Anthriscus nemorosa essential oil inhalation prevents memory impairment, anxiety and depression in scopolamine-treated rats

Anthriscus

nemorosa

essential

oil

inhalation

prevents

memory

impairment,

anxiety

and

depression

in

scopolamine-treated

rats

Eyup

Bagci

a

,

Emel

Aydin

a

,

Eugen

Ungureanu

b

,

Lucian

Hritcu

b,

*

a

DepartmentofBiology,FacultyofScience,FiratUniversity,23119Elazig,Turkey

b

DepartmentofBiology,AlexandruIoanCuzaUniversityofIasi,Bd.CarolI,No.11,700506,Romania

ARTICLE INFO Articlehistory:

Received27September2016

Receivedinrevisedform21October2016 Accepted24October2016

Keywords:

Anthriscusnemorosaessentialoil Scopolamine Memory Anxiety Depression Alzheimer’sdisease ABSTRACT

Anthriscusnemorosa(Bieb.)Sprengelisusedformedicinalpurposesintraditionalmedicinearoundthe world,including Turkey.EthnobotanicalstudiessuggestthatAnthriscusessentialoilcould improve memoryinAlzheimer’sdisease.Thecurrentstudywashypothesizedtoinvestigatethebeneficialeffects ofinhaledAnthriscusnemorosaessentialoilonmemory,anxietyanddepressioninscopolamine-treated rats.Anthriscusnemorosaessentialoilwasadministeredbyinhalationinthedosesof1%and3%for21 continuous days and scopolamine (0.7mg/kg) was injected intraperitoneally 30min before the behavioraltesting.Y-mazeandradialarm-mazetestswereusedforassessingmemoryprocesses.Also, theanxietyanddepressiveresponseswerestudiedbyelevatedplus-mazeandforcedswimmingtests.As expected,thescopolaminealone-treatedratsexhibitedthefollowing:decreasethepercentageofthe spontaneousalternationinY-mazetest,increasethenumberofworkingandreferencememoryerrorsin radialarm-mazetest,decreaseoftheexploratoryactivity,thepercentageofthetimespentandthe numberofentriesintheopenarmwithinelevatedplus-mazetestanddecreaseofswimmingtimeand increaseofimmobilitytimewithinforcedswimmingtest.However,dualscopolamineandAnthriscus nemorosaessentialoil-treatedratsshowedsignificantimprovementofmemoryformationandexhibited anxiolytic-andantidepressant-likeeffectsinscopolamine-treatedrats.

TheseresultssuggestthatAnthriscus nemorosaessential oilinhalationcanprevent scopolamine-inducedmemoryimpairment,anxietyanddepression.

ã2016ElsevierMassonSAS.Allrightsreserved.

1.Introduction

Alzheimer’sdisease(AD)isthemostcommon neurodegenera-tive disorder, accounting for 70%of all dementia cases [1].AD affectsmillionsof peopleworldwidecausing massiveeconomic burdenandthenumberofcasesisexpectedtorisedramatically [2].

Several pathological conditions characterize AD including neuronal loss, the accumulation of senile plaques formed by extracellulardepositsofamyloidbeta(A

b

)peptides,intracellular neurofibrillarytanglesformedbyhyperfosphorilatedtauproteins, proliferationofastrocytes,andactivationofmicroglia[3].

CholinergichypofunctionisoneofthehallmarksofAD.Itwas reportedtobecausedbysignificantlossofcholinergicneurons, cholinergicneuronalandaxonalabnormalities,andreductionin thenumbersofpostsynapticneuronsaccessibletoacetylcholine

(Ach). It is evidenced that cholinergic hypofunction is closely related toA

b

and taupathologies[4].According tocholinergic hypothesis, memory deficits in AD are caused by cholinergic hypofunctionleadingtoreducedhippocampalandcorticallevels ofAch.Inhealthybrain,acetylcholinesterase(AChE)isthemost importantenzymeregulatingAChlevels,while butyrylcholines-terase(BChE)hasaminorrole.Untilnow,cholinesteraseinhibitors aretheonlyapproveddrugsfortreatingpatientswithAD[5].

