Parentucellia latifolia subsp. latifolia: A potential source for loganin iridoids by HPLC-ESI-MSn technique

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ContentslistsavailableatScienceDirect

Journal

of

Pharmaceutical

and

Biomedical

Analysis

jou rn a l h om ep a g e :w w w . e l s e v i e r . c o m / l oc a t e / j p b a

Parentucellia

latifolia

subsp.

latifolia:

A

potential

source

for

loganin

iridoids

by

HPLC-ESI-MS

n

technique

Eulogio

J. Llorent-Martínez

a

_,

_María

_Luisa

_{Fernández-de}

_Córdova

a

_,

_Gokhan

_Zengin

b,∗

_,

Mir

Babak

Bahadori

c

_,

_Muhammad

_Zakariyyah

_Aumeeruddy

d

_,

_Kannan

_RR

_Rengasamy

e

_,

Mohamad

Fawzi

Mahomoodally

d,∗

a_Department_of_Physical_and_Analytical_Chemistry,_University_of_Jaén,_Campus_Las_Lagunillas_S/N,_E-23071_Jaén,_Spain b_Department_of_Biology,_Science_Faculty,_Selcuk_University,_Campus,_Konya,_Turkey

c_Research_Center_for_{Pharmaceutical}_{Nanotechnology,}_Tabriz_University_of_Medical_Sciences,_Tabriz,_Iran d_Department_of_Health_Sciences,_Faculty_of_Science,_University_of_Mauritius,₂₃₀_Réduit,_Mauritius e_REEF_{Environmental}_Consultancy,_#2_Kamaraj_Street,_S.P._Nagar,_Puducherry₆₀₅_001,_India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22September2018 Receivedinrevisedform 14December2018 Accepted16December2018 Availableonline17December2018 Keywords: Parentucellialatifolia Antioxidants Enzymeinhibition Phenolic Flavonoid Loganin

a

b

s

t

r

a

c

t

Thisstudyattemptstocomparethepharmaceuticalpotential(antioxidantandkeyenzymeinhibitionof clinicalrelevance)oforganicandaqueousextractsofParentucellialatifolia(L.)Caruelsubsp.latifolia(L.) Caruelaswellasphytochemicalcomposition.Thephytochemicalcompoundswereevaluatedby spec-trophotometricmethods(fortotalamounts)andHPLC-ESI-DAD-MSn_(for_individual_compounds)._The

extractswerescreenedforantioxidantabilitiesbyinvitroassays.Inhibitioneffectswerealsoinvestigated againstasetofenzymeslinkedtomajorhealthproblems.Generally,themethanol(MeOH)andaqueous extractsdisplayedhigherscavengingabilitiesonradicalsandreductiveeffectswhencomparedwiththe ethylacetate(EtOAc)extract.Ontheotherhand,theEtOAcextractwasthemostactiveinhibitoron cholinesterases(1.81–1.88mgGALAE/g),amylase(0.70mmolACAE/g),glucosidase(2.85mmolACAE/g) andlipase(33.24mgOE/g).ThehighestTPCwasobservedintheaqueousextract(25.07mgGAE/g) whileMeOHextractpossessedthehighestlevelofTFC(44.15mgRE/g)andTPAC(3.46mgCE/g). LC-MSn_metabolite_proﬁling_indicated_that_loganin_and_its_isomers,_rutin,_and_{luteolin-O-hexoside}_were_the

mostabundantcompounds.OurresultssuggestthatP.latifoliamaybevaluablesourceofphyto-agents forthemanagementofnoncommunicablediseases.

1. Introduction

Oxidativestressisassociated withsomeimportantdisorders

including diabetes mellitus, cancer, rheumatoid arthritis,

cere-brovascular and cardiovascular diseases, chronic inﬂammation,

aging,andotherdegenerativediseases.Theincreasinglevelof

reac-tiveoxygenspecies(ROS)isharmfulforhumanmacromolecules

suchasDNA,lipids,andproteins,leadingtomutationsand

carcino-genesis.Consequently,theregulationofcellularreactiveoxygen

speciesisvitaltomaintaincellularhomeostasisAtthispoint,

nat-uralantioxidantcompoundswithoutsideeffectsmayhinderthe

destructiveeffectsofthereactiveoxygenspeciesandplant

sec-ondarymetabolitesaregreatsourcesofnaturalantioxidants[1].

∗ Correspondingauthors.

E-mailaddresses:gokhanzengin@selcuk.edu.tr(G.Zengin), f.mahomoodally@uom.ac.mu(M.FawziMahomoodally).

