The document describes a study that investigated the antidepressant activity of the essential oil from Litsea glaucescens leaves. The essential oil was found to have antidepressant-like effects in mice based on testing in the forced swimming test. Two major compounds in the oil, β-pinene and linalool, were identified as the active principles responsible for the antidepressant effects. β-pinene and linalool also showed sedative-like activity in mice. The results support the traditional use of L. glaucescens for treating sadness and nervous conditions.

1. Antidepressantactivityof Litsea glaucescens essentialoil:Identification
of b-pineneandlinaloolasactiveprinciples
S.L. Guzma´ n-Gutie´ rrez a, R.Go´ mez-Cansino a, J.C.Garcı´a-Zebadu´ a a, N.C.Jime´nez-Pe´ rez b,
R. Reyes-Chilpa a,n
a Universidad NacionalAuto´noma deMe´xico, InstitutodeQuı´mica, DepartamentodeProductosNaturales,CiudadUniversitaria,04510Coyoaca´n Me´xico D.F.,Mexico
b Universidad Jua´rez Auto´noma deTabasco,Divisio´n deCienciasBiolo´gicas, CarreteraVillahermosa-Ca´rdenas Km0.5,Villahermosa,Tabasco86039,Mexico
a r t i c l e info
Article history:
Received 18February2012
Received inrevisedform
22 June2012
Accepted 22July2012
Available online31July2012
Keywords:
Litsea glaucescens
Essential Oil
Depression
Sedative
b-pinene
Linalool
a b s t r a c t
Ethnopharmacologicalrelevance:Litseaglaucescens (Lauraceae)hasbeenusedinMexicanTraditional
Medicinetorelieveillnessrelatedtocentralnervoussystem,suchasepilepsy,frightandsadness.
In thisstudy, L. glaucescens essentialoilpropertiesoncentralnervoussystemwereevaluatedinmice
using differentbehavioraltests.
Materialsandmethods: The essentialoilwasobtainedbyhydrodistillationandanalyzedbyGC/MS.
Identificationofmajorcompoundswasalsocarriedoutbycomparisonwithauthenticsamples.The
psychopharmacologicalprofileof L. glaucescens essentialoil,andsomeitsmajorcompounds,were
evaluatedinmiceusingseveralexperimentalmodels:forcedswimmingtest(FST:Antidepressant-like
activity),openfieldtest(OFT:Spontaneouslocomotoractivity),elevatedplus-maze(EPM:Anxiolytic-
like activity),exploratorycylinder(ECT:Sedative-likeactivity),rotarod(motorcoordination)and
tractionperformance(myo-relaxanteffect)theessentialoilandactiveprincipleswasadministered
intraperitoneally.
Results: The essentialoilshowedantidepressant-likeactivityatdosesof100and300mg/Kg.The
monoterpenes b-pineneandlinaloolwereidentifiedasthetwomainactiveprinciplesoftheessential
oil, andshowedantidepressant-likeandsedative-likeactivity.Eucalyptol,limoneneand a-pinenethey
did notshowantidepressant-likeactivity,andwerenotfurthertested.
Conclusions:L.glaucescens essentialoilshowedantidepressantactivity, b-pineneandlinaloolwere
identifiedasitsactiveprinciples.Theseresultssupporttheuseof L. glaucescens in MexicanTraditional
Medicineforthetreatmentofsadness.
& 2012 ElsevierIrelandLtd.Allrightsreserved.
1. Introduction
Increasing urbanpopulationandmetropolitanlifestylehasled
to healthproblemslikedepressionandanxiety;insomniacanbe
associated withthesesufferingsworseninghealthstatus.The
WHO estimatesthatdepressionwillbetheseconddisabling
disease intheworldin2020.Applicationofdifferentmedicinal
plant speciesfortreatmentofdepressionhasshowntobe
effective (Sarris etal.,2011). Severalessentialoilsobtainedfrom
aromatic speciesofplantsarealsousedinaromatherapytorelive
depression, forexample: Lavandula spp (lavender), Jasminum
officinale (jasmine), Rosmarinus officinalis (rosemary), Rosa spp
(rose) and Matricaria chamomile among others(Steflitsch and
Steflitsch, 2008). Themonoterpenesarethemajorconstituents
of essentialoils,andsomeofthemhaveshowncentralnervous
system activity,forinstancelinaloolissedativeinhumans
(Sugawara etal.,1998) and a-terpineol isanalgesicinmice
(Quintans-Ju´ nior etal.,2011).
