FACULTAD DE FARMACIA Y NUTRICIÓN

FACULTADDEFARMACIAYNUTRICIÓN

Cactus(Opuntiaficus-indicaMill.)cladodesasadietarysourceof

bioaccessible(poly)phenols:effectofheattreatment,gastrointestinal

digestionandhumangutmicrobiotaaction,andbioactivityincolon

Elnopal(Opuntiaficus-indicaMill.)comofuentedietéticade

(poli)fenolesbioaccesibles:efectodeltratamientotérmico,digestión

gastrointestinalyaccióndelamicrobiotaintestinal,yactividad

biológicaenelcolon

ElsyGabrielaDeSantiagoCastanedo

Pamplona,diciembrede2018

Cactus(Opuntiaficus-indicaMill.)cladodesasadietarysourceof

bioaccessible(poly)phenols:effectofheattreatment,gastrointestinal

digestionandhumangutmicrobiotaaction,andbioactivityincolon

Elnopal(Opuntiaficus-indicaMill.)comofuentedietéticade

(poli)fenolesbioaccesibles:efectodeltratamientotérmico,digestión

gastrointestinalyaccióndelamicrobiotaintestinal,yactividad

biológicaenelcolon

Memoria presentada porDña. Elsy Gabriela De Santiago Castanedo para aspirar al

gradodeDoctoraporlaUniversidaddeNavarra.

ElpresentetrabajohasidorealizadobajoladireccióndelaDra.MªPazdePeñaFariza

y la co-direcciónde laDra.Mªde laConcepciónCidCanda en elDepartamentode

CienciasdelaAlimentaciónyFisiologíayautorizamossupresentaciónanteelTribunal

quelohadejuzgar.

EnPamplona,diciembrede2018

Dra.MªPazdePeñaFarizaDra.MªdelaConcepciónCidCanda

FacultaddeFarmaciayNutrición

DepartamentodeCienciasdelaAlimentaciónyFisiología

La directora del Departamento, Dra. Diana Ansorena Artieda, CERTIFICA que el

presentetrabajodeinvestigaciónhasidorealizadoporlagraduadaDña.ElsyGabriela

DeSantiagoCastanedo,enelDepartamentodeCienciasdelaAlimentaciónyFisiología

delaFacultaddeFarmaciayNutricióndelaUniversidaddeNavarra.

Dra.DianaAnsorenaArtieda

EnPamplona,diciembrede2018

Losdatospresentadosenestetrabajosonpartedelasinvestigacionesllevadasacabo

dentrodelproyectodeinvestigación:

“Matrices alimentarias de origen vegetal con potencial actividad antioxidante

sometidasadiferentestecnologías:evaluaciónquímicaybiológica.”

Entidadfinanciadora:MinisteriodeEconomíayCompetitividad

ReferenciadelProyecto:(AGL2014-52636-P).

IP1:Dra.MªPazdePeñaFariza.

IP2:Dra.DianaAnsorenaArtieda.

Duración:1Enero2015-31Diciembre2018.

Acknowledgements/Agradecimientos

Gracias a todas las manos y corazones que contribuyeron a la realización de esteproyecto.

A mis directoras, Mª Paz de Peña y Mª de la Concepción Cid, por su confianza ydedicación en el desarrollo de esta tesis. Gracias por brindarme la oportunidad depertenecerasuequipodeinvestigación.

AlaUniversidaddeNavarra,laAsociacióndeAmigosdelaUniversidaddeNavarra,laComisióndeInvestigación, laFacultaddeFarmaciayNutriciónyalDepartamentodeCiencias de la Alimentación y Fisiología por proporcionarme los medios técnicos,humanosyeconómicospararealizaresteproyecto.Asimismo,aLaFundaciónBancaria“LaCaixa”porlabecademovilidadrecibidapararealizarunapartedemiinvestigaciónenlaUniversidaddeUlster.

AlDr.José́ManuelMorenoRojasylaDra.GemaPereiraCarodelCentroIFAPAAlamedadelObispoenCórdoba,porsuayudaycolaboraciónyacogermeensulaboratorio.

IwouldliketoexpressmygratitudetoDr.ChrisGillforwelcomingmeinhislaboratoryinUlsterUniversityandforhisexpertguidanceinmyresearchworkandhissupportandassistancereceivedduringmystay.Likewise,thanktoDr.KieranTouhyforgivingmetheopportunitytoworkinFondazioneEdmundMachandDra.IlariaCarafaforherkindhelp.

Atodaslasintegrantesde“Broma”,alasqueestuvieronyalasqueaúnsiguen,quemásquecompañerasdetrabajo,seconvirtieronengrandesamigas.GraciasportodoslosinolvidablesmomentosdentroyfueradelaUniversidad,quehicierondeestos4añoslosmejores.TambiénaGwenporsudisponibilidadyayudaenellaboratorio,asícomoenlosgrandesretosmontañeros.

ACarmen,VanessayMariana,porsermásquecompañerasdepisoyhacermesentirquealsalirdelaUniversidadteníaunhogaralcualllegar.

Amifamiliayamigos,porsucariñoypalabrasdealientodesdeladistancia,asícomoen cada vuelta a casa. Especialmente amis padres y hermana, por su comprensión,consejosygranapoyoalolargodeestosaños.

Todostenemossueños,peroparaconvertirlosenrealidad,senecesita

determinación,esfuerzoydedicación.

JesseOwens

ABSTRACT

Cactus (Opuntia ficus-indicaMill.) cladodes, commonly eaten as a fresh or cookedvegetable,presentagreatamountof(poly)phenoliccompoundswhichcanbemodifiedby cooking treatments, gastrointestinal digestion and the human gut microbiota.Likewise, (poly)phenols couldbe able to exert their biological activity into the coloncells.Therefore,themainaimofthisworkwastoevaluatetheimpactofheattreatment(boiling, microwaving, griddling, and frying in olive and soybean oils) on nutritionalcomposition,antioxidantcapacityand(poly)phenolsprofileofcactuscladodes,aswellas their bioaccessibility after an in vitro gastrointestinal digestion and colonicfermentation.Moreover, thepotentialbiologicalactivityofcolonic fermentedcactuscladodes in HT29 colon cells was assessed. Previously, the extraction method of(poly)phenolic compounds with several solvents and conditions was optimized,selectingacombinationofmethanol,acetoneandwaterasthebestone.Inaddition,acid hydrolysis conditions were also optimized in order to identify and quantify(poly)phenolaglyconesbyHPLC-DADanalysis.

Culinaryprocesses,exceptboiling,increasedsolubleandinsolublefibreupto5.0g/100g,becomingcactuscladodesagoodfibresource.Likewise,heattreatmentsincreasedthe totalphenolic contentmeasuredbyFolin-Ciocalteu in cactuscladodes,except inboiledonesduetotheleachinglosses.Atotalof45(poly)phenolswereidentifiedandquantified in rawand cooked cactus cladodesbyUHPLC-PDA-HR-MS,predominatingflavonoids (60−68% total), mainly isorhamnetin derivatives, and phenolic acids(32−40%) with eucomic acids as the predominant ones.Microwaving (1.4-fold) andgriddling (1.2-fold) increased the total amountof compoundswhileboth frying (0.6-fold)andboiling(0.9-fold)producedadecrease.Additionally,antioxidantcapacitywasmeasured,whichwasalsoincreasedaftercookingtreatmentsapplied.

Gastrointestinal digestion significantly (p<0.05) decreased (poly)phenolic compoundsshowing flavonoids a higher retention or degradation (37−63%bioaccessibility) thanphenolic acids (56−87% bioaccessibility). Furthermore, gastrointestinal digestioninducedisomerizationsamongpiscidicandeucomicacids.Thehumangutmicrobiotaalsoproducedahigherdegradationinflavonoidsthaninphenolicacids.However,somecompoundsstillremainedafter24hofcolonicfermentation,beingeucomicacidasthemost relevant. Cactus cladodes after the action of colonic microbiota showed anantigenotoxiceffectonHT29coloncells,reducingtheH2O2-inducedDNAdamage.

RESUMEN

El nopal (Opuntia ficus-indicaMill.), comúnmente consumido como vegetal fresco ococinado,presentaunagrancantidaddecompuestos (poli)fenólicosquepueden sermodificadosporlostratamientosculinarios,asícomoporladigestióngastrointestinalyla acción de la microbiota intestinal. Asimismo, los (poli)fenoles podrían ejercer suactividadbiológicaen las célulasdecolon.Por lo tanto,elobjetivoprincipaldeestetrabajo fueevaluar el impactodel tratamiento térmico (hervido, almicroondas, a laparrilla y fritura en aceites de oliva y de soja) sobre la composición nutricional, lacapacidadantioxidanteyelperfildeloscompuestos(poli)fenólicosenelnopal,asícomosu bioaccesibilidad después de la digestión gastrointestinal in vitro y fermentacióncolónica. Además, se evaluó la potencial actividad biológica del nopal posterior a lafermentacióncolónicaencélulasdecolonHT29.Previamente,seoptimizóelmétodode extracción de compuestos (poli)fenólicos con distintos solventes y condiciones,seleccionandounacombinacióndemetanol,acetonayaguacomoelmejor.Además,seoptimizaronlascondicionesdehidrólisisácidaconelfindeidentificarycuantificarlasagliconasdelos(poli)fenolesmedianteelanálisisporHPLC-DAD.

Losprocesosculinarios,exceptoelhervido,incrementaronlafibrasolubleeinsolublehasta5.0g/100g,siendoelnopalunabuenafuentedefibra.LostratamientostérmicostambiénincrementaronelcontenidototalencompuestosfenólicosmedidoporFolin-Ciocalteu en el nopal cocinado, excepto en el hervido, debido a las pérdidas porlixiviación.Untotalde45(poli)fenolesfueronidentificadosycuantificadosenelnopalcrudo y cocinadoporUHPLC-PDA-HR-MS,predominando los flavonoides (60-68%entotal),principalmentederivadosdeisorhamnetina,asícomoácidosfenólicos(32-40%)siendolosácidoseucómicoslosmayoritarios.Lacocciónalmicroondas(1.4veces)yala parrilla (1.2 veces) aumentaron la cantidad total de compuestos,mientras que lafritura (0.6 veces) y el hervido (0.9 veces) produjeron una disminución. Se evaluótambiénlacapacidadantioxidante,lacualseincrementódespuésdelostratamientosdecocciónaplicados.

La digestión gastrointestinal disminuyó significativamente (p<0.05) los compuestos(poli)fenólicos,mostrandounamayorretenciónodegradacióndelosflavonoides(37-63%bioaccesibilidad)quedelosácidosfenólicos(56-87%bioaccesibilidad).Además,ladigestióngastrointestinalindujoisomerizacionesenlosácidospiscídicoyeucómico.Lamicrobiotaintestinalprodujotambiénunamayordegradaciónenlosflavonoidesqueenlosácidosfenólicos.Sinembargo,algunospolifenolesaúnpermanecierondespuésde24hdefermentacióncolónica,siendoelácidoeucómicoelmásrelevante.Elnopal

despuésdelaaccióndelamicrobiotaintestinal,mostróunefectoantigenotóxicosobrelascélulasdecolonHT29,reduciendoeldañoenelADNinducidoporH2O2.

LISTOFABBREVIATIONS

ABTS• +2,2’-Azinobis (3-ethylbenzothiazonile-6-sulfonic acid) diammoniumsalt

ANOVA AnalysisofVariance

CID Collision-InducedDissociation

DAD Diode-ArrayDetector

dm Drymatter

DMEM Dulbecco'sModifiedEagleMedium

DMSO Dimethylsulfoxide

DNA DeoxyribonucleicAcid

DPPH• 2,2-Diphenyl-1-picrylhydrazyl

EDTA Ethylenediaminetetraaceticacid

ESI ElectrosprayInterface

FACS Fluorescence-ActivatedCellSorting

FAME FattyAcidMethylEsters

FBS FetalBovineSerum

FC Folin-Ciocalteu

FDA FoodandDrugAdministration

FID FlameIonizationDetector

FRAP FerricReducingAntioxidantPower

GA GallicAcid

GIT GastrointestinalTract

H2O2 HydrogenPeroxide

HCl HydrochloricAcid

HPLC HighPerformanceLiquidChromatography

HESI HeatedElectrosprayIonization

MRM MultipleReactionMonitoring

MS MassSpectrometry

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide

MUFA MonounsaturatedFattyAcids

nd Notdetected

•O2− SuperoxideRadicals

•OH− HydroxylRadicals

ORAC OxygenRadicalAbsorptionCapacity

PBS PhosphateBufferedSaline

PCA PrincipalComponentAnalysis

PDA PhotodiodeArray

PenStrep PenicillinStreptomycin

PUFA PolyunsaturatedFattyAcids

RDA RecommendedDietaryAllowance

ROS ReactiveOxygenSpecies

SD StandardDeviation

SFA SaturatedFattyAcids

TFC TotalFlavonoidCompounds

TPC TotalPhenolicCompounds

tr Traces

Trolox 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid

UHPLC UltraHighPerformanceLiquidChromatography

WHO WorldHealthOrganization

CONTENTS

INTRODUCTION..............................................................................................................1

OBJECTIVES/OBJETIVOS...............................................................................................19

EXPERIMENTALDESIGN................................................................................................23

RESULTS.......................................................................................................................27

OBJECTIVE1..........................................................................................................29

Paper 1 (In preparation): “Optimization of the methodology for extraction,identificationandquantificationof(poly)phenoliccompoundsincactus(Opuntiaficus-indica)cladodes”..........................................................................................31

OBJECTIVE2..........................................................................................................51

Paper2:“Impactofcookingprocessonnutritionalcompositionandantioxidantsofcactuscladodes(Opuntiaficus-indica)”............................................................53

OBJECTIVE3..........................................................................................................63

Paper3:“Digestibilityof(poly)phenolsandantioxidantactivityinrawandcookedcactuscladodes(Opuntiaficus-indica)”................................................................65

OBJECTIVE4and5................................................................................................85

Paper4(Underreview):“DigestionandcolonicfermentationofrawandcookedOpuntiaficus-indicaimpactsbioaccessibilityandbiologicalactivity....................87

GENERALDISCUSSION................................................................................................117

CONCLUSIONS/CONCLUSIONES.................................................................................133

RESEARCHDISSEMINATION.......................................................................................139

ANNEX.........................................................................................................................143

INTRODUCTION

Introduction

3

Cactus(Opuntiaficus-indicaMill.)cladodes

The cactus (Opuntia ficus-indicaMill.) is a native plant of the American continent,member of the family Cactaceae, which produces edible seeds, fruits andphotosynthetic stems (cladodes) (Figure 1). Cactus can grow in arid and semi-aridclimates and is also cultivated in theMediterranean countries (Spain, Italy, Greece,Egypt,Turkey),CentralandSouthAfrica,aswellasintheMiddleEast(Israel,Jordan),AustraliaandIndia(Stintzing&Carle,2005).

Figure1.Cactus(Opuntiaficus-indicaMill.)

Cactuscladodeshavebeenusedtopreparedifferentproductssuchasjams,juices,bodylotions, shampoos, creams, etc., as well as functional constituents for food andtraditional medicine for several health problems as metabolic diseases, diabetes,arteriosclerosis and inflammatory diseases (Osuna-Martínez, Reyes-Esparza, &Rodríguez-Fragoso,2014;Patel,2014).

Theinterest incactuscladodeshasbeenincreasingbecauseoftheirgreatnutritionalcompositionincludingahighcontentoffibresuchaspectin,lignin,mucilage,celluloseandhemicelluloseinvolvedinhealthbenefitsashypoglycemicandhypolipidemicaction(Hernández-Urbiola,Pérez-Torrero,&Rodríguez-García,2011;Jun,Cha,Yang,Choi,&Kim,2013;López-Romeroetal.,2014;Méndez,Flores,Martín,RodríguezRodríguez,&DíazRomero,2015).Likewise,ithasbeenshownthatcactuscladodescontainbioactivecompoundssuchas(poly)phenolswhichprovidepotentialantioxidantactivityrelatedto anti-inflammatory effects (Alimi et al., 2010; El-Mostafa et al., 2014; Feugang,Konarski,Zou,Stintzing,&Zou,2006).

Introduction

4

(Poly)phenoliccompoundsaresecondarymetabolitesofplantswhichhavestructurescharacterizedbythepresenceofatleastonearomaticringwithoneormorehydroxylgroupsattached. (Poly)phenolscanbeclassifiedbythenumberandpositionof theircarbonatomsintwomaingroups:flavonoidsandnon-flavonoidssuchasphenolicacids(Crozier, Jaganath,& Clifford, 2009;Manach, Scalbert,Morand, Rémésy,& Jiménez,2004). Epidemiological studies have investigated the role of (poly)phenols in healthbenefits including cancer prevention, incidence, or mortality, as well as protectionagainstdiabetes,neurodegenerationandcardiovasculardiseases (Crozier,DelRio,&Clifford,2010;DelRioetal.,2013;Tresserra-Rimbauetal.,2014).Thebeneficialeffectsof(poly)phenoliccompoundsareattributedtotheirantioxidantactivitythatreducethefree-radical oxidative stress, which is responsible for cellular damage (Crascì, Lauro,Puglisi,&Panico,2018).

Previous studies in raw cactus cladodes have been identified the presence of(poly)phenoliccompoundssuchasphenolicacids:eucomic,piscidic,ferulic,4-hydroxybenzoic, malic, citric and salicylic acids; as well as flavonoids, predominatingisorhamnetin, kaempferol and quercetin, most of them usually in their glucosides,rutinosides and xylosides forms (Astello-García et al., 2015; Ginestra et al., 2009;Guevara-Figueroaetal.,2010;Menaetal.,2018).Inaddition,antioxidantcapacityhasbeenevaluatedinrawcactuscladodesbyDPPH,FRAPandORACassays,showingagreatcorrelationwiththe(poly)phenolscontent(Astello-Garcíaetal.,2015;Corral-Aguayo,Yahia,Carrillo-Lopez,&González-Aguilar,2008;Santos-Zea,Gutiérrez-Uribe,&Serna-Saldivar,2011).Enzymatic(superoxidedismutase,ascorbateperoxidaseandcatalase)andothernon-enzymatic(carotenoids)systemsmightalsocontributetotheantioxidantcapacityofcactuscladodes(Ventura-Aguilar,Rivera-Cabrera,Méndez-Iturbide,Pelayo-Zaldívar,&Bosquez-Molina,2013).

Theextractionof(poly)phenolsfromthefoodmatrixisanimportantprocesstoobtainthecompoundsefficientlyinordertoidentifyandquantifythosethatcontributetothehealthbenefits and also to incorporate them intodifferent functional products. Theextraction methods depend on the technique conditions applied, the polarity ofsolventsandtheextractiontime.StudiesinO.ficus-indicaextractshavebeenobtainedusingmethanol(Medina-Torresetal.,2011;Menaetal.,2018;Sánchezetal.,2014),ethanol(Guevara-Figueroaetal.,2010;Lee,Kim,Kim,&Jang,2002)oracombinationof several solvents such as acetone, methanol and water (Avila-Nava et al., 2014;Ramírez-Moreno,Córdoba-Díaz,Sánchez-Mata,Díez-Marqués,&Goñi,2013).

Inaddition,aftertheextraction,cactuscladodesareinmostcaseshydrolysedtoreleasetheaglyconesfromtheirrespectiveglycosidespriortoanalysisbyHPLC-DAD.Themost

Introduction

5

common method for breakdown flavonoid glycosides is the acid hydrolysis withhydrochloricacid(HCl)undercontinuousheating(Khoddami,Wilkes,&Roberts,2013;Moussa-Ayoub,El-Samahy,Kroh,&Rohn,2011).

Although there are different conditions reported, there is not a consensus on theoptimal methodology and conditions to follow for the extraction of (poly)phenoliccompoundsandthehydrolysisofaglyconesforcactuscladodes.

In America, especially in Mexico, cladodes known as “nopales”, are part of thetraditional diet and are commonly eaten as a fresh or cooked vegetable includingboiling, griddling and frying (Stintzing & Carle, 2005). Several factors such as heattreatment, gastrointestinal digestion and the gut microbiota affect the content of(poly)phenols and their consequentbioaccesibility andbiological activity (D’Archivio,Filesi,Varì,Scazzocchio,&Masella,2010).

Heattreatment

Heattreatmentcaninducechangesinchemicalandnutritionalqualitiesoffoodssuchaswaterloss,changesinenergy,fatcontentandconsequentlyinthefattyacidprofile,as well as in the amount of bioactive compounds such as (poly)phenols (Miglio,Chiavaro, Visconti, Fogliano, & Pellegrini, 2008) and also producing some beneficialaspects,improvingpalatability,texture,flavouranddigestibility(Guillén,Mir-Bel,Oria,& Salvador, 2017; van Boekel et al., 2010). Likewise, heat treatments produce thereleaseof (poly)phenoliccompoundsfromthecellandwallsplants leadingtoobtainbetter extractability and higher concentrations in cooked than in fresh samples.Dependingonthevegetableandthecookingtechniquesapplied,aswellastemperatureandtime,(poly)phenoliccompoundsandconsequentlytheirantioxidantcapacity,couldbeincreasedordegraded(Palermo,Pellegrini,&Fogliano,2014;Pellegrinietal.,2009;Ramírez-Anaya,Samaniego-Sánchez,Castañeda-Saucedo,Villalón-Mir,&DeLaSerrana,2015).

Thehightemperatureappliedinheattreatmentsasgriddlingandfryingcanfavourtheinactivation of oxidative enzymes and the hydrolization of phenolics glycosides,inducing the formation ofMaillard reaction products,which besides giving sensorialcharacteristics,havealsoshownhighantioxidantproperties(Nunes&Coimbra,2010;Pérez-Martínez,Caemmerer,DePeña,Cid,&Kroh,2010).

The effect of heat treatments on (poly)phenolic compounds as well as on theirantioxidant activity are controversial. Previous studies in cooked vegetables such asgriddled and fried onion, pepper, cardoon (Juániz, Ludwig, Huarte, et al., 2016b),

Introduction

6

griddledeggplant (LoScalzoetal.,2016)andmicrowavedbroccoli (Turkmen,Sari,&Velioglu, 2005) have demonstrated the increase of (poly)phenols after cookingtreatments.Nonetheless,decreasesinsquash,peas,leek,(Turkmenetal.,2005),kaleand red cabbage (Murador, Mercadante, & De Rosso, 2015) after boiling andmicrowavinghavebeenreported.

