model of post-traumatic stress disorder The effect of Elettaria cardamomum extract on anxiety-like behavior in arat ScienceDirect

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Original article

The effect of Elettaria cardamomum extract on anxiety-like behavior in a rat model of post-traumatic stress disorder

Yaser Masoumi-Ardakani

^a

, Hossein Mahmoudvand

^b

, Amin Mirzaei

^c

,

Khadijeh Esmaeilpour

^d

, Hamed Ghazvini

^d

, Solmaz Khalifeh

^e

, Gholamreza Sepehri

^f,

*

aPhysiologyResearchCenter,InstituteofNeuropharmacology,KermanUniversityofMedicalSciences,Kerman,Iran

bRaziHerbalMedicinesResearchCenter,LorestanUniversityofMedicalSciences,Khorramabad,Iran

cDepartmentofBiology,FarsScienceandResearchBranch,IslamicAzadUniversity,Shiraz,Iran

dNeuroscienceResearchCenter,InstituteofNeuropharmacology,KermanUniverityofMedicalSciences,Kerman,Iran

eMedicalGenomicsResearchCenterandSchoolofAdvanced,SciencesinMedicine,TehranMedicalSciencesBranch,IslamicAzadUniversity,Tehran,Iran

fCardiovascularResearchCenter,InstituteofBasicandClinicalPhysiologySciences,KermanUniversityofMedicalSciences,Kerman,Iran

ARTICLE INFO

Articlehistory:

Received8September2016

Receivedinrevisedform17December2016 Accepted27December2016

Keywords:

PTSD E.cardamomum Anxiety-likebehavior Rats

ABSTRACT

Post-traumaticstressdisorder(PTSD)isadebilitatingpsychiatricconditionwhichdevelopsin6–8%of thegeneralpopulation.CurrentstandardpharmacologicaltreatmentsforPTSDcannotbewidelyused duetohavingvarioussideeffects.Nowadays,variouspharmacologicalpropertieshavebeenrelatedto ElettariacardamomumL.(familyofZingiberaceae).ThepresentstudyaimstoevaluatetheefﬁcacyofE.

cardamomummethanolicextractonanxiety-likebehaviorinaratmodelofPTSD.AdultmaleWistarrats (200–250gr)wereusedinthisstudy.Theratsunderwentsingleprolongedstress(SPS)orcontroland intraperitoneallyreceivedeithersalineordifferentdosages(200,400,and800mg/kg)ofE.cardamomum methanolic extractbeforeand afterstresssessions. Moreover,openfield,elevated plus-maze,and rotarod tests were used to evaluate locomotion and anxiety-like behavior in the rats. Findings demonstrated that E. Cardamomum methanolic extract, particularly at the dose of 400mg/kg, significantly(P<0.05)improvedanxiety-likebehaviorinaratmodelofPTSD,asexaminedbythe openfield,elevatedplus-maze,androtarodtests.AdministrationofE.cardamomummethanolicextract afterstressmighthelptopreventtheformationofanxiety-likebehaviorintheanimals.However,further studiesarerequiredtoclarifytheexactmechanismsinvolved.

1.Introduction

Post-traumatic stressdisorder(PTSD) is a psychiatricillness withalife-timeprevalenceof5–8%inthegeneralpopulation[1].It is one of the most debilitating conditions which significantly reduces the quality of life in the patients [2]. The disease is characterized by three main symptoms, including recurring flashbacksandnightmares,hyperarousal,andnumbing[3].PTSD isananxiety-disorder whichcouldbeclassifiedasacute(acute stressdisorder)orchronic(PTSD)[3].Severaltreatmentmodalities havebeenproposedtopreventandtreatanxietyinPTSDpatients.

Selective serotonin reuptake inhibitor (SSRI) and serotonin-

norepinephrinereuptakeinhibitor(SNRI)agentssuchasﬂuoxetine and paroxetine havebeen approved for thetreatmentof PTSD symptoms [4]. Despite their effectiveness against the PTSD symptoms,theirusearelimitedbecauseofvarioussideeffects, which highlights the need for further research on therapeutic modalities[5].

Duringrecentdecadesplant-derivedextractsandcompounds wereconsideredasavaluablesourceinordertocureawidevariety ofdiseasessuchaspsychiatricdisorders.Thisrichsourcehassome advantages including few side effects, low cost and high availability.