The leavesof Litsea glaucescens (Lauraceae), anativeshrub
species ofMe´ xico andCentralAmerica,knownas‘‘laurel’’,are
commonly usedinthisareaforseasoningfood,replacingthe
leaves oftheEuropeanspecies Laurus nobilis (Lauraceae), butthey
are alsousedinTraditionalMedicine(Jime´nez-Pe´ rez etal.,2011).
Due toitsextensiveuse, L. glaucescens constitute oneofthemain
non timberforestproductsinMe´ xico (Tapia-Tapia andReyes-
Chilpa, 2008). Regardingtoitsmedicinalapplications,theinfu-
sion oftheleavesof L. glaucescens are usedtotreatdiarrhea,
vomit, paininthebones,postpartumbaths,colicofchildren,and
illness relatedtocentralnervoussystem(Jime´nez-Pe´ rez etal.,
2011). Forinstance,thepeopleoftheMazahuaethniafrom
Atlacomulco, Me´ xico, prepareadecoctiontotreatsadness,ner-
vousness, anger,andfright(‘‘susto’’);forthesepurposesthey
boil theleavesof L. glaucescens along withothersmedicinal
plants, suchas Melissa officinalis (lemon balm), Mentha spp (mint),
Contents listsavailableat SciVerse ScienceDirect
journalhomepage: www.elsevier.com/locate/jep
JournalofEthnopharmacology
0378-8741/$ -seefrontmatter & 2012 ElsevierIrelandLtd.Allrightsreserved.
http://dx.doi.org/10.1016/j.jep.2012.07.026
n Corresponding author.Tel.: þ52 5556224430;fax: þ52 5556162203.
E-mail address: chilpa@servidor.unam.mx (R.Reyes-Chilpa).
Journal ofEthnopharmacology143(2012)673–679

2. Aloysiatriphylla (cedro´ n),and Cinnamomumzeylanicum (cinnamon).
The patientfastsovernightandinthemorningdrinksaglassof
the decoctionduring8day;thedecoctioncanalsobedrunk
instead ofdailywateras‘‘aguadeuso’’(Lozano-Mascarua,1996;
Lozano 222,HerbariumIMSSM).
Medicinal applicationsof Litsea glaucescens can alsobetracked
to manuscriptswrittenintheXVIcentury.TheSpaniardphysician
and naturalistFranciscoHerna´ndez whoexploredthecentralpart
of whatnowisMe´ xico, wroteinhis‘‘HistoriadelasPlantasdela
Nueva Espan˜ a’’ (HistoryofthePlantsoftheNewSpain,1571–
1577) aboutaplantnamed‘‘Ecapatli’’inNahuatl(theAztec
language), thefollowing:‘‘Itisakindof‘laurel’smallerthan
ours, andwithsmallerleaves,butsimilarinwhateverelse.The
natives cureparalysiswithitsodoroussmoke,itsdecoction
administered inwashingsandbathshealsfatigue,andepilepsy
of children’’(Herna´ ndez, 1959). Ecapatlihasbeenidentifiedas
Litsea glaucescens Kunth (Valde´ s andFlores,1985). Currently,in
Me´ xico theNahuatlwords‘‘ecapatli’’and‘‘ecapatle’’arestillused
in somelocalitiestonamethisplant,etymologicallyarederived
from theterms‘‘ehe´ catl’’ (wind)and‘‘pahtli’’(medicine)
(Montemayor, 2007). Theterm‘‘pahtli’’isusedtoanyplantwith
medicinal properties(Be´ jar etal.,2000).
In thepresentworkweexaminedthepotentialactivityof
Litsea glaucescens essential oil,andsomeofitsmainmonoter-
penes, incentralnervoussystemusingseveralbehavioralmodels
in mice:forcedswimmingtest(FST:Antidepressant-likeactivity),
open fieldtest(OFT:Spontaneuslocomotoractivity),rotarod
(motor coordination),tractionperformance(myo-relaxant effect),
elevated plus-maze(anxiolytic-likeactivity)andexploratory
cylinder (ECT:Sedative-likeactivity).
2. Materialsandmethods
2.1. Plantmaterialandessentialoilextraction
Litsea glaucescens leaves werecollectedinHuitzila,Veracruz,
Me´ xico, avoucherspecimenwasdepositedintheHerbariumof
Instituto deEcologı´a, A.C.atXalapa,Me´ xico. Theleaveswere
dried atroomtemperature,andground(350g),essentialoilwas
isolated byhydrodistillationwithdistilledwater(1.5L,3h).The
oil obtainedwasdriedwithanhydroussodiumsulphateand
stored inamberglassvialat4 1C.
