Trichomes micromorphology and essential oil variation at different developmental stages of cultivated and wild growing Mentha pulegium L. populations from Portugal

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Industrial

Crops

and

Products

j o u r n al hom ep a ge :w w w . e l s e v i e r . c o m / l o c a t e / i n d c r o p

Trichomes

micromorphology

and

essential

oil

variation

at

different

developmental

stages

of

cultivated

and

wild

growing

Mentha

pulegium

L.

populations

from

Portugal

Leandra

Rodrigues

_,

_Orlanda

_Póvoa

_,

_Generosa

_Teixeira

_,

_Ana

_Cristina

_Figueiredo

_,

Margarida

Moldão

_{, Ana}

_Monteiro

a_{CentrodeBotânicaAplicadaàAgricultura(CBAA),InstitutoSuperiordeAgronomia,ThecnicalUniversityofLisbon,TapadadaAjuda,1349-017Lisboa,Portugal}

b_{EscolaSuperiorAgráriadeElvas,EdifíciodoTremAuto,Avenida14deJaneiro,7350-903Elvas,Portugal}

c_{UniversidadedeLisboa,FaculdadedeFarmáciadeLisboa,CentrodeBiologiaAmbiental,Av.Prof.GamaPinto,1649-003Lisboa,Portugal}

d_{UniversidadedeLisboa,FaculdadedeCiênciasdeLisboa,DepartamentodeBiologiaVegetal,IBB,CentrodeBiotecnologiaVegetal,C2,Piso1,CampoGrande,1749-016Lisboa,}

Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11April2012

Receivedinrevisedform11July2012

Accepted31July2012 Keywords: MenthapulegiumL. Lamiaceae Trichomes Essentialoil Histochemistry GC GC–MS

a

b

s

t

r

a

c

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Theindumentum of Mentha pulegium L.,studied by lightand scanning electron microscopy, was characterizedbynon-glandularandglandulartrichomes,whichcorrespondedtothecommon arrange-mentdescribedfortheLamiaceaefamily.Histochemistryrevealedthepresenceofpectins,totallipids, acidiclipidsandessentialoilsintheglandulartrichomessecretions.Theessentialoilyieldranged from0.3%(w/d.w.)inthevegetative phaseto1.6%at fullflowering.Gaschromatographyandgas chromatography–massspectrometryessentialoilscompositionanalysisatfullfloweringrevealedmostly quantitativeratherthanqualitativevariations,withpulegoneasthemajorcompound(52–82%),followed byisomenthone(2–36%),menthone(0.1–17%),andpiperitenone(1–15%).Comparativeevaluationof cul-tivatedandwildgrowingpopulationsshoweddifferencesintherelativeamountsofthemaincomponents oftheessentialoilsisolatedfromplantsharvestedatdifferentdevelopmentalstages.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Thegenus Mentha L. (Lamiaceae), comprising more than 25

species,isresponsibleforapproximately2000tofworldessential

oil, making it thesecond most important essential oil

produc-inggenus,afterCitrus(Mucciarellietal.,2001).Menthapulegium

L., a member of this genus, commonly known as pennyroyal

(poejoinPortuguese),isanaromaticperennialherbaceousplant

reachingupto40cm height(Stengele and Stahl-Biskup, 1993).

This species grows wild in humid and damp areas and water

banks of central, southern and Western Europe, north Africa

and Asia Minor (Chalchat et al., 2000; Tutin et al., 1972). The

aerial parts are pubescent bearing glandular trichomes which

areresponsible forthe essentialoilsecretion. Themorphology,

∗ Correspondingauthorat:DCEB/Secc¸ãodeHerbologia,InstitutoSuperiorde

Agronomia,TechnicalUniversityofLisbon,TapadadaAjuda,1349-017 Lisboa,

Portugal.Fax:+351213653238.

E-mailaddresses:[email protected],[email protected]

(L.Rodrigues).

distributionandfrequencyoftheseglandulartrichomesare

dis-tinctive characteristics among the Lamiaceae species (Werker,

2000).InPortugal,theaerialpartsofM.pulegiumandthe

prepa-rationsfrom it havebeen traditionallyused inAlentejo Region

toflavourrecipes,aswellasforitsmedicinalproperties(Póvoa

etal.,2006).Infolkmedicineit isusedasaninfusion,

prevent-ingdifferentgastricdisordersandinflammationsoftherespiratory

tract (Mkaddem et al., 2007; Póvoa et al., 2006).Nevertheless,

there are noapproved medicinaluses for pennyroyal essential

oil(Barceloux,2008).This planthas alsobeen used as a spice

and flavouringagent indifferent foods (Mkaddem etal., 2007;

Monteiroetal.,2007a), despitedefactthattheessentialoilsof

M.pulegiumaregenerallyconsideredtoberichinpulegone,atoxic

compoundwithpotentiallylethalhepatotoxiceffects (Anderson

et al.,1996).Severalpublications, reports and directives ofthe

UEemphasize the needfor a bettercharacterization of

botani-calsand botanicalpreparationsand forscientific assessment of

risks from exposure of consumers to these products. The UE

directive88/388/EEChasstipulatedamaximumconcentrationfor

this oxygen-containingmonoterpene of100mg/kgin beverages

and 25mg/kg in foodstuff, with the exception of 250mg/kg in

0926-6690/$–seefrontmatter © 2012 Elsevier B.V. All rights reserved.

flavoured beverages and350mg/kg inmint confectionery(EEC, 1988).

