Metabolic Profiling of Pyrrolizidine Alkaloids in Foliage of Two Echium spp. Invaders in Australia

17 Pages·2015·2.99 MB·English

Article
Metabolic Profiling of Pyrrolizidine Alkaloids in
Foliage of Two Echium spp. Invaders in
Australia—A Case of Novel Weapons?
DominikSkoneczny1,PaulA.Weston1,XiaochengZhu1,GeoffM.Gurr1,2,
RaganM.Callaway3andLeslieA.Weston1,*
Received:7September2015;Accepted:26October2015;Published:6November2015
AcademicEditor:UteRoessner
1 GrahamCentreforAgriculturalInnovation,CharlesSturtUniversity,WaggaWagga,NSW2678,
Australia;[email protected](D.S.);[email protected](P.A.W.);[email protected](X.Z.);
[email protected](G.M.G.)
2 InstituteofAppliedEcology,FujianAgriculture&ForestryUniversity,Fuzhou350002,China
3 DivisionofBiologicalScience,UniversityofMontana,Missoula,MT59812,USA;
[email protected]
* Correspondence:[email protected];Tel./Fax:[phone]
Abstract:Metabolicprofilingallowsforsimultaneousandrapidannotationofbiochemicallysimilar
organismal metabolites. An effective platform for profiling of toxic pyrrolizidine alkaloids (PAs)
and their N-oxides (PANOs) was developed using ultra high pressure liquid chromatography
quadrupole time-of-flight (UHPLC-QTOF) mass spectrometry. Field-collected populations of
invasive Australian weeds, Echium plantagineum and E. vulgare were raised under controlled
glasshouse conditions and surveyed for the presence of related PAs and PANOs in leaf tissues at
various growth stages. Echium plantagineum possessed numerous related and abundant PANOs
(>17) by seven days following seed germination, and these were also observed in rosette and
flowering growth stages. In contrast, the less invasive E. vulgare accumulated significantly lower
levels of most PANOs under identical glasshouse conditions. Several previously unreported PAs
werealsofoundattracelevels. Field-grownpopulationsofbothspecieswerealsoevaluatedforPA
productionandhighlytoxicechimidineN-oxidewasamongstthemostabundantPANOsinfoliage
ofbothspecies. PAsinfieldandglasshouseplantsweremoreabundantinthemorewidelyinvasive
species,E.plantagineum,andmayprovidecompetitiveadvantagebyincreasingtheplant’scapacity
todeternaturalenemiesinitsinvadedrangethroughproductionofnovelweapons.
Keywords: metabolomics; UHPLC Q-TOF; Paterson’s curse; E. plantagineum; E. vulgare;
plantdefense
1. Introduction
Metabolitesaretheendproductsofgeneexpressionandareareflectionofanorganism’sstate
at a particular moment in time [1–3]. Metabolites directly reflect the interaction between a plant’s
environment and its genome, and metabolic profiling allows further investigation of the impact of
plant phenology and biotic or abiotic stressors on plant phenotype [2–4]. Metabolic profiling is a
techniquethatgeneratesauniquechemicalfingerprintforeachsampleandallowstheidentification
of diverse set of plant secondary products (PSPs) in plant extracts [3,5]. PSPs are defined as
metabolites of plant origin that are not related to housekeeping functions of an organism [3,6].
Metabolic profiling most often focuses on compounds of similar origin or chemical properties [7],
andcanbeperformedusingbothtargetedanduntargetedapproaches[3]. Bioinformaticsperformed
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Int.J.Mol.Sci.2015,16,26721–26737
tointerpretdatageneratedbymetabolicprofilingresultsinanimprovedunderstandingoftheplant’s
responsetovaryingenvironmentalconditionsatthebiochemical,cellularandorganismallevel[4,8,9].
PSPs serve in plant defense against herbivores, microbes and competing plants [6]. PSPs may
alsoplayacrucialroleinplantinvasionasphytochemicalweaponsservingtoinhibitorganismsthat
are not evolutionarily adapted to detoxify or cope with such novel compounds [10,11]. In native
ecosystems,herbivores,microbesandotherplantshaveco-evolvedandtypicallyadaptedtoexisting
plant defense mechanisms by detoxification of toxic plant constituents [12]. Naive organisms in
non-native ecosystems, however, did not co-evolve with endemic pathogens and herbivores and
therefore the PSPs produced by invaders can be highly toxic to native species. In the introduced
range,PSPsmaythereforeactasphytochemicallyunique“novelweapons”[10,11,13–15].
Alkaloids are a family of complex PSPs that often contribute to plant constitutive defense
mechanisms. Pyrrolizidine alkaloids (PAs), named for their characteristic double pyrrole ring
structure (Figure 1), are interesting from an ecological perspective and are commonly found in the
Asteraceae,Boraginaceae[16,17],Fabaceae[17]andOrchidaceae[18]. AlthoughthechemistryofPAs
hasbeenwelldocumentedandover350structuresarecurrentlyidentified, theirbiosynthesisisnot
alwayswelldescribed. Ourcurrentunderstandingoftheirbiosynthesisisgenerallybasedonseveral
well-decribed examples [16,18]. Hartman and co-authors (1999) have presented one of the most
completereviewsofthebiosythesisofPAsintheBoraginaceae.ThebackboneofPAstructureconsists
ofahydroxylatednecinebaseformingtwofive-memberedpyrroleringswithabridgeheadnitrogen
atomatposition4(Figure1)[19]. Thenecinebaseoriginatesfromprimarymetabolites, specifically
L-amino acids converted to homospermidine, the first specific PA precursor [12,16]. Necine bases
areesterifiedwithnecicacids,usuallyderivedfromaliphaticaminoacidsthataretransformedtoC
7
aliphathicacids[12].
PAs possess 1–2 unsaturated bonds which contribute to their toxicity; however, they are often
stored in a non-toxic N-oxide (PANO) form in the plant or in an adapted insect. After foliar
tissues are ingested by mammals and metabolized, oxidation can be reversible. PAs undergo
a three-step metabolic process in hepatocytes to form tertiary alkaloids (free bases, pyrrols) that
directly induce toxicity [17,18]. Pyrrole derivatives of PAs are highly reactive and can alkylate
DNA and/or induce formation of DNA cross-linkages which interrupt DNA replication and incur
mutations [20]. PAs may be hazardous, especially for non-ruminant animals, and the consumption
of high quantities of foliage can lead to occasional death of cattle, horses and sheep [21]. Toxicity
hasalsobeenobservedinpigs[22]andratswhentheirdietwassupplementedwithtoxicPAs[21].