Epidemiologicalstudiesshowthatneuropsychiatricsymptoms (NPS) are common in AD. For example, 51% of new onset AD patientswereobservedtohaveatleastoneNPS.Themostcommon symptoms weredepression (25%), apathy (17%),and irritability (17%)[6].

Scopolamine, a muscarinic receptor antagonist, crosses the bloodbrainbarrierandinducesdementiaandcognitive dysfunc-tion[7].Amnesiceffectsofscopolaminehavebeenverifiedbya varietyoflearningparadigmssuchasT-mazealternationtaskand objectrecognitiontest,respectively[8].Thismodelisfrequently employed as a quick way for testing new drugs as cognition enhancers[9].In healthyadults,scopolaminehasbeenusedto

* Correspondingauthor.

E-mailaddress:hritcu@uaic.ro(L.Hritcu).

http://dx.doi.org/10.1016/j.biopha.2016.10.075

0753-3322/ã2016ElsevierMassonSAS.Allrightsreserved.

Available

online

at

ScienceDirect

createimpairmentinmemoryandinformationprocessingspeed andefficiency that resemblesimpairmentsin patientswithAD [10].

Anthriscus (commonly known as beaked chervil, beaked parsley,rough chervil) is one of thearomatic members of the Apiaceaefamily [11] usedfor medicinalpurposesin traditional medicinearoundtheworld,includingTurkey.Anthriscusnemorosa (Bieb.)SprengelisoneofthespeciesdistributedinTurkey.Ithas beenknownasgimigimiinsomepartsofTurkey[12].Thefruitsof this plant are used to treat gastrointestinal ailments [12], for inflammationandrheumatism[13].A.nemorosaisoneofthe25 herbsusedtomake herbycheesein eastern and south-eastern partsofTurkey(especiallyinVanregion),called“Otlupeynir”in Turkish [14]. Furthermore, aerial parts of A. cerefolium, called mende,menduormendainTurkish,havebeeneatenasmealafter boiled for stomach-ache in eastern parts of Turkey [15]. In a handwrittenfolk-medicinal documentinSwitzerland a distilled beveragemadeofA.cerefoliumis indicatedtoimprovememory [16]. In addition, the roots of A. sylvestris have been used as antitussive,antipyretic,analgesic,diuretic,andcoughremedyin Chinesetraditional medicine, and theyoung aerialpart of this plantis usedfor food[17].Ethnobotanical studiessuggest that essentialoilofA.nemorosacouldimprovememoryinAD[12].To our knowledge, there is no study indicating the effects of A. nemorosaessential oil on scopolamine-induceda rat model of cholinergicamnesia.

In thisstudyweexaminedtheeffectsofAnthriscusnemorosa essential oil onmemoryprocesses, anxietyand depressive-like behaviorsin scopolamine-treatedrats.Correlation betweenthe behavioral scores of scopolamine-treated rats, as a result of inhalationofessentialoilwasalsoinvestigated.

2.Materialsandmethods

2.1.Plantmaterialsandvolatileoilpreparation

Aerial parts of Anthriscus nemorosa were collected in the floweringstageinAdiyaman,EasternAnatolia,Turkey,inJune2013 and identified by Prof. dr. Eyup Bagci at the Herbarium of DepartmentofBiology,FiratUniversitywhereavoucherspecimen (no.Hayta4829)wasregisteredanddepositedforreadyreference. Air-driedaerialpartsoftheplantsamplesweresubjectedto hydro-distillationfor3husingaClevenger-typeapparatustoobtainthe essentialoil.Thetotalessentialoilyieldwas0.7%(v/w). 2.2.Gaschromatography(GC–MS/GC–FID)analysis

GC–MSanalysisof theAnthriscusnemorosaessential oil was performedinPlantProductsandBiotechnologyResearch Labora-tory (BUBAL), Firat University, using Hewlett Packard- Agilent 5973N GC_–MS system with 6890 GC equipped with a _flame ionization detector (FID). The procedure was performed as describedpreviously[18].Individualcomponentswereidentified bycomparison withtheretention index fromthe GC_–MS NIST library[19].

