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Original Article

Antioxidant activity and phenolic content of Portuguese wine aged brandies

Sara Canas a,�, Vera Casanova b, A. Pedro Belchior a

a Estac- ao Vitivinıcola Nacional, Quinta d’Almoinha, Dois Portos 2565-191, Portugalb Departamento de Agro-Industrias e Agronomia Tropical, Instituto Superior de Agronomia, Tapada da Ajuda, Lisboa 1349-017, Portugal

a r t i c l e i n f o

Article history:

Received 18 February 2008

Received in revised form

26 June 2008

Accepted 3 July 2008

Keywords:

Antioxidant activity

Phenolic compounds

Aged brandies

Botanical species

Toasting level

Ageing time

Food composition

a b s t r a c t

The antioxidant activity and the phenolic composition (total polyphenols, phenolic acids and

hydrolyzable tannins) of Portuguese brandies were analyzed with regard to the kind of wood (chestnut

and Limousin oak), the barrels toasting level (light, medium and heavy), and the ageing time (first 4

years of ageing). In the experimental conditions, the botanical species affects significantly the

antioxidant activity of the brandies. Those aged in chestnut wood present higher antioxidant activity

than those aged in Limousin oak, which confirms the remarkable quality of the former. The antioxidant

activity of the aged brandies is not significantly influenced by the wood toasting level, owing to the

strong variability induced by this cooperage operation. During the ageing period, the antioxidant

activity of the brandies undergoes a highly significant increase. The most significant correlations

between antioxidant activity and phenolic composition of brandies were found for phenolic acids,

mainly for gallic acid and ellagic acid.

& 2008 Elsevier Inc. All rights reserved.

1. Introduction

Several studies show that the consumption of food andbeverages with high phenolic content correlates with reducedcardiovascular and neurodegenerative diseases and cancer mor-tality (Umar et al., 2003). The ‘‘French Paradox’’ demonstrates thatfor apparently the same level of risk factors, cardiovascularmortality rate is lower in France than in the European Northerncountries (Renaud and De Lorgeril, 1992). Furthermore, amongthe French regions, this phenomenon is particularly evident in thesouthwest France, a region where people do not drink more winethan elsewhere, but often drink Armagnac (Tunstall-Pedoe et al.,1999).

In fact, Armagnac, Cognac and other aged brandies are rich inphenolic compounds due to their maturation in wooden barrels(Viriot et al., 1993; Canas, 2003). Phenolic compounds have beenreported to exhibit multiple biological effects, including antiox-idant activity. Some studies demonstrate the strong antioxidantactivity of some phenolic acids, like gallic and ellagic acid(Goldberg et al., 1999; Vinson et al., 2001; Priyadarsini et al.,2002; Sroka and Cisowski, 2003) while others emphasize the roleof hydrolyzable tannins (Da Porto et al., 2000; Mullen et al., 2002).

The antioxidant activity of the phenolic compounds dependson their chemical structure, concentration and oxidation status. In

the case of brandy, the latter two are mainly determined by theageing conditions, including the wooden barrel characteristics,such as wood botanical species (Belchior et al., 2001), toastinglevel (Canas et al., 1999; Belchior et al., 2001) and barrel size(Canas et al., 2008), and the cellar environment (Cantagrel et al.,1991a). Therefore, many studies were carried out to evaluate theantiradical properties of commercial Armagnacs and Cognacs(Goldberg et al., 1999; Da Porto et al., 2000; Umar et al., 2003),highlighting the relationship between their ageing time andantioxidant power.

Concerning the wood botanical species, our previous studiesdemonstrated significant differences in the phenolic compositionbetween brandies aged in chestnut and oak wooden barrels(Canas et al., 1999; Belchior et al., 2001). Limousin oak wood istraditionally used in the ageing of brandies and chestnut woodhas been showing interesting qualities for this purpose (Canaset al., 1999; Belchior et al., 2001; Caldeira et al., 2002).

The toasting assumes a crucial role in cooperage due to itsgreat influence on the wooden barrel quality and, consequently,on the chemical characteristics of the aged brandies, namely lowmolecular weight extractable compounds that are released fromwood to the distilled during the ageing process, and also on theirsensory properties (Artajona et al., 1991; Canas et al., 1999, 2007;Belchior et al., 2001; Caldeira et al., 2002, 2006).