Numerous citations have reported that the presence of PAs in the human diet can lead to severe
healthproblemsespeciallyifconsumedinlargequantities[17,23–25]. PAsareassociatedwithweak
antileukemic and virustatic activity, carcinogenicity, embryotoxicity, hepatotoxicity, mutagenicity,
and pneumotoxicity [19]. In addition to detrimental effects in mammalian systems, PAs can act as
toxins to insect herbivores [26] as insect feeding deterrents [12,27] or stimulants for specialist insect
herbivores[27].
Echium plantagineum L. and E. vulgare L. (Borgainaceae) produce a diverse suite of PSPs
including toxic PAs, which accumulate primarily in foliar tissues [20,28,29], and root-produced
naphthoquinones localized in living roots [28,30,31]. These species became exotic invaders in
Australiashortlyaftertheirintroductioninthe1800s. IntheirnativerangeoftheIberianPeninsula,
thesespeciesareuncommonandgenerallyfoundindiversemixtureswithotherplantflora[32,33].
In Australia, E. plantagineum, also known as Paterson’s curse or Salvation Jane, is an annual weed
now naturalized over 30 M ha and dominates roadside plant communities, grazing lands and
pastures [31,34,35]. Echium vulgare, or Viper’s bugloss, is a perennial and is limited to areas of
higher rainfall and elevation in southern Australia where it typically does not have major impacts
onAustralianagriculture[33]. Indirectcontrast,theprevalenceandtoxicityofE.plantagineumhave
significantly impacted Australia’s textile and meat industries, causing an estimated annual loss of
AUD$250Mandreducedpasturequality[36].
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This study aimed to develop a useful platform for rapid and simultaneous profiling of
PAs and PANOs in plant extracts using ultra high pressure liquid chromatography quadrupole
time-of-flight (UHPLC-QTOF) mass spectrometry. This sensitive platform was used to study
Australian populations of two related Echium species, more or less invasive, in both field and
controlled glasshouse conditions. We compare the invasive statusof these two species with respect
totheproductionofPAsassociatedwithplantdefenseandherbivoretoxicity.
2. ResultsandDiscussion
2.1. ProfilingofPyrrolizidineAlkaloidsfromFoliage
High-speed,automatedextractionoffoliartissueyieldedhighquality,uniformextractsthatwere
furtherpurifiedusingsolidphaseextraction(SPE)toreducesamplematrixcomplexityforprofiling
alkaloids of interest. Concentrated samples contained alkaloids, specifically 20 more abundant
compounds along with trace quantities of additional constituents. The UHPLC-QTOF MS method
coupled to bioinformatics and statistical analysis along with the integration of published data on
similar pyrrolizidine alkaloids allowed for annotation of 17 PAs and PANOs (Table 1; Figure 2)
detected in extracts of foliar tissues of E. plantagineum. Earlier reports indicated the presence of
five[37],twelve[20,28]orfourteenPAsinfoliartissuesofthisspecies[29]. Theautomatedextraction
process reduced leaf extraction time from 48 h [29] to 27 min. The use of ultra-high pressure liquid
chromatography, coupled to sensitive and accurate LC/MS QTOF detection, allowed for improved
separationofpyrrolizidinealkaloids, particularlystereoisomersorothercloselyrelatedcompounds
(putative stereoisomers were referred to as compound A and B; Table 1; Figure 2). Seventeen PAs
andPANOs(Table1)wereidentified, mainlybasedonretentiontime(RT)andaccuratemass(AM)
asreportedbyColegateandco-authors[29]whoidentified14relatedstructuresin2005(Table1)in
E.plantagineum. TheseauthorsalsoanalysedPAswithHPLC/MSusingauthenticatedstandardsin
positiveionizationmode.
Detection of PAs was performed in the past in different matrices, including honey, which
required the use of advanced clean up procedures in addition to SPE. Several techniques were
successfully used for analysis and identification of PAs including UV-Vis, NMR, HPLC [38], TLC,
UHPLC-MS, and GC-MS [17,38]. Griffin et al. [39] developed a UHPLC ion trap MS method for
analysis based on commercial standards available that allowed for identification of 11 PAs during
a 30-min run. The constituents analysed by the present authors in plant extracts were less polar
as they were not in the oxidized (PANO) form and typically eluted with higher concentrations
of organic solvent (>20%) later in the gradient. In contrast, the method developed in this study
allowed the annotation of 17 related PAs and PANOs of relatively high polarity during 18-min
runs. MetabolicprofilingofalkaloidsusingUHPLC-QTOFMSwasalsoreportedbyJaiswaletal.[5],
who successfully identified 48 compounds including diterpene alkaloids in three separate plant
species, and Zhuetal.[23], who developed the UHPLC-QTOF MS methodology for profiling of
retronencine-typePAsinherbaceousplantextractswithoutcorrespondingstandards.
Inadditionto17knownPAsandPANOs,18additionalPAswerecharacterizedinE.vulgarein
a previously reported study (Table S1) [20]. In our study, masses corresponding to several of the
same PAs identified previously in E. vulgare were present in trace quantities or were completely
absent in analysed extracts. Structural similarity and the presence of related compounds with
identicalmasses(TableS1)madeidentificationofallPAsrelativelydifficult. Structuralconfirmation
ofrelatedPAshaspreviouslyrequiredtheuseofothertechniquesforidentificationincludingNMR
or GC-MS [20]. Although this study focused mainly on separation and profiling of compounds
previously identified in E. plantagineum, structurally similar alkaloids were indeed present in both
species,suggestingsimilarityinbiosyntheticpathwaysamongrelatedEchiumspecies. Interestingly,
we detected ions of low abundance corresponding to the presence of intermedine, lycopsamine,
acetyllycopsamine/intermedine, echiumine and 3’-acetylechiumine N-oxides in E. vulgare samples.
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ThesePANOswerenotpreviouslyreportedinE.vulgare(TableS1). Thestructuralsimilarityofless
well-described PAs observed in extracts of both Echium species further suggests that the LC/MS
InQt.I nJT.t .OM J.Fo Ml. soSylc. isS. tc2ei0.m 1250,1 w156,e ,1 p6e,a mpgeap–gpelo–apgyaeeg de was highly sensitive, but this sensitivity does not negate the need for
useofNMRforfinalstructuralconfirmationofthosealkaloidspresentintracequantities.
