A.M. Jordão *, A.C. Correia

Transcription

1 Reltionship Between Antioxidnt Cpcity, Pronthocynidin nd Anthocynin Content During Grpe Mturtion of Tourig Ncionl nd Tint Roriz Grpe Vrieties A.M. Jordão *, A.C. Correi Polytechnic Institute of Viseu, Agrrin Higher School, Deprtment of Food Industries, Estrd de Nels, Quint d Algo, Rnhdos, Viseu, Portugl Sumitted for puliction: Mrch 212 Accepted for puliction: June 212 Key words: Antioxidnt cpcity, nthocynins, grpe mturtion, pronthocynidins To investigte ntioxidnt cpcity in seeds nd skins during grpe mturtion nd its reltionship with nthocynin nd pronthocynidin content, two Portuguese red grpe vrieties, Tourig Ncionl nd Tint Roriz (Vitis vinifer L.) were studied. Two nlyticl methods were used for ntioxidnt cpcity nlysis: the DPPH nd ABTS methods. Pronthocynidins from seeds nd skins were seprted into monomers, oligomers nd polymers, while 13 individul nthocynins from the skins were lso evluted y HPLC. For oth grpe vrieties studied, ntioxidnt cpcity from the skins nd seeds ws chrcterised during grpe mturtion y generl decrese, minly in the first weeks fter vérison, followed y stilistion nd slight increse in the vlues in the lst three weeks of ripening. A similr tendency ws oserved for the mount of ll the different pronthocynidin frctions quntified. Our results lso showed tht seeds re n importnt source of pronthocynidins with respect to the grpe erry skins. Seeds were lso the grpe erry frction with the highest ntioxidnt cpcity when compred to the ntioxidnt cpcity content of the skins. For the 13 individul monomeric nthocynins quntified during grpe mturtion, evolution ws generlly chrcterised y continuous increse in the vlues. However, for some of the individul nthocynins, the continuous increse ws followed y stilistion or decrese in the vlues in the lst weeks of ripening. Finlly, there ws positive reltionship etween the different pronthocynidin frctions nd ntioxidnt cpcity of oth grpe vrieties studied; while negtive reltionship during grpe mturtion ws otined for individul nthocynins. INTRODUCTION Grpes hve long een pprecited for their rich content of phenolic compounds such s gllic cid, ctechin, nthocynins nd resvertrol, nd wide vriety of procynidins (Cheynier & Rigud, 1986; Ricrdo-d-Silv et l., 1992; Jordão et l., 1998, 1998; Kennedy et l., 2, 21; Jordão et l., 21,; Sun et l., 21). Phenolic compounds cn e clssified into two groups: flvonoids nd nonflvonoids. The min C 6 -C 3 -C 6 flvonoids in wine include conjugtes of the flvonols, quercetin nd myricetin; flvin-3-ols (+)-ctechin nd (-)-epictechin; nd nthocynins. The nonflvonoids incorporte the C 6 -C 1 hydroxy-enzoic cids, nd gllic nd ellgic cids; the C 6 - C 3 hydroxycinnmtes cffeic, cftric nd p-coumric cids, nd the C 6 -C 2 -C 6 stilenes trns-resvertrol, cis-resvertrol, nd trns-resvertrol glucoside. In the grpe erry, flvonoids such s nthocynins re locted minly in the skins (Jordão et l., 1998), while the flvn-3-ols (ctechins nd pronthocynidins) re present in the skins, seeds nd stems (Riéreu-Gyon, 1972; Ricrdod-Silv et l., 1992; Fuleki & Ricrdo-d-Silv, 1997; Kennedy et l., 2, 2; Jordão et l., 21,; Sun et l., 21). Anthocynin ccumultion in the vcuoles of the skin egins in vérison nd reches mximum concentrtion round hrvest time (Kennedy et l., 22; Cnls et l., 25). A decrese in the totl mount of nthocynins just efore hrvest nd during over-ripening hs een recorded in some works (Ryn & Revill, 23; Fournnd et l., 26). The highest levels of pronthocynidins in grpes were usully found t the onset of ripening, fter which they decresed (Jordão et l., 1998; Kennedy et l., 2, 2; Hnlin & Downey, 29). However, for some grpe vrieties nd for some procynidins, mximum is sometimes oserved t vérison (Jordão et l., 21). According to severl studies, flvn-3-ols, flvonols nd nthocynins re the most importnt compounds contriuting to the ntioxidnt proprieties of red grpes nd *Corresponding uthor: ntoniojordo@esv.ipv.pt 214

2 215 Antioxidnt Cpcity nd Grpe Phenolic Compounds wine (Wng et l., 1997; Simonetti et l., 1997; Ghiselli et l., 1998; Beecher, 23). In recent yers, severl uthors hve reported on the phenolic composition nd ntioxidnt cpcity of severl red nd white grpes from different vrieties, grpe erry frctions nd countries (Ork, 27; Bozn et l., 28; Poudel et l., 28; Yng et l., 29; Xu et l., 21; Breks et l., 21). However, in these studies the grpes were only generlly hrvested t optimum mturity or technologicl mturtion. Thus the ntioxidnt cpcity of different frctions of the grpe erry during grpe mturtion nd its reltionship with pronthocynidin nd individul nthocynin evolution hd not een investigted. The min oject of this study therefore ws to investigte the evolution of the ntioxidnt cpcity of the different frctions of grpe erry (seeds nd skins) nd its reltionship with the different pronthocynidin frctions (monomeric, oligomeric nd polymeric frctions) nd the individul nthocynin content of two importnt Portuguese red grpe vrieties during ripening (Tourig Ncionl nd Tint Roriz). MATERIALS AND METHODS Smples Tourig Ncionl nd Tint Roriz (Vitis vinifer L.) grpes were hrvested t grpe mturtion in 29 from vineyrd locted in Viseu (northern Portugl). Two hundred erries per vriety were selected from two clusters per plnt mong totl of 1 plnts. Smples were hrvested weekly (except in the lst three weeks of mturtion), strting t vérison nd continuing until technologicl mturity (56 dys