Rev. bras. farmacogn. vol.26 número6

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w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Chemical

characterization

of

two

morphologically

Espeletia

(Asteraceae)

species

and

chemometric

analysis

based

on

essential

oil

components

Guillermo

F. Padilla-González,

Jennyfer

A. Aldana,

Fernando

B. Da

Costa

∗

LaboratóriodeFarmacognosia,FaculdadedeCiênciasFarmacêuticas,UniversidadedeSãoPaulo,RibeirãoPreto,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3February2016

Accepted5May2016

Availableonline25June2016

Keywords:

Asteraceae Chemotaxonomy

Essentialoils

Espeletia

Chemometricanalysis

a

b

s

t

r

a

c

t

Inthisstudy,acomprehensivephytochemicalcharacterizationoftwomorphologicallyrelatedspecies fromthegenusEspeletiaMutisexBonpl.,namely,EspeletiagrandifloraHumb.&Bonpl.andEspeletiakillipii Cuatrec.,Asteraceae,hasbeenperformedbygaschromatographycoupledtoflameionizationdetection, gaschromatographycoupledtomassspectrometryandultra-highperformanceliquidchromatography coupledtoultravioletandhigh-resolutionmassspectrometry.Analysisofethanolextracts(70%,v/v) fromleavesandconcomitantcompounddereplicationallowedtheidentificationofmajorpeaks,most ofthemnewreportsforthegenusEspeletiaorthesubtribeEspeletiinae.Chemicalcharacterizationof resinsessentialoilsindicatedseveralsimilaritiesanddifferencesbetweenbothspeciesandfromother membersofthesubtribe.Chemometricanalysis(hierarchicalclusteringanalysisandorthogonalpartial least-squaresdiscriminantanalysis)appliedtotheessentialoilcompositionof31speciesfrom Espeleti-inaefurthermoreallowedtheidentificationofthreeprimaryclusterscorrelatedwiththetaxonomy. Hence,thisstudyunderscoredqualitativeandsemiquantitativedifferencesbetweenthechemical com-positionofleavesandresinsofE.grandifloraandE.killipii,providedinformationonchemotaxonomyand describedthepresenceofdifferenttrendsintheessentialoilcompositionfromspeciesofEspeletiinae.

Introduction

The genusEspeletia Mutis ex Bonpl., Asteraceae, constitutes

aneotropicalgroupendemictothenorthernAndeanregionsof

Venezuela, Colombia,and Ecuador. With ca. 71 species,

Espele-tiais themost diverse genus withinthe subtribe Espeletiinae,

whichalsocomprisesthegeneraEspeletiopsisCuatrec.,Coespeletia

Cuatrec.,RuilopeziaCuatrec.,LibanothamnusErnst,Carramboa

Cua-trec.,TamaniaCuatrec.,andParamiflosCuatrec.(Cuatrecasas,2013;

Diazgranados,2012a).

EspeletiagrandifloraHumb. &Bonpl. wasthefirst species in

Espeletiinaetobedescribedandcontinuestobefrequentlystudied

intermsofitsmorphology,ecophysiology,distribution,and

tax-onomy(FaguaandGonzalez,2007;Cuatrecasas,2013).However,

gapsremaininthetaxonomicdelimitationofthisspecies,which

couldpossiblyincludedifferenttaxonomicentities,givenitshigh

polymorphism and broad geographic distribution (Cuatrecasas,

2013). Its shared morphological characteristics with Espeletia

∗ _{Corresponding}_author.

E-mail:[email protected](F.B.DaCosta).

killipiiCuatrec.(e.g.,leafshape,plantheight,andoverall

appear-ance)moreoverhinderitsunequivocalidentification.Accordingto

Cuatrecasas(2013),twoprimarymorphologicalcharacteristicsthat

distinguishE.grandiflorafromE.killipiiincludethepresenceofat

leastonepairofsterileoppositeleavesintheproximalpartofthe

synflorescencesintheformerspecies—bycontrast,E.killipiilacks

suchsterileleaves—andthelengthofthesynflorescences,whichare

longerinE.grandiflora.However,thefrequenthybridizationofthe

twospeciesinzoneswhereoverlappingpopulationsoccur(e.g.,La

ChisacápáramoinColombia)furthercomplicatestheir

unambigu-oustaxonomicdelimitation(Cuatrecasas,2013).

The secondary metabolite chemistry of E.grandiflora and E.

killipii hasbeen poorly investigated. Nevertheless, classic

phy-tochemical studies of E. grandiflora resins have reported the

presenceofsixent-kauranediterpenes—kaur-16-ene,

kaur-16-en-19-al,kaur-16-en-19-ol, kaurenoicacid, grandiflorolic acid, and

grandiflorenicacid(Piozzietal.,1968,1971,1972)—whereasother

chemicalclasses(e.g.flavonoids,sesquiterpenelactones,and

triter-penes)havebeenreportedfromtheleavesofE.killipii(Torrenegra

etal.,1994;TorrenegraandTellez,1995,1996).However,no

com-prehensivephytochemicalcharacterizationthatincludestheleaves

andresinsofbothspecieshasbeenperformed,andthesimilarities

http://dx.doi.org/10.1016/j.bjp.2016.05.009

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anddifferencesbetweenthemetabolicfingerprintsofE.grandiflora

andE.killipiiremainunknown.

