Eur Food Res Technol (2005) 220:331–340DOI 10.1007/s00217-004-1109-9

O R I G I N A L P A P E R

Mar�a Monagas · Bego�a Bartolom� ·Carmen G�mez-Cordov�s

Evolution of polyphenols in red wines from Vitis vinifera L.during aging in the bottleII. Non-anthocyanin phenolic compoundsReceived: 14 September 2004 / Published online: 21 January 2005� Springer-Verlag 2005

Abstract The evolution of non-anthocyanin phenoliccompounds (as measured by high-performance liquidchromatography) in young red wines from Vitis viniferaL. cv Tempranillo, Graciano and Cabernet Sauvignon(vintage 2000) from Navarra (Spain) was studied during26 months of aging in the bottle. Hydroxybenzoic acidsand derivatives (gallic, protocatechuic, vanillic and sy-ringic acids, and methyl and ethyl gallates), hydroxycin-namic acids and derivatives (trans-caftaric, cutaric, caf-feic and p-coumaric acids, and hexose esters of trans-p-coumaric acid), stilbenes (trans- and cis-resveratrol-3-O-glucosides, and trans- and cis-resveratrol), phenolic al-cohols and other related compounds (tyrosol and tryp-tophol), flavanols [procyanidins B1 and B2, (+)-catechinand (�)-epicatechin] and flavonols (myricetin-3-O-glu-coside, quercetin-3-O-galactoside and quercetin-3-O-glucuronide, kaempherol-3-O-glucoside, myricetin andquercetin) presented different evolution patterns duringaging, in some cases also being different depending on thegrape variety studied. The changes that occurred duringaging in the bottle were the decrease of the concentrationof trans-caftaric and cutaric acids accompanied by anincrease of trans-caffeic acid and, especially of trans-p-coumaric acid. The greater increase of trans-p-coumaricacid was also associated with the disappearance of p-coumaroyl-acylated anthocyanins that occurs during ag-ing in the bottle. Flavanols registered a major decrease,with the disappearance rate being greater for the dimericprocyanidins than for the monomeric flavanols, and theorder of the disappearance rate by variety was as follows:Tempranillo<GracianoffiCabernet Sauvignon. The lowdisappearance rate of flavanols in wines from Tem-pranillo was attributed to their low reactivity with an-thocyanins, which in turn could be associated with the

particular anthocyanin-to-flavanol ratio and with the highpH value of this variety.

Keywords Red wine · Aging in the bottle ·Hydroxybenzoic acids · Hydroxycinnamic acids ·Stilbenes · Flavanols and flavonols

Introduction

Non-anthocyanin phenolic compounds present in winemainly include hydroxybenzoic and hydroxycinnamicacids and their derivatives, stilbenes, phenolic alcoholsand flavonoid compounds such as flavonols and flavanols.Together with anthocyanins, these compounds constitutean important quality parameter of wines since they con-tribute to their organoleptic characteristics. Phenolic acidsand flavanols are largely responsible for the astringencyand bitterness of young wines [29]. Hydroxycinnamicacids and flavanols, together with flavonols, also act ascopigments of anthocyanins and participate in the colorstabilization of red wine [5, 6, 19]. Moreover, in the lastfew years phenolic compounds, especially flavonoids andstilbenes, have been recognized as being responsible forseveral beneficial physiological effects associated withred wine consumption, mostly owing to their antioxidantand anti-inflammatory properties [9, 40].

It has long been recognized that the disappearance ofmuch of the bitterness, astringency and harshness thatoccurs during red wine aging is attributed to chemicalreactions of phenolic compounds which result in theformation of new colored and uncolored oligomers andpolymers that produce changes in the sensory response.Flavanols, in particular, are involved in chemical andenzymatic oxidative browning reactions, and in interac-tions with proteins that result in haze formation [2, 30].Besides, flavanols also participate in direct and acetal-dehyde-mediated condensation reactions with antho-cyanins [37, 42] and with other flavanols [10, 34].Products resulting from the flavanol–flavanol condensa-tion reaction mediated by glyoxylic acid have also been

