The document describes a study that evaluated the neuropharmacological effects of the methanol extract of Heteropterys brachiata (HbMeOH) in mice. HbMeOH produced significant antidepressant effects in the forced swimming test at 500 and 750 mg/kg doses. It also exhibited clear dose-dependent anxiolytic activity in the elevated plus maze at doses from 500 to 1500 mg/kg. A dose of 500 mg/kg showed significant anticonvulsant activity in the pentylenetetrazole-induced seizures test without sedative effects. The main compounds in HbMeOH were chlorogenic acid and chlorogenic acid methylester, as well as less abundant terpene-type compounds. The

1. Neuropharmacological in vivo effects andphytochemicalprofile
of theextractfromtheaerialpartsof Heteropterys brachiata (L.)
DC. (Malpighiaceae)
Maira Huerta-Reyes a,b,n, MaribelHerrera-Ruiz a, Manase´ s Gonza´ lez-Cortazar a, AlejandroZamilpa a,
Esther Leo´n c, RicardoReyes-Chilpa d, ArturoAguilar-Rojas a, JaimeTortoriello a
a Centro deInvestigacio´n Biome´dica delSur,InstitutoMexicanodelSeguroSocial,ArgentinaNo.1,Col.Centro,Xochitepec,MorelosC.P.62790,Mexico
b Unidad deInvestigacio´n Me´dica enFarmacologı´a, HospitaldeEspecialidades,CentroMe´dico NacionalSigloXXI,InstitutoMexicanodelSeguroSocial,
Av. Cuauhte´moc No.330,Col.Doctores,Del.Cuauhte´moc, C.P.06720MexicoD.F.,Mexico
c Herbario NacionaldeMe´xico (MEXU),InstitutodeBiologı´a, UniversidadNacionalAuto´noma deMe´xico, ApartadoPostal70-367,Del.Coyoaca´n, C.P.04510MexicoD.F.,Mexico
d Instituto deQuı´mica, UniversidadNacionalAuto´noma deMe´xico, Del.Coyoaca´n, C.P.04510MexicoD.F.,Mexico
a r t i c l e info
Article history:
Received 28September2012
Received inrevisedform
28 November2012
Accepted 29December2012
Available online10January2013
Keywords:
Heteropterys brachiata
Antidepressant
Anxiolytic
Anticonvulsant
Sedative
a b s t r a c t
Ethnopharmacologicalrelevance:Heteropterysbrachiata is aplantspeciesthathasbeenusedin
traditionalMexicanmedicineforthetreatmentofnervousdisorders.
Aim ofthestudy: To evaluatetheanxiolytic,anticonvulsant,antidepressantandsedativeeffects
producedbythemethanolicextractof Heteropterysbrachiata (HbMeOH)inICRmice.Additionally,
we determinetheacutetoxicityprofilesoftheextractandthepresenceofitsmainconstituents.
Material andmethods: The neuropharmacologicaleffectsoftheextractwereevaluatedusingavariety
of models,suchastheelevatedplusmaze(EPM),theforcedswimmingtest(FST),thepentobarbital
potentiationtest(PTBt),pentylenetetrazole-inducedseizurestest(PTZt),andtheopenfieldtest(OFT).
HPLC wasemployedforobtentionofphytochemicalprofile.
Results: HbMeOHproducedasignificantantidepressanteffectinFSTat500and750mg/kgdoses,while
doses from500to1500mg/kgexhibitedacleardose-dependentanxiolyticactivityinEPM.Adoseof
500 mg/kgshowedasignificantanticonvulsantactivityinPTZtandanabsenceofsedationeffectsin
PTBt.ThemaincompoundsofHbMeOHwerechlorogenicacidandchlorogenicacidmethylester,as
well aslessabundantterpene-typecompounds.Furthermore,theextractwaseithersafewithno
deaths inmicetreatedorallywith2000mg/kg.
Conclusions: HbMeOHextractwhichcontainsmainlyhydroxycinnamicacidsandtriterpene-type
compounds,possessesantidepressant,anxiolyticandanticonvulsivepropertiesandcanbeconsidered
safe oroflowtoxicitywhenorallyadministrated.Thesefindingslendpharmacologicaljustificationto
the traditionaluseof Heteropterysbrachiata in thetreatmentofnervousdisorders.
& 2013 ElsevierIrelandLtd.Allrightsreserved.