However, to the best of our knowledge, there is no scientificresearch on the relationship between the wood botanical speciesor the toasting level and the antioxidant activity of the agedbrandies over the ageing time.

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Journal of Food Composition and Analysis

0889-1575/$ - see front matter & 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.jfca.2008.07.001

� Corresponding author. Tel.: +351 261712106; fax: +351 261712426.

E-mail address: evn.sara.canas@mail.net4b.pt (S. Canas).

Journal of Food Composition and Analysis 21 (2008) 626–633

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Therefore, the aim of this study was to evaluate the antioxidantactivity and the phenolic composition (total polyphenols, phenolicacids and hydrolyzable tannins) of Portuguese brandies, from theLourinha region, related to the kind of wood used (chestnut andLimousin oak wood), the toasting level of the wooden barrels(light, medium and heavy) and the ageing time (1–4 years ofageing). Correlation analysis between antiradical activity andphenolic composition of the brandies was also assessed.

2. Materials and methods

2.1. Chemicals

Ellagic acid dihydrate, gallic acid monohydrate, vanillic acid,syringic acid and ferulic acid were purchased from Fluka, Buchs,Switzerland. All of them were used as standards without furtherpurification. The solutions were prepared fresh prior to use withethanol/water (75:25, v/v). 1,1-Diphenyl-2-picrylhydrazyl radical(DPPHd) was purchased from Aldrich, Steinheim, Germany.

All solvents used were HPLC gradient grade purchased fromMerck, Darmstadt, Germany.

2.2. Experimental design and brandy sampling

Experiment 1: In the study of botanical species and toastinglevel effects, a two factorial design (two wood botanicalspecies� three toasting levels) with two replications was estab-lished. The wood botanical species used in the cooperage of 250-Lbarrels were chestnut wood (Castanea sativa Mill.) from northernPortugal (CT) and oak wood (Quercus robur L.) from Limousin (CL).The barrels were submitted to heat treatment with three levels oftoasting: light (QL), medium (QM) and heavy (QF), with threereplications of each level. The toasting process was controlled bythe cooper, which is about 10 min for light toasting, 20 min formedium toasting and 25 min for heavy toasting, over a fire ofcorresponding wood offcuts.

The barrels were placed at Adega Cooperativa de Lourinha insimilar cellar conditions and were filled with the same Lourinhabrandy. After 4 years of ageing, 18 brandy samples were collected:CT QL1, CT QL2, CT QL3, CT QM1, CT QM2, CT QM3, CT QF1, CT QF2,CT QF3, CL QL1, CL QL2, CL QL3, CL QM1, CL QM2, CL QM3, CL QF1,CL QF2, CL QF3.

Experiment 2: In the study of ageing time effect, three 250-Lbarrels of CL QM were filled with the same Lourinha brandy andthe brandy sampling was made after 1 year (1A), 2 years (2A), 3years (3A) and 4 years (4A) of ageing, so 12 brandies samples withdifferent ages were compared.

2.3. Determination of total polyphenols

Total polyphenols content of brandies was determined byabsorbance at 280 nm (Ribereau-Gayon, 1970). Brandies werediluted with ethanol/water 75:25. Results were expressed as mg/Lgallic acid.

2.4. Isolation of fractions of phenolic acids and hydrolyzable tannins

The isolation of fractions of phenolic acids and hydrolyzabletannins of the brandies was performed by liquid–liquid extraction,according to a new method developed in our laboratory: (1) 10 mLof brandy were dealcoholized by means of rotary evaporator at30 1C and diluted to the original concentration by distilled water.Then they were adjusted to pH 2 with 2 M HCl and extractedfour times with 10 mL of ethyl acetate. Both the medium andthe solvent were mixed together for 5 min. The upper phase

containing the phenolic acids was decanted after eachextraction and the four solvent phases were mixed togetherand then evaporated to dryness in a rotary evaporator at30 1C. The dry residue was dissolved in 1.5 mL of methanoland injected into the chromatograph. (2) The remainingphase (10 mL) was adjusted to pH 7 with 2 M NaOH, followed bythree extractions with 10 mL of ethyl acetate in order to eliminatethe neutral phenolic compounds. (3) The aqueous phase contain-ing the hydrolyzable tannins was decanted after each extraction,and the three aqueous phases were mixed and evaporated todryness in a rotary evaporator at 30 1C. The dry residue wasdissolved in 1.5 mL of distilled water and injected into thechromatograph.