2.3.Animals

36maleWistarratsaged3–4months(25010g)uponarrival tothelaboratorywereusedinthisstudy.Theanimalswerehoused ina temperatureandlight-controlledroom(22C, a 12-hcycle startingat08:00h)andwerefedandallowedtodrinkwaterad libitum.Theratsweredividedinto6groups(6animalspergroup): (1) the Control group received 0.9% saline with 1% Tween 80 treatment;(2)theScopolamine(Sco,0.7mg/kgb.w.,i.p.)– alone-treatedgroupreceived0.9%salinewith1%Tween80treatment,as

negative control; (3) the Diazepam alone-treated group (DZP, 1.5mg/kg)received0.9%salinewith1%Tween80treatment, as positive control; (4) the Tramadol alone-treated group (TRM, 10mg/kg)received0.9%salinewith1%Tween80 treatment, as positivecontrol; (5)theScopolamine-treatedgroupreceived by inhalationAnthriscusnemorosaessentialoil1%(Sco+AEO1%)and (6)theScopolamine-treatedgroupreceivedbyinhalation Anthris-cusnemorosaessentialoil3%(Sco+AEO3%).Control,DZP,TRM-and scopolamine alone-treated groups were caged in the same conditions but in the absence of the tested essential oil. Rats weretreatedinaccordancewiththeguidelinesofanimalbioethics fromtheActonAnimalExperimentationandAnimalHealthand WelfarefromRomaniaandallprocedureswereincompliancewith Directive 2010/63/EU of the European Parliament and of the Councilof22September2010ontheprotectionofanimalsusedfor scientificpurposes.ThisstudywasapprovedbytheCommitteeon the Ethics of Animal Experiments of the Alexandru Ioan Cuza Universityof Iasi(permitnumber:2192)and also,efforts were madetominimizeanimalsufferingandtoreducethenumberof animalused.

2.4.Y-mazetest

Short-termmemorywasassessedbyspontaneousalternation behaviorintheY-mazetask.TheY-mazeusedinthepresentstudy consistedofthreearms(35cmlong,25cmhighand10cmwide) and an equilateral triangular central area. 15min after the inhalation of Anthriscus nemorosa essential oil (AEO1% and AEO3%),ratswereplacedattheendofonearmandallowedto movefreelythroughthemazefor8min.Anarmentrywascounted whenthehindpawsoftheratwerecompletelywithinthearm. Spontaneousalternation behavior was defined asentryinto all three arms on consecutive choices. The number of maximum spontaneousalternationbehaviorswasthenthetotalnumberof armsenteredminus2andpercentspontaneousalternationwas calculated as (actual alternations/maximum alternations)100 [20]. Spontaneous alternation behavior is considered toreflect spatialworkingmemory,whichisaformofshort-termmemory. Locomotoractivitywasassessesbythenumberofarmentries.The mazewascleanedwitha10%ethanolsolutionanddriedwitha clothbeforethenextanimalwastested.

2.5.Radialarm-mazetest

The radialarm-mazeusedin thepresent studyconsistedof eight arms, numbered from 1 to 8 (48cm12cm), extending radiallyfromacentralarea(32cmindiameter).Theapparatuswas placed 50cm abovethefloor,and surroundedbyvarious extra-mazevisualcuesplacedatthesamepositionduringthestudy.At theendofeacharmtherewasafoodcupthathadasingle50mg foodpellet.Priortotheperformanceofthemazetask,theanimals werekeptonrestricteddietandbodyweightwasmaintainedat 85%oftheirfree-feedingweightoveraweekperiod,withwater beingavailableadlibitum.Beforetheactualtrainingbegan,three orfourratsweresimultaneouslyplacedin theradialarm-maze andallowedtoexplorefor5minandtakethefoodfreely.Thefood was initially available throughout the maze, but was gradually restrictedtothefoodcup.Theanimalsweretrainedfor4daysto runtotheendofthearmsandconsumethebait.Toevaluatethe basal activityofratsin radialarm-maze,theratsweregiven5 consecutivetrainingtrialsperdaytoruntotheendofthearmsand consumethebait.Thetrainingtrialcontinueduntilall5baitshave beenconsumedoruntilthe5minhaveelapsedwhichhavebeen set as the performance criteria. After adaptation,all rats were trainedwith1trialperday.Briefly,15minaftertheinhalationof Anthriscusnemorosaessentialoil(AEO1%andAEO3%),eachanimal