Itisknownthatthechemicalcompositionofplantsisinfluenced

byseveralexternalfactorsincludinggrowingconditionsand

cli-mate(Figueiredoetal.,2008).TheessentialoilsfromM.pulegium

havebeencharacterizedindifferentregionsoftheworld,Table1

(andreferencestherein).Despitethesereports,andstudiesin

Por-tuguesecultivatedpopulations(Lopesetal.,2010;Monteiroetal.,

2007b;Teixeiraetal.,2012),there isnopreviousreportonthe

chemicalcompositionofwildgrowingpopulationsofM.pulegium

volatileoilsfrommainlandPortugal.Oneshouldnotforgetthat,in

cultivatedmintplantsthequalitativeoilcompositionisrelatively

stable,butinmostwildgrowingmintsagreatdiversityin

essen-tialoilconstituentshasbeenobserved(Mimica-DukicandBozin,

2008).

Inviewofthepotentialpharmacological,commercialandfood

industryvalueof thisspecies, theworkdescribedin thispaper

wascarriedoutto(1)characterize,bylightandscanningelectron

microscopy,themorphology andstructure of theindumentum,

in particular of the glandular trichomes, which are the main

responsiblefortheessentialoilssecretion,(2)typifythechemical

compositionoftheessentialoilfromPortugueseM.pulegiumwild

populations,(3)comparetheessential oilcomposition between

cultivated and wild growing populations and (4) evaluate the

evolution ofthe essentialoilcomponents throughouttheplant

developmentalstages.

2. Materialandmethods

2.1. Plantmaterial

In2009,severalfieldtripswereconductedacrossthegeographic

rangeofM.pulegium.Atotalof14populationswithdifferent

geo-graphicorigins,representativeofthedistributionofthespeciesin

mainlandPortugal,wereincludedintheanalysis(Table2,Fig.1).

Tocharacterizetheessentialoil(EO)compositionandidentify

pos-siblechemotypes,these14populationswerecollected,duringthe

floweringphase,fromnaturalhabitats.Voucherspecimenshave

beendepositedintheLISIherbarium(Table2).

InordertounderstandtheevolutioninEOcompositionandyield

alongtheplantlifecycle,andcomparecultivatedwithwild

grow-ingconditions,atime-coursestudywasundertaken.Inthisstudy,

sixpopulations(15plantsperpopulation)wherecollectedfrom

thewild,transportedincontainersandtransplantedtotheessay

fieldatInstitutoSuperiordeAgronomia,Lisbon,Portugal.Plants

wereplanted50cmapart,in2m2_{plots,anddripirrigated}

period-ically(each7–10days).Plotswerekeptweedfreebyhandhoeing.

Samplesfromthesixpopulations,inthewildandinthecultivated

essayfield,wereharvestedatthevegetative,pre-floweringandfull

floweringphases.

2.2. Morphologicalstudies

2.2.1. Lightmicroscopy(LM)

Stems,leavesandflowersatthedifferentdevelopmentalstages,

of 10 individuals for each population, were fixedwith3%

glu-taraldehyde(Merck,Germany)ina0.1Msodiumphosphatebuffer,

pH7.3,for4hat4◦C,andwashedinthesamebuffer(Ascensão

etal.,1999).Afterdehydrationinagradedseriesofethanol

solu-tions,hand-cutcross-sectionsweremadeandclarifiedwithsodium

hypochloriteandwashedindistilledwater(Evans,1996).

Obser-vationswerecarriedoutunderaNikonEclipseE400microscope

equippedwitha NikonCoolpixMDClens adapter.Imageswere

obtainedwitha Nikon Coolpix995digital camera.Quantitative

Fig.1. Map ofPortugalwith collectionsitesofMenthapulegium populations

analyzed.Symbols,accordingtotheessentialoilclusteranalysis(Fig.3).For

abbre-viations,seeTable2.

charactersaretheaverageof,at least,30 differentobservations

foreachpopulation.

2.2.2. Scanningelectronmicroscopy(SEM)

Plant material was fixed as above, critical-point dried in a

PolaronBioRad E3500,andcoated withgoldin aJeol JFC-1200

(Tokyo, Japan). Observations were carried out at 15kV, on a

Jeol JSM-5220 LV scanning electron microscope (Tokyo, Japan)

equippedwithanimageacquisitionsystem.Measuresandcounting

wereobtainedbycomputer-assistedimageanalysis.

2.3. Histochemicalstudies

Generalstaining procedures fordetecting someof themain

chemicalgroupssecretedwerecarriedoutusingfreshleavesand

flowers from 2 populations (Table 1). The histochemical tests

included:(1)Sudan IIIfortotal lipids(Johansen,1940);(2)Nile

Blueforneutralandacidiclipids(Jensen,1962);(3)Nadireagent

foressentialoilsandresinacids(DavidandCarde,1964);and(4)

RutheniumRedforpectins(Johansen,1940).Standardcontrol

pro-cedureswerecarriedoutsimultaneously.

2.4. Essentialoilanalysis

2.4.1. Isolationprocedure

Foreachsample,aerialpartsof10individualsperpopulation

were collected, grosslypulverized, and 20g were subjected to

Table1

DatafrompreviousstudiesontheessentialoilcompositionofMenthapulegiumL.

