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Analytica Chimica Acta 513 (2004) 113118

Antioxidant activity of wines and relation with their


polyphenolic composition
M.S. Fernndez-Pachn, D. Villao, M.C. Garca-Parrilla , A.M. Troncoso
Facultad de Farmacia, Universidad de Sevilla, rea de Nutricin y Bromatologa, C/P. Garca Gonzalez s/n, Sevilla 41012, Spain
Received 8 July 2003; accepted 6 February 2004
Available online 17 April 2004

Abstract
The purposes of this work comprise the assessment of the antioxidant activity of wine samples by different analytical methods. The relation
between antioxidant values and phenolic composition is also considered as well as the insight of which red wine phenolic fraction is more
effective towards quenching radicals.
In vitro antioxidant activity of wines has been examined by a number of assays including: oxygen radical absorbance capacity (ORAC),
2,2 -azinobis-(3-ethylbenzothiazoline)-6 sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH). Forty-one wine samples were
analysed (16 red, 17 white and 9 sherry wines). The total phenolic index was evaluated. In addition, red wines were fractionated using solid
phase extraction to separate three main fractions (phenolic acids, flavan-3-ol and anthocyanins, flavonols).
Antioxidant activity of red wines is higher than that of white or sherry wines with every method under study. Analysis of variance (ANOVA)
demonstrated significative differences among red and white wines; and among red and sherry wines. However, ANOVA showed no statistical
significative differences among antioxidant results for white and Sherry wines. Total phenolic content is related to antioxidant activity, the
highest correlation coefficients being obtained for ORAC method. Concerning red wines fractions activity, larger values were obtained for
Fraction 2 corresponding to flavanols and anthocyanins in every case analysed. Additionally, similar values were obtained for Fraction 1 and
white wines ORAC results.
2004 Elsevier B.V. All rights reserved.
Keywords: Antioxidant; Wines; Polyphenolic; Oxygen radical absorbance capacity; 2,2 -Azinobis-(3-ethylbenzothiazoline)-6-sulfonic;
1,1-Diphenyl-2-picrylhydrazyl; N,N-Dimethyl-p-phenylene diamine

1. Introduction
Over the last decade health effects of wine consumption have been studied in depth. Special attention has been
focused on protection against cancer and cardiovascular disease. Generally, it is established that an oxidation process is
involved in the initial development steps of these diseases.
Indeed, reactive oxygen species (ROS), naturally formed
during normal metabolism, can damage biological structures such as proteins, lipids or DNA. Human metabolism
counts on an antioxidant defensive system involving enzymes and proteins to prevent these effects. However, the
defences can be overwhelmed under certain circumstances
so that harmful effects occur. It is accepted that the intake
of antioxidant substances reinforces the defences against

Corresponding author. Tel.: +34-95-4556761; fax: +34-95-4233765.


E-mail address: mcparrilla@us.es (M.C. Garca-Parrilla).

0003-2670/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2004.02.028

ROS. Therefore, the role of antioxidant nutrients such as


Vitamins (A, E and C) and other food components has been
raised. Polyphenolic compounds, naturally present in vegetal sources, can contribute to the dietetic intake due to their
antioxidant nature. Consequently, the evaluation of antioxidant activity is of major interest to compare the potential antioxidant value of different foods and to evaluate antioxidant
intake.
There are plenty of analytical methods for assessing the
antioxidant activity of foodstuffs but unfortunately, there is
not a standardised method [1]. Their significance is determined by the experimental conditions and methodological
approach involved. Some methods evaluate the susceptibility of a sample to be oxidised, a relevant matter in fats, for
instance. On the other hand, the potential nutritional value
is related to the ability to quench free radicals or to protect a biological structure from oxidation. Sample matrix,
hydrophilic/lipophilic characteristic of compounds under