When honey from Paterson’s curse was analysed, two additional PAs were reported using
WWhheenn hhoonneeyy frfroomm PPaateterrssoonn’s’s ccuurrssee wwaass aannaalylysseedd,, ttwwoo aaddddiittiioonnaall PPAAss wweerree rreeppoorrtetedd uussiningg
GC-MS analysis following the derivatization of purified honey extracts [40,41]. Constituents included
GGCC-M-MSSa annaalylyssisisf ofolllolowwininggt htheed deerirvivaatitzizaatitoionno offp puurirfiifeieddh hoonneeyye exxtrtaracctsts[ 4[400,4,411].].C Coonnsstittiutueenntstsi ninccluluddeedd
3’-acetyl derivatives of intermedine and lycopsamine [40,41]. Recently, the use of LC-MS plus
33’-’a-acceetytyll ddeerirvivaatitviveess ooff inintetremrmededinineea nanddl ylcyocpopsasmaminiene[ 4[04,04,14]1.]. RReecceennttllyy,, tthhee uussee ooff LLCC-M-MSS ppluluss
targeted analysis with authenticated standards has resulted in identification of numerous
tatargrgeetetedda naanlyaslyisswis ithwaituht haeunttihceantetidcastteadn dasrtadnsdhaarsdrse suhlatse driensiudletendti fiicna tiiodneonftinfiucmatieorno usocf omnupmouenroduss
compounds at trace levels; however, not all of these compounds were present in extracts of foliar
actotmrapcoeulnevdesl sa;t htroawcee vleevr,elns;o thoawlleovferth, ensoet caollm opf otuhnesdes cwomerpeopurnedsesn wt einre epxrterascetnst oinf feoxltiraarcttsis souf efsoloiafr
tisEts.iuspselusa enostf a ogEfi. n Epe.lu apmnlatan[g2ta8ing,2ein9ue]m.u m[N2 [8o2,2n89,-2t]a9. r]N.g Neotnoe-dnta-traagnregateleytdes dias naoanflaylpsyiussir soi fifo efp dpuureirfxiifteirdead ce texsxtrtoarfacctEsts .o opf flEa En. .tp aplgalaninntteaauggimnineeuruemmsu rrletesesudullttieendd
int hitnhe etdh deeet detececttiteoicontnio oonff oaafcc eaetctyyelltiiynnltitenerrtmemrmeeddeiidnnieen eiinn i nllee laaeffa eef xxettxrrtaarccattcsst..s T.T Thhhiissi smm meetethhthoooddd ss uusucccccceeesssssfsfuufulllllyyly aa allllloolowwweeeddd f fofoorrr r rraaapppiididd a aannnddd
sismsimiumlutulatlnataennoeeuoousu spspr porrofiofilfilinilnigng ogof o fPfP APAsAs oso fof kfk nknonowowwnn noo roriirggiiginni nbb ubuutt taa lalssloso oaa lalllloolowwweededd ff ooforr raa addddddiittiiitooionnnaaall ldd daaattataa mm miininniininnggg t ththhrrrooouuuggghhh
unuutnantratgaregrgteeetdeted adna ananlayalyslyisssi siasas asssossocoicaciatiaetetded dww witihtiht htht htehe emm meetetaatbabobololiislsimsmm oo fof fPP PAAAss suu ussisinninggg aa a ss hshhooorrtrt trr ruuunnn tt iitmmimeee oo offf aa apppppprrroooxxxiimimmaaatteteellylyy
202 20m0m imni.ni n..

Figure 1. Structure of echimidine (image modified from [42]), the most abundant pyrrolizidine
FigFuigreu r1e. S1.t rSutcrutucrteu roef oefc hecimhiimdiidnein (ei m(imagaeg me modoidfiiefide dfr formom [4 [24]2)], )t, hteh em mosots ta babuunnddaannt tp pyyrrrorolilzizididininee aallkkaallooiidd
alkaloid (PA) in its N-oxide form in Echium plantagineum and E. vulgare. The unsaturated bond
(PA(P)A i)n iint si tNs -Nox-oidxeid feo rfomr min i nE cEhciuhimu mp lpanlatnatgaignienuemum a nadn dE E. .v uvluglagraer.e .T Thhe eu unnsasatuturaratetedd bboonndd bbeettwweeeenn
between positions 1 and 2 induces toxicity. Reversible oxidation of nitrogen at position four alters
popsiotisoitnios n1s a1n adn 2d i2n dinudcuecse tso txoicxiitcyit. yR. eRveevresribsilbel eo xoixdiadtaiotino no fo fn nitirtoroggenen a at tp poosistiitoionn f ofouurr a altleterrss cchheemmiiccaall
chemicalproperties. Sidechainsatpositions1and7varybetweenanalyzedalkaloids. Athorough
properties. Side chains at positions 1 and 7 vary between analyzed alkaloids. A thorough review of
properties. Side chains at positions 1 and 7 vary between analyzed alkaloids. A thorough review of
reviewofPAswaspublishedpreviouslyandisreferredtoforadditionalinformation[20].
PAs was published previously and is referred to for additional information [20].
PAs was published previously and is referred to for additional information [20].
Figure 2. Cont.
Figure 2. Cont.
Figure2.Cont.
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Int. J. Mol. Sci. 2015, 16, page–page
Figure 2. Overlaid total ion chromatograms (TIC) of 12 composite extracts (each represented by
Figure 2. Overlaid total ion chromatograms (TIC) of 12 composite extracts (each represented by
ddififfefererennt tccoololorr)) ooff ((aa)) EEcchhiiuumm ppllaannttaaggiinneeuumm aanndd ((bb)) EE.. vvuullggaarree,, ggrroowwnn uunnddeerr iiddeennttiiccaall ccoonntrtroollleledd
gglalassshshoouussee ccoonnddititiioonnss.. NNuummbbeerrss 11––1177 ddeennoottee PPAAss aanndd PPAANNOOssp prroofiflieleddi nint hthees tsutuddyy( T(aTbalbele1 )1.).