Consequently,this studyaimedtoperformacomprehensive

phytochemical characterization of E. grandiflora and E. killipii

leavesand resinsbyusingmodernandcomplementary

analyti-caltechniquessuchasGC–FID,GC–MSandUHPLC–UV-HRMS,all

todeterminesimilaritiesanddifferencesbetweenthespecies,as

wellasamongEOfrom31speciesofEspeletiinae,asperformedby

chemometricmethodsinvolvingmultivariatestatisticalanalysis.

Materialsandmethods

Plantmaterial

LeavesandresinsofEspeletiagrandifloraHumb.&Bonpl.and

E.killipii Cuatrec.,Asteraceae, werecollectedin theCruzVerde

(N4◦34′48.5′′W73◦59′45.3′′–3328m.)andSumapazpáramos

(N4◦ 17′ 24′′W74◦ 12′ 24.2′′–3717m.),respectively,in

Cundi-namarca,Colombia,bySandraL.Casta˜nedaandCarlosI.Suárezin

January2015.PlantidentificationwasperformedbyDr.Mauricio

DiazgranadosandCarlosI.SuarezfromJardínBotánicodeBogotá

(JBB),andavouchersampleofeachspecieswasdepositedatJBB’s

herbariumunder thecollectionnumbers SLC-192and SLC-207,

respectively.PlantcollectionsweremadewithJBBcollection

per-mits.

LeavesextractionandUHPLC–UV-HRMSanalysis

Fromeachspecies,20mgofdryleaveswerepowderedwith

liq-uidnitrogenandextractedwith2mlofEtOH:H₂O(7:3,v/v,HPLC

grade)inanultrasonicbathfor15minat25◦C.Plantextractswere

centrifugedat19,975×gfor10minandpartitionedwith0.5ml

ofhexane.Theaqueouslayerwasfilteredthrougha0.2␮mPTFE

membraneandanalyzedbyUHPLC-UV-HRMSinanAccelaUHPLC

apparatus(ThermoScientific,Carlsbad,CA,USA)coupledtoan

80-Hz photodiodearraydetector (PDA)(Thermo Scientific)and an

ExactiveTM_Plus_Orbitrap_mass_spectrometer_(MS)_(Thermo

Scien-tific).

Aheatedelectrosprayprobewasusedasanionizationsource

inMSanalysesalongwiththefollowingparameters:positiveand

negativeionizationmodes(fullscanmethod)overamassrange

of150–2000Da,resolutionof70,000fullwidthathalfmaximum

(FWHM) (m/z200), mass accuracy <3ppm, scan time of about

2.5scans/s,sprayvoltagesof+3.6kVand−3.2kVforthepositive

andnegativeionizationmodes,respectively,capillaryandheater

temperatureof300◦C,sheathgasof30arbitraryunits,andS-lens

of55arbitraryunits.

Subsequently, 10␮lof each plant extractwere injected and

chromatographed in a C18 column (Hypersil Gold, 1.9␮m,

50mm×2.1mm, Thermo Scientific) connected to a C18

Secu-rity GuardTM _Ultra_cartridge _(Phenomenex, _Torrance, _CA,_USA).

Themobilephaseinvolvedpurifiedwaterwith0.1%formic acid

and acetonitrile, and separation was performed at a flow rate

of300␮l/min.Thegradientprogramstartedwith5%acetonitrile

forthefirst3minandincreasedtheamountlinearlyto100%in

30min.Acetonitrileremainedconstantfor5minbeforeultimately

decreasingto5%after40min.Thecolumntemperaturewas

main-tainedat30◦_C,_the_tray_temperature_was_fixed_to₁₀◦_C,_and_the

PDAdetectorwassettorecordintherangeof200–600nm.

Dereplicationofleafextracts

Extractsdereplicationwasperformedbasedonspectral(UVand

accuratemass)and retentiontime(Rt)comparisonswith

refer-encecompoundspreviously isolatedin ourlaboratory—namely,

3-O-(E)-caffeoylquinic acid, protocatechuic acid, 3,5-di-O-(E

)-caffeoylquinic acid, 4,5-di-O-(E)-caffeoylquinic acid, quercetin,

3-methoxyquercetin,andent-kaurenoicacid.Compoundswhose

reference standard was unavailable were tentatively identified

by accuratemass and UV spectracomparisons with data from

theliterature,includingtheDictionaryofNaturalProducts(DNP)

and the Asteraceae Database (AsterDB), the latter of which

correspondstoanin-housedatabase(www.asterbiochem.org)

con-taininghundredsofchemicalcompoundspreviouslyreportedin

theAsteraceaefamily,includingofthesubtribeEspeletiinae.

