Rev. bras. farmacogn. vol.26 número2

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

Original

Article

Neuropharmacological

effects

of

the

ethanolic

extract

of

Sida

acuta

Dora

M. Benjumea

a,∗

_,

_Isabel

_C.

_{Gómez-Betancur}

a

_,

_Julieta

_Vásquez

a

_,

_Fernando

_Alzate

b

_,

Andrea

García-Silva

c

_,

_José

_A.

_Fontenla

c

a_{Ophidism/Scorpionism}_Program,_University_Research_Center,_University_of_Antioquia,_Medellin,_Colombia b_Institute_of_Biology,_Faculty_of_Natural_Sciences,_University_of_Antioquia,_Medellin,_Colombia

c_Laboratory_of_Pharmacology_of_the_Central_Nervous_System,_Department_of_{Pharmacology,}_Faculty_of_Pharmacy,_University_of_Santiago_de_Compostela, SantiagodeCompostela,Spain

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received7May2015 Accepted22September2015 Availableonline5January2016

Keywords:

Anxiolyticactivity Anticonvulsantactivity Rotarod

Sodiumpentobarbital-inducedsleeping time

a

b

s

t

r

a

c

t

SidaacutaBurm.f.,Malvaceae,isregardedasastringent,tonicandusefulintreatingurinarydiseases

andblooddisorders,bile,liverandastreatmentfornervousdiseases.Differentmethodsweredeveloped: sodiumpentobarbital-inducedsleepingtime,anxiolyticactivity,testformuscle-effects, pentylenetetra-zole(PTZ)-inducedseizures,effectonnormalbodytemperature.Allexperimentswereperformedinan isolatedroomwith12/12hlight/darkcyclesat22±1◦_C._The_effects_described_in_this_work_for_Sida_acuta areaccordingtowhatisknownintraditionalmedicine,whereisusedassedativeagent.Atthehigher dosesusedinthiswork(500and1000mg/kg),theSidaacutaextractreducedthelatencytime(T1)and increasedthesleepingtime(T2)inducedbypentobarbital,indicatingasedativeandhypnoticeffectof theplant’sextract.TheextractofSidaacutashowsanincreaseinopenarmexploration(anxiolytic activ-ity).Resultsobtainedintherota-rodtestshowedthatonlytheelevateddose(750mg/kg)ofSidaacuta extract,acutelyadministered,promotessignificantchanges,at60and120minpost-administration,in thetimeofpermanenceintherod.TheethanolicextractfromtheleavesandstemsofSidaacuta,causes effectsonthecentralnervoussysteminexperimentalanimals.

Introduction

Malvaceaeisafamilyoffloweringplantscontainingover200

generawithcloseto2300species.Themaineconomicuseof

Mal-vaceaeplantsisasasourceofnaturalfibers,thefamilyisalsoused

forfood,beverages,timber,intraditionalmedicineandin

horticul-ture.ThelargestgeneraareHibiscus(300species),Streculia(250

species),Dombeya(225species),Pavonia(200species)and Sida

(200species)(RizkandSoliman,2014).SidaacutaBurm.f.,locally

knownas“escobabosa”and“kuala”inCuna,“samampiisa”in

Bur-kinaFaso(Nadembegaetal.,2011),“arbreàbalai”inFrenchand

“zon-raaga”inMooré,isaperennialshrubwidelydistributedinthe

subtropicalregions,foundinbushes,infarms,aroundhabitations.

Growsabundantlyoncultivatedfields,wasteareas,roadsidesand

highways,indampordry,between0and1800masl(Mejíaetal.,

1994;Karouetal.,2007).

In Colombia, the whole plant of S. acuta is widely used in

traditionalmedicineoftheIndigenousTribesEmbera,Wounaan,

∗ Correspondingauthorat:DepartamentodeFarmacia,FacultaddeQuímica Far-macéutica,UniversidaddeAntioquia,Medellín,Colombia.

E-mail:[email protected](D.M.Benjumea).