22.2.2. .AAcccucummuulalatitoionn oof fPPAAss aanndd PPAANNOOss iinn tthhee FFiieelldd CCoonnddiittioionnss
TThhee PPAAss aanndd PPAANNOOss ooff ggeeooggrraapphhiiccaallllyy ddiissttiinncctt ppooppuullaattiioonnss ooff EE.. ppllaannttaaggiinnuueemm aanndd EE.. vvuulglgaarere
susurvrveeyyeedd aanndd ssaammppleledda carocrsossNs SNWSiWnd iicnadteicdarteelda tirveellaytilvitetllye vlaitrtilaet iovnariniaptiroond uinct iopnroodfuPcAtiso(nF igouf reP3A)s
(Faimguorneg 3p)o apmuolantigo npsopofuEla.tpiloannst aogfi nEe.u pmladnetasgpiinteeuthme idrecsopllietec ttihoenirfr coomlledcitvieorns efrcolimm adtiivcezrosne ecsl;iminaatdicd zitoionnes,;
ins oamdeditviaornia, tsioomnei nvaprliaantitonm ionr pphlaonlot gmyowrpahsoolobgsyer wveads oabmsoenrvgedp laamntopnogp pullaantito pnosp(ui.lea.,tiopnlasn (ti.eh.,e ipglhatn,t
hleeiagfhtn, ulemabf enru, mflobwere, rflocowloerr) c.oloAr).l oAn gloitnugdiitnuadlinsaulr vsueyrvepye rpfoerrmfoerdmeadcr aocsrsosths ethset astteasteos fofN NewewS Soouuthth
WWaaleless, ,VViiccttoorriiaa aanndd AAuussttrraalliiaann CCaappiittaall TTeerrrriittoorryy iinn AAuussttrraalliiaa bbyy WWeessttoonn eett aall.. [[2288]] ffoouunndd ssigignnifiifcicaanntt
ppoosistiitvivee ccoorrrreelalattiioonn bbeettwweeeenn llaattiittuuddee aanndd pprroodduuccttiioonn ooff PPAANNOOss.. HHoowweevveerr,, tthhee ccoorrrreellaattioionnw waassn noott
sisgignnifiificcaannt tfoforr eelelevvaattiioonn oorr lloonnggiittuuddee.. IInn aaddddiittiioonn ttoo 2200 ssaammpplleedd ppooppuullaattiioonnss ooff EE.. pplalannttaagginineeuummi nin
ththisi sexepxpereirmimenent,t w, we aelsaols ioncilnucdluedd esdamspamlesp olefs Eo. vfuElg.avrue ltghaarte wtheraet cwolelreectecdol flerocmted arferaosm ofa hriegahseor felhevigahtieorn
(Telaebvlaet iSo2n). (ATa sbulervSe2y) .of Afiesludr svietyes oifn fi2e0l1d3–s2it0e1s4 isnh2o0w1e3d–2 c0o1n4sisdheorwabedle cdoifnfseirdeenrcaebsl,e wdiitfhfe frieenldce ssa,mwpitlhes
sefipealdrastaemd pinletso scelpuasrtaetresd biyn tsopcelcuiestse r(Fsibgyursep e3c)i.e Psr(oFfiigliunrge 3s)h.oPwreodfi licnognssihdoewraebdlec ovnasriidaetiroanb lienv ParAiast iaonnd
PiAnNPAOss ainnd EP.A vNulOgasrein, aEn.dv ullegpatrae,nathnidnel-eNp-toaxnitdhein aen-Nd- o7x-Oid-eacaentydll7y-cOo-pascaemtyilnlye/cionptesrammeidnein/ei ndteertmeceteddin ein
Ed. eptlaenctteagdinineuEm. pwlaenrtea gnionte udmetwecetered ninot Ed.e vteuclgteadrei nsaEm.pvulelsg.a rOenslaym fpoluers .fiOelndl ysiftoeus rofif eEld. vsuitlegsaroef iEn.fevsutlagtairoen
winefrees itdaetinotnifiweder aenidd ecnotllieficetdeda nacdrocsosll Necetewd Saocuroths sWNaelews aSnodu tAhuWstarlaelsiaann CdaApiutastlr TaelirarnitoCrayp dituael tToe rlirmitoitreyd
adnude/otro spliomriatdedic aensdta/bolirshspmoernadt iocf etshtiasb slipsehcmieesn itno Af uthsitsraslpiae c[i3e3s].i nFoAliuars teraxltiraac[t3s3 ]o.f bFootlhia rspeexctireasc tws eorfe
ribcoht hin sepcehciimesidwineer eanrdic hecihniuemchinime Nid-ionxeidaensd, kencohwiunm fionre thNe-ior xpiodteesn,t ktonxoiwcinty ftoor gtrhaeziirngp ohteernbtivtooxreicsi. tLyetaost
agbruanzdinagnt hecrobmivpooruesn.dsL eians tbaobtuhn dsapnetciceosm ipnocluunddesd inlebpottahntshpinecei-eNs-oinxcidlued eadndle p7t-aOn-tahcinetey-lNly-cooxpidsaemainnde/
in7t-eOrm-aecedtiynlel,y wcohpiscahm wineree/ innotte rdmeteedcitnede, inw Eh.i cvhulgwaerer.e Onvoetradlle, tEec. tveudlgianre Ep.rovduulgcaerde. upO tvoe rfoalul,r-Efo.ldvu llogwareer
toptraold cuocnecdenutrpattioonfso oufr -PfAolsd alnodw PeAr NtoOtasl thcoannc Een. ptrlaatnitoangsinoeufmP A(Fsigaunrde 3P)A. NOs than E. plantagineum
(Figure3).
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Table 1. Chromatographic and spectrometric properties of related pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) profiled by ultra high pressure
liquid chromatography quadrupole time-of-flight (UHPLC-QTOF) mass speS. Compounds were annotated based on m/z of molecular ions and retention time
(RT) in relation to results published previously [29]. “A” and “B”—Denote potential isomers; AM—Accurate mass; * Denotes compounds which could be
intermedinederivatives.