The cardamom (Elettaria cardamomum L. (Maton) from Zingiberaceae family)is one ofthe mostimportantspice crops cultivatedwidelyinIndiaandothertropicalregionsworldwide.E.

cardamomumiscalled“QueenofSpices”inIndiaand“Hel”inIran thatusedasaﬂavoragent(spice)inavarietyoffoodstuffs[6].Inthe

* Correspondingauthor.

E-mailaddress:[email protected](G.Sepehri).

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folkmedicine,differentpartsofE.Cardamomumhavebeenusedin the treatment of gastrointestinal disorders and also used as stomachic,resolvent,retentive,digestive,antiemetic,carminative andanti-putrefactive duringembalmment [7,8]. Inrecent years andinmodernmedicinevariouspharmacologicalpropertiessuch as antimicrobial, anti-inﬂammatory, analgesic, anti-depression, anticonvulsantandantispasmodicactivitieshavebeenattributed tothisplant[9–11].

There are much evidence about E. cardamomum beneﬁcial effects against many complications and diseases as mentioned above but there is no study about E. cardamomum effect on PTSD.Considering E.cardamomumconstituents suchas quercetin,kaempferolandrutinofwhichquercetinhaseffectsonCNS function we decide to evaluate E. cardamomum methanolic extract effects on anxiety-like behavior in rat model of PTSD [12–14].

2.Materialsandmethods 2.1.Plantmaterials

DryE.cardamomumseedswerepurchasedfromthemarket.The plantseedswereidentiﬁedbyabotanistattheBotanyDepartment ofShahidBahonarUniversity,Kerman,Iran.Avoucherspecimenof theplantmaterialswasdepositedattheHerbariumofDepartment ofPharmacognosyofSchoolofPharmacy,KermanUniversityof MedicalSciences,Iran(KF1375).

2.2.Preparingmethanolicextract

Thedriedseedsoftheplant(100g)weregrindedandextracted bypercolationmethodandthroughusingmethanol(80%)for72h at room temperature. The solvents were removed in a rotary evaporator,andafterﬁltering,theextractswereconcentratedto drynessandstoredat 20Cuntiltestingbegins[15].

2.3.PhytochemicalanalysisofE.cardamomum

TheE.cardamomumseedmethanolicextractwasscreenedfor saponins,alkaloids,ﬂavonoids,and tannins [16].ATLCmethod (mobilephasecompositionwith46.15%chloroform,30.77%ethyl acetate,15.38%methanol,and 7.69%formic acid:) was usedfor separationanddetectionof differentcompoundsin theextract.

QuercetinconcentrationintheextractwasdeterminedbyHPLC/

UVdetectorthrough usingdifferentconcentrations of standard quercetin[11].

2.4.Animals

HealthyadultratsofWistarstrainweighingaround200–250g wereusedinthepresentstudy.Theanimalswerehousedinclean polypropylenecagesandmaintainedinawell-ventilatedtemper- ature controlled animal house with constant 12h light/dark schedule. The animals were fed with standard rat pellet diet andcleandrinkingwaterwasmadeavailableadlibitum.Allthe procedureswereperformedfrom12:00to16:00.Maximumeffort wasmadetominimizepainfortheanimals.

2.5.Ethicalstatement

This study was carried out in strict accordance with the recommendationsintheGuidefortheCareandUseofLaboratory AnimalsoftheNationalInstitutesofHealth(PublicationNo.85-23, revised1985).TheprotocolwasapprovedbytheCommitteeonthe Ethics of Animal Experiments at KermanUniversity of Medical

Sciences(PermitNumber:KNRC/92-7).Moreover,alleffortswere madetominimizesufferingoftherats.

2.6.Experimentaldesign

The ratsweredividedintotwo main groups.The ﬁrstmain groupwassingleprolongedstress(SPS)groupIcontaining96rats, whichwassubdividedintofoursubgroups;subgroupIa(24PTSD ratstreated with salineas vehicle), subgroup Ib (24 PTSD rats treatedwith200mg/kgoftheextract),subgroupIc(24PTSDrats treatedwith400mg/kgoftheextract),andsubgroupId(24PTSD ratstreatedwith800mg/kgoftheextract).Eachsubgroupwasalso subdividedintothreefurthersubgroups(n=8rats):(i)pre-stress (receivedsalineorextract30minbeforeestablishmentofPTSD), (ii)post-stress(receivedsalineorextract30minafterestablish- mentofPTSD),and(iii)pre-test(receivedsalineorextract30min beforebehavioraltest).Thesecondmaingroupwascontrolgroup IIcontaining96rats,whichwassubdividedintofoursubgroups;

subgroupIa(24non-PTSDtreatedwithsalineasvehicle),subgroup Ib (24 non-PTSD rats treated with 200mg/kg of the extract), subgroup Ic (24 non-PTSD rats treated with 400mg/kg of the extract)andsubgroupId(24non-PTSDratstreatedwith800mg/kg oftheextract).

