ToxicolRes.2014Dec30(4):297304.

PMCID:PMC4289931

doi:10.5487/TR.2014.30.4.297

PeppermintOilPromotesHairGrowthwithoutToxicSigns
JiYoungOh, 1MinAhPark,2andYoungChulKim2
1
DepartmentofBeauty,DaedukCollege,Daejeon,Korea
2
DepartmentofPublicHealth,KeimyungUniversity,Daegu,Korea
Correspondingauthor.
Correspondenceto:YoungChulKim,DepartmentofPublicHealth,KeimyungUniversity,1095Dalgubeoldaero,Daegu704701,KoreaEmail:
yckim@kmu.ac.kr
Received2014Dec4Revised2014Dec24Accepted2014Dec26.
Copyright2014,TheKoreanSocietyOfToxicology
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNonCommercialLicense
(http://creativecommons.org/licenses/bync/3.0/)whichpermitsunrestrictednoncommercialuse,distribution,andreproductioninanymedium,
providedtheoriginalworkisproperlycited.

Abstract

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Peppermint(Menthapiperita)isaplantnativetoEuropeandhasbeenwidelyusedasacarminativeandgastric
stimulantworldwide.Thisplantalsohasbeenusedincosmeticformulationsasafragrancecomponentandskin
conditioningagent.ThisstudyinvestigatedtheeffectofpeppermintoilonhairgrowthinC57BL/6mice.The
animalswererandomizedinto4groupsbasedondifferenttopicalapplications:saline(SA),jojobaoil(JO),3%
minoxidil(MXD),and3%peppermintoil(PEO).Thehairgrowtheffectsofthe4weektopicalapplicationswere
evaluatedintermsofhairgrowth,histologicalanalysis,enzymaticactivityofalkalinephosphatase(ALP),and
geneexpressionofinsulinlikegrowthfactor1(IGF1),knownbiomarkersfortheenhancedhairgrowth.Ofthe
4experimentalgroups,PEOgroupshowedthemostprominenthairgrowtheffectsasignificantincreasein
dermalthickness,folliclenumber,andfollicledepth.ALPactivityandIGF1expressionalsosignificantly
increasedinPEOgroup.Bodyweightgainandfoodefficiencywerenotsignificantlydifferentbetweengroups.
TheseresultssuggestthatPEOinducesarapidanagenstageandcouldbeusedforapracticalagentforhair
growthwithoutchangeofbodyweightgainandfoodefficiency.
Keywords:Alkalinephosphatase,Hairgrowth,Hairfollicle,Insulinlikegrowthfactor1,Peppermintoil
INTRODUCTION

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Hairlossisadistressingconditionthatisassociatedwithamultitudeofnatural,medical,ornutritional
conditions.Forexample,androgeneticalopeciainmen,ormalepatternbaldness,isincreasinglyrecognizedasa
physicallyandpsychologicallyseriousmedicalconditionthatoftenrequiresaprofessionalcarebygeneralist
clinicians(1).
TheonlyproductssanctionedbytheUSFDAforhairlosstreatmentareoralfinasteride(Proscar)andtopical
minoxidil(Rogaine).MinoxidilwasoriginallycreatedasahypertensionmedicationbyUpjohn
Pharmaceuticals(2).Upjohnitselfhaswarnedofpossiblenegativesideeffectsofthemedicationincluding
increasedheartrate,difficultybreathing,rapidweightgain,edema,seborrhoeicdermatitis,scalpitching,and
scaling(35).

Traditionalplantremedieshavebeenusedforcenturiesinthetreatmentforhairloss,butonlyafewhavebeen
scientificallyevaluated(5).Peppermint(Menthapiperita)extractedfrompeppermintleavesisgenerallyregarded
asanexcellentcarminativeandgastricstimulant,andalsohasbeenusedincosmeticformulationsasafragrance
componentandageneralskinconditioningagent.Theprincipalingredientofpeppermintoil,menthol,is
primarilyresponsibleforitsbeneficialeffects(6).Invitro,pepperminthasbeenreportedtoshowanti
inflammatory,antimicrobial,andantifungalactivitiesaswellasstrongantioxidantactivity,andantiallergenic
andantitumoractions(7,8).Severalclinicaltrialsexaminingtheeffectsofpeppermintoil(PEO)onirritable
bowelsyndromehavebeenreported(9).However,experimentaltrialofPEOinitshairgrowthactivityhasnot
beenfullyreported.TheaimofthisstudywastoaddressthetherapeuticpotentialofPEOforhairlossviathe
comparativeanalysisbetweenPEOandminoxidil.
MATERIALSANDMETHODS