No. Name AM Calculated[M+H] Measured[M+H] ∆ppm Approx.RT Formula
1 Leptanthine-N-oxide 331.1631 332.1704 332.1701 0.90 1.08 C H NO
15 25 7
2 Echimiplatine-N-oxide 331.1631 332.1704 332.1701 0.90 1.27 C H NO
15 25 7
3 Uplandicine-N-oxide 373.1737 374.1809 374.1808 0.27 1.91 C H NO
17 27 8
4 Intermedine-N-oxide 315.1682 316.1755 316.1754 0.32 2.35 C H NO
15 25 6
5 Lycopsamine-N-oxide 315.1682 316.1755 316.1753 0.63 2.54 C H NO
15 25 6
6 7-Angeloylretronencine-N-oxide 253.1314 254.1387 254.1384 1.18 3.42 C H NO
13 19 4
7 7-O-Acetyllycopsamine* 341.1838 342.1911 342.1907 1.16 5.30 C H NO
17 27 6
8 7-O-Acetyllycopsamine-N-oxideA* 357.1788 358.1860 358.1858 0.56 5.59 C H NO
17 27 7
9 7-O-Acetyllycopsamine-N-oxideB* 357.1788 358.1860 358.1854 1.67 5.95 C H NO
17 27 7
10 9-O-Angelylretronencine-N-oxide 253.1314 254.1387 254.1384 1.18 6.70 C H NO
13 19 4
11 Echimidine-N-oxideA 413.2050 414.2122 414.2121 0.24 8.12 C H NO
20 31 8
12 Echiuplatine-N-oxide 397.2101 398.2173 398.2170 0.75 8.18 C H NO
20 31 7
13 Echimidine-N-oxideB 413.2050 414.2122 414.2120 0.48 8.32 C H NO
20 31 8
14 3’-O-Acetylechimidine-N-oxide 455.2155 456.2228 456.2228 0.00 9.77 C H NO
22 33 9
15 Echiumine-N-oxideA 397.2101 398.2173 398.2172 0.25 10.20 C H NO
20 31 7
16 Echiumine-N-oxideB 397.2101 398.2173 398.2173 0.00 10.32 C H NO
20 31 7
17 3’-O-Acetylechiumine-N-oxide 439.2206 440.2279 440.2276 0.68 11.53 C H NO
22 33 8
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AA ffooccuusseedd eevvaalluuaattiioonn ooff lleeaaffs suurrffaacceec chheemmisistrtyryo offt htheseeset wtwoos pspeceiceisesa laslosos hsohwowededsi msimilairlarre rseuslutsl.ts.
EEcchhiiuumm ppllaannttaaggiinnuueemm lleeaaff ddiippss ttyyppiiccaallllyy ccoonnttaainineeddm moorreeP PAAssa nanddP APANNOOssin ing rgeraetaetrear baubnudnadnacnecteh tahnan
ddiidd EE.. vvuullggaarree;; lleeaaff ssuurrffaaccee ddiippss ddiiffffeerreedd qquuaanntittiatatitviveelylya annddq quuaaliltiattaitviveleyl,yw, withithe cehcihmimidiidnienbe ebineigngth tehe
mmoosstt aabbuunnddaanntt PPAAd deetteecctteeddi inne exxttrraacctstso offb boothths sppeeccieiess[ 4[433].].I nIno ouurrr erececnentts usurvrveye,yfi, efiledl-dc-oclolelcletecdtepdl panlatnsts
ooff bbootthh ssppeecciieess hhaadd llooww ccoonncceennttrraattiioonnsso offl eleppththaanntitninee-N-N-o-oxxidide,ew, whhicihchw wasasp rperveivoiuosulyslyfo fuonudndto tboe boen oene
ofthemostabundantalkaloidsdetectedinfieldsurveysacrossAustralia[28]. Differencescouldbe
of the most abundant alkaloids detected in field surveys across Australia [28]. Differences could be
associatedwithvariableweatherconditionsintheweekspriortocollection.
associated with variable weather conditions in the weeks prior to collection.

FFiigguurree 33.. VVaarriiaattiioonn iinn aabbuunnddaannccee ooff 1177 ppyyrrrroolliizziiddiinnee aalklkaaloloididss aannddt htheierirN N-o-xoixdiedses( T(aTbalbele1 )1i)n in
ccoommppoossiittee,, fifieelldd--ccoolllleecctteedd ssaammpplelesso offE Echcihuimump lpalnatnatgaignienuemuma nadndE .Ev. uvlgualrgea.re.H Hieirearracrhcihcaiclacl luclsutesrtienrging
aallggoorriitthhmm aanndd EEuucclliiddeeaann ddisistatanncceem meetrtircicw wereereu usesdedo nonn onromrmalaizliezdeda baubnudnadnacnecvea vluaelus.esD. eDnednrdorgorgamram
wwaass ggeenneerraatteedd uussiinnggM MaassssP PrroofiflielerrP Prorofefsessisoionnalasl osfotfwtwaraere(A (Agigleilnetn,tS,a SnatnatCa lCarlaa,raC, AC,AU, SUAS)A. ).
22..33.. GGrroowwtthh SSttaaggee aanndd GGeennoottyyppee--DDeeppeennddeennttA Accccuummuulaltaitoinono fofP APAsasn adndPA PNAONsOs
PPhheennoollooggiiccaall ssttaaggee oofftteenn iimmppaaccttss ththee eexxppreresssisoionno offc ocommpplelxexb iboisoysnytnhtehteicticp aptahtwhwayasysfo froPr SPPSsP.s.
IInnccrreeaasseedd pprroodduuccttiioonn oof fphpehneonloicli ccomcopmopuonudnsd hsash baesenbe reenporretpedor atet dthea tretphreodruepctriovdeu scttaigvee inst amgueltiinple
pmluanltti pgleenpelarna,t gaennde rah,iganhderh iPgShPe raPbSuPnadbaunncdea nwcaesw oafsteonft ecnocrorerrlaeltaetde dwwitihth aattttrraaccttiioonn ooffp oplolilnliantoatros,rs,
aann iimmppoorrttaanntt eeccoollooggiiccaall ffuunncctitoionn [4[444].]. TThhee pprroodduuccttiioonn ooff PPAAss aanndd PPAANNOOssv avraierisess esaesaosnoanllayllyin in
SSeenneecciioo [[4455]] aanndd aallssoo iinn EEcchhiiuumm,, wwiitthh ggrreeaateterr lelevveelslso offn naapphhththooqquuininonoenseso bosbesrevrevdedw withithin icnrceraesainsigng
ppllaanntt mmaattuurriittyy [[2288]].. IInn EE.. ppllaannttaaggiinneeuumm aanndd EE.. vvuulglgaarree,, rroooot-tp-prorodduucecded nanpahpthhtohqouqiunionnoensesw wereere
pprroodduucceedd wwiitthhiinn 4488 hhoouurrss ffoolllloowwiinnggg geerrmmininaatitoionna annddw wereerea laslosos isgingnifiifciacnatnlytlym moroeraeb aubnudnadnatnatt aptl panlatnt
mmaattuurriittyy [[3311]]..
