UHPLC high resolution orbitrap metabolomic fingerprinting of the unique species Ophryosporus triangularis meyen from the atacama desert, Northern Chile

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

Original

Article

UHPLC

high

resolution

orbitrap

metabolomic

fingerprinting

of

the

unique

species

Ophryosporus

triangularis

Meyen

from

the

Atacama

Desert,

Northern

Chile

Mario

J.

Simirgiotis

a,∗

_,

_Cristina

_Quispe

b

_,

_Andrei

_Mocan

c

_,

_José

_Miguel

_Villatoro

a

_,

_Carlos

_Areche

d

_,

Jorge

Bórquez

e

_,

_Beatriz

_Sepúlveda

f

_,

_Carlos

_{Echiburu-Chau}

g,h

a_{InstitutodeFarmacia,FacultaddeCiencias,UniversidadAustraldeChile,Valdivia,Chile}

b_{InstitutodeEtnofarmacología,FacultaddeCienciasdelaSalud,UniversidadArturoPrat,Iquique,Chile}

c_{DepartmentofPharmaceuticalBotany,IuliuHat¸ieganuUniversityofMedicineandPharmacy,Cluj-Napoca,Romania} d_{DepartamentodeQuímica,FacultaddeCiencias,UniversidaddeChile,Santiago,Chile}

e_{DepartamentodeQuímica,FacultaddeCienciasBásicas,UniversidaddeAntofagasta,Antofagasta,Chile} f_{DepartamentodeCienciasQuímicas,UniversidadAndresBello,CampusVi˜nadelMar,Vi˜nadelMar,Chile} g_{CentrodeInvestigacionesdelHombreenelDesierto(CIHDE),Arica,Chile}

h_{FacultaddeCienciasdelaSalud,UniversidaddeTarapacá,Arica,Chile}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20May2016 Accepted28October2016 Availableonline24November2016

Keywords:

UHPLC

Orbitraphighresolutionmassspectrometry Flavonoids

Chileanplants

Ophryosporus

PaposoValley

a

b

s

t

r

a

c

t

High-resolutionmassspectrometryiscurrentlyusedtodeterminethemassofbiologicallyactive com-poundsinplantsandUHPLC-Orbitrapisarelativelynewtechnologythatallowsfastfingerprintingand metabolomicsanalysis.Inthiswork,severalphenoliccompoundsincludingelevenphenolicacids,two fattyacids,twochromonesandfourteenflavoneswererapidlyidentifiedinthemethanolicextracts ofaerialpartsandflowersoftheuniqueChileanspeciesOphryosporustriangularisMeyen,Asteraceae, growingintheAtacamaDesertbymeansofultrahighresolutionliquidchromatographyorbitrapMS analysis(UHPLC-PDA-OT-MS)forthefirsttime.TheUHPLC-MSfingerprintgeneratedcanbeusedforthe authenticationofthisendemicspecies.Themethanolicextractsoftheaerialpartsandflowersshowed also antioxidant capacities (65.34±1.32 and 52.41±1.87␮g/mlin theDPPH assay, 184.88±13.22 and196.80±13.28␮mol TE/gdryweightin theferric reducingpower assayand56.17±3.03and 65.41±1.96%inthesuperoxideanionscavengingassay,respectively).

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Ophryosporus isa genusof South Americanflowering plants

belonging to the sunflower (Asteraceae, order Asterales, tribe

Eupatorieae) family. The tribe Eupatorieae Cass has two genus

growing in Chile: Eupatorium and Ophryosporus. The genus

Ophryosporus Meyen belongs to the subtribe Critoniinae R. M.

King&H.Rob,including41SouthAmericanspecieswhichgrow alongthecordilleradelosAndesdistributedinArgentina,Brazil, Bolivia, Chile, Ecuador and Peru. The aerial parts (leaves and stems)ofspeciesbelongingtothisgenusproduceseveralphenolic compounds including flavonoids, chromenones, acetophenones, coumarins,chromenes,benzofurans,andterpenoidssuchas eudes-manolides,sesquiterpenesandentandseco-labdanediterpenoids

∗ _{Correspondingauthor.}

E-mail:[email protected](M.J.Simirgiotis).

