Phenolic profile and effect of regular consumption of Brazilian red wines on in vivo antioxidant activity

Original

Research

Article

Phenolic

profile

and

effect

of

regular

consumption

of

Brazilian

red

wines

on

in

vivo

antioxidant

activity

E.F.

Gris

a,d,

*

,

F.

Mattivi

e

,

E.A.

Ferreira

b,d

,

U.

Vrhovsek

e

,

D.W.

Filho

c

,

R.C.

Pedrosa

b

,

M.T.

Bordignon-Luiz

a a_{DepartamentodeCieˆnciaeTecnologiadeAlimentosCAL,UniversidadeFederaldeSantaCatarina,Brazil}

b_{DepartamentodeBioquı´mica,BQA,UniversidadeFederaldeSantaCatarina,Brazil} c

DepartamentodeEcologiaeZoologiaBioquı´mica,Rod.AdmarGonzaga,1346,Itacorubi,Floriano´polis,SC,Brazil

d

UniversidadedeBrası´lia,FaculdadeCeilaˆndia,QNN14,Ceilaˆndia,Brası´lia,DF,Brazil

e

EdmundMachFoundation,IASMAResearchandInnovationCentre,FoodQualityandNutritionDepartment,ViaEMach1,38010SanMicheleall’Adige,TN,Italy

1. Introduction

Anumber ofepidemiologicalstudieshave demonstratedthe

correlation between an unbalanced diet and coronary heart

diseases,sometypesofcanceranddiabetes.Epidemiologistshave observedthatadietrichinpolyphenoliccompoundsmayprovidea positiveeffectduetoantioxidantproperties(RenauldandLorgeril, 1992;Frankeletal.,1995). Wineis animportantcomponentin Mediterranean dietary traditions, and it is rich in antioxidant

compounds. These compounds have a functional role, acting

againstfreeradicals,aswellasaphysiologicalrole;infact,theycan increasetheantioxidantcapacityofthehumanbodyfollowingred

wine consumption (Renauld and Lorgeril, 1992). Furthermore,

phenoliccompoundsconstituteoneofthemostimportantwine

quality parameters since they contribute to the organoleptic

characteristics,particularlycolor,astringencyandtaste(Vrhovsek, 1998).

Theflavonoidcompositionofredwinesincludesanthocyanins,

catechins, and flavonols. The main flavonols are myricetin,

quercetin, kaempferol, syringetin and laricitrin (Mattivi et al., 2006).Anthocyaninsarethemainphenoliccompoundsassociated withthecolorofredwinesandareantioxidants(Rice-Evansetal., 1996;Rossettoetal.,2004).ThesecompoundsarepresentinVitis

vinifera grapes as glycosylated anthocyanidins with glucose

attachedtothe3-hydroxylposition,which canbeesterifiedby

differentorganicacids(Mazza,1995;Rice-Evansetal.,1996).

Themainnon-flavonoidcompoundsinwinearethephenolic

acids(variousbenzoicandcinnamicacidderivatives).Theyplaya primary role in defining thesensorial characteristics of wines, givingthe‘‘oakwood’’tastetypicaloflong-agedproducts,besides being largely responsible for theastringency and bitterness of youngwines(Somersetal.,1987;Vrhovsek,1998;Monagasetal., 2005). Hydroxycinnamicacids and their tartaric esters are the

ARTICLE INFO Articlehistory:

Received13November2011

Receivedinrevisedform27February2013 Accepted22March2013

Keywords: Vitisviniferawines Qualityredwines Phenoliccompounds Anthocyanins Flavonols Phenolicacids Hydroxycinnamicacids Hydroxybenzoicacids HPLC-DADanalysis Wineconsumption Invivoantioxidantactivity Foodanalysis

Foodcomposition

ABSTRACT

Inthisstudy,VitisviniferaLwinescv.CabernetFranc,Merlot,SangioveseandSyrah,2006and2007

vintages,producedinSa˜oJoaquim,anewwine-producingregioninsouthernBrazil,wereevaluated.As

phenoliccompoundcontentisoneofthemostimportantparametersinassessingwinequalityandis

possiblypartiallyresponsibleforthebeneficialhealthpropertiesofwines,inthispaperthelevelsofthe

mainanthocyanins,flavonols,hydroxycinnamicacidandhydroxybenzoicacid(HPLC-DADand

HPLC-DAD–MSanalysis)andtheinvivoantioxidantactivityinmicearereported.Theantioxidantcapacityof

plasmawasassessedthroughthereductionofferriciron(FRAP).Lipidperoxidation(TBARS),carbonyl

protein(CP),reducedglutathione(GSH)levelsandthecatalase(CAT),superoxidedismutase(SOD)and

glutathioneperoxidase(GPx)activityweredeterminedinliversofthetestanimals.Theresultsforthe

phenoliccompoundscontentofthewinesampleswereconsideredappropriateforqualityredwines,

andthewineconsumptionpromotedasignificantincreaseinFRAPanddecreasesintheTBARSandCP

levelsandintheCAT,SODandGPxactivity.Moreover,thephenoliccontentofthewineswaspositively

correlatedwiththeinvivoantioxidantcapacitypromotedbyregularwineconsumption.

ß2013ElsevierInc.Allrightsreserved.

*Correspondingauthorat:UniversidadedeBrası´lia,FaculdadedeCeilaˆndia,QNN 14,AreaEspecial,Ceilaˆndia,Brası´lia,DF,CEP:72220140,Brazil.

Tel.:+5506131078418;fax:+5506131078420. E-mailaddresses:elianagris@unb.br,bordign@cca.ufsc.br,

elianagris@gmail.com(E.F.Gris).

ContentslistsavailableatScienceDirect

Journal

of

Food

Composition

and

Analysis

j our na l h ome p a ge : w ww . e l se v i e r. co m/ l oc a te / j f ca

0889-1575/$–seefrontmatterß2013ElsevierInc.Allrightsreserved.

main class of non-flavonoid phenolics in red wines. They are

involved in the browning reactions of must and wine and are

precursorsofvolatilephenols(Vrhovsek,1998).Gallicacidisthe

mainhydroxybenzoicacidinredwine,whichis formedmainly

throughthehydrolysisofflavonoidgallateesters.Winesmatured

in oak present high levels of hydroxybenzoic acid derivatives,

mainlyellagicacid.

Thechemicalcharacterizationofwinephenolicsiscurrentlyan

issue for several reasons: it can aid the evaluation of the

authenticityofregionalproductsandthepredictionofthesensory properties and oxidative stability of wine (Lopes et al., 2006).

Moreover,phenolicsareusedasmarkersofthewineprocessing

technologyorwineaging(Ribe´reau-Gayonetal.,1998;Vrhovsek, 1998).

Sa˜oJoaquimisarecentwine-makingregioninsouthernBrazil. ThisregionissituatedinthehighplainsofSantaCatarinaStateand isknownasthecoldestplaceinthecountry,ataltitudesranging

from1200 to1400m. Recent papers have reported interesting

resultsthatsupportthepotentialoftheregiontoproducehigh qualitywines.Falca˜oetal.(2008a,b) reportedimportantresults

regarding the sensory profile and the main aroma-impact

componentsofCabernetSauvignonwinesfromthisregion.These

authors established a positive relationship between vineyard

altitudeand pyrazine content. Gris et al. (2011a) verified that flavan-3-olandproanthocyanidinconcentrationswereinlinewith thosereportedintheliteratureforwinefromthemostrenowned productionregions.Moreover,theresultspresentedbyGrisetal. (2011b) showed that some wines from this region have an unusuallyhighcontentofstilbenes.Furthermore,theconsumption ofthesewinesbylaboratoryanimalssubmittedtoa hypercholes-terolemicdietreducedsignificantlythehypercholesterolemiaand hypertriglyceridemia,andalsodecreasedtheatherogenicleveland increasedsignificantlytheHDLlevel.