M. Monagas · B. Bartolom� · C. G�mez-Cordov�s ())Instituto de Fermentaciones Industriales,CSIC,Juan de la Cierva 3, 28006 Madrid, Spaine-mail: cgcordoves@ifi.csic.esTel.: +34-91-5622900Fax: +34-91-5644853

identified in wines [7, 11]. In addition, reaction-interme-diate derivatives such as vinylflavanols can be added tothe C-4/C-5 positions of anthocyanins, giving rise topyranoanthocyanins [8, 16]

Other chemical reactions involving phenolic com-pounds during wine aging include the transformation ofgallic acid to ellagic acid under oxidative conditions [43],and the hydrolysis of flavonol glycosides and tartaricesters of hydroxycinnamic acids to their correspondingfree forms [39, 45]. Recently, Schwarz et al. [35] dem-onstrated that hydroxycinnamic acids can also react withanthocyanins, resulting in pyranoanthocyanin-type pig-ments.

Despite the importance that the changes of phenoliccompounds constitute to the final quality and, therefore,to the consumer acceptance of red wine, there is not muchdata in the literature concerning the main non-anthocya-nin compounds that undergo changes during red wineaging under nonoxidative conditions (bottle), and moreimportant, how is the magnitude of these changes withrespect to the losses registered in anthocyanin concen-tration. The aim of the present work was to determine theevolution of non-anthocyanin phenolic compounds in redwine from different Vitis vinifera grape varieties duringaging in the bottle and to evaluate the overall changeswith respect to those of anthocyanins.

Materials and methods

Materials

Methyl gallate and ethyl gallate, were purchased from Extrasyn-th�se (France). (�)-Epicatechin, myricetin, quercetin, tryptophol,trans-resveratrol, and gallic, trans-caffeic, trans-p-coumaric, van-illic and protocatechuic acids were purchased from Sigma (USA).Syringic acid and tyrosol were purchased from Aldrich (Germany).cis-Resveratrol was obtained from the standard of trans-resveratrolafter exposure to UV light (340 nm) for 1 h.

Winemaking

Monovarietal young red wines made from grapes of V. vinifera cv.Tempranillo, Graciano and Cabernet Sauvignon grown in the samegeographical area (Navarra, Spain) were elaborated at the Viticul-ture and Enology Station of Navarra (EVENA), Spain (vintage2000), as described in part I of this work. Two wine samples fromeach variety were analyzed after 1.5, 3, 7, 9, 12, 19.5 and 26 monthsof bottling and storage at 13 �C and 80–85% relative humidity.

Extraction of phenolic compounds

A volume of 50 ml of wine was concentrated to 15 ml undervacuum at 30 �C and extracted three times with diethyl ether(15 ml) and three times with ethyl acetate (15 ml). The organicphases were combined and dried with anhydrous Na2SO4 for30 min. The extract was then taken to dryness under vacuum,dissolved in 2 ml methanol–water (1:1) and finally filtrated(0.45 mm) and injected (5–10 ml of wine extract) into the high-performance liquid chromatography (HPLC) column.

HPLC–diode-array detection analysis

A Waters (Milford, MA) liquid chromatography system equippedwith a 600-MS controller, a 717Plus autosampler and a 996 pho-todiode-array detector was used. Separation was performed on areversed-phase Waters Nova-Pak C18 (300 mm�3.9 mm, 4 mm)column at room temperature. A gradient consisting of solvent A[water–acetic acid, 98:2, (v/v)] and solvent B (water–acetonitrile–acetic acid, 78:20:2, (v/v)] was applied at a flow rate of 1.0 ml/minas follows: 0–80% solvent B linear from 0 to 55 min, 80–90%solvent B linear, from 55 to 57 min, 90% solvent B isocratic from57 to 70 min, 90–95% solvent B linear from 70 to 80 min, 95–100%solvent B from 80 to 90 min, followed by washing (methanol) andre-equilibration of the column from 90 to 120 min. Diode-arraydetection was performed from 220 to 380 nm. Quantification wascarried out by external standard calibration curves. Hydroxybenzoicacids, stilbenes, phenolic alcohols and other related compounds,flavanols and flavonols were quantified at 280 nm; caffeic acid andits derivatives at 340 nm; and p-coumaric acid and its derivatives at310 nm. Caffeic and p-coumaric acid derivatives, flavonol glyco-sides and stilbene glucosides were quantified by the calibrationcurve of their respective free form. Monomeric and dimeric flavan-3-ols were quantified using the (�)-epicatechin calibration curve.