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Materials.Thisstudyusedpeppermintoil(Sanoflore,France)certifiedas100%pureandnaturalessentialoil
byanorganicproductcertificationorganization(ECOCERTF32600)andjojobaoil(DesertWhale,USA).The
chemicalcompositionsofpeppermintoilandjojobaoilusedarelistedinTable1.The3%minoxidilwas
obtainedfromHyundaiPharmacia(Korea).
Table1.
Compositionofpeppermintoilandjojobaoil

Experimentalanimal.FiveweekoldmaleC57BL/6mice(DaehanBiolinkCo.,Korea)wereallowedtoadaptto
theirnewenvironmentforoneweek,withfoodandwaterprovidedadlibitumunder221roomtemperature,
505%relativehumidityand12hrsofalight/darkcyclebeforetheexperimentwasbegun.Thedorsalarea(2
cm4cm)ofthe6weekoldC57BL/6micewasshavedwithananimalclipper.Uponshavingthemiceallof
thehairfolliclesweresynchronizedinthetelogenstage,showingpinkcolor.Allanimalswererandomizedinto4
groupsbasedondifferenttopicalapplications:saline(SA),jojobaoil(JO),3%minoxidil(MXD),and3%
peppermintoil(PEO,dilutedinjojobaoil).Eachcompound(100l)wastopicallyappliedtotheshaveddorsal
areaonceaday,6daysaweek,for4weeks.Bothanimalcareandtheprotocolforthisstudywereinaccordance
withIACUC(InstitutionalAnimalCareandUseCommittee)andOECDguidelines.
Hairgrowthobservation.Toassessthehairgrowthineachgroup,photographsoftheanimalsweretakenat
week1,2,3,and4aftertopicalapplicationwasbegun.Thehairgrowtheffectwasscoredasfollows,0:nohair
growth1:lessthan20%growth2:20%tolessthan40%growth3:40%tolessthan60%growth4:60%to
lessthan80%growthand5:80%to100%growth.
Histologicalanalysis.Themicewereeuthanizedwithdiethyletherandextractedskintissue.Numberofmice
sacrificedatweek1,2,and4wasand3,3,and5,respectively,andtheirdermalskinsampleswerefixedin10%
bufferedformalinfor24hrs,followedbyparaffinwaxembeddingusingstandardtechniques.Generalhistology
wasvisualizedbyhematoxylineosin(H&E)staining,andwesubsequentlyobservedthenumber,elongationand
depthofhairfolliclesbyfluorescentmicroscopy(Axioimager,CarlZeiss,Germany).Thedermalthicknessand
follicledepthwerealsomeasuredbyusingthescalebartoolofthefluorescentmicroscope.
Detectionofalkalinephosphataseactivityindermalskin.Theextracteddorsalskinwasmincedand
homogenizedwithahomogenizer(T25basic,IKA,Malaysia)byadding4timesphosphatebufferedsaline(PBS)
togivea20%homogenate.Thehomogenatewascentrifugedat12,000rpm,4,for20min(AVANTI,
BeckmanCoulterInc.,USA).Thesupernatantswerekeptinadeepfreezerat80andusedfortheassay.The
activityofalkalinephosphatase(ALP)wasanalyzedwithanautobiochemistryanalyzer(Konelab20XT,
Thermo,Finland).

IsolationoftotalRNAandcDNAsynthesis.TotalRNAwasisolatedfromtheextracteddorsalskinusingthe
HighPureRNAIsolationKit(RocheAppliedScience,Penzberg,Germany)followingthemanufacturer's
protocol.ThequantityandqualityoftheisolatedtotalRNAweredeterminedbytheUV/Visspectrophotometer
(MecasysCo.,Korea).Onlysampleswith2.0>OD260/280>1.8werefurtheranalyzed.cDNAwassynthesized
from1goftotalRNA,usingAccuPowerCycleScriptRTPreMixKit(Bioneer,Korea)inafinalvolumeof205
l.
Reversetranscriptionpolymerasechainreaction.First,cDNAwasdiluted1:10withsteriledeionizedwater,and
2lofthedilutedcDNAwasaddedtoAccupowerTMPCRPreMix(Bioneer,Korea)and10pmol/Lspecific
primer.Thisreactionmixturewasfilleduptoafinalvolumeof20lwithwater.PCRwascarriedoutinaPCR
cycler(MycyclerTMthermalcycler,BioRad,USA).Cyclingprotocolforinsulinlikegrowthfactor1(IGF1)
wasasfollows:1cycle94for5min,followedby35cycles,94for30s,60for30s,72for30s,anda
finalextensionat72for5min.CyclingprotocolforGAPDHwasasfollows:1cycle94for5min,followed
by35cycles,94for30s,58for30s,72for30s,andafinalextensionat72for5min.Reaction
productswereelectrophoresedin1.5%agarosegelsandvisualizedwithusingethidiumbromide(EtBr).Each
bandwasdensitometricallyquantifiedbyimageanalyzer(Kodak1Dv3.6imageAnalysissystem,USA)and
normalizedwithGAPDHintensity.Theprimersequencesusedwereasfollows:IGF1forward5'
AGAGACCCTTTGCGGGGCTGA3',reverse5'CTTCTGAGTCTTGGGCATGT3'GAPDHforward5'
AACGGATTTGGTCGTATTGG3',reverse5'AGCCTTCTCCATGGTGGTGAAGAC3'.
Waterandfoodintakes,foodefficiencyratioandbodyweightchange.Thewaterandfoodintakesof
experimentalanimalsweremeasuredonceaweek,andtheweightwasmeasuredimmediatelybeforethe
experimentstartedandat09:00~10:00a.m.onceaweekduringtheexperimentalperiod.
Statisticalanalysis.ThedatawerestatisticallyanalyzedbyStudentsttestforthecomparisonamonggroups
usingSPSSWIN(v21.0).Theresultswereconsideredstatisticallysignificantifthepvalueswerelessthan0.05.
RESULTS