whichareoftaxonomicalrelevance(BohlmannandZdero,1979; Bohlmann et al., 1984; Zdero et al., 1990; deLampasonaet al., 1997;Favieretal.,1997,1998;Sigstadetal.,1992,1993,1996; Barrero et al., 2006; Garneau et al., 2013). The essential oils from some of the species were also studied. For instance, the essentialoilsfromleavesandstemsofOphryosporuspiquerioides

show mainly(E)-nerolidol, caryophyllene oxide,campheneand

␤-caryophylleneasprincipalcomponents,whiletheessentialoils

fromO.hepthantusshowedmainlyprecoceneandp-cimene(Arze

etal.,2004).Furthermore,severalcompoundsisolatedfromthese species showinteresting biologicalactivities.For instance,from theacetoneextractofleavesfromO.axilliflorus,knownbythelocal nameofcharruainArgentina,sevenacetophenoneswereisolated which presented anti-inflammatory activity in a carragenan-inducedmousepawedemaassay(Favieretal.,1998)whilefrom thebutanolextractofleavesofthespeciesO.heptanthusfourteen compounds, including terpenoids and flavonoids were isolated which presented antioxidant activities(Barrero et al.,2006).In

http://dx.doi.org/10.1016/j.bjp.2016.10.002

180 M.J.Simirgiotisetal./RevistaBrasileiradeFarmacognosia27(2017)179–187

addition,theethanolicextractofleavesandstemsofO.peruvianus

showedantibacterialactivityagainstbacteriaandfungiincluding

Bacillussubtilis,Candidaalbicans,Trichophytonmentagrophytesand

Sporothrixschenckii(4.3,17,27and 13mmofgrowthinhibition

zone,in a diskbioassay) (Rojaset al.,2003).Paposois a small valley onthe Chilean coast about 50km of the town of Taltal and200kmsouthofthecityofAntofagasta.Coastalfogs inthis locationcalledlocallycamanchacasallowthegrowingofseveral speciesin this special ecosystem (Squeoet al.,1998)including

Ophryosporus triangularis Meyen, locally called rabo de zorro

(foxtail),inChile.Thisisauniqueendemicspeciesgrowingina relativelysmallareainnorthernChile,intheAtacamaDesert,inthe Nortegranderegion,fromAricatoCoquimbo.FromO.triangularis

growingin thesurroundings ofLaSerena,Chile,several benzo-furans,polymethoxylatedflavonesandchromeneswereisolated including the compounds 6-acetyl-2,2-dimethyl-chromen-4-one, 5,4-dihydroxy-6-methoxyflavone, 6,5,4-trihydroxyflavone, 6-acetyl-8-senecioyl-2,2-dimethylchromene, 5-acetyl-6-hydroxy-2-isopropenyl-benzofuran, and its O-methylated-derivative (Alarconetal.,1993.)amongothers.However,tothebestofour knowledge,nomedicinalusesorbioactivitieswerereportedsofar forthisspecies.

HPLChyphenatedwithmassspectrometryistodayakeytool intheanalysisofplantsamplesforchemotaxonomyormetabolic profiling.Amongtheinstrumentsinthemarket, theQ-exactive focusmassspectrometercombinesrapidultra-highperformance liquidchromatography(UHPLC)separation,diodearraydetection (PDAorDAD)andhighmassspectrometry(MS)performanceof anorbitaltrapwiththehelpof aquadrupoleand an outstand-ingdiagnostic powerusing a highenergy collision dissociation (HCD)cell, which delivers highresolution MSn _{fragments. This}

methodologyisusefulforthefastanalysisofsmallcompounds, including toxins, pesticides, phenolics and terpenes (Zhao and Jiang,2015;Martínez-Domínguezetal.,2016; Simirgiotisetal., 2016b;Yangetal.,2016).Inthecontinuingeffortsmadebyour laboratory in the search for new interesting metabolites from Paposo flora (Simirgiotis et al., 2015, 2016b) several phenolic compoundswererapidlydetectedinthemethanolicextractfrom flowersandaerialparts(leavesandstems)ofO.triangularis(Fig.1) usinghighresolution Q-orbitraptechnologycoupledtoan PDA detector.Themethanolextractsfromflowersandaerialpartswere alsosubmitted to antioxidant activity assays (DPPH, FRAP and superoxideanionscavengingassays),plusmeasurementsoftotal phenolicandflavonoidcontentbyspectroscopy.

Materialandmethods

Chemicalsandplantmaterial

HPLC–MS Solvents, LC–MS formic acid and GR acetonitrile and methanol were purchased from Merck (Santiago, Chile). Ultrapure water was obtained from a Millipore water purifi-cation system(Milli-QMerck Millipore,Chile).HPLC standards, Isorhamnetin, caffeic acid, quercetin-3-O-glucose, p-coumaroyl galactose(allstandardswithpurityhigherthan95%byHPLC)were purchasedeitherfromSigma–Aldrich(SaintLouis,MO,USA), Chro-maDex(Santa Ana, CA,USA), orExtrasynthèse(Genay, France). Folin–Ciocalteuphenolreagent(2N),reagentgradeNa2CO3,AlCl3, HCl,FeCl3,NaNO2,NaOH,quercetin,trichloroaceticacid,sodium acetate,gallic acid, 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ),Trolox, nitrobluetetrazolium,xanthineoxidaseandDPPH (1,1-diphenyl-2-picrylhydrazyl radical) were purchased from Sigma–Aldrich ChemicalCo.