wasplacedindividuallyinthecenterofthemazeandsubjectedto workingandreferencememorytasks,inwhichsame5arms(nos. 1, 2,4,5and7),werebaitedforeachdailytrainingtrial.Theother3 arms(nos.3,6and8)wereneverbaited.Theselectionofthebaited armsisbasedonthefactthatanimalsprefertosolvethemaze usinganadjacentarmselectionstrategy.Inthiscase,wealtered adjacentarmpatterningbehaviorbyonlybaiting5arms(nos.1,2, 4,5,and7)subjectinganimalstochangetheirstrategyandavoid theunbaitedarms.Anarmentrywascountedwhenallfourlimbs oftheratwerewithinanarm.Measuresweremadeofthenumber ofworkingmemoryerrors(enteringanarmcontainingfood,but previouslyentered)andreferencememoryerrors(enteringanarm thatwasnotbaited)[20].Referencememoryisregardedasa long-termmemoryforinformationthatremainsconstantoverrepeated trials(memoryforthepositionsofbaitedarms),whereasworking memory is considered a short-term memory in which the information toberememberedchanges ineverytrial(memory forthepositionsofarmsthathadalreadybeenvisitedineachtrial). Themazewascleanedwitha10%ethanolsolutionanddriedwitha clothbeforethenextanimalwastested.

2.6.Elevatedplus-mazetest(EPM)

BehaviorinEPMisutilizedtoassessexploration,anxiety,and motorbehavior.15minaftertheinhalationofAnthriscusnemorosa essentialoil(AEO1%andAEO3%),eachratwasplacedinthecenter of themaze facingone closed arm.Behavior was observed for 5min,andthetimespentandnumberofentriesintotheopenand enclosedarmswascounted[21].Thepercentagesoftimespentin theopen arms(time spentin the openarms/time spentin all arms100)werecalculated.Inaddition,thetotalnumberof open-andenclosed-arm entries(numberofcrossing),whichindicates theexploratoryactivityofanimals[22],wasmeasured.IntheEPM, diazepamalone-treatedgroupwasusedaspositivecontrol. 2.7.Forcedswimmingtest(FST)

TheFSTisusedforassessingdepressive-likeresponse[23].The depressive-likeresponsewasassessed,basicallyusingthesame methoddescribedbyCamposetal.[24],butwithmodification.On the first day of the experiments (pretest session), rats were individually placed into cylindrical recipients (diameter 30cm, height59cm)containing25cmofwaterat261C.Theanimals wereleft toswim for 15min beforebeing removed,dried and returnedtotheircages.Theprocedurewasrepeated24hlater,ina 6minswimsession (testsession),15minaftertheinhalationof Anthriscusnemorosaessentialoil(AEO1%andAEO3%).Duringthe testsession,thefollowingbehavioralresponseswererecorded:(1) immobility(timespentfloatingwiththeminimalmovementsto keeptheheadabovethewater);and(2)swimming(timespent withactiveswimmingmovements).IntheFST,tramadol alone-treatedgroupwasusedaspositivecontrol.

2.8.Statisticalanalysis

Behavioral scores within Y-maze, radialarm-maze, elevated plus-mazeandforcedswimmingtestswereanalyzedbytwo-way analysisofvariance(ANOVA)followedbyTukeyposthoctestusing GraphPad Prism 6 software for Windows. In order toevaluate differencesbetweengroupsintheradialarm-mazetask,separate repeated-measures ANOVA were calculated on the number of working memory errors and the number of reference memory errorswithgroup(Control,Sco,Sco+AEO1%andSco+EO3%)as between-subjectfactoranddays(1–7)aswithin-subjectsfactors. Allresultsareexpressedasmeanstandarderrorofmean(S.E.M). F values for which p<0.05 were regarded as statistically

significant.Pearson’scorrelationcoefficientandregression analy-sis were used in order to evaluate the connection between behavioralmeasures.