2.5. Determination of phenolic acids

Gallic acid, vanillic acid, syringic acid, ferulic acid and ellagicacid were quantified in the isolated fraction as described byCanas et al. (2003), with a HPLC Lachrom Merck Hitachi system,Merck, Darmstadt, Germany, equipped with a quaternary pump L-7100, a column oven L-7350, a UV–vis detector L-7400, andan autosampler L-7250, coupled to a HSM D-7000 software,Merck, Darmstadt, Germany, for management, acquisition andtreatment of data. A Lichrospher RP 18 (5mm) column (250 mm�4 mm i.d.), Merck, Darmstadt, Germany, was used. Detection wasmade at 280 nm. Samples were added with an internal standard(20 mg/L of 4-hydroxybenzaldehyde), filtered through 0.45mmmembrane, Titan, Scientific Resources Ltd., Gloucester, UK, andanalyzed by direct injection of 20mL. The identification ofchromatographic peaks was made by comparison of their relativeretention times with those of external standards, as well as bytheir UV–vis spectra. The chromatographic purity of the peaks andthe UV–vis spectra (200–400 nm) were performed using a Waterssystem equipped with a photodiode-array detector (Waters 996),with the same chromatographic conditions, managed by ‘‘Millen-nium 2010’’ Software, Waters, Milford, USA.

Fig. 1 shows the HPLC chromatograms of phenolic acidsfraction of brandies aged in chestnut and Limousin oak barrels.

2.6. Determination of hydrolyzable tannins

Monogalloyl-glucose, grandinin, vescalagin, roburin E andcastalagin present in the isolated fraction were quantified byHPLC on a Lachrom Merck Hitachi system, already described inSection 2.5. A Phenomenex Synergi Polar RP (4mm) column(150 mm�4.6 mm i.d.) was used as stationary phase. Sampleswere filtered through a 0.45mm membrane, Titan, Gloucester, UK,and analyzed by direct injection of 20mL. A gradient consisting ofsolvent A, water-formic acid (98:2, v/v), solvent B, water andsolvent C, acetonitrile-water-formic acid (70:28:2, v/v/v) was asfollows: 50% solvent A and 50% solvent B over 8 min, 50–98%solvent B and 0–2% solvent C over 1 min, 98% solvent B and 2%solvent C isocratic in 16 min, 98–91% solvent B and 2–9%solvent C over 9 min, 91–89% solvent B and 9–11% solvent C over1 min, 89% solvent B and 11% solvent C isocratic in 15 min,89–20% solvent B and 11–80% solvent C over 5 min and thenisocratically for 5 min. Column temperature was 28 1C, flow ratewas 0.7 mL/min for 25 min and 0.9 mL/min after that.Compounds were detected at 280 nm. The identification of thecompounds was made by LC–MS on an Agilent (1100 series)/Bruker Daltonics (Esquire 3000plus) in the same chromatographicconditions. A Bruker Daltonics Data Analysis 3.0 was used for thedata analysis. Mass spectra were recorded from m/z ¼ 50 to 2000in a negative ionization mode. Normal scan resolution (13,000 m/z s�1) was selected. Voltages for the skimmer and capillary were

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0 5 10 15 20 25 30 35 40 45Retention Time (min)

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12 4

5

Fig. 1. HPLC chromatograms of phenolic acids fraction of brandies aged in (a) chestnut and (b) Limousin oak barrels; (1) gallic acid, (2) vanillic acid, (3) syringic acid, (4)

ferulic acid, (5) ellagic acid.

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Fig. 2. HPLC chromatograms of hydrolyzable tannins fraction of brandies aged in (a) chestnut and (b) Limousin oak barrels; (1) monogalloyl-glucose, (2) grandinin, (3)

vescalagin, (4) roburin E, (5) castalagin.