After sampling at three different growth stages, PANOs were observed to be present in all
investigated populations of E. plantagineum an2d67 l2im7 ited variation between population and growth
7
Int.J.Mol.Sci.2015,16,26721–26737
After sampling at three different growth stages, PANOs were observed to be present in all
investigated populations of E. plantagineum and limited variation between population and growth
stageextractswasobserved(Figure4).Themostabundantcompoundsatrosetteandfloweringstages
were echimidine-N-oxide B (13) and echiumine-N-oxide B (16) while 31-O-acetylechiumine-N-oxide
(17) and 7-O-acetyllycopsamine/intermedine (7) were least abundant. Echiumine-related PAs and
Int. J. Mol. Sci. 2015, 16, page–page
PANOs were significantly downregulated with increasing maturity in all studied populations.
Similarlsyt,ag7e -eOx-traaccetsty wllaysc oopbssearmveidn e(F/iginutreer 4m).e Tdhine emoBst (a9b)u,n3d1a-nOt -caocmetpyoluenchdsim ati droinseette( 1a4n)d afnlodwelreinpgt hantine
(1) N-osxtaidgeess wwereer eechsiimgindiifinec-aNn-tolxyided Bo w(1n3)r eagnud laectheidum(ipne-<N-o0x.0id5e) Bo (v1e6)r wtihmilee .3′-OH-acoewtyleevcheiru,mtihnee-No-pposite
trend woxaisdeo (b17s)e arnvde d7-Of-oarcetryelltyrcoonpesnamciinnee/-irnetelarmteeddinPeA (7N) wOesr,e lweahsti cahbunwdeanret. Elcikhieulmyinree-aredlaitleyd iPnAtse racnodn verted
PANOs were significantly downregulated with increasing maturity in all studied populations.
to other structures such as retronencine, the structural backbone of multiple PANOs in Echium
Similarly, 7-O-acetyllycopsamine/intermedine B (9), 3′-O-acetylechimidine (14) and lepthantine (1)
plantaginuem[29]. Echimidine-N-oxideB(13)[40]wasalsosignificantly(p<0.05)upregulatedover
N-oxides were significantly downregulated (p < 0.05) over time. However, the opposite trend was
timewithplantmaturationandwasmostabundantatrosetteandfloweringstagecomparedtothe
observed for retronencine-related PANOs, which were likely readily interconverted to other
seedlingstrsutcatguereisn sutchhe ams raetjroorniteyncoinfep, tohpe ustlrautcitournasl b(aFcikgbuornee o4f) m. uNltiipnlee PoAuNtOosf isne Evcehniutme epnlanctoagminpueomu [n29d]s. varied
with grEocwhitmhidsitnaeg-Ne-,oxaindde Bp (o1p3)u [l4a0t]i ownasˆ algsor oswignthificsatnatglye (dp if<f e0r.0e5n)c uepsrewgeurlaetecdl eoavrelry tiombes ewrvitehd pl(aFnitg ure 5).
BiosyntmheastuisraotifoPnA ansda wndas PmAoNst Oabsuwndaasnta asts orocsieatttee danwd iftlhowpehrienngo sltoaggeic caolmsptaagreed, two itthhe sseigednliifingca sntatgde iifnf erences
the majority of populations (Figure 4). Nine out of seventeen compounds varied with growth stage,
in chemical profiles observed between plants of flowering and seedling stage, with rosette stage
and population × growth stage differences were clearly observed (Figure 5). Biosynthesis of PAs and
intermediate. Although plant populations were generally similar in abundance of PAs, several
PANOs was associated with phenological stage, with significant differences in chemical profiles
individualcompounds(1,2,3,9and13)respondeddifferentlyovertimewithrespecttopopulation
observed between plants of flowering and seedling stage, with rosette stage intermediate. Although
differenpcleasnt( Fpiogpuurleat4io;nTsa bwleerSe 3g)e.nerally similar in abundance of PAs, several individual compounds
Ap(1r,e 2v, i3o,u 9s arnedp o13r)t roenspPoAndseidn dSieffneerceinotljya coovbeare taim(sey wn.itJha croesbpaeecat vtou lpgoapriusl)astihoon wdieffdereelnecveas t(eFdigluerve e4l;s ofPAs
Table S3).
in the leaves at the flowering stage in comparison to the rosette stage, and floral extracts possessed
A previous report on PAs in Senecio jacobaea (syn. Jacobaea vulgaris) showed elevated levels of
thehighestconcentrations[17]. SeneciospeciesbiosynthesizePAsintheirroots[12],andthereforePA
PAs in the leaves at the flowering stage in comparison to the rosette stage, and floral extracts
foliar content may increase during transport to newly opened flowers. The location of biosynthesis
possessed the highest concentrations [17]. Senecio species biosynthesize PAs in their roots [12], and
of PAs tihnerEe.foprela PnAta fgoilniaeru cmonhteanst mnoaty binecerneasset uddurieindg itrnangsrpeoartt tdoe nteawill,yb ouptenitedh faloswbeeres.n Thsue glogceastitoend oft hat site
of prodbuicotsiyonnthvesairsi oefs PaAms oinn Eg. pslpanetcaigeisne[u4m6 ]h.asW neoth baevene sdtuedteiecdte idn gsriegant idfiectaainl,t bPuAt it choans tbeenetn isnugsgteesmteda nd leaf
extractst,hawt istihte coof npsrioddeurcatibolne vcaorinecs eanmtorantgi osnpescineos t[e46d]. oWne lheaavfes duertfeaccteeds .sigWnieficaalnsto PdAe cteocntteendt rine lsatteimve ly low
and leaf extracts, with considerable concentrations noted on leaf surfaces. We also detected relatively
concentrations of the three main alkaloids in root extracts as well, suggesting production or later
low concentrations of the three main alkaloids in root extracts as well, suggesting production or later
translocationtolivingrootsovertime.
translocation to living roots over time.

Figure 4. Relative abundance of 1–17 PANOs (Table 1) in extracts of five populations of
Figure 4. Relative abundance of 1–17 PANOs (Table 1) in extracts of five populations of
Echium plantagineum harvested at three growth stages in controlled conditions experiment.
Echium plantagineum harvested at three growth stages in controlled conditions experiment.