ThreedifferentspecimensofOphryosporustriangularisMeyen, Asteraceae(aerialpartsandflowers)werecollectedinQuebradade

Fig.1. PictureofoneherborizedsampleofOphryosporustriangulariscollectedin QuebradadePaposo,AtacamaDesert,inApril2011.

PaposoinJune2011(geographicalcoordinates:lat:25◦0794S, long:70◦2727W,forspecimenI,25◦0048S,long:70◦2681W forspecimenIIand25◦0952S,long:70◦2630Wforspecimen III).ThesampleswereidentifiedbythebotanistAliciaMarticorena fromtheUniversityofConcepción,Chile.Voucherherbarium speci-mensarekeptattheNaturalProductsLaboratoryoftheUniversidad deAntofagastaunderreferencenumber:OT110403-12, OT110403-13andOT110403-14.

Extraction

Dried aerial parts (leaves and stems) and flowers of O.

tri-angularis(100mg each,for thethreedifferentspecimens)were

extracted separately withmethanol for 30min withsonication (5ml)inordertoobtainanextract.Theextractswereimmediately concentrated invacuobelow 40◦Ctogive tworesulting brown syrups for each sample (10.3 and 15, 11.2 and 17 and 13.24 and17.4mg,foraerialpartsandflowersofsamplesI,IIand III, respectively).

UHPLC-DAD-MSinstrument

TheequipmentusedwasaThermoScientificDionexUltimate 3000UHPLCsystemhyphenatedwithaThermoQexactivefocus machine.Fortheanalysisthepreparedextractswerefiltered(PTFE filter)and10␮lwereinjectedintheinstrument,withall specifica-tionssetaspreviouslyreported(Simirgiotisetal.,2016b).

LCparametersandMSparameters

Scientific,Bremen,Germany)asreportedpreviously(Simirgiotis etal.,2016b).Thedetectionwavelengthswere254,280,330and 354nm,andDADwasrecordedfrom200to800nmforpeak char-acterization.Mobilephaseswere1%formicaqueoussolution(A) andacetonitrile(B).Thegradientprogramwas:5%to30%solvent Bfrom5to10min,then,maintaining30%Bfor5min,thento70% B5minmore,thenmaintaining70Bin5moremin,thenreturning to5%Bin5moreminand12minforcolumnequilibrationbefore eachinjection.Theflowratewas1mlmin−1_{,andtheinjection} vol-umewas10␮l.TheHESIIIandOrbitrapspectrometerparameters wereoptimizedaspreviouslyreported(Simirgiotisetal.,2016b).

Antioxidantassays

DPPHFreeradicalscavengingactivity

TheDPPHfreeradicalscavengingactivitywasdeterminedas previouslydescribed(Ramirezetal.,2015).Valuesarereportedas IC50(concentrationofsamplerequiredtoscavenge50%ofDPPH freeradicals).IfIC50≤50␮g/mlthesamplehashighantioxidant capacity,if50␮g/ml<IC50≤100␮g/mlthesamplehasmoderate antioxidantactivityandifIC50>200␮g/mlthesamplehasno rele-vantantioxidantactivity.

Ferricreducingantioxidantpower

Ferricreducingability(FRAPassay)orferricreducing antiox-idantpoweror oftheextractsand standardswasperformedas recommended by(Benzie and Strain, 1996) withsome modifi-cationsas described (Ramirezet al., 2015). Thestock solutions preparedwere300mMacetatebufferpH3.6,10mM 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) solution in 40mM HCl, and 20mM FeCl3.6H2Osolution.PlantextractsorstandardmethanolicTrolox solutions(150␮l)wereincubatedat37◦Cwith2mloftheFRAP solution (25ml acetate buffer, 5ml TPTZ solution, and 10ml FeCl3·6H2Osolution) in thedark for 30min.Absorbance ofthe ferroustripyridyltriazinebluecomplexformedwasthenreadat 593nm.Quantificationwasperformedusingastandardcalibration curveofantioxidantTrolox(from0.2to2.5␮mol/ml,R2_:0.995). Sampleswereanalyzedin triplicate(n=3 foreach sample)and resultsareexpressedin␮molTE/gdrymass.