In-sourcefragmentationintheUHPLC–UV-HRMS(Orbitrap)(MS/MS)

wasadditionallyconsideredtoproposesomeofthepeak

assign-ments.

Essentialoilextraction

TheresinsofE.grandiflora(2.1g)andE.killipii(16.8g),both

col-lectedduringanthesis,werehydrodistilledusingaClevenger-type

apparatusfor3handstoredat−20◦Cforfurtheranalysis.Before

gaschromatographyanalysisEOsweredissolved(1%,v/v)inethyl

acetate.

Essentialoilanalyses

EOfromE.grandifloraandE.killipiiresinswereanalyzedusing

a Hewlett–Packard 6890N Plus GC–FID(USA) apparatus where

thepercentagecompositionofindividualcomponentswas

deter-mined.Identificationofcompoundswasperformedbycomparison

oftheretentionindicesofeachcomponentrelativetoaseriesofn

-alkanes,andbymassspectracomparisonswithNIST08andWilley

7databasesbasedonGC–MSanalysesonaShimadzuQP-2010

sys-tem(Tokyo,Japan).Toconfirmidentifications,themassspectrum

andretentionindexofeachcompoundwasfurthercomparedwith

publisheddata(Adams,2007).

DuringGC-FIDanalyses,1␮lofeach samplewasinjected(in

triplicate) using a split ratio of 1:20 and separated ona HP-5

fusedsilicacolumn(30m;0.32mmI.D.;0.25␮mfilmthickness).

Thecarriergaswashydrogenat1.3ml/min,theoventemperature

wasprogrammedfrom60to240◦_C_at₃◦_C/min,_and_the_injector

temperaturewas240◦_C._Retention_indices_were_calculated

rela-tivetoC8–C40n-alkanes.ForGC–MSanalyses,anEN5MScolumn

wasemployed(30m;0.25mmI.D.;0.25␮mfilmthickness)with

asourcetemperatureof250◦_C,_carrier_gas_adjusted_to_41.6_cm/s,

anionizationenergyof70eV,andascanrangeof40–500Da.The

temperatureprogramandremainingparameterswereidenticalto

thoseinGC–FIDanalyses.

Chemometricanalyses

TherelativeamountoftheEOcomponentsof31speciesfromthe

subtribeEspeletiinaereportedintheliteratureandinthepresent

study(Table1)wereusedtoperformmultivariatestatistical

meth-odsinthesoftwareR3.0.3(RFoundationforStatisticalComputing,

Vienna,Austria)andSIMCAP13.0.3.0(UmetricsAB,Malmö,

Swe-den).Priortoanalyses, thedatamatrixwasscaledbyusingthe

arcsinemethod,whichisappropriateandcommonlyusedfordata

expressed as percentages. Hierarchical clustering analysis with

bootstrapresampling (HCAbp)wasperformedintheRpackage

pvclust(SuzukiandShimodaira,2006),usingWard’smethodasthe

clusteringalgorithmandEuclidiandistance.Togeneratea

quanti-tativemeasureofaccuracyintheclustersobtained,approximately

unbiased(AU)pvalueswereconsideredintheanalysis,calculated

bymultiscalebootstrap,whichismoreaccuratethantraditional

bootstrapping,asSuzukiandShimodaira(2006)haveconfirmed.

Inmultiscalebootstrapping,sincethesizeofthebootstrapsample

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G.F.Padilla-Gonzálezetal./RevistaBrasileiradeFarmacognosia26(2016)694–700

Table1

SpeciesfromsubtribeEspeletiinaeusedtoperformchemometricanalysesbasedonessentialoilcompositions;speciesorganizedaccordingtothethreeclustersidentified

byhierarchicalclusteringanalysiswithbootstrapresampling.

Cluster Species Localityofcollection

(Country)

Altitude m.a.s.l.

