Rev. bras. farmacogn. vol.25 número2

(1)

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

Involvement

of

GABAergic

pathway

in

the

sedative

activity

of

apigenin,

the

main

flavonoid

from

Passiflora

quadrangularis

pericarp

Andressa

C. Gazola

a,∗

_,

_Geison

_M.

_Costa

a,b

_,

_Leonardo

_Castellanos

b

_,

_Freddy

_A.

_Ramos

b

_,

Flávio

H. Reginatto

a

_,

_Thereza

_C.M.

_de

_Lima

c

_,

_Eloir

_P.

_Schenkel

a

a_Programa_de_{Pós-graduac¸}_ão_em_Farmácia,_Universidade_Federal_de_Santa_Catarina,_{Florianópolis,}_SC,_Brazil b_Departamento_de_Química,_Universidad_Nacional_de_Colombia,_Bogotá,_Colombia

c_Programa_de_{Pós-graduac¸}_ão_em_{Farmacologia,}_Universidade_Federal_de_Santa_Catarina,_{Florianópolis,}_SC,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6September2014 Accepted13March2015 Availableonline1April2015

Keywords:

Apigenin

Passifloraquadrangularis

Passionflower Passionfruit Sedative

a

b

s

t

r

a

c

t

InthecurrentstudyweshowedthatoraladministrationofanaqueousextractofPassifloraquadrangularis L.,Passifloraceae,pericarpresultsinasignificantprolongationofthesleepdurationinmiceevaluatedin theethylether-inducedhypnosistestwhichindicatessedativeeffects.Apigenin,themainflavonoidof theextract,inducedasimilarsedativeresponsewhenappliedalone,atadoseequivalenttothatfound intheextract,suggestingthatapigeninismediatingthesedativeeffectsofP.quadrangularisextract.In addition,thesedativeeffectofapigeninwasblockedbypretreatmentwiththebenzodiazepine antag-onistflumazenil(1mg/kg),suggestinganinteractionofapigeninwithgamma-aminobutyricacidtype A(GABAA)receptors.However,apigeninatconcentrations0.1–50␮MfailedtoenhanceGABA-induced

currentsthroughGABAAreceptors(␣1␤2␥2S)expressedinXenopusoocytes.Nevertheless,basedonour

results,wesuggestthattheinvivosedativeeffectoftheP.quadrangularisextractanditsmainflavonoid apigeninmaybebeduetoanenhancementoftheGABAergicsystem.

Introduction

The genus Passiflora has the most economical significance

amongallothergenerainthefamilyPassifloraceae.Itsspeciesare

mainlydistributedthroughoutLatinAmerica.Passiflora

quadrangu-larisL.isusuallycultivatedataltitudesbelow2500m(Killip,1938).

InBrazil,theoccurrenceofthisspeciesisdescribedinthestatesof

AmazonasandMaranhão(Cervi,1997)and alsoin Colombia,in

theregionsofChocó,Meta,HuilaandSantander.Popularlyknown

as“maracujá-ac¸u”inBrazil(Cervi,1997)and“badea”inColombia

(HernándezandBernal,2000),thisspeciesiswidelyconsumedin

Colombiaasjuice.Thefruitsaremuchbiggerthantheotherfruits

ofPassifloraspeciesshowingpaleyellowishgreencolorwhenripe

(Vanderplank,2000).

Todate,onlyfewpharmacologicalandchemicalstudieswith

pericarpsofPassifloraspeciesisavailable.Intraditionalmedicine,

preparationsusingleavesofsomespeciesareusedassedativesand

mildtranquilizers(LewisandElvin-Lewis,1977;Schindler,1884).

Inparticular,sedative, anxiolytic-likeandanticonvulsanteffects

∗ _{Corresponding}_author.

E-mail:[email protected](A.C.Gazola).

ofleavesextractsfromdifferentspecies ofPassiflora havebeen

reported(forareview,seeDhawanetal.,2004).