CunasandKatíos,andinothersregionsofAntioquia,preparedas

drinks, ointements and external baths against snakebite (Otero

etal.,2000a,b,c;Vásquezetal.,2015).Itisalsousedasstomachic,

diaphoreticandantipyretic.Itisregardedasastringent,tonic,

use-fulinurinarydiseasestreatment(diuretic)andalsoblooddisorders

(stops bleeding),bile and liverand nervous diseases treatment

(sedative) inIndian traditional medicine(Sreedevi et al.,2009;

Govindarajan,2010);inMexico,smokedasmarihuanasubstitute,

and it is also used to treat asthma, renal inflammation, colds,

gonorrhea,fever,bronchitis,malaria,diarrhea,headache,

dysen-tery,abortion,breastcancer,skindiseases,hemorrhoids,insects’

bites,erectiledysfunction,elephantiasis,rheumatismandulcers

(Napralertdatabase,Bhardwajetal.,2011;Kumaretal.,2012).Itis

claimedtohaveaphrodisiacproperties(Govindarajan,2010).The

root’sjuiceisappliedtowoundsandthebarksareusedformeasles

(Adetutuetal.,2011;Allabietal.,2011).InNigeria,S.acutaisoneof

theplantsmostcommonlyusedforthetreatmentofhypertension,

usingitsleaves,seedsandstemsindifferentpreparations(Gbolade,

2012).

ThephytochemicalscreeningofS.acuta speciesrevealedthe

presenceofalkaloidssuchasvasicine,ephedrineandcryptolepine

(themainalkaloidintheplant)(Prakashetal.,1981;Karouetal.,

2005), saponosides (unspecifiedtype or hemolytic), coumarins,

http://dx.doi.org/10.1016/j.bjp.2015.09.011

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steroids(ecdysterone,␤-sistosterol,stigmaterol,ampesterol),

tan-nins,phenolic compounds(evofolin-A,andB,scopoletin,loliolid

and4-ketopinoresinol,polyphenol,sesquiterpeneandflavonoids

(Konatéetal.,2010;Napralertdatabase).

Thetested pharmacological activitiesof theS. acuta involve

stimulating smooth muscle, abortifacient, antiulcer, antiyeast,

diuretic,antiplasmodial,antimicrobial,antiophidian,antioxidant,

hepatoprotective, insecticidal, larvicidal-repellent and cytotoxic

activities(Oteroetal.,2000a;Karouetal.,2003;Banzouzietal.,

2004; Ekpo and Etim,2009; Akilandeswari et al.,2010; Pieme etal.,2010;Upadhyayetal.,2010;Adeniyietal.,2010;Ahmed etal.,2011;Koudouvoetal.,2011).Meanwhile,S.acutaextracts

impactinthecardiovascularsystemfunctioninzebrafishembryos

(KannanandPrakash,2012).Additionally,ithasbeenprovedthat

theaqueous-acetoneandethanolicextractsofS.acutaleaveshave

analgesicactivityandantidepressant-likepropertiestestedin

dif-ferentanimalmodels,provingthattheplantcontainspsychoactive

substances(Konatéetal.,2012;Ibironkeetal.,2014).

Sidacordifolia(L.)extracthasbeenreportedtohavecentral

ner-voussystemactivityinexperimentalanimals(Francoetal.,2005)

andSidatiagiiBhandarihasbeenreportedtohaveanxiolyticand

anticonvulsantactivity(Datusaliaetal.,2008).Inthepresentstudy,

assessestheneuropharmacologicalproperties ofS. acutaleaves

andstems,includingsedative,anticonvulsantandanxiolytic

activ-ities.

Materialsandmethods

Plantmaterial

Sidaacuta Burm.f.,Malvaceae,wascollectedfromMedellín,

inaUniversityofAntioquia(Colombia),at1525maltitudeabove

sea level, in February 2012. It was identified by Dr. Fernando

AlzateGuarín,InstituteofBiology,UniversityofAntioquia

(Medel-lín,Colombia),wherevoucherspecimenshavebeendepositedwith

thenumber5111inthecollection.

PreparationofSidaacutaextracts

Leavesandstems(60:40)ofS.acutawereair-driedinanoven

at40◦_C_for₄_days,_then_the_dry_plant_was_cut_and_ground_to

pow-derbymechanicalmilling.Thedriedpowderedplantmaterialwas

submittedtoacontinuousextractioninaSoxhletextractorfor5

daysusing100%ethanolassolvent.Thesolventwasthen

elimi-natedbyvacuumdistillationinarotaryvacuumevaporator(Büchi

R–124,Flawil,Switzerland),anditwaslyophilized,representing

anextractionyieldof3.1%ofthedrymaterial.