2.2. Drugs
The positivecontrolswere:Diazepam(DZP,1.5and3.5mg/Kg,
Roche) asanxiolyticandsedativedrug,imipraminehydrochloride
(IMI, 30mg/Kg,Sigma)asantidepressantdrug.Theauthentic
samples forthecharacterizationof L. glaucescens essential oilby
GC/MS, andforbiologicaltestswere:Eucalyptol,(R)-(þ)-limo-nene,
linalool,(1S)-()-b-pinene, (1S)-()-a-pinene, g-terpinene,
(R)-() carvoneand o-cymene (AldrichCo.).
2.3. Chemicalanalysis
The essentialoilinchloroformsolutionwasanalyzedby
chromatograph (GC)Agilent6890Ncoupledtomassspectro-
meter (MS)LECOmodel4D.ThecapillarycolumnwasaDB-5
10 m0.18 mmI.D.0.18 mm filmthickness,theheliumwasthe
carrier gas,flowrate,1mL/min;oventemperaturerangewas
from roomtemperatureto40/300 1C, injectortemperature,
300 1C; detectortemperature,200 1C. Massspectrawereregis-
tered over m/z 45–500, usinganionizingvoltageof70eV.
The constituentsofessentialoilwerecharacterizedbymatching
their massspectrawithcompoundlibraryNIST.Themonoterpenes
eucalyptol, o-cymene, linalool, a-pinene, b-pinene, g-terpineneand
L-carvone werealsoidentifiedbycomparisonwithmassspectraof
authenticsamplesinjectedtoGC–MSapparatus.ThealkanesC8 to
C24 wereusedtocalibratetheKovatsscale(linealretentionindexes)
forthemixturesofterpenesstudied(Macı´as-Rubalcavaetal.,2010;
Rivero-Cruzetal.,2011).Quantificationofeachcompoundwas
performed onthebasisoftheirGCpeakareas.
2.4. Animals
All experimentswereperformedwithadultmaleICRmice
(27–32 g)obtainedfromUniversidadAuto´noma Metropolitana–
Xochimilco –atMe´ xico City.Miceweremaintainedatconstant
room temperature(22171 1C) undera12hlight/darkcyclewith
free accesstofoodandwater.Proceduresinvolvinganimalcare
were conductedinconformitywiththeMexicanOfficialNormfor
Animal CareandHandling(NOM-062-ZOO-1999),andincompli-
ance withinternationalrulesoncareanduseoflaboratory
animals. Allexperimentswereperformedinaroomisolatedfrom
external noise.
2.5. Treatments
Litsea glaucescens essential oil, b-pinene, a-pinene, linalool,
limonene andeucalyptolweresuspendedin0.5%Tween80in
saline solution(0.9%).Imipraminewasdissolvedinsalinesolution
(0.9%). Thesuspensionwasintraperitoneallyinjectedinavolume
of 0.1mL/10gbodyweight.Controlanimalsreceivedthesame
volume ofvehicle.FortheFST,allsubstanceswereadministered
three times:Immediatelyaftertheinitial15-minpre-test,18and
1 hpriortotheswimmingtest(Herrera-Ruiz etal.,2006). Theoil
and itscomponentswerefirstassayedintheFST,andifactive
were furthertestedwithOF,EC,PMrota-rodandtraction
performance withthesameadministrationsequenceofFST,24,
18 and1hpriortothebehavioraltasks.TheOFT,ECandPMwere
carried outconsecutivelyinthesameexperiment.
2.6. Forcedswimmingtest(FST)
TheFSTforassessingantidepressantactivitywassimilartothat
describedby Herrera-Ruiz etal.(2006) and Martı´nez-Va´zquezetal.
(2012) on basisofthemodelproposedby Porsoltetal.(1977).
The apparatusconsistedofaglasscylinder(25cmhigh12 cm
diameter)filledwithwater(2471 1C) upto15cm.Eachanimalwas
subjectedtoapre-testsession(15min)inthevessel24hbeforethe
swimmingtestwhichlasted5min. Litseaglaucescens essentialoil,
monoterpenes, vehicleorimipraminewereadministeredthree
times:Immediatelyaftertheinitial15-minpre-test,18and1h
priortotheswimmingtest(Herrera-Ruiz etal.,2006). Alltest
sessionswerevideotaped,andanalyzedaftertheexperiment;the
immobilitytime(seconds)foreachanimalwasregistered.Mice
wereconsideredasimmobilewhentheymadenofurtherattempts
toescape,exceptingthemovementsnecessarytokeeptheirheads
above water.Adecreaseinthedurationofimmobilitytimeinthe
testgroupcomparedtothecontrolgroupindicatesanantidepres-
sant effectofthesubstancetested. Eachexperimentalgroupcon-
sisted of10–12animals(Herrera-Ruiz etal.,2006).