fter vérison). The erries were frozen t -18ºC until processing. Generl physicochemicl prmeters From the initil 2 erries, susmple of 1 erries ws tken from the Tourig Ncionl nd Tint Roriz grpes nd nlysed t technicl mturity for estimted lcohol degree, ph, titrtle cidity nd totl phenolic index using the nlyticl methods recommended y the OIV (199). Totl nthocynins were mesured s recommended y Glories (1999). All determintions were crried out on the must (except totl phenols nd totl nthocynins). Totl phenols nd totl nthocynins were determined from n extrct otined y mcerting the crushed grpes t 25ºC for 24 h in ph 3.7 uffer (Cronneu & Chmpgnol, 1993). All lortory nlyses were done in duplicte. The generl physicochemicl composition of the Tourig Ncionl nd Tint Roriz grpe cultivrs t hrvest re presented in Tle 1. The totl phenol index in the Tint Roriz grpes ws much higher thn tht in Tourig Ncionl, while Tourig Ncionl showed the highest vlues for totl nthocynins. Smple preprtion for ntioxidnt cpcity, pronthocynidins nd individul nthocynin nlyses Skins nd seeds (from the other susmple of 1 erries) were seprted y hnd from the clusters (pproximtely 6 nd 2 g respectively) of ech grpe cultivr. After this seprtion, ech frction ws wshed seprtely severl times with distilled wter to remove foreign prticles, nd moisture ws sored with lotting pper. The seeds were crushed mnully efore the extrction process. TABLE 1 Generl physicochemicl prmeters of Tourig Ncionl nd Tint Roriz grpes t technologicl mturity. Prmeters Grpe vrieties Tourig Tint Ncionl Roriz Estimted lcohol degree (%, V/V) Titrtle cidity (g/l trt. cid) ph Totl phenols index (o.d. x 1) Totl nthocynins (mg/1 g erry) According to the procedure descried y Sun et l. (1996), grpe skins nd seeds were sumitted seprtely to n extrction process using 5 ml of 8% methnol first, followed y 5 ml of 75% cetone (ech for 3 h under gittion). After centrifugtion (1 min t 3 5 rpm), n liquot of ech extrct ws filtered (Whtmn.45 µm) nd frozen t -18 ºC until processing. Antioxidnt cpcity There re vrious methods to evlute the ntioxidnt cpcity of different foods, such s wine, which involve different mechnisms. The vlues for ntioxidnt cpcity chnge ccording to the method used. The lck of strong correltion etween different methods, e.g. DPPH nd ABTS, cn proly e ttriuted to the fct tht every individul phenol compound contined in wine cuses different response to the specific rdicl used in the ssy (Lchmn et l., 27). Thus the use of single method cnnot provide comprehensive prediction of the ntioxidnt efficcy of the different compounds (Arts et l., 23). The totl ntioxidnt cpcity ws performed seprtely from the skin nd seed extrcts produced ccording to the methodology descried previously. For the nlysis of ntioxidnt cpcity, two nlyticl methods were used: ABTS (2.2 -zinois-3-ethylenzothizoline-6-sulfonic cid) nd DPPH (2.2-diphenyl-1-picrylhydrzyl). The ABTS method is sed on discolortion, which occurs when the rdicl ction ABTS.+ is reduced to ABTS (Re et l., 1999). The rdicl ws generted y recting 7 mm solution of ABTS in wter with 2.45 mm of potssium persulphte (1:1). The ssy ws mde with 98 μl of ABTS.+ solutions nd 2 μl of the diluted smple (1:5 in wter). The rection tkes plce in drkness t room temperture. Asornce mesurements t 734 nm were tken fter 15 min of rection time. The procedure used to determine ntioxidnt cpcity using the DPPH method is descried y Brnd-Willims et l. (1995). This spectrophotometric technique employs the 2.2-diphenyl-1-picrylhydrzyl free rdicl (DPPH ), which shows chrcteristic UV-vis spectrum with mximum sornce close to 515 nm in methnol. Briefly,.1 ml of different smple concentrtions ws dded to 3.9 ml of DPPH methnolic solution (25 mg/l). The DPPH solution ws prepred dily nd protected from light. Asornce t 515 nm ws mesured fter 3 min of rection t 2ºC. The rection ws crried out under shking in closed Eppendorf

3 Antioxidnt Cpcity nd Grpe Phenolic Compounds 216 tues t 2ºC. Methnol ws used s lnk reference. The ntioxidnt cpcity results were expressed s Trolox equivlents (TEAC mm) y clirtion curve otined with stndrd Trolox. All lortory mesurements were performed in duplicte. Pronthocynidins ccording to their degree of polymeristion Seed nd skin extrcts were seprted into three frctions contining flvn-3-ol monomers, oligomeric (degree of polymeristion rnging from 2 to 12 15) nd polymeric (degree of polymeristion > 12 15) frction, using the C 18 Sep-Pck column s descried y Sun et l. (1998). Thus, ech smple ws pssed through the two preconditioned neutrl C 18 Sep-Pck crtridges connected in series. To eliminte phenolic cids, 4 ml de-lcoholised medium ws djusted to ph 7. nd then pssed through the two connected C 18 Sep-Pck crtridges preconditioned with 1 ml of wter djusted to ph 7.. After drying the column with nitrogen, elutions were crried out, first with 25 ml of ethyl cette to elute ctechins nd oligomeric pronthocynidins, nd then with 1 ml of methnol to elute polymeric frction. The ethyl cette elute ws tken to dryness under vcuum, re-dissolved in 3 ml of 67 mm phosphte uffer t ph 7., nd reloded onto the sme series of crtridges tht hd een conditioned gin s descried ove. The crtridges were dried with nitrogen nd eluted sequentilly with 25 ml of diethyl ether (frction contining flvn-3-ol monomers) nd 1 ml of methnol (frction contining oligomers). The three frctions otined were evported to dryness under vcuum nd