Collectioncountry Plantpart Extractionprocedure Maincomponent(s)(%) Identificationmethod Reference

Algeria Aerialparts Hydrodistillation Pulegone4–87 GC,GC/MS Beghidjaetal.(2007)

Bulgaria Aerialparts Waterandsteam

distillation

Pulegone43–45 GC,GC/MS Stoyanovaetal.(2005)

Europeancountries Differentplant

parts

Hydrodistillation Pulegone7–85 GC,GC/MS StengeleandStahl-Biskup(1993)

Greece Aerialparts Hydrodistillation Pulegone0.1–91 GC,GC/MS Kokkinietal.(2004)

India Aerialparts Hydrodistillation Pulegone65–83 GC,GC/MS.1_HNMR,

13_CNMR

Agnihotrietal.(2005)

Iran Aerialparts Hydrodistillation Pulegone38 GC/MS Agheletal.(2004)

Iran Aerialparts Hydrodistillation Piperitone38

Piperitenone33

GC,GC/MS MahboubiandHaghi(2008)

Iran Aerialparts Hydrodistillation Menthone39 GC,GC/MS Hassanpouraghdametal.(2011)

Portugal Aerialparts Hydrodistillation Pulegone60 GC,GC/MS Monteiroetal.(2007b)

Portugal Aerialparts Hydrodistillation Pulegone65–87 GC,GC/MS Lopesetal.(2010)

Portugal Aerialparts Hydrodistillation Menthone36Pulegone

23

GC,GC/MS Teixeiraetal.(2012)

Portugal Aerialparts Hydrodistillation Pulegone35

Piperitenone27

GC/MS Mataetal.(2007)

Portugal Aerialparts Hydrodistillation Pulegone80 GC,GC/MS Reis-Vascoetal.(1999)

Spain Aerialparts Hydrodistillation Pulegone41–42 GC,GC/MS, Maroto-Diazetal.(2007)

Tunisia Aerialparts Hexaneextract Pulegone42 GC,GC/MS Mkaddemetal.(2007)

Tunisia Aerialparts Hydrodistillation Pulegone44 GC,GC/MS Hajlaouietal.(2010)

Turkey Aerialparts Hydrodistillation Pulegone72 GC,GC/MS Sarikurkcuetal.(2012)

Uruguay Leaves Hydrodistillation Pulegone73 GC-FID,GC/MS Lorenzoetal.(2002)

Yugoslavia Aerialparts SteamDistillation Menthone31 GC,GC/MS Chalchatetal.(2000)

totheEuropeanPharmacopoeia(CouncilofEurope,2007).Theoils

werekeptat4◦Cuntilfurtheranalysis.

2.4.2. Gaschromatography(GC)

GCanalysiswereperformedusingaPerkinElmer8700gas

chro-matograph(PerkinElmer, Shelton, CT, USA) equipped with two

FIDs,a data-handlingsystem,and a vaporizing injector port in

whichtwocolumnsofdifferentpolaritieswereinstalled:aDB-1

fused-silicacolumn(30m×0.25mmi.d.,filmthickness0.25␮m;J

&WScientificInc.,AgilentTechnologies,SantaClara,CA,USA);and

aDB-17HTfused-silicacolumn(30m_×0.25mmi.d.,filmthickness

0.15␮m;J&WScientificInc.).Oventemperaturewasprogrammed,

45–175◦C,at3◦Cmin−1,subsequentlyat15◦Cmin−1upto300◦C,

andthenheldisothermalfor10min;injectoranddetector

temper-atureswere280◦Cand290◦C,respectively;carriergas,hydrogen,

adjustedtoalinearvelocityof30cms−1.Sampleswereinjected

usingthesplitsamplingtechnique,ratio1:50,withavolumeof

injectionof0.1␮lofapentane–oilsolution.Thepercentage

com-positionoftheoilswascomputedbythenormalizationmethod

fromtheGCpeakareas,whichwerecalculatedasmeanvaluesof

twoinjectionsofeachoilsample,withoutusingresponsefactors.

2.4.3. Gaschromatography–massspectrometry(GC–MS)

The GC–MS unit consisted of on PerkinElmer Autosystem

XLgaschromatograph(PerkinElmer,Shelton,Connecticut,USA),

equippedwithDB-1fused-silicacolumn(30m×0.25mmi.d.,film

thickness 0.25␮m; J & W Scientific, Inc., Agilent Technologies,

SantaClara,CA,USA),andinterfacedwithaPerkinElmerTurbomass

mass spectrometer(softwareversion 4.1, PerkinElmer, Shelton,

CT,USA).Injectorandoventemperatureswereasabove;transfer

linetemperature,280◦C;iontraptemperature,220◦C;carriergas,

helium,adjustedtoalinearvelocityof30cms−1;splitratio,1:40;

ionizationenergy, 70eV; ionization current, 60␮A; scan range,

40–300␮m;scan time,1s. Theidentityofthecomponentswas

assigned bycomparison of theirretention indices, relative toa

C9–C17 hydrocarbonstandardmixture,andwithGC–MSspectra

fromahome-madelibrary,constructedbasedontheanalysesof

referenceoils,laboratory-synthesisedcomponentsand

commer-cialavailablestandards.

2.4.4. Dataanalysis

Thepercentagecompositionoftheisolatedessentialoilswas

usedtodeterminetherelationshipbetweenthedifferentsamples

Table2

DataoncollectionsiteandsampletypeofMenthapulegiumwildpopulationsstudied.