2.7. Openfieldtest(OFT)
The spontaneouslocomotoractivitywasmeasuredinanopen
area madeoftransparentacrylic(1828 cm)dividedinto12
squares ofequalarea.Eachanimalwasgentlyplacedinthecenter
of apparatus.Theobservedparameterwasthenumberofsquares
crossed (withthefourpawsinasquare)inaperiodoffive
S.L. Guzma´n-Gutie´rrez etal./JournalofEthnopharmacology143(2012)673–679 674

3. minutes. Eachexperimentalgroupconsistedof6animals
(Herrera-Ruiz etal.,2006).
2.8. Exploratorycylinder
The apparatusconsistedofaglasscylinder(30cminheight,
11 cmindiameter,and3mminthickness).Amousewasplaced
on thecylinderfloorcoveredwithpaper;thenumberofrears
performed overa5-minperiodwasrecorded.Theinnersideof
the apparatuswascleanedwithanethanolsolution(10% v/v), and
the paperchangedaftereachtest.Reducedexploratoryrearing
after placementinanunfamiliarenvironmentrevealsasedative
effect. Eachexperimentalgroupconsistedof6animals(Guzma´n-
Gutie´ rrez andNavarrete,2009; Ugalde etal.,2005).
2.9. Elevatedplus-maze(EPM)
The plus-mazeapparatuswaselevated50cmabovefloorlevel
and consistedoftwoopenarms(length30cm width 5cm)and
two enclosedarms(length30cm width 5cm height 15cm)
and acentralplatform(55 cm).Eachanimalwasplacedatthe
center ofmazefacinganopenarm.Thecumulativetimespentin
the openarmswasrecordedfor5min.Amousewasratedinside
the openarmsifallfourpawswereonthearm.Alltestswere
recorded withavideocamera,andanalyzedaftertheexperiment.
After eachtest,themazewascleanedupwithethanolsolution
(10% v/v). Eachexperimentalgroupconsistedof6animals
(Carrasco etal.,2006)
2.10. Rota-rodandtractionperformance
Motor coordinationwasassessedbyaRota-rodTreadmill7600
(Ugo Basile),4cmdiameter,atconstantspeed(16rpm).Only
mice thatapprovedaprevioustrainingremainingforatleasttwo
min ontherod,and30shangedwiththeirforelimbsona
horizontal stainlessbar(1.5mmofdiameter,40cminheight
and 60cmoflength)wereselected,andpooledingroupsofsix
animals. Thetimethateachmouseremainedontherodduring
the testperiod(maximumtwominutes)andhangedofthebar
(maximum 30s)wasrecordedat15,30,45,60,90and120min
after administration(Oliva etal.,2004).
Table 1
Constituents identifiedintheessentialoilof Litsea glaucescens by GC/MSanalysis.
Compound Retention time(s)Peakarea(%)Kovatsretention
index
a-pinene 246.3 3.86945.72
Camphene 257.75 0.24958.63
b-pinene 280.75 2.34984.56
p-mentha-1,5-diene 302.30.511014.5
Limonene 321.55 8.661050.0
o-cymene 323.8 25.861054.2
Eucalyptol 326.75 26.061059.6
g-terpinene 336.6 2.831077.8
Terpinolene 350.55 0.9231103.5
Linalool oxide 352.75 0.9231107.6
Dehydro-p-cymene 355.650.3571112.9
Linalool 362.5 3.641125.6
Fenchol, exo- 370.75 0.1441140.8
4-carene 373.7 0.1491146.2
4-acetyl-1-methylcyclohexene 376.90.7041152.1
Terpinen-4-ol 402.45 5.081199.3
a-terpineol 409.3 0.7201214.9
9,12,15-octadecatrienal 410.10.7721216.8
Bicyclo[2.2.1]heptan-2-ol,1,3,3-trimethyl-, acetate, endo- 416.90.4391232.5
2,6-octadien-1-ol, 2,7-dimethyl-424.50.1731250.1
S (þ)-carvone 431.9 1.851267.2
2-cyclohexen-1-one, 3-methyl-6-(1-methylethyl)-436.450.2511277.7
1,3-butanedione, 1-(2-furanyl)-436.60.2401278.1
2H-pyran-3-ol, 6-ethenyltetrahydro-2,2,6-trimethyl-445.350.5831298.3
Bicyclo[2.2.1]heptan-2-ol, 1,7,7-trimethyl-,acetate,(1S-endo)- 447.000.5831302.1
Phenol, 2-ethyl-4,5-dimethyl-463.50.0811340.3
2-oxabicyclo[2.2.2]octan-6-ol, 1,3,3-trimethyl-,acetate470.150.2871355.7
Neryl acetate 478.55 1.351375.1
Carveol 478.95 1.351376.1
Caryophyllene 486.65 0.3281393.9
trans-a-bergamotene 507.050.1761447.6
a-caryophyllene 516.5 0.3481472.9
