Neutral sugar side chains of pectins limit interactions with procyanidins

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with procyanidins

Aude Watrelot, Carine Le Bourvellec-Samour, Anne Imberty, Catherine Renard

To cite this version:

Aude Watrelot, Carine Le Bourvellec-Samour, Anne Imberty, Catherine Renard. Neutral sugar side

chains of pectins limit interactions with procyanidins. Carbohydrate Polymers, Elsevier, 2014, 99,

pp.527-536. �10.1016/j.carbpol.2013.08.094�. �hal-02640610�

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Carbohydrate Polymers (2014), Vol. 99, p. 527-536, DOI: 10.1016/j.carbpol.2013.08.094 Journal homepage: www.elsevier.com/locate/carbpol

Neutral sugar side chains of pectins limit interactions with procyanidins

AudeA.Watrelot^a,b,∗,CarineLeBourvellec^a,b,AnneImberty^c,CatherineM.G.C.Renard^a,b

aINRA,UMR408SécuritéetQualitédesProduitsd’OrigineVégétale,F-84000Avignon,France

bUniversitéd’AvignonetdesPaysdeVaucluse,UMR408SécuritéetQualitédesProduitsd’OrigineVégétale,F-84000Avignon,France

cCentredeRecherchessurlesMacromoléculesVégétales,CERMAV-CNRS(AffiliatedwithUniversitédeGrenoble),B.P.53,F-38041Grenoble,France

Keywords:

Polyphenol Condensedtannins Polysaccharide Rhamnogalacturonan Association ITC

a b s t r a c t

Interactions between seven hairy regions of pectins, rhamnogalacturonans II and arabinogalactan–proteins and procyanidins with different average degrees of polymerization, low (DP9)andhigh(DP30),wereinvestigatedbyisothermaltitrationcalorimetryandabsorptionanalysis tostudytheimpactofneutralsugarsidechainsofpectinsontheseassociations.Associationsbetween pecticfractionsandprocyanidinsinvolvedhydrophobicinteractionsandhydrogenbonds.Nodifference inassociationconstantsbetweenvarioushairyregionsandprocyanidinsDP9wasfound.Nevertheless, arabinanchainsshowedlowerassociationconstants,andhairyregionsofpectinswithonlymonomeric sidechainsshowedhigherassociationwithprocyanidinDP30.Onlyverylowaffinitieswereobtained withrhamnogalacturonansIIandarabinogalactan–proteins.Aggregationcouldbeobservedonlywith theprocyanidinsofDP30andtheprotein-richarabinogalactan–protein.Associationswereobtainedat bothdegreesofpolymerizationoftheprocyanidins,butdiffereddependingonneutralsugarcomposition andthestructureofpecticfractions.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Pectinsarecomplexpolysaccharidesofplantcellwalls,present mostly in themiddle lamellaand primary wall. Theycomprise smooth and hairy regions. The “smooth regions”, also known as homogalacturonans (HGs),consist of a long chain of (1→4) linked␣-d-galacturonic acid (GalpA) with various degrees of methylationand/oracetylation.Thehairyregions,namedrhamno- galacturonans(RGs),areoftwotypes(IandII)(Caffall&Mohnen, 2009;DeVries,Rombouts,Voragen,&Pilnik1982;Thomas,McNeil, Darvill,&Albersheim,1987).RhamnogalacturonansIareformed by the repeating unit →4)-␣-d-GalpA-(1→2)-␣-l-Rhap-(1→), the rhamnose residues of the RG fractions being decorated by long side-chains composed mostly of galactose and arabinose (Ridley, O’Neill, &Mohnen, 2001; Voragen,Coenen, Verhoef, &

Schols, 2009). RG I presents this strictly alternating RG back- bone (De Vrieset al.,1982; Renard,Crépeau,&Thibault,1995;

Schols, Posthumus, & Voragen, 1990), while the misleadingly namedRGIIpossessesabackboneof(1→4)linked␣-d-GalpAas

∗Corresponding authorat: INRA, UMR408Sécurité et Qualité desProduits d’OrigineVégétale,DomaineSaint-Paul,SiteAgroparc,84914AvignonCedex9, France.Tel.:+330432722537;fax:+330432722492.