Regardingthechemicalcomposition,mostofthestudiesisalso

relatedtoleavesextractsandreportedthepresenceofflavonoids

(Petry etal.,1998;Ulubelenetal.,1982;Zucolottoetal.,2012)

andsaponins(Orsinietal.,1985;Reginattoetal.,2001).In this

respect,duethelackintheliteratureaboutneuropharmacological

andchemicaldataofpericarpfromPassifloraspeciesand

consid-eringthatthesedativeandanxiolytic-likeactivitiesofP.edulisvar.

flavicarpapericarpwerepreviouslydescribed(Senaetal.,2009),

theaimofthepresentstudywastoevaluatethesedativeactivity

ofP.quadrangularispericarpaqueousextracts,thecorrelationofthe

sedativeactivitytoitsflavonoidsconstituentsbesidestostudythe

involvementofthegamma-aminobutyricacidsystemintheaction

ofthesecompounds.

Materialsandmethods

Plantmaterialandextraction

Fruits of Passiflora quadrangularis L., Passifloraceae, were

collectedinColombia[Neiva,Huila(2◦₅₉′₅₅′′_,₋₇₅◦₁₈′₁₆′′_)],

iden-tifiedbyProf.LuisCarlosJimenez(InstitutoNacionaldeCiencias,

http://dx.doi.org/10.1016/j.bjp.2015.03.009

(2)

Universidad Nacional de Colombia) and a voucher specimen

wasdepositedintheHerbariumoftheUniversidadNacionalde

Colombia(COL572634).

Extractfortheethylether-inducedhypnosistestaswellasthe

HPLC-DADanalyseswasobtainedasfollows.Freshpericarpwas

directlyextractedbyinfusionwithhotwater(90◦_C;_{plant:solvent,}

1:3,w/v)for10min.Theaqueousextractsweresubsequently

fil-teredandfreeze-dried.

HPLC-DADanalyses

TheanalyseswereperformedonaPerkinElmerSeries200

high-performanceliquidchromatography(HPLC)systemequippedwith

diodearraydetection (DAD),quaternary pump,onlinedegasser

and auto-sampler (injectionvolume was20␮l). The separation

wasperformedwithaPerkinElmerBrownleeAnalyticalC18

col-umn(250mm×4.6mmi.d.;5␮m).Theflowwasmaintainedata

constantrateof1.2ml/min.Themobilephaseusedconsistedof

solventA(acetonitrile)andsolventB(0.5%formicacidinwater)

inalineargradientelution(0–20min–15%–35%ofA;20–25min

–isocratic35%A).Thechromatogramsweremonitoredat340nm

andUVspectrawererecordedinthe200–400nmrange.Thedata

were processed with Chromera software® _(Version _3.2.0.4847)

(PerkinElmer®

,Waltham,MA,USA).

Qualitative and quantitative analyses were performed with

the extract in a concentration of 5000␮g/ml. For qualitative

analyses the reference standards co-eluted with the extract

wereapigenin(≥95%–Sigma–Aldrich®_,_USA),_isoorientin₍

≥98%

– Extrasynthèse®_, _France), _vitexin-2′′_-_O_-rhamnoside ₍

≥98% –

Sigma–Aldrich®_,_USA),_vitexin-2′′_-_O_-xyloside₍_Costa_et_al.,₂₀₁₃_),

isovitexin(≥98%–Fluka®_,_USA)_and_swertisin₍_Santos_et_al.,₁₉₉₆_).

Thequantitativeanalysesweredevelopedusingtheexternal

standardmethod,beingapigeninusedasareferencesubstanceto

createacalibrationcurvewithsixpointsina0.5–40␮g/ml

con-centrationrange.Allofthestandardsolutionswereanalyzedin

triplicate,andtheaveragepeak areasweremeasured.The

vali-dationoftheanalyticalprocedureswasperformedinaccordance

withICHguidelines(2005).Inparticular,thefollowing

parame-terswereevaluated:specificity,linearity,accuracy,precision(both

repeatabilityandintermediateprecision),thelimitof

quantifica-tion(LOQ)andthelimitofdetection(LOD).