However,dataonthedetailedphenoliccompositionandinvivo

antioxidant capacity (in Swiss mice) have not previously been

reported. The aim of this study was therefore to evaluate the

association between in vivo antioxidant capacity and phenolic

contents.Tothisendtheconcentrationofthemainflavonoidand

non-flavonoidcompoundsand theircontributiontothe

antioxi-dant activity in Cabernet Franc, Merlot, Sangiovese and Syrah

wines,ofthe2006and2007vintages,producedinSa˜oJoaquim, SantaCatarinaState,southernBrazilwereinvestigated.

2. Materialsandmethods

2.1. Standardsandreagents

All chromatographic solvents were HLPC grade and were

purchased from Carlo Erba (Rodano, Italy). Pure, HPLC grade

myricetin,quercetin, laricitrin,kaempferol,isorhamnetin,

syrin-getin and malvidin 3-glucoside chloride, were purchased from

Extrasynthe`se (Genay, France); ellagic acid, gallic acid, proto-catechuicacid,p-hydroxybenzoicacid, syringicacid,caffeicacid andtrans-p-coumaricacidwerepurchasedfromSigmaChemicalCo. (Steinheim,Germany);trans-caftaricacid,trans-coutaricacidand trans-fertaricacidwereisolatedfromGrenachegrapesasdescribed byMeyeretal.(1998);2,5dihydroxybenzoicacid,vanillicacid,and

ferulic acid were purchased from Fluka (Steinheim, Germany);

trichloroacetic acid, 2,4-dinitrophenylhydrazine (DNPH), 2-thio-barbituricacid(TBA),5,50_{-dithiobis(2-nitrobenzoicacid)(DTNB),}

butylatedhydroxytoluene(BHT),epinephrine,hydrogenperoxide, tert-butyl hydroperoxide, 2,4,6-tripyridyl-s-triazina (TPTZ), TRO-LOX(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid),

b

-nicotinamideadeninedinucleotidephosphate(NADPH),

glutathi-onereductase(GR)andreducedglutathione(GSH)werepurchased

fromSigma–AldrichCo.

2.2. Characterizationofthewine-growingregion

Sa˜oJoaquimislocatedinthehighplainsofSantaCatarinaState ataltitudesrangingfrom1200to1400m,andistheviticulture region situatedatthehighest altitudein Brazil.Thesoilofthis regionisofthetypeInceptisolaccordingtoU.S.D.A.classifications andtheclimateoftheSa˜oJoaquimregionis‘‘Cool,Coolnightsand

Humid’’ according to the Geoviticulture Multicriteria Climatic

Classification System, and as ‘‘Region I’’ (<1389 GDD), a ‘‘cold region’’,intermsoftheWinklerRegions(Grisetal.,2011a,b). 2.3. Samples

2.3.1. Grapes

The wines were produced from Cabernet Franc, Merlot,

SangioveseandSyrahgrapes,2006and2007vintages,fromyoung commercialvineyardsinSa˜oJoaquim,SantaCatarinaState,Brazil. Thesevineyardsarelocatedatanaltitudeof1290m,latitudeof288 150_{andlongitudeof49850}0_{.Thevinesinvestigatedinthisstudy}

wereplantedin2003andtheclonesusedwere986,181,VCR23

and VCR1, respectively. The rootstock was Paulsen 1103 (Vitis

berlandieri PlanchVitis rupestris Scheele), the vertical shoot

positioning trellis system was used,the row and vine spacing

were 3.0 and 1.2m, respectively, and the vineyard yield was

between6and7t/ha.Thegrapeswereharvestedatthestageof technicalmaturityandhadsugarreadingsof19.1–24.28Brix,0.57–

0.81mgL1 _{titratable acidityand pH values between 3.31and}

3.58.

2.3.2. Winesamples

Thewineswereallproducedunderthesameconditionsina

commercial winery in Sa˜o Joaquim as described by Gris et al.

(2011a,b).The wine samplesfromthe2007 and 2006vintages

were analyzed after one and two years of aging in the bottle,

respectively.Thewineswerestoredat108Cpriortoanalysis. 2.4. Determinationofphenoliccompounds

2.4.1. Spectrophotometricanalysisoftotalphenols(TP)

Total phenols (TP) weredirectly measured using the

Folin-Ciocalteu reagent method (Singleton and Rossi, 1965) and

concentrationsweredeterminedbymeansofacalibrationcurve

asmggallicacid/Lofwine. 2.4.2. Flavonolscontent

The flavonols (myricetin, quercetin, laricitrin, kaempferol,

isorhamnetinandsyringetin)contentsofthewinesampleswere

determinedbyHPLCafteracidhydrolyticcleavageoftheflavonol conjugates(Mattivietal.,2006).HPLCseparationand quantifica-tionofflavonolswascarriedoutaccordingtoMattivietal.(2006)

usingaWaters2695HPLCsystemequippedwithaWaters2996

DAD (Waters, Milford, MA), using a Purospher RP18

reversed-phase column of 250mm4mm (5

m

m), with a precolumn

(Merck, Germany). The following solvents were used: (A) 0.3%

HClO4inwater;(B)methanol.Thelineargradientwasfrom40to

90%Bin30minandtheflowratewas0.45mL/min.Thecolumn

equilibrationtimewas5minandtheinjectionvolumewas5

m

L.

Thepresenceofflavonolaglyconswasconfirmedbyco-injection

withthecorrespondingstandards.Eachflavonolwasquantifiedat 370nmandexpressedasmgmL1_{ofwinebymeansofcalibration}

withexternalstandards. 2.4.3. Freeanthocyaninscontent

Thesamplepreparationforthedeterminationofthemainfree

anthocyaninsinthewinesampleswascarriedoutaccordingto

were filteredthrough 0.22

m

m, 13mm PTFE syringe tip filters (Millipore)intoLCvialsandimmediatelyinjectedemployingthe

samesystem,columnandeluentsusedfortheHPLC-DADanalysis

of flavonols. Separation of the 15 main free anthocyanins was

obtained at 408Cwith a flow rate of 0.45mL/min. The binary

gradientwasappliedasfollows:from27to44.5%ofBin32min, thento67.5%ofBin13min,to100%Bin2min,isocratic100%Bfor

3min; total analysis time, 50min. Delphinidin 3-glucoside,

cyanidin3-glucoside,petunidin3-glucoside,peonidin3-glucoside,

malvidin 3-glucoside, and their relevant acetic acid and

p-coumaric acid esters were identified according to Castia et al.

(1993)and quantifiedat 520nm usingacalibrationcurvewith malvidin3-glucosidechloride.