Dateof collection

Plant stage

Plant part

Reference

1 Co.moritziana1 LaCulata(VEN) 3750 9/2/1998 Anthesis Leaves Aparicioetal.(2002)

1 Co.moritziana2 Pi˜nago(VEN) 3800 12/9/1999 Anthesis Leaves Aparicioetal.(2002)

1 Co.moritziana3 LosOsos(VEN) 3700 5/25/2004 Anthesis Leaves Ibá˜nezandUsubillaga(2006a)

1 Co.moritziana4 PicodelÁguila(VEN) 4100 9/22/2002 Anthesis Leaves Ibá˜nezandUsubillaga(2006a)

1 Co.spicata1 LaCulata(VEN) 3500 11/22/1998 Anthesis Leaves Aparicioetal.(2002)

1 Co.spicata2 LasCruces(VEN) 3850 12/9/1999 Anthesis Leaves Aparicioetal.(2002)

1 Co.timotensis PicodelÁguila(VEN) 4000 n.r. Anthesis Aerialparts Rojasetal.(1999)

1 E.nana ParamodeOrtiz(VEN) 3085 5/1/2010 n.r. Leaves Pe˜naetal.(2012)

1 E.batata PiedrasBlancas(VEN) 4200 11/1/1998 n.r. Leaves Usubillagaetal.(2001b)

1 E.grandiflora ParamodeCruzverde(COL) 3328 1/22/2015 Anthesis Resin Presentstudy

1 E.killipii ParamodeSumapaz(COL) 3717 1/26/2015 Anthesis Resin Presentstudy

1 L.occultusssphumbertii LagunaNegra(VEN) 3200 12/2/1999 Anthesis Leaves Usubillagaetal.(2001a)

2 L.neriifolius ElBatallón(VEN) 2800 11/6/1998 Anthesis Leaves Usubillagaetal.(2001a)

2 L.occultus ElBatallón(VEN) 2800 11/6/1998 Anthesis Leaves Usubillagaetal.(2001a)

2 L.lucidus LaAguada(VEN) 3400 11/24/1999 Anthesis Leaves Usubillagaetal.(2001a)

2 E.schultzii1 LaCulata(VEN) 2800 9/12/2002 Anthesis Leaves Ibá˜nezandUsubillaga(2006b)

2 E.schultzii2 LosOsos(VEN) 3700 9/22/2002 Anthesis Leaves Ibá˜nezandUsubillaga(2006b)

2 E.schultzii3 PicodelÁguila(VEN) 4100 10/22/2002 Anthesis Leaves Ibá˜nezandUsubillaga(2006b)

2 E.weddellii LasCruces(VEN) 4080 n.r. Anthesis Leaves Khourietal.(2000)

2 E.semiglobulata PicodelÁguila(VEN) 3800 n.r. n.r. Leaves Usubillagaetal.(1999)

2 E.xaurantia PicodelÁguila(VEN) n.r. 11/15/2003 Anthesis Leaves Ibá˜nezandUsubillaga(2008)

3 R.marcescens1 ElBatallón(VEN) 3000 6/22/1999 Anthesis Leaves Aparicioetal.(2001)

3 R.marcescens2 ElBatallón(VEN) 3000 1/27/2000 Anthesis Leaves Aparicioetal.(2001)

3 R.atropurpurea1 ElBatallón(VEN) 3400 11/6/1998 Anthesis Leaves Aparicioetal.(2001)

3 R.atropurpurea2 ElBatallón(VEN) 3400 6/22/1999 Anthesis Leaves Aparicioetal.(2001)

3 R.lindenii SanJosé(VEN) 3100 8/10/1999 Anthesis Leaves Aparicioetal.(2001)

3 R.floccosa PicodelÁguila(VEN) 3800 5/25/1999 Anthesis Leaves Aparicioetal.(2001)

3 Ca.badilloi LaAzulita(VEN) 1700 n.r. n.r. Leaves Corderoetal.(2011)

3 Ca.badilloivar.pittieri Bailadores(VEN) 1800 n.r. n.r. Leaves Rojasetal.(2008)

3 Co.thyrsiformis Lanegra(VEN) 3000 6/22/1999 Anthesis Leaves Aparicioetal.(2002)

3 Es.angustifolia SanJosé(VEN) 2870 6/1/2006 n.r. Leaves Mecciaetal.(2007)

n.r.,notreported.Generaabbreviations:Co.,Coespeletia;E.,Espeletia;L.,Libanothamnus;R.,Ruilopezia;C.,Carramboa;Es.,Espeletiopsis.

VEN,Venezuela;COL,Colombia.

traditionalbootstrappingvaluecausedbytheconstantsamplesize

(SuzukiandShimodaira,2006).

Toidentifythecompoundsresponsiblefor speciesclustering

inthethreeprimary clustersobservedin theHCAbp,an

ortho-gonalpartialleast-squaresdiscriminantanalysis(OPLS-DA)was

performed with SIMCA P. This method separates the

system-aticvariationin X intocorrelated (predictive)anduncorrelated

(orthogonal)variablestoY(Eriksson etal.,2012).Thevariables

responsiblefor theclustering pattern observed were identified

accordingtoavariableimportanceintheprojection(VIP)value

>1.0,a significantcontributiontothe loadingsplot, anda high

magnitude and high reliability confidence intervals in the

coefficientsplot.

Resultsanddiscussion

LeavescharacterizationbyUHPLC–UV-HRMS

Themetabolicfingerprintingofcrudeethanol(70%v/v)extracts

from E. grandiflora and E. killipii leaves by using

UHPLC–UV-HRMS (Fig. 1) and concomitant dereplication allowed the

identificationofseveralsimilaritiesanddifferencesbetweenthe

100

90

80

70

60

50

40

30

20

10

0

0 2 4 6 8 10 12 14 16 18 20

Time (min)

Relativ

e ab

undance

14∗

7∗

5∗

4∗

3 2∗

1∗

8∗

15 13

12 11

10

9 6

22 24 26 28 30

E. killipii E. grandiflora

32 34 36 38 40

Fig.1.Totalioncurrent(TIC)chromatogramsofE.grandifloraandE.killipiileavesextractsobtainedinnegativemode.(1)3-O-(E)-caffeoylquinicacid,(2)protocatechuicacid,