Populations Sampletypea _{Specificsamplecollectionsites}

Localization Altitude(m) Latitude Longitude Hidrograficbasin Voucher

Mp2 W/C/DS Ouguela,CampoMaior 207 39◦₄_54.96_{N 7}◦₀_4.33_{W Guadiana 1059/2010}

Mp32 W/C/DS ValênciadeAlcântara 313 39◦₂₈_1.17_{N 7}◦₁₂_24.16_{W Tejo 1060/2010}

Mp33 W/C/DS Alburquerque 234 39◦₁₁_0.69_{N 7}◦₁_59.03_{W Guadiana 1061/2010}

Mp34 W Montalvão,Nisa 116 39◦₃₉_50.86_{N 7}◦₃₂_19.27_{W Tejo 1062/2010}

Mp36 W Bagaúste,PesodaRégua 50 41◦₉_0.41_{N 7}◦₄₅_2.24_{W Douro 1063/2010}

Mp40 W GomesAires,Almodôvar 200 37◦3058.11N 8◦115.17W – 1064/2010

Mp41 W/C/DS LaCodosera 298 39◦1648.08N 6◦5220.89W Guadiana 1065/2010

Mp54 W/C/DS Segura,Idanha-a-Nova 235 39◦₄₉_11.06_{N 6}◦₅₈_52.99_{W Tejo 1066/2010}

Mp55 W/C/DS SalvaterradoExtremo,Idanha-a-Nova 253 39◦₅₃_37.50_{N 6}◦₅₄_18.38_{W Tejo 1067/2010}

Mp59 W Monfortinho,Idanha-a-Nova 255 39◦₅₉_9.96_{N 6}◦₅₂_50.23_{W Tejo 069/2010}

Mp60 W Entradas,CastroVerde 154 37◦₄₄_36.51_{N 7}◦₅₈_44.60_{W Guadiana 1070/2010}

Mp62 W Póvoa,MirandadoDouro 750 41◦3422.71N 6◦1917.53W Douro 1071/2010

Mp63 W Escarigo,FigueiradeCasteloRodrigo 560 40◦5034.73N 6◦4933.62W Douro 1072/2010

Mp64 W Vilarseco,MirandadoDouro 725 41◦₃₁_25.48_{N 6}◦₂₄_5.56_{W Douro 1073/2010}

byclusteranalysisusingNumericalTaxonomyMultivariate

Anal-ysis System (NTSYS-pc software, version 2.2, Exeter Software,

Setauket,NewYork)(Rohlf,2000).Forclusteranalysis,correlation

coefficientwasselectedasameasureofsimilarityamongall

acces-sions,andtheUnweightedPairGroupMethodwithArithmetical

Averages(UPGMA)wasusedforclusterdefinition.Thedegreeof

correlationwasevaluatedaccordingtoPestanaandGageiro(2000)

and classified as very high (0.9–1), high (0.7–0.89), moderate

(0.4–0.69),low(0.2–0.39)andverylow(<0.2).

3. Resultsanddiscussion

3.1. Morphologicalstudies

Theindumentumof M.pulegiumincludesnon-glandularand

glandulartrichomesscatteredalloverthevegetativeand

repro-ductiveorgans.Thenon-glandulartrichomesareofthreedifferent

types:(i)unicellular,withawartysurface,aswollenbasal

epider-malcellandacuteapices(Fig.2A),whichisseenonstemsandsepals

andonbothleafsurfaces,butmoreabundantontheadaxial

sur-face;(ii)shortmulticellular,2–4cells, uniseriate,wartysurface,

supportedbya cellularpedestal formedby two tofive

epider-malcellsarranged aroundthebaseand acuteapices,sparseon

adaxialleafsurfacebutcommononsepalsinnerandouterfaces

(Fig. 2A); (iii) longmulticellular, upto 8cells, with biggercell

dimensions,thin,uniseriate,acuteapices,wartysurface, always

leanedtowardtheapexandsupportedbyacellularpedestalformed

bytwotofiveepidermalcells,onlyseenonthepetalapexouterface

(Fig.2B).Theglandulartrichomesbelongtotwomorphologically

differenttypes,peltateandcapitate,whichareconsideredasthe

commonglandulartrichomearrangementintheLamiaceaefamily

(Werkeretal.,1993)andwereaswelldescribedin Mentha

spi-cata,M.spicata×suaveolens(Martins,2002),M.cervina(Rodrigues

etal.,2008)andotherLamiaceaespecies(Ascensãoetal.,1999;