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�40 and +40,000 V, respectively. Other MS conditions were:nebulizer gas (N2), 10 psi; drying gas (N2) 10 L/min; and drytemperature, 250 1C.

Fig. 2 shows the HPLC chromatograms of hydrolyzabletannins fraction of brandies aged in chestnut and Limousin oakbarrels.

2.7. Measurement of the antioxidant activity

The DPPHd method (Da Porto et al., 2000) was modified in ourlaboratory and used to determine the antioxidant activity.Firstly, 10mL of methanol was added to 3 mL of a 8.5�10�5 MDPPHd methanol solution (Ainitial) and secondly, 10mL of brandy orisolated fraction was added to 3 mL of a 8.5�10�5 M DPPHd

methanol solution. In both steps, a pure methanol solutionwas used as reference. The decrease in absorbance, measuredat 515 nm, was determined continuously until the reactionreached a plateau (Afinal). The inhibition percentage ofthe DPPHd solution was calculated according to the followingequation:

Inhibition ð%Þ ¼ ðAinitial � AfinalÞ � 100.

Besides, to assure the repeatability of the method, the samplesand the DPPHd methanol solution were kept in a bath at 30 1Cbefore the analysis.

2.8. Statistical analysis

The two-way analysis of variance was performed to evaluatethe effects of the wood botanical species and the toasting level.The one-way analysis of variance was performed to evaluate theeffects of the ageing time. Calculation of least-significantdifference (LSD) was applied for comparison of the differentaverages. It was also performed the correlation analysis betweenantioxidant activity and phenolic composition of the brandies(total polyphenols, phenolic acids and hydrolyzable tannins). Allthe calculations were carried out using Statistica v. ‘98 edition,Statsoft Inc., Tulsa, USA.

3. Results and discussion

3.1. Wood botanical species effect

3.1.1. Total polyphenols and antioxidant activity of aged brandies

The analysis of variance shows a very significant effect ofthe botanical species on the antioxidant activity of the studiedbrandies, independently of the toasting level (there is nosignificant interaction between wood and toasting level).The brandy aged in chestnut wood presents higher antioxidantactivity (% DPPHd inhibition ¼ 93.570.91) than the brandyaged in Limousin oak wood (% DPPHd inhibition ¼ 45.772.38;Fig. 3).

Correspondingly, the total polyphenols of the brandies aged inchestnut wood are significantly higher than those of the brandiesaged in Limousin oak wood (Table 1), as observed in our previousstudies (Belchior et al., 2001; Canas, 2003). Thus, as expected,there is a very significant correlation between the antioxidantactivity and the total polyphenolic content of the brandies(r ¼ 0.9990). Similar correlations were obtained with Cognacs(Da Porto et al., 2000) and Sherry brandies (Alonso et al., 2004).

It is interesting to point out that the phenolic content of thebrandies aged in Limousin oak wood is 15-fold less than thephenolic content of the brandies aged in chestnut wood while theantioxidant activity of the first brandies is only two-fold less. Thisphenomenon could be explained on the basis of synergistic(Vinson et al., 2001; Psarra et al., 2002) and antagonisticeffects (Pinelo et al., 2004) together with other compoundspresent in the brandies, such as volatile compounds (Lee andShibamoto, 2001).

3.1.2. Phenolic acids and antioxidant activity of aged brandies

There is a very significant effect of the wood botanical specieson the antioxidant activity of the phenolic acids fraction of thebrandies, independently of the toasting level (Fig. 4). Althoughthis activity is slightly higher in the brandies aged in chestnutwood (% DPPHd inhibition ¼ 14.270.02) than in the brandies agedin Limousin oak wood (% DPPHd inhibition ¼ 14.070.07), the lowstandard deviation makes the difference very significant.

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60

80

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% D

PPH

inhi

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n

b

a

CT CL QL QM QF

Fig. 3. Mean values of antioxidant activity of the brandies aged in different kinds

of wood (CT ¼ chestnut, CL ¼ Limousin oak), and in wood with different toasting

levels (QL ¼ light toasting, QM ¼medium toasting, QF ¼ heavy toasting); wood:

nine samples; toasting level: six samples; error bars ¼ standard deviation;

columns with different letters indicate a very significant difference (po0.01).