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M.S. Fernandez-Pachon et al. / Analytica Chimica Acta 513 (2004) 113118

study and heterogenicity of substrates determine the results obtained. In addition, phytochemicals usually present
different antioxidant features simultaneously. In this way,
some authors have pointed out the need of developing a
reliable protocol which involves the measure of more than
one relevant property [2].
Recently, antioxidant activity of wines has been determined by different methods. For instance: the oxidation of
human low-density lipoprotein [3], 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay [4],2,2 -azinobis-(3-ethylbensothiazoline)-6-sulfonic acid (ABTS) radical assay [5], N,NDimethyl-p-phenylene diamine (DMPD) assay [6]. However, multiple and variable experimental conditions performing the assays (radical generator, time of measure, expression
of results, . . . ) makes it difficult to compare reported data.
The purposes of this work include an insight into the
validity of existing methodologies for the evaluation of
antioxidant features of wines. Their feasibility, advantages
and disadvantages, practical limitations and usefulness are
considered. Selected methods include the measurement of
scavenging capacity against a given radical (ABTS, DPPH,
DMPD) and the protection of a protein from suffering oxidation the oxygen radical absorbance capacity (ORAC). They
have been applied to different types of wine (red, white and
sherry wines) in order to evaluate possible differences. In
addition, this work will explore the relation between phenolic composition and antioxidant values by searching for
correlations with the total polyphenolic index as well as examining the contribution of the different polyphenolic families to the total antioxidant values. Red wines were fractionated into phenolic acids, anthocyanins and Flavonols, and
Flavonols and the antioxidant values of the three fractions
have been determined by the four methods under study.

2. Materials and methods


2.1. Samples
A total of 41 wine samples purchased in the market place
were analysed including 16 red wines, 17 white wines and
9 sherry wines. Samples were selected to be representative
of the most consumed wines in the south of Spain.
2.2. Chemicals
Folin Ciocalteu reagents was provided by Merck.
Radical scavenging assay reagents were purchased from
Sigma: ABTS, hydrogen peroxide and peroxidase type VIA from horseradish, and Iron(III) anhydrous chloride.
-Phycoerithrin (-PE) from Porphyridium cruentum were obtained from Sigma (St. Louis, MO). Before
use, its suitability for the assay was tested by verifying the loss of 95% fluorescence within 30 min in the
presence of 4 nmol l1 of 2,2-azobis(2-amidinopropane)
dihydrochloride (AAPH) as described elsewhere [7].

AAPH and 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid


(TROLOX) were purchased from Wako Chemicals and
Aldrich, respectively.
2.3. Instrumental
Spectrophotometric measurements were performed on a
UV-Vis double beam Milton Roy Spectronic 3000 spectrophotometer. Fluorimetric measurements in the ORAC assay were recorded in a F-2500 Hitachi fluorimeter equipped
with a microcuvette (10 mm pathlength) provided by Hellma
and connected to a device which maintained the temperature at 37 . Other equipment employed in this study is as
follows: VisiprepTM Solid Phase Extraction Vacuum Manifolds from Supelco, Sorwall Centrifuge, VV-micro Heidolph evaporator and a Selecta-P thermostatic bath.
2.4. Procedure (ORAC)
The procedure is based on a previously reported method
with slight modifications [7]. Briefly, it is described as follows: 150 l of wine sample is mixed with 150 l of -PE
(68 mg l1 ) and 75 l of AAPH (160 mM). Fluorescence is
recorded for 60 min until the final value is <5% of the initial value (excitation wavelength is set at 540 nm; emission
wavelength at 565 nm). The Fluorimetric cuvettes are kept
at 37 C. One hundred and fifty microliters of phosphate
buffer (PBS) (75 mM, pH 7) is used instead of the sample
to assay the blank. TROLOX solution (20 M) is used as a
standard. A duplicate was performed in every case.
Results are calculated as ORAC values using the differences of areas under the -PE decay curve between the blank
and the sample and are expressed as TROLOX equivalent.
TROLOX fluorescence decay curves are registered for every
new solution of -PE.
Wine samples are dealcoholised under vacuum at 38 C
in order to avoid ethanol interference in the assay as our
previous experience shows that ethanol content stabilises
-PE fluorescence avoiding normal decay. Indeed, different
ethanol-PBS solutions ranging from 0.2 to 2 ml l1 were
submitted to the test and increasing areas were found as the
ethanol concentration increased. A linear correlation (r =
0.9999, n = 6) was found for the ethanol concentration
versus -PE fluorescence decay curve.
PBS is added to reconstitute an evaporated wine sample
to reach the initial volume. White and sherry wines were
diluted 1:100 and red wines 1:500 to achieve an adequate
response.
2.5. Radical scavenging assays
ABTS, DPPH and DMPD are used for post-addition assays (commonly known as decoloration methods) in which
the sample is added after the radical is formed. The decrease of absorbance of the radical is proportional to the
concentration and activity of the sample analysed. In all as-