Animals

MaleadultSwissmice(3–4months)weighing 35–50gwere

usedfor behavioral evaluations.Animalsweremaintainedona

12-hlight-darkcycle(lightsonat7a.m.)ataconstantroom

temper-ature(23±2◦_C)._Mice_were_housed_in_groups_of_twenty_per_plastic

cage(30cm×37cm×16cm)withfoodandwaterprovidedad

libi-tumexceptduringtheexperiments.Allanimalswereallowedto

adapttothelaboratoryconditionsforatleastoneweekpriorto

thebehavioralassessmentsofthestudy.Onthedayofthe

exper-iment,animalswerehousedintheexperimentalroomforatleast

1hpriortothestartofthetestingprocedures.Eachanimalwas

usedonlyonce.Experimentswereconductedinaccordancewith

nationalandinternationalstandardsofanimalwelfare(Brazilian

Law#11,794,10/08/2008;NIHpublication#85-23,revisedin1996)

andapprovedbythelocalCommitteeforAnimalCareinResearch

(#23080.044085/2009-37/CEUA/UFSC).Allefforts weremadeto

minimizethenumberofanimalsusedandtheirsuffering.

Drugsandtreatments

Micewereorallytreated(p.o.)throughanintragastriccannula,

inaconstantvolumeof0.1ml/10gofanimalweight,withP.

quad-rangularisaqueousextractorapigenin,bothdissolvedindistilled

water.Intheethylether-inducedhypnosistest,theextractwas

testedatthedosesof100,300and600mg/kgandapigeninwas

testedatthedosesof0.1,0.3,0.6and1.0mg/kg.Anequalvolume

ofdistilledwater(vehicle,p.o.)treatedmiceofthecontrolgroups.

Diazepam(DZP–Hoffman-LaRoche®_,_Switzerland)_was_used_as

positivedrugcontrol,dissolvedinwaterwith10%ofpropylene

glycol,andadministeredp.o.atadoseof1mg/kginthesedative

test(Senaetal.,2009).Animalswereindividuallytested1hafter

theirtreatmentswiththeextractorapigeninand30minaftertheir

treatmentwithDZP.

InordertoassesstheinvolvementoftheGABAergicsystem,a

groupofanimalswerepretreated(i.p.)withphysiologicalsaline

solution(QuimibrásIndústriasQuímicasS.A.,Brazil)or

flumaze-nil(Flumazen®

,UniãoQuímicaLab.,Brazil–1mg/kg–Carvalho

etal.,2011)15minpriortheoraltreatmentwiththetest

com-poundapigenin(0.6mg/kg),thereferencedrugDZP(1mg/kg)or

distilledwater(controlgroup).

Ethylether-inducedhypnosistest

Animalswereplacedinanethylether-saturatedglasscage(6ml,

13minofsaturation,30cm×20cmglasscage)aftertreatments.A

stopwatchwasusedtorecordthedurationofhypnosisinseconds

(s).Thehypnosistimewasmeasuredbythelossoftherighting

reflex,andtherecoveryofthisreflexwasconsideredasthe

end-pointofthehypnosis(Vieira,2001).

Voltageclampanalysis

Preparation of stages V–VIoocytes from Xenopus laevis and

synthesis of capped off run-offpoly(A+) cRNA transcripts from

linearizedcDNAtemplates(pCMVvector)wereperformedas

pre-viouslydescribed(Khometal.,2006).Briefly,femaleXenopusLaevis

frogs(Nasco,FortAtkinson,USA)wereanaesthetizedbyexposing

themfor15mintoa0.2%solutionofMS-222(methanesulfonate

salt of3-aminobenzoicacidethyl ester;Sigma,Vienna,Austria)