Table 1Total polyphenols (g/L gallic acid), phenolic acids contents (mg/L) and hydrolyz-

able tannins contents (mg/L gallic acid) of the brandies aged in chestnut wood and

Limousin oak wooda

Effect Limousin oak Chestnut

Total polyphenols ** 0.6370.09 a 9.3470.01 b

Phenolic acids

Gallic acid ** 38.1970.14 a 302.58734.45 b

Vanillic acid ** 2.0370.13 a 5.5670.19 b

Syringic acid * 4.3170.11 a 7.4670.59 b

Ferulic acid ns 3.6170.21 2.7470.80

Ellagic acid ** 56.2671.04 a 79.5371.03 b

Total ** 104.4170.87 a 397.88737.07 b

Hydrolyzable tannins

Monogalloyl-glucose ns nd 5.1670.79

Grandinin ns 0.1270.05 0.1470.02

Vescalagin ns 0.1170.08 0.1770.07

Roburin E ns 0.1470.05 0.1270.04

Castalagin ns 12.0777.25 6.3373.52

Total ns 12.7973.24 14.1071.57

a Data are mean value7S.D. (nine samples for each kind of brandy); mean

values in the same row with different letters (a, b) are significantly (*po0.05) or

very significantly (**po0.01) different; ns: without significant difference; nd: not

detected.

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It is possible to significantly distinguish the studied brandiesbased on the total content of phenolic acids, and all of thesecompounds contribute to the observed effect, except for ferulicacid (Table 1).

In accordance with the results obtained for the antioxidantactivity, the brandies aged in chestnut wood are about four-foldricher in phenolic acids than the brandies aged in Limousin oakwood. In the former, except for ferulic acid, all the acids presenthigher concentration, whereas in the latter, ellagic acid is the mostabundant compound, followed by gallic acid, syringic acid and,finally, vanillic acid. These results are consistent with those ofother works (Goldberg et al., 1999; Da Porto et al., 2000). In Frenchspirits, which are aged in French oak wood (Q. robur L. fromLimousin forest), the content of gallic acid varies between 31 and38 mg/L (Puech et al., 1994). Conversely, in the brandies aged inchestnut wood, gallic acid predominates, followed by ellagic acid,syringic acid and vanillic acid. Similar results were also obtainedin our previous studies (Belchior et al., 2003; Canas, 2003). Thedifferences in gallic acid and ellagic acid contents are particularlyimportant for the discrimination of the brandies.

Significant correlation was found between antioxidant activityand total content of phenolic acids (r ¼ 0.9573). Strong correla-tions were observed between antioxidant activity and gallicacid content (r ¼ 0.9555), ellagic acid content (r ¼ 0.6138) andvanillic acid content (r ¼ 0.5836), indicating that these com-pounds can greatly contribute to the overall antioxidant power ofthe brandies. Hence, the higher antioxidant activity exhibited bythe brandies aged in chestnut wood could be partially due to therichness in gallic acid, ellagic acid and vanillic acid. Thishypothesis is supported by some studies that proved the strongantiradical activity of gallic and ellagic acids even at very lowconcentrations (Peiwu et al., 1999; Priyadarsini et al., 2002; Srokaand Cisowski, 2003). In contrast, Peiwu et al. (1999) reported theweak antioxidant capacity of vanillic acid and the significantcorrelation between antioxidant activity and syringic acid con-tent. According to Ou and Kwok (2004), ferulic acid presentshigher antiradical activity than gallic acid. These discrepanciescould be justified by the different kind of matrix analyzed, whichcould have a distinct influence on the synergistic and/orantagonistic effects between the phenolic acids.

3.1.3. Hydrolyzable tannins and antioxidant activity of aged brandies

Regarding the inhibition of DPPHd by the hydrolyzable tanninfraction, there is no significant difference between the brandiesaged in barrels from different botanical species (Fig. 5):chestnut ¼ 4.572.13%, Limousin oak ¼ 1.370.18%. This statisticalresult derives from the high standard deviation that could expressthe strong variability of the brandy composition in each wood. Infact, the good repeatability of the methods used in the isolation ofthe tannin fraction and in the determination of the antioxidantactivity (data not shown) points to the conclusion that thereferred effect could be related to the wood intraspecificvariability (Doussot et al., 2000) and to the variability inducedby the toasting of the barrel (Chatonnet, 1995).

