

SMZandECG definedtheexperimentaldesignof thisstudy. ASFSJA,LLSA,TCSandGMCcontributedbyperformingthe biolog-icalandphytochemicalassays.FHR,FAR,LCandEPScontributed

topreparetheextracts.ASFSJA,SMZandECGdraftedthepaper, whileEPS,VPSRcontributedtocriticalreadingofthemanuscript. Allauthorsreadandapprovedthefinalmanuscriptsubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Thiswork wassupportedby funds fromCNPq/Brazil (Grant No. 304453/2012-9toECG)and fromthe FederalUniversity of RioGrandedo Norte.ASFSJAhelda PhDfellowship foundedby CAPES/Brazil.

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