Statistical analysis

Analysis of variance (ANOVA) was performed using the PC soft-ware package Statgraphics Plus 2.1 (Graphics Software Systems,Rockwille, MD, USA).

Results and discussion

Non-flavonoid and flavonoid phenolic compounds wereidentified in the wines from V. vinifera L. cv Tempranillo,Graciano and Cabernet Sauvignon, according to themethod of Monagas et al. [22]. Figures 1 and 2, respec-tively, illustrate their evolution during the 26 months ofaging in the bottle. Similarly, Fig. 3 shows the evolutionof the compounds grouped (i.e., S) as hydroxybenzoicacids and their derivatives, hydroxycinnamic acids andtheir derivatives, stilbenes, phenolic alcohols and otherrelated compounds, flavanols and flavonols. Some datacorresponding to certain compounds are missing from thegraphs owing to the poor resolution between chromato-graphic peaks at some relative compound concentrations.Finally, Table 1 presents the probabilities associated withthe statistical significance for the factors “time” and“variety” resulting from the two-way ANOVA analysis.

Evolution of non-flavonoid phenolic compounds

Hydroxybenzoic acids and their derivatives

The evolution of gallic, protocatechuic, vanillic and sy-ringic acids, and of methyl and ethyl gallates during agingin the bottle is presented in Fig. 1a. These compoundsexhibited different trends according to the grape variety.For instance, gallic acid and ethyl gallate presented aslight increase in Graciano and Cabernet Sauvignonwines, whereas a decrease was registered in Tempranillowine (Fig. 1a). However, as a group (i.e., S), the totalcontent of hydroxybenzoic acids and their derivatives

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Fig. 1 Evolution of individual non-flavonoid phenolic compoundsduring aging in the bottle. a Hydroxybenzoic acids and their

derivatives; b hydroxycinnamic acids and their derivatives; c stil-benes; d phenolic alcohols and other related compounds

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Fig. 1 (continued)

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Fig. 2 Evolution of flavonoid phenolic compounds during aging in the bottle. a Flavanols; b flavonols

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(Fig. 3a) did not show significant differences (p>0.05)with aging time in the bottle, with the differences beingsignificant (p<0.05) only for the variety factor (Table 1).

There is not much literature data concerning the evo-lution profile of hydroxybenzoic acids during wine aging.Gallic acid, the only native hydroxybenzoic acid of V.vinifera grapes, is extracted from the seeds during themaceration and fermentation processes, as confirmed byZou et al. [46]. Its esterification with ethanol and meth-anol also appears to occur during fermentation, as nosignificant changes were registered for these esters underthe aging conditions employed, even in presence of highlevels of gallic acid. The same situation can also beconsidered for the other hydroxybenzoic acids (protocat-echuic, vanillic and syringic acids) studied in the differentwines. Revilla and Gonz�lez-San Jos� [27] also found nosignificant changes in the total (S) concentration of gallic,protocatechuic and syringic acids in Tempranillo wineselaborated from vinifications treated with pectolitic en-zymes during 24 months of bottle-aging.

Hydroxycinnamic acids and their derivatives

The evolution of trans-caftaric, trans-cutaric, trans-caf-feic and trans-p-coumaric acids, and of hexose esters oftrans-p-coumaric acid is presented in Fig. 1b. In general,a decrease in the concentration of trans-caftaric and cu-taric acids was observed together with an increase of thecorresponding free acid forms, trans-caffeic and p-cou-maric acids (Fig. 1b). For trans-caffeic acid, the incre-ment in concentration was lower and mainly occurredduring the first 3 months of aging, being stable during therest of the aging period. However, in the case of trans-p-coumaric acid a progressive and greater increase wasregistered during the whole aging process. The mostmarked changes for both tartaric esters took place in thewine from Tempranillo, in which a linear increase oftrans-p-coumaric acid was registered as the aging timeprogressed. Other derivatives, such as the hexose esters oftrans-p-coumaric acid, only showed slight fluctuationsduring the 26 months of aging in the bottle (Fig. 1b).