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Hairgrowthpromotion.Fromweek2,PEOgrewhairmorerapidlythanSAandJO.Atweek3,PEO
remarkablypromotedhairgrowththanSAandJO,evengreaterthanMXD.Atweek4,PEOshowedhairgrowth
about92%,whereasMXDabout55%(Fig.1).
Fig.1.
ComparisonofthehairgrowtheffectinC57BL/6mice.Thebackskins
ofthemicewereshavedandtestcompoundsweretopicallyappliedfor
4wks.Thehairgrowtheffectwascalculatedusingscoringindex:
0~19%(1),20~39%(2),40~59%(3),60~79%(4),...
Hairgrowthpromotionwasevaluatedbyobservingthedarkeningoftheskincolor,whichindicatedtelogento
anagenconversion,brightpinkintelogenandgrey/blackinanagen.Atweek1,PEOchangedthedorsalskin
colorfrompinktogrey/blackandfromweek2,itshowedaconsiderablyrapidincreaseinhairgrowth(Fig.2).
TheseresultsclearlydemonstratethatthetopicalapplicationofPEOinducesrapidanagenhairgrowthintelogen
mouseskin.
Fig.2.
GrossobservationofbackskinsinC57BL/6mice.Thebackskinsof
themicewereshavedandtestcompoundsweretopicallyappliedfor4
wks.SAsaline,JOjojobaoil,MXD3%minoxidil,PEO3%
peppermintoil.

Increaseofdermalthickness,hairfolliclenumberandhairfollicledepthinhistologicalanalysis.Histological
analysisshowedthat4wktopicalapplicationofPEOandMXDinducedverythickandlonghairgrowthand
promotedtheelongationofhairfolliclesfromdermistosubcutis(Fig.3).Theseresultsindicatethatthehair
folliclesofPEOandMXDgroupsatweek4wereintheanagenstage.Wealsoobservedaslightincreaseof
epidermalthicknessinPEOgroup.
Fig.3.
HistologicalobservationofhairfolliclesinC57BL/6mice.Theback
skinsofthemicewereshavedandtestcompoundsweretopically
appliedfor4wks.H&Estain,100.Scalebar200m.(A)transverse
viewofhairfollicles,(B)...