M.S. Fernandez-Pachon et al. / Analytica Chimica Acta 513 (2004) 113118

says, wines are appropriately diluted in order to achieve a


linear response. Absorbance measurements are transformed
in antioxidant activity using TROLOX as reference. Results
are expressed as TROLOX equivalent antioxidant capacity
(TEAC). Each value is the mean of five or six determinations
of different dilutions of wine which gives a linear response.
2.5.1. ABTS assay
This decoloration method consists of an enzymatic system containing a peroxidase enzyme, its substrate of oxidation (hydrogen peroxide) and the chromophore (ABTS). A
radical is generated from ABTS and has a characteristic absorption spectrum with a maximum at 414 nm. The reaction
mixture contains 1.5 mM ABTS, 15 M H2 O2 , 0.25 M peroxidase, to obtain 30:M ABTS+ , in a 50 mM glycineHCl
buffer pH 4.5. Once the radical is formed, 2 ml of ABTS+
are mixed with 100 l of sample and the reaction is monitored at 414 nm for 15 min.
2.5.2. DPPH assay
In this method, 100 l of appropriately diluted wine is
added to 3.9 ml of DPPH+ (25 ppm) in methanol and the
absorbance is measured until the reaction reaches an equilibrium.
2.5.3. DMPD assay
In the presence of an oxidant solution of (iron(III) chloride), DMPD, forms a coloured radical cation, DMPD+ .
The reaction is as follows: 1 ml of 100 mM DMPD is added
to 100 ml of 0.1 M acetate buffer, pH 5.25, and the radical is
obtained by adding 0.2 ml of 0.05 M ferric chloride. Scavenging assay proceeds by adding 0.1 ml of sample to 2 ml
of radical solution and measuring the absorbance at 505 nm
for 10 min after the addition of the sample.
2.6. Phenolic fractionation of wine
The procedure involves a solid phase extraction using Waters C18 cartridges [8]. Initially, the wine is adjusted to pH 7.
Thus phenolic acids are not retained by the stationary phase
(Fraction 1). Fraction 2 (flavanols and anthocyanins) and
Fraction 3 (flavonols) are then adsorbed onto the column.
Their separation is accomplished by modifying the polarity
of the mobile phase eluting first with aqueous acetonitrile
followed by ethyl acetate.
2.7. Total phenolic index
The Folin-Ciocalteu method was applied and results were
expressed as gallic acid.
2.8. Statistical analysis
Analysis of variance and linear correlations test were performed with Statistica software.