Hierarchical clustering was performed using MPP Software on normalized values and Euclidean
Hierarcdhiisctaanlccel umsetterricin. *g–dwenaosteps esrigfonrifmicaendceu osfi npgopMulaPtPionS, ogfrtowwathre stoagne naondrm thaeliirz iendtervaactliuoens (raenpdeatEedu clidean
distancemmeaseutrreics. A*N–OdVenAo; tpe <s 0s.i0g5n). iKfiecya ntoc peoopfulpaotiopnusl:a Bt–ioBnen,dgigroo,w CtMh—stCaogoemabnadh, tChRe—irCionbtearr,a GctGio—nG(orle peated
measureGsol,A WNWO—VWAa;gpga <Wa0g.g0a5 ()n.umKbeerys intdoicaptoe pbulolcakt ioof nresp:licBat–ioBne)n. digo, CM—Coombah, CR—Cobar,

GG—GolGol,WW—WaggaWagga(numbersindicateblockofreplication).
8
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Int.J.Mol.Sci.2015,16,26721–26737
Int. J. Mol. Sci. 2015, 16, page–page
EEcchhiiuumm pplalannttaagginineeuumm acaccucummuulaltaetdedm modoedreartealtyelyh ighhighle vleelvseolsf PoAf NPOAsNwOist hiwnitahinw eeak wafeteekr
gafetremr ingaetrimonin.ationA.c cAucmcuumlatuiolantioonf oPfA PsAos f olfo wlowa baubnudnadnacnecea tatt htehe seseededlinligng stsataggee,, ssuucchh aass
lleeppttaanntthhiinnee--NN--ooxxiiddee ((11)) aanndd 77--OO--aannggeellooyyllrreettrroonneenncciinnee ((66),), ininccrreeaasseedd oovveerr titmimee (F(Figiguurere4 )4.). TThhee
bbiioossyynntthheessiiss ooff PPAAss ooccccuurrrreedd rraappiiddllyy ppoosstt ggeerrmmiinnaattiioonn,, lliikkeellyy aass aa mmeeaannss ttoo pprrootteecctt tthhee ppllaanntt aanndd
ddeetteerr ggeenneerraalliisstt hheerrbbiivvoorreess [[2277]].. PPAAss aanndd PPAANNOOss ppootteennttiiaallllyy ccoonnttrriibbuuttee ttoo aaccttiivvee ppllaanntt ddeeffeennssee bbyy
ssuupppprreessssiinngg tthhee ffeeeeddiinngg ooff iinnsseeccttss aanndd ootthheerr hheerrbbiivvoorreess aass wweellll aass ssuupppprreessssiinngg cceerrttaaiinn ppllaannttss;; ttooggeetthheerr
wwiitthh aannttiimmiiccrroobbiiaall nnaapphhtthhooqquuiinnoonneess pprroodduucceedd iinn tthhee rroooott ppeerriiddeerrmm [[3311]],, tthheeyy ffoorrmm aa ssuucccceessssffuull
bbaarrrriieerr aanndd eennhhaannccee ppllaanntt rreessiilliieennccee ttoo ppaatthhooggeennss aanndd pprreeddaattoorrss bbootthh aabboovvee aanndd bbeellooww tthhee ggrroouunndd..

Figure 5. Principal component analysis (PCA) of controlled condition experiment, evaluated over five
Figure 5. Principal component analysis (PCA) of controlled condition experiment, evaluated over
populations of E. plantagineum. (a) PCA of 17 PAs and PANOs and growth stage as independent
fivepopulationsofE.plantagineum. (a)PCAof17PAsandPANOsandgrowthstageasindependent
variable. Component 1, 2 and 3 contribute separation by 23.39%, 21.04% and 12.45%, respectively.
variable. Component 1, 2 and 3 contribute separation by 23.39%, 21.04% and 12.45%, respectively.
Discriminating metabolites for component 1 (PCA loadings > 0.3): 6, 11, 13 and for component 2
Discriminating metabolites for component 1 (PCA loadings > 0.3): 6, 11, 13 and for component 2
(PCA loadings > 0.3): 5, 8, 16, 17. Key to plant stages: seedling, brown; flowering plants, red and
(PCA loadings > 0.3): 5, 8, 16, 17. Key to plant stages: seedling, brown; flowering plants, red and
rosette, blue; (b) PCA of 17 PAs and PANOs with population as an independent variable. Component
rosette,blue;(b)PCAof17PAsandPANOswithpopulationasanindependentvariable.Component
1, 2 and 3 contribute to separation by 23.39%, 21.04% and 12.45%, respectively. Key to populations:
1,2and3contributetoseparationby23.39%,21.04%and12.45%,respectively. Keytopopulations:
Bendigo, red; Coombah, brown; Cobar, blue; Gol Gol, grey and Wagga Wagga, green.
Bendigo,red;Coombah,brown;Cobar,blue;GolGol,greyandWaggaWagga,green.
Under a controlled glasshouse environment, variation in accumulation of PAs and PANOs was
Under a controlled glasshouse environment, variation in accumulation of PAs and PANOs
occasionally observed between populations (Figure 5). Six compounds were differentially expressed
was occasionally observed between populations (Figure 5). Six compounds were differentially
among populations. These included uplandicine-N-oxide (3), lycopsamine-N-oxide (5) and
expressedamongpopulations.Theseincludeduplandicine-N-oxide(3),lycopsamine-N-oxide(5)and
7-O-acetyllycopsamine/intermedine-N-oxide B (9) which were significantly downregulated (p < 0.05)
7-O-acetyllycopsamine/intermedine-N-oxideB(9)whichweresignificantlydownregulated(p<0.05)
in the Coombah population (Figure 4; Table S3). Interestingly, the Coombah population also
intheCoombahpopulation(Figure4;TableS3).Interestingly,theCoombahpopulationalsoexhibited
exhibited reduced shikonin biosynthesis in its roots in a separate study and these differences were
reduced shikonin biosynthesis in its roots in a separate study and these differences were attributed
attributed to possible genetic differences in PSP biosynthesis [31]. Leptanthine-N-oxide (1) was
to possible genetic differences in PSP biosynthesis [31]. Leptanthine-N-oxide (1) was significantly
significantly downregulated in seedlings of the Wagga Wagga population.
downregulatedinseedlingsoftheWaggaWaggapopulation.