1. Introduction
Mental disordersaffect450millionpeopleworldwide,with121
millionsufferingfromdepression,andapproximately50million
people haveepilepsy(World HealthOrganization(WHO),2011). In
Mexico, approximately15millionindividualsexperiencesometype
of mentalhealthproblem,withdepressionandanxietybeingthe
mostcommon(Valencia,2007); however,theuseofmedical
national healthservicesbyMexicanpopulationispoor(o25%)
(Medina-Moraetal.,2003). Indevelopedcountries,70%to80%of
the populationresorttotraditionalmedicinetotreatillnesses,and
nearly53%ofpatientswithdepressionandanxietyutilizecomple-
mentaryandalternativeplant-relateddrugs(World Health
Organization(WHO),2008).Thisfactmaybeduetoseveralfactors,
includingthedesiretoavoidadverseeffectsaswellastotake
advantage ofthesafety,effectivenessandbetterqualitycontrolof
thephytomedicinesavailableonthemarkettoday(De Souzaetal.,
2009). Medicinalplantshavebeenanimportantresourceinthe
developmentofdrugs.Indeed,nearly25%oftoday’sconventional
drugsoriginateddirectlyorindirectlyfromplants(Zhang, 2004). A
considerablenumberofherbalmedicinesarerecognizedasactivein
Contents listsavailableat SciVerse ScienceDirect
journalhomepage: www.elsevier.com/locate/jep
JournalofEthnopharmacology
0378-8741/$ -seefrontmatter & 2013 ElsevierIrelandLtd.Allrightsreserved.
http://dx.doi.org/10.1016/j.jep.2012.12.049
n Corresponding authorat:CentrodeInvestigacio´n Biome´ dica delSur,Instituto
Mexicano delSeguroSocial,ArgentinaNo.1,Col.Centro,Xochitepec,MorelosC.P.
62790, Mexico.Tel.: þ52 7773612155;fax: þ52 7773612194;Unidadde
Investigacio´n Me´ dica enFarmacologı´a, HospitaldeEspecialidades,CentroMe´ dico
Nacional SigloXXI,InstitutoMexicanodelSeguroSocial.Av.Cuauhte´moc No.330,
Col. Doctores,Del.Cuauhte´moc, C.P.06720Me´ xico D.F.,Mexico.
Tel.: þ52 5556276900x21368.
E-mail address: chilanguisima@yahoo.com (M.Huerta-Reyes).
Journal ofEthnopharmacology146(2013)311–317

2. the centralnervoussystem(CNS),andtheyhaveatleasta
hypothetical therapeuticpotential toaffectchronicconditionssuch
as anxiety,depression,headachesorepilepsyincaseswhere
patients donotrespondwelltoconventionaltreatments
(Quintans-Ju´ nior etal.,2008).
Heteropterys brachiata (L.) DC.isawoodyvinebelongingtothe
Malpighiaceae familyandiswidelydistributedthroughoutthe
tropics andsub-tropicsfromMexicotoSouthAmerica(Anderson,
1993). InMexico,itispopularlyknownas‘‘BejucodeMargarita’’,
‘‘tsak tsaah’’or‘‘tipite-ek’’andhasbeenusedintraditional
medicine mainlyforthetreatmentofnervousdisorders(Argueta
et al.,1994). Someothersreportsindicatethat Heteropterys
brachiata has alsobeenemployedascontraceptiveaswellasin
the treatmentoffever,scabiesandrabies(Monroy-Ortiz and
Castillo-Espan˜ a, 2007). Despitethewidespreaduseofthisplantin
Mexican folkmedicine,thereisnoscientificallyinvestigation
reported abouttheplantanditsbiologicalproperties.Ourstudy
focuses ontheneuropharmacologicalactivitiesof Heteropterys
brachiata with respecttounderstandingitstraditionalmedicinal
applications andpotentialusesindrugdevelopment.Thepresent
study wasconductedtoevaluatetheanxiolytic,antidepressant,
sedative, anticonvulsantandinfluenceonthemotoractivity
effects producedbytheextractof Heteropterys brachiata in ICR
mice usingavarietyofmodels,suchastheelevatedplusmaze,
the forcedswimmingtest,thepentobarbitalpotentiationtest,
pentylenetetrazole-inducedseizurestest,andtheopenfieldtest.
Furthermore, theextractof Heteropterys brachiata was evaluated
to determineitsacutetoxicityprofileandanalyzedtoquantifyits
main constituents.
2. Materialsandmethods
2.1. Plantcollectionandidentification
The aerialpartsof Heteropterysbrachiata (L.)DC.werecollected
fromthestateofMorelos,Mexico(atlatitude18147024.500 north
andlongitude99110012.10 0 west)inSeptember2007.Theidentifica-
tion oftheplantwasauthenticatedbyanexpertinthefieldofplant
taxonomy,whoisalsooneoftheauthors(E.Leo´ n). Avoucherwas
depositedasreferenceattheMexicanInstituteofSocialSecurity
MedicinalHerbarium(IMSSM)underthenumber15442.