Duringthelastyears,therehasbeenincreasinginterestinthe

explorationofherbsinthepharmaceuticalindustriesforpotent

noveldrugs.Nonetheless,oftheestimatednumberofplantspecies

globally, just a few has been chemically investigated, whereas

a muchsmallerfractionhasbeensubjectedtopharmacological

screening[2].Despitethelackofinformationontherapeuticeffects

ofsomeplants,theirmedicinalusesarebecomingmore

accept-ableascomplementaryandalternativemedicinesduetotheirlow

costandlessadverseeffects[3–5].Withthedevelopmentof

mod-ernandrapidisolationtechniques,bioassayscreeningprocedures,

andspectroscopicmethods,phytochemistryhasnowbecomean

importantbranchinpharmaceuticalstudies[2].Recently,therole

ofphytoconstituentshasbeenextendedtotherapeuticuses,

pro-vidingconsiderablehealthbeneﬁts[6].

ThegenusParentucellia(Orobanchaceae)comprisesofannual

herbs,withsessileandgenerallyoppositeupperleavesand

ﬂow-erswhicharearrangedinracemesorspike-like[7].Fourspecies

are distributed in Western Europe, Mediterranean,Central and

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SouthwesternAsia.Morphologically,P.latifoliasubsp.latifoliacan

beseparatedfromP.latifoliasubsp.ﬂaviﬂorabyitspurplecorolla

andrelativelythickstem[7].

Fewphytochemicalstudieshavebeenperformedonthegenus

Parentucellia. For instance,Biancoet al. [8] isolated iridoid

glu-cosidesfrom Parentucelliaviscosa. Terpenoidsand malonic acid

derivativeswerealsoidentiﬁedinP.latifolia[9,10].Furthermore,

deUrbina,etal.[11]puriﬁedtheantispasmodicperacetylated

pen-stemonoside,aucubin,andcatalpolfromP.latifolia.Nonetheless,

wefoundthatthereisstilladearthofpharmacological

exploita-tionofplantsfromthisgenus.Inthiscontext,thepresentstudy

aimedtoinvestigatethebiologicalandchemicalpropertiesof

Par-entucellialatifoliasubsp.latifoliainanattempttoobtainvaluable

phytocompoundsfromthisspeciesforthemanagementofchronic

disorders. Thebioactivitieswere assessedbased onantioxidant

activity(reducing, chelating, and radical scavengingpotentials)

and enzymeinhibitory effects against enzymesassociated with

some noncommunicable diseases including neurodegenerative

disorders(acetylcholinesterase(AChE)andbutyrylcholinesterase

(BChE)),hyperpigmentation(tyrosinase),hyperglycemiaand

dia-betes (␣-amylase and ␣-glucosidase),and obesity(lipase). The

phytochemicalproﬁleoftheplantwasalsodeterminedtoidentify

anypossiblecorrelationwiththeobservedbiologicalactivities.

2. Materialsandmethods

2.1. Plantmaterial

Plantmaterials(n=20)werecollectedfromsamepopulation

duringﬁeldstudy(spring2016)inKonya,Turkey(between

Alaed-din Keykubat Campus and Yukselen villageroad, 1100m). The

taxonomicidentiﬁcationwasperformedbythebotanistDr.Murad

Aydin Sanda and one voucher specimen was deposited at the

herbariumofSelc¸ukUniversity,Konya,Turkey.

2.2. Extractionprocedures

Toprepareethylacetateandmethanolextracts,theplant

sam-ples (dried-powdered aerial parts (about 2mm)) were stirred

overnight (24h) at room temperature (10g in 200mL solvent

(EtOAcandMeOH)).Afterﬁltration,theextractswereconcentrated

usingarotaryevaporatorundervacuumat40◦C.Forwaterextract,

infusionwasprepared(10gofplantswerekeptin200mLofboiled

waterfor20min).Afterthat,theinfusionwasﬁlteredanddriedby

usingalyophilizator.Thesampleswerestoredat+4◦Cuntilfurther

analysis.

2.3. Totalmetabolitecontentsdetermination

Similartoourpreviousstudies,thetotalamountofphenolics

(TPC)(bystandardFolin-Ciocalteumethod),ﬂavonoids(TFC)(by

AlCl3method)andtotalphenolicacidcontent(TPAC)(byArnow

assay)weredetermined.Theﬁnalresultswereexpressedas

equiv-alentsofstandardcompounds(gallicacid(mgGAE/g)forTPC;rutin

(mgRE/g) forTFCand caffeicacid(mgCE/gextract)for TPAC),

respectively).Theexperimentaldetailsweresummarizedin

pre-viouspapers[12,13].