2.6.1.Stressprocedure

Theratswerebroughttothelaboratory24hbeforecommence- mentofstressprocedure togethabituatedtotheenvironment.

Theywereindividuallycagedonedaypriortothestresssession.On thestressday,theratswerebroughttothelaboratory.TheSPS procedure was performed on them as previously described elsewhere [17]. Three consecutive stressors, including restraint stress(2h),swimmingstress(20min)inacylindricaltank(with 40cmdiameter,50cmheight,35cmwaterheight,and26Cwater temperature),andlossofconsciousnesswithDiethylEtherwere administeredtotheratsaftera15minrest[18].Sevendayspost PTSD,theratswereagainbroughttothelaboratoryandundergone behavioraltestsafteronehourhabituation.

2.6.2.Openﬁeldtest

The rats werebrought to thetesting environment after 1h acclimationperiod.Theratswerethenputinthemiddleofanopen ﬁeld.Theopenﬁeldapparatuswasasquarearena[909030[H]

cm]whichwasmadeofPlexiglas,anditsfloorwasdividedinto16 squares so the field was divided into central and peripheral squares.The verticaland horizontal activitiesof therats were recorded duringa five minperiod and then analyzed using an EthoVision software[version7.1], a video trackingsoftwarefor automation of the behavioral paradigms [Noldus Information Technology,theNetherlands].Parametersincludingtotaldistance moved in cm (TDM), number of grooming and rearing (as a measureofverticalactivity),andthetimespentinperipheryand centerwererecordedforeachrat.Attheendofeachtest,therats wereremovedfromthechamberandthefieldwascleanedwitha dampcloth[19].

2.6.3.Elevatedplus-maze

Theelevatedplus-mazecomprisedablackwoodenapparatus witharmshavingequaldimensions.Twoofitsarmswereenclosed bywalls(30155cm)andarrangedinlinewith2oppositeopen arms(305cm).Themazewas elevated50cmabovetheﬂoor.

Theratswerethenplacedatthecenterofthemaze,facingtheopen arms.Two100Wlampsbrightlyilluminatedthearena.Therats were allowed to explore the maze, and their behavior was monitoredfor10minusingtheEthoVisionsoftware[version7.1]

[NoldusInformationTechnology,theNetherlands].Aftereachtest, the apparatus was cleaned with 70% ethanol to eliminate the

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remainingodors.Subsequently,thetimespentintheopenarms, thenumberofentriesintotheopenarms,andthetotalnumberof entriesintothearmswasrecorded[20,21].

2.6.4.Rotarodtest

ToanalyzetheeffectsofE.cardamomumonmotorcoordination andbalance skills,weusedtheaccelerating rotatingrod[Hugo SachsElectronik,Germany].The rotarodspeed which usedwas from10to60rpm.Therewerethreetrialsupto300sfollowedby 30minintertrialrest.Werecordedthedurationwhicheachrat wasabletomaintainitsbalancewalkingontopofthemovingrod [22].

2.7.Statisticalanalysis

Theobtainedresultsareexpressedas themeanSEM.Data analysiswascarriedoutbyusingSPSSstatisticalpackageversion 17.0(SPSSInc., Chicago,IL,USA).One-wayANOVAwithTukey’s post-hoctestwasusedtoassessdifferencesbetweenexperimental groups.In addition,p<0.05 was consideredstatisticallysigniﬁ- cant.

3.Results

3.1.PhytochemicalanalysisofE.cardamomum

Preliminary phytochemical screening of E. cardamomum showed that the methanolic extract of seeds contains tannins andﬂavonoids.However,thetestsforalkaloidsandsaponinswere negative. Moreover, larger amounts of kaempferol, rutin, and quercetinwereobservedusingtheTLCmethod.Accordingtoour previous experiments,thepreciseconcentrationofquercetinin theextractwas0.51

m

^g/ml^[11].

3.2.Openﬁeldtest

As shown in Fig. 1, in pre-stress subgroups, anxiety-like behaviors such as grooming, rearing, time spent in perimeter, andtimespentincenterweresigniﬁcantly(P<0.05)alteredinSPS group receiving the E.cardamomum extract,particularly at the 400mg/kgdose,comparedtothoseinthecontrolgroups,which indicated improvement of anxiety-likebehaviors in this group.