Animals

MaleCD1albinoSwissmice(weighing22±2g)wereobtained

fromtheCentralAnimalHouse(UniversityofSantiagode

Com-postela) and housed in groups of four, eight or twelve in

standardMakrolon®_cages₍₂₁₅_mm

×465mm×145mm).The

ani-malsreceivedstandard laboratorychow(ScientificAnimalFood

andEngineering(SAFE),Augy,France)andtapwateradlibuntilthe

beginningoftheexperiments.Threedaysafterlaboratoryarrival,

boththehousingandhandlingduringexperimentationwere

car-riedoutaccordingwiththestandardsestablishedinthedirective

2010/63UEoftheEuropeanParliamentand theCouncilandthe

Galician(Decreto296/2008,DOGAnumber11,January16,2009)

andSpanish(RealDecreto53/2013,BOEnumber34,February8,

2013)legislationonanimalexperimentation.

Drugs

AlldrugswereboughtfromSigma–Aldrich(USA)orMerck

(Ger-many).Drugsor thecrude extract ofSida acuta wereprepared

immediatelybeforeuseandorallyadministered(peros)inatotal

volumeof0.1ml/10gbodyweight(b.w.).Diazepam(DZP),sodium

pentobarbital(PTB)andPentylenetetrazole(PTZ)weredissolvedin

salinesolution(NaCl0.9%).ThecrudeextractofS.acutawas

sus-pendedina1%(weight/volume)sodiumcarboxymethylcellulose

(NaCMC)dispersionatdosesof50,100,300,500and1000mg/kg.

Pharmacologicalevaluation

All experiments were performed in an isolated room with

12/12hlight/darkcyclesat22±1◦_C,_at_the_same_time_of_day,_in

ordertoavoidpotentialvariationscausedbycircadianrhythms.

Theevaluationoftimeinopenarms,numberofentries,traveled

distance,velocity,and anotherevaluatedparameterin elevated

plusmaze(EPM)testandtheopenfieldtest(OFT)weremadewith

thevideocomputerizedanimalobservationsystemEthoVisionV.

3.16(NoldusInformationTechnology,Wageningen, The

Nether-lands).

Alltheproceduresfollowtheguidelinesoftheresearchethics

committeeattheUniversityofSantiagodeCompostela,according

withtheguidelinesoftheEuropeanCommunityCouncilDirective

86/609.

Sodiumpentobarbital-inducedsleepingtime

TheeffectofS.acutaextractonpentobarbitalsleepingtimewas

performedinsixgroupsofmice(n=4).Fourgroupsreceivedgraded

dosesoftheextract(100,300,500and1000mg/kgp.o.).Onegroup

receiveddiazepam(5mg/kgi.p.),whileanimalsinthecontrolgroup

wereadministeredNaCMC(0.1ml/10gp.o.).Thirtyminutes

post-treatment,sodiumpentobarbital(40mg/kgi.p.)wasadministered

toeachmouse.Theelapsedtimebetweentheadministrationof

pentobarbitaluntilthelossoftherightingreflexwasrecordedas

thesleeplatency(T1),andthetimeelapsedbetweenthelossand

recoveryoftherightingreflex(T2)wasrecordedasthesleeptime

(CarliniandBurgos,1979;Ramírezetal.,1998;Wambebe,1985).

Anxiolyticactivity

Elevatedplusmaze(EPM):TheEPMtestwasintroducedinthe

researchofnewdrugswithpotentialanxiolyticactivity(Hanledy

andMithani,1984).Experimentalgroupsoffourmiceweretreated

withvehicle(NaCMC1%,0.1ml/10gp.o.),Sidaacuta(50,100,300

and500mg/kgp.o.)ordiazepam(1mg/kgi.p.).

Theelevatedplusmaze(EPM)ismadeofwoodpaintedblack.It

hastwoarms60cm×10cm,arrangedoppositetoeachother,and

enclosedbywalls35cmheight.Italsohastwoopenarmsofthe

samesizeandwithoutflanges.Thefourarmsareinterconnected

byacentralsquareof10cm×10cm,formingacross,andelevated

toadistanceof75cmfromtheground.Thedeviceisilluminatedby

fourindependenttubes,arrangedinacross,locatedintheceiling

oftheroom,andtheanimalbehaviorwasobservedwithavideo

cameradisposedonthecenter.