2.2. Preparationofextracts
The plantmaterialwasdriedinadarkatroomtemperature
and powered(1.64kg).Afterdewaxingwith n-hexane, theplant
material wassuccessivelyextracted(3) overnightwithmetha-
nol (100%).Theextractionvolumeusedwas7.5Lofsolventper
each kgofplantmaterial.Theliquidextractwasdriedbyremoval
of thesolventundervacuum.TheHbMeOH(175.7g)wasthen
used inthepharmacologicalexperiments.
2.3. Drugsandchemicals
Imipramine hydrochloride(IMI)andpentylenetetrazole(PTZ)
were purchasedfromSigma-AldrichChemicalCo.(St.Louis,MO,
USA). Diazepam(DZP)andcarboxymethylcellulose(CMC)were
obtained fromCryopharmaS.A.deC.V.(Guadalajara,Jal,Mexico).
Sodium pentobarbital(PEN)waspurchasedfromPfizerInc.(New
York, NY,USA).
2.4. Animals
The animalexperimentswereperformedinstrictadherencetothe
official requirementsoftheMexican RegulationsofExperimental
AnimalCare(NOM-062-ZOO-1999).Theexperimentalprotocolwas
approvedbytheinstitutionalresearchandethicscommittee(Registry
number2007-1701-8).Foreachneuropharmacologicalassay,groups
ofeightICRalbinomiceweighing30–36geachwereutilized(Harlan
Me´xico S.A.deC.V.,MexicoCity,Mexico).Theanimalswerehoused
incommunitycagesandmaintainedunderregularlaboratorycondi-
tions (2572 1C, 12-hlight-darkcycle,freeaccesstowaterand
standardrodentchow:2018S,HarlanTekland).Allanimalswere
acclimatizedfor3weekspriortoinitiationofthetest.Theexperi-
ments wereconductedinaspecialadjacentnoise-freeroomwith
controlledillumination.Each animal wasusedonlyonceinthe
experiment.
2.5. Neuropharmacologicalassays
HbMeOH wasadministrated p.o. at adoseof500mg/kg.
In thoseassaysthatexhibitedactivityattheinitialdoseof
500 mg/kg,thedose-dependenteffectsweredeterminedusing
750, 1000and1500mg/kgdoses.
2.5.1. FST
The FSTisthemostwidelyusedpharmacological in vivo model
for assessingantidepressantactivity.Thedevelopmentofimmo-
bility whenmiceareplacedinaninescapablecylinderfilledwith
water reflectsthecessationofpersistentescape-directedbehavior
(Porsolt etal.,1977). TheapparatusutilizedtoperformtheFST
consisted ofaclearglasscylinder(20cmhigh12 cmdiameter)
with waterfilledtoadepthof15cm(2471 1C). Themicewere
treated withHbMeOH(500mg/kg,experimentaltreatment, n¼8)
and CMC1%(vehicle, p.o., controlgroup, n¼8) at48,36,24,18,
and 1hpriortothetest.IMI(15mg/kg, i.p., positivecontrol, n¼8)
was administered24h,18h,and30minbeforethetest.Priorto
the administrationschedule,themiceweresubjecttoapre-test
session inwhicheachanimalwasindividuallyplacedinthe
cylinder for15min.Duringthetestsession,atrainedobserver
recorded theimmobilitytime.
2.5.2. EPM
The EPMtestisthemostfrequentlyemployedmodelforthe
assessment oftheanxiolyticactivityofnovelsubstances(Lister,
1987). ThemazewasconstructedofPlexiglasandconsistedofa
central platform(55 cm)withtwoopen(305 cm)andtwo
closed arms(305 cm)and25cmhighwalls.Themazewas
elevated 38.5cmfromtheroom’sfloor.Themiceweretreated
30 minpriortothetestwithDZP(1mg/kg, i.p., positivecontrol,
n¼8), whileCMC1%(vehicle, p.o., controlgroup, n¼8) and
HbMeOH (500mg/kg,experimentaltreatment, n¼8) wereadmi-
nistrated 1hpriortothetest.Eachanimalwasplacedatthe
center ofthemazefacingoneoftheopenarms.Thenumberof
entries andthetimespentintheenclosedandopenarmswere
recorded duringthe5mintest.Allofthetestsessionswere
recorded byvideocamera.Aftereachtest,themazewascarefully
cleaned withwettissuepaper(10%ethanolsolution).
2.5.3. OFT
The openfieldareawascomprisedoftransparentacrylicwalls
and ablackfloor(303015 cm)dividedintoninesquaresof
equal size.Onehourbeforethetest,themiceweretreatedwith
HbMeOH (500mg/kg,experimentaltreatment, n¼8) andCMC1%
(vehicle, p.o., controlgroup, n¼8). Theopenfieldtestwasusedto
evaluate thelocomotoractivityofmicethathadpreviouslybeen
subjected totheFSTandEPMtests.Theobservedparameters
included thenumberofsquarescrossed(withfourpaws)(cross-
ings) andthenumberofrearings(Archer, 1973).