Other authors have also observed changes in the hy-droxycinnamates during wine aging, although certain ex-ceptions have been reported depending on fermentationand aging conditions. According to Somers et al. [39], therapid appearance of free hydroxycinnamic acids during thefirst hours of fermentation of a Chardonnay must be due tothe enzymatic hydrolysis of tartaric esters. In the sameway, these authors found that the hydrolysis reactions canalso take place during wine aging in stainless steel tanks orin oak barrels. However, during the aging of sparklingwines over yeast lees, no significant changes have beenregistered in the hydroxycinnamate composition [26]. Onthe other hand, Zafrilla et al. [45] only found a slightdecrease in the concentration of trans-caftaric acid inMonastrell wine after 7 months of aging in the bottle at20 �C. Schwarz et al. [36] described similar behavior fortrans-caftaric acid in wines from Pinotage (vintages 1998–

2001), finding relatively constant levels of trans-caffeicacid in wines from different vintages. In contrast, G�mez-Plaza et al. [12] reported a large decrease of both trans-caftaric and cutaric acids during 12 months of bottle-agingat temperatures between 10 and 35 �C.

The results found in this study indicate that the in-crease in free acids, especially in trans-p-coumaric acid,not only originated from the hydrolysis of the respectivetartaric esters, since the loss of trans-cutaric acid was0.63 mg/l for Tempranillo, 0.12 mg/l for Graciano and0.23 mg/l for Cabernet Sauvignon, whereas the increasein the concentration of trans-p-coumaric acid was1.24 mg/l for Tempranillo, 0.36 mg/l for Graciano and0.43 mg/l for Cabernet Sauvignon during 19.5 months ofaging. This indicates an increase of 0.61, 0.24 and0.20 mg/l, respectively, over the content expected to arisefrom the simple ester hydrolysis, assuming the same re-sponse factor between conjugated and free forms. Anadditional source of trans-p-coumaric acid is most likelyto come from the hydrolysis of p-coumaroyl-acylatedanthocyanins during aging in the bottle (results presentedin part I of this work), confirming that the disappearanceof acylated anthocyanins during wine aging is in part dueto the hydrolysis of the acylated group. Considering thatanthocyanins in Graciano and Cabernet Sauvignon winesexhibited a faster disappearance rate than in Tempranillo(results presented in part I of this work), the hydrolysis ofp-coumaroyl-acylated anthocyanins seems to be a moreimportant source of free p-coumaric acid than that of thep-coumaroyltartaric ester in the former two varieties,whereas in Tempranillo the opposite situation seems moreimportant, as described earlier (Fig. 1a). Finally, the totalconcentration of the group (i.e., S) of hydroxycinnamicacids and their derivatives (Fig. 3b) did not present sig-nificant changes (p>0.05) with aging time in the bottleowing to the interconversion of the different forms.However, significant differences (p<0.05) were found forthe grape variety employed (Table 1).

Stilbenes

The evolution of cis- and trans-resveratrol and of theirrespective glucosides during aging in the bottle did notfollow defined trends, although the trans forms seem to

Table 1 Probabilities associated with the factors “time” and “va-riety” from the two-way analysis of variance analysis.

Group p value

Time Variety

Non-flavonoidsHydroxybenzoic acids and their derivatives 0.9290 0.0007Hydroxycinnamic acids and their derivatives 0.3026 0.0001Stilbenes 0.0653 0.0000Phenolic alcohols and other related compounds 0.0073 0.0000FlavonoidsFlavanols 0.0002 0.0000Flavonols 0.0481 0.0000

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Fig. 3 Evolution of the different groups (i.e., S) of non-anthocy-anin phenolic compounds during aging in the bottle. a Hydroxy-benzoic acids and their derivatives; b hydroxycinnamic acids and

their derivatives; c stilbenes; d phenolic alcohols and other relatedcompounds; e flavanols; f flavonols

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decrease during the first months of aging (Fig. 1c). Cis-and trans-resveratrol were only detected at trace levels inCabernet Sauvignon wines. Considering the stilbenes as agroup (i.e., S) (Fig. 3c), the ANOVA only reflected sig-nificant differences (p<0.05) depending on the varietyfactor but not in relation to the time factor (Table 1).

Although the evolution of stilbenes during vinificationhas been studied [17, 25], there is not much literature dataconcerning the evolution of these compounds in postfer-mentation stages (i.e., aging in the barrel or the bottle).The hydrolysis of glucosides give rise to aglucones, areaction known to occur during the winemaking process[17], and is likely to occur during aging in the bottlealthough in to a lesser extent.