re-dissolved in 3 ml of methnol. For ech frction otined previously, the quntifiction of flvnols ws performed y the modified vnillin ssy s descried y Sun et l. (1998). Thus, the vnillin rection with the ctechin frction ws crried out in 3ºC wter th for 15 min, nd mesurement of 5 nm ws lso tken t 3ºC. For oligomeric nd polymeric frctions, oth the vnillin rection nd the mesurement of 5 nm were performed t room temperture, nd the mximum of 5 nm ws tken s the mesured vlue. Oligomeric nd polymeric pronthocynidins isolted from grpe seeds previously isolted y column chromtogrphy on Lichroprep RP-18, in ccordnce with wht ws descried erlier y Sun et l. (1998), were dissolved in methnol to prepre the stndrd solutions used to uild the stndrds curves. All lortory nlyses were done in duplicte. Chromtogrphic nlysis of individul nthocynins For the nlysis of individul skin nthocynins, n HPLC Dionex Ultimte 3 Chromtogrphic System (Sunnyvle, Cliforni, USA) equipped with quternry pump Model LPG-34 A, n uto smpler Model ACC- 3, thermostted column comprtment (djusted to 25ºC) nd multiple Wvelength Detector MWD-3 ws used. The column (25 x 4.6 mm, prticle size 5 μm) ws C 18 Acclim 12 (Dionex, Sunnyvle, Cliforni, USA) protected y gurd column of the sme mteril. The solvents were (A) 4% formic cid, (B) pure cetonitrile nd (C) i-distilled wter. The individul nthocynins were nlysed y HPLC using the method descried y Dlls nd Lureno (1994). Thus, initil conditions were 25% A, 1% B nd 65% C, followed y liner grdient from 1 to 3% B, nd 65 to 45% C for 4 min, with flow rte of.7 ml/ min. The injection volume ws 4 µl. Detection ws done t 52 nm nd Chromeleon softwre progrm version 6.8 (Sunnyvle, Cliforni, USA) ws used. The individul nthocynins were quntified y clirtion curve otined with stndrd solutions of mlvidin-3-glucoside chloride (> 95% purity) from Extrsynthese (Geny, Frnce). The chromtogrphic peks of nthocynins were identified ccording to reference dt previously descried y Dlls nd Lureno (1994). All HPLC nlyses were done in duplicte. Sttisticl nlysis Anlysis of vrince (ANOVA, one-wy) nd comprison of tretment mens y Duncn s test (α =.5) were crried out using the SPSS softwre progrm version 11. (SPSS Inc. Hedqurters, Chicgo, IL, USA). In ddition, the correltion coefficient etween ntioxidnt cpcity vlues nd the content of the different phenolic compounds ws determined using the Microsoft Office Excel 23 softwre progrm. RESULTS AND DISCUSSION Evolution of pronthocynidin frction Pronthocynidins from the seeds nd skins of Tint Roriz nd Tourig Ncionl were seprted into monomers (ctechins), oligomers nd polymers, nd then quntified during grpe erry development (Figs 1 nd 2). In the seeds nd skins of oth grpe vrieties, generl decrese in the vlues ws oserved for the three frctions nlysed, followed y stilistion in the lst weeks of ripening. The decrese in pronthocynidin frctions oserved for oth grpe frctions in oth grpe vrieties hs previously een reported for other grpe vrieties nd in other regions y vrious uthors (Jordão et l., 21,; Downey et l., 23; Ó-Mrques et l., 25). In study on condensed tnnin ccumultion in the skin during erry development in Shirz nd Cernet Suvignon grpe vrieties, Hnlin nd Downey (29) reported mximum tnnin concentrtion just fter fruit set, with lower tnnin concentrtions t vérison nd hrvest. The tendency for higher concentrtions of condensed tnnins in the erly stges of erry development my e relted to their metolistion throughout ripening. For Kennedy et l. (2), the decrese in pronthocynidin content in the seeds fter vérison could e explin y oxidtion rections, while for Cheynier et l. (1997) the decrese could e ttriuted to reduced extrctility resulting from the conjugtion of pronthocynidins with other cell components. Vlero et l. (1989) considered tht the concentrtion decrese during grpe mturtion is only consequence of the incresing weight of the erries or seeds. However, for Bogs et l. (25) the level of pronthocynidins detected in the grpes represents lnce etween the ccumultion of pronthocynidins through synthesis nd decresed extrctility, nd my not e true reflection of iosynthesis in the fruit. Finlly, Bordig et l. (211) recently reported progressive pronthocynidin decrese

4 217 Antioxidnt Cpcity nd Grpe Phenolic Compounds (with the exception of epiglloctechin) during grpe ripening nd, t sme time, n increse in the percentge of prodelphinidins etween the first nd the lst smpling. These uthors lso reported n inter-conversion etween ctechin nd epiglloctechin during the mturtion of the different red grpes nlysed. During grpe erry ripening, in oth grpe vrieties, the seeds represented the highest concentrtion of ll pronthocynidin frctions (from 72. mg/g of seeds t the eginning of vérison to 3. mg/g of seeds t the end of mturtion for the polymeric frction in the Tint Roriz grpe vriety, nd from 56. mg/g of seeds t the eginning of vérison to 8. mg/g of seeds t the end of mturtion for the oligomeric frction in the Tourig Ncionl grpe vriety), followed y the skins (from 15.4 mg/g of skins t the eginning of vérison to 7.8 mg/g of skins t the end of mturtion for the polymeric frction, nd from 17.1 mg/g of skins t the eginning of vérison to 8.2 mg/g of skins t the end of mturtion for the oligomeric frction in the Tint Roriz grpe vriety). The quntifiction of high pronthocynidin concentrtions in the seeds in reltion to the skins is in ccordnce with the results otined y other