E-mailaddresses:[email protected],[email protected] (A.A.Watrelot),[email protected](C.LeBourvellec).

homogalacturonans with side chains containing rhamnose and a varietyof raremonosaccharides (Pérez,Rodríguez-Carvajal, &

Doco,2003).Arabinogalactan–proteins(AGPs)areoftenassociated withpectinsbecausebothmoleculescontainasimilarstructural motifoftypeIIgalactans,i.e.(1→3)and(1→6)linkedgalactans ramifiedwitharabinanoligomers(Voragenetal.,2009).However, inAGPsthearabinogalactandomainsareconnectedviaaspecific hydroxyproline-richprotein(Clarke,Anderson,&Stone,1979).

Procyanidins are flavonoids, major polyphenols found in vacuolesoffruitsandvegetables.Theyareasubclassofproantho- cyanidinsderivedfromtheoligomerizationofflavan-3-olsunits andformthecondensedtannins.Theyarecharacterizedbytheir constitutiveunits,theirinterflavaniclinkagesandtheirdegreeof polymerization(Hemingway&Karchesy,1989;Quideau,Deffieux, Douat-Casassus,&Pouységu,2011).Specifically,appleprocyani- dinsarecharacterizedbythepredominanceof(−)-epicatechinand ofC4–C8interflavaniclinkages(Guyot,Marnet,Laraba,Sanoner,

&Drilleau,1998;Sanoner,Guyot,Marnet,Molle,&Drilleau,1999;

Wojdyło,Oszmianski,&Laskowski,2008).Theycanbeoligomersor polymersandinciderapples,theirnumber-averagedegreeofpoly- merizationrangebetweenDPn4.5–50forvarietiesJeanneRenard andAvrollesrespectively(Sanoneretal.,1999).Theyhavebeen reportedtoexhibitseveralhealtheffectsasantioxidantsandanti- carcinogens,andtoprotectagainstcardiovasculardiseases.

Fruitandvegetableprocessingrupturescells.Thisdisruption leadstoassociationbetweenconstituentsofcellwallsandvacuoles,

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www.elsevier.com/locate/carbpol

withaprominentroleofpectinsandprocyanidins(LeBourvellec, Bouchet,&Renard, 2005). The bioavailability of polyphenols is dependentontheirstructureandtheirassociationwithothercom- pounds suchas sugars (Parada &Aguilera, 2007).Procyanidins associatedwithdietaryfiberarenot bioaccessiblein theupper partofthehumanintestine,andtraveltothecolon,wherethey becomefermentablesubstratesforbacterialmicroflora(Cheynier, Sarni-Manchado,&Quideau,2012;Palafox-Carlos,Ayala-Zavala,

&González-Aguilar,2011;Saura-Calixtoetal.,2010), producing metabolitesthat arebothabsorbable andbioactive(Williamson

& Clifford, 2010). In vitro, the interaction between procyani- dins and dietaryfiberreduces thepolysaccharide fermentation andincreasestheproductionofprocyanidincolonicmetabolites (Aura et al., 2012; Saura-Calixto et al., 2010). To understand thehealtheffectofpolyphenolsafterbindingtopolysaccharides suchaspectins,earlierworkdealtwithmechanismsofinterac- tionsbetweenthesetwomacromolecules.Inourpreviouswork (Watrelot, LeBourvellec, Imberty, &Renard 2013), interactions betweenprocyanidinsandpectinswereanalyzed,andtheimpact ofthedegreeofmethylationofsmoothregionsandofthedegreeof polymerizationofprocyanidinscouldbedemonstrated.However, theroleofthehairyregionsofpectinshasnotbeendetermined.