2.4.4. Hydroxycinnamicacids(HCAs)

SamplepreparationandHPLCseparationandquantificationof

HCAs(withmodifications)wereperformedaccordingtoVrhovsek

etal.(2004).Thesamesystemandcolumnusedfortheflavonols wereusedforthedeterminationofHCAs.Thefollowingsolvents wereused:(A)0.5%formicacidinwaterand(B)2%formicacidin

methanol.Thegradientswereasfollows:from16%to19% Bin

7min,from19%to53%Bin12min,from53%to100%Bin0.1min,

100% B for 5min, back to 16% B in 0.1min. The column was

equilibratedfor7minpriortoeach analysis.Theflowratewas 0.4mL/minandtheinjectionvolume10

m

L.ThedetectionofHCAs

was carried out at 320nm. Each compound wasquantified as

mgmL1 _{of wine by means of calibration with an external}

standard. The %RSD values obtained experimentally from six

consecutivedeterminationsofthesamewinewereasfollows: cis-caftaric acid, 0.89%; trans-caftaric acid, 0.64%; cis-cutaric acid, 0.57%;trans-cutaricacid,0.32%;fertaricacid,0.43%;trans-caffeic acid,0.64%;trans-p-coumaricacid,0.53%;andtrans-ferulicacid, 0.82%.

2.4.5. Hydroxybenzoicacids

Samplepreparationforp-hydroxybenzoic,syringicandvanillic

acids wascarried outas follows: 10mL of wine and 1.0mLof

internalstandard (2,5-dihydroxybenzoicacid, 100mgL1_)were

concentratedtoapproximately7mLusingrotaryevaporationand reducedpressureat358C.Afterconcentration,thesamplepHwas

adjustedto8.0withNaOHandthesamplewasmadeuptothe

initialvolume(10mL)withdistilledwater.Analiquotof5mLwas addedtoa C18-SPEcartridge(1g,Waters),previouslyactivated withmethanol(5mL)andwater(10mL).Theeluatewascollected, itspHvaluewasadjustedto2.7withformicacidandthevolume wascompletedto10mLwithdistilledwater.Thefinalsamplewas filteredthrough0.22

m

m,13mmPTFEsyringetipfilters(Millipore, Bedford,MA)intoLCvialsandimmediatelyinjectedintothe HPLC-DAD.

2.4.6. HPLC-DADanalysisofp-hydroxybenzoic,syringicandvanillic acids

The HPLCsystem consisted of a Waters2695 HPLC system

equipped with a Waters 2996 DAD (Waters, Milford, MA).

ChromatographicseparationswereperformedonaGeminiRP18

(Phenomenex)column(250mm2.0mm,5

m

m),protectedby

aprecolumn.SolventAwas1%formicacidinwaterandsolvent

Bwasacetonitrile.Thelineargradientwasasfollows:from0to

20% Bin 40min, 20to 100%Bin 0.10min, 100%Bfor 2min,

back to 0% B in 0.1min. The column equilibration time was

5min,theinjectionvolumewas10

m

Landthetemperaturewas

408C. The compounds detected were identified at 280nm by

comparingtheir retention timeswiththose of purestandards

and quantified by means of the external standard method

(resultswerecorrectedonthebasisofrecoveryoftheinternal standard).

2.4.7. HPLC-DAD–MSanalysisofellagic,gallicandprotocatechuic acids

The wine samples, without prior preparation, were filtered

through PTFE syringe tip filters (0.22

m

m pore size, 13mm;

Millipore, Bedford,MA)prior toinjection intotheWaters2690

HPLCsystem(Waters,Milford,MA,USA)equippedwithaWaters

996DADand MicromassZQelectrosprayionization-mass

spec-trometer.TheUV–VISspectrawererecordedfrom210to400nm,

withdetectionat280nm.TheMSdetectoroperatedatacapillary voltageof3000V,extractorvoltageof3V,sourcetemperatureof 1058C,desolvationtemperatureof2008C,conegasflow(N2)of

61Lh1_{anddesolvationgasflow(N}

2)of460Lh1.Electrospray

ionization-massspectrometry(ESI-MS)wascarriedoutfromm/z

100to1500witharesidencetimeof0.1s.

Thesamechromatographicseparationconditionsusedfor

p-hydroxybenzoic, syringic and vanilic acids wereapplied in the

determination of ellagic, gallic and protocatechuic acids. The

identificationwasperformedonthebasisoftheirretentiontime,

molecularionandmainfragmentbyMSthroughcomparisonwith

the values for the pure standards. The optimal coefficient of

variation(CV)forallionswas20.Themolecularions(MH)_for

ellagicacid(m/z301.19)andgallicacid(m/z169.12)aswellasthe

(M+H)+ _{for protocatechuic acid(m/z 153.12)wereusedfor the}

quantificationbasedonexternalstandardcalibrationcurves. 2.4.8. Methodrepeatability

Methodrepeatabilitywasbasedonsixconsecutive

determina-tions (ellagic, gallic and protocatechuic acids) by six SPE

purifications (p-hydroxybenzoic, syringic and vanillic acids)

applied tothesame wine. The%RSD values obtained werethe

following:ellagicacid6.75%;gallicacid1.26%;protocatechuicacid

1.61%; p-hydroxybenzoic acid 4.32%; syringic acid 3.23%; and

vanillicacid4.76%.

2.4.9. Detectionandquantificationlimits(phenolicacids)

The experimental limit of detection (LOD) and limit of

quantification(LOQ)fortheHPLC-DAD–MSmethodwereestimated

atasignal-to-noiseratioof3and10,respectively,andthevalues

obtained were as follows: ellagic acid 0.031 and 0.098mgL1

(R2_{=0.9956); gallic acid 0.172 and 0.568mgL}1 _(R2_=0.9945);

protocatechuic acid 0.148 and 0.490mgL1 _(R2

=0.9953); p-hydroxybenzoicacid0.039and0.130mgL1_(R2_{=0.9993);syringic}

acid0.027and0.084mgL1_(R2_{=0.9989);andvanillicacid0.034}

and 0.113mgL1 _(R2

=0.9986), respectively. All results were consideredacceptableforresearchpurposes.

2.5. Invivoantioxidantactivity 2.5.1. Animals

All animal used in this study received humane care in

accordancewiththeprinciplesofthelegalrequirements appro-priateforthespecies(GuidingPrinciplesfortheCareandUseof LaboratoryAnimals,NIHpublication#85.23,revisedin1985)and withtheapprovalofInstitutionalEthicsCommitteeofUniversity

of Santa Catarina (PP005422010/CEUA/UFSC). Male Swiss mice

weighing222gwerehousedundercontrolledconditions(12-h light–darkcycle,2228C,60%airhumidity)andhadfreeaccessto standardlaboratorychowandwater.

Micewererandomlydividedinto10groups(n=6eachgroup) asdetailedbelow:onecontrolgroup,treatedwithwaterdaily;one

control ethanol group, treated with vehicle (hydroalcoholic

solution 12%) daily and eight test groups, treated with one of

the 8 wine samples daily. The treatment (7.0mLkg1_,

20mgkg1_day1_{totalpolyphenols)wasperformed bygavage}

for30daysandonthe31stdaytheanimalswereeuthanizedby

2.5.2. Oxidativestressbiomarkers–invivoantioxidantactivity Thetotalantioxidantcapacity(FRAP)oftheanimalsplasmawas

determined and liver samples were analyzed to evaluate the

endogenous lipid peroxidation (TBARS), oxidative damage to

proteinsbycarbonylation(PC)andcatalaseactivity(CAT)aswell asthereducedglutathione(GSH),superoxidedismutase(SOD)and glutathioneperoxidase(GPx)content.