uthors (Ricrdo-d-Silv et l., 1992; Jordão et l., 21; Ó-Mrques et l., 25). In ddition, the results show (Fig. 1 nd 2) tht, during the entire mturtion process nd for ll erry frctions nd for oth vrieties, the monomeric Content (mg/g seeds) Monomeric frction Dys fter verison,15,12,9,6,3 Monomeric frction Dys fter verison Content (mg/g seeds) Oligomeric frction Dys fter verison 3 2,5 2 1,5 1,5 Oligomeric frction Dys fter verison Content (mg/g seeds) Polymeric frction Polymeric frction Dys fter verison FIGURE 1 Evolution of the different seed pronthocynidin frctions during the ripening of two red grpe vrieties for the sme smpling dy. Different letters show significnt differences etween mens ccording to the Duncn test (α =.5); Error rs indicte stndrd devition. (O) Tint Roriz; ( ) Tourig Ncionl Dys fter verison FIGURE 2 Evolution of the different skin pronthocynidin frctions during the ripening of two red grpe vrieties for the sme smpling dy. Different letters show significnt differences etween mens ccording to the Duncn test (α =.5); Error rs indicte stndrd devition. (O) Tint Roriz; ( ) Tourig Ncionl

5 Antioxidnt Cpcity nd Grpe Phenolic Compounds 218 frction represented the lowest concentrtion nd the polymeric frction the highest concentrtion of condensed tnnins. These results confirm reserch y Sun et l. (21) nd Ó-Mrques et l. (25) on pronthocynidin evolution in skins nd seeds during grpe erry ripening in the Cernet Suvignon nd Tint Roriz vrieties. Finlly, the results show significnt differences etween the two vrieties, nmely significnt high concentrtion of pronthocynidins in the seed nd skins (except for the polymeric frction) for the Tint Roriz grpe vriety in contrst to Tourig Ncionl. In ddition, the vlues otined for Tint Roriz re similr to those otined y Ó-Mrques et l. (25) for the sme grpe vriety, ut from other growing regions with other vineyrd mngement prctices nd environmentl conditions. This fct my led us to consider tht it is the cste fctor tht most influences pronthocynidin content in the different grpe erry frctions with respect to other fctors such s climtic nd geogrphicl fctors, culturl prctices nd the plnts vegettive vigour. Individul nthocynin evolution Figs. 3 nd 4 show the evolution of different individul monomeric nthocynins extrcted from the skins during ripening. In oth grpe vrieties, generl increse followed y slight oscilltion in the individul nthocynins ws oserved during the ripening process. However, for the mjority of individul nthocynins in the Tourig Ncionl vriety (delphinidin-3-glucoside, petunidin-3-glucoside, mlvidin-3-glucoside, delphinidin-3-cetylglucoside, petunidin-3-cetylglucoside, mlvidin-3-p-coumroyl glucoside nd petunidin-3-p-coumroyl glucoside), slight decrese in the vlues ws quntified during the lst two weeks of the ripening process. For oth vrieties, the results indicte tht the group of nthocynin-3-glucoside pigments were the most undnt, followed y the 3-cetyl-glucoside forms nd finlly the coumroyl-glucoside pigments. Considering the individul nthocynin content, mlvidin-3-glucoside (from.4 to 7.79 mg/g skin t the eginning of vérison nd t the end Delphinidin-3-glucoside Cynidin-3-glucoside,8,6,4,2, Dys fter verison,3,2,1 Dys fter verison,2,15,1,5 Petunidin-3-glucoside Dys fter verison,4,3,2,1 Peonidin-3-glucoside Dys fter verison Mlvidin-3-glucoside 1, 8, 6, 4, 2,, Dys fter verison FIGURE 3 Evolution of skin nthocynin glucoside derivtives during the ripening of two red grpe vrieties for the sme smpling dy. Different letters show significnt differences etween mens ccording to the Duncn test (α =.5); Error rs indicte stndrd devition. (O) Tint Roriz; ( ) Tourig Ncionl

6 219 Antioxidnt Cpcity nd Grpe Phenolic Compounds,8 Delphinidin-3-cetylglucoside,8 Mlvidin-3-cetylglucoside,6,4,2 Dys fter verison,6,4,2 Dys fter verison,16,12,8,4 Peonidin-3-cetylglucoside Dys fter verison,1,8,6,4,2 Petunidin-3-cetylglucoside Dys fter verison,6,4,2 Mlvidin-3-p-coumroyl glucoside Dys fter verison,12,9,6,3 Peonidin-3-p-coumroyl glucoside Dys fter verison Petunidin-3-p-coumroyl glucoside Cynidin-3-p-coumroyl glucoside,1,4,8,6,4,2 Dys fter verison,3,2,1 Dys fter verison FIGURE 4 Evolution of skin nthocynin cetyl nd coumroyl glucoside derivtives during the ripening of two red grpe vrieties for the sme smpling dy. Different letters show significnt differences etween mens ccording to the Duncn test (α =.5); Error rs indicte stndrd devition. (O) Tint Roriz; ( ) Tourig Ncionl

7 Antioxidnt Cpcity nd Grpe Phenolic Compounds 22 of mturtion respectively), followed y delphinidin-3- glucoside (from.3 to.62 mg/g of skins t the eginning of vérison nd t the end of mturtion respectively) nd mlvidin-3-cetylglucoside (from.5 to.57 mg/g of skins t the eginning of vérison nd t the end of mturtion respectively), ws the most undnt in oth vrieties. In ddition, cynidin-3-glucoside (from. to.19 mg/g of skins t the eginning of vérison nd t the end of mturtion respectively) nd cynidin-3-p-coumroyl-glucoside (from. to.29 mg/g of skins t the eginning of vérison nd t the end of mturtion respectively) were the lest undnt pigments in oth vrieties. According to Roggero et l. (1986), cynidin derivtives re one of the primry pigments in the iosynthetic pthwy, constituting the smllest group during mturtion, while for other uthors mlvidin-3- glucoside represents the ultimte form in iosynthesis trnsformtion chins (Jordão et l., 1998). Severl uthors (Bkker & Timerlke, 