Phenolic alcohols and other related compounds

The concentration of the phenolic alcohol tyrosol and ofthe non-phenolic alcohol tryptophol, compounds formedduring yeast fermentation from tyrosine [3-(4-hydrox-yphenyl)-alanine] and tryptophan [2-amino-3-(3-indolyl)-propionic acid], respectively [1, 13] presented a slightdecrease during the first months of aging in the bottle(Fig. 1d). Tryptophol could not be quantified in Gracianowines owing to its coelution with an unknown compound[22]. As a group (i.e, S) (Fig. 3d), the changes registeredwere significant (p<0.05) in relation to both the time andthe variety factors (Table 1). According to the studies ofSapis and Rib�reau-Gayon [33], tryptophol is practicallyabsent in aged wines, while the tyrosol concentrationseems to remain relatively constant during aging.

Evolution of flavonoid phenolic compounds

Flavanols

(+)-Catechin, (�)-epicatechin, as well as the procyanidinsdimers B1 and B2, progressively decreased during the26 months of aging in the bottle (Fig. 2a). As a group (i.e,S) (Fig. 3e), the ANOVA analysis revealed significantdifferences (p<0.05) depending on both the time and thevariety factors (Table 1). Other authors have also foundlosses of flavanols during aging in the bottle [12, 27] andin oak barrels [24].

The decrease in the concentration of the different fla-vanols during aging in the bottle, which is associated withtheir participation in several chemical reactions, followeda second-order polynomial trend and not an exponentialtrend (first-order kinetics), as observed for the antho-cyanins (results reported in part I of this work). However,in model solutions containing acetaldehyde or no acetal-dehyde and incubated at 22 �C, Dallas et al. [4] reportedfirst-order kinetics for modeling the disappearance offlavanols. Similar results have been found in model so-lutions containing malvidin-3-glucoside and procyanidinB2-30-O-gallate, incubated at 30 �C [31]. In the case un-der study, the temperature (13 �C) may have affected the

reactivity of the flavanols, influencing their rate of dis-appearance and the evolution trend during aging in thebottle.

Table 2 summarizes the time required for a 25% re-duction of the initial concentration of the different mo-nomeric flavanols and dimeric procyanidins in the winesfrom the three grape varieties studied. Dimeric pro-cyanidins presented a much faster disappearance rate (lesstime) than monomeric flavanols in the different wines(threefold higher in Tempranillo and Graciano, and two-fold in Cabernet Sauvignon). In Tempranillo wine, thedifferent flavanols presented the slowest disappearancerate independently of their chemical structure. CabernetSauvignon wine presented a slightly superior disappear-ance rate of monomeric flavanols than Graciano wine,whereas for the dimers the disappearance rate was prac-tically identical for both varieties (Table 2). The highdisappearance rate of dimeric procyanidins in comparisonwith that of momeric flavanols is mainly attributed to theacid-catalyzed C–C bond-breaking of procyanidins duringaging [14, 31].

The total loss of flavanols at the end of the agingperiod was 35% for Tempranillo, 40% for Graciano and52% for Cabernet Sauvignon, approximately 2 timeslower than that registered for anthocyanins (results re-ported in part I of this work). Wulf and Nagel [44] foundthat the loss of monomeric flavanols was approximately4 times lower than that of anthocyanins in wines fromMerlot and Cabernet Sauvignon after 200 days of aging at22 �C. Similarly, Sun et al. [41] recently reported lowerlosses of flavanols in comparison with losses of antho-cyanins in 5–8-year-old wines aged in the bottle. Salas etal. [31] also described similar results in model solutionscontaining malvidin-3-glucoside and procyanidin B2-30-O-gallate (pH 3.8, 30 �C).

Flavonols

The evolution of myricetin-3-O-glucoside, quercetin-3-O-galactoside, quercetin-3-O-glucuronide, kaempferol-3-O-glucoside, myricetin and quercetin is presented in Fig. 2b.Quercetin-3-O-galactoside was not given in Tempranilloand Graciano wines owing its low values. During aging inthe bottle, a slight decrease in the concentration of theglycosilated forms together with a slight increase of thecorresponding aglycones was registered, possibly owing

Table 2 Time (months) required for a 25% reduction of the initialflavanol concentration.