Sixtyminutesafterthetreatment, animalswereplaced

indi-viduallyatthecenteroftheelevatedplusmazewiththeirnose

facingthedirectionof one oftheenclosed arms,and observed

for5min(Pellowetal.,1985;Lister,1987;YemitanandAdeyemi,

2003).Themazeplatformsandwallswerethoroughlycleanedwith

70%ethanolbetweensessionsandallowedtodry.Totalresidence

timeinopenorclosedarms,timeratiospentinopenarms,and

thenumberofentries(frequency)intheopenorclosedarmswere

recorded.

Openfieldtest(OFT):Thismethodisusedtoevaluatepossible

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throughtheevaluationofambulatorybehaviorinmice;alsouseful

todetectbothanxiolyticandanxiogenicagents.30minafterthe

treatment(n=4)withvehicle(1%NaCMC0.1ml/10g,p.o.),S.acuta

atdosesof 50,100, 300and500mg/kg p.o.ordiazepam1 and

5mg/kgi.p.,theanimalswereplacedatthecenterofanopenfield

arena.Theapparatusconsistedoffouridenticalarenasmeasuring

eacharena50cm(width)×50cm(length)30cm(height).Traveled

distance(cm),velocity (cm/s)andnumber of rearingbehaviors

wererecordedandanalyzedwithEthoVisionina30minsession

(Siegel,1946;Archer,1973;Carlinietal.,1986).Aftereachtrial,the

open-fieldapparatuswaswipedcleanwithethanol(10%)solution.

Testformuscle-effects

Rota-rodtest(RRT):Itistheclassicmethodusedforthe

assess-mentofmotorcoordinationinmiceorrats(Dunham andMiya,

1957;Ozturketal.,1996;Pérezetal.,1998).Animalsweretrained

previously (72hbefore experiment)based on theircapacity to

maintaintheirbalance(for3min)ontherotatingbar(16rpm)with

a3cm diameteraxis.Arota-rodtreadmilldevice(Rotamex4/8,

ColumbusInstruments,OH,USA)wasusedforthispurpose.

Attime 0 (0min)the pre-trainedanimalswere placed ona

horizontalrotatingrodsetataspeedof16revolutionspermin.

Micethatwereabletoremainontherodlongerthan180swere

selectedandclassifiedintosixgroupsoffourmicepergroup.Four

groupsoftheselectedanimalsreceivedgradeddosesoftheS.acuta

extract(100,300,500and750mg/kg,p.o.),whiletheremaining

groupsreceivedNaCMC(0.1ml/10gb.wp.o.)ordiazepam(5mg/kg

i.p.).Sixtyminuteslater,eachmousewasplacedontherota-rod,

forevaluationofmotor-coordination(cut-offtimewas180s).Two

complementarymeasurementsweremade60minapart(120and

180min).

Tractiontest(TT):Themouse’sforepawswereplacedonasmall

twistedwire rigidly supportedabove(20cm high) a laboratory

benchtop.Normalmicegraspedthewirewiththeforepaws,and

whenallowedtohangfree,placedatleastonehindfootonthewire

within5s.Inabilitytoplaceatleastonehindfootmarkedfailure

inthetractiontest–lowmuscularstrength(Rudziketal.,1973).

Groupsoffourmalemicewereusedforthetestat0and60,120

and180minaftertheadministrationofSidaacuta(500,750and

1000mg/kg,p.o.),diazepam(5mg/kg,i.p.)orNaCMC(0.1ml/10g,

p.o.)tofivedifferentgroups.

Chimneytest (ChT):The chimneytest measurestheanimals’

motorcoordinationandmuscledysfunctionanditwasperformed

accordingtoamethoddescribedbyBoissieretal.(1960).Amouse

wasintroducedatoneendandallowedtomovetotheotherend

ofa Pyrexglasstube(30cmlong×3.0cm diameter),markedat

20cmfrombase.Whentheanimalreachedtheendofthetube,

thetubewasmovedtotheverticalpositionandimmediately,the

mousetriedtoclimbthetubewithabackwardmovement.The

micethatcrossthemarkin30sorlessare“normal”unlessare

“affected”.Thetestwasrepeatedat0,60,120and180minafter

theadministrationofS.acutaextract(500,750and1000mg/kg,

p.o.),diazepam(5mg/kg,i.p.)orNaCMC(10ml/kg,p.o.)to

differ-entgroups(n=4).Motor/muscleimpairmentwasexpressedasthe

percentageofanimalsunabletoperformthistestwithin30s.The

averagetimenecessarytoclimbbackwardsupthechimneywas

alsocountedforeachexperimentalgroup.