Moreover, there is only a slight difference in hydrolyzabletannins content between brandies aged in chestnut and inLimousin oak wood (Table 1). Nevertheless, dimeric forms(roburins A, B, C and D) were not detected in the analyzedbrandies. Their absence in the brandies could result from theirthermal degradation in the wood during the heat treatment of thebarrel in the cooperage process (Chatonnet, 1995).

Considering the hydrolyzable tannin and phenolic acid con-tents in both brandies, there is reliable experimental evidence toconsider that the antioxidant activity of each fraction and itscontribution to the overall antioxidant activity depends on theirconcentration.

No significant correlations were found between antioxidantactivity and individual and total hydrolyzable tannin content,which contradicts the results obtained by other authors (Da Portoet al., 2000). This finding does not necessarily imply thathydrolyzable tannins do not contribute to the antioxidant activityof the studied brandies since their activity could be determined bysynergistic phenomena.

3.2. Toasting level effect

3.2.1. Total polyphenols and antioxidant activity of aged brandies

The analysis of variance demonstrates that the antioxidantactivity of the aged brandies is not significantly influenced by thewood toasting level (Fig. 3): QL ¼ 60.679.33%, QM ¼ 69.67

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5

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inhi

bitio

n

CT CL QL QM QF

b a

Fig. 4. Mean values of antioxidant activity of the phenolic acid fraction of brandies

aged in different kinds of wood (CT ¼ chestnut, CL ¼ Limousin oak), and in wood

with different toasting levels (QL ¼ light toasting, QM ¼ medium toasting,

QF ¼ heavy toasting); wood: nine samples; toasting level: six samples; error

bars ¼ standard deviation; columns with different letters indicate a very

significant difference (po0.01).

0

2

4

6

8

10

% D

PPH

inhi

bitio

n

CT CL QL QM QF

Fig. 5. Mean values of antioxidant activity of hydrolyzable tannins fraction of the

brandies aged in different kinds of wood (CT ¼ chestnut, CL ¼ Limousin oak), and

in wood with different toasting levels (QL ¼ light toasting, QM ¼medium toasting,

QF ¼ heavy toasting); wood: nine samples; toasting level: six samples; error

bars ¼ standard deviation.

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10.63%, QF ¼ 63.5712.56%. The observed statistical effect ismainly explained by the high variability associated with thetoasting operation (Cantagrel et al., 1991b; Canas et al., 1999). Forthe same reason, the toasting level does not affect significantly thetotal polyphenols of the aged brandies (Table 2).

The different evolution pattern of antioxidant activity and totalpolyphenols with the increase of the toasting level seems toindicate that the heavy toasting provokes considerable modifica-tion of some wood phenolic compounds, which have an importantcontribution to the antioxidant activity of the brandies (Canas,2003).

3.2.2. Phenolic acids and antioxidant activity of aged brandies

In the same manner, the antioxidant activity of the phenolicacids fraction does not depend on the toasting level (Fig. 4):QL ¼ 13.571.05%, QM ¼ 14.170.13%, QF ¼ 13.970.26%.

However, according to the analysis of variance, the phenolicacid content, except for gallic and ferulic acids, allows distinguish-ing the aged brandies as a function of the toasting level of thebarrel (Table 2). The behavior of gallic acid probably results fromthe higher thermal sensitivity of this acid (Chatonnet, 1995) andthe high variability in the composition of brandies from barrel tobarrel with the same toasting level, which derives from theprotocol of this cooperage operation (Cantagrel et al., 1991b;Canas et al., 2007). Since gallic acid is the most plentiful phenolicacid, its behavior determines the toasting effect on the totalcontent of phenolic acids fraction.

The ellagic acid content increases very significantly with therise of the toasting level, as verified in other works (Sarni et al.,1990), as a consequence of its high fusion point (4450 1C) andellagitannins degradation. Syringic acid presents similar evolutionwhile vanillic acid content decreases between the medium andthe heavy toasting, owing to their specific thermal resistance andto lignin degradation.