M. Huerta-Reyesetal./JournalofEthnopharmacology146(2013)311–317 312

3. 2.5.4. PTZt
HbMeOH (500mg/kg,experimentaltreatment, n¼8) andCMC
1% (vehicle, p.o., controlgroup, n¼8) wereadministered1h
before thePTZ(75mg/kg, i.p.), whileDZP(1mg/kg, i.p., positive
control, n¼8) wasadministeredonly30minpriortothePTZ.
Following theadministrationofPTZ,micewereplacedinseparate
transparent Plexiglascages(251510 cm)andwereobserved
for theoccurrenceofseizuresovera30mintimeperiod.Latency
of convulsions(thetimepriortheonsetofclonicconvulsions),
convulsions number(tonicorclonicconvulsionsof5sdurationat
least), andmortalityprotection(percentageofdeathsin24h)
were recorded(Williamson etal.,1996).
2.5.5. PTBt
HbMeOH (500mg/kg,experimentaltreatment, n¼8) andCMC
1% (vehicle, p.o., controlgroup, n¼8) wereadministrated1hprior
to thetest,andDZP(1mg/kg, i.p., positivecontrol, n¼8) was
administered 30minbeforethetest.Inthisexperiment,afterthe
administration ofPEN(30mg/kg, i.p.), themicewereplaced
separately intransparentPlexiglascages(251510 cm)to
observe thehypnoticeffect,whichwasconsideredtobethetime
interval betweenthedisappearance(latency)andreappearance
(duration) oftherightingreflex(Williamson etal.,1996).
2.6. Toxicityassay
The acutetoxicitytestwasperformedaccordingtotheOECD
guidelinesforthetestingofchemicals(OECD,2001).NineICR
femalemice(26–28g,8weeksold)wereacclimatizedunderregular
laboratoryconditions(thesameasthosedescribeabove).Foodwas
withheldfor1hbeforeadministration.Theanimalswerecagedin
groups ofthree,anddosesof500and2000mg/kgofHbMeOHand
100 ml/10gofCMC1%(vehicle, p.o., controlgroup)wereadminis-
tered orallybygavage.Thirtyminutesafteradministration,the
animalsweresubjectedtoaninitial periodofobservationofseveral
CNS activity-associatedbehavioral parameters:locomotoractivity,
tremors,gripstrength,bizarrebehavior,convulsions,abdominal
contortions,gaitincapacity,piloerection,palpebralclosure,and
constipation. Thisprocedurewasperformedthreetimesweekly
for 2weeksduringwhichanimaldeaths,animalweights,andfood
consumptionwerealsorecorded.
2.7. HPLCanalysis
We performedanHPLCanalysisofHbMeOHfordetectionand
quantification ofitsmajorconstituents.HPLCanalysiswascon-
ducted onaWaters2695liquidchromatographerequippedwith
a Waters2996photodiodearraydetector.Separationwascarried
out usingaRPC-18Superspher(Merck)column(1204 mm;
5 mm) withthefollowingsolventratiosforthemobilephase
where solventAiswaterandsolventBisacetonitrile:A:B¼100:0
(0–1 min);90:10(2–4min);80:20(5–9min);70:30(10–15min);
60:40 (16–18min);40:60(19–20min);0:100(21–30min);and
100:0 (31–32min).Thesampleinjectionvolumewas10 ml witha
1.0 ml/minflowrate.Thedetectionwavelengthwasscannedat
190–400 nm.Quantificationofthemaincompoundswas
achieved usingcalibrationcurvesthatwereseparatelycon-
structed withpurestandards(Herrera-Ruiz etal.,2006).
Purification ofthemaincompoundsdetectedintheextract
was carriedoutbysuccessiveextraction(3) of20gofHbMeOH
with n-hexane andethylacetate.Theremainingsolidwas
dissolved inmethanol(Hb-bip-MeOH,13.1g)andsubjectedto
Si gelchromatography,elutingwithCH2Cl2:(CH3)2CO¼100:0 to
50:50; 7fractionswerecollected.Fraction5(1.2g)wassubjected
to Sigelchromatography,elutingwithCH2Cl2:MeOH¼100:0 to
0:100, and10fractionswerecollected.Chlorogenicacidmethyl
ester precipitatedinFraction3(33.3mg),whilechlorogenicacid
was detectedinFraction4(31.7mg).