2.4. Antioxidantcapacitymeasurements

Differentspectrophotometricexperimentswereusedtoscreen

theantioxidantcapacityoftheParentucelliaextractsas

phospho-molybdenum,quenchingofradicals(DPPHandABTS),reduction

potentials(FRAPandCUPRAC),andferrousionchelating.The

ﬁnd-ingswereexpressedasstandardcompoundsequivalents(mgTE/g

andmgEDTAE/g).Theproceduresofassayswerealready

summa-rizedreportedinourearlierwork[12].

2.5. Keyenzymeinhibitoryeffects

The Parentucelliaextractswere tested as sourcesof enzyme

inhibitorsonsomeenzymes,including␣-amylase,␣-glucosidase,

cholinesterases, tyrosinase and lipase. The procedures of these

assays were already summarized in our earlier work[12]. The

enzymeinhibitoreffectswereevaluatedasequivalentsoforlistat

(forlipase), acarbose (for␣-amylase and␣-glucosidase),

galan-tamine(forAChEandBChE),andkojicacid(fortyrosinase).

2.6. Chromatographicconditions

An Agilent HPLC system (Series1100 with a G1315B diode

arraydetector)wasemployedforchromatographicanalysis.Itwas

connected to an Esquire 6000, Bruker Daltonicsion trapmass

spectrometer with an electrospray interface.A reversed phase

LunaOmegaPolarC18analyticalcolumn(150×3.0mmand5␮m

particlesize(Phenomenex))anda PolarC18SecurityGuard

car-tridge (Phenomenex)of 4×3.0mm wereused foranalysis.The

HPLC-DAD-MS parameters have been previously reported [14].

10␮Lofsample(5mg/mLinMeOH)wasﬁlteredthrough0.45␮m

PTFEmembraneﬁlters(AnálisisVínicos,Madrid,Spain)andthen

injected.

2.7. Quantiﬁcationofpolyphenols

Calibration curves were prepared using the following

stan-dardreagents:ferulicacid,apigenin,kaempferol,loganin,luteolin,

quercetin,rutin,andvicenin2.Quantiﬁcationwasperformedby

UVsignal,recordingchromatogramsat320and350nmfor

phe-nolicacidsandﬂavonoids,respectively.Whentheexactanalytical

standard wasavailable,it wasusedfor thequantiﬁcation.

Oth-erwise, thecorrespondinganalytical standard wasused for the

(semi)quantiﬁcationofitsderivatives(forinstance,theaglycone

forthecorrespondingglycosides).

2.8. Statisticalanalysis

Allassaysresultswereexpressedasmeanofthreeexperiments

withstandarddeviation(S.D).ANOVAassay(withTukey’s)were

performedtoevaluatethedifferencesintheextracts.Thestatistical

analysisweredonebyusingSPSSv.17.0program.

3. Resultsanddiscussion

3.1. Phytochemicalscontent

Extraction of plantmaterial is theﬁrst step in

phytochemi-calstudiesandplaysa crucialrole.In theconventional method

of extraction, several techniques have been employed suchas

hydrodistillation,soxhletextractionandmaceration.Amongthese,

macerationisinexpensiveandstraightforwardtoextractphenolic

components.Thistechniquehasbeenperformedatroom

tempera-tureandhasawiderangeofapplicationincludingpharmaceutical

and foodareas.Secondly, thesolvent selectionis another

criti-cal step which frequentlyuses most commonsolvents suchas

methanol,ethanol,ethylacetateandwater[15].Takentogether,

weselectedthemacerationtechniquewiththreesolvents(ethyl

acetate,methanol,andwater)toprepareP.latifoliasubsp.latifolia

extracts.

ThetotalbioactiveamountsoftheextractsofP.latifoliawere

measuredasTPC,TFC,andTPAC(Table1).ThehighestTPCwas

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Table1

TotalbioactivecompoundsofthesamplesA_.

Extracts TPC(mgGAE/gextract) TFC(mgRE/gextract) TPAC(mgCE/gextract)

EtOAc 20.47±0.41c _7.14_±_0.13c _nd

MeOH 22.63±0.30b _44.15_±_0.22a _3.46_±_0.57a

Water 25.07±0.17a _34.22_±_0.22b _1.31_±_0.19b

A_Values_expressed_as_means_±_S.D._of_three_parallel_{measurements.}_CE:_caffeic_acid_equivalents;_GAEs:_Gallic_acid_equivalents;_REs:_Rutin_equivalents;_TFC:_Total_ﬂavonoids

content;TPAC:Totalphenolicacidscontent;TPC:Totalphenoliccontent.nd:nondetected.Differentlettersreferstatisticallysigniﬁcantdifferencesintheextracts(p<0.05).