CorsiandBottega, 1999;Rodriguesetal.,2006).InM.pulegium

peltatetrichomesareseenalloverbothleafsurfaces,dominanton

theabaxialsurface,onthestem,ontheinnerandoutersurfaces

ofsepals,andontheouterfaceapexofpetals.Theyhaveashort

stalkandasmoothlargehead,withavariablenumberof

secre-torycellsarrangedinoneortwocircles.Ourresultsshowthatin

M.pulegium12cellsareprobablythemostcommonnumberina

peltatetrichomeofmatureleaves,usually4intheinnercircleand

4–8intheouter(Fig.2G).Thatnumbercanreachupto16cells

inthepetalspeltatetrichomes,usually4intheinnercircleand

8–12intheouter.AccordingtoTurneretal.(2000)andMartins

(2002),thenumberofcells formingtheheaddiscinpeltate

tri-chomesdependsonthedevelopmentstageaswellasoftheplant

species,amatureleafpeltatetrichomeexhibited10disccellsin

M.pulegium,8inM.piperita(Turneretal.,2000),12inM.spicata

andM.spicata×suaveolens(Martins,2002),and16inProstanthera

ovalifolia(Gersbach,2002).Uponmaturation,theseglandular

tri-chomesaresunkeninepidermaldepressionsandthecuticleofthe

cellsofthesecretoryheadlifts,formingasubcuticularspacethat

enclosessecretions.Theheaddimensionsofpeltatetrichomesare

variable,butbiggeronthereproductivestructures:diameterupto

109␮m(±9␮m)onthecorolla,comparedto88␮m(±10␮m)on

theadaxialleafsurfaceand92␮m(±9␮m)ontheabaxialleaf

sur-face.Morphologicallywelldevelopedpeltateglandulartrichomes,

werealsoobservedoncotyledons(Fig.2C),asinotherLamiaceae,

suchasSalviaofficinalisandOcimumbasilicum(Croteauetal.,1981;

Werkeretal.,1993).Thepeltatetrichomesaretheonlykindof

glan-dulartrichomesseenonreproductivestructures,occurringalong

thelowersideoftheconnectivetissue,betweenthetwoanther

lobes(Fig.2D).Thisreportonthepresenceofpeltatetrichomes

betweenthetwoantherlobesisnoteworthybutwasalsoreported

forotherLamiaceaespecies(Ascensãoetal.,1995;Rodriguesetal.,

2008).

Itiswithinthepeltatetrichomesthatmostoftheessentialoil

isbelieved tobesynthesized(Turneretal.,2000).Thematerial

secretedbytheglandularheadcellspassesthroughtheapicalcell

wallsandaccumulateswithinalargespaceformedbythe

detach-mentofthecuticletogetherwiththepectinlayerofthesecretory

cellwalls.Thesecretoryproductsremainin thisspace,givinga

sphericalshape toeach maturepeltatetrichome.Asimilar

pat-ternofsubcuticularspaceformationwasdescribedinoilglandsof

otherLamiaceaespecies(Turneretal.,2000;Werkeretal.,1993).

Measurementsoftheglandularsecretoryheadcellsandofthe

sub-cuticularfillingsshowthatthemaximumdiameterofthesecretory

headcellsisachievedduringanearlierstageofdevelopment,and

thattheincreaseintotaldiameterofthepeltateglandulartrichome

isduetofurthersecretionduringleafgrowthanddependentof

theorganinwhichitispresent.Becausetheaccumulationofthe

secretedmaterialcontinuesduringthegrowthoftheorgansthat

bearthem,theyareconsideredlong-termtrichomes(Werkeretal.,

1993;Werker,2000andreferencestherein).

CapitatetrichomesarealsowidespreadinLamiaceae.Thetypes

ofcapitatetrichomesfounddifferinstalklengthandheadshape

andinclude:(i)capitatetypeI,withonestalkcell10␮m(±0.1␮m)

inlength,andaround/ovalsecretoryheadcell,withasmooth

sur-face(Fig.2E),27␮m(±2.2␮m)inlengthand21␮m(±1.7␮m)in

diameteratthehead,uniformlydistributedonbothleafsurfaces,

calyxandstems;ii)capitatetypeII,withalowerconicalstalkcell,

28␮m(±6␮m)inlengthand1to2elongatedneckcells,12␮m

(±0.7␮m)andaroundsecretoryheadcell,withasmoothsurface

(Fig.2F),and13␮m(±0.1␮m)indiameteratthehead,onlyonthe

adaxialpetalsurface.Thecapitatetrichomesfoundcorrespondto

thecapitatetypesIandIIdescribedbyWerkeretal.(1985).In

cap-itateglandulartrichomesmuchlessmaterialisaccumulatedinthe

celllumenandnoruptureofthecuticlewasobserved.

OnfullyexpandedmatureleavesofM.pulegium,thecapitate

glandulartrichomesweredensely distributed whilethepeltate

werescatteredamong them.Similarresultswerefoundalsoin

M.pulegiumbyKarray-Bouraouietal.(2009)andinbothM.

spi-cataandM.spicata×suaveolensbyMartins(2002).Thedensities

observedwere:(i)2.7and3.9peltatetrichomes/mm2_onthe

adax-ialand abaxial leafsurfacerespectively, (ii)10 and 13 capitate

trichomes/mm2 _{ontheadaxial andabaxial leafsurface,}

respec-tively.Theseresultsshowedalargerdistributionontheabaxial

surface, a common featurefor several otherLamiaceae species

(Ascensãoetal.,1995;Rodriguesetal.,2006,2008;Turneretal., 2000;Werkeretal.,1993).Nevertheless,thedensitiesfoundinM.

pulegiumwerethelowestvaluescomparedtootherstudiesinM.

pulegium(Karray-Bouraouietal.,2009)andothermints(Turner

etal.,2000).

3.2. Histochemicalstudies

Datafromhistochemicaltestsrevealedthatthesecreted

mate-rialcompositionwassimilarinbothleavesandflowersandhada

complexnature,containinglipophilicaswellashydrophilic

com-ponents.Thepresenceofthesecompounds,althoughindependent

oftheorganandofitsdevelopmentalstage,wasdependentonthe

trichometype(Table3).Thechemicalcompositionoftrichomes

secretionsseemstobedependentonthetypeoftrichomeandbe

independentoftheorganand itsdevelopment.Secretions from

peltatehairsstainedpositivewithRutheniumRed(pectins),Sudan

III(totallipids),NileBlue(acidiclipids)andNadireagent

(essen-tialoils),whilesecretionsfromcapitatehairsonlystainedpositive

withRutheniumRed,SudanIIIandNadireagent.