Naphthalene, 1,2,4a,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-532.60.3761516.0
Naphthalene, 1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-,(1S-cis)- 539.750.1611535.2
cis-a-Bisabolene 547.75 0.3951556.6
a-Calacorene 549.45 0.3951561.2
1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-556.150.2741579.1
Caryophyllene oxide564.450.5511601.6
Ledene oxide-(II) 574.9 0.2151634.6
2-Isopropenyl-4a,8-dimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalene 582.20.1861657.6
t-cadinol 584.95 0.4421666.35
Dispiro[2.1.2.4]undecane, 8-methylene-586.350.4421670.7
2-naphthalenemethanol, decahydro,4a-trimethyl-8-methylene-,[2R-(2,4a,8a)]- 590.80.1631684.8
a-bisabolol 1717.9 0.0411717.9
Kaurene 712.45 0.9942089.4
S.L. Guzma´n-Gutie´rrez etal./JournalofEthnopharmacology143(2012)673–679 675

4. 2.11. Statisticalanalysis
The datawereanalyzedbyone-wayANOVAfollowedby
Tukey’s test,unlessotherwisestated.Intheindicatedcasesthe
data weretransformedforperformingtheBartlett’stest.The
differences wereconsideredsignificantif pr0.05. Thedatawere
expressed asmean7S.E.M.
3. Results
3.1. CompositionofL.glaucescensessentialoil
The L. glaucescens essential oilwasobtainedina0.85%yield.
A totalof45compoundswereidentified representing95.23%ofthe
yield. Themajorcomponentwaseucalyptol (26.06%),followedby
o-cymene(25.86%),limonene(8.66%),tepinen-4-ol(5.08%), a-pinene
(3.86%),linalool(3.64%), g-terpinene (2.83%), b-pinene(2.34%),
(S)-(þ)-carvone(1.85%),andnerylacetate(1.35%),andcarveol
(1.35%)(Table1). Thesecompounds(11)accountedfor82.88%of
the yield,whileotheridentifiedconstituentsrepresented o1%.Ina
previous study,wedidnotdetect o-cymene intheessentialoilof L.
glaucescens, this canbeattributedtoadifferentextractionmethod
(hydrodistillation),andanalyticalconditionshereused,whichallowed
to abetterseparationofthecomponentsthatelutebetween321.5
and 326.7s(limonene, o-cymene,andeucalyptol).Thisalsomay
account toareductionintheyieldofeucalyptol(26.06%),ascompared
withthepreviousanalysis(36.29%)(Jime´nez-Pe´rez etal.,2011).
3.2. EffectofL.glaucescensessentialoilinbehavioraltests
When theanimalsweresubjectedtotheFSTtheessentialoil
administered atthedosesof100and300mg/Kgsignificantly
decreased theimmobilitytime(F4,45¼23.27; pr0.05) ascom-
pared withthevehiclegroup(Fig. 1A). However,noneofboth
doses reachedtheeffectofthepositivecontrolimipramineat
30 mg/Kg.Themicetreatedwiththedoseof300mg/Kgpresented
a lightabdomencontractionaftertheadministrationofthe
essential oil,conductthatweinterpretedaspain,butremained
alive andingoodconditionsduring24h(beforesacrifice).The
OFT wascarriedouttodeterminetheeffectofessentialoilonthe
spontaneous motoractivityofmice;howeveranyofthetested
doses modifiedthenumberofcrossingsofmice,while,diazepam
caused asignificantdecreaseinthemotoractivityofthemice
(F4,25¼22.9; pr0.05) (Fig. 1B). IntheEPMtest,theessentialoil
Treatment mg/Kg
Immobility (s)
0
50
100
150
200
250
a
ab
Vehicle
Imipra 30 54.8 100 300
L. glaucescens essential oil
ab
b
b
Number of crossings
0
10
20
30
40
a
Treatment mg/Kg
Vehicle
DZP 3.5 54.8 100 300
L. glaucescens essential oil
b b
b b
Fig. 1. Effect producedbyi.p.administrationofdifferentdosesof L. glaucescens essential oilinmice.(A)ImmobilitytimeofmiceintheFST.Datarepresentthemean7SEM
(n¼10). ANOVAfollowedbyTukey’stest.ThedataweretransformedfortheBartlett’stest.Imipra¼imipramine. (B)TotalcrossingsofmiceexposedtoOFT.Datarepresent
the mean7SEM (n¼6). ANOVAfollowedbyTukey’stest.DZP¼diazepam. Lettersaandbindicatesignificantdifferencewiththevehicleandpositivecontrol,respectively,
po0.05.