Fig.1.HPLC-ESI-MSn_base_peak_{chromatograms}_(BPC)_of_the_methanolic_extract_of_P._latifolia.

theMeOHandEtOAcextracts(22.63and20.47mgGAE/g,

respec-tively).However,MeOHextractcontainedthehighestlevelofTFC

(44.15mgRE/g)followedbytheaqueousextract.Withregardsto

TPAC,asimilarpatternwasobservedanditwasnotdetectedinthe

EtOAcextract.

3.2. HPLC-ESI-MSn

HPLC-ESI-MSn_(with_the_negative_ion_mode)_was_carried_out_to

identifyphytochemicalsinanalyzedextracts.Theobtained

chro-matogramfortheMeOHextractofP.latifoliaisshowninFig.1and

theidentiﬁedcompoundscouldbeseeninTable2.

3.2.1. Iridoidandphenylethanoidglycosides

Compound2,with[M−H]−_at_m/z_375,_exhibited_MSn_fragment

ionsatm/z213,169,151,and125.Thisexactfragmentationpattern

hasbeenpreviouslyreportedfortheiridoidglycoside

dihydroco-minicacid[16].

Compound6wasidentiﬁedasloganin(formateadduct).

Com-pound5and7presentedthesamefragmentationpattern,sothey

werecharacterizedasloganinisomers. Theseiridoid glycosides

werethemostabundantcompoundsintheanalyzedextracts.

Threephenylethanoid glycosideswerecharacterized.On one

hand,compound22,with[M−H]−_at_m/z_651,_exhibited_fragment

ionsatm/z475,457,and329,inagreementwiththe

fragmenta-tionofmartynoside[17].Ontheotherhand,compounds10and

13presentedthesamefragmentationpatternasverbascoside[18]

andweretentativelycharacterizedasisomers.

3.2.2. Flavonoids

Compound4sufferedthesequentialneutrallossesof176Da

(glucuronide)and308Da(rutinoside)toyieldquercetinatm/z301

(themassspectrum ofquercetinwasconﬁrmedby comparison

withananalyticalstandard).Thisfragmentationpatternisin

agree-mentwithquercetin-O-glucuronide-O-rutinoside.Compound12

wasidentiﬁedasrutinbycomparisonwithananalyticalstandard.

Compound25wasidentiﬁedasapigenin,andthreerelated

gly-cosideswerealsocharacterized(compounds8,9,and18).Inall

cases,apigeninwasobservedatm/z269,andtheattachedsugars

wereglucuronidemoieties(neutrallossesof176Da).

Compound11,with[M−H]−_at_m/z_609,_suffered_two

consecu-tivelossesofhexosidemoieties(162Da),yieldingtheaglyconeat

m/z285(kaempferol).Hence,itwascharacterizedas

kaempferol-O-dihexoside.

Compound23,luteolin,presentedatypicalfragmentionatm/z

243andcompound14,withanadditional162Daloss,was

charac-terizedasluteolin-O-hexoside.

Compound15presentedthedeprotonatedmolecularionatm/z

491and, after theneutral lossof 176Da, yielded theaglycone

isorhamnetinatm/z315(characteristic315→300fragmentation).

Therefore,itwascharacterizedasisorhamnetin-O-glucuronide.

Finally,compound26wastentativelycharacterizedasa

methy-latedﬂavonoidduetothelossof15Da(methylgroup)observed.

Compounds17,19,and20hadadditionalrutinoside,hexoside,and

glucuronidemoietiesintheirstructures,whichindicatedthatthey

wereprobablyglycosidesofcompound26.

3.2.3. Othercompounds

Compound3couldnotbefullyidentiﬁed,butitseemsa

sac-charidederivative,asitpresentedfragmentionsatm/z179,161,

143, 119, and 113, typicalof hexosides [19].Compound 16

dis-played ferulic acid at m/z 193 (fragment ions at m/z 149 and

134),soitwascharacterizedasaderivative.Similarly,compound

21 was characterized as a coumaric acid (163→119

fragmen-tation) derivative. Finally, compound 24 was characterized as

oxo-dihydroxy-octadecenoicacid[20].Thestructuresofthemain

compoundscouldbefoundinFig.2.