1985; Roggero et l., 1986; Jordão et l., 1998; Romero-Cscles et l., 25; Mulincci et l., 28) hve reported tht, during ll stges of ripening, nthocynin-3-glucosides re the most undnt pigment group, while mlvidin-3-glucoside is the most undnt individul nthocynin, regrdless of the interntionl wine grpe vriety. However, the min nthocynin in pool of red tle grpes ws peonidin-3-glucoside (Cntos et l., 22). For some indigenous Spnish Vitis vinifer L. red grpe vrieties in dnger of extinction, Gómez-Alonso et l. (27) reported tht delphinidin-3-glucoside (Tinto Velsco vriety), peonidin-3-glucoside (Gorder Roj nd Tet de Vc Tint vrieties) nd cynidin-3-glucoside (Rojl vriety) were the predominnt individul nthocynins. Furthermore, some uthors elieve tht the rtio of totl nthocynins to cetyl nd coumroyl derivtes cn e usefully correlted to the specificity of ech grpe cultivr (Mulincci et l., 28). It is commonly ccepted tht the nthocynin composition of ech cultivr is closely linked to its genetic inheritnce nd, from qulittive point of view, is quite independent of sesonl conditions or production re (Férnndez-Lopez et l., 1998). In our study, nd in generl, the Tourig Ncionl vriety presented the highest content of cetyl nd coumroyl derivtives, ut the lowest content of the 3-glucosides derivtives during grpe ripening. The evolution of ntioxidnt cpcity nd its reltionship with pronthocynidins nd nthocynins. The evolution of ntioxidnt cpcity in the seeds nd skins mesured y the ABTS nd DPPH methods during grpe mturtion of the Tourig Ncionl nd Tint Roriz grpe cultivrs is presented in Figs. 5 nd 6 respectively. For oth grpe vrieties nd for ll grpe frctions, generl decrese ws oserved in the erly stges of mturtion, followed y stilistion of the vlues in the lst weeks of ripening. However, tendency for slight increse in the vlues in the skins of Tint Roriz ws oserved in the lst two weeks of ripening. These results were independent of the ntioxidnt cpcity method used. In ddition, the seeds represented the highest ntioxidnt cpcity during grpe mturtion in oth vrieties. In generl, these results re ccording to others tht nlysed the ntioxidnt cpcity of seeds nd skins in severl red grpe vrieties (Poudel et l., 28; Xu et l., 21). However, these works only presented the ntioxidnt cpcity results t technicl grpe mturtion, nd not over the grpe mturtion process. It is lso importnt to consider tht the mjority of the cited works only report the results of the totl ntioxidnt cpcity from the grpe must, without the contriution of seeds during the extrction process or without n individul nlysis for ech grpe erry frction. Thus, it is difficult to compre our results with the cited results otined for other grpe vrieties. It is importnt to highlight tht, in the literture, there generlly re lrge vritions in vlues of ntioxidnt cpcities otined for different grpe vrieties s result of different nlyticl methods, different smple preprtion nd extrction processes, nd different grpe vrieties nd species studied (Vitis vinifer, Vitis lruscn, Vitis murensis nd severl Euro-Americn nd Asin hyrids). In ddition, the impct of climtic nd soil conditions on the phenolic composition of grpes should e considered (Yokotsuk et l., 1999; Mteus et l., 22). In generl, the ntioxidnt cpcity of Tint Roriz during mturtion ws significntly higher thn tht of Tourig Ncionl (Figs. 5 nd 6). This ws more evident for the results otined using the ABTS method. At hrvest, the content of ntioxidnt cpcity (mesured y the ABTS method) of Tint Roriz ws nd 72. µmol trolox/g for seeds nd skins respectively, while for Tourig Ncionl the vlues in the seeds nd skins were nd 52.5 µmol trolox/g respectively. However, t technicl mturity the Antioxidnt cpcity (µmol trolox/g seeds) Antioxidnt cpcity (µmol trolox/g skins) Seeds ABTS Method Dys fter verison Skins Dys fter verison FIGURE 5 Evolution of the ntioxidnt cpcity in seeds nd skins mesured y the ABTS method during the ripening of two red grpe vrieties for the sme smpling dy. Different letters show significnt differences etween mens ccording to the Duncn test (α =.5); Error rs indicte stndrd devition. (O) Tint Roriz; ( ) Tourig Ncionl

8 221 Antioxidnt Cpcity nd Grpe Phenolic Compounds quntittive vlues otined for the seeds nd skins of the two Portuguese vrieties re lower thn the vlues reported for the Cernet Suvignon grpe vriety y Xu et l. (21), ut similr to the results reported for severl other grpe vrieties y Poudel et l. (28). The dt in Tle 2 show the liner correltion coefficients (R 2 ) etween the different pronthocynidin frctions nd the ntioxidnt cpcity quntified in the seeds nd skins during grpe mturtion in the two vrieties. The correltion coefficients generlly indicted n importnt role of the different pronthocynidin frctions of the skins nd seeds in the ntioxidnt cpcity during grpe mturtion. The correltion coefficient vlues for skins rnged from.72 to.82, from.72 to.86 nd from.52 to.82 for the monomeric, oligomeric nd polymeric frction of pronthocynidins respectively, while for seeds the correltion vlues rnged from.57 to.79, from.72 to.86 nd from.47 to.79 for the monomeric, oligomeric nd polymeric frction of pronthocynidins respectively. Severl uthors hve reported considerle correltions etween ntioxidnt cpcity nd the totl polyphenolic content of lrge numer of grpe seed nd skin extrcts from different vrieties (Bkklse et l., 25; Yng et l., 29; Xu et l., 21). However, other uthors (Bozn