2.8. Statisticalanalysis
The datawereanalyzedusingaone-wayANOVAfollowedbya
post hocDunnetttestusingtheSPSS11.0program.Differences
between theexperimentalgroupswereconsideredstatistically
significant when po0.05.
3. Results
3.1. FST
HbMeOH inducedasignificantantidepressanteffectintheFST
because itsignificantlydiminishedtheimmobilitytimecompared
with thecontrol(po0.05) (Fig. 1). Inadditiontotheeffects
observed forthe500mg/kgdose,the750mg/kgdosecauseda
significant increaseinimmobilitytime(po0.0) intheFSTwith
respect tocontrolgroup(Veh).However,the1000and1500mg/
kg dosesdidnotinduceasimilarbehavior(Fig. 1).
3.2. EPM
The anxiolyticDZPinducedasignificantlyincreasedofthe
percentage ofentriestoopenarms(EOA)andthepercentageof
time spentinthearms(TOA)intheEPM,whencomparingto
control group(po0.05). MicetreatedwithHbMeOHinthedose
range from500to1500mg/kg,provokedsignificantlychangesof
EOA andTOA,withrespecttothecontrolgroup(Veh)(po0.05)
(Fig. 2).
3.3. OFT
Mice treatedwithHbMeOH(500mg/kg),didnotshowedany
change intheparametersoftotalcrossingandrearsintheOFT,
comparing withthecontrolgroup(Veh)(p40.05). Inthecaseof
animals treatedwithdosesof750,1000and1500mg/kgof
HbMeOH exhibitedadecreaseinthenumberofthetotalcrossing
(po0.05), butnotinthenumberofrears(p40.05) (Fig. 3).
3.4. PTZt
The doseof500mg/kgofHbMeOHshowedatotalprotection
against deathinmiceaswellassignificantdiminutionofthe
number ofconvulsions(3.271.3, po0.05) (Table 1) while
Fig. 1. Effect oforaladministrationof Heteropterys brachiata methanol extracton
immobility timeofICRmiceexposedtoFST. npo0.05 withANOVAfollowedbya
post hoc Dunnett’s test(mean7S.D.). Veh,vehicle;IMI,imipraminehydrochloride;
HbMeOH, methanolicextractof Heteropterys brachiata.
M. Huerta-Reyesetal./JournalofEthnopharmacology146(2013)311–317 313

4. 750 mg/kgofHbMeOHdiminishedtheconvulsionsnumber
(po0.05) withaprotectiononlyofthe57.1%.Sincetreatments
with 1000and1500mg/kgofHbMeOHofferednoanticonvulsant
protection anddidnotchangetheonsetofseizures,theseeffects
are notdifferentascomparedwiththoseobservedforthecontrol
group (Table 1).
3.5. PTBt
The treatmentof500mg/kgofHbMeOHexhibitednochanges
in micebehaviorintheparametersoflatencyanddurationas
compared withthecontrolgroup(p40.05) (Table 2).
3.6. Toxicity
Normal behaviorwasobservedinCMC1%-treatedmice
because animalsdidnotexhibitanyalterationsintheparameters
analyzed. Nochangesintheweightoftheanimalsandinthefood
consumption weredetected.Therewerenodeathsduringthe14
days ofobservationaftertheacute i.p. treatmentwith2000mg/kg
of HbMeOH.
3.7. HPLCanalysis
WeanalyzedHbMeOHbyHPLCfordetectionandquantification
of majorconstituentsofactiveextract. Fig.4 showstheHPLCprofile
of HbMeOHrecordedat220nm,inwhichfourpeaksweredetected.
The maincompoundsofHbMeOH,peak1(tR¼3.9 min)andpeak2
(tR¼8.1min),wereisolated,identifiedandquantifiedaschlorogenic
acid (3.2mg/kg)andchlorogenicacidmethylester(60mg/kg),
respectively(Fig. 5),sincethespectroscopicdatawerefoundtobe
in goodagreementwithvaluesreportedintheliterature(Jang etal.,
2010; Sari,2010).Quantificationwasestablishedwithcalibration
curves(linearregressionwhere r240.9850).