The absence of statistical effect of the toasting on theantioxidant activity of the aged brandies could be attributed togallic acid which has the best correlation with the antioxidantactivity of the brandies. The gallic acid behavior could alsocontribute to the different evolution pattern of antioxidant

activity and total polyphenols with the increase of the toastinglevel.

3.2.3. Hydrolyzable tannins and antioxidant activity of aged

brandies

The antioxidant activity of the hydrolyzable tannins fraction isalso independent of the wood toasting level (Fig. 5), due to thehigh variability associated with the toasting (Cantagrel et al.,1991b; Canas et al., 1999): QL ¼ 4.871.20%, QM ¼ 2.970.90%,QF ¼ 3.670.50%.

The total content of hydrolyzable tannins of the aged brandiesis not affected by the toasting level (Table 2), but shows atendency to decrease from the light toasting to the medium andstrong toasting levels that could be explained by their degradationwith the rise of temperature (Chatonnet, 1995). Conversely, thehydrolyzable tannins contents do not present a clear trend as afunction of the toasting level.

3.3. Ageing time effect

3.3.1. Total polyphenols and antioxidant activity of aged brandies

The analysis of variance indicates that the ageing time has ahighly significant influence on the antioxidant activity of thebrandies (Fig. 6). The antioxidant activity increases with theageing time, despite the untypical behavior in the third year ofageing. This fact was already verified in the low molecular weightextractable compounds (Canas, 2003), and is probably due to anyproblem in the brandy sampling procedure in this particular year.

The total polyphenols of the brandies increase continuouslyduring the ageing period and permit to distinguish significantlythe aged brandies (Table 3). It is important to point out that thegreat increment occurs in the first year owing to the highestconcentration gradient between the wood and the distilled (Canaset al., 2002). Then, there is an increase of 30% in the totalpolyphenols between the first and the second year, 13% betweenthe second and the third year and 19% from the third to the fourthyear. This type of evolution is coherent with that observed in ourprevious works (Belchior et al., 2001; Canas, 2003).

So, the antioxidant activity and the total polyphenol tends toincrease with the ageing time, being highly correlated with thisfactor (r ¼ 0.9283), as referred by other authors (Goldberg et al.,1999; Alonso et al., 2004).

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Table 2Total polyphenols (g/L gallic acid), phenolic acids contents (mg/L) and hydrolyz-

able tannins contents (mg/L gallic acid) of the brandies aged in wooden barrels

with different toasting levelsa

Effect Light toasting Medium toasting Heavy toasting

Total polyphenols ns 3.3371.70 4.9871.36 5.1172.17

Phenolic acids

Gallic acid ns 159.89716.58 170.39712.73 224.96755.73

Vanillic acid * 1.2470.27 a 3.7970.75 b 3.4870.46 b

Syringic acid ** 2.1870.19 a 5.8971.85 b 6.8571.86 b

Ferulic acid ns 2.8670.23 3.1870.70 2.0970.82

Ellagic acid ** 37.0071.97 a 67.89713.46 b 73.96713.55 b

Total ns 203.16719.24 251.14710.78 311.34712.43

Hydrolyzable tannins

Monogalloyl-glucose ns 1.2870.81 2.5871.45 1.7670.89

Grandinin ns 0.1170.05 0.1370.03 0.1370.02

Vescalagin ns 0.1470.04 0.1470.04 0.1370.01

Roburin E ns 0.1170.01 0.1370.02 0.1470.03

Castalagin ns 12.4578.30 9.2074.06 10.2875.70

Total ns 14.0977.41 12.1876.08 12.4374.25

a Data are mean value7S.D. (six samples for each kind of brandy); mean

values in the same row with different letters (a, b) are significantly (*po0.05) or

very significantly (**po0.01) different; ns: without significant difference, nd: not

detected.

01 A 2 A 3 A 4 A

10

20

30

40

50

% D

PPH

inhi

bitio

n

brandies acids tannins

b

a a

c

a b b c

Fig. 6. Evolution of antioxidant activity of brandies, phenolic acids fraction and

hydrolyzable tannins fraction of brandies aged in Limousin oak wood during the

first 4 years of ageing. Mean values of three samples/year; error bars ¼ standard

deviation; points signed with different letters indicate very significant differences

(po0.01).