Superoxideanionscavengingassay

Thesuperoxideanionscavengingactivitiesofextractand stan-dardswere measuredasreported previously(Simirgiotiset al., 2016a).Thesuperoxideanion(SAA)radical(O2•−)producedby theenzymexanthineoxidasereducesthenitrobluetetrazolium (NBT), producing a chromophore measured by spectroscopy at 520nm.TheSAscavengersreducethespeedofformationofthis chromophore.Extractswereevaluatedat100␮g/ml.Valuesare presentedasmean±standarddeviationofthreedeterminations.

Polyphenol,flavonoidandanthocyanincontents

Thetotalpolyphenoliccontents(TPC)weredeterminedby spec-troscopy using the Folin–Ciocalteau reagent (Simirgiotis et al., 2013b).Analiquotofeachextract(200␮l,approx.2mg/ml)was addedtotheFolin–Ciocalteaureagent(2ml,1:10,

v

/

v

inpurified water)andafter5minofreactionat25◦C,2mlofa100g/l solu-tionofNa2CO3wasadded.Then,after60min,theabsorbancewas measuredat710nm.Thecalibrationcurvewasperformedwith gal-licacid(concentrationsrangingfrom16to500␮g/ml,R2_=0.999) and theresultswere expressedas mg gallic acidequivalents/g drymatter.Determinationoftotalflavonoidcontent(TFC)ofthe methanolicextractswasperformedasdescribedusingtheAlCl3 method(Simirgiotisetal.,2013b).Quantificationwasexpressedby reportingtheabsorbanceinthecalibrationgraphofquercetin,used asastandardflavonoid(from0.1to65␮g/ml,R2_{=0.994).Results}

areexpressedasmgquercetinequivalents/gdryweight.All mea-surementswereperformedusingaHaloMPR-96microplatereader (DynamicaGmbH,Dietikon,Switzerland)oraSpectroquantPharo UV-Visspectrophotometer(Merck,Darmstadt,Germany).

Statisticalanalysis

Thestatisticalanalysiswascarriedoutusingthesoftware orig-inPro9.1(OriginlabCorporation,Northampton,MA,USA).Dataare reportedasmean±SDforatleastthreereplications.Analysisof variancewasperformedusingANOVA.Tukey’stestwascomputed forallassays(95%significancelevel).

Resultsanddiscussion

PhenoliccontentandantioxidantcapacitiesofOphryosporus

triangularis

Consumptionofplants,fruitsandvegetableswithantioxidant activityhasbeenrelatedtoavarietyofbeneficialbiological activ-ities such as anticancer,diabetes and heart disease prevention (Owenetal.,2000;Caoetal.,2014).Inthisstudy,the methano-lic extracts of aerial parts and flowers of the endemic desert ChileanspeciesO.triangularisshowedgoodantioxidantactivity. Severalantioxidanttestswithdifferentmechanismofactionwere employedinthisstudy,sincesomeauthorsconsiderthat differ-enttestsshouldbeperformedtoassuretheantioxidantcapacity. ForinstancetheFolin–Ciocalteau’sassayfrequentlyoverestimates the phenolic contentbecause thereagentis able toreact with alsoproteinsandinorganicions.Theextractsfromflowersand aerialpartsofthreedifferentspecimenscollectedinPaposoValley (Figs.S1andS2,supplementarymaterial)weretestedin antioxi-dantactivityassays(DPPH,FRAP,andsuperoxideanionscavenging assay,SAA,Table1),plus measurementsof totalphenolic (TPC) andtotalflavonoid(TFC)contents(Table1).Therewereonlysome smalldifferencesbetweenthespecimens(Table1)however,the differencescouldbeattributedtootherantioxidantcompounds ratherthanphenolicssincethechromatogramsforphenolic com-poundswerealmostidenticalforthethreespecimensunderstudy (Fig.S3,Supplementarymaterial).Sinceitisdifficulttocompare thetestswithothersperformedindifferentconditions,wehave compared ourvalues withpreviousantioxidant plantmaterials analyzedbyus andalsotothevaluespublishedfortherelated speciesO.hepthantus(Barreroetal.,2006).Thus,theTPCvalues oftheaerialpartsforspecimenIwerelowertothatreportedfor theleavesoftheChileanspeciesCryptocariaalba,(100.12±0.83mg GAE/gdryweight)butthevaluesfortheflowerswereclosetothat value(Simirgiotis,2013).TheTPCoftheaerialpartsforspecimen onewerealsoalmosthalftothatobtainedfortheaerialpartsof