3.Results

3.1.ChemicalcompositionoftheAnthriscusnemorosaessentialoil TheGC–MS/GC–FIDanalysisofthevolatileprofilesinAnthriscus nemorosaessentialoilislisted inTable1.Atotalof18different compoundswereisolatedwhichconstituted85.9%(w/w)ofthe total essentialoil. Asa resultof GC_–MC analysis,caryophyllene (23.6%) was the major compound for the sample studied. Additionally, other major compounds were trans-pinocarveol (9.8%), germacrene D(5.6%),

b

-elemene (4.2%) and

a

-terpineol (2.7%).Sesquiterpenesrepresented66.50%oftheessentialoiland consistedofcaryophyllene(23.6%),

b

-elemene(4.2%),

a

-farnesene (1%),germacreneD(5.6%),bergamotene(0.9%),valencene(0.6%),

d

-cadinene(12.1%),caryophylleneoxide(12.3%)andcarotol(6.2%). Monoterpenes represented 19.40% of the essential oil and consisted of

a

-pinene (0.5%), sabinene (1.7%),

b

-pinene (0.5%),

b

-phellandrene(0.4%),

b

-ocimene(1%),trans-pinocarveol(9.8%), 3-cyclohexen-1-ol(0.8%),

a

-terpineol(2.7%)andpregeijerene(2%). 3.2.EffectoftheAnthriscusnemorosaessentialoilonspatialmemory inY-mazetest

Analysesofthespontaneousalternationpercentagewithin Y-maze task showed significant overall differences between all groups(F(3,20)=9.73),p<0.0001(Fig.1b).Bothdosesofinhaled Anthriscusnemorosaessentialoil,significantlyimprovedmemory formation in scopolamine-treated rats as compared to scopol-amine alone-treated rats. The changes in the spontaneous alternation percentages of scopolamine-treatedratsexposed to Anthriscusnemorosaessentialoilarenotrelatedtothechangesin motor activity, as evidenced by the number of arm entries as comparedtocontrolrats(Fig.1a).

3.3.EffectoftheAnthriscusnemorosaessentialoilonspatialmemory inradialarm-mazetask

To investigate whether exposure to Anthriscus nemorosa essentialoil ofscopolamine-treatedratsaffectsspatialmemory,

Table1

Chemicalcomposition(%)ofidentifiedcompoundsintheessentialoilofAnthriscus nemorosaaerialparts.

No. Compounds RI Concentration(%) 1 a-pinene 1021 0.5 2 sabinene 1051 1.7 3 b-pinene 1056 0.5 4 b-phellandrene 1077 0.4 5 b-ocimene 1107 1 6 trans-pinocarveol 1176 9.8 7 3-cyclohexan-1-ol 1203 0.8 8 a-terpineol 1214 2.7 9 pregeijerene 1290 2 10 b-elemene 1369 4.2 11 caryophyllene 1390 23.6 12 a-farnesene 1399 1 13 germacreneD 1432 5.6 14 bergamotene 1438 0.9 15 valencene 1442 0.6 16 d-cadinene 1458 12.1 17 caryophylleneoxide 1496 12.3 18 carotol 1508 6.2 Total 85.9

theratswerefurtherevaluatedintheradialarm-mazetask.For workingmemoryerrors, repeated-measures ANOVA revealed a significanttimedifference(F(6140)=2.30,p<0.01)andasigni fi-cant group difference (F(3140)=3.11, p_<0.01) (Fig. 2a). For referencememoryerrors,repeated-measuresANOVArevealeda significant time difference(F(6140)=3.06,p<0.001) and group difference(F(3140)=7.60,p<0.0001)(Fig.2b).

3.4.EffectoftheAnthriscusnemorosaessentialoilonelevated plus-mazebehavior

AscanbeseeninFig.3a,intheelevatedplus-mazetaskANOVA revealeda significant overalldifferences between allgroups (F (4,25)=10.80,p<0.0001)onthepercentageofthetimespentin theopenarms.BothdosesoftheAnthriscusnemorosaessentialoil, but especially 3%,significantlyincreased thepercentage of the time spent in the open arms in scopolamine-treated rats as comparedtoscopolaminealone-treatedrats. Ascanbeseen in Fig. 3b, in the elevated plus-maze task ANOVA revealed a signi_ficantoveralldifferencesbetweenallgroups(F(4,25)=4.87, p<0.01)onthenumber ofopen-arm entries.Inhalation ofthe Anthriscusnemorosaessentialoil,butespecially3%,significantly increasedon thenumber of open-arm entriesof scopolamine-treatedratsascomparedtoscopolaminealone-treatedgroup.As canbe seen in Fig. 3c,in theelevated plus-maze task ANOVA revealeda significant overalldifferences between allgroups (F