Even though the nutritional composition, identification of some (poly)phenoliccompounds and antioxidant capacity in raw cactus cladodes have been studied,researchabouttheeffectofheattreatmentonthemisverylimited.Ramírez-Morenoetal.(2013)andJaramillo-Floresetal.(2003)foundthatthetotalcontentofphenoliccompoundsandconsequently their antioxidant capacitydecreased significantlyafterboiling.However,changesinthe(poly)phenolsprofileincactuscladodesaftercookingtreatmentsstillremainunknown.

Bioaccessibilityof(poly)phenoliccompounds

Bioaccessibilityhasbeendefinedastheamountorfractionofafoodcompound,whichisreleasedfromthefoodmatrixinthegastrointestinaltract(GIT),becomingavailableforabsorption(Cilla,Bosch,Barberá,&Alegría,2018).

Bioaccessibilityofbioactivecompoundsdependsonboththecharacteristicsofthefoodmatrix and the gastrointestinal digestion conditions, leading to the structuralbreakdown of cellwallmatrix and their release them from fibres and proteins. Thestabilityofthecompoundscanbealsomodified,tendingtobemoresusceptibilitiestodegradation and isomerization (Acosta-Estrada, Gutiérrez-Uribe, & Serna-Saldívar,2014;Almingeretal.,2014).

Invitrogastrointestinaldigestionmethodsarewidelyusedtoevaluatebioaccessibilityoffoodcomponents,simulatingthephysiologicalconditionstakingplaceinthehumanGIT throughout the mouth, stomach and intestine, including enzymatic, pH andtemperatureparameters(Cardoso,Afonso,Lourenço,Costa,&Nunes,2015;Hur,Lim,Decker,&McClements,2011;Minekusetal.,2014).Thecomplexphysicochemicalandphysiologicalconditionssuchasdigestiveenzymes,pHchangesandtemperatureoftheGITcanmodify (poly)phenolsand, consequently, theirbioaccessibility (Saura-Calixto,Serrano,&Goñi,2007).

Studies in vegetables after an in vitro gastrointestinal digestion applied arecontroversial,showingthat(poly)phenoliccompoundsdecreaseinartichoke(Garbettaetal.,2014),pepper(Juániz,Ludwig,Bresciani,etal.,2016a)andcardoon(Juánizetal.,2017), whereas others reports demonstrate that compounds are not affected by

Introduction

7

digestion process (D’Antuono, Garbetta, Linsalata, Minervini, & Cardinali, 2015;Kamiloglu&Capanoglu,2014).However,theevaluationof(poly)phenolsofbothrawand cooked cactus cladodes after gastrointestinal digestion and their antioxidantactivitieshavenotyetbeenreported.

Mostphenoliccompoundshavecovalentinteractionswiththefibrefromthecellwall,formingesterlinkageswhicharenothydrolysedbyphaseIandIIenzymes,limitingtheirrelease into the small intestine and can reach the colon to be metabolized by themicrobiota.Likewise,thefibrecanprotect(poly)phenolsfromthedigestiveconditionsfavouring their bioaccessibility and maintaining their antioxidant properties(Domínguez-Avilaetal.,2017).

Mostofthe(poly)phenolspresentintheirglycosideformsarenotabsorbedduringthegastrointestinaltractandreachthecolontobehydrolysedbycolonicmicrobiotabeforeabsorption. The colonic microflora, inhabited by a diverse population ofmicroorganisms, hydrolyses glycosides into aglycones and degrades them producingmetabolitestosmallerandmoreabsorbablemolecules(Rodriguez-Mateosetal.,2014;Serraetal.,2012;Williamson&Clifford,2017).

(Poly)phenols biotransformations by the large intestinemicrobiota enzymes includehydrolysis, dehydroxylation, deconjugation, demethylation, ring cleavage anddecarboxylationactivities.Therefore,gutmicrobiotametabolismcanalsomodulatetheeffects of dietary (poly)phenolic compounds by altering their absorption and theirbiologicalactivity(Espín,González-Sarrías,&Tomás-Barberán,2017;Scalbert,Morand,Manach,&Rémésy, 2002). In fact, studies in Ileostomypatientshavedemonstratedthat,despitechangesduringtheGIT,agreatamountof(poly)phenoliccompoundsinberriesarenotabsorbedintocirculationfromthesmallintestineandareabletopassintothecolontobedegradedby thecolonicmicrobiota(Brownetal.,2012;Brown,Nitecki,etal.,2014).

Invitrocolonicfermentationmodelshaveperformedusinganaerobic,stirred,pHandtemperaturecontrolledfaecalbacterialculturestosimulatetheconditionslocatedinthe proximal region of the human large intestine (Low,Hodson,Williams,D’Arcy,&Gidley,2016;Rechneretal.,2004;Vollmeretal.,2017).Effectsofcolonicmicrobiotaonthephenolic profilehavedemonstrated in somevegetables aspepper (Juániz et al.,2016a),cardoon(Juánizetal.,2017),fruitsasapples(Koutsosetal.,2017)andincoffee(Ludwig, de Peña, Cid, & Crozier, 2013), showing a degradation of most of thecompounds, but at the same time, the formation of new metabolites. Hence,antioxidant and beneficial properties could be attributed not only to the nativecompounds,butalsototheirmetabolites.Furthermore,metabolitesgeneratedmight

Introduction

8

dependon the foodmatrix, culinaryprocesses,aswellas the specific (poly)phenoliccompositionofeachfood.

Biologicalactivityof(poly)phenoliccompounds

The remaining bioaccessible (poly)phenols which reach the colon could exert theirantioxidantactivityandmodulatemetabolicpathwaysrelatedtothegutmicrobiotaandcellprocesses,asinflammation,immunity,cellproliferationandoxidativestress(Espínetal.,2017;Heyman-Lindénetal.,2016;Marín,Miguélez,Villar,&Lombó,2015).

Potentialantioxidanteffectsof(poly)phenolsmaybeduetothechelatingredoxactivemetals or by directly scavenging ROS (including H2O2), hydroxyl radicals (•OH−) andsuperoxideradicals(•O2−),bybeingeffectiveelectrondonorsandmodulatingbiologicalprocesses(Cemeli,Baumgartner,&Anderson,2009).

Invitrocellculturestudiesareoneofthemosteffectivewaytoinvestigatetheimpactofthegutmicrobiotaonthetransformationsof(poly)phenols,andtherefore,ontheirbiological activity. Some researches have focused on the effect of (poly)phenolicsagainstcoloncancercellproliferation,intestinalinflammationandoxidativestressusingintestinalcellmodelssuchascolonicHT29andCaco2cells(Brown,Latimer,etal.,2014).Antigenotoxicityof(poly)phenoliccompoundsincellcultureshasbeenalsoevaluatedtomeasuretheDNAdamageafterchallengingwithhydrogenperoxide(H2O2),toshowifthecompoundshaveapotentialprotectionreducingtheoxidativedamage(Azqueta&Collins,2016).

Studies in some fruits such as apples, berries and cherries (Olsson, Gustavsson,Andersson,Nilsson,&Duan,2004)andinflavonoidssuchasrutin(benSghaieretal.,2016)andquercetin(Min&Ebeler,2009)havereportedanticytotoxicandantigenotoxiceffects onHT29 andCaco2 cells, demonstrating thepotential antioxidant propertiesinvolved. Although studies have shown the biological activity of (poly)phenols, fewinvestigationshaveconsideredtheeffectof intestinaldigestionandtheactionofthehumanmicrobiota.Recently,studiesinberriesafterpreviousgastrointestinaldigestionandcolonicfermentation,haveshowntheeffectof(poly)phenolsinthereductionofH2O2-inducedDNAdamage(Brownetal.,2012;Coatesetal.,2007;LópezdelasHazas,Mosele, Macià, Ludwig, & Motilva, 2016; McDougall et al., 2017). However, thebiological activity could be different depending on the food extracts or isolatedcomponents.

RegardingtoO.ficus-indica,studieshavesuggestedthatthecactuspearfruithaveapotential antioxidant effects against cervical, ovarian and bladder cancer cell lines(Osuna-Martínezetal.,2014).Likewise,extractsfromjuicesofcactuspearfruits(Serra,

Introduction

9

Poejo, Matias, Bronze, & Duarte, 2013). have also demonstrated anti-proliferativeeffectsonHT29cells.Nevertheless,gastrointestinaldigestionandgutmicrobiotaactionwerenotconsidered.

Introduction

10

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15

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OBJECTIVES/OBJETIVOS

Objectives

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CactuscladodeisoneofthenativefoodsofthetraditionalMexicandietproposedtopreventdiseasesrelatedtooxidativestress,suchascancer,diabetesandcardiovasculardiseases due to the presence of bioactive antioxidant compounds. Among themainbioactivecomponentspresentincactuscladodes,(poly)phenoliccompoundsarefound.However,thebioaccessibilityofthesecompoundsdependsonseveralfactorssuchastheinteractionwithotherfoodcomponents(proteins,carbohydrates,fatsandfibre),aswellastheculinarytechnologicalprocessesapplied.Moreover,afteringestion,thestructureof(poly)phenolscouldbemodifiedalongthegastrointestinaltract.Likewise,upon reaching the colon, these compounds could be transformed by the intestinalmicrobiota into smaller and more absorbable metabolites which could exert theirbiologicalactivityatleastinthecoloncells.

Therefore, the main aim of this PhD thesis was to evaluate the bioaccessibility of(poly)phenolic compounds of raw and cooked cactus cladodes using simulatedgastrointestinaldigestionmodelandcolonicmicrobiotaaction,aswellastoevaluatethepotentialbiologicalactivityincoloncells.

Toachievethisaim,thefollowingpartialobjectivesincactus(Opuntiaficus-indicaMill.)cladodeswereestablished:

1. To optimize the methodological conditions for extraction of (poly)phenoliccompounds.

2.Toevaluatetheinfluenceofheattreatment(boiling,microwaving,griddling,fryinginolive and soybean oils) on nutritional composition, antioxidant capacity and(poly)phenoliccompounds.

3.Toevaluatethebioaccessibilityof(poly)phenoliccompoundsandtheirantioxidantcapacityafterinvitrogastrointestinaldigestion.

4.Toevaluatetheactionofcolonicmicrobiotaon(poly)phenoliccompounds.

5. To evaluate the biological activity of colonic fermented cactus cladodes on HT29humancoloncancercells.

Objetivos

22

Elnopalesunodelosalimentosautóctonosdeladietamexicanatradicionalsugeridospara prevenir enfermedades relacionadas con el estrés oxidativo, como cáncer,diabetes y enfermedades cardiovasculares debido a la presencia de compuestosbioactivos antioxidantes. Entre los principales componentes bioactivos presentes seencuentran los compuestos (poli)fenólicos.Noobstante, la bioaccesibilidadde estoscompuestosdependedediversosfactorescomolainteracciónconotroscomponentesde los alimentos (proteínas, hidratos de carbono, grasas y fibra), así como de losprocesos tecnológicos culinarios aplicados. Por otra parte, la estructura de loscompuestos (poli)fenólicos se puedemodificar a lo largo del tracto gastrointestinal.Asimismo, al llegar al colon, estos compuestos podrían ser transformados por lamicrobiota intestinal en metabolitos, que son moléculas más pequeñas y másabsorbiblescapacesdeejercersuactividadbiológicaalmenosenlascélulasdelcolon.

Porlotanto,elobjetivogeneraldeestaTesisDoctoralfueevaluarlabioaccesibilidaddeloscompuestos(poli)fenólicosdelnopaltratadotérmicamenteenunsistemamodelodedigestióngastrointestinalyacciónde lamicrobiota,asícomoevaluarsupotencialactividadbiológicaaniveldelcolon.

Paraellosepropusieron lossiguientesobjetivosparcialesenelnopal (Opuntia ficus-indicaMill.):

1.Optimización de lametodología de análisis para la extracción de sus compuestos(poli)fenólicos.

2.Evaluacióndela influenciadeltratamientotérmico(hervido,microondas,plancha,fritura en aceite de oliva y de soja), sobre su composición nutricional, capacidadantioxidanteycompuestos(poli)fenólicos.

3.Evaluaciónde labioaccesibilidaddesuscompuestos (poli)fenólicosysucapacidadantioxidantetrasladigestióngastrointestinalinvitro.

4. Evaluación de la acción de la microbiota intestinal sobre sus compuestos(poli)fenólicos.

5.EvaluacióndelaactividadbiológicadenopalposterioralafermentacióncolónicaencélulasdecáncerdecolonHT29.

EXPERIMENTALDESIGN

Experimentaldesign

25

Extraction methodology

Solvents Successiveextractions

Objective 1

Heat treatment

Raw Boiled Microwaved Griddled Fried inoliveoil Fried insoybeanoil

Bioaccessibility

Objective 2

Invitrogastrointestinaldigestion

Invitro faecal fermentation

(Poly)phenolic compounds (UHPLC-ESI-MS/MS)- Flavonoids

- Phenolic acids

Objective 3

Objective 4

Biological activity

HT29humancoloncancercells

Objective 5

Cactus cladodes(Opuntia ficus-indica)

MoistureProteinAsh

Fat(Fattyacids)Carbohydrates

Fibre(soluble and insoluble)Energy

Totalphenolic compoundsAntioxidant capacity(DPPH,

ABTS)(Poly)phenolic compounds

(HPLC-DAD)

(Poly)phenolic compounds (UHPLC-ESI-MSn)- Flavonoids

- Phenolic acidsAntioxidant capacity(DPPH)

Cytotoxicity (MTT assay)Geno-protective effect (Comet assay)

Cellcycle analysis

Total(poly)phenolic contentTotalflavonoid content

Antioxidant capacity(DPPH, ABTS)(Poly)phenolic compounds (HPLC-DAD)

Effect ofcolonic microbiota

Paper1(inpreparation)Paper2

Paper3Paper4(underreview

)

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Objective1

To optimize the methodological conditions for extraction of (poly)phenoliccompoundsofcactus(Opuntiaficus-indicaMill.)cladodes.

Optimización de la metodología de análisis para la extracción de los compuestos(poli)fenólicosdenopal(Opuntiaficus-indicaMill.).

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Paper1

Extraction of (poly)phenolic compounds of cactus (Opuntia ficus-indica Mill.)cladodes:impactofsolventsandconditions

ElsyDeSantiago,IsabelJuániz,ConcepciónCidandMaría-PazDePeña.(Inpreparation).

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TITLE:Extractionof (poly)phenoliccompoundsofcactus (Opuntia ficus-indicaMill.)cladodes:impactofsolventsandconditions

AUTHORS:ElsyDeSantiagoa,IsabelJuániza,ConcepciónCidaandMaría-PazDePeñaa*

aUniversidaddeNavarra,FacultaddeFarmaciayNutrición,DepartamentodeCienciasdelaAlimentaciónyFisiología,C/Irunlarrea1,E-31008Pamplona,Spain.IdiSNA,NavarraInstituteforHealthResearch.Pamplona,Spain.

*Correspondingauthor:María-PazdePeña.Tel:+34948425600(806580);Fax:+34948425740.E-mailaddress:[email protected]

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ABSTRACT

Researchincactus(Opuntiaficus-indicaMill.)cladodesasafunctionalfoodisincreasingbecause of their (poly)phenolic compounds and health-promoting ability. Extractionconditions are the basis to obtain the highest amount of bioactive compoundswithantioxidant properties. Therefore, several extraction solvents and conditions werecarried out. Even ethanol (80 and 100%) extracts high amount of (poly)phenols,successive extractionwithmethanol, acetone andwater, favoured the extraction offlavonoidsandantioxidantcapacity(DPPHandABTS).Additionally,inordertoquantifythe most abundant (poly)phenols by HPLC-DAD, aglycones were released fromflavonoidglycosidesbyacidhydrolysisthrougharefluxat90°Catdifferenttimes(2hand3h)andHClconcentrations(0.6,1.2,1.5and1.7M).Afterhydrolysis,3flavonoidsaglycones(isorhamnetin,quercetinandkaempferol)and2phenolicacids(ferulicandhidroxybenzoicacids)wereidentifiedandquantified.Comparingdifferentprocedures,acid hydrolysis with 1.5M HCl during 2h was the best for the release of flavonoidaglycones, but a less concentrationofHCl in a shorter timeof reflux showedbetterresultsforphenolicacids.KEYWORDS: Phenolics, flavonoids, phenolic acids, antioxidant capacity, cactus,extraction,hydrolysis,aglycones.

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1.Introduction

Studieshavedemonstratedthataconsumptionoffruitsandvegetables isassociatedwithalowerriskofchronicdiseasessuchascancer,diabetesorcardiovasculardiseasesduetotheantioxidantpropertiesofbioactivecompoundsthatinhibitreactiveoxygenspecieswhichareresponsibleforoxidativestressandcellulardamage(DelRioetal.,2013).

Thecactus(Opuntiaficus-indicaMill.),anendemicAmericanplant,hasbeenusedfreshorprocessedforhumanconsumption,aswellasafunctionalconstituentforfoodandpharmaceutical products due to the important content of bioactive compounds like(poly)phenols(El-Mostafaetal.,2014).

Cactusstems,knownascladodes,havebeencharacterizedbythepresenceofphenolicacids and flavonoids such as isorhamnetin, kaempferol, quercetin and isoquercitrin,whichmost of them, are usually in their glucosides, rutinosides and xylosides forms(Astello-Garcíaetal.,2015;Ginestraetal.,2009;Weirong,Xiaohong,&Tang,2010).

The (poly)phenolic compoundsextraction is themost important stepbeforeanalysisand, consequently, characterization of bioactive compounds of cactus cladodes. Acommonissueregardingto(poly)phenolsextractionisthelinkageswithdifferentfibresstructures such as pectin, lignin, mucilage, cellulose, hemicellulose where thecompounds can be retained (Hernández-Urbiola, Pérez-Torrero, & Rodríguez-García,2011). Several studies with O. ficus-indica have used different solvents for theirextractionlikemethanol(Sánchezetal.,2014),ethanol(Guevara-Figueroaetal.,2010)oracombinationofseveralsolventssuchasacetone,methanolandwater(Avila-Navaet al., 2014; Ramírez-Moreno, Córdoba-Díaz, Sánchez-Mata, Díez-Marqués, & Goñi,2013). It is also important to control the conditions as temperature, time, andconcentrations used in the extraction methods to avoid the degradation of thecompoundsorunwantedreactionslikeoxidation.

Inaddition, after theextraction, theglycosidesof cactus cladodesare inmost caseshydrolysedtotheirrespectiveaglyconespriortoanalysisbyHPLC-DADbecauseofthelack of pure standards. The most common method for breakdown the flavonoidglycosidesistheacidhydrolysiswithhydrochloricacid(HCl)undercontinuousheating(Moussa-Ayoub,El-Samahy,Kroh,&Rohn,2011,Khoddami,Wilkes,&Roberts,2013).Although there are different conditions reported, there is not a consensus on theoptimalmethodology and conditions to follow for the extraction and hydrolysis forcactuscladodes.

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Therefore, the main aim of this work was to optimize the extraction conditions of(poly)phenolic compounds and antioxidant capacity from cactus cladodes usingdifferentsolvents.Afterselectingthebestextractionmethod,differentconditionsofacid hydrolysis were developed to release phenolic aglycones and to identify andquantifythembyHPLC-DAD.

2.Materialandmethods

2.1Chemicalandreagents

Ethanol,methanol,acetoneandhydrochloricacid(HCl)wereofanalyticalgradefromPanreac (Barcelona, Spain). The acetonitrile and formic acid (HPLC grade)were alsopurchasedfromPanreac(Barcelona,Spain).Folin–Ciocalteureagent,trolox(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid),2,2ʹ-azinobis(3-ethylbenzothiazonile-6-sulfonicacid)diammoniumsalt(ABTS),2,2-diphenyl-1-picrylhydrazyl(DPPH·)andrutin;as well as the standards used for identification and quantification of phenoliccompounds (isorhamnetin, kaempferol, quercetin, ferulic acid and hydroxybenzoicacid),werepurchasedfromSigma-Aldrich(Steinheim,Germany).

2.2Samplespreparation

Freshcactus(O.ficus-indica)cladodeswereobtainedfromBioArchenCompanylocatedinMurcia,Spain.Cladodeswerewashedandthethornswereremovedmanually.Then,werecutintoequalsmallpieces,mixedwellandlyophilizedinafreezedryerCryodos-80(Telstar,Terrasa,Spain),andstoredat-18°Cuntilanalysis.

2.3Extractionmethods

Fourdifferentextractswerepreparedasfollows:

a)100%water:TheextractwaspreparedaccordingtoSiddiqmethod(Siddiq,Roidoung,Sogi, & Dolan, 2013) with somemodifications. Briefly, 30mL of distilledwater wasaddedto2gof lyophilizedcactuscladodes.Thecontentwasmixedonamechanicalshaker for 1 h at room temperature and then centrifuged at 4,000 rpm for 10min.Supernatantwascollectedandresidueswerere-extractedtwiceusing10mLofdistilledwater by vortexing (1 min) and centrifuged at 4,000 rpm for 5 min. All threesupernatantswerecombinedandfrozenat-18°Cbeforeanalysis.

b)80%ethanol (EtOH):Asecondextractwasperformedaspreviouslydescribed,butinsteadofadding100%ofdistilledwater,thirtymLofethanol/water(80/20)wasused.Likewise,aftermixingthethreesupernatants,theextractwasfrozenat-18°C.

c)100%EtOH:Leeetal.(2002)methodwasusedforthisextract.Fivegoflyophilizedcladodesweremixedwith50mLofEtOHandshakenatroomtemperaturefor72h.

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Then,itwasfilteredandcentrifugedat4,000rpm10min.Supernatantwasstoredat-18°Cbeforeanalysis.

d)Successiveextractions:ThelastextractwaspreparedbasedonmethodofAvila-Navaetal.(2014)withmodifications.Amixof50mLofmethanol/watersolution(50/50)wasaddedto4goflyophilizedcladodes.Thismixturewasstirredfortwohandthenvacuumfiltered. The resulting filtratewas saved and stored. The residuewas subjected to asecondextractionwith50mLofacetone/water(70/30)solutionandagitationfortwoh. The mixture was vacuum filtered and stored. A third extraction was performedstirringtheresiduein50mLofdistilledwaterforthirtyminandalsovacuumfiltered.Resultingfiltratesweremixedtogetherandstoredat-18°Cbeforeanalysis.