Atweek2,PEOshoweddermalthicknessto95%and66%greaterthanSAandJO,respectively(p<0.01).At
week4,PEOshoweditto120%and81%greaterthanSAandJO,respectively(p<0.01),comparabletoMXD(
Fig.4).
Fig.4.
ChangeofdermalthicknessinC57BL/6mice.Thebackskinsofthe
micewereshavedandtestcompoundsweretopicallyappliedfor4
wks.Thedermalthicknesswasmeasuredbytheaverageoftwo
differentpointsinthefieldwithaneyepieceof100...
Fig.5showsthegrowthpromotingactivityofhairfolliclenumber.Atweek2,thehairfolliclenumberofPEO
groupwas473%and218%greaterthanSAandJOgroups,respectively(p<0.05).Atweek4,PEOgrouphad
740%and307%morehairfolliclesthanSAandJOgroups,respectively(p<0.001),comparabletoMXDgroup.
Wealsofoundthatthenumberofhairfolliclesincreasedashairregrew.
Fig.5.
ChangeoffolliclenumberinC57BL/6mice.Thebackskinsofthe
micewereshavedandtestcompoundsweretopicallyappliedfor4
wks.Thefolliclenumberwasdeterminedbytheaverageoftwo
differentpointsinthefieldwithaneyepieceof100...
Fig.6showsthegrowthpromotingactivityofhairfollicledepth.Atweek2,thedepthofhairfolliclesofPEO
groupwas172%and133%greaterthanSAandJOgroups,respectively(p<0.01).Atweek4,thedepthofhair
folliclesofPEOgroupwas236%and182%greaterthanSAandJOgroups,respectively(p<0.001),comparable
toMXD.HistologicalstudiesrevealedthatPEOmarkedlystimulatedtheskinandthickenedit.Thedepth,size,
andnumberofhairfollicleswerealsomarkedlyincreasedinPEOtreatedskin.Theseresultsclearlydemonstrate
thattopicalapplicationofPEOmarkedlystimulatedhairgrowthandinducedrapidanagenhairgrowthintelogen
mouseskin.
Fig.6.
ChangeoffollicledepthinC57BL/6mice.Thebackskinsofthemice
wereshavedandtestcompoundsweretopicallyappliedfor4wks.The
follicledepthwasmeasuredbytheaverageoftwodifferentpointsin
thefieldwithaneyepieceof100...

ChangeofALPenzymeactivitywithhaircycle.Atweek2,PEOshowed253%(p<0.05),35%,and13%
greaterALPactivitycomparedtoSA,JO,andMXD,respectively(Fig.7).Atweek4,PEOshowed192%(p<
0.05),90%,and13%greaterALPactivitycomparedtoSA,JO,andMXD,respectively.Aftertopicalapplication
onthebacksofC57BL/6micefor4wks,PEOinducedtheearliesttelogentoanagenconversion,withMXD,JO
andSAfollowinginorder.TheincreaseinALPactivityofPEOgroupwasfastcomparedtoMXDgroup,with
remarkablysignificantcomparedtoSAandJOgroups.
Fig.7.
ChangeofskinALPactivityinC57BL/6mice.Thebackskinsofthe
micewereshavedandtestcompoundsweretopicallyappliedfor4
wks.ValuesarethemeanSDof3,3,and5miceatweek1,2,and4,
respectively.Thevalueissignificantly...
ComparisonofIGF1mRNAexpression.Atweek2,PEOshowed33%and21%greaterIGF1mRNA
expressioncomparedtoSAandJO,respectively(p<0.05)(Fig.8).Atweek4,PEOshowed89%(p<0.001)
and34%(p<0.01)greaterIGF1mRNAexpressioncomparedtoSAandJO,respectively,comparabletoMXD.
PEOshowedremarkablyincreasedIGF1mRNAexpression,evenbetterthanMXD.
Fig.8.
ChangeofIGF1expressioninC57BL/6mice.Thebackskinsofthe
micewereshavedandtestcompoundsweretopicallyappliedfor4
wks.ValuesarethemeanSDof3,3,and5miceatweek1,2,and4,
respectively.Thevalueissignificantly...
Changeofwaterandfoodintakes,foodefficiencyratio,andbodyweight.Bodyweightgain,foodefficiency,
andweightofMXDgroupwerehigherthantheothergroupsbutdidnotshowsignificantdifference(Table2and
Fig.9).
Table2.
Dailywaterintake,foodintake,bodyweightgain,andfoodefficiency
ratioinC57BL/6miceappliedwithtestcompoundsfor4wks
Fig.9.
ChangeofbodyweightinC57BL/6mice.Thebackskinsofthemice
wereshavedandtestcompoundsweretopicallyappliedfor4wks.
ValuesarethemeanSDof11mice.SAsaline,JOjojobaoil,MXD
3%minoxidil,PEO3%peppermintoil.
DISCUSSION

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MXDhasbeenwidelyusedtotreatandrogeneticalopecia,butlittleisknownaboutitspharmacologicalactivity
orabouttheidentityofitstargetcellsinhairfollicles(10).TopicallyappliedMXDwasbelievedtostimulatehair
growthbyindirectdrugaction,i.e.byinducingvasodilatationandincreasingbloodflowtothefolliculardermal
papillacells,orbycreatingalocalirritation(11).Thefolliculardermalpapillacellsarethemostlikelytargetsite
fortheactionofMXD(12).MoriandUno(13)reportedthattopicalapplicationofMXDspecificallystimulates
thesecondarygermofthetelogenfollicles,resultingintheirrapidprogressiontoanagenfollicles.Thus,hair
follicleisusefulmarkerthatisassociatedwithhaircycle(14).AnagenIVIdevelopmentischaracterizedby
increasinglengthofthehairfollicleandcatagenIVIIbydecreasinglength.Duringtelogen,thehairfollicle