Lumaapiculata(179.83±0.38mgGAE/g)(Simirgiotisetal.,2013a),

whilethetotalflavonoidcontentoftheaerialparts(Fig.2)was eight times lower than those of theaerial parts of L.apiculata

182 M.J.Simirgiotisetal./RevistaBrasileiradeFarmacognosia27(2017)179–187

Table1

Scavengingofthe1,1-diphenyl-2-picrylhydrazylradical(DPPH),ferricreducingantioxidantpower(FRAP),superoxideanionscavengingactivity(SAA),totalphenoliccontent (TPC),totalflavonoidcontent(TFC)ofmethanolicextractsofthreedifferentsamplesofOphryosporustriangularisfromthePaposoValley(IIRegion)ofChile.

Plantparts DPPH−a _FRAPb _SAAc _TPCd _TFCe _Yieldf

O.triangularisaerialparts-1 65.34±1.32l _{184.88±13.22}k _56.17±3.03l _74.25±3.94m _17.89±1.34◦ _10.30

O.triangularisflowers-1 52.41±1.87j _{196.82±13.28}k _{65.41±1.96 94.71±3.50}n _29.44±1.62r _15.02

O.triangularisaerialparts-1I 61.22±1.16 181.45±11.10k _52.23±4.10l _72.18±3.23m _{13.41±1.22 11.20}

O.triangularisflowers-1I 51.33±1.28j _{193.73±11.21}k _{62.45±1.86 91.29±2-87}n _{25.19±1.47 17.00}

O.triangularisaerialparts-1II 69.79±1.46 197.23±12.87k _55.24±2.87l _75.44±3.12m _19.37±136o _13.24

O.triangularisflowers-1II 63.72±1.48l _{201.84±15.32}k _{69.68±1.68 98.98±3.43}n _32.26±1.54r _17.41

Querceting _{31.43±1.24 449.11±11.31 80.05±3.37 – – –}

Gallicacidg _{21.78±1.39 565.49±14.65 93.24±2.63 – – –}

Valuesinthesamecolumnmarkedwiththesameletterarenotsignificantlydifferent(atp<0.05).

a_{AntiradicalDPPHactivitiesareexpressedasIC50in␮g/mlforextractsandcompounds.} b _{Expressedas␮Mtroloxequivalents/gdryweight.}

c _{Expressedinpercentagescavengingofsuperoxideanionat100␮g/ml.} d _{Totalphenoliccontent(TPC)expressedasmggallicacid/gdryweight.} e_{Totalflavonoidcontent(TFC)expressedasmgquercetin/gdryweight.} f _{Yieldisexpressedaspercentageinadryweightbasis.}

g_{Usedasstandardantioxidants.}

Fig.2. TIC(totalioncurrent,negativemode)UHPLCchromatogramsofOphryosporustriangularisextract.(A)Flowersand(B)aerialparts.

2015).TheFRAPvalues (Table1)oftheflowersandaerialparts forspecimenonewerehighertothatoftheethylacetateextract

ofNolanaramosissima(116.07±3.42␮MTroloxequivalents/gdry

weight) (Simirgiotis etal., 2015)and tothatof themethanolic extractofthefruitsofLumachequen(76.2±0.042␮MTrolox equiv-alents/gdry weight) (Simirgiotis et al., 2013a).The superoxide anionscavengingactivity(SAA)oftheaerialpartsandflowersfor specimenonewaslowerthanthoseofthetestedphenolic stan-dards(93.24and80.05%forgallicacidandquercetin,respectively). TheSAAoftheflowers(65.41%)werealsoclosethanthosereported forthemethanolicextractoftheblueberriesVacciniumcorimbosum

(72.61_±1.91%)(Ramirezetal.,2015).

Comprehensiveidentificationofthecompounds

Thirty-ninecompoundsweredetectedandtwenty-eight iden-tifiedortentativelyidentifiedbymeansofhighresolutionorbitrap MSandPDAdetection(Table2)bycomparisonwithHR-MSand PDAdatafoundinliterature.Someofthecompoundswere iden-tifiedbyco-elutionwithauthenticstandards.Amongthetwenty eight compoundsidentified, eleven werephenolic acids(peaks 2,8–12,14,15,18,20and35),fourteenwereflavonoids(peaks

23–36),twowerechromones(peaks 31and39),twowere sug-ars (peaks 6 and 7), and two were fatty acids (peaks 37 and 38).Somesevencompoundsremainedunknown(peaks3–5,13, 16, 21, and 22). The UHPLC chromatogram for specimen one (Fig.2)anddetailedidentificationofthecompoundsareexplained below.