2.4Totalphenoliccompounds(TPC)

TPCweremeasuredusingtheFolin–CiocalteureagentaccordingtoSingleton'smethod(Singleton&Rossi,1965).Eachextractwasproperlydilutedindemineralizedwaterand100μLweremixturewith500μLofFolin–Ciocalteureagentand7.9mLofdemineralizedwater. After 2 min, 1.5 mL of a 7.5% sodium carbonate solution was added. Then,sampleswereincubatedindarknessfor90minatroomtemperature.Theabsorbanceof the samplewasmeasured at 765 nm in a spectrophotometer Lambda 25UV/VIS(PerkinElmerInstruments,Madrid,Spain).GallicAcid(GA)wasusedasreference,andtheresultswereexpressedasmilligramsofGAequivalentpergramofdrymatter(mgGA/gdm).

2.5Totalflavonoidcontent(TFC)

Thealuminumchloridemethod(Lamaison&Carnet,1990)wasusedforestimationofTFCoftheextractedsamples.Analiquotof100µLofeachextractproperlydilutedwasaddedto1mLofa2%AlCl3·6H2Omethanolsolution.Themixturewasshaken,andafter10minof incubationatroomtemperature, theabsorbancewasreadat430nminaspectrophotometerLambda25UV/VIS (PerkinElmer).Rutinwasused forcalibrationcurveandresultswereexpressedasmilligramofrutinequivalentpergramofdrymatter(mgrutin/gdm).

2.6AntioxidantcapacitybyABTSassayTheABTSantioxidantcapacitywasperformedaccording to themethodof (Reetal.,1999). The radicals ABTS•+ were generated by the addition of 0.36 mM potassiumpersulfatetoa0.9mMABTSsolutionpreparedinphosphatebufferedsaline(PBS)(pH7.4),andtheABTS•+solutionwasstoredindarknessfor12h.TheABTS•+solutionwasadjustedwithPBS toanabsorbanceof0.700 (±0.020) at734nm ina3mL capacitycuvette (1cm length)at25 °C (Lambda25UV–VIS spectrophotometer,Perkin-Elmer

Results

38

Instruments, Madrid, Spain). An aliquot of 100 μL of each extract sample properlydilutedindemineralizedwater,wasaddedto2mLofABTS•+solution.After18min,theabsorbancewasmeasuredat734nm.CalibrationcurvewasperformedwithTroloxsolution(awater-solublevitaminEanalog),andresultswereexpressedasmicromolesofTroloxequivalentpergramofdrymatter(μmolTrolox/gdm).

2.7AntioxidantcapacitybyDPPHassay

Theantioxidantcapacitywasalsomeasuredusing2,2-diphenyl-1-picrylhydrazyl(DPPH•)decolorization assay (Brand-Williams, Cuvelier, & Berset, 1995) with somemodifications. A 6.1×10−5 M DPPH• methanolic solution was prepared immediatelybeforeuse.Ina3mLcapacitycuvette(1cmlength)theDPPH•solutionwasadjustedwith methanol to an absorbance of 0.700 (±0.020) at 515 nm (Lambda 25 UV–VISspectrophotometer, Perkin-Elmer Instruments,Madrid, Spain). All the extracts wereproperlydilutedindemineralizedwaterand50μLofsampleswereaddedto1.95mLoftheDPPH•solution.Theabsorbancewasmeasuredat515nmafter18min.CalibrationwasperformedwithTroloxsolutionandexpressedasmicromolesofTroloxequivalentpergramofdm(μmolTrolox/gdm).

2.8AcidHydrolysis

For the identification and quantification of (poly)phenolic aglycones by HPLC-DAD,previous acid hydrolysis was carried out through a reflux at 90 °C with differentconcentrationsofHClandtimes:0.6Mfor3h,1.2Mfor3h,1.5Mfor2and3h,and1.7M for 2 and 3 h. The resulting extract was filtered and frozen at -18 °C forchromatographicanalysis.

2.9(Poly)phenoliccompoundsbyHPLC-DAD

(Poly)phenoliccompoundswereanalysedbyHPLCfollowingthemethoddescribedbyJuániz et al. (2016) with somemodifications. HPLC analysis was performedwith ananalyticalHPLCunitmodel1200 (AgilentTechnologies,PaloAlto,CA,USA)equippedwith a binary pump and an auto-sampler injector. The column used was a C18 5UKinetex 100A (250 x 4.60mm) and the volumeof each sample injectionwas 20 µL.Chromatographicseparationwascarriedoutusingagradientofacidwaterwithformicacid1%(solventA)andacetonitrile(solventB)ataconstantflowof1mL/minstartingwith95%solventA.ThensolventAwasdecreasedto60%within55min,maintaineduntil60minanddecreasedto10%.Then,itremaineduntil67minandreturnedtotheinitial conditions (solventA:95%solventB:5%).Detectionwasaccomplishedwithadiodearraydetector(DAD),at360nmforflavonoids;at325nmforferulicacid,andat256 nm for hydroxybenzoic acid. Chromatograms were analysed using the Agilent

Results

39

ChemStationsoftwareandtheidentificationofphenoliccompoundswasperformedbycomparisonofretentiontimesandUVspectrawithreferencestandards.Acalibrationcurvewith reference standardsof identified compoundswas carriedout toquantify(poly)phenols.Resultswereexpressedasmilligramsofeachcompoundpergramofdrymatter(mg/gdm).

2.9Statisticalanalysis

Eachparameterwasanalysedintriplicate.Resultsareshownasthemean±standarddeviation(SD).One-wayanalysisofvariance(ANOVA)wasappliedforeachparameter.A Tukey testwas applied asa posteriori testwith a level of significance of 95%. AllstatisticalanalyseswereperformedusingtheSTATAv.12.0softwarepackage.

3.Resultsanddiscussion

3.1TotalPhenolicContent(TPC)andTotalFlavonoidContent(TFC)

Figure1showstheTPCandTFCofthedifferentextractsobtainedfromcactus(O.ficus-indica)cladodes.ThehighestamountofTPCcorrespondedtothe80%EtOHextractwith39.26mgGA/gdm(250.86mgGA/100g),followedbythesuccessiveextractionsextractwith36.62mgGA/gdm(234.01mgGA/100g).Theextractwiththeleastamountofphenoliccompoundswasthe100%waterextractwith14.18mgGA/gdm(90.62mgGA/100g).Thereisagreatvariationintheresultsreportedbyotherauthorsdueinparttothedifferentexpressionofresults(freshweight,drymatter,mililiterofextract)andthemethodology applied.Nevertheless, the amount of TPC found in 80% EtOH andsuccessive extractions extracts were higher than those previously reported in theliterature.Sánchezetal.(2014)reportedatotalphenolicamountof4.27mgGAE/gdmusing70%methanol,whilstGuevara-Figueroaetal.(2010)reportedatotalcontentof19.9 mg GAE/g with 100% ethanol used for extraction. Similar processes withsuccessivesextractionswerecarriedoutbyDib,Beghdad,Belarbi,Seladji,&Ghalem(2013)andRamírez-Moreno,Córdoba-Díaz,deCortesSánchez-Mata,Díez-Marqués,&Goñi(2013).However,theyreportedlessamountthaninthepresentstudy(26.7and4.58mgGA/gdm,respectively),maybeduetothemethodologyappliedbutalsotosamplesdifferencesinthecladodematurity,cultivationregionandclimate.

Ontheotherhand,theextractwihthehighestamountofTFCwastheseobtainedbysuccessiveextractionswith4.83mgrutin/gdm(30.89mgrutin/100g).Inthiscase,the80%EtOHextractpresentedthelowestamountofTFCwith2.85mgrutin/gdm(18.22mg rutin/100 g). Flavonoid results are also difficult to compare becauseof differentmethodologiesandstandardcompoundsusedsuchasrutin,quercetinandcathechineforreportingresults.

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3.2Antioxidantcapacity

AntioxidantcapacitymeasuredbyABTSandDPPHradicalsofcactuscladodesextractsare shown in Figure 2. In both cases, the extract obtainedby successive extractionspresented thehighest antioxidant capacitywith778.03and72.17µmol trolox/gdm(4791.63 and 461.14 µmol trolox/100g) respectively. The extract with the leastantioxidant capacity was that obtained with 100% water, accounted for 1.13 µmoltrolox/gdm(7.25µmoltrolox/100g)forABTSand1.44µmoltrolox/gdm(9.21µmoltrolox/100g)forDPPHradical.Incontrast,theamountfoundinsuccessiveextractionsextractishigherthanthatreportedbyothersauthors:13.97µmoltrolox/gdmforABTSradical (Ramírez-Moreno,Córdoba-Díaz, Sánchez-Mata,Díez-Marqués,&Goñi,2013)and90µmoltrolox/100gforDPPH(Corral-Aguayo,Yahia,Carrillo-Lopez,&González-Aguilar,2008).

Polysaccharides present in cactus cladodes, such as pectins, form a water-solublecomplexwith(poly)phenols,aswellascellulosewiththenon-covalenthydrogenbondsandhydrophobicinteractionsmakingextractionmoredifficult(Bordenave,Hamaker,&Ferruzzi,2014).

Resultssuggestthattheuseofdifferentsolventsfacilitatestheantioxidantextractionprocess, obtained an optimal polarity conditions, because of the retention of thecompoundsinthecellwallsofthecactus.Hence,thecombinationofethanol,acetoneandwatercouldimprovetheextractionofbioactivecompoundslike(poly)phenols.

3.3(Poly)phenoliccompoundsbyHPLC-DAD

Becauseof thehighestDPPHandABTSantioxidantcapacityand thehighamountoftotalphenolicandflavonoidcontent,successiveextractionmethodwasselectedasthebesttofurtheridentifyandquantify(poly)phenoliccompoundsbyHPLC-DADpreviousacid hydrolysis. Five acid hydrolysiswere carried out through a reflux at 90 °CwithdifferentconcentrationsofHClandtimes:0.6Mfor3h,1.2Mfor3h,1.5Mfor2and3h,and1.7Mfor2and3h.

Afteracidhydrolysis,3flavonoidsaglycones(quercetin,kaempferolandisorhamnetin)and2phenolicacids(ferulicandhidroxybenzoicacids)wereidentifiedandquantifiedincactus cladodes (Table 1). Results from HPLC-DAD measurements showed the(poly)phenolic aglycones, indicating that after acid hydrolysis, compounds werereleasedfromtheglycosides,butalsofromthefoodmatrixboundtoorassociatedwithpolysaccharidessuchasthefibre.ChromatogramsobtainedbyHPLC-DADbeforeandafter hydrolysis show the release of the quercetin, kaempferol and isorhamnetinaglycones(Figure3).

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Flavonoids were the main compounds accounting for around 85% of total(poly)phenolics.Isorhamnetinwasthemostabundantflavonoidcompound,whichisinagreementwithliterature(Avila-Navaetal.,2014;Ginestraetal.,2009;Jun,Cha,Yang,Choi,&Kim,2013),followedbysmalleramountofquercetinandkaempferol.Previousstudies without hydrolysis have identified isorhamnetin glycosides as the mainflavonoid glycosides in cactus cladodes (Ginestra et al., 2009;Moussa-Ayoub et al.,2014), although kaempferol and quercetin glycosides are also present (Guevara-Figueroa et al., 2010). Santos-Zea, Gutiérrez-Uribe & Serna-Saldivar (2011) havereportedlessamountofisorhamnetin(0.6mg/gdm)andhigheramountofkaempferol(0.4mg/gdm)thaninthisstudy,buttheydidnotfindquercetin.

Otherwise, ferulic acid was the predominant phenolic acid, followed by 4-hydroxybenzoicacid.Protocatechuic,chlorogenic,salicylic,gallic,andcaffeicacidshavebeenpreviously reported inOpuntia cactus cladodes (Guevara-Figueroaet al., 2010;Jun,Cha,Yang,Choi,&Kim,2013),buttheywerenotdetectedinthepresentstudy.

Comparingdifferenthydrolysisconditions,thehighestamountoftotal(poly)phenoliccompoundswasthatobtainedafter2hofrefluxat90°Cwith1.5MHCl(3.97mg/gdm).Likewise,thedeglycosylationofflavonoidsandthusthehighestamountofaglyconeswasalsoobservedwith1.5MHClduring2hofreflux.Resultsshowthatlessacidity(0.6MHCl),doesnotcompletelyreleasetheaglycones.Otherwise,ahigheracidity(1.7M)andalongertimeofreflux(3h),resultedinthedegradationofflavonoidaglycones.Aprevious study in cactus cladodes which performed an acid hydrolysis with higherconcentration of HCl (3.2M) during 20min, showed less amount than in this study(Santos-Zea,Gutiérrez-Uribe,&Serna-Saldivar,2011),demonstratingthedegradationofthecompounds.Likewise,Moussaetal(2014),identifiedisorhamnetinastheonlyaglycone presented after performing an enzymatic hydrolysis; but the amount wasmuchsmaller(0.003mg/gdm)thanthatobtainedinthepresentstudy.

Regardingtototalphenolicacidscontent,the0.6MHClhydrolysis,showedthehighestamount,followingbythe1.2MHClconcentration.Itcanbeobservedthatwhentheacidconcentrationandtherefluxtimewereincreased,ferulicacidwasdegraded.However,the highest content of 4-hydroxybenzoic acid was measured after 3h of 1.5M HClhydrolysis, and starting to decrease after longer time and higher acid concentrationapplied.Inanycase,theamountofeachphenolicacidfoundinthepresentstudywashigherthanthatfoundbyGuevara-Figueroaetal.(2010).Asimilarworkcarriedoutinonionandspinachshowedthat the2hrefluxat80°Cwith1.2MHClgavethebestresultsforquercetinandkaempferolaglycones,whereasphenolicacidsweredegradedunder the same conditions (Nuutila, Kammiovirta, & Oksman-Caldentey, 2002).

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Therefore,becausephenolicacidsaremoresusceptiblethanflavonoidstobedegraded,it is necessary to choose the acid hydrolysis conditions that favoured the release ofaglyconeswithoutputatrisktheidentificationandquantificationofphenolicacids.

(Poly)phenols are bound to the polymeric compounds like fibre, carbohydrates orproteins, thusthestabilityoftheglycosidesdependsonthekindandpositionofthesugar,aswellasthecompoundunion.Likewise,thenumberofaromaticmoietiesandhydroxylgroupspresentinphenoliccompoundsareimportantfactors,becausemorearomaticringstendedtobindmorestronglytocellulosethanthosecompoundswithasinglearomaticring(Bordenave,Hamaker,&Ferruzzi,2014).Eventhoughthereleaseofaglyconesfromthecellstructureenhancetheantioxidantcapacity,iftimeandHClconcentrationsarenottheoptimalandoxidationreactionscouldtakeplacedegradingthecompounds.

4.Conclusions

Thepresent studyevidenced that themethodology and conditionsof extractionarecrucialtoobtainbioactivecompoundsandthereforethepotentialantioxidantcapacityofcactuscladodes.Resultssuggeststhatamixtureofmethanol,acetoneandwaterarerequired for an optimal polarity conditions to extract biological compounds like(poly)phenols.Ontheotherhand,toallowtheidentificationofphenoliccompoundsbyHPLC-DAD,itisoftennecessarytocarryoutapreviousacidhydrolysis,demonstratingthat 1.5M HCl during 2 h showed the highest amount of (poly)phenols, especiallyflavonoidsaglycones.Thisstudyalsoshowedthatacidhydrolysisatlongtimeperiodsor at high temperature leads to the degradation of (poly)phenolic compounds.Therefore,thechoiceofthebestmethodofextractionisveryimportantforobtainingthe highest amount of bioactive compounds, especially antioxidants, from the plantmaterial,buttoxicologicalandsustainablecriteriashouldalsobeconsideredinordertousecactuscladodesextractsforthedevelopmentoffunctionalfoods.

Acknowledgments

This researchwas funded by the SpanishMinistry of Economy and Competitiveness(AGL2014-52636-P).E.DeSantiagoandI.JuánizwishtoexpresstheirgratitudetotheAssociationofFriendsoftheUniversityofNavarraforthegrantsreceived.

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Laursen,B.B.,Lin,L.W.,DeLeón-Rodríguez,A.,Fomsgaard, I.S.,&BarbadelaRosa, A. P. (2010). Proximate composition, phenolic acids, and flavonoidscharacterization of commercial and wild nopal (Opuntia spp.). Journal of FoodCompositionandAnalysis,23,525–532.

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Juániz,I.,Ludwig,I.A.,Huarte,E.,Pereira-Caro,G.,Moreno-Rojas,J.M.,Cid,C.,&DePeña, M.-P. (2016). Influence of heat treatment on antioxidant capacity and(poly)phenoliccompoundsofselectedvegetables.FoodChemistry,197,466–473.

Jun, H. Il, Cha, M. N., Yang, E. I., Choi, D. G., & Kim, Y. S. (2013). PhysicochemicalpropertiesandantioxidantactivityofKoreancactus(Opuntiahumifusa)cladodes.HorticultureEnvironmentandBiotechnology,54(3),288–295.

Lamaison, J., & Carnet, A. (1990). Teneurs en principaux flavonoids des fleurs deCrataegeusmonogynaJacqetdeCrataegeuslaevigata(PoiretD.C)enfonctiondelavegetation.PharmaceuticaActaHelvetiae,65,315–320.

Lee, J.C.,Kim,H.R.,Kim, J.,&Jang,Y.S. (2002).AntioxidantpropertyofanethanolextractofthestemofOpuntiaficus-indicavar.saboten.JournalofAgriculturalandFoodChemistry,50(22),6490–6496.

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Moussa-Ayoub,T.E.,El-Samahy,S.K.,Kroh,L.W.,&Rohn,S.(2011).Identificationandquantificationofflavonolaglyconsincactuspear(Opuntiaficusindica)fruitusinga commercial pectinase and cellulase preparation. Food Chemistry,124, 1177–1184.

Nuutila, A. M., Kammiovirta, K., & Oksman-Caldentey, K. M. (2002). Comparison ofmethods for the hydrolysis of flavonoids and phenolic acids from onion andspinachforHPLCanalysis.FoodChemistry,76(4),519–525.

Ramírez-Moreno, E., Córdoba-Díaz,D., Sánchez-Mata,M. de C., Díez-Marqués, C.,&Goñi, I. (2013).Effectofboilingonnutritional,antioxidantandphysicochemicalcharacteristics in cladodes (Opuntia ficus indica). LWT - Food Science and

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Sánchez,E.,Dávila-Aviña,J.,Castillo,S.L.,Heredia,N.,Vázquez-Alvarado,R.,&García,S. (2014). Antibacterial and antioxidant activities in extracts of fully growncladodesof8cultivarsofcactuspear.JournalofFoodScience,79(4),659–664.

Santos-Zea, L., Gutiérrez-Uribe, J. A., & Serna-Saldivar, S. O. (2011). Comparativeanalyses of total phenols, antioxidant activity, and flavonol glycoside profile ofcladodefloursfromdifferentvarietiesofOpuntiaspp.JournalofAgriculturalandFoodChemistry,59,7054–7061.

Siddiq,M.,Roidoung,S.,Sogi,D.S.,&Dolan,K.D.(2013).Totalphenolics,antioxidantpropertiesandqualityoffresh-cutonions(AlliumcepaL.)treatedwithmild-heat.FoodChemistry,136,803–806.

Singleton,V.,&Rossi,J.(1965).Colorimetryoftotalphenolicswithphosphomolybdic-phosphotungsticacidreagents.AmericanJournalofEnologyandViticulture,16(3),144–158.

Weirong, C. A. I., Xiaohong, G. U., & Tang, J. (2010). Extraction, purification, andcharacterisationoftheflavonoidsfromOpuntiamilpaaltaskin.CzechJournalofFoodSciences,28(2),108–116.

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Figure1.Totalphenolic(a)andflavonoid(b)concentrationofcactuscladodesextracts.Differentlettersindicatesignificantdifferences(p≤0.05).a)

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Figure2.AntioxidantcapacitymeasuredbyABTS(a)andDPPH(b)ofcactuscladodesextracts.Differentlettersindicatesignificantdifferences(p≤0.05).

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Figure3.HPLC-DADchromatogramsofcactuscladodesextractsa)beforeandb)afteracidhydrolysis(1.5MHCl2h)at360nm.

a)Unhydrolysed

b)Hydrolysed

Q:quercetin,K:kaempferol,I:isorhamnetin.

I

K Q

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Table1. (Poly)phenoliccompounds incactuscladodesafterdifferenthydrolysis (HCl)conditions.Resultsareexpressedasmean±standarddeviation(mg/gdm).

Compound 0.6M3h 1.2M3h 1.5M2h 1.5M3h 1.7M2h 1.7M3hIsorhamnetin 0.92±0.07a 1.34±0.03b 2.58±0.27d 2.15±0.16c 2.08±0.03c 1.29±0.13abKaempferol 0.25±0.00b 0.11±0.00a 0.36±0.07c 0.29±0.01bc 0.32±0.00bc 0.06±0.01aQuercetin 0.36±0.04c 0.19±0.00b 0.42±0.03c 0.37±0.01c 0.38±0.01c 0.11±0.00aTotalflavonoids 1.53±0.04a 1.64±0.04a 3.36±0.29c 2.81±0.18b 2.78±0.05b 1.47±0.15aFerulicacid 0.63±0.03c 0.58±0.01c 0.43±0.07b 0.41±0.03ab 0.34±0.00ab 0.32±0.02a4-Hydroxybenzoicacid

0.16±0.05c 0.15±0.00abc 0.18±0.01c 0.15±0.00abc 0.12±0.00ab 0.10±0.00a

Totalphenolicacids

0.79±0.08d 0.73±0.01cd 0.61±0.08bc 0.56±0.03ab 0.46±0.00a 0.42±0.02a

Totalcompounds 2.32±0.07a 2.37±0.05a 3.97±0.37c 3.37±0.17b 3.24±0.05b 1.89±0.17a

Differentlettersforeachrowindicatesignificantdifferences(p≤0.05)amongsamples.

51

Objective2

Toevaluatetheinfluenceofheattreatment(boiling,microwaving,griddling,fryinginolive and soybean oils) on nutritional composition, antioxidant capacity and(poly)phenoliccompoundsofcactus(Opuntiaficus-indicaMill.)cladodes.

Evaluación de la influencia del tratamiento térmico (hervido, microondas, plancha,fritura en aceite de oliva y de soja), sobre la composición nutricional, capacidadantioxidanteycompuestos(poli)fenólicosdenopal(Opuntiaficus-indicaMill.).