Thepresenceoftotallipidsinsecretionsofthetwotypesof

Fig.2. (A–G)SEMmicrographsshowingdistributionandtypesofMenthapulegiumtrichomes.(A)Abaxialleafsurfaceexhibitingunicellular(1)andshortmulticellular(2)

non-glandulartrichomeswitharegulardistribution.Alsovisiblearethepeltate(3)andthecapitatetypeI(4)glandulartrichomessunkeninepidermaldepressions.(B)

Petalapexouterfaceshowinglargemulticellularnon-glandulartrichomes,leanedtowardtheapex.(C)Cotyledonsexhibitingmorphologicallywelldevelopedpeltateand

capitateglandulartrichomes.(D)Stamensshowingpeltatetrichomesbetweenthetwoantherlobes.(E)CapitatetypeIglandulartrichomewithonestalkcellandanoval

secretoryheadcell.(F)CapitatetypeIIglandulartrichomewithalowerconicalstalkcell,exhibiting1–2elongatedneckcells.(G)Peltateglandulartrichomeswithtwelve

secretorycells,arrangedintwocircles(bar=20␮m).

mentioned inpreviousstudies withTymbracapitata(Rodrigues

etal.,2006).Thesecretionsfrombothtrichomestypesshoweda

bluecolourwiththeNadireagent,assumingapredominanceof

essentialoils.SimilarresultsalsohavebeenverifiedbyHuangetal.

(2008)inLavandulapinnataandalsothepresenceofpectinsinthe

cellwallsbyRutheniumRedisreferencedinPlectranthusornatus

(Ascensãoetal.,1999)andSaturejasubspicata(Marinetal.,2010).

3.3. Essentialoilcomposition

Theessentialoilyield,inthe14wildpopulationsofM.pulegium,

collected at full flowering ranged from 0.7% to 1.6% (w/d.w.)

(Table4).Theaverageessentialoilyield(1.1%)achievedatthe

flow-eringphaseisinaccordancewithsomereportedoilyieldsatfull

floweringforwildM.pulegiumplants(1.2%,Hassanpouraghdam

et al.,2011), M. arvensis,M. piperita,M. spicataand M.

longifo-lia(1.7%,1.2%,1.2%,1.0%,respectively,Hussainet al.,2010)but

therehavebeenstudiesreportingtwicetheyieldinM.pulegium

(3.8%,Kokkinietal.,2004;3.9%,Cooketal.,2007).Severalstudies

suggestthatoilyieldisassociatedwithclimaticfactors;higher

tem-peratures,summerwaterdeficitandhighersummersunshineare

factorsthatseemtofavourtheoveralloilyield(Kokkinietal.,2004;

Voirinetal.,1990).AlthoughPortugalhashighertemperaturesand

Table3

Histochemistryofthesecretionsoftheglandulartrichomespresentonthe

vegeta-tiveandreproductiveorgansofMenthapulegium.

Histochemicaltest Typeof com-pounds/reaction colour Peltate trichomes Capitatetrichomes TypeI TypeII

SudanIII Totallipids/Red + + +

NileBlue Neutrallipids/Pink − − −

Acidiclipids/Blue + − −

Nadi Essentialoils/Blue + + +

Acidicresins/Red − − −

RutheniumRed Pectins/Red + + +

characterizedbysummerwaterdeficit,whichmayexplainthelow

yieldsfound.

Thirty-ninecomponentswereidentifiedintheEOsisolatedfrom

theM.pulegium populationsstudied,rangingfrom92 to99%of

thetotaloilcomposition.Theidentifiedoilcomponentsarelisted

inTable4inorderoftheirelutionontheDB-1column,arranged

accordingtothefourtypesofessentialoilsobtainedby

agglomer-ativeclusteranalysis,withthelowestandthehighestpercentages

foundforeachcomponentineachvolatileoiltype.

Mostly quantitative rather than qualitative variation was

observed in all the essential oils analyzed. Oxygen-containing

monoterpenes(83–98%)weredominantinalloils,Table4.

Pule-gonewasthemajorcompoundinallofthepopulations(52–82%)

at full flowering, followed by isomenthone (2–36%), menthone

(0.1–17%),andpiperitone(1–15%).Despitesomevariabilityamong

the evaluated populations, cluster analysis (Fig. 3), confirmed

a high chemical correlation among all accessions (Scorr≥0.9%)

Table4

Minimumandmaximumpercentagerangeofcomponentsidentifiedintheessentialoil,isolatedfromtheaerialpartsof14Menthapulegiumwildpopulationscollectedat

full-floweringphase.ForsamplesgroupedoneachoftheclustersI–IIandsubclustersa–c,seeFig.3.