Theaimofthisstudywastoquantifythebindingofaspecificclass ofproanthocyanidins,theprocyanidins,tohairyregionsofpectins.

Isothermaltitrationcalorimetryandaspectrophotometricmethod wereusedtoprobethermodynamicparametersandaggregation phenomenaininteractionsbetweenhairyregionfractionsandpro- cyanidinswithdifferentaveragedegreesofpolymerization:low (DP9)andhigh(DP30).

2. Materialsandmethods

2.1. Chemicals

Methanol,acetonitrile,andacetoneofchromatographicquality werefromBiosolve(Distribio,Evry,France).Hexane(Merck,Darm- stadt,Germany)wasofanalyticalquality.Ethanolandacetonewere fromFisherScientific(Strasbourg,France).

SilvernitratewasfromMerck(Darmstadt,Germany).Sodium borohydride (NaBH₄), N-methylimidazole, acetic anhydride, toluene-␣-thiolandpectinfromapplewerefromSigma-Aldrich (Deisenhofen, Germany). Chlorogenic acid, (+)-catechin, and (−)-epicatechinwerefrom Sigma-Aldrich.4-Coumaricacidwas fromExtrasynthèse(Lyon,France).Phloridzinandsugarstandards were from Fluka (Buchs, Switzerland). Methanol-d3 was from Acros Organics (Geel, Belgium). Enzymatic cocktail Endozym polifruitliq⁺wasfromSpindalgroupeAEB(Gretz-Armainvilliers, France).Modifiedhairyregions(MHR1)fromapplewerekindly supplied and characterized by Dr. M. C. Ralet (INRA-UR1268 BiopolymèresInteractionsAssemblages,Nantes,France).Modified hairyregionsMHR2,MHR3andMHR4frompear,onionandsoy- bean(SSPS)respectivelywerekindlysuppliedbyDr.HenkSchols (WUAgrotechnology&FoodSciences,Wageningen,Netherlands).

Rhamnogalacturonans of type II monomer and dimer (mRG-II anddRG-II)andarabinogalactan–protein(AGP0andAGP4)were kindlysuppliedby Dr.ClaireDufour(INRA-UMR408 Sécuritéet QualitédesProduitsd’OrigineVégétale,Avignon,France).

2.2. Plantmaterial

Applefruits(MalusdomesticaBorkh.)oftheAvrollesandMarie Ménardvarietieswereharvestedatcommercialmaturityduring the2000seasonintheexperimentalorchardoftheCenterTech- niquedesProductionsCidricoles(CTPC,Sées,France).Fruitswere mechanicallypeeledandcoredaspreviouslydescribedbyGuyot,

Doco,Souquet,Moutounet,andDrilleau(1997)andcortextissues werefreeze-dried.ApplepomacewasfromValdeVireBioactives (Condé-sur-Vire,France).Fibrex615(Sugarbeetpulp)wasfrom NordicSugar(Copenhagen,Denmark).

2.3. Procyanidinextractionandpurification

Procyanidins were extracted from “Marie Ménard” and

“Avrolles”freeze-driedpulpsbysuccessivesolventextractionsand purifiedasdescribedbyWatrelotetal.(2013).Thepurifiedpro- cyanidinfractionsweredesignatedDP9(from“MarieMénard”)and DP30(from“Avrolles”).Inthetwofractions,(−)-epicatechinwas thepredominantconstitutiveunit,accountingformorethan95%

and88%inDP30andDP9respectively.Fordetails,seeWatrelot etal.(2013).