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Paper2

Impact of cooking process on nutritional composition and antioxidants of cactuscladodes(Opuntiaficus-indica)

ElsyDeSantiago,MaiteDomínguez-Fernández,ConcepciónCidandMaría-PazDePeña

FoodChemistry,2018,(240)1055–1062.https://doi.org/10.1016/j.foodchem.2017.08.039.

Qualityindices:• Impactfactor(JCR,2017):4.946• JournalRankincategories:

o FoodScience&Technology:7/133(Q1)(D1)o Chemistry,applied:7/71(Q1)(D1)o Nutrition&Dietetics:11/81(Q1)

The final Published Journal Article (PJA) is included with the permission of Elseviereditorial.

De Santiago E, Domínguez-Fernández M, Cid C, De Peña MP. Impact of cooking

process on nutritional composition and antioxidants of cactus cladodes (Opuntia ficus-

indica). Food Chemistry, 2018, 240:1055-1062.

https://doi.org/10.1016/j.foodchem.2017.08.039

https://doi.org/10.1016/j.foodchem.2017.08.039

63

Objective3

To evaluate the bioaccessibility of (poly)phenolic compounds and its antioxidantcapacityafterinvitrogastrointestinaldigestionincactus(Opuntiaficus-indicaMill.)cladodes.

Evaluación de la bioaccesibilidad de sus compuestos (poli)fenólicos y su capacidadantioxidantetrasladigestióngastrointestinalinvitroenelnopal(Opuntiaficus-indicaMill.).

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Paper3

Digestibility of (poly)phenols and antioxidant activity in raw and cooked cactuscladodes(Opuntiaficus-indica)

ElsyDeSantiago,GemaPereira-Caro,JoséManuelMoreno-Rojas,ConcepciónCidandMaría-PazDePeña

JournalofAgriculturalandFoodChemistry,2018,66,5832−5844.https://doi.org/10.1021/acs.jafc.8b01167.

Qualityindices:• Impactfactor(JCR,2017):3.412• JournalRankincategories:

o Agriculture,multidisciplinary:2/56(Q1)(D1)o FoodScience&Technology:18/133(Q1)o Chemistry,applied:17/71(Q1)

Reprintedwithpermissionfrom.J.Agric.FoodChem.66,23,5832-5844.Copyright2018AmericanChemicalSociety.

De Santiago E, Pereira-Caro G, Moreno-Rojas JM, Cid C, De Peña MP. Digestibility of

(Poly)phenols and Antioxidant Activity in Raw and Cooked Cactus Cladodes (Opuntia f

icus-indica). Journal of Agricultural and Food Chemistry, 2018, 66, 5832-5844.

http://doi.org/10.1021/acs.jafc.8b01167





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SUPPLEMENTARY INFORMATION

TITLE: Digestibility of (poly)phenols and antioxidant activity in raw and cooked

cactus cladodes (Opuntia ficus-indica)

AUTHORS: Elsy De Santiagoa, Gema Pereira-Carob, José Manuel Moreno-Rojasb,

Concepcion Cida, M. Paz de Peñaa*

a Universidad de Navarra, Facultad de Farmacia y Nutrición, Departamento de Ciencias

de la Alimentación y Fisiología, C/ Irunlarrea 1, E-31008 Pamplona, Spain.

IdiSNA, Navarra Institute for Health Research. Pamplona, Spain.

b Department of Food Science and Health. Andalusian Institute of Agricultural and

Fisheries Research and Training (IFAPA). Alameda del Obispo, Avda. Menéndez Pidal,

s/n, 14071, Córdoba. Spain

*Corresponding author: María-Paz de Peña. Tel: +34 948 425600 (806580); Fax: +34

948 425740. E-mail address: [email protected]

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2

Table S1. Concentrations of electrolytes Simulated Salivary Fluid (SSF), Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF)

Stock solution SSF (250mL) SGF (250mL) SIF (250mL)

Constituent g/50mL mol/L mL mL mL

KCl 1.87 0.5 9.44 4.31 4.25

KH2PO4 3.40 0.5 2.31 0.57 0.50

NaHCO3 4.20 1 4.25 7.81 26.56

NaCl 5.90 2 ----- 7.38 6.00

MgCl2(H2O)6 1.53 0.15 0.31 0.25 0.69

(NH4)2CO3 2.40 0.5 0.04 0.31 0.69

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Table S2. Mass spectrometric characteristics of native (poly)phenolic compounds identified in this study.

Compound Chemical formula

Rt

(min)

[M-H]¯

(m/z) δppm Fragment ions under low collision

energy (m/z) Piscidic acid I C11H12O7 5.4 255.0501 0.78 193.0499 / 179.0340 / 165.0546 Piscidic acid II C11H12O7 6.1 255.0501 0.78 193.0499 / 179.0340 / 165.0546 Piscidic acid III C11H12O7 6.6 255.0501 0.78 193.0499 / 179.0340 / 165.0546 Eucomic acid I C11H12O6 11.8 239.0550 -0.46 195.0658 / 179.0342 / 149.0599 /

107. 0490 Eucomic acid II C11H12O6 12.8 239.0550 -0.46 195.0658 / 179.0342 / 149.0599 /

107. 0490 Eucomic acid III C11H12O6 13.4 239.0550 -0.46 195.0658 / 179.0342 / 149.0599 /

107. 0490 Ferulic acid derivatives

Ferulic acid C10H10O4 27.8 193.0497 1.04 1-O-feruloylglucose I C16H20O9 15.1 355.1037 3.94 239.0558 / 193.0503 / 175.0391 1-O-feruloylglucose II C16H20O9 16.4 355.1037 3.94 239.0558 / 193.0503 / 175.0391 1-O-feruloylglucose III C16H20O9 17.5 355.1037 3.94 239.0558 / 193.0503 / 175.0391 1-O-feruloylglucose IV C16H20O9 18.6 355.1037 3.94 239.0558 / 193.0503 / 175.0391 Dihydroferulic acid -O-glucuronide I C16H20O10 9.4 371.0982 2.69 239.0558 / 179.0554 / 133.0135 Dihydroferulic acid -O-glucuronide II C16H20O10 9.9 371.0982 2.69 239.0558 / 179.0554 / 133.0135

Isorhamnetin derivatives Isorhamnetin C16H12O7 72.0 315.0504 1.59 151.0027 Isorhamnetin hexose rhamnose hexoside

C34H42O21 32.4 785.2152 2.29 503.1777 / 371.0984 / 315.0503 / 151.0025

Isorhamnetin di-hexoside C28H32O17 34.6 639.1574 2.97 477.2342 / 361.1868 / 315.0503 Isorhamnetin rutinoside rhamnoside C34H42O20 36.4 769.2199 1.82 315.0503 / 145.0495 Isorhamnetin hexose pentoside C27H30O16 39.1 609.1462 1.97 477.1982 / 315.0508 Isorhamnetin rutinoside I C28H32O16 42.1 623.1627 1.76 477.2346 / 315.0501 Isorhamnetin rutinoside II C28H32O16 43.5 623.1627 1.76 477.2346 / 315.0501 Isorhamnetin 3-O-beta-(6-O-coumaroylglucoside)-7-O-beta-glucoside I

C37H38O19 59.2 785.1940 2.16 315.0507 / 179.0554 / 145.0496

Isorhamnetin 3-O-beta-(6-O-coumaroylglucoside)-7-O-beta-glucoside II

C37H38O19 61.8 785.1940 2.16 315.0507 / 179.0554 / 145.0496

Isorhamnetin 3-O-beta-(6-O-coumaroylglucoside)-7-O-beta-glucoside III

C37H38O19 62.5 785.1940 2.16 315.0507 / 179.0554 / 145.0496

Isorhamnetin 3-O-beta-(6-O-coumaroylglucoside)-7-O-beta-glucoside IV

C37H38O19 64.4 785.1940 2.16 315.0507 / 179.0554 / 145.0496

Isorhamnetin 3-O-beta-(6-O-coumaroylglucoside)-7-O-beta-glucoside V

C37H38O19 66.1 785.1940 2.16 315.0507 / 179.0554 / 145.0496

Isorhamnetin 3-ferulylrobinobioside C38H40O19 63.9 799.2096 2.00 315.0509 Quercetin derivatives

Quercetin C15H10O7 56.4 301.0354 3.98 178.9978 / 151.0027 Quercetin hexosyl pentosyl rhamnoside C32H38O20 30.4 741.1895 3.10 301.0351 / 151.0391 Quercetin hexose pentoside C26H28O16 31.5 595.1311 3.02 463.0881 / 433.2078 / 415.0884 Quercetin 3-O-rutinoside (rutin) C27H30O16 35.6 609.1470 3.27 301.0353 / 145.0496 Quercetin hexose dirhamnoside C33H40O20 36.8 755.2040 1.46 609.1467 / 301.0349

Kaempferol derivatives Kaempferol C15H10O6 68.9 285.0404 3.85 Kaempferol hexoside dirhamnoside I C33H40O19 34.3 739.2102 2.98 431.2286 / 285.0402 Kaempferol hexoside dirhamnoside II C33H40O19 35.3 739.2102 2.98 431.2286 / 285.0402 Kaempferol hexose pentose rhamnoside C32H38O19 35.9 725.1945 3.03 285.0401 Kaempferol hexose pentoside C26H28O15 37.5 579.1362 3.11 496.2458 / 285.0402 Kaempferol rutinoside I C27H30O15 41.7 593.1520 3.22 496.2455 / 285.0403 Kaempferol rutinoside II C27H30O15 47.3 593.1520 3.22 496.2455 / 285.0403 Kaempferol rutinoside III C27H30O15 48.0 593.1520 3.22 496.2455 / 285.0403

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Kaempferol acetyl arabinopyranosyl hexoside

C28H30O16 42.5 621.1450 2.74 503.2504 / 285.0402

Methoxy kaempferol hexoside C22H22O12 44.6 477.1027 2.52 314.0434 / 285.0406 Kaempferol acetyl hexoside C23H22O12 45.8 489.1039 2.46 445.1141 / 285.0406 Kaempferide 3,7 – dirhamnoside C28H32O14 58.3 591.1708 2.37 285.0402 Kaempferol coumaryl glucoside glucoside I

C36H36O18 60.5 755.1834 2.25 285.0404 / 179.0554 / 161.0446

Kaempferol coumaryl glucoside glucoside II

C36H36O18 61.4 755.1834 2.25 285.0404 / 179.0554 / 161.0446

Rt, retention time; m/z, mass-to-charge ratio; [M-H]¯, Negatively charged molecular ion

85

Objective4

Toevaluatetheactionofcolonicmicrobiotaon(poly)phenoliccompoundsofcactus(Opuntiaficus-indicaMill.)cladodes.

Evaluacióndelaaccióndelamicrobiotaintestinalsobreloscompuestos(poli)fenólicosdelnopal(Opuntiaficus-indicaMill.).

Objective5

Toevaluatethebiologicalactivityofcolonicfermentedcactus(Opuntiaficus-indicaMill.)cladodesonHT29humancoloncancercells.

Evaluacióndelaactividadbiológicadenopal(Opuntiaficus-indicaMill.)posterioralafermentacióncolónicaencélulasdecáncerdecolonHT29.

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Paper4

DigestionandcolonicfermentationofrawandcookedOpuntiaficus-indica impactsbioaccessibilityandbioactivity

ElsyDeSantiago,ChrisI.R.Gill,IlariaCarafa,KieranM.Touhy,María-PazDePeñaandConcepciónCid.

JournalofAgriculturalandFoodChemistry.(Underreview).

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TITLE: Digestion and colonic fermentation of raw and cookedOpuntia ficus-indicaimpactsbioaccessibilityandbioactivity

AUTHORS:ElsyDeSantiagoa,ChrisI.R.Gillb,IlariaCarafac,KieranM.Touhyc,María-PazDePeñaaandConcepciónCida*

aUniversidaddeNavarra,FacultaddeFarmaciayNutrición,DepartamentodeCienciasdelaAlimentaciónyFisiología,C/Irunlarrea1,E-31008Pamplona,Spain.

IdiSNA,NavarraInstituteforHealthResearch.Pamplona,Spain.

bNutrition InnovationCentreforFoodandHealth,Centre forMolecularBiosciences,University of Ulster, Cromore Road, Coleraine, Northern Ireland, BT52 1SA, UnitedKingdom.

cNutrition&NutrigenomicsUnit,DepartmentofFoodQualityandNutrition,Researchand InnovationCentre, FondazioneEdmundMach (FEM),Via E.Mach1, 38010, SanMicheleall’Adige,Trento,Italy.


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ABSTRACT

Thebioactivityof(poly)phenolsfromafoodareinterplaybetweenthecookingmethodsapplied and its interactionwith the gastrointestinal tract. (Poly)phenolic profile andbiological activity of cactus (Opuntia ficus-indica Mill.) cladodes following in vitrodigestionandcolonicfermentationwereevaluated.Twenty-seven(poly)phenolswereidentified and quantified by HPLC-ESI-MS, with piscidic acid the most abundant.Throughout the colonic fermentation, flavonoids showed more degradation thanphenolicacids,remainedeucomicacidthemostrelevantafter24h.Thecatabolite3-(4-hydroxyphenyl)propionicacidwasgeneratedafter24hoffermentationincubation.Cytotoxicity,genotoxicityandcellcycleanalyseswereperformedinHT29cells.Cactuscolonic fermentates showed higher cell viability (80%) compared to the controlfermentationwithnocactus.Cactuscolonicfermentatessignificantly(p<0.05)reducedH2O2-inducedDNAdamageinHT29cells,comparingtothecontrolone.Resultssuggestthat,althoughphenoliccompoundsweredegradedduringthecolonicfermentation,thebiologicalactivityisretainedincoloncells.

KEYWORDS: Opuntia ficus-indica, Polyphenols, Gut microbiota, Antioxidant activity,DNAdamage,cytotoxicity.

CHEMICAL COMPOUNDS STUDIED IN THIS ARTICLE: Ferulic acid (PubChem CID:445858); Piscidic acid (PubChem CID: 6710641); Eucomic acid (PubChem CID:23757219); Kaempferol (PubChem CID: 5280863); Isorhamnetin (PubChem CID:5281654).

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1.Introduction

Cactus(Opuntiaficus-indicaMill.)cladodesisanAmericannativeplant,whichfarmingisslowlyextendingthroughouttheWorld.InMexico,itsannualconsumptionis6.4kgper capita 1,which couldbeeatenasdifferent cooking styles (i.e., boiling, griddling,frying and microwaving). It is being recognized as a great source of (poly)phenols,predominating flavonoids as isorhamnetin, kaempferol and quercetin derivatives, aswellasphenolicacidslikepiscidicandeucomicacids2–4.

Thedietaryintakeof(poly)phenoliccompoundshasbeenstudiedfordecadesbecauseoftheirbeneficialandprotectiveeffectsondifferentchronicdiseasessuchascancer,diabetes and cardiovascular disease due to their antioxidant properties to reduceinflammationcausedbyoxidativestress5.Afterconsumption,digestiveconditionsandenzymesaffect(poly)phenolsbioaccessibilityofcactuscladodes6.Then,bioaccessiblecompoundscanbeabsorbedthroughthestomachandsmallintestine,ormostofthemcouldreachthecolonandbeextensivelycatabolisedbythehumangutmicrobiota7.

(Poly)phenolsbiotransformationscanoccurinthecolon,inwhichtheenzymesofthegut microbiota act to breakdown complex polyphenolic structures by hydrolysis,dehydroxylation, deconjugation, demethylation, ring cleavage and decarboxylationreactions.Somestudieshaveshowntheinfluenceofcolonichumanmicrobiotaonthephenolicprofileandcatabolitesproducedinsomevegetablesaspepper8,andcardoon9aswellasincoffee10andlingonberries11.

Otherwise,theremainingpolyphenolswhichreachthecoloncouldmodulatemetabolicpathwaysrelatedtothegutmicrobiotaandcellprocesses,asinflammation,immunity,cellproliferationandoxidativestress12.Recently,studieshavereportedthebiologicalandantioxidantactivityof(poly)phenolsfromraspberriesaftercolonicfermentationonHT29humancoloncellsreducingDNAdamage13.Furthermore,extractsfromO.ficus-indica juicesofcactuspearfruitshavealsodemonstratedantiproliferativeeffectsonHT29cells14,evenintestinaldigestionandgutmicrobiotaactionwerenotconsidered.

Inlightofthescientificevidenceabouttheinfluenceofgutmicrobiotaon(poly)phenoliccompounds,andsubsequentlytheirbiologicalactivity,aswellastheincreasinginterestoncactuscladodespotentialbioactivity,theaimofthecurrentstudywastoevaluatetheeffectofthehumancolonicmicrobiotaon(poly)phenoliccompoundsofrawandcookedcactus(O.ficus-indica)cladodes,aswellastheirbiologicalactivityonHT29coloncells.

2.Materialandmethods

2.1Chemicalandreagents

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O. ficus-indica cactus cladodeswere purchased from BioArchen company located inMurcia,Spain(August2015).Oliveandsoybeanoilswereobtainedfromlocalmarkets.Methanol, acetone, acetonitrile (HPLC grade) and formic acid (HPLC grade) werepurchased fromPanreac (Barcelona, Spain). Potassiumchloride and sodiumchloridewereobtainedfromMerck(Darmstadt,Germany).Humansalivaα-amylase(852U/mgprotein), pepsin (674 U/mg), pancreatin (4xUPS), bile salts (for digestion), sodiumhydrogen carbonate, potassium phosphate monobasic, magnesium sulfatemonohydrate,werepurchasedfromSigma-Aldrich(Steinheim,Germany).

Chemicalsforthebatchculturenutrientmediumfortheinvitrofermentationasbilesalts,yeastextract,bufferedpeptonewater,sodiumchloride,Tween80,hemin,vitaminK, L-cysteinhydrochloridemonohydrate, resazurin redox indicator, sodiumhydrogencarbonate, potassium phosphate monobasic, magnesium sulfate monohydrate,potassiumhydrogen phosphate, calcium chloride hexahydratewere purchased fromSigma-Aldrich(St.Louis,MO,USA)andApplichem(Darmstadt,Germany).

Dulbecco'sModified EagleMedium (DMEM), fetal bovine serum (FBS) and penicillinstreptomycin (Pen Strep) were obtained from Gibco Life Technologies Ltd (Paisley,Scotland,UK).MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)wasacquired from Promega (Madison, USA). All other chemicals for tissue culture andbiological activity assays were purchased from Sigma-Aldrich Company Ltd (Dorset,England,UK)unlessotherwisespecified.

2.2Samplespreparation

Cactuscladodeswerewashedandthethornsweremanuallyremoved.Then,theywerecutintosmallpiecesanddividedintosixportions(300gforeachone).OneportionwasusedasrawsampleandtheotherfivewereprocessedbythedifferentcookingmethodsasshowninTable1.Afterwards,eachrawandcookedsamplewaslyophilizedinafreezedryerCryodos-80(Telstar,Terrasa,Spain)andstoredat-18°C.

2.3Invitrogastrointestinaldigestionandcolonicfermentation

AsimulateddigestionmodelwasperformedaccordingtoMinekusetal.(2014)15andMonenteetal. (2015) 16 adapted toour laboratory.Three stepswerecarriedout tosimulate theoral (withα-amylase), gastric (withpepsin at pH3) and small intestine(withpancreatinandbilesaltsatpH7)conditions.DetailsoftheinvitrogastrointestinalprocessaredescribedinDeSantiagoetal.(2018)6.Eachcactussamplewasdigestedinduplicateandthenthetworepetitionsweremixedandhomogenized.Afterintestinaldigestion,sampleswerelyophilizedinafreezedryerCryodos-80(Telstar),andstoredat-18°Cuntilinvitrofermentationprocess.

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ThedigestarawandcookedcactuscladodesweresubjectedtoaninvitrofermentationwithhumanfaecalsamplestosimulatetheconditionpresentinthecolonfollowingthemethoddescribedbyKoutsosetal.201717andbrieflydescribedbelow.

Thecompositionfor1Lofgrowthmediumwas2gofpeptone,2gofyeastextract,0.1gofNaCl,0.04gofK2HPO4,0.04gofKH2PO4,0.01gofMgSO4x7H2O,0.04gofCaCl2x6H2O,2gofNaHCO3,2mLofTween80,0.05gofhemindissolvedin1mLof4MNaOH,10mLof vitaminK, 0.5 gof L-cysteineHCl, 0.5 gofbile salts and4mLof resazurinsolution(0.025%,w/v)asananaerobicindicator.Thegrowthmediumwassterilizedat121°Cfor15minglassvessels(280mL)beforesamplepreparation.

Thefermentationprocessofdigestarawandcookedcactusaswellasthecontrol(withno cactus cladodes) was developed using anaerobic, stirred, pH (5.3-5.7) andtemperature controlled faecal batch cultures. Glass water-jacketed vessels weresterilizedandfilledasepticallywith67.5mLofpre-sterilizedbasalnutrientmedium.Themediumwasthengassedovernightwithoxygenfreenitrogentomaintainanaerobicconditions.Thefollowingdayandbeforetheinoculation,eachvesselwasdosedwith0.75 g of digesta cactus, for a final concentration of 1% (w/v). Fresh human faecalsampleswerecollectedinananaerobicjarfromthreehealthyfaecaldonorswhowerefreeofanyknownmetabolicandgastrointestinaldiseases,werenottakingprobioticorprebioticsupplements, followedapolyphenol-freediet for2daysandhadnottakenantibiotics 3 months before faecal collection. Faecal slurry was prepared byhomogenizing the faeces in pre-reduced phosphate buffered saline (PBS). Thetemperature was set to 37 °C using a circulating water-bath and the vessels wereinoculatedwith7.5mLfaecalslurry(10%w/voffreshhumanfaeces)foraperiodof24h,duringwhichsampleswerecollectedat4timepoints(0,5,10and24h).Sampleswere centrifuged at 13,500 rpm at 4 °C for 5min and stored at −80 °C for furtheranalysis.

2.4Identificationandquantificationof(poly)phenoliccompoundsbyHPLC-ESI-MS

(Poly)phenolic compounds from fermented raw and cooked cactus cladodes wereextracted following themethoddescribed in Juánizet al. (2017) 9.Briefly, 0.1mLofmethanol/acidified water (0.1% formic acid) (80:20 v/v) was added to 0.1 mL offermented samples, vortexed and centrifuged at 14,000 rpm for 10 min. Then,supernatantswere transferred tovials to carryoutHPLC-ESI-MSanalysis,whichwasperformedusinganHPLCunitmodel1200(AgilentTechnologies,PaloAlto,CA,USA)equippedwithaTripleQuadrupoleLinear IonTrapMassSpectrometer3200Q-TRAP

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(ABSciex,Framingham,MA,USA).ThecolumnusedwasaCORTECS®C18(3x75mm,2.7µm)fromWaters(Milford,MA,USA).