Components RI ClusterI ClusterII

Min Max a b c

Min Max Min Max Min Max

3-Methylcyclohexanone 914 t t t t t t t t ␣-Thujene 924 t t t t t t t t ␣-Pinene 930 t 0.3 0.5 0.7 0.3 0.8 0.4 0.7 Camphene 938 t t t t t t t t Sabinene 958 t 0.1 0.2 0.2 t 0.3 0.1 0.2 1-Octen-3-ol 961 t t t t t t t t ␤-Pinene 963 0.3 0.4 0.4 0.5 0.2 0.6 0.3 0.5 3-Octanol 974 1.0 1.4 1.5 1.8 1.1 1.8 1.2 2.1 ␤-Myrcene 975 t t t t t t t t p-Cymene 1003 t t t t t t t t 1,8-Cineole 1005 t t 0.3 0.3 t 0.4 t 0.2 Limonene 1009 t t 0.3 0.4 0.2 0.5 0.3 1.2 ␥-Terpinene 1035 t t t t t t t 0.2 Linalool 1074 t t t t t 0.1 t 0.1 3-Octanolacetate 1086 0.1 0.2 t 0.1 t 0.1 t t trans-Verbenol 1114 t t t t t t t t Menthone 1120 1.5 4.2 0.9 1.1 0.1 4.0 6.5 17.0 Isomenthone 1126 28.6 36.0 17.7 22.7 8.1 22.7 1.9 10.8 Menthofuran 1134 t t t t t t t t cis-Isopulegone 1134 0.7 0.7 0.7 0.8 0.8 1.2 0.8 0.9 Menthol 1148 t t t t t t t t Terpinen-4-ol 1148 t t t t t 0.1 t t ␣-Terpineol 1159 t 0.2 t 0.2 t 0.2 t 0.2 Myrtenol 1168 t t t t t 0.1 t 0.1 Pulegone 1210 52.0 55.5 60.3 61.4 61.4 81.8 57.0 69.8 Piperitoneepoxide 1210 t t t t t t t t Piperitone 1211 1.0 3.1 1.1 2.2 t 1.1 0.5 2.2 Menthylacetate 1278 t t t t t t t t Isomenthylacetate 1288 t t t t t t t t Piperitenone 1289 3.0 6.6 6.8 13.1 0.6 6.8 5.0 14.9 Nepetalactone 1291 t t t t t t t t Piperitenoneoxide 1315 t t t t t t t 0.3 ␤-Bourbonene 1379 t t t 1.1 t 5.7 t 1.1 ␤-Caryophyllene 1414 t t t t t 0.1 t 0.1 ␣-Humulene 1447 t t t t t t t t 2-Methoxy-6-methylacetophenonea _{1447 t t t t t t t t} ␤-Caryophylleneoxide 1561 t t t t t t t t Humuleneepoxide 1580 t t t t t t t t 2-Methyljasmonate 1634 t t t t t t t t %Identification 97.4 99.5 98.0 99.3 92.1 98.9 95.0 99.0 Groupedcomponents Monoterpenehydrocarbons 0.5 0.6 1.5 1.7 0.7 2.2 1.1 2.6 Oxygen-containingmonoterpenes 95.4 97.7 94.4 95.2 82.5 96.9 90.4 95.7 Sesquiterpenehydrocarbons t t t 1.1 t 5.7 t 1.2 Oxygen-containingsesquiterpenes t t t t t t t t Othersb _{1.2 1.5 1.5 1.9 1.1 1.9 1.2 2.1} Oilyield(w/d.w.) 0.7 1.2 1.1 1.1 0.9 1.6 0.7 1.1

RI,retentionindexrelatitetoC9–C17n-alkanesontheDB-1column;t,traces(<0.05%).

a_{Identificationbasedonmassspectraonly.}

Mp2 Cluster I Mp34 Mp32 Mp41 Cluster I a Mp33 Mp40 M55 Mp55 Mp59 Mp60 Cluster II b Mp54 Mp36 Mp63 0.90 0.95 1.00 Mp64 Mp62 c Correlation Coefficient

Fig.3.DendrogramobtainedbyclusteranalysisofthepercentagecompositionofessentialoilsfromtheMenthapulegiumsamplesexamined,basedoncorrelationandusing

theunweightedpair-groupmethodwitharithmeticaverage(UPGMA).Forabbreviations,seeTable2.

even thoughdefining two clusters, and 3sub-clusters, in total.

Althoughsomeclustersoverlapped,otherswereclearlyseparated

andgroupedpopulationsaccordingtotheirgeographicalcollection

site(Fig.1).Themaindifferencesbetweenthetwoclusterswere

thepulegone(≤55%)andisomenthone(>28%)relativeamountsin

clusterI.Menthonerelativeamount(>6%)separatedsub-clusterIIc,

andisomenthonerelativeamount(>17%)sub-clusterIIa,fromthe

otherEOinsub-clusterIIb.

Aliteratureassessment(Table1)showedthat inpennyroyal

pulegone-typeessentialoils,pulegoneusuallyrangesfrom60to

90%.InPortugal, arecent studyononeM.pulegiumpopulation

describedmenthone(36%)andpulegone(23%)asthemain

essen-tialoilcomponents(Teixeiraetal.,2012).Inthepresentstudy,all

theessentialoilsbelongtothepulegonechemotype,supporting

theresultsofMonteiroetal.(2007b)andLopesetal.(2010),on

nineteencultivatedPortuguesepopulationscollectedduringthe

floweringphase;Reis-Vascoetal.(1999)inonepopulation

col-lectedinSintra,andMataetal.(2007),inonesampleboughtina

localmarketinAlentejoRegion.M.pulegiumstudiedoilsshowed

a differentbehaviourfromtheoils of mostof theothermints,

sincetheexistenceofdifferentchemotypesisacommonfeature

inMenthaspeciesandhybrids(KokkiniandVokou,1989).