(3)di-caffeoylquinicacidisomer,(4)3,5-di-O-(E)-caffeoylquinicacid,(5)4,5-di-O-(E)-caffeoylquinicacid,(6)tri-caffeoylaltraricacidisomer,(7)quercetin,(8)3-methoxy

quercetin,(9)8,8′′_{-methylene-bisquercetin,}₍₁₀₎_ent_{-12-oxo-kaura-9(11),16-dien-18-oic}_acid,₍₁₁₎_ent_{-12-hydroxy-kaura-9(11),16-dien-18-oic}_acid,₍₁₂₎_ent_-15_␣

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species.Asobservedintotalioncurrentchromatograms(Fig.1),

bothspecieshavehighlysimilarfingerprintswithgreater

semi-quantitative instead of qualitative differences. All dereplicated

compounds (Fig. 1) were detected in both species, though E.

killipii showed a greater relative abundance of peaks 6 and

10–15(Fig.1).Fourmainchemicalclassesofsecondary

metabo-lites were identified in the leaves extracts of both species:

trans-cinnamicacidderivatives, flavonoids, diterpenes,and one triterpene.

Amongflavonoids,bothspeciesproducehighquantitiesof

3-methoxyquercetin(peak8,Fig.1).Thisflavonoid,identifiedby

itsRt,UV,andaccuratemasscomparisonwithareference

com-pound,hasbeenpreviouslyreportedonlyintheleavesofE.killipii

(Torrenegraetal.,1994)andE.barclayana(Gutierrezetal.,1998).Its

presenceinE.grandiflorathereforeconstitutesanewreportforthis

species.Quercetin(peak7,Fig.1),alsoidentifiedinbothspeciesby

itscomparisonwithareferencesubstance,constitutesanewreport

forE.grandifloraaswell.Peak9(Fig.1)wastentativelyproposed

to be 8,8′′-methylene-bisquercetin (flavonoid). This compound

showedadeprotonatedmoleculeat615.07815m/zanda

proton-atedoneat617.09174m/zforC₃₁H₂₀O₁₄.Themassspectrumin

thenegativemodealsoshowedanintensepeakat299.01935m/z,

originatedbyin-sourcefragmentationofaquercetinunitand a

methylgroupin accordancewiththeliterature(Martuccietal.,

2014).Althoughthiscompoundhasnotbeenpreviouslyreported

inthesubtribeEspeletiinae,Martuccietal.(2014)reporteditinthe

genusVernonia(Asteraceae).

Six trans-cinnamic acid derivatives were identified (Fig. 1).

Amongthem,3-O-(E)-caffeoylquinicacid(peak1,Fig.1),

protoca-techuicacid(peak2,Fig.1),3,5-di-O-(E)-caffeoylquinicacid(peak

4,Fig.1),and4,5-di-O-(E)-caffeoylquinicacid(peak5,Fig.1)were

identifiedbasedonspectral(UVandaccuratemass)andRt

com-parisonswithreferencesubstances.Peak3(Fig.1)wastentatively

proposedtobeanotherdicaffeoylquinicacidisomer.This

com-poundshowedadeprotonatedmoleculeat515.11884m/zanda

protonatedone at 517.13385m/z forC25H24O12.Its mass

spec-truminthepositivemodeshowedin-sourcefragmentationwith

twomainpeaks:oneat499.12311m/z,which correspondswith

thelossofawatermolecule,andanotherat163.03882m/z,which

correspondswithanionizedresidueofcaffeicacid(Santosetal.,

2008).TheUVspectrumof thispeakexhibited twoabsorptions

atapproximately300and325nm,whichsuggestchlorogenicacid

derivatives.

By contrast, peak 6 (Fig. 1) was proposedto be a possible

tricaffeoylaltraric acidisomer based onaccurate mass

compar-isons with compounds reported in AsterDB and maximum UV

absorptions.Thiscompounddisplayedadeprotonatedmoleculeat

695.12531m/zforC33H28O17andtwoUVmaximaatapproximately

300and329nm.Thepresenceofthepreviouslyreportedtrans

-cinnamicacidderivativesinE.grandifloraandE.killipiiconstitutes

thefirstreportforthischemicalclassinthesubtribeEspeletiinae.

However,theirpresenceisunsurprising,fortheyconstituteahighly

commonclassofsecondarymetaboliteswidespreadinAsteraceae

(Chagas-Paulaetal.,2011).

IntheleavesofE.grandifloraandE.killipii,fiveent-kaurane

diter-peneswerealsoidentified,amongwhichent-kaurenoicacid(peak

14,Fig.1)wasunambiguouslyidentifiedbyRtandaccuratemass

comparisonwithareferencesubstance.Theidentitiesoftheother

four(peaks10–13,Fig.1)weretentativelyproposedbasedonlyon

accuratemassandchemotaxonomicinformation.Allofthese

diter-peneshave beenpreviouslyreportedinEspeletiinae (Bohlmann

etal.,1980;Usubillagaetal.,2003).