S. Canas et al. / Journal of Food Composition and Analysis 21 (2008) 626–633 631

Author's personal copy

The decrease of the antioxidant activity and the rise of totalpolyphenols in the third year of ageing suggest the existence ofsome degradation phenomena that involved the most determin-ing compounds for antiradical activity (Pellegrini et al., 2000;Landrault et al., 2001).

3.3.2. Phenolic acids and antioxidant activity of aged brandies

In the experimental condition, there is a highly significantincrement of the antioxidant activity of phenolic acids fractionduring the ageing period (Fig. 6). The untypical behavior in thethird year of ageing is also observed.

The results of analysis of variance demonstrate that all of thephenolic acids are involved in the differentiation of brandies as afunction of the ageing time. The most discriminant phenolic acidsare syringic acid and ellagic acid (Table 3).

The total content gradually increases with the ageing time, butwithout significant difference between the second and the thirdyear. Ellagic acid content increases during the ageing period, likelyby wood ellagitannins hydrolysis and direct extraction of freeellagic acid from the wood (Viriot et al., 1993; Peng et al., 1991;Canas, 2003). Gallic acid content presents a tendency to increasewith the ageing time (Viriot et al., 1993). Ferulic acid contentincreases continuously with the ageing time, while vanillic andsyringic acid contents increase up to the third year and thendecrease.

Gallic acid content seems to determine the evolution of theantioxidant activity of the brandies, since these two variablesshow high significant correlation.

3.3.3. Hydrolyzable tannin and antioxidant activity of aged brandies

During the first 4 years of ageing, the antioxidant activity of thebrandies associated with their hydrolyzable tannins fractionundergoes significant variation (Fig. 6), but without an obvioustendency. In fact, the antioxidant activity of the hydrolyzabletannins fraction of 1- and 3-year-aged brandies is similar, andlower than that of 2- and 4-year-aged brandies.

In addition, the total content of hydrolyzable tannins variesirregularly with the ageing time (Table 3). Castalagin seems todetermine this evolution, representing more than 90% of hydro-lyzable tannins found in the brandies. These results are in

accordance with those of other authors (Viriot et al., 1993; Penget al., 1991).

4. Conclusions

In the experimental conditions, the results obtained confirmthe remarkable quality of the brandies aged in chestnut woodenbarrels owing to their higher antioxidant activity. This conclusionreinforces those of our previous studies which demonstrated thatchestnut wood is suited for cooperage (Carvalho, 1998), and it alsohas positive repercussions on the chemical composition andsensory properties of the corresponding aged brandies, thuscontributing to increase their quality and to shorten the ageingperiod (Canas et al., 1999; Belchior et al., 2001; Caldeira et al.,2002; Canas, 2003).

Further experiments are required to fully elucidate therole of individual phenolic compounds, namely phenolic acidsand hydrolyzable tannins, in the antioxidant activity of agedbrandies. More detailed studies are also necessary to understandthe synergistic and the antagonistic phenomena between theseconstituents which may allow a deeper knowledge of thereactions involved in the brandy’s ageing process.

Acknowledgments

The authors thank Maria Isabel Spranger and Maria Conceic- aoLeandro for technical assistance, Cristina Matos for manuscriptrevision and Project PARLE for the financial support.

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Table 3Total polyphenols (g/L gallic acid), phenolic acids contents (mg/L) and hydrolyzable tannins contents (mg/L gallic acid) of the brandies aged in Limousin oak wood during

the first four years of ageinga

Effect 1st Year 2nd Year 3rd Year 4th Year

Total polyphenols * 0.3670.03 a 0.4770.05 ab 0.5370.04 bc 0.6370.09 c

Phenolic acids

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Vanillic acid ** 1.1270.04 a 1.2470.02 a 2.4670.06 c 2.0370.13 b

Syringic acid *** 2.0270.02 a 2.8070.07 b 4.9570.12 d 4.3170.11 c

Ferulic acid *** 1.1670.21 a 2.1470.23 b 2.5770.21 b 3.6170.29 c

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