Compound Tempranillo Graciano CabernetSauvignon

Monomeric flavanols(+)-Catechin 13.1 8.6 6.1(�)-Epicatechin 12.0 9.7 5.5Dimeric flavanolsProcyanidin B1 4.1 2.8 2.9Procyanidin B2 4.9 2.9 2.9

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to hydrolysis reactions (Fig. 2b). Zafrilla et al. [45] alsoreported this behavior for flavonols in wines fromMonastrell stored for 7 months at 20 �C. Considering theaglycones, myricetin presented the major increment after12 months of aging in the bottle, whereas the incrementfor quercetin was observed during the first months ofaging. The total concentration of the group (i.e., S) wassignificant (p<0.05) depending on the time and varietyfactors (Fig. 3f, Table 1).

Evolution of the pigment-to-copigment ratioduring wine aging

Anthocyanins and flavanols were the group of compoundsthat presented the major losses during aging in the bottle.In Tempranillo wine both groups of compounds exhibitedslower kinetics than in Graciano and Cabernet Sauvignonwines, which also presented very similar kinetics profiles.The loss of anthocyanins and flavanols during wine agingis mainly attributed to their participation in numerouschemical reactions, as described in the Introduction.Considering the nonoxidative conditions present in thebottle, the anthocyanin–flavanol direct condensation re-action, together with hydrolytic and degradative reactionsin the case of anthocyanins [31, 32], are most likely to beresponsible for the decrease in the concentration of bothtypes of compounds during aging in the bottle. Acetal-dehyde and glyoxylic acid mediated condensation reac-tions are less likely to occur during bottle-aging as theyrequire the presence of oxygen to convert ethanol intoacetaldehyde and tartaric acid into glyoxylic acid, re-spectively. Finally, anthocyanin losses do not appear to beattributed to pyranoanthocyanin formation under the ag-ing conditions employed in this study.

Evidence for the occurrence of the anthocyanin–fla-vanol direct condensation reaction was the detection ofthe dimer malvidin-3-glucoside-(epi)catechin [21]. How-ever, the detection of polymeric pigments resulting fromthis reaction is very difficult to achieve owing to the lackof resolution of reversed-phase HPLC. The increment ofthe base line (hump) as well as the increase of a very widepeak that elutes during the washing phase of the columnwith methanol indicate the formation of polymeric pig-ments during wine aging [15, 23]. Both indicators havebeen observed in the wines studied.

Under identical fermentation (temperature, yeast strainand yeast pitching rate), and aging conditions (13 �C; 80–85% relative humidity), as in the case under study, theprogress of the anthocyanin–flavanol direct condensationreaction mainly depends on the anthocyanin-to-flavanolratio and on the pH [3, 28], factors which are conditionedby the initial composition and, therefore, by the grapevariety. The initial anthocyanin-to-flavanol ratio of thewines studied (8.9 for Tempranillo, 3.2 for Graciano and3.9 for Cabernet Sauvignon) revealed a substantiallyhigher proportion of anthocyanins by unit of flavanol inTempranillo wine in comparison with Graciano andCabernet Sauvignon wines, which presented a similar

ratio. Monagas et al. [20] demonstrated that the lowproportion of flavanols in Tempranillo wine arose fromthe low content of monomeric flavanols and oligomericprocyanidins present in the seeds from this grape variety.Figure 4 shows the evolution of the ratio of “disappearedanthocyanins-to-disappeared flavanols” during aging, aparameter which can be taken as a measure of the reac-tivity associated with the wines from each variety. Ini-tially the three wines presented very close values, indi-cating that from 1.5 to 3 months of aging both groups ofcompounds disappeared in the same proportion in thedifferent wines. From 3 to 7 months of aging, a veryreactive phase was observed, characterized by an incre-ment in the disappeared anthocyanins-to-disappearedflavanols ratio, which then remained relatively stable upto 12 months of bottle-aging. Later on (12–26 months), itwas observed that in Tempranillo wine a higher amountof anthocyanins was lost for each unit of flavanol incomparison with the wines from Graciano and CabernetSauvignon. These results revealed that the initial unbal-anced anthocyanin-to-flavanol ratio of Tempranillo winecould affect the reactivity of anthocyanins and flavanols,finally explaining their slower disappearance kinetics incomparison with the kinetics in Graciano and CabernetSauvignon wines. Other authors have also found thatwines containing a low concentration of non-anthocyaninphenols or low levels of flavanols exhibited slower ratesof anthocyanin disappearance [24, 38]. Similarly, Rib�r-eau-Gayon [28] has described the importance of a bal-anced proportion of anthocyanins and flavanols for thecorrect progress of condensation reactions during aging.