Time after injection (min)
Performance (s)
0
20
40
60
80
100
120
140
Vehicle
L. glaucescens 100 mg/Kg
Linalool 100 mg/Kg
β-pinene 100 mg/Kg
Diazepam 3.5 mg/Kg
a
a
a
Time after injection (min)
0 20406080100120140 0 20406080100120140
Performance (s)
0
5
10
15
20
25
30
35
Vehicle
L. glaucescens 100 mg/Kg
Linalool 100 mg/Kg
β-pinene 100 mg/Kg
Diazepam 3.5 mg/Kg
a a a
a
a
Fig. 2. Effect producedbyi.p.administrationof L. glaucescens essential oil, b-pinene, linaloolanddiazepamonmice.(A)Motorcoordinationinrotarodtest.(B)Traction
test. Datarepresentthemean7SEM (n¼6). ANOVAfollowedbyTukey’stest.Letteraindicatesignificantdifferencewiththevehicle, po0.05.
S.L. Guzma´n-Gutie´rrez etal./JournalofEthnopharmacology143(2012)673–679 676

5. tested atthesamedosesasintheFSTdidnotmodifysignificantly
the numberofentriesintotheopenandclosedarms,neitherthe
time spentintheopenarmascomparedwiththevehicle(data
not shown),indicatinglackofanxiolyticactivity.Themicetreated
with theessentialoildidnotexhibitedlackofcoordinationor
muscle relaxation,asassessedintherotarod,orinthetraction
performance tests,respectively;incontrastwiththediazepam,a
positive control,(F4,25¼15.96; pr0.05 and F4,25¼16.95; pr0.05)
(Fig. 2A andB).
3.3. IdentificationoftheactivemetabolitesofL.glaucescens
essential oil
In ordertoidentifythecompoundsresponsibleofantidepres-
sant activityofessentialoil,weevaluatedtheeffectofsomeofits
major components(eucalyptol,limonene,linalool, a-pinene and
b-pinene) inmiceusingtheFSTat100mg/kgi.p.Onlythe
b-pinene andthelinaloolsignificantlydecreasedtheimmobility
time (F6,63¼13.48; pr0.05) ofmiceascomparedwiththevehicle
(Fig. 3), theeffectofthetwomonoterpenesatthisdosewas
similar tothepositivecontrolimipraminetestedat30mg/Kg.
b-pinene andthelinaloolwerefurthertestedatahigherand
lower doseintheFST.Onlythe b-pinene showedadecreaseinthe
time ofimmobilitywiththedosesof173.2mg/kg(F4,55¼6.699;
pr0.05) (Fig. 4A andB).However,the b-pinene (F4,25¼18.74;
pr0.05) andthelinalool(F4,25¼52.12; pr0.05) decreasedthe
spontaneous motoractivityofmiceinOFT,butnotatthesame
level asdiazepamat3.5mg/Kg(Fig. 5A andB).Bothmonoter-
penes inducedareductionofexploratoryactivityassessedinthe
ECT (Fig. 6A andB)asthesamelevelasdiazepam(F4,25¼11.08;
pr0.05 and F4,25¼34.78; pr0.05). Itisinterestingtonotethat
b-pinene andlinaloolatactivedosesintheFST(100mg/kg)didnot
produce areductioninperformanceofmiceintherotarodand
traction tests,incontrastwiththediazepam,apositivecontrol,
(F4,25¼15.96; pr0.05 and F4,25¼16.95; pr0.05)(Fig.2A andB).
4. Discussion
The useofaromaticplantstoreliefdifferentillnessisnota
new therapy,actuallyaromaticplantshavebeenusedsincemany
centuries agobydifferentculturesaroundtheworld(Linck etal.,
2009); however,untilrecently,modernsocietieshaveoverlooked
this ancientknowledge,butachangeinattitudehasoccurred,and
the notionthataromatherapycanrelieveillnesssuchasdepres-
sion, anxietyandinsomnia,commonhealthproblemsinthe
society,hasspread,especiallyinbigcitieswithastressedlifestyle.