beforesurgicallyremovingpartsoftheovaries.Folliclemembranes

from isolated oocytes were digested with 2mg/ml collagenase

solution (Type 1A).Selected stages V–VIoocytes wereinjected

withabout10–50nlofDEPC-treatedwater(diethyl

pyrocarbon-ate) containing thecRNAs at a concentration of approximately

300–3000pg/nl.TheamountofcRNAwasdeterminedbymeans

ofaNanoDropND-1000(KiskerBiotech,Steinfurt,Germany).To

ensureexpressionofthegamma-subunit,cRNAsforexpressionof

˛1ˇ22sreceptorsweremixedinaratioof1:1:10,respectively.

Afterinjection,oocyteswerestoredat18±8◦_C_for_24–48_h_in_ND96

solutioncontaining penicillinG (10,000IU/100ml)and

strepto-mycin(10mg/100ml).ElectrophysiologicalexperimentsonGABAA

receptorswereperformedusingthe

two-microelectrode-voltage-clampmethodataholdingpotentialof−70mV,makinguseofa

TURBOTEC01Camplifier(npielectronic,Tamm,Germany)andan

AxonDigidata1322Ainterface(MolecularDevices,Sunnyvale,CA).

DataacquisitionwasdoneusingpCLAMPv.9.2.Thebathsolution

contained90mMNaCl,1mMKCl,1mMMgCl2.6H2O,1mMCaCl2

and5mMHEPES(pH7.4).Microelectrodeswerefilledwith3MKCl.

GABAandapigeninwereappliedbymeansofafastperfusion

system(ScreeningTool,npielectronic,Tamm,Germany)(Baburin

etal.,2006)tostudytheGABA-inducedchloridecurrent(IGABA)

modulation.ToelicitIGABA,thechamberwasperfusedwith120␮l

ofGABAcontainingsolutionatavolumeratebetween300and

1000ml/s.TheIGABArisetimerangedfrom100to250ms(Khom

etal.,2006).Toexcludevoltage-clamperrors,oocyteswith

maxi-malcurrentamplitudes>3mAwerediscarded.

PotentiationoftheIGABA inpercentwasdefinedaccordingto

the formula:IGABA (%)=[I(GABA + apigenin)/IGABA−1]×100, where

(3)

Water 0 50 100 150 200

100

*

300

mg/Kg

Time (s)

600 DZP

Fig.1.EffectsoftheoraltreatmentwiththeaqueousextractfromthepericarpofP. quadrangularis(100,300and600mg/kg)onthedurationofthehypnosisinducedby ethyletherinmice.Diazepam(DZP)(1mg/kg,p.o.)wasusedascontroldrug.n=7–8 animals/group.Dataareexpressedasmean±S.E.M.*p≤0.05comparedwiththe controlgroup.Comparisonswereaccomplishedwithone-wayANOVAfollowedby Dunnett’stestorStudent’sttest(DZPgroup×controlgroup).

concentrationsofapigenin,andIGABAisthecontrolGABA-induced

chloridecurrent.

Statisticalanalysis

Results of the in vivo experiments are expressed as

means±S.E.M. Data of the experiments without flumazenil

pre-treatmentwere analyzed by Student’s t test (positive

con-trolgroups X control groups) or one-way analysis of variance

(ANOVA)followedbyDunnett’stest.Influmazenilpretreatment

protocol data were analyzed by two-way ANOVA followed by

Newman–Keuls’test.Allofthestatisticalanalyseswereperformed

usingGraphPad Prism®

5.0 (La Jolla,CA,USA) and/or Statistica

Statsoft® _7.0_(Tulsa,_OK,_USA)._Differences_were_considered_to_be

statisticallysignificantwhenp≤0.05.

Forinvitroexperiments,OriginSoftware®

(OriginLab

Corpora-tion)wasused.Responsesweregraphedasmean±S.E.M.fromat

leastthreeoocytesoutof≥2differentbatches.Statistical

signifi-cancewascalculatedusingone-wayANOVAfollowedbyTukey’s

testwithaconfidenceintervalofp≤0.05.