Thetotalantioxidantcapacity(FRAP)wasdeterminedusingthe

animals plasma according to Benzie and Strain (1996). In this

analysistheantioxidantpresentinthesamplereducesFe2+_toFe3+

whichischelatedby2,4,6-tri(2-pyridyl)-s-triazine(TPTZ)andthe bluecolorismonitoredat593nm.Peroxidationofhepatictissue lipids,invivo,wasmeasuredbythemethoddescribedbyOhkawa etal.(1979).Theamountofthiobarbituricacid-reactivesubstances

(TBARS)was expressed as nmoles of TBARS formedper mg of

protein, using a molar coefficient (

e

) of 153mmolL1_cm1_.

Oxidativedamageofproteinswasquantifiedascarbonylprotein

content(CP)accordingtoLevineetal.(1990).Thismethodisbased onspectrophotometricdetectionoftheendproductofthereaction

of 2,4-dinitrophenylhydrazine with carbonyl protein to form

protein hydrazones detected at 370nm. The results were

expressedasnmolesofcarbonylgrouppermgofprotein,using

e

=22mmolL1_cm1_{. Liver GSH levels were measured by a}

spectrophotometric method (Anderson, 1985), in acid hepatic

homogenatescombinedwithadisodiumhydrogenphosphateand

DTNBsolution,andtheyellowchromophoreformedwasdetected

and quantified at 412nm using a molar coefficient (

e

) of

14.1mmolL1_cm1_{.The resultswereexpressed in mmolmg}1

ofprotein.CATactivitywasdeterminedbymeasuring,at240nm,

the decrease in H2O2 in a freshly prepared 10mM hydrogen

peroxidesolutionandexpressedinmmolmin1_mgprotein1_and

e

=40mmolL1_cm1_{(Aebi,1984).SODactivityinhomogenates}

was determined by measuring the inhibition of the rate of

autocatalyticadrenochromeformationandexpressedinUSODmg

protein1_{(MisraandFridovich,1972).GPxactivitywasquantified}

by a coupled assay with glutathione reductase (GR)-catalyzed

oxidationof NADPH.Measurementswere takenat 340nm and

expressedinmmolmin1_mgprotein1_{(Flohe´ andGunzler,1984).}

ProteinwasmeasuredbythemethodofLowryetal.(1993)using

bovineserumalbuminasthestandard.

2.6. Statisticalanalysis

Analysisof variance(ANOVA),TukeyHSDTestandprincipal

componentsanalysis (PCA) were carried out using Statistica 7

(2001) (StatSoft Inc., Tulsa, OK, USA). Values of p0.05 were consideredstatisticallysignificant.

3. Resultsanddiscussion

3.1. Chemicalanalysis

3.1.1. Flavonoidphenoliccompounds

Table1showstheresultsfortheflavonolcontentsofthewine samples.Thesixflavonolsdeterminedinthepresentstudy,after acidhydrolysis,includedallaglyconsexpectedonthebasisofthe

diagram for the biosynthesis of grape flavonols proposed by

Mattivietal.(2006).Ingeneral,themainflavonolsinthesewine

sampleswerequercetinand myricetin.Thesecompounds were

especially highin Sangiovese 2007(quercetin)and Syrah 2007

(myricetin).Lower concentrations of laricitrin, kaempferol, iso-rhamnetinandsyringetinwerefound.However,despitetheirlow

concentrations, the identification and quantification of such

compoundsisimportantsincetheyallowabettercharacterization forthisfamilyofcompounds.Moreover,accordingtoMattivietal. (2006) all of these flavonols are required for a more detailed

classificationofredandwhitegrapevarietiesonthebasisoftheir flavonolprofile.Theconcentrationsoftotalflavonols(sumofthe

individualcontents) inthe winesamples rangedfrom20.81to

46.79mgL1_{, and are similar to those reported for regions}

renownedforwineproduction(Simonettietal.,1997;Monagas

etal.,2005).

The levels of delphinidin-3-glucoside, cyanidin-3-glucoside,

petunidin-3-glucoside, peonidin-3-glucoside,

malvidin-3-gluco-side and their respective derivatives (esters of acetic and

p-coumaricacids)inthewinesamplesarepresentedinTable2.The meantotalanthocyaninscontent(sumoftheindividualcontents) was47.43mgL1_{,andrangedfrom12.94to125.60mgL}1_.Ofthe

anthocyanins evaluated, malvidin-3-glucoside presented the

highest concentrations (as expected since this is the main

anthocyaniningrapesandwines)andtherelativeamountofthis anthocyanininthecultivarsstudiedrangedfrom33.4%(Cabernet Franc,2006vintage)tomorethan47%(Syrah,2007vintage).In

general, acetylated anthocyanins werethederivatives foundin

highest concentrations in both vintages, withrelative amounts

rangingfrom17% to25% ofthetotalanthocyanins.Thehighest

valueswerefoundforCabernetFranc2007(22.83mgL1_).The

p-coumaratedanthocyanincontentcorrespondedto4.3–11%ofthe

totalanthocyanins.

Two-wayANOVAanalysisrevealedthat thetotalcontents of

flavonolsandanthocyaninswereinfluencedbytwofactors:variety andvintage(p<0.05).Itisknownthatthebiosyntheticpathways

involved in flavonoids production in plant tissues are greatly

influencedbymanyclimaticfactorsincludingsunlightexposure, temperatureandUVexposure.Thus,inthisstudy,thedifferences

found between the two vintages were to be expected since

significantdifferenceswereobservedbetweenclimatesinwhich

they were grown (Gris et al., 2010). However, an alternative

hypothesis to explain the differences observedin thephenolic

compoundscontentofthetwovintagesisthetimeofwineaging.

Since wine composition is in constant evolution, the aging in

bottles also seems to contribute to changes in the flavonols

content,throughtheinteractionofflavonolswithother constitu-ents(Ribe´reau-Gayonetal.,1998).

Itwasfoundthattheeffectofvintagewassignificantlymore

pronounced on the anthocyanins than on the flavonols, the

anthocyaninsconcentrationinthe2006vintagebeingsignificantly

lower for all varieties when compared to the later vintage

(p<0.05). This can be explained by the fact that, although

weatherconditionshaveastronginfluenceontheanthocyanins

concentration,accordingtoMonagasetal.(2005)theseareoneof

the groups of phenolic compounds that present major losses

during wine aging in the bottle. This is attributed to their

participationinnumerouschemicalreactions which,ingeneral,

lead tothe disappearance of monomericanthocyanins and the

formation to more stable oligomeric and polymeric pigments.

Moreover,Rossettoetal.(2004)alsoaffirmthatthepolymerization processwhichoccursduringwineagingcausesthedisappearance ofthefreeformofanthocyanins.Onceawineisbottled,changes

are determined mainly by non-oxidative reactions (

Ribe´reau-Gayonetal.,1998).However,recentstudiesindicatethatwinesare

also subjected to oxidative reactions (Lopes et al., 2006).

Considering thenon-oxidative conditions present in thebottle,

the direct condensation reaction of anthocyanins with other

phenolic compounds, and hydrolytic and degradativereactions

(SomersandEvans,1986)aremostprobablyresponsibleforthe

decrease in the monomeric anthocyanins concentration during

aging in the bottle. Thus, a combination of these factors can

influencethephenoliccompositionofwineand,consequently,the levelsofanthocyaninsandflavonols.