Lastly, the identity of an oleane-type triterpene (peak 15,

Fig. 1) was tentatively proposed based on accurate mass and

databasesearch.Thiscompoundshowedadeprotonatedmolecule

at 655.42114m/z and a protonated one at 657.43524m/z for

C39H60O8. An accurate mass search in DNP afforded three

possiblecompounds:brevenal(CAS:776331-34-1),

olean-12-ene-3,16,21,22,28-pentol,16,28-diacetate22-(2-methyl-2-butenoate)

(CAS: 124641-09-4), and olean-12-ene-3,16,21,22,28-pentol,

22,28-diacetate 21-[2(or 3)-methyl-2-butenoate] (CAS:

55949-26-3). Amongthem, onlythe two oleane-typetriterpenes have

previously been isolatedfrom species belongingto plant

fami-lies (Apiaceaeand Polemoniaceae),whereas brevenal has been

reportedonlyin themarinedinoflagellate Kareniabrevis(DNP).

Further analyses are therefore necessary to unambiguously

identifythiscompoundinEspeletiinae.

ResincharacterizationbyGC–FIDandGC–MS

ThehydrodistillationofE.grandifloraandE.killipiiresins

pro-vided yellowish EOs with yields of 14.29% and 14.88% (v/w),

respectively,basedontheirfreshweight.Inbothspecies,the

iden-tifiedsubstances constitute95–97%ofthetotaloilcomposition

(Table2).

AcomparisonoftheEOcompositionofE.grandifloraandE.

kil-lipiiresinsrevealednosignificantdifferences;bothspecieswere

characterizedbya predominanceofmonoterpene hydrocarbons

with ␣-pinene as the major component (Table 2), which

rep-resented morethan 61% of thetotal oil composition (Table 2).

However,importantdifferenceswereobserved.Forexample,␤

-pinene represented the second major compound (3.4%) in E.

grandiflora resin EO, whereas sabinene constituted the second

major peak in E. killipii (6.5%), as Table 2 shows. E.

grandi-florapossessesagreaterproportionofoxygenatedmonoterpenes

(18.1%)thanE.killipii(12.5%),butalesserproportionof

sesquiter-penehydrocarbons(Table2).Remarkably,onlyonesesquiterpene

hydrocarbon was detected in E. grandiflora resin EO—namely,

␦-cadinene—whereas several others, including ␣-copaene, ␤

-bourbonene, ␦-selinene, and ␥-muurolene, were detectedin E.

killipii resin EO (Table 2).In general,E. killipii presentsa more

complex EO composition than E. grandiflora, since more

com-poundsanddifferentchemicalclassesweredetectedinthisspecies

(Table2).

Monoterpene hydrocarbons constitute the main EO

compo-nentsfromallspeciesofEspeletiinaeinvestigatedthusfar(Table1).

Amongthem,␣-pineneusuallyconstitutesthechiefEOcomponent

innumerousspecies(Aparicioetal.,2002;Ibá˜nezandUsubillaga,

2006a, 2008; Meccia et al., 2007; Pe˜naet al., 2012). However,

some species produceother compounds suchas limonene and

␣-phellandreneasthemajorpeaks(Aparicioetal.,2001;Ibá˜nez

and Usubillaga, 2006b). Based on this observation of different

terpenoidspredominanceinEOsfromEspeletiinae,chemometric

analysesusingGCdatahasbeenperformedtoidentifytrendsin

theEOcompositionof31speciesfromEspeletiinae,asdiscussed

below.

Chemometricanalysesofessentialoils

Percentage EOcompositions of 31 species fromEspeletiinae

wereusedasinputdatatoperformchemometricanalyses(HCAbp

andOPLS-DA).Table1showsthespeciesnamesandtheirlocality,

altitudeanddateofcollection,aswellastheplantpartand

repro-ductivestage.Inthisstudy,weconsideredonlyspeciescollectedin

theanthesisperiod,includingafewcasesinwhichthatinformation

wasnotspecified(Table1).

Results from HCAbp (Fig. 2) demonstrated trends in EO

composition of species from Espeletiinae, and three primary

clusters were determined based on chemical similarities and

high support values (≥83%). As Fig. 2 also shows, intraspecies

(5)

Table2

PercentageofessentialoilcomponentsidentifiedinE.grandifloraandE.killipiiresins.