(4,25)=11.30,p_<0.0001)onthenumberofcrossing(exploratory activity).Inhalation oftheAnthriscusnemorosaessentialoil,but especially 3%,significantlyincreasedthenumber of crossingof scopolamine-treated rats as compared to scopolamine alone-treated rats. The diazepam treatment, as positive control, significantly increasedthe percentage of thetime spent in the openarms,thenumberofopen-armentriesandthenumberof crossingintheelevatedplus-maze,actingasanxiolyticagent. 3.5.EffectoftheAnthriscusnemorosaessentialoilintheratforced swimmingtest

In the forced swimming test, ANOVA revealed a significant overalldifferencesbetweenallgroupsontheswimmingtime(F (4,25)=46.18,p<0.0001)(Fig.4a)andontheimmobilitytime(F (4,25)=43.01, p<0.0001) (Fig. 4b). Both doses of Anthriscus nemorosaessential oil,but especially3%, signi_ficantlyincreased swimmingtimeanddecreasedimmobilitytimeof scopolamine-treatedratsascomparedtoscopolaminealone-treatedrats.The tramadoltreatment,aspositivecontrol,increasedtheswimming timeanddecreasedtheimmobilitytimeintheforcedswimming test,actingasantidepressantagent.

More importantly, when linear regression was determined, significantcorrelationsbetweenthepercentageofthe spontane-ousalternationvs.thepercentageoftheopenarmstime(n=24, r=0.529,p<0.001)(Fig.5a),thepercentageofthespontaneous

Fig.1.EffectsoftheinhaledAnthriscusnemorosaessentialoil(AEO1%andAEO3%)intheY-mazeonthenumberofarmentries(a)andspontaneousalternation%(b)inthe scopolamine(Sco)-treatedrats.ValuesaremeansS.E.M.(n=6animalspergroup).ForTurkey’sposthocanalyses–#_{Scovs.Sco+AEO1%:p<0.001and}$_{Scovs.Sco+AEO3%:}

p<0.0001.

Fig.2.EffectsoftheinhaledAnthriscusnemorosaessentialoil(AEO1%andAEO3%)ontheworkingmemoryerrors(a)andthereferencememoryerrors(b)during7days trainingintheradialarm-mazeinthescopolamine(Sco)-treatedrats.ValuesaremeansS.E.M.(n=6animalspergroup).ForTurkey’sposthocanalyses–#

Scovs. Sco+AEO1%:p<0.01and$

alternation vs. swimming time (n=24, r=0.562, p<0.001) (Fig. 5b), the percentage of the spontaneous alternation vs. immobility time (n=24, r= 0.556, p<0.001) (Fig. 5c), the referencememoryerrors vs.swimming time (n=24,r= 0.595, p<0.001) (Fig. 5d) and the reference memory errors vs. immobility time (n=24, r=0.585, p<0.001) (Fig. 5e) were evidenced.

4.Discussion

Inthepresentstudy,weinvestigatedwhetherinhalationofthe Anthriscusnemorosa essential oil (1%and 3%for 21 continuous days)causesbehavioraleffectsasmemory-enhancing,anxiolytic andantidepressant-likeeffectsinscopolamine-treatedratsbased

Fig.3.EffectsoftheinhaledAnthriscusnemorosaessentialoil(AEO1%andAEO3%)intheelevatedplus-mazetestonthepercentageofthetimespentintheopenarms(a),the numberofopen-armentries(b)andnumberofcrossing(c)inthescopolamine(Sco)-treatedrats.ValuesaremeansS.E.M.(n=6animalspergroup).ForTurkey’sposthoc analyses–#

Scovs.Sco+AEO1%:p<0.01and$

Scovs.Sco+AEO3%:p<0.001(a),#

Scovs.Sco+AEO1%:p<0.001and$

Scovs.Sco+AEO3%:p<0.0001(b)and#

Scovs. Sco+AEO1%:p<0.001and$

Scovs.Sco+AEO3%:p<0.0001(c).