Regardingthesignificantdifferencesobservedinthecontents offlavonoid phenolic compoundsofthedifferentvarieties,itis

known that, although theenvironmental conditions where the

fruit is grown influence greatly the synthesis of phenolic

compounds, their nature and different relative amounts are

consequencesofageneticdeterminant, specificforeachvariety (Mazza,1995).

3.1.2. Non-flavonoidphenoliccompounds

The results for the determination of hydroxycinnamic and

hydroxybenzoicacidsinthewinesamplesarepresentedinTable3. Inwines,themostabundanthydroxycinnamicacidsarepresentin

the conjugated form (tartaric esters; hydroxycinnamates), as

foundinthisstudy(Table3).Thepresenceoffreeformsofthese acidsin wines,since theyarenot present ingrapes(Vrhovsek, 1998),accordingtoSomersetal.(1987),isduetothehydrolysisof

hydroxycinnamatesbycinnamoylesterases.

Atwo-wayANOVAcarriedoutconsideringtwofactors,variety andvintage,revealedthatthesefactorsinfluencethetotalcontent of hydroxycinnamic acids (p<0.05). Regarding the varieties, CabernetFrancpresentedthehighestcontentofhydroxycinnamic acidinbothvintagesanalyzed(p<0.05).Itwasverifiedthatthe

total hydroxycinnamic acid content was higher for the 2007

vintage(p<0.05).

Itwasfoundthatthevaluesfortrans-caftaricandcisand trans-coutaricacidsinthewinesofthe2006vintagewerelowerthan

those found for the 2007 vintage, with the opposite being

observedfor the trans-caffeicandtrans-p-coumaric acids.One

hypothesiswhichmayexplainthisobservationistheconstant

evolutionofthewinecompositionandtheseveralreactionsthat

occur duringwineproduction, storagein barrelsandagingin

bottles.ThistrendwasalsoverifiedbyMonagasetal.(2005)who notedthattheincreaseinfreeacidsinwinesmayoriginatenot onlyfromthehydrolysisoftherespectivetartaricesters(Somers etal.,1987),butalsofromthehydrolysisofp-coumaroyl-acylated anthocyaninsduringaginginthebottleandthatthe disappear-anceofacylatedanthocyaninsduringwineagingis,inpart,dueto

the hydrolysis of the acylated group. This explanation is also

consistentwith thelower concentrationsofthese anthocyanic

derivativesverifiedinthe2006comparedwiththe2007vintage

(Table2).

Theresults forthehydroxybenzoicacidsconcentrationsare

presentedinTable3.Oftheseacids,gallicacidwaspredominant,

representing, onaverage, 76%of allhydroxybenzoic acids.The

presence of high amounts of gallic acid in red wines is to be

expected since it is formed mainly through the hydrolysis of

flavonoidgallateesters.Thevarietyandvintageinfluencedthe

total hydroxybenzoicacidsas determinedbytwo-way ANOVA

(p<0.05).The totalhydroxybenzoicacidscontent(sumofthe individualcontents)ofthe2007vintagewashigherthanthatof the 2006 vintage (p<0.05) for all varieties, except for Syrah

2007. Theindividual analysisof hydroxybenzoic acidsshowed

thatthecontentsvariedamongvintagesandvarieties(p<0.05).

The evolution of such acids during aging in bottles is very

variable. Accordingto Monagas et al.(2005),although

hydro-xybenzoic acids are susceptible to changes during enological

Table1

Contentofmainflavonolsandtotalphenols(TP)inwinesamples.

Vintage 2006 2007

CabernetFranc Merlot Sangiovese Syrah CabernetFranc Merlot Sangiovese Syrah Myricetin 14.490.18 11.590.23 9.330.13 10.420.06 13.830.13 14.040.10 11.830.21 16.470.14 Quercetin 22.590.23 19.010.23 7.490.20 14.820.19 17.800.17 20.500.23 27.440.22 13.880.16 Laricitrin 3.390.14 2.400.12 1.470.12 2.320.10 2.860.11 2.500.09 1.670.08 3.410.13 Kaempferol 1.650.08 1.250.06 0.230.05 0.430.05 0.380.08 0.760.04 0.740.07 0.690.05 Isorhamnetin 2.720.04 2.020.06 0.740.02 2.120.07 2.000.08 2.000.07 0.480.08 3.280.10 Syringetin 1.940.08 2.060.04 1.570.09 1.730.04 2.290.09 1.990.06 0.600.05 2.210.08 Totalflavonols 46.790.9a _38.32 1.4b _20.81 0.7d _31.85 0.3e _39.15 0.2b _41.79 0.3b,c _42.76 0.4a.c _39.93 0.3b,c Totalphenols 2680.412.4a _2692.1 40.3a _2287.6 31.1b _2732.2 37.2a _2691.4 26.2a _2813.7 16.2c _2732.1 27.9a _2790.5 34.2a ValuesinunitsofmgL1

standarddeviationoverthreereplicationsinonewinesample.Differentletters(totalcontents)withineachrowaresignificantlydifferent(Tukey’stest, p0.05).

Table2

Contentofmainanthocyaninsandderivatives(estersofaceticandp-coumaricacids)inwinesamples.

Vintage 2006 2007

CabernetFranc Merlot Sangiovese Syrah CabernetFranc Merlot Sangiovese Syrah Delphinidin-3-glucoside 1.770.07 2.160.06 1.500.06 2.010.06 14.830.34 11.230.29 2.690.16 7.510.06 Cyanidin-3-glucoside 0.490.07 0.690.05 0.460.05 0.550.03 2.740.05 2.720.04 2.760.03 1.070.06 Petunidin-3-glucoside 1.480.05 1.740.06 1.240.05 1.940.03 13.260.25 8.490.18 3.300.08 9.520.25 Peonidin-3-glucoside 1.320.03 1.490.08 1.120.08 1.710.06 10.500.10 6.610.08 2.580.06 6.910.06 Malvidin-3-glucoside 5.580.06 6.020.08 4.530.07 7.620.08 53.481.01 25.250.78 8.450.47 41.541.02 Delphinidin-3-glucoside-acetate 0.380.04 0.340.04 0.160.02 0.340.01 2.390.05 2.070.06 0.210.03 1.350.05 Cyanidin-3-glucoside-acetate 0.120.03 0.170.06 0.070.014 0.090.02 0.840.03 0.740.03 0.160.03 0.260.04 Petunidin-3-glucoside-acetate 0.170.06 0.350.03 0.170.03 0.220.02 2.520.13 1.480.08 0.220.03 1.400.03 Peonidin-3-glucoside-acetate 0.740.03 0.720.03 0.470.03 0.580.06 3.390.09 1.520.04 1.780.08 2.200.03 Malvidin-3-glucoside-acetate 2.820.05 2.370.06 1.890.10 2.730.07 13.680.49 6.270.18 4.620.21 9.740.24 Delphinidin-3-glucoside-cumarate 0.190.03 0.060.02 0.170.04 0.160.03 0.680.06 0.410.03 0.350.05 0.510.06 Cyanidin-3-glucoside-cumarate 0.420.03 0.270.04 0.200.04 0.370.06 1.070.08 0.730.05 n.d. 0.970.06 Petunidin-3-glucoside-cumarate 0.060.02 0.050.01 0.040.01 0.110.02 0.460.03 0.330.03 0.070.01 0.380.04 Peonidin-3-glucoside-cumarate 0.320.02 0.290.04 0.230.05 0.290.03 1.570.06 0.310.03 0.300.02 1.810.06 Malvidin3-glucoside-cumarate 0.850.07 0.850.06 0.680.05 1.010.08 4.170.12 2.410.06 0.460.06 3.180.03 Totalglucoside 10.650.3a _12.09 0.3a _8.85 0.3a _13.84 0.3a _94.81 0.5b _54.31 1.2c _19.78 0.8d _66.54 1.3e