ForHPLCseparation,mobilephaseAwas0.1%(v/v)formicacid inwaterandmobilephaseBwasacetonitrile.Separationswerecarriedoutwithaninjectionvolumeof4μL,columnoventemperatureof30°Candelutionflowrateof0.35mL/min.Themobilephasescomprisedaprogramof0–1.20min,5%B;1.20–8.80min,5–11.4%B;8.80-10min,11.4–11%B;10–30min,11-30%B;30-32min,28-100%Bandthenreturnto5%Bin2minandmaintainedisocraticuntiltheendoftheanalysis(38min)tore-equilibratethecolumn.Massanalyseswereperformedinnegativeionizationmode,withtheturboheatermaintainedat500°CandIonSprayvoltagesetat-3500.Nitrogenwasusedasnebulizing,turboheaterandcurtaingasandwassetatthepressureof40,50and35psi,respectively.ChromatogramswereacquiredusingAnalystsoftware1.6.3(ABSciex,Framingham,MA,USA).

Fortheidentificationofthephenoliccompounds,apreliminaryanalysiswascarriedoutina full scan from100to1000m/z,andaconsecutivelyselectiveproduct ionmodeanalysiswithspecificm/z.Identificationwasachievedbymultiplereactionmonitoring(MRM).Twotransitionswerestudiedforeachphenoliccompound.Thefirsttransition,corresponding to themost abundant fragment, was used as quantifier ion, and thesecond as qualifier ion. Details of the (poly)phenols identification are shown in theSupportingInformationTableS1(SupplementaryMaterial).

Phenolicacidswereexpressedas ferulicacidequivalents,whereas isorhamnetinandkaempferolderivativeswerequantifiedwiththeirrespectiveaglyconesusingcalibrationcurves.Resultswereexpressedasmilligramsofeachcompoundpermillilitreofbatchculture(µg/mL).

2.5Biologicalactivityassays

2.5.1Tissueculture

Biological activity of fermented cactus cladodes was performed using HT29 humancolorectaladenocarcinomacellsacquiredfromtheEuropeanCollectionofCellCultures(ECACC). Cells were grown in tissue culture flasks and maintained in DMEMsupplementedwithFBS(10%)andPenStrep(1%).Theywereincubatedat37°Cwith5%CO2andsub-culturedevery2daysbytheadditionoftrypsin(0.25%trypsin-EDTA)for10minat37°Candcentrifugedat1,200rpmfor3min.Afterthat,thesupernatantwasdecantedandcellswerere-suspendedintheappropriatemedium.Twenty-fourhwasselectedastheexposuretimeforallinvitrostudieswithfermentedsamples,asitisgenerallyconsideredtoreflecttheaveragecolonictransittime.

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2.5.2Cytotoxicityassay

RawandcookedcactuscladodescolonicfermentatesweredilutedinDMEMat10%and20%(v/v)toevaluatecytotoxicitybytheMTTcolorimetricassay,followingthemethoddescribedbyMcDougalletal.(2017)13.First,HT29cellswereseededin96multi-wellplates(Costar,Cambridge,MA,USA)ataconcentrationof1.5x104cellsperwell.After2 days of incubation at 37 °C,mediawas replacedwith fermented raw and cookedcactus samples at 10% and 20% (v/v) and then incubated for 24 h. Thewells werewashedandrefreshedwithmedia.Thereafter,15µLofMTT(5mg/mL)wereaddedtoeachwell.After4hat37°C,lysiswascarriedoutwith100µLofsolubilizingsolutionand measured using a plate reader (Alpha, SLT Rainbow Thermo, Antrim, UK) at awavelengthof570nm,reference650nm.Eachsamplewasmeasuredinoctupleandresults are presented as percentage cell viability compared to untreated cells. Theexperimentwasrepeatedindependentlythreetimes.

2.5.3Genotoxicityassay(Cometassay)

TheeffectofcolonicallyfermentedrawandcookedcactusoncolonocyteDNAdamagewas determined using the Comet assay according to the method described byMcDougalletal.(2017)13.HT29cellswerepre-incubatedwiththesamplesfor24h.Theanti-genotoxiceffectwasassessedbytreatingpre-incubatedHT29cellswithhydrogenperoxide(75µMH2O2),orwithPBSastheirrespectivecontrol.Briefly,thecellswerereconstitutedin85μLof0.85%lowmeltingpointagaroseinPBSandmaintainedinawaterbathat37°C.Thissuspensionwasmixedwith1%normalagarosetopreviouslypreparedgelsonfrostedslidesandcoverslipswereadded.Theslidesweresubjectedtolysisbuffer(2.5MNaCl,100mMNa2EDTA,10mMTRIS)for1hat4°Candplacedinelectrophoresisbufferfor20minat25V300mA.Subsequently,slideswerewashed(3×5min)inneutralisationbuffer(0.4MTRISHCl,pH=7.5)at4°C.Allslideswerestainedwithethidiumbromide(20μLof20μg/mL)priortoscoring.Imageswereanalysedat400 × magnification using a Nikon eclipse 600 epifluorescence microscope. Thepercentage(%)tailDNAwasrecordedusingKomet5.0imageanalysissoftware(KineticImaging Ltd, Liverpool, UK). For each slide, 50 cellswere scored and themeanwascalculated.Resultsarepresentedasmeanpercentage(%)tailDNA.Theexperimentwasrepeatedindependently3timesandpositive(H2O2)andnegativecontrols(PBS)wereincludedinallexperiments,aswellascellswithoutfermentedtreatment.

2.5.4Cellcycleanalysis

Cellcycleanalysisoffermentedrawandcookedcactuswasdeterminedusingpropidiumiodidestainingandmeasuredusingflowcytometry(Omerod,2000).Briefly,HT29cells

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wereseededatadensityof1x106cellspermLfor24hbeforetreatmentwitheachfermentedextractat10%.Afterfermentatetreatmentexposure,thesuspensionwascentrifuged at 1,200 rpm for 5 min, the supernatant discarded and the pellet re-suspendedin1mLoficecoldPBS.Then,thesuspensionwascentrifugedat4,000rpmfor5min,thesupernatantwasdiscardedandthecellswerere-suspendedin1mLoficecoldethanol/PBS(90:10)andincubatedat-20°Covernight.Afterincubation,cellswerewashedtwicewithicecoldPBSandthestainingbuffer(100µL/mLofRNaseA,10µL/mLofpropidiumiodide,0.1%NP40and50µL/mLtrisodiumcitrateinPBS)wasadded.Thecellswerethenincubatedat37°C,5%CO2for30minbeforebeinganalysedonaFACSCaliburflowcytometer(BectonDickinson,UK)andthefluorescenceemissionspectraofpropidium iodide was collected at 585 nm, using Beckman-Coulter Gallios flowcytometer.SubsequentlytheseemissionspectrawereanalysedforDNAcontentusingWinMDIsoftware(J.Trotter,ScrippsInst.).Theexperimentwasperformedastriplicateindependentexperimentsandresultsarepresentedasapercentage(%)ofcellcount.2.11Statisticalanalysis

Themeanofeachdatasetwasusedforstatisticalanalysis.TheShapiro-Wilktestwasused to test for normality. Analysis of variance was applied to test for significantdifferencesbetweenmeansusingone-wayanalysisofvariance(ANOVA,DunnettTandT3tests).ABonferronitestwasappliedasaposteriori testforsignificantdifferencesamongfermentationtimepoints.Significancewasacceptedatp<0.05.AnalysiswascarriedoutusingSPSS(version20forWindows).

3.Resultsanddiscussion

The effect of the human gut microbiota on (poly)phenolic compounds of raw andcookedcactus (O. ficus-indica)cladodes,aswellastheirbiologicalactivityoncoloniccells,hasbeenevaluatedforthefirsttime,tothebestofourknowledge.

3.1Coloniccatabolismof(poly)phenoliccompounds

The(poly)phenoliccompositionoffermentatesovertimeisreportedinTable2.Twenty-seven (poly)phenolic compoundswere identified and quantified in baseline (0 h) offermented cactus cladodes, being phenolic acids themost abundant in all samples,accountedfor78-88%ofthetotalcontent.Piscidicacidwasthemaincompound(52-65% total (poly)phenolic compounds), followed by eucomic acid (15-36% total(poly)phenoliccompounds).Griddledcactuscladodespresentedthehighestamountofpiscidicacid,andmicrowavedsampleshadhigheramountofeucomicacidthantherestof cooking treatments. Flavonoids were also present in cactus cladodes colonicfermentates,beingkaempferolderivatives(6-13%total)andisorhamnetinderivatives(4-7%total)mainly intheirglycosidesforms.Griddledcactuscladodespresentedthe

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highestamountofkaempferolderivativeswhilstmicrowavedsamplescontributedwiththehighestisorhamnetinderivativescontentatthebeginningofcolonicfermentation(0h).These(poly)phenolicprofilesareconsistentwiththoseofpreviousstudiesinO.ficus-indicacactuscladodeswithoutgastrointestinaldigestion2,4.Inapreviousworkincactus cladodes after gastrointestinal digestion, quercetin derivatives were alsoidentified6.Nevertheless,inthisstudytheywerenotdetectedatanytimepointofthefermentation, may be due to the rapid metabolism of quercetin glycosides at thestartingofcolonicfermentation.Ithasbeenpreviouslydemonstratedafastdegradationwithinthefirst15minaftercolonicfermentationinquercetinderivativesfromrawandcookedpepper8aswellasinapples17.

Changes in the amount of (poly)phenols throughout the faecal fermentations wereobserved. Even though a degradation of the compounds was shown, some(poly)phenolswerestillpresentafter24hoffaecalfermentation.Bothkaempferolandisorhamnetinderivativeswererapidlycatabolisedbyhumanmicrobiota,showinglowamounts,tracesorevennodetectedattheendofthe24hofcolonicfermentation(Fig.1a). Inparticular, isorhamnetinhexoserhamnosehexosideIIandIII, isorhamnetindi-hexoside II, isorhamnetinhexose rhamnosehexoside I, kaempferol rutinoside III andkaempferolglucosideIIwerecompletelydegradedinmostofthesamplesafter24hofcolonicfermentation,exceptingriddledandmicrowaved,inwhichremainedavailableorintraces.

Sincethefirststepofthefaecalincubation,thehydrolysisofflavonoidglycosidesandtheconsequent formationofaglycones tookplace,especially fromthe isorhamnetinderivatives.Thehighestamountofisorhamnetinwasdetectedinmicrowavedsamplesafter10hoffaecalincubationbutthenitdecreased.Kaempferolderivativeswerealsodegradedafter24hfermentation,buttheirrespectiveaglyconewasnotdetectedduetothelowamountofitsnative(poly)phenols.Someincreasesinflavonoidsduringthefermentationwere also observed,may be due to the release from the foodmatrix.Particularly,methoxykaempferolhexosideIincreasedafter5and10hofincubationinallsamples,except inbothfriedsamples.Moreover,althoughattheendofthe24hfermentationitdecreased,thefinalamountwashigherthaninthebeginninginraw,microwavedandgriddledcactuscladodes.

Withrespect tophenolicacids,eucomicacidwasthemoststableduringthe24hoffaecalincubation(Fig.1b).Althoughitwasdegraded,itstillremainedinhighamountsinallsamples,especiallyinmicrowavedandgriddledones(Table2).Conversely,piscidicacid increased after 5 h of incubation in all samples; but was almost completelydegradedafter24h.However,thedegradationwasloweringriddledsamples.Likewise,

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feruloylglucose I and IIwere rapidlydegradedafter24hof faecal fermentation,butremainedintracesingriddledandmicrowavedcactuscladodes.

Eucomicacidweremorestableundercolonicconditionsprobablyduetotheresistancetogastrointestinalandcolonicmicrofloraenzymes,aswellasthereleasefromthecellwalls by the microbiota action 18. However, ferulic acid derivatives were rapidlydegraded in all samplesmay be due to the catabolism related tomicrobial feruloylesteraseswhicharepresentinthecolonicinoculumanddegradedthecompounds19.

After24hcolonicfermentation,thecatabolite3-(4-hydroxyphenyl)propionicacidwasfound in trace amounts maybe produced via demethoxylation from ferulic acidderivatives10,aswellasviadehydroxylationof3-(3,4-dihydroxyphenyl)propionicacidderivedfromferulicacidandtheringfissionofquercetin,isorhamnetinandkaempferolderivatives8,9,20.

Metabolic products depend on (poly)phenolic compounds present and theinterindividual variation in the gut microbial composition and enzymatic capacitiesleading to different catabolite profile 10. However, in the present study 3-(3,4-dihydroxyphenyl)propionicacidwaspresentinallthevolunteers,evenintraces.Furtherinvestigationisneededtoevaluatetheabsorptionofbioactivecompoundsfromcactuscladodestakingintoaccountthegastrointestinaldigestionandmicrobiotaconditions.

3.4Biologicalactivityincoloncells

The biological activity of cactus cladodes submitted to the most common culinarytechniques(boiling,griddlingandfrying)inhumancolorectalcellswasevaluatedaftergastrointestinal digestion and the action of gutmicrobiota inHT29 cells to simulatephysiologicalconditions.Rawsamplesandcontrol(withnocactus)fermentateswereusedaspositiveandnegativecontrols.

NocytotoxicactivitywasobservedinHT29cellstreatedwithfermentedrawandcookedcactuscladodesat10%and20%(v/v)after24hofincubation,comparedtountreatedcells(Fig.2).Likewise,nosignificantdifferences(p<0.05)wereobservedamongcookingtreatments. Nevertheless, cell viability was significant lower (p<0.05) in controlfermentation(withnocactuscladodes)at20%(v/v)thaninuntreatedcellsanddidnotpresentacellviabilityhighertan80%.Therefore,a10%(v/v)concentrationwasusedforthecometandcellcycleassays.Uptoourknowledge,cytoxicityofcactuscladodesafter colonic fermentation has not been previously evaluated. Similarly, Serra et al.(2013)14reportednocytotoxicactivityofO.ficus-indicafruitjuicesinaCaco-2humancoloncellmodel.

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The effect of cactus cladodes colonic fermentates against DNA damage induced byoxidationincolonocyteswasassessedbythecometassay(Fig.3).ThepercentageofDNAinthetailinducedbyH2O2wassignificantly(p<0.05)reducedbyrawandcookedcactus cladodes colonic fermentates (34-38%) in comparison with the controlfermentate (withnocactuscladodes) (45%).Thereduction in theH2O2-inducedDNAdamage of fermented cactus cladodes may be attributed to the (poly)phenoliccompoundswhichstillremainedaftercolonicfermentation,especiallyeucomicacids.Similarly,studiesindigestedblackcurrant21andelderberry22haveobservedareductioninH2O2-inducedDNAdamageinNCM460(non-tumorigeniccoloncellline)andinCaco-2cells.However, the roleof fermentationon thebioactivityof theextractswasnotconsidered. A similar consideration to this study was those reported with colonicfermentedraspberry,strawberryandblackcurrantwhichexertedasignificant(p<0.05)reduction of H2O2-induced DNA damage in HT29 cell line 23,24, demonstrating thatantioxidantactivity,andspecificallypotentialantigenotoxicactivityof(poly)phenolsisretainedaftercolonicfermentation.

Finally, theeffectof cactuscladodescolonic fermentateson theHT29cell cyclewasevaluated(Fig.4).Nosignificanteffect(p<0.05)wasobservedineachstageoftheHT29cellcycleafter24hofincubationwithcolonicfermentedcactuscladodes.Nosignificantdifferences (p<0.05) were also observed among fermented samples. Results areconsistentwithCoatesetal.(2007)whoreportednotsignificantchangesincellcycleproliferation of HT29 cells pre-treated with colonic fermented berries. In contrast,extractsfromO.ficus-indicafruitjuiceinducedacellcyclearrestedintheG1phaseinHT29cells14,attributingthiseffecttothebetalains.

In summary, results of the in vitro colonic fermentation of raw and cooked cactuscladodesshowedthatflavonoidstendedtobedegradedmorethanphenolicacids,buttheyarestillavailableafter24hofcolonicincubation,especiallyeucomicacid.Resultsalsosuggestthatremained(poly)phenolsaftertheactionofcolonicmicrobiotastillhaveantioxidant and genoprotective properties which can exert into the intestinal cells.Therefore,thecurrentstudyprovidesabasisforfurtherinvestigationsaboutthecactuscladodesbioactivecomponentspotentiallyavailabletobeabsorbed,andtoexerttheirbeneficialeffects incolonandother targetorgansafter ingestionandtounderstandtheirmechanismofactioninvivo.

Acknowledgments

This researchwas funded by the SpanishMinistry of Economy and Competitiveness(AGL2014-52636-P). E. De Santiago expresses their gratitude to the Association of

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Friendsof theUniversityofNavarraand LaCaixaBanking Foundation for thegrantsreceived.WethankKeithThomasforhiskindhelp.

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Figurecaption

Figure1.Degradationof(poly)phenoliccompoundsofgriddledcactuscladodesduring24 h colonic fermentation. a) Total kaempferol derivatives, total isorhamnetinderivatives and isorhamnetin aglycone. b) Total phenolic acids, piscidic acid andeucomicacid.

Figure2.Thecytotoxiceffectof20%(v/v)and10%(v/v)fermentedcactuscladodesonHT29cells.Dataarepresentedasmeansof3independentexperiments+/-SDcomparedto the untreated cells as a control. One-way ANOVA andPost Hoc test Dunnett’s T*p<0.05.

Figure3.Antigenotoxicactivityoffermentedcactuscladodes10%(v/v)onDNAdamagein HT29 cells challenged with 75 μM H2O2. Data are presented as means of 3independent experiments +/- SD compared to the control fermentation. One-wayANOVAandPostHoctestDunnett’sT*p<0.05.

Figure4.Effectsoffermentedcactuscladodes10%(v/v)onHT29cellcycle.Dataarepresentedasmeansof3independentexperiments+/-SDcomparedtotheuntreatedcellsasacontrol.

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Figure1a)

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Figure2

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Table1.CookingConditionsAppliedtoCactusCladodes.Cookingtechnique

Time Temperature Amountofwateroroil

Equipment

Boiling 10min 100°C 600mL Stainlesssteelpot

Microwaving 5min - - Siliconecase(Lékué,Barcelona,Spain)andmicrowaveoven(Whirlpool,Michigan,USA)at900W

Griddling 5min&5min 150°C&110°C - Non-stickgriddle(JataElectro,Vizcaya,Spain)

Fryinginolive/soybeanoil

10min&5min 100°C&90°C 30mL Non-stickfryingpan

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109

Results

Table1.Native(Poly)ph

enolicCom

poun

dsand

Catab

olite

sProdu

cedDu

ringCo

lonicFerm

entatio

nofRaw

and

Coo

kedCa

ctus

Clad

odes.R

esultsareExpressed

asM

ean±Stan

dardDeviatio

n(µg(Poly)ph

enolicCom

poun

d/mL)(n

=3).