et l., 28) reported no significnt correltions etween individul flvnols nlysed y HPLC or totl polyphenols nd ntioxidnt cpcity vlues in seed extrcts from Antioxidnt cpcity (µmol trolox/g seeds) Antioxidnt cpcity (µmol trolox/g skins) DPPH Method Seeds Dys fter verison Skins 5 Dys fter verison FIGURE 6 Evolution of the ntioxidnt cpcity from seeds nd skins mesured y the DPPH method during the ripening of two red grpe vrieties for the sme smpling dy. Different letters show significnt differences etween mens ccording to the Duncn test (α =.5); Error rs indicte stndrd devition. (O) Tint Roriz; ( ) Tourig Ncionl TABLE 2 Correltion coefficients etween the levels of different pronthocynidin frctions nd their ntioxidnt cpcity from seeds nd skins during the mturtion of Tourig Ncionl nd Tint Roriz grpe vrieties. Correltion coefficient (djusted R 2 ) Antioxidnt methods Pronthocynidin frctions DPPH ABTS Tint Roriz Monomeric Seeds Oligomeric Polymeric Monomeric Skins Oligomeric Polymeric Tourig Ncionl Monomeric Seeds Oligomeric Polymeric Monomeric Skins Oligomeric Polymeric severl grpe vrieties. Thus, there is conflicting evidence in the literture out the correltion etween polyphenol content nd the ntioxidnt cpcity of grpes. Furthermore, it is importnt to consider tht there is quntittive nd qulittive chnge in the phenolic profile of the grpes during the mturtion process nd this consequently will ffect the evolution of ntioxidnt cpcity vlues. Anthocynins re considered very good ntioxidnt gents, with their high ctivity eing ttriuted to their peculir structure, nmely the oxonium ion in the C ring. The ntioxidnt functions of nthocynins hve een ttriuted to the glycone moiety, nd this ws demonstrted for cynidin nd some of its glycosides (Wng et l., 1999). The numer of sugr residues t the 3-position, the oxidtion stte of the C ring (Lpidot et l., 1999), the hydroxyltion nd methyltion pttern (Wng et l., 1997), s well s cyltion y phenolic cids (Degenhrdt et l., 2) re considered crucil fctors for the expression of ntioxidnt effects. Correltion coefficients etween the levels of individul nthocynins nd the ntioxidnt cpcity from the skins during grpe mturtion of the two grpes vrieties studied re presented in Tle 3. There ws negtive reltionship etween individul nthocynins nd ntioxidnt cpcity during grpe skin mturtion. These results were independent of the ntioxidnt cpcity method used nd the grpe vrieties studied, suggesting tht nthocynins were not the most powerful rdicl scvengers tht occur in grpe erry skins during the mturtion process. Thus, grpe skins contin other molecules, like pronthocynidins, ut perhps lso flvonols

9 Antioxidnt Cpcity nd Grpe Phenolic Compounds 222 TABLE 3 Correltion coefficients etween the concentrtion of individul nthocynins nd their ntioxidnt cpcity during the skin mturtion of Tourig Ncionl nd Tint Roriz grpe vrieties. Correltion coefficient (djusted R 2 ) Antioxidnt methods Individul nthocynins DPPH ABTS Tint Roriz Delphinidin-3-glucoside Cynidin-3-glucoside Petunidin-3-glucoside Peonidin-3-glucoside Mlvidin-3-glucoside Delphinidin-3-cetylglucoside Petunidin-3-cetylglucoside Peonidin-3-cetylglucoside Mlvidin-3-cetylglucoside Peonidin-3-p-coumroyl glucoside.5.29 Mlvidin-3-p-coumroyl glucoside Cynidin-3-p-coumroyl glucoside Petunidin-3-p-coumroyl glucoside Tourig Ncionl Delphinidin-3-glucoside.41.8 Cynidin-3-glucoside Petunidin-3-glucoside Peonidin-3-glucoside Mlvidin-3-glucoside.58.9 Delphinidin-3-cetylglucoside Petunidin-3-cetylglucoside Peonidin-3-cetylglucoside Mlvidin-3-cetylglucoside.39.8 Peonidin-3-p-coumroyl glucoside Mlvidin-3-p-coumroyl glucoside.72.9 Cynidin-3-p-coumroyl glucoside.48.8 Petunidin-3-p-coumroyl glucoside Negtive liner correltions vlues. nd phenolic cids, tht hve more powerful effect on the ntioxidnt cpcity of grpe skins during mturtion. Meyer et l. (1997) showed tht nthocynins in grpe extrcts re rther modertely relted to the inhiition of low-density lipoprotein oxidtion, which hs een ttriuted minly to totl phenol content. In ddition, Kllithrk et l. (25) reported low nd sttisticlly insignificnt correltion for the totl nthocynin content nd ntioxidnt cpcity of skin extrcts from severl Greek grpe cultivrs t hrvest. According to Ork (27), the ntioxidnt cpcity of red grpe cultivrs does not lwys hve reltionship with the presence of their nthocynin content. Despite their low contriution to the ntioxidnt power of the grpe, nthocynins hve een climed to exhiit vrious ctivities of iologicl importnce, such s n ntioxidnt nd nti-inflmmtory function (Wng et l., 1999) nd peroxynitrile scvenging (Tsud et l., 2). CONCLUSIONS The ntioxidnt cpcity of oth Portuguese grpe vrieties studied showed tendency to decrese in ll the grpe erry frctions studied during grpe mturtion. A similr tendency ws oserved for the different pronthocynidin frction contents nlysed in the skins nd seeds. These tendencies were prticulrly more highly pronounced in the first five weeks fter vérison. At the sme time, our results show tht seeds re n importnt source of pronthocynidins nd consequently present high ntioxidnt cpcity with respect to the grpe erry skin. As for the different individul monomeric nthocynins quntified during grpe mturtion, continuous increse in the vlues ws generlly oserved during the ripening process. However, for some of the individul nthocynins nlysed, this continuous increse ws followed y slight stilistion or decrese in the vlues in the