Pentylenetetrazole(PTZ)-inducedseizures

Thecapacityof S.acutatoprovideprotectionwasmeasured

inchemical seizuretestsbydetermining modulationof

charac-teristics of theseizures induced bythe i.p. injectionof PTZ. In

agreementwithSwinyardetal.(1952),thisdose(75mg/kg)was

givenintraperitonealy30minafterS.acutaextractadministration

(50,100, 300and 500mg/kg p.o.)or diazepam(1mg/kgi.p.)to

groupsofeightmice.Anothergroupofeightmiceservesascontrol

(NaCMC1%p.o.).AfterPTZinjectionmicewereplacedseparately

intocircularandtransparentplexiglassdevices,measuring25cm

(diameter)×14cm(height)andobservedfor30minforthe

occur-renceofseizures,andweretakenasthemaximumtimeofonsetof

seizures(Tmax)(VogelandVogel,2002).Thetimetakenbeforethe

onsetofclonicconvulsionsandthepercentageofseizureprotection

wereindividuallyrecorded.

Effectonnormalbodytemperature

Therectaltemperatureofeach mousewasmeasured30min

afteradministrationofeachdoseofSidaactua(SA)(100,300and

500mg/kg,p.o.)usingathermometermodel0331(PanLab

Instru-ments,Barcelona,Spain).

Resultspresentation,plottingandstatisticalanalysis

Results areexpressedas mean±standard errorof themean

(SEM). All data were analyzed using one-tailed test analysis

of variance (ANOVA) followed by Dunnet’s test post hoc. The

graphicalrepresentationandstatisticalanalysiswereperformed

using GraphPad Prism (V 4.3) (San Diego, USA). Statistically

significantdifferencesweredeterminedbyone-wayANOVA

(treat-ment) followed by the Dunnet’s test or by two-way ANOVA

(treatment–time)followedbyBoferronitest.Statistically

signifi-cantdifferencesweredeterminedwithp<0.05.

Results

Sodiumpentobarbital-inducedsleepingtime

Treatment with SA at high doses (500 and 1000mg/kg)

increasedthesleepingtime(T2)anddecreasedthesleepinglatency

(T1);theeffectwassignificantlydifferentfromthecontrolgroup

(p<0.05).Diazepam5mg/kgincreasedthesleepingtime(T2)and

decreasedthesleepinglatency(T1)(Fig.1).Theeffectsofgraded

dosesofSAshowedadosedependenteffect.

Anxiolyticactivity

Elevatedplusmaze(EPM)

Administration of SA (at dose of 500mg/kg) significantly

increasedtheamountoftimespentandthepercentageofentries

intheopenarmsoftheEPM(p<0.05),comparedtovehicle

admin-istration.Similarly,animalstreatedwithdiazepam(1mg/kg,i.p.)

demonstrated a significantly increased number of entries and

increased time in the open arms, as compared with controls

(p<0.01)(Fig.2).

Thehigherthepercentagesofentriesandtimeinopenarms

means, rodents havelower levelsof anxiety(Park et al.,2005;

Primeauxetal.,2006).

Openfieldtest

IntheOFT,SAatdosesof50,100,300and500mg/kgdecreased

thedistancemoved(cm),velocity(cm/s)andrearings(f)as

com-pared tocontrol(NaCMC 1%)(p<0.01).Diazepam, as expected,

showednoeffectatthedoseused(1mg/kg,i.p.),butatdoseof

5mg/kgshowsdiminutionoftheseparameters(Table1).

Testsformuscleeffects

Therota-rod,chimneyandtractiontestswereusedtoassess

motoractivitycoordination,musclerelaxantandmusclestrength

inexperimentalanimalsunderstimulantsordepressantscentral

nervoussystemdrugs.SAshowsnodecreaseinmotorcoordination

atalluseddoses,except750mg/kgat60and120min(p<0.05vs.