Phenolicacids

Mostofthephenolicacidsarereportedforthefirsttimeinthis species.Peak2witha[M−H]− ionatm/z191.0190was identi-fiedasquinicacid(C7H11O6−)whilepeak11witha[M−H]−ionat

m/z179.0342wasidentifiedascaffeicacid(C9H7O4−)(Simirgiotis et al., 2015). Accordingly, peak 12 with a [M−H]− ion at m/z

323.1340producingacaffeicacidionatm/z179.0341was iden-tifiedasanunknowncaffeicacidderivative(C13H23O9−).Peak8 witha [M−H]− ion at m/z 473.2015 wasidentified as thymol-diglucoside (2-isopropyl-5-methylphenol-diglucose) asreported (Farag et al., 2014). Peaks 9(Fig.3A),10 and 15 with[M−H]− ionsatm/z325.0923,325.0924and325.0923wereidentifiedas coumaroyl-hexoseisomers(Faragetal.,2013),p-coumaroylhexose

M.J.

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Revista

Brasileira

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183

UHPLC-PDAandhighresolutionmassspectraldataforOphryosporustriangularisfromtheAtacamaDesert.

Peak number

Retention time(min)

UVmax(nm) Theoretical mass(m/z)

Measured mass(m/z)

Otherions(m/z) Formula Identification

1 1.64 – – 377.0891 191.0553(quinicacid) – Unknownquinicacidderivative

2 2.36 – 191.0190 191.0190 111.0078 C7H11O6− Quinicacida

3 2.64 – – 197.8073 115.0027 – Unknown

4 10.25 – – 283.1544 481.1835 – Unknown

5 10.73 – – 350.1370 211.0637,170.0185,161.0810 – Unknown

6 10.81 – 161.0819 161.0810 113.0233 C7H14O4− _Cymarose

7 10.87 – – 381.1313 161.0809 – Unknowncymaroseconjugate

8 11.32 269 473.2028 473.2015 463.1730 C22H33O11− _{Thymol-O-diglucoside}

9 11.76 227–277–320 325.0929 325.0923 651.1906[2M−H]−_{,163.039(coumaricacid),}

119.0491

C15H17O8− _{O-Coumaroylglucose}a

10 11.97 227–277–320 325.0929 325.0924 651.1907[2M−H]−,163.0390(coumaricacid),

119.0491

C15H17O8− p-Coumaroylglucose

11 12.40 244–290–324 179.0348 179.0342 135.0441 C9H7O4− _Caffeicacida

12 12.66 244–290–324 – 323.1340 179.0341 C13H23O9 Caffeicacidderivative

13 13.42 – – 313.0712 131.0704 C17H13O6− _Unknown

14 13.67 265 327.1085 327.1076 165.0547(phenyllacticacid) C15H19O8− _{Phenyllacticacid-2-O-glucoside}

15 14.93 227–277–320 325.0929 325.0923 651.1907[2M−H],163.0391

(hydroxycinnamicacid),119.0491

C15H17O8− _{p-Coumaroylgalactose}a

16 15.43 – – 471.1861 461.1576 – Unknown

17 15.72 257–355 447.0933 447.0959 241.0016,205.0862 C21H19O11− _{Quercetin-3-O-rhamnose}

18 17.70 265 165.0557 165.0549 121.0650 C9H9O3− _{Phenyllacticacid}

19 18.43 257–348 463.0882 463.0876 301.0632 C21H19O12− _{Quercetin-3-O-glucose}a

20 18.89 235–325 515.1195 515.1185 353.0870,191.0552,179.0341 C25H23O12− _{Dicaffeoyl-quinicacid}

21 18.89 237–277–325 – 651.1984 493.0394,330.0373 – Unknown

22 19.71 237–277–325 – 695.1223 515.1183 – UnknowndiQAderivative

23 19.85 257–342 425.0184 425.0176 345.0606,330.0372,315.0139,195.0290 C17H13O11S− _{7,3-di-O-Methylmyricetin-5-O-sulfate}

24 19.94 257–341 425.0184 425.0173 345.0605(syringetin),330.0372,315.0501,

287.0189

C17H13O11S− _{Syringetin-7-O-sulfate}

25 20.15 258–342 477.1038 477.1028 447.0924,284.0320 C22H21O12− _{Isorhamnetin-3-O-glucoside}

26 20.24 258–342 507.1144 507.1132 344.0530(syringetin),329.0295 C23H23O13− _{Syringetin-3-O-galactoside}