Compo

unds

Raw

Boiled

Microwaved

Griddled

Friedinoliveoil

Friedinso

ybeanoil

µg/m

Lµg

/mL

µg/m

Lµg

/mL

µg/m

Lµg

/mL

Phen

olicacids

Piscidicacid

0h

5h

10h

24h

250.59

±76.17

26

5.01

±33.27

24

9.05

±86.67

0.46

±0.15

140.53

±24.75

15

9.95

±23.06

14

0.77

±73.26

0.12

±0.05

333.09

±32.90

42

2.39

±21.15

43

1.91

±11.54

1.47

±0.56

355.75

±11.86

38

4.77

±17.70

30

2.27

±93.99

32

.31±6.42

182.60

±8.31

205.54

±12.26

13

7.83

±85.08

0.16

±0.09

177.79

±46.77

21

2.97

±96.91

14

7.98

±98.80

0.45

±0.12

FeruloylglucoseI

0h

5h

10h

24h

0.95

±0.51

0.15

±0.20

0.01

±0.00

nd

0.54

±0.30

0.07

±0.03

nd

nd

1.17

±0.29

0.76

±0.03

0.03

±0.01

tr

0.84

±0.13

0.28

±0.20

0.60

±0.23

tr

0.41

±0.18

0.06

±0.09

tr

tr

0.54

±0.29

tr

tr

tr

FeruloylglucoseII

0h

5h

10h

24h

1.14

±0.43

0.48

±0.57

0.01

±0.00

nd

0.70

±0.27

0.28

±0.36

tr

nd

1.12

±0.19

1.06

±0.41

0.24

±0.29

tr

1.33

±0.25

0.77

±0.43

0.60

±0.23

tr

0.77

±0.17

tr

nd

nd

0.81

±0.24

0.16

±0.21

nd

nd

Eucomicacid

0h

5h

10h

24h

178.06

±51.20

16

3.38

±43.33

10

5.72

±16.66

71

.50±30

.68

33.01±17

.84

56.99±27

.89

58.21±25

.39

42.98±2.31

161.75

±12.80

18

4.41

±19.23

20

8.89

±11.54

15

4.08

±2.88

100.91

±15.31

10

5.63

±30.81

11

8.23

±25.60

81

.09±18

.66

54.86±3.96

57

.13±13

.20

52.14±16

.07

35.29±12

.68

64.91±10

.16

55.89±15

.63

53.66±10

.49

36.31±19

.81

TOTA

LPH

ENOLICAC

IDS

0h

5h

10h

24h

430.75

±126

.65

429.01

±130

.27

354.92

±117

.91

71.96±50

.23

174.78

±66.34

21

7.29

±75.34

19

8.98

±58.37

43

.10±30

.31

497.12

±158

.46

608.63

±199

.85

641.07

±206

.10

155.55

±107

.92

458.83

±167

.45

491.44

±181

.50

421.18

±142

.59

113.40

±34.50

238.64

±85.86

25

9.72

±104

.41

189.97

±60.59

35

.45±24

.84

244.05

±83.53

26

9.02

±110

.35

201.64

±66.69

36

.77±25

.36

Isorha

mne

tinderivates

Isorhamne

tin

0h

5h

10h

24h

0.11

±0.14

1.37

±0.86

0.84

±0.69

0.73

±0.93

0.18

±0.21

1.30

±0.86

1.38

±0.58

0.68

±0.55

0.28

±0.42

0.78

±0.80

4.74

±2.01

2.89

±2.49

0.22

±0.35

1.17

±0.64

1.85

±1.50

1.63

±0.26

0.20

±0.28

0.75

±0.35

1.58

±1.68

1.77

±1.24

0.15

±0.26

0.75

±0.56

1.08

±0.34

0.49

±0.46

Results

110

Results

109

Isorhamnetinhexoserham

nosehexosideI

0h5h10h24h

0.02±0.00trtrnd

trtrndnd

0.02±0.010.03±0.000.01±0.00

tr

0.02±0.000.01±0.00

trtr

0.01±0.00trtrtr

0.01±0.00trtrnd


nosehexosideII

0h5h10h24h

trtrtrnd

trtrndnd

trtrtrnd

trtrtrnd

trtrtrnd

trtrtrnd


nosehexosideIII

0h5h10h24h

trtrtrnd

trtrtrnd

0.01±0.00trtrnd

0.01±0.00trtrnd

trtrtrnd

trtrtrnd

Isorhamnetindi-hexosideI

0h5h10h24h

0.15±0.090.14±0.120.09±0.110.06±0.08

0.12±0.060.13±0.040.07±0.070.01±0.02

0.24±0.0250.35±0.020.27±0.070.15±0.12

0.22±0.020.22±0.070.14±0.100.01±0.01

0.12±0.010.10±0.060.06±0.050.04±0.05

0.13±0.050.15±0.010.14±0.01

nd

Isorhamnetindi-hexosideII

0h5h10h24h

trtrtrnd

trtrtrnd

0.01±0.00trtrnd

0.01±0.00trtrnd

trtrtrnd

trtrtrnd

Isorhamnetinrutinosiderham

noside

0h5h10h24h

0.17±0.100.09±0.110.08±0.07

tr

0.10±0.03trtrnd

0.25±0.110.35±0.000.25±0.070.07±0.05

0.24±0.080.20±0.080.10±0.07

tr

0.14±0.050.10±0.03

trtr

0.13±0.070.08±0.070.06±0.05

nd

Isorhamnetinrutinosiderham

nosideII

0h5h10h24

0.18±0.110.14±0.120.09±0.110.02±0.03

0.12±0.040.12±0.09

trtr

0.23±0.100.38±0.010.34±0.010.20±0.11

0.22±0.070.23±0.090.19±0.090.04±0.06

0.12±0.030.08±0.070.03±0.03

tr

0.13±0.090.15±0.000.15±0.00

nd

Isorhamnetinhexosepentoside

0h5h10h24h

2.36±1.831.73±1.571.25±1.480.30±0.48

2.29±1.401.63±1.850.85±1.16

tr

4.63±2.128.08±1.226.54±1.712.71±0.22

4.39±1.883.92±1.761.69±0.270.94±0.77

2.57±1.191.95±1.600.49±0.63

tr

2.77±2.063.40±1.262.84±1.13

tr

Results

111

Results

Isorham

netin

rutin

osideI

0h

5h

10h

24h

0.71

±0.52

0.82

±0.76

0.86

±1.18

0.30

±0.48

0.83

±0.65

0.72

±0.20

0.53

±0.01

tr

0.58

±0.13

1.39

±0.56

2.02

±1.12

2.11

±0.01

0.60

±0.17

1.06

±0.52

1.17

±1.01

1.03

±0.83

0.42

±0.14

0.80

±0.36

0.59

±0.53

0.22

±0.23

0.42

±0.15

0.66

±0.67

0.91

±0.78

0.02

±0.03

Isorham

netin

rutin

osideII

0h

5h

10h

24h

19.12±9.09

6.15

±5.72

1.76

±1.05

0.07

±0.01

12.00±6.72

1.97

±0.95

0.20

±0.07

tr

34.58±7.56

30

.61±8.67

12

.80±10

.27

0.51

±0.51

30.53±9.75

24

.51±11

.16

10.65±4.06

2.02

±1.41

13.26±3.40

8.36

±2.98

0.79

±0.28

tr

17.02±5.31

7.57

±3.53

3.42

±3.13

tr

Unknow

niso

rham

netin

derivative

0h

5h

10h

24h

0.75

±0.53

0.33

±0.42

0.22

±0.13

nd

0.41

±0.26

0.20

±0.27

tr

nd

1.00

±0.58

1.37

±0.15

1.07

±0.22

0.24

±0.17

0.89

±0.49

0.71

±0.36

0.31

±0.15

0.16

±0.08

0.54

±0.32

0.40

±0.03

0.05

±0.03

tr

0.56

±0.44

0.49

±0.30

0.35

±0.20

nd

TOTA

LISO

RHAM

NETINDE

RIVA

TES

0h

5h

10h

24h

23.57±6.23

10

.78±2.04

5.19

±0.63

1.49

±0.26

16.04±3.90

6.06

±0.77

3.03

±0.53

0.70

±0.48

41.81±10

.29

43.34±9.99

28

.03±4.29

8.87

±1.24

37.33±9.09

32

.02±7.95

16

.11±3.40

5.84

±0.80

17.36±4.32

12

.53±2.68

3.61

±0.55

2.03

±0.95

21.32±5.56

13

.25±2.62

8.97

±1.31

0.52

±0.33

Kaem

pferolderivates

Kaem

pferolhexosidedirham

nosid

eI

0h

5h

10h

24h

0.24

±0.16

0.16

±0.21

0.17

±0.18

nd

0.18

±0.09

0.16

±0.21

tr

nd

0.40

±0.21

0.65

±0.06

0.58

±0.08

0.32

±0.18

0.32

±0.14

0.30

±0.15

0.17

±0.13

0.12

±0.16

0.20

±0.07

0.23

±0.05

0.05

±0.04

tr

0.20

±0.12

0.23

±0.00

0.05

±0.04

nd

Kaem

pferolhexosidedirham

nosid

eII

0h

5h

10h

24h

0.14

±0.10

0.07

±0.07

0.05

±0.07

tr

0.09

±0.05

tr

tr

nd

0.17

±0.11

0.29

±0.02

0.20

±0.01

0.04

±0.05

0.15

±0.07

0.13

±0.08

0.06

±0.05

0.04

±0.05

0.10

±0.05

0.06

±0.04

0.13

±0.22

nd

0.10

±0.06

0.09

±0.01

0.05

±0.04

nd

Kaem

pferolhexosepe

ntoside

0h

5h

10h

24h

1.28

±0.88

0.69

±0.71

0.35

±0.41

0.08

±0.14

0.83

±0.49

0.51

±0.67

0.26

±0.36

0.01

±0.02

1.91

±1.08

2.63

±0.12

2.06

±0.05

0.93

±0.18

1.38

±0.66

1.09

±0.50

0.55

±0.36

0.12

±0.16

0.77

±0.36

0.50

±0.44

0.20

±0.20

tr

0.88

±0.67

1.02

±0.34

0.68

±0.26

tr

Kaem

pferolru

tinosideI

0h

5h

0.10

±0.07

0.18

±0.07

0.07

±0.06

0.19

±0.01

0.11

±0.06

0.20

±0.11

0.09

±0.04

0.14

±0.08

0.07

±0.05

0.10

±0.06

0.08

±0.04

0.11

±0.09

Results

112

Results

111

10h24h

0.21±0.21tr

0.16±0.14nd

0.33±0.240.58±0.01

0.15±0.140.07±0.10

0.06±0.070.08±0.11

0.16±0.09tr

KaempferolrutinosideII

0h5h10h24h

0.06±0.030.13±0.100.11±0.166

tr

0.07±0.020.16±0.080.02±0.00

nd

0.05±0.000.23±0.130.23±0.180.08±0.08

0.06±0.030.13±0.080.15±0.160.08±0.11

0.10±0.050.06±0.040.13±0.22

tr

0.06±0.040.16±0.080.28±0.08

tr

KaempferolrutinosideIII

0h5h10h24h

0.17±0.050.28±0.300.02±0.02

nd

0.20±0.140.04±0.03

ndnd

2.58±3.692.51±2.820.19±0.240.01±0.02

1.53±1.801.91±2.510.35±0.140.08±0.07

0.64±0.910.14±0.120.04±0.05

nd

1.28±1.931.43±1.990.63±0.90

nd

KaempferolrutinosideIV

0h5h10h24h

4.20±4.021.10±1.360.53±0.240.01±0.01

2.58±2.67trtrnd

8.18±6.6214.04±2.492.44±3.270.11±0.14

6.34±5.215.55±4.311.45±0.850.34±0.57

1.78±1.461.23±0.260.17±0.08

nd

3.25±2.902.08±1.130.96±1.13

tr

KaempferolglucosideI

0h5h10h24h

0.22±0.230.14±0.200.08±0.130.06±0.09

0.21±0.170.19±0.270.14±0.20

nd

0.27±0.240.46±0.430.54±0.500.38±0.21

0.26±0.140.32±0.180.32±0.180.19±0.06

0.11±0.080.15±0.100.07±0.090.05±0.03

0.10±0.080.19±0.150.11±0.012

nd

KaempferolglucosideII

0h5h10h24h

0.10±0.100.05±0.060.04±0.06

nd

0.10±0.09trtrnd

0.22±0.190.15±0.200.01±0.01

tr

0.23±0.020.22±0.090.12±0.070.02±0.03

0.06±0.040.05±0.06

trnd

0.07±0.090.02±0.010.04±0.05

nd

Methoxykaem

pferolhexosideI

0h5h10h24h

11.71±7.3614.89±21.4718.08±27.2913.84±21.34

15.45±12.4018.79±25.6215.47±19.710.51±0.08

22.13±16.0735.27±25.5443.48±35.1636.44±15.75

24.01±17.0832.53±16.6531.11±27.4830.42±15.00

10.04±7.484.03±2.032.09±0.531.37±0.58

4.99±1.363.61±2.402.35±0.821.51±0.94

Methoxykaem

pferolhexosideII

0h5h10h24h

12.41±10.604.08±5.520.29±0.260.02±0.03

12.30±9.941.71±2.390.50±0.70

tr

25.96±26.4421.44±28.510.60±0.070.26±0.37

28.14±23.5225.31±19.4810.93±12.620.16±0.12

8.81±6.116.29±6.701.20±0.96

tr

7.56±10.301.97±2.340.33±0.290.03±0.04

TOTALKAEM

PFEROLDERIVATES

0h5h10h24h

30.63±4.7521.77±4.4419.92±5.4014.01±5.63

32.08±5.5121.75±6.5216.55±6.230.53±0.35

61.98±9.4477.89±11.6850.66±12.9239.16±10.91

62.51±10.2867.64±11.4945.35±9.5031.63±9.14

22.67±3.6912.87±2.064.04±0.691.51±0.66

18.56±2.5310.92±1.175.83±0.671.55±0.87

Results

113

Results

3-(4-hydroxyph

enyl)propion

icacid

0h

5h

10h

24h

nd

nd

nd

tr

nd

nd

nd

tr

nd

nd

nd

tr

nd

nd

nd

tr

nd

nd

nd

tr

nd

nd

nd

tr

TOTA

L(PO

LY)PHE

NOLICCO

MPO

UNDS

0h

5h

10h

24h

484.95

±60.93

46

1.56

±63.22

38

0.03

±55.29

87

.46±18

.51

222.90

±28.98

24

5.10

±38.03

21

8.55

±40.59

44

.32±17

.44

600.91

±70.90

72

9.85

±91.64

71

9.75

±95.72

20

3.58

±34.00

558.67

±71.30

59

1.10

±79.95

48

2.64

±64.89

15

0.86

±19.76

278.67

±38.15

28

5.13

±43.11

19

7.61

±32.26

38

.99±11

.63

283.94

±37.80

29

3.19

±46.12

21

6.44

±33.60

38

.83±13

.58

nd=node

tected

tr=traces

Results

114

SUPPLEMENTARYINFORMATION

TITLE: Digestion and colonic fermentation of raw and cookedOpuntia ficus-indicaimpactsbioaccessibilityandbioactivity

AUTHORS:ElsyDeSantiagoa,ChrisI.R.Gillb,IlariaCarafac,KieranM.Touhyc,María-PazDePeñaaandConcepciónCida*

aUniversidaddeNavarra,FacultaddeFarmaciayNutrición,DepartamentodeCienciasdelaAlimentaciónyFisiología,C/Irunlarrea1,E-31008Pamplona,Spain.

IdiSNA,NavarraInstituteforHealthResearch.Pamplona,Spain.

bNutrition InnovationCentre forFoodandHealth,Centre forMolecularBiosciences,University of Ulster, Cromore Road, Coleraine, Northern Ireland, BT52 1SA, UnitedKingdom.

cNutrition&NutrigenomicsUnit,DepartmentofFoodQualityandNutrition,Researchand InnovationCentre, FondazioneEdmundMach (FEM),Via E.Mach1, 38010, SanMicheleall’Adige,Trento,Italy.


Results

115

TableS1.Massspectrometriccharacteristicsof(poly)phenoliccompoundsidentifiedinthisstudy.

Compound Chemicalformula

Rt(min)

[M-H]¯

(m/z)Fragment

ionsPhenolicacids

Piscidicacid C11H12O7 3.1 255.1 165/179FeruloylglucoseI C16H20O9 7.7 355 193/179FeruloylglucoseII C16H20O9 11.08 355 193/179EucomicacidI C11H12O6 9.5 239.2 179/195

Isorhamnetinderivatives Isorhamnetin C16H12O7 33 315 151Isorhamnetin hexose rhamnosehexosideI

C34H42O21 17.5 785 315/151

Isorhamnetin hexose rhamnosehexosideII

C34H42O21 27.9 785 315/151

Isorhamnetin hexose rhamnosehexosideIII

C34H42O21 28.15 785 315/151

Isorhamnetindi-hexosideI C28H32O17 18.19 639 477/315Isorhamnetindi-hexosideII C28H32O17 19.25 639 477/315IsorhamnetinrutinosiderhamnosideI C34H42O20 18.8 769 315/145IsorhamnetinrutinosiderhamnosideII C34H42O20 19.1 769 315/145Isorhamnetinhexosepentoside C27H30O16 20.25 609 477/315IsorhamnetinrutinosideI C28H32O16 20.1 623 315/299IsorhamnetinrutinosideII C28H32O16 21.99 623 315/299UnknownIsorhamnetinderivaive 19 755 315/299

Kaempferolderivatives KaempferolhexosidedirhamnosideI C33H40O19 18 739 285KaempferolhexosidedirhamnosideII C33H40O19 18.5 739 285Kaempferolhexosepentoside C26H28O15 19.7 579 285/255KaempferolrutinosideI C27H30O15 18.98 593 285/255KaempferolrutinosideII C27H30O15 19.45 593 285/255KaempferolrutinosideIII C27H30O15 20.03 593 285/255KaempferolrutinosideIV C27H30O15 21.5 593 285/255KaempferolglucosideI C22H22O12 21 447 285/255KaempferolglucosideII C22H22O12 22 447 285/255Methoxykaempferolhexoside C22H22O12 22 477 314/285MethoxykaempferolhexosideII C22H22O12 22.55 477 314/285

Catabolite 3-(4-hydroxyphenyl)-propionicacid C9H10O3 12.18 165 93

Rt,retentiontime;m/z,mass-to-chargeratio;[M-H]¯,Negativelychargedmolecularion

GENERALDISCUSSION

GeneralDiscussion

119

ExtractionandHPLCanalysisof(poly)phenols

Cactus(Opuntiaficus-indicaMill.)cladodeshavebeenproposedasapotentialsourceof (poly)phenolic compoundswithhealth relatedproperties (El-Mostafaetal.,2014;Stintzing&Carle,2005).Theirextraction isacrucialstepfromtheanalyticalpointofviewforthecharacterizationof(poly)phenoliccompoundsprofile,butalsoforafurtherdevelopment of ingredients for functional foods. Polysaccharides present in cactuscladodes, such as pectins, form a water-soluble complex with (poly)phenols. Alsocellulose with non-covalent hydrogen bonds and hydrophobic interactions makes(poly)phenols extraction more difficult (Bordenave, Hamaker, & Ferruzzi, 2014).Therefore,several individualsolventsasethanolandmethanol,aswellassuccessiveextractionswithmethanol(50%),acetone(80%)andwaterwereusedfortheextractionofcactuscladodes(poly)phenoliccompounds.Evenethanol(80%)extractedthehighesttotal phenolic content, successive extraction with methanol, acetone and waterfavoured the extraction of antioxidants (measured byDPPH andABTS radicals), andspecificallyflavonoids.

Onceselectingsuccessiveextractionsasthebestextractionmethod,the(poly)phenoliccompoundswereextractedandanalysedbyHPLC-DAD.Even though theanalysisbyHPLC-MSiswidelyextended,theavailabilityinmanylaboratoriesislimitedduetothehighprice.Otherlimitationintheanalysisof(poly)phenoliccompoundsisthelackofstandardsortheirhighprice.Forthesereasons,achromatographicmethodforHPLC-DADwasdeveloped.

TheanalysisofthecactuscladodesextractbyHPLC-DADshowedthepresenceofseveralunidentifiedpeaks,buttheabsenceofthefreeflavonoidaglyconesandfreephenolicacids. In fact, subsequent analyses in HPLC-MS (De Santiago, Pereira-Caro,Moreno-Rojas,Cid,&DePeña,2018b)confirmedtheabsenceofflavonoidaglyconesincactuscladodes. Therefore, acidhydrolysiswas applied tohydrolyse the flavonoids in theircorrespondingaglyconesandthusbeabletoquantifythem.AfteracidhydrolysiswithdifferentconcentrationsofHCl(0.6-1.7M)andtimes(2-3h),threeflavonoidsaglycones(isorhamnetin, quercetin and kaempferol) and two phenolic acids (ferulic andhidroxybenzoic acids) were identified and quantified by HPLC-DAD using thecorrespondingstandards.Acidhydrolysiswith1.5MHClduring2hwasshownas theoptimalfordeglycosilationofflavonoidswithoutadegradationofphenolicacids.

Aftertheoptimizationof(poly)phenolsextractionandHPLCanalysis,theresearchstudyabout the content of (poly)phenolic compounds in cactus cladodesbefore and afterculinarytechniques,aswellastheirbioaccessibilitywasperformed.

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Influenceofheattreatment

Cactus cladodes can be consumed both raw and after cooking techniques such asboiling, microwaving, griddling and frying. The influence of different culinary heattreatments on the nutritional and fatty acid composition, antioxidant capacity and(poly)phenolicprofileofcactuscladodeswasevaluatedforthefirsttime.TheobtainedresultswerepublishedinFoodChemistryandintheJournalofAgriculturalandFoodChemistry(DeSantiago,Domínguez-Fernández,Cid,&DePeña,2018a;DeSantiagoetal.,2018b).

Cactuscladodeswerecharacterizedbyahighmoisturecontent(79.7-93.7g/100g),lowprotein (1.1-2.0 g/100 g) and mineral content (0.5 g/100 g) and high dietary fibrecontent(3.1-5.0g/100g),showinghigheramountofinsolublefibrethansolubleone.Dietary fibre is an essential part of the composition of cactus cladodes, includingcellulose, hemicelluloses, pectin, lignin and gums (Ayadi, Abdelmaksoud, Ennouri, &Attia, 2009; Bensadón, Hervert-Hernández, Sáyago-Ayerdi, & Goñi, 2010).RecommendedDietaryAllowance(RDA)suggestsanintakebetween20and25goffibreperday(WHO,2003).Aportionof100gofcactuscladodesprovidesbetween15%(forrawandboiledsamples)and20–25%(formicrowaved,griddledandfriedsamples)oftheRDAoffibreperday.AccordingtotheFoodandDrugAdministration(FDA),foodscanbeconsideredasahighsourceoffibreifitscontributionisatleast20%ofthedailyvalue per serving (Food and Drug Administration, 2013). Thereby, raw and cookedcactuscladodesareagoodsourceoffibre.

After cooking processes, soluble and insoluble fibre, and consequently total fibrecontent,increased,exceptinboiling.Heattreatmentinducesdepolymerisationandtherupture of some glycosidic linkages in dietary fibre polysaccharides that favour theobserved increase in carbohydrates (Ramírez-Moreno, Córdoba-Díaz, Sánchez-Mata,Díez-Marqués,&Goñi,2013).

Cactus cladodes can be considered as a fat-free vegetable, except when they weresubmittedtoafryingprocess(8.3–11.0gfat/100g).Inconsequence,friedvegetablesalsohadthehighestcaloricvalue.Whencactuscladodesaresubmittedtofrying,theuseofoliveoilispreferableduetoitswell-knownhealthycharacteristicsandalowerω-6/ω-3ratio(4.586)thansoybeanoil(10.224).

Heattreatmentincreasedthetotalphenoliccontentincactuscladodes,exceptinboiledonesduetotheleachinglossesfavouredbythebreakdownofcellularstructurescausedat high temperatures (Ramírez-Anaya, Samaniego-Sánchez, Castañeda-Saucedo,Villalón-Mir,&De La Serrana, 2015; Turkmen, Sari,&Velioglu, 2005).Griddling andfrying apparently higher increased the total phenolic content measured by Folin-

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Ciocalteumethodthanmicrowaving.However,when(poly)phenoliccompoundsprofilewasdeeplyanalysedbyHPLC,microwavingandgriddlingwereshownastheculinarytechniques that favoured the release of (poly)phenolic compounds from the foodmatrix.

Theidentificationandquantificationof(poly)phenolicprofileofrawandcookedcactuscladodes by HPLC-DAD showed the predominance of flavonoids (80-85%) versusphenolic acids (20-25%). Three flavonoid aglycones (isorhamnetin, quercetin andkaempferol)andtwophenolicacids(ferulicand4-hydroxybenzoicacids)werefound.The analysis performed by UHPLC-PDA-HR-MS showed a total of 45 (poly)phenoliccompounds, with flavonoids accounted for 60-68% and phenolic acids for 32−40%.Among flavonoids, 13 isorhamnetin derivatives, 5 quercetin and 14 kaempferolderivativeswereidentified.Regardingphenolicacids,3stereoisomersofeucomicacidwerethemostabundant,followedby3ofpiscidicacidsand7ferulicacidderivativesinlower amounts. Results of total (poly)phenolic compounds by UHPLC-PDA-HR-MS(72.77-158.21mg/gdm)aremuchhigherthanHPLC-DAD(1.42-5.76mg/gdm)analysismaybedueto(1)apartialdegradationof(poly)phenolsbyacidhydrolysis,eventhoughitwaspreviouslyoptimized;and(2)thehigherdetectioncapacityofUHPLC-PDA-HR-MSthanHPLC-DAD.Then,besidesagreaternumberofcompoundsidentified(45),smalleramounts could be quantified by UHPLC-PDA-HR-MS than by HPLC-DAD. Specifically,eucomicandpiscidicacidscouldbeidentifiedandquantifiedonlybyUHPLC-PDA-HR-MS.