# Compoundname RI RI-lit E.grandiflora E.killipii

1 Santolinatriene 908 906 0.3 0.9

2 ␣-Thujene 927 924 – 0.3

3 ␣-Pinene 937 932 69.7 61.8

4 Camphene 950 946 0.4 0.3

5 Thuja-2,4(10)-diene 955 953 1.2 0.5

6 Sabinene 975 969 0.6 6.5

7 ␤-Pinene 979 974 3.4 4.0

8 o-Cymene 1026 1022 2.1 0.6

9 Limonene 1029 1024 1.0 1.4

10 1,8-Cineole 1033 1026 0.8 2.1

11 ␥-Terpinene 1058 1054 t 0.2

12 Fencholenicaldehydea ₁₀₉₂ _– _0.6 _t

13 Linalool 1101 1095 t 0.1

14 ␣-Campholenal 1128 1122 2.8 1.2

15 trans-Pinocarveol 1141 1135 2.3 1.2

16 cis-Verbenol 1143 1137 0.8 0.3

17 trans-Verbenol 1147 1140 2.9 1.1

18 trans-Pinocamphone 1162 1158 0.2 t

19 Pinocarvone 1164 1160 0.6 0.2

20 p-Mentha-1,5-dien-8-ol 1169 1166 2.9 2.3

21 Terpinen-4-ol 1178 1174 t 0.8

22 p-Cymen-8-ol 1186 1179 – 0.2

23 ␣-Terpineol 1187 1186 t 0.4

24 Myrtenal 1188 1193 0.2 t

25 Myrtenol 1199 1194 1.6 1.0

26 Verbenone 1212 1204 1.4 1.2

27 trans-Carveol 1221 1215 0.8 0.2

28 Carvone 1246 1239 0.1 t

29 Linaloolacetate 1253 1254 t 0.2

30 Lavandulylacetate 1293 1288 0.1 t

31 ␣-Copaene 1376 1374 t 1.7

32 ␤-Bourbonene 1385 1387 t 0.7

33 ␥-Muurolene 1477 1478 t 1.0

34 ␦-selinene 1491 1492 t 0.2

35 ␣-Muurolene 1500 1500 – 0.3

36 ␦-Cadinene 1524 1522 0.2 1.5

37 Spathulenol 1577 1577 t 0.3

38 Copaborneol 1604 1613 – 0.9

39 kauranediterpeneb ₁₉₈₁ _t _0.3

40 kauranediterpeneb ₁₉₈₈ _t _0.7

Monoterpenehydrocarbons 78.7 76.5

Oxygenatedmonoterpenes 18.1 12.5

Sesquiterpenehydrocarbons 0.2 5.4

Oxygenatedsesquiterpenes – 1.2

Totalidentified 97.0 95.6

–notdetected;t,traces.

a_{Identification}_based_only_on_mass_spectra_comparison_with_Willey₇_database.

b _Tentative_{identification}_based_on_{fragmentation}_pattern_comparison_with_Adams₍₂₀₀₇₎_and_Usubillaga_and_Nakano₍₁₉₇₉₎_;_The_two_most_abundant_compounds_in_E.

grandifloraandE.killipiiareemphasizedinbold.

different localities (e.g., Coespeletia moritziana and Espeletia

schultzii)wasnotsignificantenoughtopromote species

group-inginadifferentcluster,whichprovidessupportfortheproposed

groups.

Notably,thethreeclustersidentifiedshouldnotbeconsidered

tobedifferentchemotypes,sinceweanalyzeddifferentspecies,and

bydefinition,achemotypecorrespondsto“organismscategorized

undersamespecies,subspeciesorvarietieshavingdifferencesin

quantityandqualityoftheircomponentsintheirwholechemical

fingerprintthatisrelatedtogeneticorgeneticexpression

differ-ences”(Polatoglu,2013).

Supervised OPLS-DA displayed a good separation

(R2Ycum=0.985; Q2cum=0.789), in which 22% of the

varia-tioninXrelatestotheseparationbetweenthethreeclusters(Fig.

3)and afforded the identificationof discriminating substances.

Cluster 1 was characterized by high ␣-pinene, ␤-pinene, and

␤-phellandrene contents, cluster 2 by high␣-phellandrene, ␣

-thujene,p-cymene,andsabinenecontents,andcluster3mainly

byhighgermacreneD,limonene,and␤-caryophyllene contents

(Fig.3).

AdditionalOPLS-DAanalysesconsideringspecieslocalityor

ele-vationrangeasYvariables(Table1)revealednocorrelationwith

EOcompositions(datanotshown,butareevidentfromTable1).

Thisresultsuggeststhatintraspeciesvariationduetogeography

orelevation,asreportedbyAparicioetal.(2002)andIbá˜nezand

Usubillaga(2006b),islessthaninterspeciesvariationin

Espeleti-inae.Therefore,whenconsideringseveralspeciesfromdifferent

generainasingleanalysis,evenspeciescollectedatdifferent

local-itiesclusteraccordingtotheirtaxonomicrelatednessratherthan

theirgeographicorigin(Fig.2).AsshowninTable1,acertain

cor-relationbetweenspeciesEOsandtheirgenericclassificationswas

evident.Forexample,cluster1iscomposedprimarilyofspecies

belongingtothegenusCoespeletiaandafewspeciesofEspeletia,

whereascluster2presentsspeciesmostlyofEspeletiaandafew

ofLibanothamnus.Bycontrast,cluster3isprimarilycomposedof

speciesofRuilopeziaandafewofCarramboa(Table1).Nevertheless,

theclusteringofEspeletiinaespeciesbasedontheirEOcomponents

doesnotfollowstrictgenericboundaries(basedonmorphology),

sincenoevidentcorrelationwasobservedwhenconsideringthe

(6)