A numberofstudiesreportthatessentialoilshaveactivityon
centralnervoussysteminanimalmodels,forexampleshowinganti-
inflammatory andantinociceptive(Raymundo etal.,2011),antic-
onvulsant(de Almeidaetal.,2011), sedative,anxiolyticeffects
(Hajhashemi etal.,2010), amongothers.Also,someconstituents
of essentialoils,forinstancelinalool(Linck etal.,2010), and
a-terpineol(Quintans-Ju´ nior etal.,2011) havesedativeactivityin
humans andantinociceptiveactivityinmice,respectively.Pharma-
cologicalstudiesprovidescientificsupporttothetraditionaluseof
aromatic medicinalplants,andaromatherapy;nevertheless,there
are stillrequiredmoreclinicaltrialsregardingtotheireffectiveness
in ordertoestablishaguidancetotheiruseinroutinehealthcare
(Cooke andErnest,2000).
Immobility (s)
0
50
100
150
200
250
b
a
b
b b
a a
Treatment mg/Kg
100
Vehicle
Imipra 30
Eucalyptol
Limonene
α-pinene
β-pinene
Linalool
Fig. 3. Immobility timeofmiceproducedbyi.p.administrationoffivemono-
terpenes (100mg/Kg)of L. glaucescens essential oilintheFST.Datarepresentthe
mean7SEM (n¼10). ANOVAfollowedbyTukey’stest.Imipra¼imipramine.
Letters aandbindicatesignificantdifferencewiththevehicleandpositivecontrol,
respectively, po0.05.
Treatment mg/Kg
Immobility (s)
0
50
100
150
200
250
β-pinene
54.8100 173.2
a
a
a
Vehicle
Imipra 30
b
b
Immobility (s)
0
50
100
150
200
250
a
a
Treatment mg/Kg
Linalool
54.8 100 173.2
Vehicle
Imipra 30
b
b b
Fig. 4. Immobility timeofmiceproducedbyi.p.administrationofdifferentdosesof b-pinene andlinalool.(A) b-pinene. Datarepresentthemean 7 SEM (n¼12) ANOVA
followed byTukey’stest, po0.05. ThedataweretransformedfortheBartlett’stest.(B)Linalool.Datarepresentthemean 7 SEM (n¼12) ANOVAKruskal-Wallisfollowed
by Dunn’stest.Imipra ¼ imipramine. Lettersaandbindicatesignificantdifferencewiththevehicleandpositivecontrol,respectively, po0.05.
S.L. Guzma´n-Gutie´rrez etal./JournalofEthnopharmacology143(2012)673–679 677

6. The chemicalanalysisofessentialoilof L. glaucescens here
reported, showedthatitsmajorconstituentiseucalyptol,whichis
also themainconstituentoftheEuropean‘‘laurel’’, Laurus nobilis
(Yalcin etal.,2007). Currently,theleavesofbothspeciesareused
in Me´ xico andCentralAmerica(Jime´nez-Pe´ rez etal.,2011)
principally ascondiment,buthavealsoapplicationsinfolk
medicine; inthecase L. nobilis, ithasbeenpreviouslyreported
the anticonvulsantactivityofitsessentialoil(Sayyah etal.,2003).
In thepresentwork, L. glaucescens essential oilshowedanti-
depressant activityinmiceusingtheFST;furthermore,itdidnot
affect thespontaneouslocomotoractivityinOFT(Fig. 1A andB).
These resultsindicatethatincreasedmotoractivitywasnot
involved intheeffectobservedintheFST,andareinagreement
with thetraditionaluseof L. glaucescens in Me´ xico totreat
sadness. Inordertoidentifytheactiveprinciples,fivecompounds
were tested,andtwoofthem, b-pinene andlinalool,presented
antidepressant activity,theireffectsdidnothavesignificant
differences withimipramine(Fig. 3). Theyieldsofthesecom-
pounds were2.34%and3.64%,respectively,oftheoil(Table 1).
The majorcomponenteucalyptol(26.06%),limonene(8.66%),and
a-pinene (3.86%)wereinactiveintheFST(Fig. 2). Inspitethat
these compoundswereinactiveinthistesttheyremaintobe
tested inotherexperimentalmodel,forinstancetheTailSuspen-
sion Test,todetermineiftheycouldexertanyantidepressant
and/or anxiolytic-likeeffects.