However,inthepre-stresssubgroups,noalternationwasobserved

Fig. 1.EffectofvariousdosesofE.cardamomummethanolicextractonlocomotionandanxiety-likebehaviorsusingwithopenﬁeldtestinthepre-stresssubgroups.*p<0.05,

**p<0.01and***p<0.001incomparisontothecontrolgroups.

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invelocityduration(Fig.1C).Further,mobilityincreasedintherats ofSPSsubgroupsthatreceivedtheE.cardamomumextractatthe doseof 400mg/kg (P<0.05) (Fig. 1D). Similarly, in post-stress subgroups,theanxiety-likebehaviorsmentionedabovewereall signiﬁcantly(P<0.05) alteredin theSPSgroupreceiving theE.

cardamomum extract, especially at the dose of 400mg/kg, indicatingimprovementofanxiety-likebehaviorsinthissubgroup.

In the pre-stress subgroups, although velocity and mobility increasedin the ratstreated with the E. cardamomumextract, thedifference was not signiﬁcant compared to therats in the controlgroups(Fig.2).

3.3.Elevatedplus-maze

AsshowninFigs.3and4,theratsinboththepre-andpost- stresssubgroupsreceivingtheE.cardamomumextractshoweda signiﬁcantdifferenceintermsofanxietybehaviorsusingelevated plus-mazeincomparisonwiththoseinthecontrolgroups.

In the pre-stress subgroups, the rats treated with the E.

cardamomumextractat thedoses of200 and 400mg/kg had a

signiﬁcant (P<0.05) increased time spent in the open arms comparedwiththoseintheothergroups,implicatingareduced anxiety-like behavior in them (Fig. 4A). However, the rats in thesesubgroupstreatedwiththeE.cardamomumextractatthe dosesof200and400mg/kgshowedareducedtimespentinthe close arms in comparison with those in the control groups (P<0.05) (Fig.4B).

Similarly,treatmentwiththeE.cardamomumextractimproved theanxiety-likebehaviorsinthepost-stresssubgroupsastherats treatedwiththeextractatthedoseof400mg/kghadasigniﬁcant (P<0.05) increasedtime spent in theopenarms(Fig.4A)and decreasedtime spentintheclosearms(Fig.4B)incomparison withthoseintheothergroups.

3.4.Rotarodtest

In both the pre- and post-stress subgroups, a signiﬁcant reductionwasobservedinthetimespentbytheratstreatedwith theE.cardamomumextractatthedoseof800mg/kg(P<0.05)on revolvingrodwhencomparedtothecontrolrats(P<0.05)(Fig.5).

Fig.2.EffectofvariousdosesofE.cardamomummethanolicextractonlocomotionandanxiety-likebehaviorsusingwithopenﬁeldtestinthepost-stresssubgroups.* p<0.05,and***p<0.001incomparisontothecontrolgroups.

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4.Discussion

Naturalproductssuchasplantscrudeextractsortheirpurified compounds provide unlimited opportunities for new drug discoveries because of the unmatched availability of chemical diversity [23]. According to World Health Organization (WHO) morethan80%oftheworldpopulationreliesonthetraditional medicinefortheirprimaryhealthcareneeds.Recently,studyinthe areaofherbalpsychopharmacologyhasdemonstratedavarietyof promisingmedicinesthatcanprovidebenefitinthetreatmentof stressandanxietydisorders.Consideringadvantageouswiderang flavonoideffects such as cognitive, anxiolytic, anti-depressants andsedativeeffectandalsopresenceofalargescaleofflavonoids includingquercetin in cardamom, we assessedE. cardamomum methanolicextracteffectsonPTSDratmodel[11–14].

We demonstrated that E. cardamomum methanolic extract significantly improvedanxiety-like behaviorsin a rat model of PTSDexaminedbytheopenfield,elevatedplus-mazeandrotarod tests.Theopenfieldtestisusedtodeterminetheanimalemotional state [24]. Our findings revealed that the E. cardamomum methanolic extract caused anxiolytic-like activity in the rats, especiallyatthedoseof400mg/kg,asitseffectshowedsignificant alternation in their anxiety-likebehaviors,including grooming, timespentinperimeterandtimespentincenterascomparedto thoseoftheratsinthecontrolgroups.

The elevated plus-maze test is used to assess psychomotor performance and emotional aspects of rodents. The obtained resultsrevealedthattheE.cardamomummethanolicextractatthe doseof400mg/kghadanxiolyticactivity,sinceincreasesinopen armentryparameters[25].