Totalacetylatedanthocyanins 4.230.08a

3.960.06a 2.760.1a 3.970.9a 22.830.1b 12.090.6c 6.990.07d 14.950.7e

Totalp-coumaratedAnthocyanins 1.830.03a

1.510.02a 1.330.05a 1.940.04a 7.960.07b 4.190.06c 1.190.09a 6.860.05d

Totalanthocyanins 16.710.5a,b

17.560.9a,b 12.940.2b 19.750.9a 125.602.1c 70.581.8d 27.961.4e 88.352.3f ValuesinunitsofmgL1

standarddeviationoverthreereplicationsinonewinesample.Differentletters(totalcontents)withineachrowaresignificantlydifferent(Tukey’stest, p0.05).LODofcyanidin-3-glucoside-cumarate=0.09mgL1

practices,itseemsthattheirevolutionprofileduringwineaging inthebottledoesnotchangesignificantly.

3.2. Antioxidantactivity

Theresultsfortheinvivostudyontheantioxidantactivityof

wine samples are presented in Fig. 1. It was found that the

ethanolcontrolgroupdidnotdiffersignificantlyfromthewater

controlgroup, indicatingthat theconsumption ofethanol did

not modify theparameters of thein vivoantioxidant activity.

Theeffectofethanolonanorganismisstillverycontroversial.

Epidemiologicalevidencesuggeststhatmoderateconsumption

ofalcoholicbeverages(20–30galcoholperday),suchaswine,is

associated with a reduced risk of death from cardiovascular

disease(Renauld and Lorgeril, 1992). However, the results of

some studies suggest that ethanol consumption promotes

toxic effects such as the generation of damaging freeradical

species in various tissues (Mantle and Preedy, 1999) and an

increased activity of CAT and SOD enzymes (Rodrigo et al.,

2002).

Thelevelsofplasmaantioxidantcapacity(FRAP)arepresented inFig.1.Whencomparedtothecontrolgroups,theFRAPincreased withadministrationofredwinesamples,withtheexceptionofthe grouptreatedwiththeSangiovese2006wine(p<0.05).Increases intheplasmaantioxidantactivityrangedfrom17.8%to70.7%with significantdifferencesfoundonlyamongvarietiesandnotamong

vintages (two-way ANOVA; p<0.05). Increases in plasma

antioxidantcapacityduetowineconsumptionhavebeenreported inseveralstudies(Grisetal.,2011a,b;Rodrigoetal.,2005).Inthis regard,itshouldbenotedthattheFRAPindicates thereducing abilityoftheplasma,butitisnotexactlyequivalenttothewhole antioxidanteffectof plasma,since inthemethodthechelating abilityisnotmeasured.

Wine consumption promoted a significantdecreasing in the

TBARS and CP levels when compared to the control groups

(p<0.05),demonstratingtheprotectiveeffectofwine consump-tionagainstlipoperoxidationandproteiccarbonylation.Itwasalso

observedthatthevarietyandvintageinfluencedboththeTBARS

andtheCPvalues(two-wayANOVA;p<0.05).Inthecaseofthe TBARSlevels,themostsignificantdecreaseswerefoundforSyrah (43.2%)andMerlot(27.6%)fromthe2007vintage.CabernetFranc andSyrahvarietiespresentedthehighestCPdecreases forboth vintagesevaluated,causinganinhibitionthatrangedfrom30.5to 42.9%.

TheincreaseinFRAPprobablyoccurredduetheabsorptionof

wine phenolic compounds,which are known tohave reducing

power(Burinetal.,2011;Baronietal.,2012).Anotherpossibilityis thatthewineconsumptionpromotedanincreaseinthelevelsof

urates in the plasma, one of the most abundant free radical

scavengersinhumans,andthiscouldleadtoprotective antioxi-danteffectsandanincreasedantioxidantpotentialoftheplasma (Modunetal.,2008).Rodrigoetal.(2005)suggestedthat wine-inducedincreasesintheantioxidantcapacityofplasmacontribute totheenhancementoftheantioxidantdefensesystemsoforgans likethekidneys, liverand lungs,due totheir well-knownhigh perfusionrate.Ithasbeenpostulatedthattheincreaseinplasma antioxidantcapacityandinhibitionoflipoperoxidationandproteic carbonylation,asobservedinthepresentstudy,promotedbywine

ingestionisdue tothehighcapacity ofphenoliccompoundsto

offer protection againstfree radicals (Frankel et al.,1995). The phenoliccompoundscanactasmetalchelantswhich,

theoretical-ly, preventiron-dependentlipidperoxidation inmembranes by

renderingironinactiveandbyscavengingchain-initiatingperoxyl radicalsattheliquid-aqueousinterface.

Inthisstudy,asignificantdifferenceinhepaticGSHlevelsofthe

different experimental groups (p<0.05) was not observed,

suggestingthatwineingestiondidnotaffectthehomeostasisof theanimalorganism.Thisisaninterestingresultsinceanincrease inGSHlevelsmaysuggestanantioxidantresponsetoanincrease instressingagents. Similarresultshavebeenreported byother authors(Grisetal.,2011a,b;Ferreiraetal.,2010).

InrelationtotheactivityofSOD,CATandGPxenzymes(Fig.1),

it was found in this study that wine ingestion decreased

significantlytheexpressionofscavengerenzymeswhencompared

tocontrolgroups(p<0.05).Statisticalanalysis(two-wayANOVA) revealedthatthevintageinfluencedtheSODandCATactivity,but

presented no significant effect for GPx (p<0.05). However,

conversely, GPxwasinfluenced by thevariety,while theother

enzymes(CATandSOD)didnotpresentasignificantassociation

withthisfactor(p<0.05).

TheSODactivitydecreasedbyaround15%inthewinesofthe 2006vintage,particularlyforSyrah(p<0.05).Thisdecreasewas morepronouncedforthewinesofthe2007vintage(43%),withno significantdifferenceamongvarieties(p<0.05).Wine consump-tioncausedadecreaseinCATactivityrangingfrom12.1to36%for

Merlot2006and2007,respectively.Cabernet Franc andMerlot

winesofthe2007vintageweremoreeffectiveatdecreasingthe

CAT activity when compared to wines of the 2006 vintage

Table3

Contentofmainhydroxycinnamicandhydroxybenzoicacidsinwinesamples.