Althoughtherearenodatarelatedto(poly)phenolsincookedcactuscladodes,similarprofileshavebeenreportedinrawcactuscladodesofO.ficus-indicaandotherOpuntiacultivars with isorhamnetin glycosides as predominant flavonoids (Avila-Nava et al.,2014;Ginestraetal.,2009;Guevara-Figueroaetal.,2010).

PiscidicandeucomicacidshavebeenpreviouslyidentifiedinO.ficus-indicaextractsasunique compounds (Ginestra et al., 2009;Mena et al., 2018). In the present study,eucomicacidswerethemostabundant(50−60%totalphenolicacids),incontrastwithGinestraetal.(2009)whoreportedpiscidicacidasthemajorone.Piscidicacidisrarelyfound in nature and is restricted to thosewith crassulacean acidmetabolism, beingOpuntiaspeciesoneofthesesucculentplants(Stintzing&Carle,2005).

(Poly)phenolicamount incactuscladodescanvarydependingonthematuritystage,origin place, harvest season and environmental conditions. In early stages phenoliccompounds are higher because they are a defencemechanism against environmentsince the product has not developed thorns to protect themselves (Figueroa-Pérez,Pérez-Ramírez, Paredes-López, Mondragón-Jacobo, & Reynoso-Camacho, 2018;

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Ventura-Aguilar, Bosquez-Molina, Bautista-Baños, & Rivera-Cabrera, 2017).Additionally, the season of the year and themoment for harvesting also influence,becauseearlyinthemorningandduringthewinter,cladodesaremoreacidduetotheircrassulaceanacidmetabolism(Ventura-Aguilaretal.,2017).Inthepresentstudycactuscladodeswerealreadymaturebecausetheycontainedthornsandtheywereharvestedinsummer.

Heattreatmentappliedtovegetablesinducesseveralstructuralandchemicalchanges,whichturninto(poly)phenoliccompoundslossesandgainsdependingonthecookingtechnique, technological parameters, as well as the food matrices. Increases aftermicrowaving(1.4-fold)andgriddling(1.2-fold)processescouldbeduetothereleaseof(poly)phenoliccompoundsfromthecellwallsandsubcellularcompartmentscausedbythermaldestruction,butalsoduetotheirliberationfrompectins,mucilages,andotherdietaryfibrecompounds(Jaramillo-Floresetal.,2003).Thisthermalhydrolyticactionmightexplainthedetectionintracesorinlowamountofaglyconesonlyafterculinaryheat treatments. Furthermore, both cooking techniques are carried outwithout theadditionofwater,avoidingtheleaching,orat leastminimizeditusingfastercookingtime(5min)inmicrowavingthaninboilingtreatment(10min).Additionally,Maillardreactionscanbefavouredbythehightemperaturesreachedduringgriddling(110−150°C), and the melanoidins formed could retain (poly)phenolic compounds into theirstructures. Besides, the inactivation of the enzyme systems (as polyphenoloxidases)leads to inhibit degradation of the (poly)phenolic compounds (Girgin & El, 2015).Similarly,fryingprocessinducesadecreaseprobablyduetoalongercookingtime(15min) than in the other heat treatments (5−10 min) making it a more deteriorativeprocess.

Regarding the (poly)phenolic compounds profile, isorhamnetin derivatives showed ahigher increment when cactus cladodes were submitted to microwaving, whilequercetin and kaempferol derivatives increased higher after griddling. Microwavedcactuscladodesalsopresentthehighestamountoftotalphenolicacids,particularlyinpiscidic and ferulic acid derivatives. These results are in agreement with thosepreviously reported inmicrowaved broccoli and cauliflower (Ramos Dos Reis et al.,2015)aswellasingriddledonion,pepper,andcardoon(Juániz,Ludwig,Huarte,etal.,2016b).

In other cactus species like Xoconostle (Opuntia joconostle) pear fruit, boiling andgriddlinghaveshownsignificantlydecreasesinflavonoidsandphenolicacids,whereasmicrowaving has preserved them (Cortez-García, Ortiz-Moreno, Zepeda-Vallejo, &Necoechea-Mondragón,2015).However,thephenolicprofileinthiscactuspearfruitis

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quitedifferentfromthatshowninthepresentwork,becauseisorhamnetinderivativeswerenotdetected.

Theincrease(orevendecrease)ofantioxidantcapacityafterheattreatmentsincactuscladodes is the result of the predominant effects (release, hydrolysis, degradation,leaching,etc)ondifferentantioxidants(phenolicandnon-phenoliclikeascorbicacid).Individualcompoundsmayrespondinadifferentmannertodifferentradicals,andtheycan also be differently affected by heat treatment. Thermal chemical changes in(poly)phenols structuresmightbecrucial tooverall antioxidant capacity. In fact, it isknownthattheglycosylationofflavonoidsreducestheiractivitywhencomparedtothecorrespondingaglycones(Buchner,Krumbein,Rohn,&Kroh,2006).Then,theirreleasefrom the cell structure, or their thermohydrolyzation into more simple glycosidesresulting triglycosides into diglycosides and monoglycosides might enhance theantioxidant capacity of cooked cactus cladodes and counterbalance the losses ofthermolabile andwater soluble antioxidants. In addition, theantioxidant capacityofphenolicacidsandtheirestersmainlydependsonthenumberofhydroxylgroupsinthemolecule(Rice-Evans,Miller,&Paganga,1996).

Bioaccesibilityof(poly)phenoliccompounds

(Poly)phenolic compounds must be released from the food matrix into thegastrointestinaltracttobeabsorbedandfurtherreachtheorganstoexerttheirhealthbenefits. Therefore, bioaccesibility as defined as the amount or fraction of a foodcompound which remain available for absorption in the gastrointestinal tract, wasevaluated in raw and cooked cactus cladodes samples after the application of asimulated 3 steps (oral, gastric and intestinal) digestion in vitro model previouslyadaptedinourlaboratory(Minekusetal.,2014;Monenteetal.,2015).

Gastrointestinaldigestionsignificantly(p<0.05)decreasedbothflavonoidsandphenolicacids of cactus cladodes, remained bioaccessible 55−64%of the total (poly)phenoliccompounds of cooked cactus cladodes, while final bioaccessibility was 44% in rawsamples(DeSantiagoetal.,2018b).

Again, microwaved cactus cladodes contributed with the highest amount of total(poly)phenoliccontent,aftertheinvitrogastrointestinalprocess,followedbygriddledsampleswhile cactus cladodes friedwith soybeanor oliveoil had the lowest.Otherauthors have also demonstrated a higher bioaccessibility of total (poly)phenoliccompoundsafterheattreatmentinboiledandsteamedcauliflower(Girgin&El,2015),griddledgreenpepper(Juániz,Ludwig,Bresciani,etal.,2016a)andcardoon(Juánizet

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al., 2017). Likewise, Antunes-Ricardo, Gutiérrez-Uribe & Guajardo-Flores (2017) alsoobservedasignificantdecreaseinthebioaccessibilityofisorhamnetinglycosidesaftertheintestinaldigestionofrawO.ficus-indicaextracts.

Thehighamountofpectinsandmucilages inOpuntia species,which includesbound(poly)phenolic compounds, alongwith thoseattached to themelanoidins formedbyMaillard reactions after intensive heat treatment like griddling, might favour aprotectiveeffectagainstenzymaticaction(Prior,Wu,&Schaich,2005).

Flavonoidsandphenolicacidswereunevenlyaffected,beingthefirstmoresensitivetogastrointestinalconditionsthanthelatter.Inthisway,digestiveenzymesandconditionshigher retainedordegraded flavonoids (37−63%bioaccessibility) thanphenolicacids(56−87% bioaccessibility), changing the ratio between them by increasing thepercentageofphenolicacids(40-54%ofthetotalcontent).

Flavonoid aglycones were detected in traces or very low amount in cooked cactuscladodesafterinvitrogastricdigestionandundetectedaftertheintestinalphase.Thisconfirmsthattheamylasesaddedtosimulatethesalivaryactionandthosepresentinpancreatin in the intestinal phase,whichnormally cleaveα-linkages, arenot able tobreak the β-glycosidic linkage between the flavonoid aglycones and their glycosidicmoieties (Gonzales et al., 2015). Thedeglycosilationof flavonoids takesplace in thebrushbordercellsofthesmallintestineandalsobecauseoftheactionofthehumanmicrobiota,sothedegradationofflavonoidscouldbemainlybecauseoftheaffinitywithdigestiveenzymes(Dayetal.,2000;Némethetal.,2003).

Moreover,isomerizationreactionsofferulic,piscidicandeucomicacidswereinducedthroughthegastrointestinaldigestion,especiallyafterthegastricphase,maybeduetotheacidpHanddigestiveconditions.

Inaddition,antioxidantcapacityevaluatedbyDPPHradicaldecreasedafterbothgastricand intestinal phases in all cactus cladodes samples. However, in microwaved andgriddledones, thedecreasewas lower than inothercooking treatments,which is inaccordance with the (poly)phenolic profile remained after the gastrointestinaldigestion.

Therefore,thecurrentstudyconfirmsthatheatingprocessesmaysignificantlyinfluencethedigestibilityofdietary(poly)phenolsofcactuscladodesfromthefoodmatrix.Thus,even(poly)phenolsareretainedordegradedbydigestiveenzymesandpHconditionsduring gastrointestinal digestion, most of them remain bioaccessible when cactuscladodesarecooked,especiallyphenolicacidsaftermicrowavingandgriddling.

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Effectofcolonicmicrobiota

Becausemost(poly)phenoliccompoundsarenotabsorbed inthesmall intestineandreach the colon, they can be metabolised by the action of human gut microbiotainducingtheformationof(poly)phenoliccatabolites(Espín,González-Sarrías,&Tomás-Barberán,2017;Juánizetal.,2017;Juánizetal.,2016a;Ludwig,dePeña,Cid,&Crozier,2013).Hence, to evaluate the effect of thehumangutmicrobiotaon (poly)phenoliccompounds of digesta cactus cladodes, a 24 h in vitro colonic fermentation wasdeveloped, simulating theanaerobic conditions located in thehuman large intestine(Koutsosetal.,2017).

At the starting of colonic fermentation (0 h), 27 (poly)phenolic compounds wereidentifiedandquantifiedinfermentedcactuscladodes,beingphenolicacidsthemostabundantwith78-88%ofthetotalcontent.Piscidicacidwasthemaincompound(52-65% total (poly)phenols), followed by eucomic acid (15-36% total (poly)phenols).Flavonoidssuchaskaempferolderivatives(6-13%total(poly)phenols)andisorhamnetinderivatives(4-7%total(poly)phenols)weremainlyfoundintheirglycosidesforms.Noquercetin derivatives were detected at any time point of the colonic fermentationmaybeduetoarapidcatabolismofquercetinglycosides,evenwithinthefirst15minafterfaecalincubation,whichhavebeenpreviouslyreported(Juánizetal.,2016a).

Mostof(poly)phenolsweredegradedduringcolonicfermentation,althoughsomeofthem were still present after the 24 h. Intestinal microbiota produced a higherdegradationofflavonoidsthanphenolicacids.Eucomicacidwasthemoststableduringthe colonic fermentationmaydue to the resistance to thehumanmicrobiota,whilepiscidic acid and both kaempferol and isorhamnetin derivatives were rapidlycatabolised. At the end of the 24 h of colonic fermentation, the catabolite 3-(4-hydroxyphenyl)propionic acid was detected in trace amounts maybe produced viademethoxylation from ferulic acid derivatives (Ludwig et al., 2013), as well as viadehydroxylationof3-(3,4-dihydroxyphenyl)propionicacidderivedfromferulicacidandthe ring fission of quercetin, isorhamnetin and kaempferol derivatives (Juániz et al.,2017;Juánizetal.,2016a;Kay,Pereira-Caro,Ludwig,Clifford,&Crozier,2017).

Biologicalactivity

Evenmany (poly)phenolic compounds or their catabolites in cactus cladodes canbebioaccessibletocrossthroughtheintestinalbarrier,theymightalsoexerttheirhealthbenefits into the colon cells. Therefore, the biological activity of colonic fermentedcactuscladodescookedbythemostcommonculinarytechniques(boiling,griddlingandfrying)was evaluated inHT29human colon cells, including cytotoxicity (MTT assay),antigenotoxicity(Cometassay)andcellcycleanalyses.

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No cytotoxicity was observed in HT29 cells after 24 h of incubation with colonicfermented raw and cooked cactus cladodes at 10% and 20% (v/v), compared tountreatedcells.However,cellviabilitywaslessthan80%inmostsamplesat20%(v/v)concentration.Basedonthat,a10%(v/v)concentrationwasusedfortheCometandcellcycleassays.

Antigenotoxicityeffectoffermentedcactuscladodeswasevaluatedforthefirsttime,showingasignificantreduction(p<0.05)intheH2O2-inducedDNAdamageinHT29cells,comparingtothecontrol fermentate(withnocactuscladodes).Thereduction intheDNA damage was observed in all the cactus cladodes samples, maybe due to thepresenceof(poly)phenoliccompoundsaftercolonicfermentation.

EucomicacidwasthemaincompoundafterthefaecalincubationandcouldstillhaveantioxidantpropertiescontributingtothetendencytoreducetheDNAdamage(Cemeli,Baumgartner,&Anderson, 2009). Similarly, colonic fermented raspberry, strawberryandblackcurrantpresentedasignificant(p<0.05)reductionofDNAdamageinHT29cellline(Brownetal.,2012;Coatesetal.,2007).

Finally, the fermented raw and cooked cactus cladodes at 10% (v/v) showed nosignificant (p<0.05)effectsatanystageof theHT29cellcycle.ResultsareconsistentwithCoatesetal.(2007),whoobservednotsignificantchangesincellcycleofHT29cellswhen they were pre-treated with fermented berries. However, Serra et al. (2013)observedthatextractsfromO.ficus-indicafruitjuiceinducedacellcyclearrestedintheG1phaseinHT29cells,attributingthiseffecttothebetalains,whichareabsentincactuscladodes.

Even the antigenotoxicity and antioxidant activity of cactus cladodes in colon cells,furtherinvestigationsareneededinalivingsystemorbyhumaninterventionstudiestocorroboratetheroleof(poly)phenoliccompoundspresentincactuscladodes.

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Monente,C.,Ludwig,I.A.,Stalmach,A.,dePeña,M.P.,Cid,C.,&Crozier,A.(2015).Invitro studies on the stability in the proximal gastrointestinal tract andbioaccessibility in Caco-2 cells of chlorogenic acids from spent coffee grounds.InternationalJournalofFoodSciencesandNutrition,66(6),657–664.

Németh,K.,Plumb,G.W.,Berrin,J.G.,Juge,N.,Jacob,R.,Naim,H.Y.,Williamson,G.,Swallow,D.M.,&Kroon,P.A.(2003).Deglycosylationbysmallintestinalepithelialcellβ-glucosidases isacriticalstep intheabsorptionandmetabolismofdietaryflavonoidglycosidesinhumans.EuropeanJournalofNutrition,42(1),29–42.

Prior,R.L.,Wu,X.,&Schaich,K.(2005).Standarizedmethodsforthedeterminationofantioxidantcapacityandphenolics infoodsanddietarysupplements.JournalofAgriculturalandFoodChemistry,53(10),4290–4302.

Ramírez-Anaya,J.D.P.,Samaniego-Sánchez,C.,Castañeda-Saucedo,M.C.,Villalón-Mir,M., & De La Serrana, H. L. G. (2015). Phenols and the antioxidant capacity ofMediterranean vegetables prepared with extra virgin olive oil using differentdomesticcookingtechniques.FoodChemistry,188,430–438.

Ramírez-Moreno, E., Córdoba-Díaz,D., Sánchez-Mata,M. de C., Díez-Marqués, C.,&Goñi, I. (2013).Effectofboilingonnutritional,antioxidantandphysicochemicalcharacteristics in cladodes (Opuntia ficus indica). LWT - Food Science andTechnology,51(1),296–302.

RamosDosReis,L.C.,DeOliveira,V.R.,Hagen,M.E.K.,Jablonski,A.,Flôres,S.H.,&DeOliveira Rios, A. (2015). Effect of cooking on the concentration of bioactivecompoundsinbroccoli(Brassicaoleraceavar.Avenger)andcauliflower(Brassicaoleraceavar.AlphinaF1)growninanorganicsystem.FoodChemistry,172,770–777.

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131

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Stintzing, F. C., & Carle, R. (2005). Cactus stems (Opuntia spp.): A review on theirchemistry, technology,anduses.MolecularNutritionandFoodResearch,49(2),175–194.

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CONCLUSIONS/CONCLUSIONES

Conclusions

135

1.Successiveextractionswithmethanol,acetoneandwaterarerequiredforanoptimalextraction of (poly)phenolic compounds of cactus cladodes, as well as for theantioxidantcapacitymeasuredbyDPPHandABTS.

2.Acidhydrolysiswith1.5MHClduring2histheoptimalconditionforthereleaseofflavonoidaglycones(isorhamnetin,quercetinandkaempferol)withoutputtingatrisktheidentificationandquantificationofphenolicacids(ferulicandhydroxybenzoicacid)byHPLC-DAD.

3.Cookingtreatments (boiling,microwaving,griddlingandfrying) inducechangesonnutritionalcompositionofcactuscladodes,increasingsolubleandinsolublefibreupto5.0g/100gbecomingagoodfibresource,aswellaslossesandgainson(poly)phenoliccompoundsandantioxidantcapacitydependingonthecookingtechnique.

4.Microwavingandgriddlingprocessesincrease1.4-foldand1.2-foldtheamountofthe45 (poly)phenolic compounds identified in cactus cladodes by UHPLC-PDA-HR-MS,respectively, due to their release from the cellwalls causedby thermal destruction,whereasbothfryinginsoybeanandoliveoilsdecrease0.6-foldandboiling0.9-foldthetotalcompounds.

5.Afterinvitrogastrointestinaldigestion,55−64%ofthetotal(poly)phenolsofcookedcactus cladodes remains bioaccessible versus 44% in raw samples. Furthermore,digestive conditions and enzymes degrade or retain more flavonoids (37−63%bioaccessibility)thanphenolicacids(56−87%bioaccessibility).

6. Microwaved cactus cladodes contribute to the highest amount of (poly)phenols(143.54mg/gdm)aftergastrointestinaldigestion,followedbygriddledsamples(133.98mg/gdm),showingalsothehighestantioxidantcapacity.Additionally,gastrointestinaldigestion induces isomerizations among the 3 stereoisomeric forms of piscidic andeucomicacids.

7. The human gut microbiota produces a higher degradation of (poly)phenoliccompounds in flavonoids than inphenolicacids.However, somecompoundsarestillremained after 24 hours of colonic fermentation, being eucomic acid as the mostrelevant.

8.(Poly)phenoliccompoundsofcactuscladodesthatremaininthecolonaftertheactionofgutmicrobiotamightexerttheirantigenotoxiceffectbyreducingtheH2O2-inducedDNAdamageintheHT29coloncells.

Conclusions

136

Inconclusion,microwavingandgriddlingarethepreferableculinarytechniquesinorderto increase bioaccessible flavonoids and phenolic acids, as well as the antioxidantcapacity in cactus cladodes. Despite of the degradation of (poly)phenols after thegastrointestinal digestion and the action of the human gut microbiota, somecompoundsarestillavailableinthecolonwheretheycanexerttheirbiologicalactivity.However, further research isneeded toevaluate thebioavailabilityof (poly)phenoliccompoundsusinginvivomodelsorhumaninterventionstudies.

Conclusiones

137

1.Serequierenextraccionessucesivasconmetanol,acetonayaguaparaobtener lascondiciones óptimas para la extracción de compuestos (poli)fenólicos del nopal, asícomoparalamedicióndelacapacidadantioxidanteporDPPHyABTS.

2.LahidrólisisácidaconHCl1,5Mdurante2heslacondiciónóptimaparalaliberaciónde agliconas de flavonoides (isorhamnetina, quercetina y kaempferol) sin poner enriesgo la identificación y cuantificación de los ácidos fenólicos (ácido ferúlico ehidroxibenzoico)porHPLC-DAD.

3.Lostratamientosdecocción(hervido,almicroondas,alaparrillayfritura)producencambios en la composición nutricional del nopal, aumentando la fibra soluble einsoluble hasta 5.0 g/100 g convirtiéndose en una buena fuente de fibra, así comopérdidas y ganancias en compuestos (poli)fenólicos y su capacidad antioxidantedependiendodelatécnicadecocciónempleada.

4.Losprocesosdemicroondasyalaparrillaaumentan1.4vecesy1.2veceslacantidaddelos45compuestos(poli)fenólicosidentificadosenelnopalporUHPLC-PDA-HR-MS,respectivamente, debido a su liberación de las paredes celulares causada por ladestruccióntérmica,mientrasqueambosfritosenaceitedesojayolivadisminuyen0.6vecesyelhervido0.9veceseltotaldecompuestos.

5.Despuésdeladigestióngastrointestinalinvitro,el55-64%deltotalde(poli)fenolesdenopalcocinadopermanecebioaccesiblefrenteal44%ennopalsincocinar.Además,lascondicionesenzimáticasydigestivasdegradanoretienenmásflavonoides(37-63%debioaccesibilidad)queácidosfenólicos(56-87%debioaccesibilidad).

6. Nopal cocinado al microondas contribuye a una mayor cantidad de (poli)fenoles(143.54mg / g dm) después de la digestión gastrointestinal, seguido de nopal a laparrilla (133.98mg / g dm), que tambiénmuestra lamayor capacidad antioxidante.Además, la digestión gastrointestinal induce isomerizaciones entre las 3 formasestereoisoméricasdelácidopiscídicoyeucómico.

7. La microbiota intestinal produce una mayor degradación de compuestos(poli)fenólicos en los flavonoides que en los ácidos fenólicos. Sin embargo, algunoscompuestosaúnpermanecendespuésde24horasdefermentacióncolónica,siendoelácidoeucómicoelmásrelevante.