The rotarod test used to evaluate the neurotoxicity of anticonvulsants and later used to report motor dysfunction producedbycentrallyactingdrugstodeterminepossiblechanges inthemotorcoordinationabilityoftheanimal[26].Weshowed thattheE.cardamomummethanolicextract(800mg/kg)signiﬁ- cantlyreducedthefall-offtimefromtherotatingrodinthetreated rats,implicatingtheskeletalmusclerelaxantactivity.

Reviewshavedemonstratedpreclinicalevidenceofanxiolytic activityinvariousplantmedicinesincludingAchilleamillefolium, Coriandrum sativum, Stachys lavandulifolia, Magnolia spp., Eschscholzia californica,Panax ginseng,Zizyphus jujuba, Tiliaspp.

andsomeotherplants[27,28].Moreover,severalhumanclinical trialsprovidepreliminary positiveevidenceofanxiolyticactivity for Matricaria recutita, Ginkgobiloba, Passifloraincanata,Echium amoenumandScutellarialateriflorainchronicuses(i.e.greaterthan oneday);whereasacuteanxiolyticactivitywasfoundforCentella asiatica,Salviaspp.,Melissaofficinalis,PassifloraincarnataandCitrus aurantium[29].

Many studies about chemical constituents of plants extract suggestthatplantscontainingﬂavonoids,alkaloids,phenolicacids, saponinsandtanninshavebeneﬁcialeffectsagainstawiderange of CNS disorders [30]. The phytochemical screening of the E.

cardamomummethanolicextractshowedthepresenceoftannins andflavonoidsinthisplant[11].Therefore,theanxiolyticactionof E.cardamomumcouldbeattributedtophytoconstituentsinthis plant. Other studies also showed that plants which have high contents of flavonoids suchas quercetin, kaempferol and rutin showed anti-depressant and anxiolytic effects. Quercetin is a flavonoidwhichhavebeenfoundinE.cardamomum,tea,apples and etc[11,31].It hasbeendescribed beforethat quercetinhas Fig.3.EffectofvariousdosesofE.cardamomummethanolicextractonanxiety-likebehaviorsusingwithelevatedplus-mazetestinthepre-stresssubgroups.**p<0.01and

***p<0.001incomparisontothecontrolgroups.

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neuroprotective effectsand atdoses 20–40mg/kgshowedanti- depressantandanti-anxietyeffect[32].Inourpreviousstudywe showedthatE.cardamomumisagoodsourceofquercetin[11].

The exact mechanism of E. cardamomum is not identiﬁed.

Previously ithasbeenprovedthat GABAAreceptoragonists are anxiolytic.Also,itshowedthatquercetininfluencedcorticotrophin releasingfactor(CRF)inducedanxietythroughGABAAreceptors [32].ThereforewecanassumethatE.cardamomumeffectswhich obtainedin this studycan beattributedto quercetin mediated effectsviaGABAAreceptorswhichmustbeanattractivepointfor moreevaluation.WeassumedthattheseeffectsofE.cardamomum canbeduetoeitherquercetinorsynergiceffectsofquercetinalong with other flavonoid, but it needs more investigation to be clarified. Other study demonstrated that ethanol extract of E.

cardamomumstronglyinhibitedacetylcholinesteraseactivityand enhancedcognitioncondition[33].Wealsoshowedthatmethanol extract of E. cardamomum promotes cognition condition in rat model of PTSD. Finally, data about anxiolytic properties of E.

cardamomumarepooranditneedstobemoreinvestigated.

5.Conclusion

The ﬁndings of the present investigation suggest that the methanolic extract of E. cardamomum possesses anxiolytic properties. We conclude that high ﬂavonoids contents of E.

cardamomumandespeciallyquercetinisresponsibleforanxiolytic effects.However,furtherstudiesarewarrantedforelucidatingthe exactmechanismandbioactivecompounds.

Fig.4.EffectofvariousdosesofE.cardamomummethanolicextractonanxiety-likebehaviorsusingwithelevatedplus-mazetestinthepost-stresssubgroups.*p<0.05in comparisontothecontrolgroups.

Fig.5. EffectofvariousdosesofE.cardamomummethanolicextractinratsinthepre andpost-stresssubgroupsonrotarodperformance.*p<0.05,and**p<0.01in comparisontothecontrolgroups.

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Conﬂictofinterest

Theauthorsdeclarenoconﬂictofinterest.

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