On the other hand, the differences in reactivity be-tween wines could also be related to the high pH valuepresented in Tempranillo wine (4.3) in comparison withthat in Graciano (3.5) and Cabernet Sauvignon (3.6)wines. Other authors [18, 24] have also reported the lowacidity of Tempranillo wines. An acidic pH assures ahigher proportion of anthocyanins in flavylium form and,therefore, a higher amount of anthocyanins reactive spe-cies in electrophilic form. In addition, an acidic pH is

Fig. 4 Evolution of the ratio “disappeared anthocyanins-to-disap-peared flavanols” during aging in the bottle.

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highly important to promote the C–C bond cleavage ofprocyanidins from which intermediate-sized carbocationsare liberated to participate in anthocyanin–flavanoland flavanol–flavanol direct condensation reactions [14,31].

Conclusion

In conclusion, this paper reports valuable data about theevolution of non-anthocyanin phenolic compounds duringaging in the bottle. Different evolution patterns wereobserved among the diverse groups of non-anthocyaninphenolic compounds during aging, in some cases alsobeing different depending on the grape variety studied.The increase found in free hydroxycinnamic acids, espe-cially in trans-p-coumaric acid, during aging in the bottlewas associated with the disappearance of p-coumaroyl-acylated anthocyanins (results reported in part I of thiswork). This fact is very important considering the rolethat hydroxycinnamic acids play as anthocyanin copig-ments and in the stabilization of wine color. As in the caseof anthocyanins, the disappearance rate of flavanols dur-ing aging was largely influenced by the grape variety. Theslow rate of disappearance of both anthocyanins andflavanols in wines from the Tempranillo grape variety ismost likely to be due to the low reactivity of both com-pounds in this wine, which in turn could be associatedwith its particular anthocyanin-to-flavanol ratio and withits high pH value. Parts I and II of this work togetherprovide an overall view of the evolution of anthocyaninand non-anthocyanin phenolics during wine aging in thebottle. Finally, the influence that the changes registered inthese compounds during aging in the bottle may have onthe color characteristics of the wines is currently understudy.

Acknowledgements The authors are grateful to Juli�n Suberviola(EVENA, Navarra, Spain) for providing the wine samples and toPedro J. Martn (CSIC, Madrid, Spain) for his kind assistance withthe graphs. The authors also thank the Agencia Espaola deCooperaci�n International for a MUTIS predoctoral scholarship toM.M. and the Spanish Comisi�n Interministerial de Ciencia yTecnologa (projects AGL2000-1427-C02-02 and AGL2003-07394-C02-02) for funding.

References

1. Barcenilla J, Estrella I, G�mez-Cordov�s C, Hern�ndez T,Hern�ndez L (1989) Food Chem 31:177–187

2. Cheynier V, Ricardo da Silva JM (1991) J Agric Food Chem39:1047–1049

3. Cheynier V (2001) Polyph�nols Actualit�s 21:3–104. Dallas C, Hip�lito-Reis P, Ricardo da Silva JM, Laureano O

(2003) Am J Enol Vitic 54:119–1235. Eiro MJ, Heinonen M (2002) J Agric Food Chem 50:7461–

74666. Escribano Bail�n T, Dangles O, Brouillard R (1996) Phyto-

chemistry 41:1583–15927. Es-Safi N, Le Guernev� C, Labarde B, Fulcrand H, Cheynier V,

Moutounet M (1999) Tetrahedron Lett 40:5869–5872

8. Francia-Aricha EM, Guerra MT, Rivas-Gonzalo JC, Santos-Buelga V (1997) J Agric Food Chem 45:2262–2266

9. Frankel E, Kanner J, German JB, Parks E, Kinsella JE (1993)Lancet 341:454–457

10. Fulcrand H, Doco T, Es-Safi N-E, Cheynier V, Moutounet M(1996) J Chromatogr A 752:85–91

11. Fulcrand H, Cheynier V, Oszmianski J, Moutounet M (1997)Phytochemistry 46:223–227

12. G�mez-Plaza E, Gil-Muoz R, L�pez-Roca JM, Martnez A(2000) J Agric Food Chem 48:736–741

13. Gil C, G�mez-Cordov�s C (1986) Food Chem 22:59–6214. Haslam E (1980) Phytochemistry 16:1625–167015. Hayasaka Y, Kennedy JA (2003) Aust J Grape Wine Res