Fig.4.EffectsoftheinhaledAnthriscusnemorosaessentialoil(AEO1%andAEO3%)onswimmingtime(a)andimmobilitytime(b)inthescopolamine(Sco)-treatedratsduring the6minperiodintheforcedswimmingtest.ValuesaremeansS.E.M.(n=6animalspergroup).ForTurkey’sposthocanalyses–#

Scovs.Sco+AEO1%:p<0.0001and$

Sco vs.Sco+AEO3%:p<0.001(a)and#

Scovs.Sco+AEO1%:p<0.0001and$

onspecificbehavioraltests(Y-maze,radialarm-maze,EPMand FST).

TheGC–MS/GC–FIDanalysesindicatedcaryophyllene(23.6%), followed by trans-pinocarveol (9.8%), germacrene D (5.6%),

b

-elemene(4.2%)and

a

-terpineol(2.7%),asthemaincomponents of our Anthriscus nemorosa essential oil suggesting that these constituents could be responsible for the observed behavioral effects in scopolamine-treated rats. It has been reported that

b

-caryophyllenepreventedcognitiveimpairmentinAPP/PS1mice, and this positive cognitive effect was associated with reduced

b

-amyloid burden in both the hippocampus and the cerebral cortex. Also, it has been suggested that

b

-caryophyllene is an attractive molecule for the development of new drugs with therapeuticpotentialforthetreatmentofAD[25].Inthelightof this study, we presume that our high-caryophyllene (23.6%) containing essential oil sustains spatial memory formation in

scopolamine-inducedamnesiainthespecificbehavioraltests (Y-maze,radialarm-maze).

BothdosesofAnthriscusnemorosaessentialoilin scopolamine-treated ratssignificantly improved spatial workingmemory, as evidencedbytheincreaseofspontaneousalternationpercentage as compared to scopolamine alone-treated rats. This result suggeststhatbothdosesofAnthriscusnemorosaessentialoilused in this study displays an improvedeffect onacquisition of the short-term memory of scopolamine-treated rats within the Y-mazetask.However,nodifferenceswereobservedbetweenboth doses of Anthriscus nemorosa essential oil on spatial working memory within the Y-maze task. The improvement of spatial working memory within Y-maze task cannot be attributed to locomotoractivity,becausesignificantchangesinthenumberof entries of the groups treated with the Anthriscus nemorosa essentialoilascomparedwithcontrolratswereobserved.

Fig.5.Pearson’scorrelationbetweenspontaneousalternation%vs.%openarmstime(a),spontaneousalternation%vs.swimmingtime(b),spontaneousalternation%vs. immobilitytime(c),referencememoryerrorsvs.swimmingtime(d)andreferencememoryerrorsvs.immobilitytime(e)incontrolgroup(*),scopolamine(Sco) alone-treatedgroup(&),Sco+AEO1%group(~)andSco+AEO3%group(^).

Thescopolamine-treatedratsexposedtodoseof1%Anthriscus nemorosa essential oil exhibited an improvement of working memory, along with an improvement of long-term memory, explored byreferencememorywhen exposed toboth dosesof Anthriscusnemorosaessentialoil(1%and3%)ascomparedwith scopolaminealone-treatedratswithintheradialarm-mazetask. ThesefindingssuggestthatinhalationoftheAnthriscusnemorosa essentialoilplaysanimportantroleinspatialmemoryformation, especially on both working memory and reference memory. However,non-significantdifferenceswereobservedbetweenboth dosesoftheAnthriscusnemorosaessentialoilonworkingmemory andreferencememoryintheradialarm-mazetask.

Thepresentstudyalsoevaluatedwhethermemoryimpairment inducedby scopolamineisrelated withanxietyand depressive behaviorsasassessedinspecificbehavioraltests(elevated plus-mazeandforcedswimmingtest).Scopolaminealone-treatedrats havedecreasedthepercentageofthetimespentintheopenarms, the number of open-arm entries and the number of crossing (exploratoryactivity)intheelevatedplus-mazetest.Thisindicates thatthescopolamine-alonetreatedratsexperiencedhighlevelsof anxietyandweresuitableforevaluatingthepresumedanxiolytic substancesasouressentialoil[21].