3.3. QuantiﬁcationbyHPLC-DAD

Calibration graphsfor each analytical standard (Section2.8)

were constructed using six concentrations (0.3–100␮g/mL in

MeOH), plotting peak area versus concentration, obtaining

R≥0.997inallcases.Quantitationlimitsvariedbetween0.3and

1.0mgL−1,andthecalibrationgraphwaslinearupto100mgL−1

forallanalyticalstandards.Boththerepeatability(sameday,n=3)

andtheintermediateprecision(3differentdays,n=9)were

eval-uated,obtainingR.S.D.lowerthan8%inallcases.Therobustness

of the method was assayed considering possible variations in

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Table2

CharacterizationofthecompoundsfoundintheanalyzedextractsofParentucellialatifolia.

No. tR(min) [M-H]−m/z m/z(%basepeak) Assignedidentiﬁcation

1 1.9 495 MS2_[495]:₄₇₇_(18),₃₇₉₍₁₀₀₎ MS3_[495→379]:₁₆₉₍₁₀₀₎ Unknown 2 4.3 375 MS2_[375]:₂₁₃_(100),₁₆₉_(31),₁₂₅₍₁₆₎ MS3_[375→213]:₁₆₉_(100),₁₅₁_(18),₁₂₅₍₄₇₎ MS4_{[375→213→169]:}₁₅₁_(100),₁₂₅₍₃₎ Dihydrocominicacid 3 5.5 433 MS2_[433]:₃₈₇_(59),₂₂₅_(74),₁₇₉₍₁₀₀₎ MS3_[433→179]:₁₆₁_(100),₁₄₃_(34),₁₁₉_(17),₁₁₃₍₁₀₀₎ Saccharidederivative 4 8.8 785 MS2_[785]:₆₀₉₍₁₀₀₎ MS3_[785→609]:₃₀₁₍₁₀₀₎ MS4_{[785→609→301]:}₂₅₅_(44),₁₇₉_(100),₁₅₁₍₁₀₎ Quercetin-O-glucuronide-O-rutinoside 5 9.7 435 MS2_[435]:₂₂₇₍₁₀₀₎ MS3_[435→227]:₁₀₁₍₁₀₀₎

Loganinisomer(formateadduct)

6 11.3 435 MS2_[435]:₃₈₉_(100),₂₂₇₍₇₀₎

MS3_[435→389]:₂₂₇₍₁₀₀₎

MS4_{[435→389→227]:}₁₀₁₍₁₀₀₎

Loganina_(formate_adduct)

7 12.8 435 MS2_[435]:₃₈₉_(29),₂₂₇₍₁₀₀₎

MS3_[435→227]:₁₀₁₍₁₀₀₎

Loganinisomer(formateadduct)

8 14.9 643 MS2_[643]:₄₆₇₍₁₀₀₎ MS3_[643→467]:₂₆₉₍₁₀₀₎ MS4_{[643→467→269]:}₂₂₅₍₁₀₀₎ Apigenin-O-glucuronidederivative 9 14.9 621 MS2_[621]:₄₄₅_(100),₂₆₉₍₄₇₎ MS3_[621→445]:₂₆₉₍₁₀₀₎ Apigenindiglucuronide 10 17.5 623 MS2_[623]:₄₆₁₍₂₄₎ MS3_[623→461]:₃₁₅_(100),₂₈₅_(48),₁₆₁_(11),₁₄₃_(27),₁₃₅₍₇₁₎ Verbascosideisomer 11 17.9 609 MS2_[609]:₄₄₇_(90),₂₈₅₍₁₀₀₎ MS3_[609→447]:₂₈₅₍₁₀₀₎ MS4_{[609→447→285]:}₂₅₅_(82),₂₄₁₍₁₀₀₎ Kaempferol-O-dihexoside 12 19.5 609 MS2_[609]:₃₀₁₍₁₀₀₎ MS3_[609→301]:₁₇₉_(100),₁₅₁₍₆₃₎ Rutina 13 20.5 623 MS2_[623]:₄₆₁₍₁₀₀₎ MS3_[623→461]:₃₁₅_(49),₁₆₁_(54),₁₄₃_(8),₁₃₅₍₁₀₀₎ Verbascosideisomer 14 20.8 447 MS2_[447]:₂₈₅₍₁₀₀₎ MS3_[447→285]:₂₄₃_(100),₁₇₅₍₆₇₎ Luteolin-O-hexoside 15 21.5 491 MS2_[491]:₃₁₅₍₁₀₀₎ MS3_[491→315]:₃₀₀₍₁₀₀₎ Isorhamnetin-O-glucuronide 16 23.2 399 MS2_[399]:₃₁₁_(61),₂₈₅_(47),₁₉₃₍₁₀₀₎ MS3_[399→193]:₁₄₉_(47),₁₃₄₍₁₀₀₎