0.0

Cluster 1 Cluster 2 Cluster 3

91 92 Co . timotensis Co . mor itziana 2 Co . mor itziana 4 E. g randiflor a E. killipii L. occultus ssp . humber tii Co . mor itziana 1 Co . mor itziana 3 E. nana E. batata Co

. spicata 1

Co

. spicata 2 E.

semiglob ulata L. ner iif olius L. lucidus L. occultus E. schultzii 1 E. schultzii 2 E. w eddellii E. schultzii 3 E. x aur antia Ca. badilloi Ca. badilloi v ar . pittier i R. atropur purea 1 R. atropur purea 2 R. marcescens 1 R. marcescens 2 Co . th yrsif or mis R. floccosa Es . angustif olia R. lindenii 100 96 92 83 94 87 84 72 98 85 86 86 84 94 81 100 99 83 86 88 85 80 96 81 100 94 0.5 1.0 1.5 2.0 2.5 3.0

Fig.2. Hierarchicalclusteringanalysiswithbootstrapresamplingof31speciesofthesubtribeEspeletiinaebasedonpercentageofessentialoilcompositions;dottedboxes

correspondtothethreeprimaryclusters.

1.5 1 0.5 0 –0.5 –1 –1.5 –2

–2.5 –2 –1.5

R. marcesc R. floccos Co. thyrsi

R. lindeni

Ca. badillR. marcesc Es. angust

R. atropur Ca. badill

R. atropur Co. moritz E. nanaCo. moritz Co. moritz Co. moritz Cluster 1 Cluster 2 Cluster 3 Co. spicat Co. timote E. grandif E. batata Co. spicat L. occultu E. x auran

L. lucidus L. neriifo E. schultz E. schultz E. schultz E. semiglo L. occultu E. weddell E. killipi

–1 –0.5 0

1.0036 ∗ t[1]

R2X[1]=0.117 R2X[2]=0.102 Ellipse: Hotelling’s T2 (95%)

1.00371

∗ t[2]

0.5 1 1.5 2

Fig.3.Orthogonalpartialleast-squaresdiscriminantanalysisscoreplotof31speciesofthesubtribeEspeletiinaebasedonpercentageofessentialoilcompositions;Clusters

1–3,determinedbyhierarchicalclusteringanalysiswithbootstrapresampling,astheYvariable.Discriminatingvariablesofeachclusterinorderofrelevanceasfollows,

Cluster1:␣-pinene,␤-pinene,␤-phellandrene;cluster2:␣-phellandrene␣-thujene,p-cymene,andsabinene;cluster3:germacreneDlimonene,␤-caryophylleneand

ent-kaur-16-en-al.

Conclusions

This research represents the first comprehensive study to

reportthechemical compositionof aEspeletiaspecies by

mod-ernandcomplementaryanalyticaltechniques,followedbyproper

chemometric analyses. The similarities observed between the

chemicalfingerprintsofE.grandifloraandE.killipiiagreewiththeir

morphologicalandphylogeneticrelatedness.However,important

qualitative and quantitative differences were identified in the

leavesand resins of both species, which suggest that chemical

markers can be potentially used to assist in their taxonomy.

Among the fifteen dereplicated compounds, ten correspond to

newreportsforeitherorbothspecies,whereaseightcorrespond

to new reports for the genus Espeletia and subtribe

Espeleti-inae,indicating thatthesecondarymetabolitechemistry ofthis

groupofplantsremainslargelyunexplored.Thisstudymoreover

afforded the identification of different trends in the

accumu-lation of EOs from 31 species of Espeletiinae, which seem to

correlate withtheirtaxonomy and thusoffer abasis for future

(7)

Authorscontributions

GFPG(Ph.D.student)contributedin collectingplantsamples

andidentification,runningthelaboratoryexperiments,analysis

ofthedataanddraftedthepaper.JAAMcontributedtolaboratory

workandcriticalreadingofthemanuscriptandFBDCdesignedthe

experiments,supervisedthelaboratoryworkandcontributedto

criticalreadingofthemanuscript.Alltheauthorshavereadthe

finalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsgratefullyacknowledgeCAPESandFAPESP(grant

#2014/17702-0)forfinancialsupport.GFPGthanksDr.Mauricio

Diazgranados,M.Sc.SandraL.Casta˜nedaandMr.CarlosI.Suarez

fromJBBfortheirvaluablehelpcollectingplantmaterialand

dur-ingbotanicalidentificationsandMrs.NubiaParraforhertechnical

assistanceinconfectionofherbariumvouchers.Specialthanksto

Prof.Dr.NorbertoP.Lopes(FCFRP-USP),forexperimental

labora-toryfacilitiestoperformGC-MSanalyseswithtechnicalsupportof

Mrs.IzabelC.C.Turatti,andtoM.Sc.GariCcana-Ccapatinta

(Aster-BioChem,FCFRP-USP)forenlighteningdiscussions.

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