27 20.50 258–346 331.0459 331.0453 316.0216,271.0140 C16H11O8− 3-O-Methylmyricetin

28 20.66 258–346 439.0341 439.0331 359.3074,329.0294,316.0579,195.0687 C18H15O11S− 3,3,7-tri-O-Methyl-5-sulfatemyricetin

29 20.84 255–348 359.0772 359.0756 329.0652,316.0216,287.0188 C18H15O8− _{3,3-7-tri-O-Methylmyricetin}

30 21.03 255–346 329.0667 329.0662 299.0189,271.0240,255.0291 C17H13O7− _{3-O-Methylisorhamnetin}

31 21.24 239–325 311.0561 311.0554 265.0449 C17H11O6−

Methyl-4[(7-hydroxy-4-oxo-4H-chromen-3-yl)oxy]benzoate

32 21.70 254,354 345.0616 345.0608(4.8) 330.0370,315.0136,285.0400 C17H13O8− _{7,3-di-O-Methylmyricetin}

33 21.87 258–346 315.0510 315.0504 271.0241,243.0291 C16H11O7− Isorhamnetina

34 21.98 258–346 345.0611 285.0399 C17H13O8- 3,5-di-O-Methylmyricetin(Syringetin)

35 22.19 417.1555 417.1546 360.210 C22H25O8− _{Syringaresinol}

36 22.46 254,354 345.0616 329.0660 299.0190,271.0240,227.0341 C17H13O8− _{3,4-Dimethylmyricetin}

37 22.59 318 327.2177 327.2171 325.1836,211.1331 C18H31O5− _{Tri-hydroxyoctadecadienoicacid}

38 23.45 275 329.2333 329.2327 325.1836,2111331 C18H33O5− _{Tri-hydroxyoctadecaenoicacid}

39 23.95 239–325 363.1238 363.1231 325.1834 C22H19O5− _{Chromonederivative}

184 M.J.Simirgiotisetal./RevistaBrasileiradeFarmacognosia27(2017)179–187

Fig.3. (Continued)

p-coumaroyl hexose (trans-2-hydroxy-galactosyl-cinnammate), respectively.Peak14(Fig.3B)witha[M−H]−ionatm/z327.1076 (C15H19O8−) producing a phenyllactic acid MS2 ion at around

m/z165.0547(C9H9O3−)wasidentifiedasphenyllacticacid-2-O -glucoside (Beelders et al.,2014).Peak 18 wasidentified asthe respectiveaglyconephenyllacticacid(Lietal.,2015)(C9H9O3−). Peak35witha[M−H]−ionatm/z417.1546wasidentifiedasthe ligninsyringaresinol(Sharpetal.,2001)(C22H25O8−).Peak20with a[M−H]−ionatm/z515.1182anddaughtercharacteristicionsat

m/z353.0870,191.0552(quinicacidC7H11O6−)and179.0341was identifiedasdicaffeoyl-quinicacid(CQA)(Narváez-Cuencaetal., 2012).TheCQAwasalsoidentifiedaccordingtoitsUVspectra(max at235–314–325nm).

Flavonols

SeveralO-methylatedflavonoidshavepreviouslybeendetected

inO.triangularisandtheirpresenceisoftaxonomicalrelevance

for this species and several species among this genus (Alarcon et al., 1993; Sigstad et al., 1996; Barrero et al., 2006). These types of compounds were also detected in the present work, confirming these flavonoids as chemotaxonomic markers. Iso-mer compounds detected by peaks 23 and 24 (Fig. 3C) with [M−H]− ionsat m/z425.0176and m/z425.0177(C17H13O11S−) producingionsatm/z345.0605(di-O-methylmyricetin),330.0372

(O-methylmyricetin), 315.0138 (myricetin), were identified as 3,7-di-O-methylmyricetin-5-O-sulfateandsyringetin-7-O-sulfate (Harborne and Boardley, 1984), respectively. Peak 25 with an [M−H]−ionatm/z477.1028wasidentifiedasisorhamnetin glu-coside(C22H21O12−)andpeak 26(Fig.3D)producing aMS2 ion at m/z 344.0530 (syringetin) was identified as syringetin 3-O -galactoside([M−H]−ionatm/z507.1132,C23H23O13−)(Suetal., 2016).Peak28(Fig.3E)withan[M−H]−ionatm/z439.0331andMS fragmentsatm/z359.3074([M−H-sulfate]−),329.0294,195.0687 was tentatively identified as myricetin 3,37-tri-O-methyl-5-O -sulfate(HarborneandBoardley,1984)(C18H15O11S−),whilepeak 29 with an [M−H]− ion at m/z 359.0756 as the related com-pound 3,3,7-tri-O-methylmyricetin (C18H15O8−), peak 32 with an [M−H]− ion at m/z 345.0608 was identified as 7,3di-O -methylmyricetin(C17H13O8−)andpeak36asitsisomersyringetin (3,5di-O-methylmyricetin).Peak33withan[M−H]−ionatm/z