Resultsanddiscussion

Inthecurrentstudy,weevaluatedthesedativeactivityofthe

aqueousextract obtainedfrompericarpsofP.quadrangularisby

meansofethylether-induced hypnosistest.Thistestallowsthe

assessmentofsedativeeffectwithoutintroducingpossible

phar-macokinetic biases caused by hepatic metabolism of the sleep

inductor,suchasinthecaseofpentobarbital-inducedhypnosistest

(Duarteetal.,2007;Carvalhoetal.,2011).

Theethylether-inducedhypnosistestofPassifloraextract

As depicted in Fig. 1, the pericarp extract of P.

quadran-gularis increased the hypnosis duration (100 and 300mg/kg –

[F3.27=3.859, p=0.0203]), an effect similar to DZP. The

seda-tive activity of P. quadrangularis pericarp extract was not

previously reported. In the literature was found just one

neuropharmacological study with this species describing the

anxiolytic-likeeffectofthehydroethanolicextractfromleavesin

ratsevaluatedintheelevatedplus-mazetest(Castroetal.,2007).

Athigherdoses(600mg/kg),however,theextractdidnotshowa

sedativeactivity.Thereasonforthisfindingshouldbeduetothe

factthatathigherdosesallchemicalcompoundspresentinthis

extractcouldbepresentathigheramountandthelackofresponse

Time (min)

Absorbance (mA

U

)

0 0 10 20 30 40 50 60 70 80 90

2 4 6 8 10 1

*

12 14 16 18 20 22 24

Fig.2. Chromatogramof theaqueous extractfrom P.quadrangularispericarp (5000␮g/ml)at340nm.Co-elutionwithstandardsshowedthepresenceof vitexin-2′′_-_O_-xyloside₍₁₎_and_apigenin_(*)._See_subsection_HPLC-DAD_analysis_in_Materials

andMethodssectionforthedescriptionofthechromatographicconditions.

maybeattributedtothecompetitionbetweenseveralcompounds

oftheextractactingindifferentreceptors,evenexcitatoryones.

Thequalitativeanalysisoftheflavonoidconstituentsintheextract

Regardingthequalitativeanalysis,moststudiesareindicating

flavonoidsasthemainchemicalconstituentsresponsibleforthe

CNSactivitiesofPassifloraspecies(Petryetal.,2001;Lietal.,2011;

Senaetal.,2009).Therefore,thepresenceofflavonoidconstituents

wasinvestigatedinP.quadrangularispericarpextractbyco-elution

withapigenin,isoorientin,vitexin-2′′_-_O_-rhamnoside,_vitexin-2′′_-_O

-xyloside,isovitexinandswertisin standards.Previousstudiesof

ourresearchgroupidentifiedtheflavonoidvitexin-2′′_-_O_-xyloside

asthemajorcompoundintheleavesextractofthisplantspecies

(Costaetal.,2013).Nevertheless,thequalitativeHPLC-DAD

anal-ysisofthepericarpextractinco-elutionwithreferencestandards

showedthatthemain flavonoidisnota glycoside,butan

agly-coneidentifiedasapigenin(Fig.2).Thepresenceofotherminor

flavonoids wasvisible in thechromatogram but their contents

wereverylowandcouldnotbeisolated.Theco-elutionprocedures

employedallowedtheidentificationofoneofthemasthesame

gly-cosidefoundintheleaves,i.e.vitexin-2′′_-_O_-xyloside,_and_excluded

thepresence ofisoorientin, vitexin-2′′_-_O_-rahmnoside, _isovitexin

andswertisin(Fig.2).Therefore,asapigeninispresentinamuch

higher amount, quantitative analysiswas focused in this main

flavonoid.