lst weeks of ripening. Finlly, for oth grpe vrieties studied, negtive reltionship ws otined etween individul nthocynins nd ntioxidnt cpcity during grpe skin mturtion. Menwhile, lthough the dt presented my contriute towrds n understnding of the role of grpe mturtion in the ntioxidnt cpcity of the different grpe erry frctions, these spects need to e studied in other grpe vrieties nd tking severl other fctors into ccount, such s the influence of vintge conditions nd culturl prctices. LITERATURE CITED Arts, M.J.T.J., Dlling, J.S., Voss, H.P., Henen, G.R.M.M. & Bst, A., 23. A criticl pprisl of the use of the ntioxidnt cpcity (TEAC) ssy in defining optiml ntioxidnt structures. Food Chem. 8, Bkklse, E., Yemis, O. & Aslnov, D., 25. Mjor flvn-3-ol composition nd ntioxidnt ctivity of seeds from different grpe vrieties grown in Turkey. Eur. Food Res. Technol. 221, Bkker, J. & Timerlke, C.F., The distriution of nthocynins in grpe skin extrcts of Port wine cultivrs s determined y high performnce chromtogrphy. J. Sci. Food Agric. 36, Beecher, G.R., 23. Overview of dietry flvonoids: nomenclture, occurrence nd intke. J. Nutr. 133, Bogs, J., Downey, M.O., Hrvey, J.S., Ashton, A.R., Tnner, G.J. & Roinson, S.P., 25. Pronthocynidin synthesis nd expression of genes encoding leuconthocynidin reductse nd nthocynidin reductse in developing grpe erries nd grpevine leves. Plnt Physiol. 139, Bordig, M., Trvgli, F., Loctelli, M., Coïsson, J.D. & Arlorio, M., 211. Chrcteristion of polymeric skin nd seed pronthocynidins during ripening in six Vitis vinifer L. cv. Food Chem. 127, Bozn, B., Tosun, G. & Özcn, D., 28. Study of polyphenol content in the seeds of red grpe (Vitis vinifer L.) vrieties cultivted in Turkey nd their ntirdicl ctivity. Food Chem. 19, Brnd-Willims, W., Cuvelier, M.E. & Berset, C., Use of free rdicl method to evlute ntioxidnt ctivity. Food Sci. Technol. 28, Breks, A.P., Tkeok, G.R., Hidlgo, M.B., Vilches, A., Vsse, J. & Rmming, D.W., 21. Antioxidnt ctivity nd phenolic content of 16 risin grpe (Vitis vinifer L.) cultivrs nd selections. Food Chem. 121,

10 223 Antioxidnt Cpcity nd Grpe Phenolic Compounds Cnls, R., Lludy, M.C., Vlls, J., Cnls, J.M. & Zmor, F., 25. Influence of ethnol concentrtion on the extrction of color nd phenolic compounds from the skin nd seeds of temprnillo grpes t different stges of ripening. J. Agric. Food Chem. 53, Cntos, E., Espiän, J.C. & Tomás-Brerán, F.A., 22. Vrietl differences mong the polyphenol profiles of seven tle grpes cultivrs studied y LC-DAD-MS-MS. J. Agric. Food Chem. 5, Cronneu, A. & Chmpgnol, F., Nouveux systèmes de culture integré du vignole. Progrmme AIR-3-CT 93. Cheynier, V. & Rigud, J., HPLC seprtion nd chrcteriztion of flvonols in the skins of Vitis vinifer vr. Cinsult. Am. J. Enol. Vitic. 37, Cheynier, V., Prieur, C., Guyot, S., Rigud, J. & Moutounet, M., The structures of tnnin in grpe nd wines nd their interctions with proteins. In: Wtkins, T.R. (ed). Wine nutritionl nd therpeutic enefits. ACS Symposium Series 661. Americn Chemicl Society, Wshington DC. pp Dlls, C. & Lureno, O., Effects of ph, sulphur dioxide, lcohol content, temperture nd storge time on colour composition of young Portuguese red tle wine. J. Sci. Food Agric. 65, Degenhrdt, A., Knpp, H. & Winterhlter, P., 2. Seprtion nd purifiction of nthocynins y high-speed countercurrent chromtogrphy nd screening for ntioxidnt ctivity. J. Agric. Food Chem. 48, Downey, M.O., Hrvey, J.S. & Roinson, S.P., 23. Anlysis of tnnins in seeds nd skins of Shirz grpes throughout erry development. Aust. J. Grpe Wine Res. 9, Férnndez-Lopez, J.A., Almel, L., Muñoz, J.A., Hidlgo, V. & Crreño, J., Dependence etween colour nd individul nthocynin content in ripening grpes. Food Res. Int. 31, Fournnd, D., Vicens, A., Sidhoum, L., Souquet, J.-M., Moutounet, M. & Cheynier, V., 26. Accumultion nd extrctility of grpe skin tnnins nd nthocynins t different dvnced physiologicl stges. J. Agric. Food Chem. 54, Fuleki, T. & Ricrdo-d-Silv, J.M., Ctechin nd procynidin composition of seeds from grpe cultivrs grown in Ontrio. J. Agric. Food Chem. 45, Ghiselli, A., Nrdini, M., Bldi, A. & Scccini, C., Antioxidnt ctivity of different phenolic frctions seprted from Itlin red wine. J. Agric. Food Chem. 46, Glories, Y., Sustnces responsile for stringency, itterness nd wine colour. J. Int. Sci. Vigne Vin 33, Gómez-Alonso, S., Fernández-González, M., Men, A., Mrtínez, J. & Grcí-Romero E., 27. Anthocynin profile of Spnish Vitis vinífer L. red grpe vrieties in dnger of extinction. Aust. J. Grpe Wine Res. 13, Hnlin, R.L. & Downey, M.O., 29. Condensed tnnin ccumultion nd composition in skin of Shirz nd Cernet Suvignon grpes during erry development. Am. J. Enol. Vitic. 6, Jordão, A.M., Ricrdo-d-Silv, J.M. & Lureno, O., Evolution of nthocynins during grpe mturtion of two vrieties (Vitis vinifer L.), Cstelão Frncês nd Tourig Ncionl. Vitis 37, Jordão, A.M., Ricrdo-d-Silv, J.M. & Lureno, O., Influênci d reg n composição fenólic ds uvs tints d cst Tourig Frnces (Vitis vinifer L.). J. Food 2, Jordão, A.M., Ricrdo-d-Silv, J.M. & Lureno, O., 21. Evolution of ctechin nd procynidin composition during grpe mturtion of two vrieties (Vitis vinifer L.) Cstelão Frncês nd Tourig Frnces. Am. J. Enol. Vitic. 