Vintage 2006 2007

CabernetFranc Merlot Sangiovese Syrah CabernetFranc Merlot Sangiovese Syrah cis-Caftaricacid 2.000.03 2.370.01 1.840.02 2.050.01 2.140.01 2.360.02 1.410.021 2.800.01 trans-Caftaricacid 92.163.17 73.593.20 68.681.48 73.822.19 150.974.79 92.302.97 99.853.61 69.763.11 cis-Coutaricacid 4.210.18 3.610.29 3.610.23 4.540.21 6.610.23 5.790.14 17.891.50 11.671.04 trans-Coutaricacid 20.941.87 17.580.95 14.691.01 19.110.90 32.491.80 18.660.93 35.281.51 27.491.49 Fertaricacid 3.390.11 3.090.15 2.970.09 3.030.12 5.210.15 3.610.13 4.040.10 1.460.10 trans-Caffeicacid 9.470.32 8.550.30 8.090.25 9.200.23 8.660.13 9.160.21 4.320.12 10.480.12 trans-p-Coumaricacid 6.610.11 7.730.23 6.540.11 6.300.20 5.590.13 5.310.12 2.730.06 5.860.19 trans-Ferulicacid 2.600.03 2.780.11 2.320.07 2.610.10 2.750.11 2.960.08 2.060.08 2.390.09 Gallicacid 40.481.31 41.601.61 34.530.49 39.940.54 45.031.41 54.441.19 32.850.83 39.330.79 Protocatechuicacid 0.870.08 1.430.08 1.910.06 2.920.09 9.730.22 5.740.17 12.660.13 10.120.25 p-Hydroxybenzoicacid n.d. 1.260.06 0.590.08 0.860.09 n.d. n.d. 1.710.08 2.100.11 Vanillicacid 3.480.070 2.130.04 1.660.08 3.670.05 3.500.11 3.780.10 2.980.07 3.220.08 Syringicacid 1.480.06 1.230.08 1.150.05 3.530.08 4.110.08 0.990.17 1.560.09 4.220.11 Ellagicacid 0.930.08 0.890.07 0.760.09 0.500.08 3.190.06 0.130.04 3.860.07 0.110.02 Totalhydroxycinnamicacids 141.404.7a _119.32

4.3b,c _108.78 3.8b _120.63 3.4b,c _214.48 7.9e _140.27 4.8a,d _167.57 6.2f _131.86 3.7a,c,d

Totalhydroxybenzoicacids 47.232.2a

48.552.7a 40.591.7b 51.411.6a 65.562.2c,d 65.081.8c 55.621.6a 50.102.1d

ValuesinunitsofmgL1_{standarddeviationoverthreereplicationsinonewinesample.Differentletters(totalcontents)withineachrowaresignificantlydifferent(Tukey’stest,}

p0.05).n.d.=notdetected.LODofp-hydroxybenzoicacid=0.039mgL1

Fig.1.Valuesfortotalantioxidantcapacityofmiceplasma(A–FRAP,TEACmM),lipidperoxidation(B–TBARS,nmolmgprotein1_{),carbonylprotein(C–CP,nmolmg}

protein1_{),hepaticGSH(D–nmolmgprotein}1_{)andcatalase(E–CAT,mmolmin}1_mgprotein1_{),superoxidedismutase(F–SOD,SODUmgprotein}1_{)andglutathione}

peroxidase(G–GPx;mmolmin1

mgprotein1

)activityinmiceliver.Controlgroups–C:water;Cet:12%hydroalcoholicsolution.Testgroups:CF06:CabernetFranc2006 wine;M06:Merlot2006wine;Sa06:Sangiovese2006wine;Sy06:Syrah2006wine;CF07:CabernetFranc2007wine;M07:Merlot2007wine;Sa07:Sangiovese2007wine; Sy07:Syrah2007wine.ANOVAtocomparedata;valueswithdifferentletterswithineachcolumnaresignificantlydifferent(Tukeytest,p0.05).

(p<0.05).TheoppositeeffectwasobservedfortheSyrahvariety (p<0.05).SuppressionofGPxactivityrangedfrom34.7to65.4%.

Syrah(2006and2007)andSangiovese(2007)presentedthemost

significantdecreaseinGPxactivity(62.4and65.4%,respectively).

These observations are of great interest since the main

enzymaticantioxidantdefensesystemiscomposedofthecellular

enzymessuperoxidedismutase(SOD),catalase(CAT)and

gluta-thioneperoxidase(GPx).Thisrepresentsthefirstlineofdefensein

theneutralizationof endogenous ROS, throughwhich the cells

attempttomaintainlowerquantitiesofO2andH2O2andthus

avoidtheformation of_{OH which,althoughshort-livedevenin}

verylowconcentrations,isextremelyreactiveanddamagingto

cells(HalliwellandGutteridge,1999).

CAT and GPx are thetwo enzymesthat have the ability to

convertcellH2O2toH2OandO2(HalliwellandGutteridge,1999).

Since CAT and GPx are also involved in the elimination of

hydroperoxides,thedeclineinTBARSlevelsobservedinthisstudy mightberelatedtotheobserveddecreaseinCATandGPxactivity. Thedecreaseintheactivityoftheseenzymesisprobablydueto suppressionoftheROSmediatedtriggerofantioxidantenzymesat

thetranscriptome level, due to the presence of wine phenolic

compounds.

3.3. Associationbetweenantioxidantactivityandconcentrationof

phenoliccompounds

Under physiological conditions,ROSareeliminatedby

enzy-matic and nonenzymatic antioxidant defense systems. The

scavenging of ROS, binding of metal ions and degradation of

peroxides are common mechanisms to prevent ROS-induced

damage propagation. An increase in ROS production and a

deficientantioxidantstatus may causesevere oxidative stress

incells,leadingtoseveraldiseasesandtoxicity.Inthiscontext, ingestionofredwinehasbeenthetargetofseveralstudiesand representsanareaofintensiveresearchinpreventingdisorders relatedtooxidativestress.Inthisstudyitwasverifiedthatwine

consumption increased the plasma antioxidant capacity and

decreasedtheTBARSandCPlevels.Areductionintheantioxidant

activitiesoftheenzymesCAT,SODandGPxwasalsoobserved.

Thus,theinfluenceofthemainphenoliccompoundsontheinvitro antioxidantactivityofthewinesamplesstudiedwasassessedby principalcomponentsanalysis(Fig.2),sinceitisbelievedthatthe

antioxidant potential of red wine is related to its phenolic

compoundscontent.

Thefirstthreeprincipalcomponentsexplained83.27%ofthe

totalvariance(Fig.2).Factor1wasnegativelyinfluencedbythe mainchemicalandantioxidantanalysis.GSH,GPx,kaempferoland

trans-p-coumaric and p-hydroxybenzoic acids positively

influ-encedFactor1.Factor2wasnegativelyinfluencedbyCATandSOD antioxidantactivity,and bytotalhydroxycinnamicacid(THCA), totalhydroxybenzoicacid(THBA)andtotalflavonols(TFLA).Some

hydroxycinnamicand hydroxybenzoicacids,cyanidinand

quer-cetinalsonegativelyinfluencedFactor2.

Fig. 2 showsthat increasesin thetotalantioxidant capacity

(FRAP) and inhibition of lipid peroxidation (TBARS) were

associatedwithmanyofthephenoliccompoundsevaluated,such

asTP, TFLA, THBA,vanillic acid, myricetinand themajority of

anthocyanins.The inhibition of proteic carbonylation (CP) was

associatedwitht-ferulic,trans-caftaric,syringicandgallicacidsas wellasisohramnetin,laricitrin,myricetinandTP.

Withregardtotheantioxidantenzymes,Fig.2showsthat a

decreasein theSOD activitywas associatedwith THCA,THBA,

TFLA, trans-caftaric acid, protocatechuic acid and the main

anthocyanins.AdecreaseintheCATactivitywasassociatedwith fertaric,protocatechuic,cis-cutaric,trans-caftaricandelagicacids, cyanidin,quercetinandTHCA.