2.4. Rhamnogalacturonanpreparation

RhamnogalacturonanswerepreparedasdescribedbyRenard and Jarvis (1999) using 15g/L of apple pectins saponified at 4^◦C, pH>12 and left overnight. The solution was then brought to pH 6 and precipitated with three volumes of an ethanol/water/hydrochloricacidmixture(96:3:1,v/v/v)withstir- ringat4^◦Candleftovernight.Thissuspensionwasfilteredona G0sinteredglassfilter.Afterfiltrationthepecticacidprecipitate waswashed thoroughlywithanethanol/water mixture (70:30, v/v)untilallremainingchlorideionswereeliminated(asverified bysilvernitrateprecipitation).Theresultingpowderwasdriedby solventexchange(ethanol96%andacetone),followedbydrying at40^◦Cinanovenovernight.Thepecticacidwasdissolved(6g/L) andhydrolyzedwithhydrochloricacid(0.25N)for 72hat80^◦C (Thibault,Renard,Axelos,Roger,&Crepeau,1993).Thesupernatant wasconcentratedonarotaryevaporatorbeforedialyzingagainst 0.1MNaClfollowedbywater toeliminatemonomersand neu- traloligomers(galactoseandarabinose).Therhamnogalacturonan oligomers(RG)werefinallyfreeze-dried.

Applehairyregionswereobtainedbyamodifiedalkalineper- oxidetreatmentasdescribedbyRenard,Lahaye,Mutter,Voragen,

&Thibault(1997).Applepomacewassuspendedovernightatroom temperaturewithstirringat50g/L inwater,and pHwasstabi- lizedat>13.Polyphenolsweretheneliminatedbyadding200mL ofhydrogenperoxide(30%).Themediumwaslefttoreactwith stirringfor2h.ThepHwasadjustedto5withaceticacid,andenzy- maticcocktailEndozympolifruitliq⁺(1mL)wasaddedandleftto actovernightwithstirringatroomtemperature.Thesuspension wasfilteredonanylonmembrane(250␮mporesize)undervac- uum.Thefiltratewasdialyzedagainst0.1MNaCl,andthenwater at4^◦C.Finally,thefiltratewasfreeze-driedtogivetheapplehairy regionfraction(AHR).

Acid-treatedhairyregions(HR-H)wereobtainedfromsugar beet pulp as described by Ralet, Tranquet, Poulain,Moïse, and Guillon(2010).

Modifiedhairyregions(MHR1)fromapplewerepreparedby themethodofScholsetal.(1990)andwereakindgiftfromDr.M.- C.Ralet.Modifiedhairyregions(MHR2andMHR3)frompearand onionwerepreparedbythemethodofScholsandVoragen(1994).

MHR4fromsoybeanwaspreparedbyCoenen(2007).MHR2,MH3 andMHR4wereakindgiftfromDr.HenkSchols.AllMHRsresulting fromdrasticenzymichydrolysisofplantcellwallswereisolatedas highmolarmasspolymerswitharhamnogalacturonanbackbone.

However,thestructureofthesidechaindependsonthebotanical origin(Table1).MHR1fromapplewasmostlycomposedof␤- (1→4)linkedgalactansandarabinans(Renard,Voragen,Thibault,&

Pilnik,1991;Schols&Voragen,1994).Theywerecharacterizedby ahighxylosecontentwithamolarratioXyl:GalAof0.4,whichcor- respondstoxylogalacturonanchains(Schols,Bakx,Schipper,and

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Carbohydrate Polymers (2014), Vol. 99, p. 527-536, DOI: 10.1016/j.carbpol.2013.08.094

Journal homepage: www.elsevier.com/locate/carbpol^{A.A.Watrelotetal./}CarbohydratePolymers99 (2014) 527–536 529 Table1

Composition(mg/gdrymatter)andpartitioncoefficientofthedifferentrhamnogalacturonansubfractions.