Ferulicacidderivative

17 24.9 607 MS2_[607]:₂₉₉_(100),₂₈₄₍₄₁₎ _{Methyl-flavonoid-O-rutinoside} 18 25.4 445 MS2_[445]:₂₆₉₍₁₀₀₎ MS3_[445→269]:₂₂₅_(100),₁₅₁₍₂₅₎ Apigenin-O-glucuronide 19 25.8 461 MS2_[461]:₂₉₉₍₁₀₀₎ MS3_[461→299]:₂₈₄₍₁₀₀₎ Methyl-flavonoid-O-hexoside 20 26.2 475 MS2_[475]:₂₉₉_(100),₂₈₄₍₈₎ _{Methyl-flavonoid-O-glucuronide} 21 28.2 783 MS2_[783]:₆₁₉_(22),₄₈₃₍₁₀₀₎ MS3_[783→483]:₃₃₇_(46),₃₁₉_(79),₁₆₃₍₁₀₀₎ MS4_{[783→483→163]:}₁₁₉₍₁₀₀₎

Coumaricacidderivative

22 30.5 651 MS2_[651]:₆₅₁_(100),_505(3),₄₇₅_(23),₄₅₇_(9),₁₉₃₍₈₎ MS3_[651→475]:₃₂₉_(100),₁₆₁₍₅₇₎ Martynoside 23 35.8 285 MS2_[285]:₂₄₃_(84),₂₄₁₍₁₀₀₎ _Luteolina 24 39.2 327 MS2_[327]:₂₉₁_(45),₂₂₉_(24),₂₁₁_(33),₁₇₁₍₁₀₀₎ _{Oxo-dihydroxy-octadecenoic}_acid 25 39.9 269 MS2_[269]:₂₆₉₍₁₀₀₎ _Apigenina 26 40.5 299 MS2_[299]:₂₈₄₍₁₀₀₎ _{Methyl-ﬂavonoid}

a_Identiﬁed_by_comparison_with_analytical_standards.

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Table3

QuantiﬁcationofphenoliccompoundsinP.latifolia(mg/gDE).

No. Assignedidentiﬁcation Concentration(mg/gDE) Iridoidglycosides 5 Loganinisomer 7.1±0.5 6 Loganin 24±1 7 Loganinisomer 3.5±0.3 Total 34.6±1 Flavonoids 4 Quercetin-O-glucuronide-O-rutinoside 0.21±0.02 8+9 Apigenin di-glucuronide+derivative 1.1±0.1 11 Kaempferol-O-dihexoside 0.71±0.05 12 Rutin 6.2±0.1 14 Luteolin-O-hexoside 6.3±0.1 18 Apigenin-O-glucuronide 0.40±0.03 23 Luteolin 0.84±0.05 25 Apigenin 0.39±0.02 Total 15.8±0.1 Phenolicacids

16 Ferulicacidderivative 0.61±0.08

21 Coumaricacidderivative 0.06±0.01

Total 0.67±0.01

TIPCa ₅₁_±₁

a_Total_individual_phenolic_content.

solutions (stored at -20◦C for at least 1 month), observing no

signiﬁcantvariationsintheanalyticalsignal.

Totalindividualphenoliccontent(TIPC)(51±1mg/gDE)was

deﬁnedasthesumofallthephenolicsthatwerequantiﬁedby

liquidchromatography.TheresultsareshowninTable3.Iridoid

glycosidesaccountedforapproximately66%oftheTIPC,followed

byﬂavonoids,whichrepresentedapproximately33%ofTIPC.On

theotherhand,theamountofphenolicacidderivativeswasalmost

residual(1.3%ofTIPC).Amongtheiridoidglycosides,onlyloganin

(compound6)anditstwoisomers(compound5and7)were

char-acterized.Loganinwasthemostabundantcompoundintheextract,

accountingfor 47%of TIPC(24±1mg/g DE).Loganin is ausual

ingredientofmanytabletsorpillsduetoitsbioactivity.Its

con-centrationhasbeenreportedindifferentplants,suchasdifferent

LoniceraspeciesandCorniFructus[21–23].Inallofthem,the

lev-elsofloganinwerelowerthan10mg/g,whichmakesP.latifoliaa

potentialsourceofloganinforpharmaceuticalapplications.