186 M.J.Simirgiotisetal./RevistaBrasileiradeFarmacognosia27(2017)179–187

Oxylipins

Thisis thefirstreportoftheseantioxidantfattyacidsinthe genus.Peak38withan[M−H]−ionatm/z327.2171(Fig.3G)was identifiedastheantioxidantfattyacidtri-hydroxyoctadecadienoic acid(Oxylipin)(Jiménez-Sánchezetal.,2016)andpeak37with an[M−H]−ionatm/z329.2327wasidentifiedastherelated tri-hydroxyoctadecaenoicacid(Jiménez-Sánchezetal.,2016).

Chromenes

ChromeneswerealsodetectedinotherOphryosporusspecies (Ferracini et al., 1989; Sigstad et al., 1992; Alarcon et al., 1993) andthose compounds arealso chemical markersfor the genus.For O. triangularis collected in LaSerena, Chile, Alarcon et al. reported several chromones and benzofuranes includ-ing 5-acetyl-6-methoxy-2-isopropenyl benzofurane, 6-acetyl-2,2-dimethyl-chromenone, 6-acetyl-5-methoxy-8-senecioyl-2,2-dimethylchromene, 6-acetyl-8-senecioyl-2,2-dimethylchromene, 6-acetyl-2,2-dimethyl-chromen-4-one(Alarconetal.,1993).Peak 31 (Fig. 3F) withan [M−H]− ion at m/z311.0554 (C17H11O6−) was identified as methyl-4[(7-hydroxy-4-oxo-4H -chromen-3-yl)oxy]benzoatewhilepeak39 (Fig.3H)withan[M−H]− ionat

m/z363.1231wasidentifiedasarelatedchromone(Mazzeietal., 2003)derivativeofmolecularformulaC22H19O5−.

Sugars

Thesugarcymarose(Franz,1971)([M−H]−ionatm/z161.0810) wasidentified(peak6,molecularformulaC7H14O4−).Anunknown compound (peak 7) producing a cymarose residue was also detected(peak7,[M−H]−ionatm/z381.1313).

Conclusions

Themethanolicextractsoftheaerialpartsand flowersofan endemic species from the Atacama Desert showed antioxidant capacities(65.34±1.32and52.41±1.87␮g/mlintheDPPHassay, 184.88±13.22and196.80±13.28␮molTE/gdryweightinthe fer-ricreducingpower(FRAP)assayand56.17±3.03and65.41±1.96% inthesuperoxideanion scavengingassay,respectively). Thirty-nine compounds responsible for the antioxidant activity were detectedinthemethanolicextractoftheaerialpartsand twenty-fourweredetectedinthemethanolicextractoftheflowersusinga hybridUHPLC-PDA-OT-MSinstrumentinthreedifferentsamples collectedin PaposoValley, AtacamaDesert.Severalcompounds wereidentifiedforthefirsttimeinthisspecies.Theflowersand aerialparts of O. triangularis canbe useful for the preparation of nutraceuticals or phytomedicines.Furthermore, the hyphen-atedUHPLC-Q-exactivefocusmachineequippedwithorbitrap-PDA detectors and high resolution collision cell provide a powerful methodfor accurateandfastmetabolomicsanalysisofthe Ata-camaDesertfloraand theUHPLC–MS fingerprintgeneratedcan beusedfortheauthenticationofthisspecieswhileseveral com-poundsincludingsomeO-methylatedflavonesandphenolicacids suchasp-coumaroyl-glucosidecanbeusedaschemotaxonomic markers.FurtherstudiesonthisChileanplantshouldbe under-takenwithmoresamplestoobtainamorecompletepictureonthe constituentsandpotentialofthisspecies.

Author’scontributions

MSandAMwroteandrevisedthemanuscript,CQ,JMVandCA analyzedthedata,BSandJBperformedtheantioxidantassays,CEC helpedinthecollectionoftheplantmaterialananalysisofthedata.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsappreciatefinancialsupportbyFONDECYT(Grant 1140178)andfondequip(EQM140002).WethankCONAF (Corpo-raciónNacionalForestaldeChile)forthesupportinthecollection ofPaposoValleyflora,inthenationalprotectedarea.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2016.10.002.

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