Samples(mg/g) Kav Rha Fuc Ara Xyl Man Gal Glc GalA Characteristicstructure

RG 0.26 49 3 45 15 10 236 128 458

AHR 0.28 23 2 251 16 – 30 19 167 Arabinan

HR-H 0.13 143 – 25 – – 199 – 437 RGwithmonomericgalactans

PooledSD – 1.7 0.1 9.6 0.7 0.9 9.5 5.7 4.5

aMHR1 −0.06 89 9 83 113 – 134 – 385 ArabinanTypeIgalactanXylogalacturonan

aMHR2 −0.03 79 – 240 42 – 101 – 209 ArabinanTypeIIgalactan

aMHR3 0.01 132 – 40 17 7 208 – 283 TypeIgalactan

bMHR4 nd 41 33 188 52 – 351 27 165 GalactanXylogalacturonan

cAGP0 0.08 11 1 358 2 18 583 16 28 TypeIIArabinogalactan

cAGP4 0.02 93 2 250 18 23 354 3 180

RG,rhamnogalacturonans;AHR,applehairyregions;HR-H,acid-treatedhairyregions;MHR,modifiedhairyregions(1fromapple,2frompear,3fromonionand4from soybean);AGPs,arabinogalactan–proteins;Kav,partitioncoefficient;Rha,rhamnose;Fuc,fucose;Ara,arabinose;Xyl,xylose;Man,mannose;Gal,galactose;Glc,glucose;

GalA,galacturonicacid.PooledSD:pooledstandarddeviation,n=11.a:ScholsandVoragen(1994);b:Coenen(2007);c:Pellerinetal.(1995).

Voragen,1995).MHR2frompearwasrichinarabinosepresentas (1→5)-linkedchains(withproportionof71%).Itsgalactanswere predominantlyhighlyramified␤-(1→3)and␤-(1→6)typeIIgalac- tanandgalactoseresidueswereterminallylinked(withproportion of36%)(Schols&Voragen,1994).MHR3fromonionwasrichin galactosewithhighproportionofterminalgalactoseresidues(58%) and in ␤-(1→4) linkedtype Igalactan(19%) (Schols& Voragen, 1994),aswasMHR4fromsoybeanwiththeadditionnalpresenceof

␤-d-Xylsidechainssubstitutedto(1,4)-linkedgalacturonicacid,i.e.

alsoxylogalacturonans(Coenen,Bakx,Verhoef,Schols,&Voragen, 2007;Nakamura,Furuta,Maeda,Nagamatsu,&Yoshimoto,2001).

Polysaccharidesisolatedfromredwine werekindlydonated byDr.C.Dufour(INRA-UMR408SécuritéetQualitédesProduits d’OrigineVégétale,Avignon,France). Rhamnogalacturonans,RG- IIs,werepurifiedasdescribedinPellerinetal.(1996).Fractions mRG-IIanddRG-IIwerepredominantlymonomeric(90%pure)and dimeric(87%pure).Monomeranddimer(mRG-IIanddRG-II)were richinarabinoseandcomposedofrareneutralsugars.Thedimer wasdistinguishedbythepresenceofboron.

TypeIIarabinogalactan–proteinfractions,AGP0andAGP4,were isolatedbyanionexchangechromatographyaccordingtoPellerin, Vidal, Williams, and Brillouet (1995).Fractions weresimilar in termsofneutralsugarcompositionexceptforarabinoseandgalac- tosewithhighercontentinAGP0(35.2%and57.3%,respectively) thanAGP4(27.1%and38.3%,respectively),whileuronicacidcon- tentwashigherinAGP4thanAGP0.Also,non-reducingarabinose and rhamnosewereobserved in bothAGPs, but rhamnosewas 2,4-linkedinAGP4.Galactosewas3,6-linkedmoreinAGP4,but 3,4,6-linkedmoreinAGP0(Pellerinetal.,1995).