In the present study we demonstrated that inhalation of Anthriscus nemorosa essential oil in scopolamine-treated rats produces anxiolytic-likeactivity.Furthermore, afterthe scopol-amine-treatedratsbeingexposedtoAnthriscusnemorosaessential oil,thepercentageof timespentintheopenarmssignificantly increased,especiallyinthegroupexposedto3%essentialoilas compared to scopolamine-alone treated rats. Additionally, the number of open arms entries and the number of crossing (exploratory activity) increased, especially in the group of scopolamine-treatedratsexposedtoAnthriscusnemorosaessential oil3%ascomparedtoscopolamine-alonetreatedrats.

Asexpected,diazepam(DZP)asabenzodiazepinedrugusedas positivecontrolproducedsignificantincreasesinthepercentageof timespentintheopenarms,thenumberofopen-armentriesand the number of crossing (exploratory activity) as compared to scopolamine-alonetreatedrats.Thesedataareconsistentwiththe resultsofnumerouspreviousstudies,whichhaveshownthatDZP andotherbenzodiazepinesproducesignificantanxiolyticeffectsin a varietyof anxiolyticscreeningprocedures, includingelevated plus-maze test procedures [26]. The pharmacological action of diazepamenhancestheeffectoftheneurotransmitterGABAby bindingtothebenzodiazepinesiteontheGABAAreceptor(viathe constituent chlorine atom) leading to central nervous system (CNS)depression[27].Theanxietyindicatorsintheelevated plus-maze(thepercentageofthetimespentintheopenarmsandthe numberof open-armentries)showed upbeingsensitive tothe agentswhichwerethoughttoactviatheGABAAreceptorcomplex [28].

Ithasbeenreportedthat

b

-caryophylleneproducedmultiple behavioralchangesrelevanttoanxietyanddepressioninC57BL/6 micebymodulatingtheGABAAreceptorsactivity[29].Inlightwith thisreport,ourhigh-caryophyllene(23.6%)containingAnthriscus nemorosaessentialoilhasincreasedanxiolytic-likebehaviorand anti-depressive-likeresponseinscopolamine-treatedrats.

Within the forced swimming test, the swimming time decreased and the immobility time increased in scopolamine alone-treatedratsascomparedtocontrolrats.Thisindicatesthat the scopolamine alone-treated rats exhibited depression. After beingexposedtobothdosesofAnthriscusnemorosaessentialoil (1%and3%),theswimmingtimesignificantlyincreasedandthe immobility time significantly decreased, especially in group exposedto3% essentialoil ascompared toscopolamine alone-treatedrats.

TheseresultssuggestedthatAnthriscusnemorosaessentialoil possessesastrongantidepressant-likeresponsetoaninescapable stress.Inourstudy,tramadol(TRM),aspositivecontrol,produced significant increases in the swimming time and decreases the immobilitytimeascomparedtoscopolamine-alonetreatedrats. Tramadolisauniquedrugwithmultiplemodesofaction.Itisa weak agonist of the

m

-opioid receptor but it also inhibits the reuptakeofserotoninaswellasnorepinephrine.Itisananalgesic anditisalsoconsideredasanantidepressant[30].

Moreover, we found a significant correlation between the percentageof thespontaneousalternationvs.thepercentageof theopenarmstime,thepercentageofthespontaneousalternation vs.swimmingtime,thepercentageofthespontaneousalternation vs.immobilitytime,referencememoryerrorsvs.swimmingtime and referencememory errors vs. immobility time when linear regressionwasdetermined.

Thesedatasuggestthatmemory-enhancementscoreswithin Y-mazeandradialarm-mazetestsalongwithdecreaseofanxietyand depressive-likebehaviorswithinelevatedplus-mazeand forced swimming tests couldbe related with the involvement of the inhaled Anthriscus nemorosa essential oil against scopolamine-inducedmemoryimpairment,anxietyanddepressionin labora-toryrats.

5.Conclusion

Consideringtheneedfornovelcompoundsthatcouldimprove conventional therapies aswellasprovidenewagents targeting psychiatricdisorders,thepresentstudyhasclearlydemonstrated thememory-enhancer,anxiolyticandantidepressanteffectsofthe inhaledAnthriscusnemorosaessentialoilinscopolamine-treated rats.

Conflictofinterest

Theauthorsdeclarenoconflictsofinterest. Acknowledgments

Emel Aydin was supported by LLP Erasmus (2013/2014), AlexandruIoanCuzaUniversity,Iasi,Romania.

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