Thequantitativeanalysisofapigeninintheextract

Inordertoquantifythecontentofapigeninintheextract,a

methodologywasdevelopedandvalidated.Theparameters

speci-ficity,linearity,accuracy,precision,limitofquantification(LOQ)

andlimitofdetection(LOD)wereevaluated(Tables1and2).The

quantificationdemonstrated agoodlinearrelationship between

peakareaandconcentration(r2_>_0.999)._LOQ_and_LOD_were_defined

bytherelativestandarddeviation(RSD>5%)andbyasignal:noise

ratioof3:1,respectively.TheRSDvaluesforrepeatabilityand

inter-mediateprecisionrangedfrom0.5to1.9%. Therecoveryofthe

spikedsamplesrangedfrom99to103%,showingthattheanalytical

approachusedinthisstudyisrepeatableandaccurate.

Sincetheproceduresofthequantitativeanalysiswereaccurate

allowingthecorrectquantificationofapigeninintheextract,they

wereemployedandthisflavonoidwasfoundas2.449±0.004mg/g

(4)

Table1

ValidationdataforapigeninquantificationbyHPLC-DAD.

Flavonoid Linearityrange(␮g/ml) Calibrationequationa _Correlation_factor_(r2₎ _LODb₍_␮_g/ml) _LOQb₍_␮_g/ml)

Apigenin 0.5–40 y=112834x–23037 0.9996 0.0625 0.25

a_Six_data_points₍_n₌_3).

b_LOQ,_limit_of_{quantification;}_LOD,_limit_of_detection.

Table2

ApigeninquantificationinPassifloraspeciesasevaluatedbyHPLC-DAD.

Flavonoid Repeatabilitya _Intermediate_precisiona _Recoverya,b

Concentration(␮g/ml) RSD(%) Concentration(␮g/ml) RSD(%) P.quadrangularis(5000␮g/ml) Mean(%) RSD(%)

Apigenin 1 1.9 10 0.5 103.7 0.1

10 0.7

40 1.1

RSD,relativestandarddeviation.

a_Limits:_RSD:_<5%.

b_Spiked_samples_with_apigenin_standard_solution_at₅_␮_g/ml.

Apigeninintheethylether-inducedhypnosistest

Apigeninwasevaluatedintheethylether-inducedhypnosistest

atdosesof0.1,0.3,0.6and1.0mg/kg.Thesedoseswereselected

consideringtheapproximatedquantityofapigeninpresentinthe

active dosesof theextract, in viewof thequantification result

forthiscompound. Thus,forthedoses100and 300mg/kgof P.

quadrangularis pericarpextract,the correspondingquantitiesof

apigeninare0.24and0.73mg/kg,respectively.

Among all the administered doses of apigenin, 0.6mg/kg

demonstratedasedativeeffect(F4.35=2.657,p=0.0490),resulting

inasignificantincreaseinthesleepingduration(Fig.3).

Theresultsobtainedsuggesta positiverelationship between

apigeninlevelsandthesedativeeffectoftheextract.This

assump-tionissupportedbyotherauthorswhodemonstratethatapigenin

atdifferentdosespossessessedativeeffect.Forexample,Avallone

etal.(2000)described thiseffectwhen apigenin(i.p.)was

eval-uatedbyspontaneouslocomotorbehavior inrats.Moreover,by

telemetrytests, Chowetal. (2011)demonstrated thatapigenin

(p.o.)promotedsedativeeffectinC57BL/6Jmice.