2.5. Phasediagram

Toidentifyaggregateformationduringpectin-tannininterac- tions,aspectrophotometricmethodwasusedat25^◦C.Systematic variationofconcentrationsandrelativeamountswascarriedout ona96-well“microplate”.Thiswasdonebyvaryingtheconcen- trations of procyanidins (0, 0.015,0.03, 0.06, 0.117,0.23, 0.47, 0.94,1.875,3.75,7.5,15mmol/L(−)-epicatechinequivalent)along the rows, and varying the concentrations of hairy regions (0, 0.12, 0.23, 0.47, 0.94, 1.875, 3.75, 7.5mmol/Lgalacturonic acid equivalent) along thecolumns. Solutions wereprepared in cit- rate/phosphate buffer at pH 3.8 and ionic strength 0.1mol/L.

Equal amounts (50␮L) of procyanidin and hairy region frac- tion solutions weremixed before each spectrum measurement for 20s. Absorbance spectra (200–650nm) were recorded, and absorbance at 650nm was analyzed for each solution using a SAFAS flx-Xenius XM spectrofluorimeter (SAFAS, Monaco).

Control spectra were obtained using wells containing hairy regions alone in bufferand procyanidins alone in buffer. After spectra recording, microplates were centrifuged for 10min at

2100×g, and supernatants of control wells (hairy regions at 7.5mmol/L in buffer (S1A) and procyanidins at 15mmol/L in buffer(S1B))andsupernatantsofwellscontainingprocyanidins at a concentration of 15mmol/L with hairy regions at a con- centration of 7.5mmol/L (S2) were analyzed by high pressure sizeexclusionchromatography(HPSEC)andhigh-performanceliq- uidchromatography–diodearraydetection(HPLC-DAD)todefine changes in partition coefficient(Kav=S2–S1A) ofhairy region fractionsandinaveragedegreeofpolymerizationofflavan-3-ols (DPn=S2-S1B).

2.6. Isothermaltitrationmicrocalorimetry

To measure enthalpy changes associated with hairy region- tannininteractionsat25^◦C,aVP-ITCmicrocalorimeter(Microcal^®, GEHealthcare,Northampton,MA,USA)wasused.Toallowcom- parison between the different pectic substrates, and given the polydispersityin molecularweightforbothpecticfractionsand procyanidins,allconcentrationsareexpressedrelativetothemain monomers,i.e.galacturonicacidforpectins and(−)-epicatechin forprocyanidins.Procyanidinsandhairyregionfractionsweredis- solvedinthesamecitrate/phosphatebufferpH3.8,ionicstrength 0.1mol/L,andfilteredon0.45␮mmembrane.Allthesolutionswere degassedbeforemeasurements.Thereferencecellwasfilledwith water.Toobtainahyperboliccurveasrecommendedforlowaffin- itysystems(Turnbull&Daranas,2003),differentconcentrationsof compoundsweretested.Thepecticfractionsolutionwasplacedin the1.448mLsamplecellofthecalorimeter,andtheprocyanidin solutionwasloadedintotheinjectionsyringeandtitratedintothe samplecellby30injectionsof10␮Laliquots.Thedurationofeach injectionwas20s,withaseparatingdelayof5min.Thecontentsof thesamplecellwerestirredthroughouttheexperimentat307rpm toensuremixing.

Rawdata obtainedasa plot ofheat flow (microcaloriesper second) against time (minutes) were then integrated peak-by- peakandnormalizedtoobtainaplotofobservedenthalpychange permoleofinjectant(H,kJmol⁻¹)againstthemolarratio((−)- epicatechin/galacturonic acid). Peak integration was performed usingMicrocalOrigin7.0(MicrocalSoftware,GEHealthcare).Con- trolexperiments includedthetitration of procyanidinfractions intobuffer,andweresubtractedfromtitrationexperiments.The experimental data were fitted to a theoretical titration curve usingMicrocalOrigin7.0,withH(enthalpychange),Ka(asso- ciationconstant),andn(numberofbindingsitespermolecule)as adjustableparameters,fromtherelationship

Q_i= nPtHV0

2

1+At

nPt+ 1

nKaPt −

1+ At

nPt + 1 nKaPt

2

−4 At

nPt

(1)