The most abundant ﬂavonoids were rutin and

luteolin-O-hexoside (compounds12 and 14), which accounted for almost

80%oftheamountofﬂavonoids.Ingeneral,loganinandits

iso-mers, rutin, and luteolin-O-hexoside were the most abundant

compounds: ∼92% of TIPC. Hence, theymay consideredas the

responsiblecomponentsforthebioactivityfoundintheanalyzed

extract,althoughloganinisthemostabundantcompoundofallof

them.

3.4. Antioxidantactivity

Inanefforttoreduceoxidativedamage,therehasbeenextensive

researchconductedonmanyplantextractsandsecondary

metabo-lites.Phenoliccompoundscanterminatetheoxidationdamagesof

freeradicalsinhumanbodybydonatinghydrogenatom,followed

bythestabilisation and delocalization ofthe unpairedelectron

withinitsaromaticring[24].

Inthiswork,theantioxidantcapacityofP.latifoliawas

inves-tigatedin terms of itsfree radicalscavenging, reducing power,

phosphomolybdenum,andmetalchelatingproperties.Asitcould

beseeninTable4,theaqueousextractshowedthebestscavenging

activity(37.80(inDPPH)and69.92mgTE/g(inABTS))whilethe

EtOAcextractdisplayedthelowestactivity.Similarly,theaqueous

extractexertedthehighesteffect(61.73mgTE/g)whiletheEtOAc

extractwastheleasteffective(24.94mgTE/g)inFRAPassay.The

highscavengingandFRAPactivityoftheaqueousextractismost

probablyduetoitshighestTPCasobservedinthisstudy[25].

Withregardstothereducing poweragainstCu(II)(CUPRAC

assay),theMeOHextract(89.08mgTE/g)exhibitedthebesteffect

followed by the water extract (87.62mg TE/g). EtOAc extract

depictedtheleastactivity(57.59mgTE/g).HighTFCandTPCinthe

MeOHextractmightberesponsibleforitsstrongreducingactivity.

Interestingly,theEtOAcextractdisplayedthehighesteffectin

phosphomolybdenum(1.82mmolTE/g)andmetalchelatingassays

(19.29mg EDTAE/g) and the aqueousextract showed theleast

activity.Ontheotherhand,theMeOHextract hadtheweakest

metalchelatingeffect(12.17mgEDTAE/g).Itisimportantto

high-light that theobserved biologicalactivity ofEtOAc extract was

notrelatedtotheleveloftotalbioactivecompoundssinceit

con-tained thelowest TPC,TFC, andTPAC amongthe threesolvent

extracts.Therefore,otherphytochemicalsareresponsibleforthese

activities.Inaddition,differentclassesofphenolicsandﬂavonoids

have different biological effects and it is possible that speciﬁc

compounds,responsiblefor thestrongactivityinthe

phospho-molybdenumand metalchelatingassay,arepresent inahigher

amountin theEtOAcextract.Thesecompoundsmayalsoactin

combination toproduce a synergisticor additiveeffectto

con-tributetotheiroverallobservedbioactivity[26].

3.5. Enzymeinhibitoryactivity

Currently,enzymeinhibitionhasbecomeanattractiveapproach

in the treatment of various noncommunicable diseases. The

enzymeinhibitoryactivityofP.latifoliaextractsaresummarized

inTable5.Inthetestedextracts,theEtOAcextracthadthe

supe-riorenzymeinhibition,displayingthehighestanticholinesterase

(1.88and1.81mgGALAE/gagainstAChEandBChE,respectively),

anti-amylase (0.70mmol ACAE/g), anti-glucosidase (2.85mmol

ACAE/g),and lipaseinhibitory activity(33.24mgOE/g).In

addi-tion,thehighesttyrosinaseinhibitionwasexhibitedbytheMeOH

extract(60.84mgKAE/gextract).Thehighcontentofﬂavonoids

in the MeOH extract could have contributed to its tyrosinase

inhibitoryactivitysinceastudybyWangetal.[27]founda

pos-itive correlationbetweenTFCand tyrosinase inhibition. Onthe

otherhand,theweakenzymeinhibitoryeffectsweredisplayedby

theaqueousextract.Inaddition,noBChEandlipaseinhibitionwas

observedbytheaqueousextract.

ThehighestcholinesteraseactivityexertedbytheEtOAcofP.

latifoliatendstojustifyitspotentialtherapeuticuseinthe

manage-mentofneurodegenerativediseases.Acetylcholineishydrolyzed

by AChE. So, its inhibition could increase acetylcholine level

andimprovement ofmemoryand brainfunction.Latestagesof

Alzheimer’sisrelatedwithincreasingofBChEactivity[28].Also,

thehighestlipaseinhibitiondisplayedbytheEtOAcextract

indi-catesitspotentialusetoreplacethesyntheticdrugorlistatinthe

managementofobesity.Indeed,orlistathasbeenlinkedtosome

importantadversegastrointestinaleffects[29].