Ontheotherside,whenapigeninwasadministeredinthedoses

of0.1 and0.3mg/kg, i.e.,theapproximatedamountofapigenin

in100mg/kgofP.quadrangularispericarpextract,thisflavonoid

didnotshowanysedativeactivity(Fig.3),showingaUinverted

Water 0 50 100 150 200

0.1

*

0.3

mg/kg

Time (s)

0.6 1.0 DZP

Fig.3.Effectsoftheoraltreatmentwiththeflavonoidapigenin(0.1,0.3,0.6,and 1.0mg/kg)onthedurationofethylether-inducedhypnosisinmice.Diazepam(DZP) (1mg/kg,p.o.)wasusedasastandarddrug.n=8animals/group.Dataareexpressed intermsofmeans±S.E.M.*p≤0.05comparedwiththecontrolgroup.Comparisons wereaccomplishedwithone-wayANOVAfollowedbyDunnett’stestorStudent’st

test(DZPgroup×controlgroup).

dose-effectcurve.Somefindings indicatethat, as occursin our

results,thehormesismodelcouldpredictsresponsesinthelow

doses zone very wellin biological tests(Calabrese, 2009). This

factindicatesthattheeffectobservedforpericarpextractisnot

dueonlytothecontentofapigenin.Actually,thiscompoundcan

bethemainresponsible,butprobablythereisalsoasignificant

contributionofothercompounds.

StudyingapotentialinvolvementoftheGABAergicsysteminthe

sedativeactivityofapigenin

Losietal.(2004)reportedaninteractionofapigeninwiththe

GABAergicandglutamatergicneurotransmissionincultured

cor-ticalneurons.Theseneurotransmittersystemsplayakeyrolein

controllingtheexcitabilityofthebrainandarethustargetedby

many psychoactive substances as wellas clinically used drugs

(Mihicand Harris,2011).In addition,Lollietal. (2007)showed

thatmethanolandhydromethanolextractsfromP.actiniaexerts

anxiolytic-likeandsedativeeffectsinmiceafteroral

administra-tion.Authorsalsoshowthatpreviousadministrationofflumazenil

intheelevatedplusmazetestindicatethattheanxiolytic-like

activ-ityisrelatedtotheinfluenceoftheGABAergicactivation(Lollietal.,

2007)pointingthatthis pathwaycanbemodulatebyPassiflora

extracts.

Water 0 50

100 &

150

_*

Time (s)

DZP DZP Water API FMZ Saline

Fig.4. Effectsoftheoraltreatmentofapigenin(API–0.6mg/kg),diazepam(DZP– 1.0mg/kg)andwaterinanimalspre-treated(i.p.)withflumazenil(FMZ–1.0mg/kg) orsalineonthedurationofhypnosisinducedbyethyletherinmice.DZPwasusedas controldrug.n=7–8animals/group.Dataareexpressedasmean±S.E.M.*,&_p_≤_0.05

(5)

0

–10

–20

–30

–40

–50

–60

–70

–80

–90

–100

100

90

80

70

60

0.1 1 10 50

Concentration (μM)

*

Inhibition of l

GABA

(%)

P

otention of l

GABA

(%)

A

B

50

40

30

20

10

0

0.1 1 10 50

Fig.5. ModulationofIGABAthroughGABAAreceptors(˛1ˇ22s)atGABAconcentrationseliciting3–7%ofthemaximalGABAresponse(EC3–7)byapigenin(A)andluteolin

(B)attheindicatedconcentrations.Eachbarrepresentsthemean±S.E.M.fromatleast3differentoocytesoftwodifferentoocytebatches.Statisticalsignificance(p<0.05, one-wayANOVAfollowedbyTukey’stest)isindicatedby(*).

AsdepictedinFig.3,0.6mg/kgofapigeninincreasedsedationin

miceintheethylether-inducedhypnosistest.Thissedativeeffect

wasblockedbyapplicationofflumazenilatadoseof1.0mg/kg

(Fig.4).Sinceflumazenilisabenzodiazepineantagonist,thisresult

indicatesthatapigeninmightinteractwiththe

benzodiazepine-bindingsiteofGABAAreceptorstopromoteitssedativeeffect.

WehavethereforeexpressedGABAAreceptorscomposedof˛1,

ˇ2and2SsubunitsinXenopuslaevisoocytesandstudieda

poten-tialmodulationofGABA-inducedcurrents(IGABA)byapigeninand

luteolinmakinguseofthe2-microelectrodevoltage-clamp

tech-niqueandafastperfusionsystem.