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CONTROL DZP 5

SA 100 SA 300 SA 500 SA 1000 SA 300 SA 500 SA 1000

0 200 400 600 800

1000 T1

*

**

** **

* *

**

Sleeping latency (s)

T2

SA 100

0 500 1000 1500 2000 2500 3000 3500 4000 4500

Sleeping time (s)

Fig.1. EffectsoftheethanolicextractofSidaacuta(SA)atdosesof100,300,500and1000mg/kg,diazepam5mg/kgandcontrol(vehicle)onpentobarbital(40mg/kgi.p.) inducedsleepingtimeinmice.T1=timeoflatencyandT2=timeofdurationofsleep,n=4.Valuesareexpressedasmean±SEM.*p<0.05,**p<0.01and***p<0.001,as comparedtonormalcontrol.ANOVAandDunnet’sastheposthoctestwereperformed.

Control DZP PTZ SA 50 SA 100 SA 300 SA500 Control DZP PTZ SA 50 SA 100 SA 300 SA 500

0 5 10 15 20 25 30 35

*

* *

*

* *

# entries into open arms

0 50 100 150 200

Time spent on open arms (s)

Fig.2.InfluenceoftheethanolicextractofSidaacuta(SA)atdosesof50,100,300and500mg/kg,diazepam1mg/kgandcontrol(vehicle),expressedbypercentageofentries inopenarm(a)andthetimespentinopenarms(b)intheelevatedplusmaze.Resultsareexpressedasmean±SEM,n=4eachgroup;*p<0.05vs.control;**p<0.01vs. control;ANOVAwithStudent–Dunnet’sposthoctest.

Table1

EffectsofSidaacuta(SA)ontheopenfieldtestinmiceatdosesof50,100,300and 500mg/kg,anddiazepamat1and5mg/kg.Valuesrepresentthemean±SEMof dis-tancemoved(cm),velocity(cm/s)andrearing(f).Valuesrepresentthemean±SEM, medium±E.S.ofdistancemoved(cm),velocity(cm/s)andrearing(f).ANOVAand Dunnet’sastheposthoctestwereperformed.

Group Distancemoved(cm) Velocity(cm/s) Rearing(f)

Control 11,268±466.4 6.26±0.26 350.5±28.9 Diazepam1mg/kg 14,862±2334.0 8.26±1.30 312.3±38.6 Diazepam5mg/kg 980±11.7** _0.77

±0.16** _22.5 ±3.8**

SA50mg/kg 2901±938.7** _1.61_±_0.52** _26.5_±_8.7**

SA100mg/kg 2726±385.0** _1.52_±_0.21** _14.8_±_5.4**

SA300mg/kg 1513±146.3** _0.84_±_0.08** _11.8_±_4.6**

SA500mg/kg 1614±254.4** _0.89

±0.14** _20.0 ±14.4** ** _p_<_0.01_compared_with_control_group_(CMC_1%).

Inthetractiontest(TT)S.acuta(500,750and1000mg/kg)did

notimpairtheabilitytoplaceatleastonehindfootonthewire

within5s,unlikediazepam(Table2).

Inthechimneytest(ChT)nosignificanteffectwasobservedin

micepretreatedwithS.acutaextract(500,750and1000mg/kg)in

thetimetakentoclimbbackwardinthePyrexglasstube.However,

micepretreatedwithdiazepamcouldnotclimbbackwardsinthe

Pyrexglasstube(Table3).

Pentylenetetrazole(PTZ)-inducedseizures

SAextractat50mg/kgdosesshowsadiminutionofthenumber

ofmicethatpresentseizures(0%),atdosesof100mg/kgshows

de 50% of the number of mice that present seizures. At doses

of300mg/kg justdemonstrated a25%ofprotectiontheseizure

Rota-rod test (RRT)

Measurement time (min)

Time spent in the rod (s)

200

160

120

80 *

*

***

*** 40

60 120 180

0

Control

DPZ

SA 100 SA 750 SA 500 SA 300

Fig.3. Effectsonrota-rodinmiceat0,60,120and180afteradministrationofSida acuta(SA),atdosesof100,300,500and750mg/kgordiazepam(DZP)at5mg/kg,

n=4.Valuesareexpressedasmean±SEM.*p<0.05,**p<0.01and***p<0.001,as comparedtonormalcontrol(NaCMC1%).TwowaysANOVAandBonferroniasthe posthoctestwereperformed.

inducedPTZ.Meanwhile,dosesof500mg/kgdonotshow

protec-tionagainstthePTZinducedseizure(Table4).