Additionally,theEtOAcextractalsoexhibitedthemostpotent

anti-diabeticactivity.␣-Glucosidaseand␣-amylaseinhibitorshave

gaineda wide applicationinthecontrol ofpostprandial

hyper-glycemia which results in a decline of carbohydrate digestion

toabsorbablemonosaccharides.␣-Amylaseisresponsibleforthe

hydrolysisoflargepolysaccharidestosmallercarbohydrates,which

arethendigestedtoliberateglucosebyintestinal␣-glucosidase.

Inthisway,inhibitionoftheseenzymeswillultimatelydiminish

thepostprandialhyperglycemia.Currently,acarboseandmiglitol

aretheleadingglucosidaseinhibitors.Nonetheless,theyareoften

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Table4

AntioxidantpropertiesofthesamplesA_. Extracts Phosphomolybdenum (mmolTE/gextract)

DPPH(mgTE/g extract) ABTS(mgTE/g extract) CUPRAC(mgTE/g extract) FRAP(mgTE/g extract)

Metalchelatingactivity (mgEDTAE/gextract)

EtOAc 1.82±0.11a _2.50_±_0.71c _14.05_±_0.95c _57.59_±_0.57b _24.94_±_0.87c _19.29_±_0.19a

MeOH 1.45±0.12b _33.71_±_0.43b _47.43_±_0.82b _89.08_±_1.77a _50.61_±_1.11b _12.17_±_0.52b

Water 0.96±0.01c _37.80_±_0.84a _69.92_±_0.47a _87.62_±_2.25a _61.73_±_1.14a _18.35_±_0.05a

A_Values_expressed_are_means_±_S.D._of_three_parallel_{measurements.}_TE:_Trolox_equivalent;_EDTAE:_EDTA_equivalent._Different_letters_refer_{statistically}_signiﬁcant_differences

intheextracts(p<0.05). Table5

EnzymeinhibitoryeffectsofthesamplesA_. Extracts AChEinhibition

(mgGALAE/g extract) BChEinhibition (mgGALAE/g extract) Tyrosinase inhibition(mg KAE/gextract) Amylase inhibition(mmol ACAE/gextract) Glucosidase (mmolACAE/g extract) Lipase(mgOE/g extract) EtOAc 1.88±0.09a _1.81_±_0.09a _48.74_±_1.11b _0.70_±_0.03a _2.85_±_0.30a _33.24_±_0.71a MeOH 1.30±0.11b _0.83_±_0.13b _60.84_±_0.85a _0.41_±_0.03b _1.08_±_0.08b _12.78_±_2.15b Water 0.28±0.03c _na _15.54_±_0.93c _0.10_±_.0.01c _0.87_±_0.05c _na

A_Values_expressed_are_means_±_S.D._of_three_parallel_{measurements.}_GALAE:_Galantamine_equivalent;_KAE:_Kojic_acid_equivalent;_ACAE:_Acarbose_equivalent;_OE:_orlistat

equivalent;na:notactive.Differentlettersreferstatisticallysigniﬁcantdifferencesintheextracts(p<0.05).

ItisimportanttonotethatalthoughtheaqueousextractofP.

latifoliashowedthehighestactivityinmostantioxidantassays,it

displayedtheweakestenzymeinhibitoryeffects.

4. Conclusion

Thisistheﬁrstreportontheenzymeinhibitor(onkeyenzymes

linkedtoglobalhealthproblems)andantioxidanteffectsof

Paren-tucellialatifoliasubsp.latifolia.Theextractsexhibitedsigniﬁcant

free radicalscavenging, reductive,chelating,as wellasenzyme

inhibitoreffects.Metaboliteproﬁlingoftheplantindicatedthat

loganinanditsisomers,rutin,andluteolin-O-hexosidewerethe

mostabundantcompounds.TheresultssuggestthatP.latifoliacan

beagreatpotentialasasourceofbioactivecompoundsfor

possi-bleusesasfunctionalfoodsandnaturalpharmaceuticals.However,

furtherworks(invivoorbioavailability)onthisspecieswouldhelp

usestablishagreaterdegreeofaccuracyonthismatter.

Acknowledgments

Authorsgratefullythankthetechnicalsupportandhumanhelp

ofCICTofUniversidaddeJaén(UJA,MINECO,JuntadeAndalucía,

FEDER).

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