However,asillustratedinFig.5A,apigeninfailedtoenhance

IGABA throughthis receptorsubtypeatalltested concentrations

(0.1–50␮M).Actually,as notexpected,it inhibitedIGABA atthe

higherconcentration,aresultthathasfurtherinvestigated.

Takingintoaccount thatthe sedativeeffect ofapigeninwas

observedafteroralapplicationitistemptingtospeculatethatnot

onlyapigenin,butanyofthemetabolitesformedafter

gastroin-testinaland/orhepaticmetabolismmightinvivomodulateGABAA

receptors.Gradolattoetal.(2004)reportedthatatleastseven

com-poundsareoriginatedfromhepaticmetabolismofapigeninwith

luteolinbeingidentifiedasthemainmetabolite.Therefore,wehave

alsoanalyzed themodulationof IGABA through ˛1ˇ22S GABAA

receptorsexpressedinXenopusoocytesbyluteolin.Asillustrated

inFig.5B,luteolinalsodidnotelicitedasignificantenhancement

ofIGABA.Thus,thisresultcouldnotexplainthesedativeeffectof

apigeninobservedafteroraltreatmentofmice,butitshowsthe

importanceofbetterunderstandthemetabolismofinvivo

admin-isteredcompounds.

Otherpossibleexplanationfor ourinvivofindings mightbe

thatapigenincanbeactingasa secondordermodulator.

Previ-ousstudiesshowedthat apigenindidnotmodulateIGABA when

appliedalonetothereceptor,butitshowedstrongeffectswhen

co-appliedwithanotherGABAAreceptormodulatorsuchasdiazepam

(Campbelletal.,2004).

Inaddition,theinfluenceofotherGABAAreceptorsubtypesin

thesedativeactivityofapigenincouldnotbediscarded. In this

sense,itwaspreviouslyshowedthat˛5subunitofGABAAreceptors

exertsinfluenceofthesedativeactivity(Savi ´cetal.,2008).

Inconclusion,thisstudyprovidesevidencesthattheaqueous

extractfromthepericarpofP.quadrangularisinducessedationin

mice.Inaddition,apigenin,themainflavonoidfoundintheextract,

alsoinducedsedativeeffect,suggestingthatthisflavonoidplaysan

importantroleinthesedativeeffectoftheextract.Furthermore,a

potentialinvolvementofGABAAreceptorsinmediatingthe

seda-tiveeffectofapigeninwasdemonstratedinvivobutnotinvitroand

themolecularmechanismremainstobeelucidated.

Conflictofinterest

Allauthorshavenonetodeclare.

Authors’contributions

ACG (PhDstudent) contributedin runningall thelaboratory

work,analysisofthedataanddraftedthepaper.GMCcontributedin

phytochemicalanalyses.LCandFARcontributedincollectionofthe

plant,confectionofvoucherspecimen,supervisionoflaboratory

workandcriticalreadingofthemanuscript.FHRsupervisedthe

lab-oratoryworkandcontributedtocriticalreadingofthemanuscript.

TCMLandEPSdesignedthestudy,supervisedthelaboratorywork

andcontributedtocriticalreadingofthemanuscript.Alltheauthors

havereadthefinalmanuscriptandapprovedthesubmission.

Acknowledgments

ThisresearchwasfundedbyCNPq,CAPES,FAPESC,COLCIENCIAS

andtheDIB-UniversidadNacionaldeColombia.ACGisrecipient

ofaPhDscholarshipfromFAPESCandreceivedaPDSE

scholar-shipfromCAPESinherforeignstageattheUniversityofVienna

underthesupervisionofDr.StephenHering.WealsothankCNPq,

whichprovidedresearchgrantstoEPS,FHRandTCML.Theauthors

aregratefultoProf.LuisCarlos Jimenezfor theidentificationof

plantmaterialandSophiaKhomandStephenHeringfortechnical

(6)

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