4. Discussion
Despite itsuseintraditionalMexicanmedicinetotreatsome
mental diseases, Heteropterys brachiata has notyetbeenevalu-
ated foritsactivityontheCNS.Thepresentstudyinvestigatedfor
the firsttimetheCNSeffectsofamethanolicextractoftheaerial
parts of Heteropterys brachiata (HbMeOH) inmice.Ourresults
showed thatHbMeOHstandardizedinitscontentofhydroxycin-
namic acidswithdosesofchlorogenicacid(3.2mg/kg)and
chlorogenic acidmethylester(60mg/kg)inducedasignificant
antidepressant effectintheFST.Inthistest,anexperimental
model fortestingtheefficacyofantidepressantdrugs,theanimal
develops alearnedhelplessnesssyndromecharacterizedbya
lowered motivationforescapingasevidencedbyincreased
periods ofimmobility(Porsolt etal.,1977). Severalauthorshave
proposed thatimmobilityduringthetestcouldbeanefficient
adaptive responsetothisstress(Porsolt etal.,1977; Tolardo etal.,
2010). Interestingly,thehigherdosesofHbMeOH(1000and
1500 mg/kg)werenoteffectiveintheFST.Itispossiblethatthis
effect showedonFSTmaybeinfluencedbytheactionof
neurotransmittersorreceptorsbecausesimilarsaturateddose-
responsecurveshavebeenalreadyreportedafteroraladministrations
Fig. 2. Effect oforaladministrationof Heteropterys brachiata methanolic extract
on thepercentageoftimespentinopenarms(TOA)andentriesintoopenarms
(EOA) bymiceexposedtoEPM. npo0.05 inANOVAfollowedbya post hoc
Dunnett’s test(mean7S.D.). Veh,vehicle;DZP,diazepam;HbMeOH,methanolic
extract of Heteropterys brachiata.
Fig. 3. Effect oforaladministrationof Heteropterys brachiata methanolic extract
on thenumberoftotalcrossingsandrearingsofICRmiceexposedtotheopenfield
paradigm. npo0.05 withANOVAfollowedbya post hoc Dunnett’s test
(mean7S.D.). Veh,vehicle;DZP,diazepam;HbMeOH,methanolicextractof
Heteropterys brachiata.
Table 1
Anticonvulsant effectofmethanolicextractfrom Heteropterys brachiata on PTZ-
induced seizuresinICRmice.
Treatment
(mg/kg)
Latency of
convulsions (s)
Convulsions
number
Mortality
protection
(%)
Veh (100 ml/10 g)91.0718.6 7.373.7 0.0
DZP (1)0.070.0n 0.070.0n 100.0
HbMeOH (500)61.8718.9n 3.271.3n 100.0
HbMeOH (750)95.07 47.8 3.372.5n 57.1
HbMeOH (1000)79.6736.1 4.873.1 14.2
HbMeOH (1500)61.1713.0 4.371.5 28.5
Data presentedasthemean7S.D. with n¼8.
n po0.05 comparedtovehicleusingANOVAanda post hoc Dunnett’s test.
Veh, vehicle;DZP,diazepam;HbMeOH,methanolicextractof Heteropterys
brachiata.
Table 2
Effect ofthemethanolicextractof Heteropterys brachiata in thePTBtestonICR
mice.
Treatment (mg/kg)LatencyDuration
Veh (100 ml/10 g)0.070.0 0.070.0
DZP (1)246.3752.5n 2690.07644.0n
HbMeOH (500)0.070.0 0.070.0
Data presentedasthemean7S.D. with n¼8.
n po0.05 comparedtovehicleusingANOVAanda post hoc Dunnett’s test.
Veh, vehicle;DZP,diazepam;HbMeOH,methanolicextractof Heteropterys
brachiata.
M. Huerta-Reyesetal./JournalofEthnopharmacology146(2013)311–317 314

5. of differentcrudeplantextracts,suchastheextractof Allium
macrostemon (Alliaceae)(Lee etal.,2010). Otherpossiblemechanism
for thiseffectshowedonFSTmaynotinvolvereceptoroccupation.In
non-competitive antagonism,thethresholddoseofagonistisnot
markedly increased,butthemaximumresponseisdepressedinthe
presenceoftheantagonist.Crudeplantextractsoftenproducethis
kind ofeffect.Itariseswhentheblockingactiontakesplace,asit
were, beyondthereceptor,suchaseffectsonintracellularsecond
messengers (Williamsonetal.,1996). Indeed,ourresultsarein
agreementwithotherstudieswherethebesteffectwasshownon
themiddledosage(750mg/kg)oftheextract,whilethehighdosage
(1500mg/kg)didnotshowntheantidepressantactivity,asthecase
of theherbalformulaeKaiSinSanusedinthetraditionalChinese
medicine (Zhou etal.,2012)
On theotherhand,inthepresentwork,theoraladministration
of HbMeOHexhibitedaclearanxiolyticactivityinadose-
dependent manner.Becausenumerousneuralpathwaysare
involved inthepathophysiologyofdepressionandanxietystates,
false positiveresultscanbeobtainedforagentsthatstimulate
locomotor activity(Bourin etal.,2001). Therefore,theobservation
that HbMeOHdidnotincreasethenumberofcrossingsand
rearings intheOFTatthe500mg/kgdoseconfirmstheassump-
tion thattheanxiolyticeffectoftheextractdoesnotinfluencethe
motor system(Sa´nchez-Mateo etal.,2005). However,although
higher dosesofHbMeOHdonotexhibitchangesinthenumberof
rears, theregistereddecreaseofthetotalcrossingsuggeststhat
there isageneraldepressoreffectonthenervoussystem(dos
Santos etal.,2006). Thisobservationisinagreementwiththose
reports inwhichhigherdoses 4500 mg/kgfromacrudeplant
extract usedwidelyinfolkmedicinenotonlyexhibitedanxiolytic
properties butalsoelicitedsedationandmyorelaxanteffects
(Galani andPatel,2011). Moreover,inthisstudy,ourresults
obtained inthePTBtassayalsoconfirmtheabsenceofsedation
effects atthelowerdoseofHbMeOHbecausethetreatmentof
500 mg/kgexhibitednochangesinthebehaviorofthemice.