reachesitsminimallength.Synchronizedhairfolliclecyclinginmiceisalsoassociatedwithstagedependent
changesindermalthickness.Ahairfollicleintelogen,anagenIoranagenIIhasnotyetreachedthesubcutis.
DuringtheanagenIIIstage,thefolliclesmovefromthedermisdowntothesubcutis.
WefoundthatPEOremarkablypromotedhairgrowthcomparedtoSAandJO,evenfasterthanMXDwithout
significantchangeofbodyweightgainandfoodefficiency.Chenetal.(15)reportedthatMXDtookonlyabout
10daysforthehairofmicetofullyregrewafterthetopicalapplicationofMXD,indicatingtheenhancingeffect
ofMXDintheproliferativerateofhairgrowth.Inourstudy,histologicalanalysisshowedthatMXDpromoted
hairgrowthintermsofhairfolliclenumber,follicledepth,anddermalthicknessatweek2.
Mentholisamajorconstituentofpeppermintoil,whichisacyclicalcohol.Mentholhasbeenwidelyusedasa
componentoffoodandcosmetics.Ithasbeenreportedthatmentholincreasesthesensitivityofcutaneouscold
receptorsbymodulatingCa2+currentsofneuronalmembranes(16).Mentholisthemosteffectivepenetration
enhancerthat,alongwithlimonene,canbeconsideredtheprototypefortheuseofterpenesaspenetration
enhancers(17).Foryearsterpenes(e.g.,menthol,pinene,terpinene4ol,pinene,1,8cineole)havebeen
usedaloneorasconstituentsofessentialoilsinmedicine,cosmeticsandhouseholdproducts.Intheexperimental
dermopharmacyandtechnologyoftransdermaldrugforms,terpeneshavealsobeenintensivelyexploredas
penetrationenhancers(18).Whenskinistreatedwithterpenes,theexistingnetworkofhydrogenbondsbetween
ceramidesmayloosenbecauseofcompetitivehydrogenbonding(19).Thehighaccumulationofmostofthe
terpenesintheskinlayersprovesthatthesecompoundseasilypermeatethestratumcorneumandthattheymay
easilypenetrateintobloodcirculationinvivo(20).
Inourstudy,wefoundthatPEOinducedverythickandlonghairafter4weektopicalapplicationandpromoted
theelongationofhairfolliclesfromtheepidermisdowntothesubcutisinaverticalsection(Fig.3),showingin
thestageofanagenIII.ApplicationofMXDcausedsimilarresults.Weobservedthatthisincreaseinhairfollicle
lengthwasnotassociatedwithanylossofhairfolliclearchitectureandthattheincreaseinhairfolliclelength
wasassociatedwithanincreaseinthelengthofthekeratinizedhairshaft.
Thedrugsforalopeciatreatmenthavebeendevelopedtomaintainorinducetheanagenstageofhaircycle.ALP
activitywasparticularlydetectedinthedermalpapilla.ALPactivityinthedermalpapillawasmoderateinvery
earlyanagen,reachedamaximallevelinearlyanagen,andwaskeptatalowlevelduringcatagen(21).The
bulbardermalsheathshowedintenseALPactivityonlyinearlyanagen(22).Althoughresultsfromclinicaltrials
vary,themajorityoftheevidenceindicatesthatthereisadirectcorrelationbetweenthehairfollicledepthand
thelevelofALPactivity.Inourstudy,PEOinducedsignificantlyhighALPactivityatweek2,evengreaterthan
MXD.ThisstudydemonstratesthatPEOstimulatesbothdermalpapillaandALPactivity,whichpromotesblood
circulationbyrelaxingvascularsmoothmuscle(8).
Tobetterunderstandtheinfluenceoftheendocrinesysteminhairgrowth,weanalyzedthemRNAexpressionof
IGF1gene.Itisapotentmitogensupportingcellgrowthandsurvival(23)andalsoplaysaroletoincreasehair
thickness(24).Inourstudy,PEOshowedremarkablyincreasedIGF1mRNAexpressionatweek2,whereas
MXDatweek4.
Inconclusion,ourexperimentaldatasuggestthat3%PEOfacilitateshairgrowthbypromotingtheconservation
ofvascularizationofhairdermalpapilla,whichmaycontributetotheinductionofearlyanagenstage.In
addition,PEOeffectivelystimulatedhairgrowthinananimalmodelviaseveralmechanismsandthuscouldbe
usedasatherapeuticorpreventivealternativemedicineforhairlossinhumans.
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