Ingeneral,theassociationbetweenphenoliccompoundsandin vivoantioxidantactivityfoundinthisstudywastobeexpected sincethesecompoundsaremainlyresponsiblefortheantioxidant activityinredwine.However,themechanismofactionofthese compoundsintheorganismisstillnotfullyunderstoodrequires furtherstudy.

Thecorrelation/association betweenantioxidant activityand phenoliccompoundshasbeenverifiedbymanyauthors(Grisetal., 2011a,b;Ferreiraetal.,2010;Ale´n-Ruizetal.,2009;DiMajoetal.,

2008). The antioxidant properties of phenolic compounds are

probablydue tothestructure ofthesecompounds.Invitro,the degreeandpositionofhydroxylandmethylgroupsintheBringof theflavonoidsaffecttheirstabilityandreactivity.Ingeneral,higher

antioxidant capacity is observed in compounds presenting the

ortho-dihydroxystructure intheBring,withthesecompounds

being effective hydrogen donors. The antioxidant activity of

phenolic acids andtheir esters is dependenton the number of

hydroxylgroupsinthemoleculethatwouldbestrengthenedby

steric hindrance. The electron-withdrawing properties of the

carboxylategroupinbenzoicacidshaveanegativeinfluenceonthe H-donatingabilitiesofthehydroxybenzoates,thus,theoretically,

hydroxylated cinnamates are more effective antioxidants than

benzoate(Rice-Evansetal.,1996).

Thebeneficialeffectsoftheingestionofphenoliccompounds, especiallyinwines,havefrequentlybeenreported.However,data

concerning the absorption mechanism and bioavailability of

polyphenols in organisms are scarce, since the issue of the

biologicaldestinationofflavonoidsortheirdietaryglycosideforms ishighlycomplexanddependentonalargenumberofprocesses.

Phenoliccompounds,suchassomehydroxycinnamicand

hydro-xybenzoicacids,quercetin,kaempferolandmalvidin-3-glucoside,

Cy -1,0 -0,5 0,0 0,5 1,0 Factor 1 : 44,23% -1,0 -0,5 0,0 0,5 1,0 Factor 2 : 26,04% GSH Galic TAacil TAglu Mv/Df/Pl/Pt GPx TP Protocatechuic Cyanidin t-cutaric TAcum T FLA FRAP TBARS TA t-p-coumaric t-caffeic Syringic CP t-ferulic Laricitrin Myricetin Quercetin Kaempferol Isorhamnetin c-caftaric Vanillic p-hydroxybenzoic T HCA t-caftaric SOD CAT Fertaric c-cutaric Elagic T HBA

Fig.2. Principalcomponent analysisofantioxidantanalysisandthephenolic compounds. THBA:total hydroxybenzoic acids; THCA: total hydroxycinnamic acids; TFLA: total flavonols; TA:total anthocyanins; TAacil: total acetylated anthocyanins;TAglu:totalglucoside;TAcum:totalp-coumaratedanthocyanins; TP: total phenols;Mv: malvidin-3-glucoside; Df: delphinidin-3-glucoside; Pl: perlagonidin-3-glucoside;Pt: petunidin-3-glucoside; PC:protein carbonylation inhibition;TBARS:lipidperoxidationinhibition;FRAP:totalantioxidantcapacity; SOD:decreaseactivityofsuperoxidedismutase;CAT:decreaseactivityofcatalase; GPx:decreaseactivityofglutathioneperoxidase;GSH:reducedglutathione.

havebeendetectedandquantifiedinhumanbiologicalfluidsafter bothacuteandsustainedwineingestion(Pignatellietal.,2006; Nardini et al., 2009). It appears that after wine ingestion

polyphenolsundergoarapidandextensivemetabolismresulting

intraceamountsofunchangedphenolsinthecirculatorysystem.

Themajorityofpolyphenolsabsorbedarepresentinplasmaand

urine in conjugated forms (methylated, glucuronated and

sulfated) indicating an extensive first-pass intestinal/hepatic

metabolismoftheingestedprimaryphenoliccompoundforms.

Thus,thebiologicalactivityofpolyphenolscanbeattributedto their metabolites (Pignatelli et al., 2006; Vanzo et al., 2007; Nardini et al., 2009; Garcia-Alonso et al., 2009; Sancho and Pastore,2012).

Initially,itwasconsideredthatonlyfreeflavonoidswithouta sugarmolecule(aglycones)wereabletopassthroughtheintestine

wall and be absorbed (Griffiths, 1982). However, more recent

studiescontestthisassumption.Apparently,quercetinglucosides fromthedietaremostlyhydrolyzedtotheiraglycones,followedby conjugationtoglucuronidesand/orsulfates(Garcia-Alonsoetal., 2009;Wuetal.,2011).Changetal.(2005)foundthat

quercetin-3-O-glucoside could be rapidly absorbed and transformed into

glucuronidatedquercetin.According toreportsintheliterature,

anthocyanins are absorbed in the glycosylated form after oral

ingestion(Nielsenetal.,2003;Garcia-Alonsoetal.,2009),andthe

stomachseemstobethesiteofabsorptionforthesecompounds

(Passamonti et al., 2003). A recent study demonstrated that

hydroxycinnamic acids in white wine are absorbed in the

gastrointestinaltractandcirculateinthebloodafterbeinglargely

metabolized to glucuronide and sulfate conjugates in humans

(Nardinietal.,2009).

Itshouldbenotedthatalthoughtheantioxidantactivityofwine isacceptedasoneofthemainbiologicalmechanismsrelatedtored

wine-derived phenolic compounds, many others have been

proposedincludingnitricoxide-mediatedvasorelaxation(Diebolt etal.,2001;Dell’Aglietal.,2005),estrogenicactivity(Klingeetal., 2003;Varadinovaetal.,2009),inhibitionofplateletaggregation (Corder,2008)andmodulationoflipidmetabolism(Frankeletal., 1993;Rouanetetal.,2010).

4. Conclusions

Theresultsofthisstudyshowedthatthewinesunderstudyhad

adequatephenolic contentand composition,demonstrating the

potentialofthisregionofsouthernBraziltoproducehighquality wines.Significantinvivoantioxidantactivitywasalsoverifiedafter

wineingestion,throughincreasedFRAPanddecreasedTBARSand

PClevels,andthesuppressionofCAT,SODandGPxenzymes.In

general,theantioxidantactivitypromotedbywineingestionwas

associated with the main phenolic compounds quantified,

suggestingthepossibleinvolvementofthesecompounds,ortheir

metabolites, in the mechanism of action. Therefore, the data

presentedinthisstudyprovideevidencethatredwineingestion contributes to increasing the in vivo antioxidant activity, and

suggest that this effect can be attributed to the phenolic

compositionofthewine.Ourresultswouldthusseemtosupport

therecommendation that moderatewine consumption may be

beneficialforhealth. Acknowledgements

WeextendourwarmgratitudetoDanielePerenzoni,Domenico

Masuero,MattiaGasperotti,EduardoBenedetti,KarinaFelipeand LuizWeisefortheirassistancewiththechemicalandbiochemical analysisandtoPauloJose´ Ogliariforassistancewiththestatistical analysis.TheauthorsacknowledgealsotheBraziliangovernmental

agency the National Council for Scientific and Technological

Development(CNPq)forprovidingadoctoralscholarship(Brazil/ Italy).

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