The effectofHbMeOHasananticonvulsantbythePTZtmodel
wasalsoexaminedinthisstudybecausethismodeliswidelyused
to investigateanticonvulsivedrugswithahighpredictivevaluefor
the detectionofclinicallyeffectivedrugs(Galani andPatel,2011).
GABA isthemajorinhibitoryneurotransmitterimplicatedinepi-
lepsy. TheenhancementandinhibitionGABAneurotransmission
willattenuateandenhanceconvulsion,respectively(de Oliveira
et al.,2011). BecausethereisevidencethatPTZmaybeexertingits
proconvulsant effect by inhibitingtheactivityofGABAatGABAA
receptors, itisprobablethatthedelayedoccurrenceofPTZconvul-
sionobservedbytheadministrationof500mg/kgofHbMeOHmay
beinterferingwiththeGABAergic mechanismtoexertitsantic-
onvulsant effect(Quintans-Ju´ nior etal.,2008).
Our findingssuggestaCNS-depressantactionofHbMeOHat
doses 4500 mg/kg.Becausetheeffectivedoseofsuchas Valeri-ana
officinalis, Crataegus monogyna and several Hypericum species
(Sa´nchez-Mateo etal.,2002), isbetween500and1000mg/kgof
plant extracts, Heteropterys brachiata appears tobenotonly
within asimilarrangeofpotencybutalsointerestingtoexplore
for itspotentialneuropharmacologicalpropertiesatdoses
o500 mg/kg,thelowestdoseemployedinthepresentstudy.
Althoughsomeotherspeciesbelongingtothe Heteropterys genus
have beenstudiedinsomeneuropharmacologicalaspects,the
literatureislackingonpharmacologicalstudiesoftheplant Het-eropterysbrachiata,
thespeciessubjectofourstudy.Theextractof
the Brazilianspecies Heteropterys aphrodisiaca improved learning
and memorydeficitsinagedratsinthepassiveavoidancetestand
in theTmaze,respectively(Galv~aoetal.,2002). Heteropterys
aphrodisiaca alsoshowedastrongantioxidativeactivity in vitro
and inducedanincreaseinsuperoxide dismutaseactivitiesinold
rats (Mattei etal.,2001).Inthecaseof Heteropterys glabra, the
ethanolicextractinducedareductioninmotoractivityandaltera-
tions inEEGparameters(Galietta etal.,2005). However,thereis
scarce informationaboutthechemicalconstitutionoftheseextracts.
The onlyreportonthistopicisapartialphytochemicalscreening
that revealedthepresenceofglycosides,polyphenols,tannins,
alkaloids, saponinsandanthracenederivativesasmajorcomponents
in thespecies Heteropterys aphrodisiaca (Galv~aoetal.,2002). Then,to
the bestofourknowledge,thepresentstudyisthefirstthat
comprises pharmacologicalandquantitativephytochemicaldataof
the genus Heteropterys and evermore,oftheplantspecies Heterop-
terysbrachiata. Thus,themaincompoundsidentifiedinHbMeOH
are thehydroxycinnamicacidschlorogenicacidandchlorogenic
acid methylester.Althoughhydroxycinnamicacidsarepresentina
largevarietyoffruitsandvegetables,chlorogenicacidisthemost
abundanthydroxycinnamicacidinfoodandhasbeenreportedto
prevent differentcancers,cardiovascular diseasesandtype2dia-
betes mellitus(Suzuki etal.,2006). Likeotherdietarypolyphenols,
chlorogenic acidandchlorogenic acidmethylesterarealso
Fig. 4. HPLC chromatogramoftheantidepressant,anxiolyticandanticonvulsiveHbMeOHextract.Thepeakswereidentifiedas(1)chlorogenicacid,(2)chlorogenic acid
methyl ester,(3)unknowntriterpene,and(4)unknowntriterpene.