52, Jordão, A.M., Ricrdo-d-Silv, J.M. & Lureno, O., 21. Evolution of pronthocynidins in unch stems during erry development (Vitis vinifer L.). Vitis 4, Kllithrk, S., Mohdly, A.A., Mkris, D.P. & Kefls, P., 25. Determintion of mjor nthocynin pigments in Hellenic ntive grpe vrieties (Vitis vinifer sp.): ssocition with ntirdicl ctivity. J. Food Comp. Anl. 18, Kennedy, J.A., Hysk, Y., Vidl, S., Wters, E.J. & Jones, G.P., 21. Composition of grpe skin pronthocynidins t different stges of erry development. J. Agric. Food Chem. 49, Kennedy, J.A., Mtthews, M.A. & Wterhouse, A.L., 2. Chnges in grpe seed polyphenols during fruit ripening. Phytochemistry 55, Kennedy, J.A., Mtthews, M.A. & Wterhouse, A.L., 22. Effect of mturity nd vine wter sttus on grpe skin nd wine flvonoids. Am. J. Enol. Vitic. 53, Kennedy, J.A., Troup, G.J., Pilrow, J.R., Hutton, D.R., Hewitt, D., Hunter, C.R., Ristic, R., Ilnd, P. & Jones, G.P., 2. Development of seed polyphenols in erries from Vitis vinifer L. cv. Shirz. Aust. J. Grpe Wine Res. 6, Lchmn, J., Šulc, M. & Schill, M., 27. Comprison of the totl ntioxidnt sttus of Bohemin wines during the wine-mking process. Food Chem. 13, Lpidot, T., Hrel, S., Akiri, B., Grnit, R. & Knner, J., ph-dependent forms of red wine nthocynins s ntioxidnts. J. Agric. Food Chem. 47, Mteus, N., Mchdo, J.M. & De Freits, V., 22. Development chnges of nthocynins in Vitis vinifer grpes grown in the Douro Vlley nd concentrtion in respective wines. J. Sci. Food Agric. 82, Meyer, A.S., Yi, O.S., Person, D.A., Wterhouse, A.L. & Frnkel, E.N., Inhiition of humn low-density lipoprotein oxidtion in reltion to composition of phenolic ntioxidnts in grpes (Vitis vinifer). J. Agric. Food. Chem. 45, Mulincci, N., Sntmri, A.R., Giccherini, C., Innocenti, M., Vllett A., Ciolfi, G. & Psqu, G., 28. Anthocynins nd flvn-3-ols from grpes nd wines of Vitis vinifer cv. Cesnese d Affile. Nt. Prod. Res. 22, Ó-Mrques, J., Reguing, R., Lureno, O. & Ricrdo-d-Silv, J.M., 25. Chnges in grpe seeds, skin nd pulp condensed tnnins during erry ripening: effect of fruit pruning. Ciênci Téc. Vitiv. 2, OIV, 199. Recueil des méthodes interntionles d nlyse des vins et moûts. Ed. Officielle, Pris. Ork, H.H., 27. Totl ntioxidnt ctivities, phenolics, nthocynins, polyphenoloxidde ctivities of selected red grpe cultivrs nd their correltions. Sci. Hortic. 111, Poudel, P.R., Tmur, H., Ktok, I. & Mochiok, R., 28. Phenolic compounds nd ntioxidnt ctivities of skins nd seeds of five wild grpes nd two hyrids ntive to Jpn. J. Food Comp. Anl. 21, Re, R., Pellegrini, N., Proteggente, A., Pnnl, A., Yng, M. & Rice-Evns, C., Antioxidnt ctivity pplying n improved ABTS rdicl ction decoloriztion ssy. Free Rdic. Biol. Medi. 26, Riéreu-Gyon, P., The tnnins. In: Heywood, P.H. (ed). Plnt phenolics. Olivier & Boyd, Edinurgh, UK. pp Ricrdo-d-Silv, J.M., Belchior, A.P., Sprnger, M.I. & Bourzeix, M., Oligomeric procynidins of three grpevine vrieties nd wines from Portugl. Sci. Aliments 12, Ricrdo-d-Silv, J.M., Rosec, J.P., Bourzeix, M., Mourgues, J. & Moutounet, M., Dimer nd trimer procynidins in Crignn nd Mouvèdre grpes nd red wines. Vitis 31,

11 Antioxidnt Cpcity nd Grpe Phenolic Compounds 224 Roggero, J.P., Coen, S. & Rgonnet, B., High performnce liquid chromtogrphy survey on chnges in pigment content in ripening grpes of Syrh. An pproch to nthocynins metolism. Am. J. Enol. Vitic. 37, Romero-Cscles, I., Orteg-Regules, A., López-Roc, J.M., Fernández- Fernández, J.I. & Gómez-Plz, E., 25. Differences in nthocynin extrctility from grpes to wines ccording to vriety. Am. J. Enol. Vitic. 56, Ryn, J.-M. & Revill, E., 23. Anthocynin composition of Cernet Suvignon nd Temprnillo grpes t different stges of ripening. J. Agric. Food Chem. 51, Simonetti, P., Piett, P. & Testolin, G., Polyphenolic content nd totl ntioxidnt potentil of selected Itlin wines. J. Agric. Food Chem. 45, Sun, B.S., Sprnger, I. & Ricrdo-d-Silv, J.M., Extrction of grpe seed procynidins using different orgnic solvents. Proceedings of the XVIII Interntionl Conference of the Groupe Polyphenols - Polyphenols Comunictions 96, Bourdeux, Frnce. pp Sun, B.S., Lendro, C., Ricrdo-d-Silv, J.M. & Sprnger, I., Seprtion of grpe nd wine pronthocynidins ccording to their degree of polymeriztion. J. Agric. Food Chem. 46, Sun, B.S., Ricrdo-d-Silv, J.M. & Sprnger, I., Criticl fctors of vnillin ssy for ctechins nd pronthocynidins. J. Agric. Food Chem. 46, Sun, B.S., Ricrdo-d-Silv, J.M. & Sprnger, I., 21. Quntifiction of ctechins nd pronthocynidins in severl Portuguese grpevine vrieties nd red wines. Ciênci Téc. Vitiv. 16, Tsud, T., Kto, Y. & Osw, T., 2. Mechnism for the peroxynitrile scvenging ctivity y nthocynins. FEBS Letters 484, Vlero, E., Sánchez-Ferrer, A., Vrón, R. & Grcí-Crmon, F., Evolution of grpe polyphenol oxidse ctivity nd phenolic content during mturtion nd vinifiction. Vitis 28, Wng, H., Co, G. & Prior, R., Oxygen rdicl soring cpcity of nthocynins. J. Agric. Food Chem. 45, Wng, H., Nir, M.G., Strsurg, G.M., Chng, Y.C., Booren, A.M., Gry, J.I. & De Witt, D.L., Antioxidnt nd nti-inflmmtory ctivities of nthocynins nd their glycone, cyniding, from trt cherries. J. Nt. Prod. 62, Xu, C., Zhng, Y., Co, L. & Lu, J., 21. Phenolic compounds nd ntioxidnt properties of different grpe cultivrs grown in Chin. Food Chem. 119, Yng, J., Mrtinson, T.E. & Liu, R.H., 29. Phytochemicl profiles nd ntioxidnt ctivities of wine grpes. Food Chem. 116, Yokotsuk, K., Ngro, A., Nkzw, K. & Sto, M., Chnges in nthocynins in erry skins of Merlot nd Cernet Suvignon grpes grown in two soils modified with limestone or oyster shell versus ntive soil over two yers. Am. J. Enol. Vitic. 5, 1-12.