Bodytemperature

S.acutaatdosesof100mg/kg(p<0.05)and300mg/kg(p<0.01)

causedasignificanthypothermiarelativetocontrolgroup,overthe

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Table2

EffectofSidaacuta(SA)onthemusclestrengthinthetractiontest.Themuscle strengthinmicewasmeasuredat60,120and180min.Eachvaluerepresentsthe mean±SEM,n=4.Fordiazepamallvalues>5s–cutofftime.ANOVAandDunnet’s astheposthoctestwereperformed.

Treatment Dose(mg/kg) Meantimeittakestoputonefootbackon thewire(±SEM)[s]

Time 60 120 180

Control – 2.1±0.6 3.9±0.7 3.6±0.6

Diazepam 5 >5.0** ₅

±0.0** _>5**

SA 500 3.1±0.7 2.9±0.5 3.5±0.8 SA 750 4.0±0.6 5.0±0.0 4.1±0.9 SA 1000 3.4±0.9 4.3±0.8 4.0±0.6

**_p_<_0.01_compared_with_the_control_group.

Table3

MusclerelaxantactivityofSidaacuta(SA)inthechimneytest.Themuscle relax-ationinmicewasmeasuredat60,120and180min.Eachvaluerepresentsthe mean±SEM,n=4.Fordiazepamallvalues>30s–cutofftime.ANOVAandDunnet’s astheposthoctestwereperformed.

Treatment Dose(mg/kg) Meantimetoclimbupthechimney (±SEM)[s]

Time 60 120 180

Control – 12.5±31 11.7±2.7 11.8±2.3 Diazepam 5 >30.0** _>30.0** _>30.0**

SA 500 11.3±3.2 20.7±5.5 22.0±2.8 SA 750 17.6±5.4 22.0±5.2 17.8±5.8 SA 1000 17.1±4.4 21.2±3.3 20.0±2.0

**_p_<_0.01_compared_with_the_control_group.

Table4

EffectofSidaacuta(SA)onpentylenetetrazole(PTZ)inducedseizuresinmice.Values areexpressedasmean±SEMoftimeofonsetseizures.=numberofmicethathave

seizures,%ofprotectionagainstPTZ(75mg/kg),tconv=timeofonsetofseizures.

N=8.ANOVAandDunnet’sastheposthoctestwereperformed.Themaximumtime ofonsetofseizureswas1800s(Tmax).

Group %protection tconv(s)X±SEM %mortality

Control 8/8 0.0 151.5±10.7 0 Diazepam1mg/kg 0/8 100.0 1800.0±0.0** ₀

SA50mg/kg 0/8 100.0 1800.0±0.0** ₀

SA100mg/kg 4/8 50.0 986.0±310* ₀

SA300mg/kg 2/8 75.0 1009.0±259.8** ₀

SA500mg/kg 7/8 12.5 405.5±200.9 12.5

*_p_<_0.05_as_compared_to_normal_control. **_p_<_0.01_compared_with_the_control_group.

Control SA 100 SA 300 SA 500

25 26 27 28 29 30 31 32 33 34 35

*

* *

T ºC

Fig.4.EffectsoncorporaltemperatureinmiceofSidaacuta(SA)atdosesof100, 300and500mg/kg,30minafter administration,n=4.Valuesareexpressedas mean±SEM.*p<0.01and**p<0.01,ascomparedtonormalcontrol(NaCMC1%).

Discussion

Thisworkrepresentsthefirststeptowardtheunderstanding

oftheeffectsinthecentralnervoussystemofthecrudeextract

obtainedfromtheleavesandstemsofS.acuta,onrodents.The

plantshoweddepressive,anxiolyticandanticonvulsanteffects;in

addition,apotentiationofhypnosisinducedbypentobarbital.The

effectsdescribedinthisworkforS.acutaareaccordingtowhat

isknownintraditionalmedicine,whereisusedassedativeagent

(Oteroet al.,2000a;Sreedevi etal.,2009; Govindarajan,2010),

alsoforplantswithinthesamespeciesS.tiagiiandSidacordifolia

(Datusaliaetal.,2008;Francoetal.,2005).