Inplantdevelopmentalterms,theessentialoilyieldhada

dif-ferent behaviouraccordingto thegrowingconditions.For wild

growingpopulations,theessentialoilyieldincreasedfromthe

veg-etativestage(meanvalue0.5%w/d.w.)untilfullflowering,Juneand

July(meanvalue1.1%w/d.w).Oppositebehaviourwasobservedin

thecultivatedones(1.9%w/d.watthevegetativestagefor1.1%

w/d.watthefloweringstage). Ingeneral,wildgrowing

popula-tionsshowedaloweroilyieldcomparedwiththecultivatedones.

TheanalysisofthemainEOconstituentsrevealedthatpulegone

remainedthemajorconstituent,alongthelifecycleoftheplant,

forbothgrowingconditions,althoughthebehaviourofthemain

componentswasslightlydifferent(Fig.4).

IntheEOisolatedfromcultivatedpopulations,pulegone

rela-tiveamountincreasedfromthevegetativeuntilthefull-flowering

phaseandthenitstartedtodecreasetowardstheendofthecycle.

In the EOisolated from wildgrowingpopulations, the relative

amountinpulegoneincreaseduntilthevegetativephaseandthen

decreasedanticipatingthepre-floweringphase.Towardsthefull

floweringthepulegonerelativeamountsufferedanewincrease.

Thesechangeswerefollowedbychangesintheisomenthoneand

menthonerelativeamounts,wheneverthepulegonedecreased,the

isomenthoneandmenthonetendedtoincrease.IntheEOisolated

fromcultivatedpopulations,menthoneandisomenthonereached

theirmaximumatthe vegetativephase and decreasedtowards

theendofthecycle(Fig.4).Thesevariationsmaybeduetothe

influenceofthedevelopmentalstageand environmental

condi-tionsontheregulationofthebiosynthesisofessentialoil,sinceitis

80 a l OE ) 60 Crop - pulegone Wild - pulegone Crop - isomenthone Wild i th o u n d (% of tot a 20 40 Wild - isomenthone Crop - menthone Wild - menthone Co m p o 0

Vegetative Pre-flowering Flowering Full-Flowering

Fig.4.Time-coursestudyofthemaincomponentsoftheMenthapulegium

essen-tialoilsisolatedinwild(opensymbols)andcultivated(closedsymbols)growing

conditionsatdifferentdevelopmentalstages.Giventheextendedperiodof

flower-ing,twosamplingpoints,onemonthapart,wereconsideredatthisdevelopmental

stage.Floweringcorrespondingto50%oftheplantsbloomingandFull-flowering

when100%oftheplantswhereblooming.Thevaluesarethemeanvaluesfrom6

knownthatthebiosynthesisoftheessentialoilsisaffectedby

phys-iologicalvariations (i.e.organand leafposition), environmental

conditions(i.e.harvestdateandplantingtime),geographic

vari-ationsandgeneticfactorsandevolution(Figueiredoetal.,2008).It

canbehypothesizedthatwildgrowingplantsaresubjecttomore

stress variables (overgrazing, human disruption, water deficit),

andthatundertheseconditionstheyprioritizethemetabolism,

whichresultsinthereductionofpulegone,favouringisomenthone

andmenthone.InMenthaxpiperitaleaves,pulegoneisreduced

byaNADPH-pulegonereductasetoyield(−)-menthoneand

(+)-isomenthone,inanapproximately10:1ratio(Davisetal.,2005).

Inthisstudymenthonewasalsoclearlydominant,butinahigher

ratio,exceptforthewildgrowingpopulationsinthepre-flowering

phase.Thechangeobservedinthepresentstudy,betweenthe

rel-ativeamountsofmenthoneandisomenthonetroughthelifecycle

mayreflectsomedegreeofsubstratespecificityorother

environ-mentaland/orphysiologicalcondition.

4. Conclusions

M.pulegiumaerialpartsshoweddifferenttypesofglandularand

nonglandulartrichomessimilartothosepreviouslydescribedfor

Lamiaceae.Histochemistrystudiesrevealedthepresenceofpectins

inthecell wallsand total lipids,acidic lipidsand essentialoils

inthesecretionsofthepeltateandcapitateglandulartrichomes.

Theattainedessentialoilyieldforthisspecieswasinaccordance

withthosereportedinpreviousstudies.M.pulegiumpopulations

studiedshowedapulegonechemotypeessentialoil,althoughthe

menthoneandisomenthonerelativeamountscouldfurther

differ-entiatetheseoils.Theevaluationoftheeffectofthedevelopmental

stageandgrowingconditionsonessentialoilcompositionshowed

mostlyquantitativeratherthanqualitativevariations,supporting

theviewthatbothfactorstogethercaninfluencetheregulationof

thebiosynthesisofessentialoils.Ourresultsalsoshowedthat

cul-tivationonlyseemstoaffecttheessentialoilyield,increasingits

content,notaffectingtheessentialoilcompositionthatseemsto

bemorestableanduniform.Thesearefeaturesthatturnthisspecies

intointerestingproductsforcultivationandcommercialization.

Acknowledgements

LeandraRodriguesisgratefultotheFoundationforScienceand

Technology (FCT) for the PhD grant SFRH/BD/38143/2008. This

studywaspartially funded bythePortuguese Ministryof

Agri-cultureandRuralFisheriesDevelopment,AgroProgram,Project

n◦522.

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