8.Loscompuestos(poli)fenólicosdelnopalquepermanecenenelcolondespuésdelaaccióndelamicrobiotaintestinalpuedenejercersuefectoantigenotóxicoreduciendoeldañodelADNinducidoporH2O2enlascélulasdecolonHT29.

Conclusiones

138

En conclusión, elmicroondas y la parrilla son las técnicas culinarias preferibles paraaumentarlabioaccesibilidaddeloscompuestosflavonoidesyácidosfenólicos,asícomola capacidad antioxidante en el nopal. A pesar de la degradación de (poli)fenolesdespués de la digestión gastrointestinal y de la acción de la microbiota intestinal,algunos compuestos están aún disponibles en el colon donde pueden ejercer suactividad biológica. Sin embargo, se necesitan más investigaciones para evaluar labiodisponibilidaddeloscompuestos(poli)fenólicosusandomodelosinvivooestudiosdeintervenciónenhumanos.

RESEARCHDISSEMINATION

ResearchDissemination

141

Publications

E. De Santiago, M. Domínguez-Fernández, C. Cid, M.P. De Peña. Impact of cookingprocessonnutritionalcompositionandantioxidantsofcactuscladodes(Opuntiaficus-indica).FoodChemistry,2018,(240)1055–1062.(Q1)(D1).

E.DeSantiago,G.Pereira-Caro,J.M.Moreno-Rojas,C.Cid,M.P.DePeña.Digestibilityof(poly)phenolsandantioxidantactivityinrawandcookedcactuscladodes(Opuntiaficus-indica).JournalofAgriculturalandFoodChemistry,2018,66,5832−5844.(Q1).

E.DeSantiago,C.I.R.Gill,I.Carafa,K.Touhy,C.Cid,M.P.DePeña.Digestionandcolonicfermentation of raw and cooked Opuntia ficus-indica impacts bioaccessibility andbioactivity.JournalofAgriculturalandFoodChemistry.Underreview.

E.DeSantiago,I.Juániz,C.Cid,M.P.DePeña.Extractionof(poly)phenoliccompoundsof cactus (Opuntia ficus-indicaMill.) cladodes: impact of solvents and conditions. Inpreparation.

Conferencecommunications

E.DeSantiago,C.Cid,M.P.dePeña.Poster:“Seleccióndeunmétododeextraccióndecompuestos fenólicos en el nopal”. In VIII Jornada de Investigación en CienciasExperimentalesydelaSalud.UniversidaddeNavarra.2015.Libroderesúmenes,página56.(Seeannex).

E.DeSantiago,I.Juániz,C.Cid,M.P.dePeña.Poster:“Optimizationofthemethodologyfor identification andquantification of (poly)phenolic compounds in cactus cladodes(Opuntiaficus-indica)”.Inthe7thInternationalConferenceonPolyphenolsandHealth.Tours,France.BookofAbstracts2015;page271.(Seeannex).

E.DeSantiago,C.I.R.Gill,I.Carafa,K.Touhy,C.Cid,M.P.dePeña.Oralcommunication:“Antioxidantactivityof (poly)phenolicextracts fromrawandcookedcactuscladodes(Opuntiaficus-indica)afterfaecalfermentationinhumancoloncarcinomaHT29cells”.In the 2nd International Conference on Food Bioactives & Health. Lisbon, Portugal.Awardedasthebestoralcommunicationinthe(poly)phenolsarea.BookofAbstracts2018;page119.(Seeannex).

ANNEX

Annex

VIII JORNADA DE INVESTIGACIÓN EN CIENCIAS EXPERIMENTALES Y DE LA

SALUD

UNIVERSIDAD DE NAVARRA

Libro de resúmenes

PAMPLONA, 26 DE MARZO DE 2015

Annex

VIII Jornada de Investigación en Ciencias Experimentales y de la Salud de la Universidad de Navarra

56

Póster nº 41 Alimentación, Estilo de Vida y Salud SELECCIÓN DE UN MÉTODO DE EXTRACCIÓN DE COMPUESTOS FENÓLICOS EN

EL NOPAL

Elsy Gabriela De Santiago Castanedo, Mª Concepción Cid Canda, Mª Paz de Peña Fariza

Departamento de Ciencias de la Alimentación y Fisiología, Universidad de Navarra

e-mail: [email protected]

La OMS insta a un mayor consumo de vegetales por ser fuente de compuestos bioactivos implicados en la

prevención de enfermedades relacionadas con el estrés oxidativo. El nopal (Opuntia ficus-indica) es uno de

los alimentos de mayor consumo en México y además un alimento rico en compuestos antioxidantes. Sin

embargo, la extracción de dichos compuestos puede verse dificultada por la capacidad de retención de

agua propia de un cactus. El objetivo de este trabajo fue comparar la eficacia de extracción de antioxidantes

de 3 métodos:

1. 100% etanol

2. 100% etanol con posterior evaporación y redisolución en agua

3. Extracciones sucesivas con metanol al 50%, acetona al 70% y agua al 100% y posterior hidrólisis

ácida.

En todos los extractos se midió la capacidad antioxidante mediante DPPH y ABTS, así como el total de

compuestos fenólicos y de flavonoides.

Los diferentes procesos empleados extrajeron cantidades semejantes de compuestos fenólicos totales. Sin

embargo, la cantidad de flavonoides extraídos con el método de extracciones sucesivas resultó inferior,

probablemente debido a la hidrólisis ácida aplicada. Por el contrario, la capacidad antioxidante fue

significativamente superior, hasta un 87% en el ABTS y un 98% en el DPPH. Esto podría deberse a la

ruptura de los glucósidos de flavonoides dando lugar a su forma aglicona, pudiendo ser estas estructuras

más bioactivas.

En conclusión, el método de extracciones sucesivas y posterior hidrólisis ácida resultó el más efectivo para

la extracción de los compuestos antioxidantes del nopal.

Annex

SELECCIÓN DE UN MÉTODO DE EXTRACCIÓN DE COMPUESTOS FENÓLICOS EN EL NOPAL

E. De Santiago, C. Cid, M. P. De Peña *Dpto. Ciencias de la Alimentación y Fisiología, Facultad de Farmacia.

Universidad de Navarra, 31008 Pamplona España. * [email protected]

INTRODUCCIÓN

OBJETIVO

MATERIAL Y MÉTODOS

BIBLIOGRAFÍA

La Organización Mundial de la Salud insta a un mayor

consumo de vegetales por ser fuente de compuestos

bioactivos implicados en la prevención de enfermedades

relacionadas con el estrés oxidativo (OMS, 2004). El nopal

(Opuntia ficus-indica) es uno de los alimentos de mayor

consumo en México y además un alimento rico en compuestos

antioxidantes. Sin embargo, la extracción de dichos

compuestos puede verse dificultada por la capacidad de

retención de agua propia de un cactus.

El objetivo de este trabajo fue comparar la eficacia de tres

métodos de extracción con la finalidad de seleccionar aquel

que permitiera obtener la mayor cantidad de antioxidantes.

100% etanol72 h

100% etanol72 h

50% metanol1 h

Liofilización

Ávila-Nava A, Calderón-Oliver M, Medina-Campos O, Tao Zou, Liwei Gu. Extract of cactus (Opuntia ficus indica) cladodes scavenges reactive oxygen species in vitro and enhances plasma antioxidant capacity in humans. J Funct Foods 2014; 10:13–24. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. LWT – Food Sci Technol 1995; 28: 25-30.Chang H, Huang GJ, Agrawal DC, Kuo CL, Wu CR, Tsay HS. Antioxidant activities and polyphenol contents of six folk medicinal ferns used as ¨Gusibu¨. Bot Stud 2007; 48: 397- 406.Lee JC, Kim HR, Kim J, Jang YS. Antioxidant Property of an Ethanol Extract of theStem of Opuntia ficus-indica var. Saboten. J Agric Food Chem 2002; 50: 6490-6496.OMS 2004, World Health Organization (WHO). Global strategy on diet, physicalactivity and health. In the 57th World Health Assembly (2004). URLhttp://www.who.int/dietphysicalactivity/strategy/eb11344/strategy_english_web.pdf.Re R, Pellegrini N, Proteggente A, Pannala A, Yang P, Rice-Evans C. Antioxidantactivity applying an improved ABTS radical cation decolorization assay. Free RadicalBio Med 1999; 26 (9-10): 1231-1237.Singleton, V.L.; Rossi, J.A. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Viticult 1965; 16: 144-158.

RESULTADOS Y DISCUSIÓN

CONCLUSIONES

En conclusión, el método de extracciones sucesivas y posterior

hidrólisis ácida resultó ser el más efectivo para la extracción

de los compuestos antioxidantes del nopal.

Los extractos de nopal con mayor cantidad de compuestos

fenólicos totales fueron el 2 (249 mg AG/100g) y el 3 (234 mg

AG/100g) (Figura 1). La cantidad de flavonoides totales

extraídos con el método 3 resultó inferior, probablemente

debido a la ruptura de parte de estos compuestos por la

hidrólisis ácida aplicada. Sin embargo, la capacidad

antioxidante de este extracto fue significativamente superior a

los otros dos, hasta un 98% en el DPPH y un 87% en el ABTS.

Lo anterior puede deberse a la ruptura de los glucósidos de

flavonoides que dan lugar a su forma aglicona, pudiendo ser

estructuras más bioactivas, o también a la presencia de otros

compuestos diferentes a los flavonoides.

AGRADECIMIENTOS

Este trabajo ha sido financiado por el Plan de Investigación de la Universidadde Navarra (PIUNA). E. De Santiago agradece a la Asociación de Amigos dela Universidad de Navarra por la beca recibida.

Compuestos bioactivos• Compuestos fenólicos totales(Singleton & Rossi, 1995)• Flavonoides totales(Chang et al., 2007)

Capacidad antioxidante• DPPH(Brand-Williams et al., 1995)• ABTS(Re et al., 1999)

Extracto 1Lee et al., 2002

Extracto 2Lee et al., 2002

Extracto 3Ávila-Nava et al., 2014

Evaporación disolvente

Redisolución en agua

70% acetona1 h

100% agua15 min

Hidrólisis ácida3 h

Compuestos fenólicos totales

Figura 1. Contenido de compuestos fenólicos totales en los extractos de nopal

Flavonoides totales

Figura 2. Contenido flavonoides totales en los extractos de nopal

DPPH

Figura 3. Actividad reductora del radical DPHH del los extractos de nopal

ABTS

Figura 4. Actividad reductora del radical ABTS de los extractos de nopal

Annex

Annex

October 2015, 27-30th - Tours (France)

271

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99%) than fl avonoids, which were even increased in some cooked samples. Thus, the higher bioaccesibility of phenolic

compounds in cooked vegetables suggests that changes during cooking process, including Maillard Reaction Products

formation, might have a protective effect against digestive enzyme activity.

Financial support: Spanish Ministry of Economy and Competitiveness (AGL2014-52636-P) and PIUN(Plan Inves-

tigación Universidad de Navarra). I.Juániz is grateful to ‘‘Asociación de Amigos de la Universidad de Navarra’’ for the

grant received.

P920Optimization of the methodology for identifi cation and quantifi cation of (poly)phenolic compounds in cactus cladodes (Opuntia fi cus-indica)

E. De Santiago1, I. Juaniz1, C. Cid1, M.P. de Peña1*

1 Department of Nutrition, Food Science and Physiology, School of Pharmacy. University of Navarra. IdiSNA, Navarra Institute

for Health Research. 31008,Pamplona, Spain.

*[email protected]

Consumption of vegetables is related with lower risks of chronic diseases associated with oxidative stress such

as cancer, cardiovascular diseases or diabetes. The cactus Opuntia fi cus-indica is one of the most consumed foods in

Mexico and a vegetable rich in antioxidant compounds, like (poly)phenolics. Most (poly)phenolic compounds, par-

ticularly fl avonoids, are in their glycosides forms, so that for their quantifi cation with HPLC-DAD it is necessary to

apply a previous hydrolysis to release their respective aglycones. The acid hydrolysis under continuous heating is the

traditional method, but it should be standardized and optimized for each food because it is dependent on the binding

sites of the glycosides in the fl avonoid nucleus. The aim of this work was to optimize hydrolysis conditions for a better

identifi cation and quantifi cation of (poly)phenolic aglycones. Additionally, antioxidant capacity was evaluated in the

selected extract.

Cactus cladodes were successive extracted with methanol, acetone and water. Hydrolysis was carried out using HCl

at 90ºC at different concentrations (0.6, 1.2, 1. and 1.7M) and times (2 and 3 hours). After hydrolysis, (poly)phenolic

aglycones and phenolic acids were identifi ed and quantifi ed using HPLC–DAD. Antioxidant capacity was measured

by DPPH and ABTS spectrophotometric assays.

Three fl avonoids (isorhamnetin, quercetin and kaempferol) and two phenolic acids (ferulic and hidroxybenzoic

acids) were identifi ed and quantifi ed. The 1.5M HCl hydrolisis during 2 hours showed the disappearance of glycosides

peaks yielding the highest amount of (poly)phenols, especially fl avonoids aglycones. Furthermore, there was an in-

crease in the antioxidant capacity measured by DPPH after hydrolisis, which might release the fl avonoids aglycones

from their glycosides forms in the food matrix.

Financial support; Spanish Ministry of Economy and Competitiveness (AGL2014-52636-P) and PIUN(Plan Inves-

tigación Universidad de Navarra). E. De Santiago and I. Juaniz thank to‘‘Asociación de Amigos de la Universidad de

Navarra’’ for the grants.

Annex

Optimization of the methodology for identification and quantification of (poly)phenolic compounds in cactus cladodes

(Opuntia ficus-indica) E. De Santiago1, I. Juániz1, C. Cid1, M. P. De Peña1*

1Department of Nutrition, Food Science and Physiology, School of Pharmacy. University of Navarra. IdisNA, Navarra Institute for Health Research. 31008, Pamplona Spain.

*[email protected]

Consumption of vegetables is related with lower risks of chronic

diseases associated with oxidative stress such as cancer,

cardiovascular diseases or diabetes. The cactus (Opuntia ficus-indica)

is one of the most consumed foods in Mexico and a vegetable rich in

antioxidant compounds like (poly)phenols, particularly flavonoids,

which are in their glycosides forms. For their quantification with HPLC-

DAD it is necessary to apply a previous hydrolysis to release their

respective aglycones. The acid hydrolysis under continuous reflux

heating is the traditional method, but it should be standardized and

optimized for each food because it is dependent on the binding sites of

the glycosides in the flavonoid nucleus.

The aim of this work was to optimize the hydrolysis conditions for a

better identification and quantification of (poly)phenolic aglycones and

phenolic acids. Additionally, antioxidant capacity was evaluated in the

selected extract.

Lyophilization

Ávila-Nava A, Calderón-Oliver M, Medina-Campos O, Tao Zou, Liwei Gu. J Funct Foods 2014; 10:13–24. Brand-Williams W, Cuvelier ME, Berset C. LWT – Food Sci Technol 1995; 28: 25-30. Juániz I, Ludwig IA, Huarte E, Pereira-Caro G, Moreno-Rojas JM, Cid C, De Peña MP. Food Chemistry (under 2nd review). Re R, Pellegrini N, Proteggente A, Pannala A, Yang P, Rice-Evans C. Free Radical Bio Med 1999; 26 (9-10): 1231-1237.

Results from HPLC-DAD measurements indicate that after acid

hydrolysis, (poly)phenolic aglycones were released from the glycosides

forms of the food matrix and could be identified and quantified.

After acid hydrolysis, three flavonoids aglycones (isorhamnetin,

quercetin and kaempferol) and two phenolic acids (ferulic and

hidroxybenzoic acids) were identified and quantified in cactus cladodes

extracts (Opuntia ficus-indica), being isorhamnetin the most abundant.

The 1.5M HCl hydrolysis during 2 hours showed the disappearance of

glycosides yielding the highest amount of (poly)phenols, especially

flavonoids aglycones. This may be partially explained by the release of

those phenolics bound to cell structures during reflux. Higher

concentrations of HCl and longer time of reflux could cause the

degradation of the (poly)phenolic aglycones.

Cactus cladodes have antioxidant capacity due to the presence of

phenolic compounds. An increase in DPPH and a decrease in ABTS after

acid hydrolysis (1.5M HCl 2h) are shown (Figure 2 and 3). The release

of (poly)phenolic aglycones probably seems favored the DPPH radical

scavenging, whereas their glycosides forms are more active against

ABTS radical.

Spanish Ministry of Economy and Competitiveness (AGL2014-52636-P) and PIUNA (Plan Investigación Universidad de Navarra). E. De Santiago and I. Juaniz thank to ‘‘Asociación de Amigos de la Universidad de Navarra’’ for the grants.

Antioxidant capacity • DPPH (Brand-Williams et al., 1995) • ABTS (Re et al., 1999)

0.6M 1.2M

DPPH

Figure 2. DPPH radical activity of cactus cladodes extracts.

ABTS

Succesive extraction: methanol (50%), acetone (70%) and water

HPLC-DAD

Unhydrolyzed Hydrolyzed

Hydrolyzed (1.5M HCl 2h) Unhydrolyzed

INTRODUCTION

OBJECTIVE

MATERIAL AND METHODS

Cactus (Opuntia ficus-indica)

1.5M 1.7M

2 h 3 h

2 h 3 h 3 h 3 h

RESULTS AND DISCUSSION

Table 1. (Poly)phenols of cactus cladodes before and after different hydrolysis (HCl) conditions

(mg phenolic compound/100g vegetable).

Figure 1. HPLC-DAD chromatograms of cactus cladodes extracts before and after acid hydrolysis

(1.5M HCl 2h) at 360 nm.

Q: quercetin, K: kaempferol, I: isorhamnetin.

Figure 3. ABTS radical activity of cactus cladodes extracts.

An acid hydrolysis with 1.5M HCl during 2h reflux has been selected in

order to further identify and quantitate (poly)phenolic aglycones and

phenolic acids by HPLC-DAD. Moreover, scavenging capacity should be

measured by using different radicals due to the fact of the similar

behavior of (poly)phenolic compounds against DPPH and ABTS

radicals.

AKNOWLEDGEMENTS

REFERENCES

CONCLUSIONS

Hydrolysis (HCl) at 90ºC (reflux)

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0

1

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(Ávila-Nava et al., 2014. with modifications)

(Juániz I et al., under 2nd review)

Compound 0.6M 3h 1.2M 3h 1.5M 2h 1.5M 3h 1.7M 2h 1.7M 3h

Isorhamnetin 5.91 ± 0.46 8.57 ± 0.30 16.49 ± 0.71 13.73 ± 1.01 13.31 ± 0.23 8.24 ± 1.20 Kaempferol 1.57 ± 0.06 0.68 ± 0.03 2.28 ± 0.48 1.89 ± 0.08 2.05 ± 0.06 0.41 ± 0.10 Quercetin 2.30 ± 0.28 1.19 ± 0.02 2.71 ± 0.21 2.39 ± 0.07 2.44 ± 0.08 0.72 ± 0.07 Ferulic acid 4.02 ± 0.21 3.69 ± 0.07 2.75 ± 0.21 2.63 ± 0.21 2.16 ± 0.03 6.54 ± 0.40 Hidroxibenzoic acid 1.05 ± 0.47 1.01 ± 0.01 1.17 ± 0.08 0.96 ± 0.03 0.77 ± 0.02 1.25 ± 0.02 Total 14.84 ± 0.09 15.13 ± 0.26 25.41 ± 1.39 21.60 ± 1.11 20.72 ± 0.38 17.16 ± 1.01

Annex

2nd International Conference on

September 2018, 26-28th . Lisbon (Portugal)

FOOD BIOACTIVES & HEALTH

Program and Abstracts

Annex

Antioxidant activity of (poly)phenolic extracts from raw and cooked cactus cladodes (Opuntia ficus-indica) after faecal fermentation in human colon carcinoma HT29 cells

De Santiago, E.1; Gill, C.2; Touhy, K.M.3; Cid, C.1; De Peña, M.P.1

1 Universidad de Navarra, Dept. Ciencias de la Alimentación y Fisiología. IdiSNA. Pamplona, Spain; 2University of Ulster, NICHE, Coleraine, UK; 3Nutrition & Nutrigenomics Unit, Fondazione Edmund Mach, Trento, Italy.

[email protected]

Cactuscladode(Opuntia ficus-indica)isaplanteatenasafreshorcookedvegetable,whichcontainsbioactivecompounds,suchasflavonoidsandphenolicacidsprovidingantioxidantcapacity.(Poly)phenolic compoundscouldbemodifiedbycooking treatmentsandgastrointestinal conditions.Theyarenotefficientlyabsorbedinthesmallintestineandpassingintothecolonandexerttheirbiologicalactivityintothecoloncells.Forthispurpose,biologicalactivityof(poly)phenolicextractsfromrawandcookedcactuscladodesafterin vitro-simulatedupperintestinaltractdigestionandsubsequentfaecalfermentation(24h)wasevaluatedinhumancoloncarcinomaHT29cell line.Cytotoxic activitywasmeasured byMTT assay, showing no cytotoxicity in fermented raw andcookedcactuscladodesat10and20%v/v,exceptincontrolfermentationat20%.Then,a10%v/vconcentrationwasusedfortheevaluationofprotectionagainstDNAoxidativedamageusingtheCometassay.Fermentedrawandcookedcactuscladodesdecreasedthe%tailDNA(34.5–38.5%)inhydrogenperoxide(75µMH2O2)challengedHT29cellscomparedtothecontrolfermentation(44.2%).Nosignificantdifferencesamongcookingtreatmentswerefound.WhencellsweretreatedwithPBS(negativecontrol),theDNAdamagewasalsolessinfermentedcookedcactussamplesthancontrolfermentation.Additionally,cellcycleanalysiswithfermentedsampleswascarriedout,showingnoalterationsduringthecellcyclephases.Therefore,resultssuggestthatcolonic(poly)phenolicmetabolitesofcactuscladodesformedbytheactionofhumangutmicrobiotapotentiallyprotectagainstDNAoxidativedamage.

Financialsupport:ThisresearchwasfundedbytheSpanishMinistryofEconomyandCompetitiveness(AGL2014-52636-P).E.DeSantiagoexpressestheirgratitudetotheAssociationofFriendsoftheUniversityofNavarraandLaCaixaBankingFoundationforthegrantsreceived.

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