Atthehigherdosesusedinthiswork,theS.acutaextract

indi-cated a sedativeand hypnoticeffectof theplant.This effectis

probablycausedbyvasicineorcryptolepine,whicharealkaloids

isolatedfromthis plant(Ahmedet al.,2011);even though,the

effectsofthesecompoundsonthecentralnervoussystemhave

notbeenstudied.ThesedativeeffectwasalsoevidentwithS.tiagii

(Datusaliaetal.,2008).

Anxiolyticcompoundsreducethenaturalanimalaversiontothe

openarmsandpromotetheexplorationthereofinthe

elevated-plusmazetest.Ontheotherhand,theforcedorvoluntarypassages

ofanimalsintotheclosedarmsoftheEPMare associatedwith

hormonalandbehavioralchangesindicativeofincreasedanxiety

(Adebesinetal.,2015).Avoidanceoftheopenarmportraysa

mani-festationoffearandanxiety.Basedontheseassertions,theelevated

plus-mazetestisareliablemeanforidentifyingselectiveanxiolytic

effectofdrugs(Adebesinetal.,2015).TheextractofS.acutashows

anincreaseinopenarmexploration(anxiolyticactivity),reflected

byanincreaseinthepercentageofentriesintoandtimespenton

theopenarms.But,alldosesoftheextractofSAdecreased the

distancemoved,velocityandrearingmeasuredintheopenfield,

similarlytoDZP.Thissuggestsanxiolyticeffectoftheextractatthe

doseof500mg/kg,givingcredencetotheindicationinthe

hole-boardtest.

Resultsobtainedintherota-rodtest(RRT)showedthat

treat-mentwithS.acuta,at100,300and500mg/kg,didnotmodifythe

spontaneousactivityofthemice,nonesignsthatdenote

depres-santeffectswereobserved,withcomparisontothemicetreated

withDZ,aclassicbenzodiazepinewithanxiolyticeffectsthat

pro-duceda decrease oflocomotor activityin comparison withthe

controlgroup.Thus,theseresultssuggestthatthisplantdidnot

producemyorelaxation,neitheraltersthemotorcoordinationof

miceexperimental,whichareundesirablesedativesideeffectsof

somebenzodiazepines.

Inthepresentstudy,resultsshowedthatS.acuta leavesand

stemsextracthasanticonvulsanteffectsonPTZmodelofepilepsy

inmice.S.acutaatthedoseof50mg/kgwasmosteffectiveagainst

PTZinduced seizure(100%protection), followedbythedoseof

300mg/kgwitha75%ofprotection.Theanti-epilepticeffectsof

drugs such as benzodiazepines are accompanied by decreased

motoractivityandsedation(Guptaetal.,2012).Therefore,S.acuta

mightpossiblybeproducinganti-epilepticactionbyincreasingthe

effectofGABA,theprincipalinhibitorytransmitterinthecentral

nervoussystem.Thisisinaccordance withthepharmacological

effectsofbenzodiazepineandhighlightstherelevanceofthe

puta-tiveanti-epilepticeffectsofS.acuta(Guptaetal.,2012).However,

furtherstudiesonneurotransmitterorneuromodulators

involve-mentsarenecessaryforcompleteunderstandingofanticonvulsant

effectsofS.acuta.

Conclusion

Inconclusion,thesedatashowedthatmicetreatedwithcrude

extract of the leavesand stems of S. acuta presented sedative

effect.Also,theextractprotectsagainstseizuresinducedbyPTZ

anditshowsanxiolyticeffect.Therefore,itisimportanttofurther

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Authors’contributions

Theidentificationandcollectionofplantmaterialweremade

byFAGincompanyofJVandIC.G-Bpreparedtheextracts.The

pharmacologicalevaluationwasperformedbyDMB,AG-SandJAF

ThemanuscriptwaselaboratedbyDMBAlltheauthorshaveread

thefinalmanuscriptandagreedtoitssubmissionforappraisal.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Dr.NormanFransworth(RIP)forgivingusinformationofthe

databaseNAPRALERT.CarolinaFoundation,UniversityofAntioquia

–ColombiaandUniversityofSantiagodeCompostela–Spainfor

financethescholarshipoftheProf.Benjumeainthe

Pharmacol-ogyLaboratoryofCentral NervousSystem(GI-1684-USC).CODI

Sustainabilityproject2014–2015.

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