2.4. Comparative Profiling of Echium Plantagineum and E. Vulgare
2.4. ComparativeProfilingofEchiumPlantagineumandE.Vulgare
Metabolic profiles of both Echium species in Australia were recently compared with respect to
Metabolic profiles of both Echium species in Australia were recently compared with respect to
production of PSPs, specifically quinone-containing shikonins in the roots. These studies revealed
production of PSPs, specifically quinone-containing shikonins in the roots. These studies revealed
that shikonin biosynthesis is highly conserved across species, with limited differences in metabolic
that shikonin biosynthesis is highly conserved across species, with limited differences in metabolic
profiles [31]. Five populations of each species were used in this evaluation of PAs and PANOs. Plants
profiles [31]. Five populations of each species were used in this evaluation of PAs and PANOs.
were harvested and extracted after 6–8 weeks at the rosette stage and at 27–29 weeks when
Plants were harvested and extracted after 6–8 weeks at the rosette stage and at 27–29 weeks when
E. plantagineum was fully flowering; interestingly, we observed in this study that E. vulgare did not
E. plantagineum was fully flowering; interestingly, we observed in this study that E. vulgare did not
flower and remained in the vegetative growth stage, likely due to its perennial growth habit and lack
of exposure to vernalizing conditions in the greenhouse [33].
26729
9
Int.J.Mol.Sci.2015,16,26721–26737
flowerandremainedinthevegetativegrowthstage,likelyduetoitsperennialgrowthhabitandlack
Int. J. Mol. Sci. 2015, 16, page–page
ofexposuretovernalizingconditionsinthegreenhouse[33].
Metabolicprofilingrevealedthat15outof17PAsorPANOsweresignificantlydownregulated
Metabolic profiling revealed that 15 out of 17 PAs or PANOs were significantly downregulated
in E. vulgare (p < 0.05) (Figure 6; Table S4); however, 7-Oacetyllycopsamine/intermedine (7)
in E. vulgare (p < 0.05) (Figure 6; Table S4); however, 7-Oacetyllycopsamine/intermedine (7) and
and uplandicine (3), lycopsamine (5), 7-angeloylretronencine (6), 9-O-angelylretronencine (10),
uplandicine (3), lycopsamine (5), 7-angeloylretronencine (6), 9-O-angelylretronencine (10),
echimidine A (11) and 3’-O-acetylechimidine (14) N-oxides were expressed differentially in both
echimidine A (11) and 3’-O-acetylechimidine (14) N-oxides were expressed differentially in both
species over time. Echimidine-N-oxide B (13) was the most abundant PANO in both species, and
species over time. Echimidine-N-oxide B (13) was the most abundant PANO in both species, and has
hasbeendescribedasresponsibleforacutealkaloidtoxicityinrats[40]. Echimidinewaspreviously
been described as responsible for acute alkaloid toxicity in rats [40]. Echimidine was previously found
found in high quantities in E. vulgare and E. setosum [47]. Rare or less common PAs observed
in high quantities in E. vulgare and E. setosum [47]. Rare or less common PAs observed included
included 7-O-acetyllycopsamine/intermedine (7) > leptanthine-N-oxide (1) > echiuplatine-N-oxide
7-O-acetyllycopsamine/intermedine (7) > leptanthine-N-oxide (1) > echiuplatine-N-oxide (12).
(12). 7-O-acetyllycopsamine/intermedine(7)was absent inE.vulgare whilelepthantine-N-oxide(1)
7-O-acetyllycopsamine/intermedine (7) was absent in E. vulgare while lepthantine-N-oxide (1) was
was found in only one sample of this species. Echiuplatine-N-oxide (12) was uncommon and was
found in only one sample of this species. Echiuplatine-N-oxide (12) was uncommon and was
identifiedinonesampleofeachspeciesofplantsgrownincontrolledconditions(Figure6). Variation
identified in one sample of each species of plants grown in controlled conditions (Figure 6). Variation
ininphenologyassociatedwithgrowthhabitmaybeimportantinregulatingPAexpression.
in in phenology associated with growth habit may be important in regulating PA expression.

FFiigguurree 66.. CCoommppaarirsiosonno foafb uabnudnadncaencoef soefl esceteledctpeydr roplyizrridoilnizeidailnkea loaildksalaoniddst haenird Nt-hoexiird eNs-ionxiEdcehsi uimn
pElcahnituamgi npleaunmtagainndeumE. avnudlg Ea.r veuplglaanret splgarnotws gnroinwnu ninif uonrmiforcmon cdointidointsionins itnh ethge lgalsasshsohuosues.e. HHiieerraarrcchhiiccaall
cclluusstteerriinngg aallggoorriitthhmm aanndd EEuucclliiddeeaann ddiissttaannccee mmeettrriicc wweerree uusseedd oonn nnoorrmmaalliizzeedd aabbuunnddaannccee vvaalluueess..
DDeennddrrooggrraamm wwaass ggeenneerraatteedd uussiinngg MMPPPP ssooffttwwaarree.. **--ddeennootteess ssiiggnniiffiiccaannccee ooff ppooppuullaattiioonn,, hhaarrvveesstt ddaattee
aanndd tthheeiirr iinntteerraaccttiioonn ((rreeppeeaatteedd mmeeaassuurreess AANNOOVVAA,, pp << 00..0055)).. KKeeyy ttoo ppooppuullaattiioonnss ((aatt ttoopp ooff ddiiaaggrraamm))::
PPAA,, EE..p lpalnatnatgaignienuemumfr ofmromAd Aamdainmabinya;bPyS;, EP.Sp, laEn.t apgliannetuamginfreoumm Sfirlovmer toSnil;vVeArto,En.; vVulAga, rEe.f rovumlgAardee lforonmg;
VACde,lEo.nvgu;l VgaCre, Efr.o vmulCgaoroe mfrao.mK Ceyootompal.a Knteyst taog epsl:anTt1 ,stfiargsetsh: aTr1v,e fsitr;sTt 2h,asrevceosnt;d Th2,a srveceostn.d harvest.
Metabolic profiles of both species were compared using a comprehensive personal compound
Metabolic profiles of both species were compared using a comprehensive personal compound
database (Table 1) based on accurate mass (AM) and retention time (RT) of compounds earlier
database (Table 1) based on accurate mass (AM) and retention time (RT) of compounds earlier
reported in E. plantagineum. Several compounds profiled in this study were found with high mass
reported in E. plantagineum. Several compounds profiled in this study were found with high mass
accuracy in both species and were not previously reported in E. vulgare extracts. However, as
accuracy in both species and were not previously reported in E. vulgare extracts. However, as
previously mentioned, the complexity and structural similarity of many PAs and PANOs
renders positive identification of metabolites a complex task, particularly for those present in trace
26730
quantities. The concentration of PANOs and PAs in E. vulgare was significantly lower in
glasshouse-grown plants when both species were produced under uniform conditions (Figures 2 and
10