9:210–22016. Mateus N, Silva AMS, Santos-Buelga C, Rivas-Gonzalo JC, De

Freitas V (2002) J Agric Food Chem 50:2110–211617. Mattivi F, Reniero F, Korhammer S (1995) J Agric Food Chem

43:1820–182318. May�n M, M�rida J, Medina M (1995) Am J Enol Vitic

46:255–26119. Mistry TV, Cai Y, Lilley TH, Haslam E (1991) J Chem Soc

(Perkin Trans II) 1287–129620. Monagas M, G�mez-Cordov�s C, Bartolom� B, Laureano O,

Ricardo da Silva JM (2003) J Agric Food Chem 51:6475–648121. Monagas M, Nfflez V, Bartolom� B, G�mez-Cordoves C

(2003) Am J Enol Vitic 54:163–16922. Monagas M, Su�rez R, G�mez-Cordov�s C, Bartolom� B

(2004) Submitted23. Peng Z, Iland PG, Oberholster A, Sefton MA, Waters EJ (2002)

Aust J Grape Wine Res 8:70–7524. P�rez-Magario S, Gonz�lez-San Jos� ML (2004) J Agric Food

Chem 52:1181–118925. Pezet R, Cuenat P (1996) Am J Enol Vitic 47:287–29026. Pozo-Bay�n MA, Hern�ndez MT, Martn-�lvarez PJ, Polo MC

(2003) J Agric Food Chem 51:2089–209527. Revilla I, Gonz�lez-San Jos� ML (2003) Food Chem 80:205–

21428. Rib�reau-Gayon P (1982) In: Markakis P (ed) Anthocyanins as

food colors. Academic Press, New York, pp 209–24429. Ribichaud JL, Noble AC (1990) J Sci Food Agric 53:343–35330. Ricardo da Silva JM, Cheynier V, Souquet JM, Moutounet M,

Cabanis JC, Bourzeix M (1991) J Sci Food Agric 57:111–12531. Salas E, Fulcrand H, Meude E, Cheynier V (2003) J Agric Food

Chem 51:7951–796132. Santos-Buelga C, Francia-Aricha EM, De Pascual-Teresa S,

Rivas-Gonzalo JC Eur Food Res Technol 209:411–41533. Sapis JC, Rib�reau-Gayon P (1969) Ann Technol Agric

18:221–22934. Saucier C, Little D, Glories Y (1997) Am J Enol Vitic 48:370–

37335. Schwarz M, Wabnitz TC, Winterhalter P (2003) J Agric Food

Chem 51:3682–368736. Schwarz M, Hofmann G, Winterhalter P (2004) J Agric Food

Chem 52:498–50437. Somers TC (1971) Phytochemistry 10:2175–218638. Somers TC, Evans ME (1986) Vitis 25:31–3939. Somers TC, V�rette E, Pocock KF (1987) J Sci Food Agric

40:67–7840. Stoclet JC, Kleschyov A, Andriambeloson E, Dielbolt M, An-

driantsitohaina R (1999) J Physiol Pharmacol 50:535–54041. Sun B, Spranger I, Concei ¼o L, Belchior P (2003) In: First

International Conference on Polyphenols and Health. Vichy(France), pp 198

42. Timberlake CF, Bridle P (1976) Am J Enol Vitic 27:97–10543. Tulyathan V, Boulton R, Singleton VL (1989) J Agric Food

Chem 37:844–84944. Wulf LW, Nagel CW (1980) J Food Sci 45:479–48445. Zafrilla P, Morillas J, Mulero J, Cayuela JM, Martnez-Cach�

A, Pardo F, L�pez Nicol�s JM (2003) J Agric Food Chem51:4694–4700

46. Zou H, Kilmartin PA, Inglis MJ, Frost A (2002) Aust J GrapeWine Res 8:163–174

340