To ourbestknowledgethisisthefirstreportontheanti-
depressant activityof b-pinene andlinalool.Incontrasttothe
essential oil, b-pinene andlinalooldecreasedthespontaneous
locomotor activityofmiceintheOFT,although,thesecompounds
did notreachtheintensityoftheeffectproducedbydiazepam
(Fig. 5A andB).Thediminutionoflocomotoractivityduetothe
administrationofbothmonoterpeneswasduetoasedativeeffect,
since theyalsoproducedadecreasedofexploratoryactivityof
mice intheECT(Fig. 6). TheeffectsobservedintheOFTandECT
Number of crossings
0
5
10
15
20
25
30
35
a
ab
Treatment mg/Kg
β-pinene
54.8100 173.2
Vehicle
DZP 3.5
ab ab
b
Number of crossings
0
10
20
30
40
ab
a
Treatment mg/Kg
Linalool
54.8100 173.2
Vehicle
DZP 3.5
ab
b
b
Fig. 5. Effect producedbyi.p.administrationofdifferentdosesof b-pinene andlinaloolonthetotalcrossingsnumberofmiceexposedtotheOFT.(A) b-pinene.
(B) Linalool.TotalcrossingsofmiceexposedtoOFT(n¼6). Datarepresentthemean7SEM. ANOVAfollowedbyTukey’stest.DZP ¼ diazepam. Lettersaandb
indicate significantdifferencewiththevehicleandpositivecontrol,respectively, po0.05.
Number of rearings
0
5
10
15
20
25
30
a a
a
Treatment mg/Kg
β-pinene
54.8
a
Vehicle
DZP 3.5
b
Number of rearings
0
5
10
15
20
25
30
35
a
Treatment mg/Kg
Linalool
a a
b
b
100 173.254.8 Vehicle
DZP 3.5 100 173.2
Fig. 6. Effect producedbyi.p.administrationofdifferentdosesof(A) b-pinene and(B)linaloolinmiceusingECT.Datarepresentthemean7SEM (n¼6). ANOVAfollowed
by Tukey’stest, po0.05. DZP¼diazepam. Lettersaandbindicatesignificantdifferencewiththevehicleandpositivecontrol,respectively, po0.05.
S.L. Guzma´n-Gutie´rrez etal./JournalofEthnopharmacology143(2012)673–679 678

7. can notbeattributedtoareductioninstrengthmuscle,orlackof
motor coordination,asindicatedbymiceperformanceinthe
rotarod, andtractiontests(Fig. 2).
Regarding tolinalool,ourdataareinagreementwiththedata
reported by Linck etal.(2009), whoindicatethatinhaledlinalool
reduces locomotioninmicewithoutaffectingsignificantlythe
motor coordination.Ithasalsobeenreportedthatlinaloolhas
antagonistic actiononNMDAreceptors,whichmayexplainits
sedative effect(Silva Brumetal.,2001). Inthecaseof b-pinene, its
possible mechanismofactionremainstobestudied.Itis
important tohighlightthatattheactivedose(100mg/Kg),the
structural isomer a-pinene didnotshowantidepressantactivity,
but itreducedthemotoractivity(datanotshown)asthe b isomer
did. Apreviousstudyreportednodifferencebetweenthepoten-
tiated responseofGABAonGABAA receptors inpresenceof a-pinene
and b-pinene (Aoshima andHamamoto,1999), which
could explainwhybothhavesedativeeffect.Otherwise,isomers
can presentdifferencesinthebiologicalactivity,forexample(S)-
(þ)-carvone hasanticonvulsant-likeactivity,whiletheRisomer
does not(de Sousaetal.,2007).
In summary,ourresultsshow that theessentialoilof
L. glaucescens, andtwoofitsconstituents,linalooland b-pinene,
show antidepressantactivity,however thesecompoundsbythem-
selveshavesedativeactivitytoo,thereforeitcanbeconcludedthat
the essentialoilisbetterthanthetwoactivecompounds,sinceit
does nothavesedativeeffect.Regardingtolinaloolourresultsarein
agreement withthosepreviously reportedonthesedativeproper-
ties foundinrodents,andhumans.Thisstudyprovidespreliminary
evidence thatsupporttheuseof L. glaucescens to relievesadness;
however,morestudiesarenecessarytosupportitspossibleclinical
applications.
Acknowledgments
Dr. SilviaLauraGuzma´n Gutierrezwassupportedbyapost-
doctoral fellowshipfromInstitutodeCienciayTecnologı´a del
Distrito Federal(ICyTDF).TheauthorsalsothankAntonioNieto
Camacho, GeorginaDuarteLisci,andJavierPe´ rez Floresfor
technical assistanceandAuraMontemayorLaraforanimalcare.
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