Fig. 5. Chemical structureofthemaincompounds,chlorogenicacidandchloro-
genic acidmethylesterisolatedfromtheantidepressant,anxiolyticandantic-
onvulsive HbMeOHextract.
M. Huerta-Reyesetal./JournalofEthnopharmacology146(2013)311–317 315

6. antioxidantswithantiviral,antibacterial andantifungalproperties
(Lafay etal.,2006; Chen etal.,2010).Additionally,chlorogenicacid
has beenconsideredtobeaneuroactivesubstancebecauseit
improved theimpairmentofshort-termorworkingmemory
inducedbyscopolamineintheY-mazeandsignificantlyreversed
cognitive impairmentsinmiceas measured bythepassiveavoid-
ance test(Kwon etal.,2010). Moreover,chlorogenicacidhasbeen
shown tohaveananxiolyticeffectproducedbyactivationofthe
benzodiazepine receptor(Bouayedetal.,2007).Inthissense,there
is apossibilitythatthechlorogenicacidduelargelytoitscontent
may beinvolvedatleastinpartintheanxiolyticeffectshownby
HbMeOH,sincethepresenceofthishydroxycinnamicacidhadbeen
responsibletoexercisetheanxiolyticeffectintheEPMmodel
(Bouayedetal.,2007).
In thecaseofpeak3(tR¼22.9min;UVabsorption:192.3nm)
and peak4(tR¼23.5 min;UVabsorption:192.3nm)alsodetectedin
HbMeOH,althoughtheiridentitiesremainunknown,their tR and UV
absorptionvaluessuggestthepresenceofacomplexmixtureof
terpene-typecompounds(Harborne,1984), whichappeartobethe
secondmostabundantchemicalfamilyinHbMeOHafterthe
hydroxycinnamicacids.Previously, terpeneshavebeenreportedto
be partlyresponsibleforthesedative,anxiolytic,anticonvulsantand
analgesicactivitiesinseveralspeciesofmedicinalplants(Arag~ao
et al.,2006; Kubacka etal.,2006; Wijeweeraetal.,2006; Taviano
et al.,2007). Furthermore,thegalphimines, agroupoftriterpenes
describedforfirsttimein1993,havebeenrecognizedasthe
compoundsresponsibleforthepotentanxiolyticpropertiesshowed
by Galphimiaglauca extractsinbehavioralmodelsandinclinical
studiesonpatientswithgeneralizedanxietydisorder(Herrera-
Arellanoetal.,2007).Itisinterestingtonotethat Galphimiaglauca
is aspeciesthatalsobelongstothesameplantfamilyas Heterop-terys:
Malpighiaceae.Nonetheless,throughthechromatographic
analysesperformedusingHPLCin the presentstudy,thegalphi-
minesA,BandEwerenotdetectedinHbMeOH.Thelackof
galphiminesdoesnotprecludethepossibilityoftheactiveinvolve-
mentoftheterpene-typecompoundsontheneuropharmacological
propertiesexhibitedbyHbMeOH,sinceinonesense,thisextractnot
onlyexertedanxiolyticactivities,andontheotherhand,as
we mentionedbefore,terpene-typecompoundshadbeendetected
as responsibleofCNSeffectsinothermedicinalplantsextracts.
Nevertheless,furtherstudiesare needed toconfirmthishypothesis.
In thepresentacutetoxicitystudy,ourresultsshowedthat
HbMeOH couldbeconsiderassafe,oroflowtoxicity,whenorally
administrated (Adeneye etal.,2006). Thislackofsignsofacute
toxicity supportstheuseof Heteropterys brachiata in folkloric
medicine byanoralrouteandencouragesfurtherstudiesforthe
development ofHbMeOHasatherapeuticagent.
5. Conclusions
Themethanolicextractof Heteropterysbrachiata possesses anti-
depressant,anxiolyticandanticonvulsivepropertiesandcanbe
consideredsafeoroflowtoxicitywhenorallyadministrated.Tothe
best ofourknowledge,thisstudypresentsthefirstreportonthe
quantitativephytochemicaldataofthisextractwhichcontains
mainlyhydroxycinnamicacidsandterpene-typecompounds.These
findingslendpharmacologicaljustificationtothetraditionaluseof
Heteropterysbrachiata in thetreatmentofnervousdisorders.
Acknowledgments
This workwassupportedbygrant82588fromSEP-CONACyT-
Ciencia Ba´ sica 2007,Mexico(toM.Huerta-Reyes)andgrantFIS/
IMSS/PROT/C2007/040fromtheFIS,InstitutoMexicanodel
Seguro Social,Mexico(toM.Huerta-Reyes).Theauthorswishto
thank JonathanOrdun˜o andArturoPe´ rez fortechnicalassistance.
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