115

3. Results and discussion


Antioxidant activity results expressed as TROLOX equivalents (TEAC index) of different types of wines (red wine,
white wine and sherry wine) determined by all the methods
under study are shown in Table 1. Absorbance measurements involved in the ABTS method have been performed
at 2 and 15 min as more time is required for some phenols
to react [9]. As can be observed, red wines values are
higher than those of white or sherry wine in every antioxidant test used. The magnitude of the difference depends
on the method employed. Antioxidant activity determined
as the capacity of quenching radicals (ABTS, DPPH methods) is 10- or 15-fold higher for red wines than for white
ones. However, if protection from oxidation of proteins is
assessed (ORAC assay) red wines are five-fold more active
than white wines. ANOVA showed no statistical differences
between white and sherry wine antioxidant activities though
sherry wines are known to suffer oxidation rather easily.
High correlation coefficients were found between the total
polyphenolic index (TPI) and antioxidant activity measured
by ABTS, DPPH and ORAC methods when the whole set
of samples is considered (Table 2). However, the correlation
coefficient of DMPD values with TPI is lower (r = 0.7771).
Red wines TPI values range from 1262 to 2389 mg l1 (mean
value is 1877 mg l1 ) whilst sherry and white wines values
range from 200 to 400 mg l1 (mean value is 256 mg l1 ). As
can be seen, the red wines TPI is seven times higher than that
of the analysed white wines. Therefore, differences observed
in antioxidant quenching activities cannot be explained just
by a direct quantitative point of view.
Qualitative chemical differences have been explored by
examining wines phenolic composition. For this purpose,
red wines were fractionated by solid phase extraction. Fraction 1 collected phenolic acids; Fraction 2 anthocyanins and
flavanols and Fraction 3 flavanones. The results obtained are
summarised in Table 3. It can be noted that the wines values are higher than the sum of the three fractions in every
analysed sample. The sum of the three fractions values accounts for the following percentages of total wines antioxidant values: 49% (ABTS); 46% (DPPH), 70% (DMPD),
70% (ORAC), respectively. Polyphenolic compounds (complex and polymeric phenolic) retained in the cartridge could
explain the difference in activity. Moreover, synergic effects among different fractions could be considered. In order
to reveal the contribution of these two effects, the following experiment was performed. The three fractions of a selected sample were gathered and the antioxidant activity of
the mixture determined by ORAC, ABTS and DPPH tests.
Antioxidant values of the mixtures were not higher than
those previously calculated (Table 4). Indeed, their values
were similar to those theoretically calculated by summing
the results of the individual fractions measured separately.
These results were identical for all the tests under study
for every analysed sample. No synergic action could, be
proved.

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M.S. Fernandez-Pachon et al. / Analytica Chimica Acta 513 (2004) 113118

Table 1
Total phenol index (TPI) and antioxidant activity of wines determined by ORAC, ABTS, DMPD and DPPH methods
TPIa

ORACb

ABTS2 minc

ABTS15 minc

DMPDc

DPPHc

W1
W2
W3
W4
W5
W6
W7
W8
W9
W10
W11
W12
W13
W14
W15
W16
W17

218
231
247
239
213
278
297
222
227
239
201
89
251
159
234
407
331

2899
840
425
1168
717
2135
1096
609
953
1019
570
122
1660
1105
1334
1564
1727

208
28
59
136
51
313
397
8
185
156
133
25
45
382
239
281
592

0.40
0.59
0.54
0.29
0.53
0.37
0.69
0.30
0.21
0.28
0.14
0.08
0.33
0.27
0.82
1.45
0.58

0.10
0.06
0.05
0.03
0.07
0.03
0.06
0.02
0.02
0.05
0.01
0.01
0.03
0.02
0.22
0.29
0.06

0.52
0.82
0.78
0.41
0.69
0.56
0.93
0.47
0.31
0.40
0.21
0.16
0.45
0.48
1.22
2.18
0.80

0.15
0.07
0.06
0.04
0.07
0.05
0.04
0.03
0.03
0.08
0.01
0.01
0.03
0.05
0.33
0.54
0.04

7.22
6.89
8.29
8.08
8.86
8.49
9.50
9.06
6.45
8.99
2.97
3.09
7.15
2.29
11.01
11.36
8.09

0.72
1.92
1.41
1.04
0.66
1.02
1.79
1.95
0.57
1.24
0.57
0.66
0.48
0.84
2.00
4.16
0.75

0.76
0.62
0.65
0.62
0.58
0.53
0.80
0.54
0.47
0.60
0.39
0.30
0.63
0.79
2.68
2.21
0.95

0.07
0.01
0.02
0.02
0.02
0.02
0.03
0.03
0.04
0.02
0.04
0.04
0.04
0.36
1.37
0.33
0.10

S1
S2
S3
S4
S5
S6
S7
S8
S9

283
280
240
251
207
446
297
284
290

1300
1391
1118
1336
1158
1782
1291
1304
995

150
47
200
103
109
96
9
71
102

0.21
0.57
0.26
0.57
0.19
0.27
0.08
0.11
0.11

0.05
0.04
0.03
0.05
0.02
0.03
0.01
0.01
0.02

0.29
0.70
0.31
0.74
0.28
0.38
0.11
0.16
0.17

0.04
0.03
0.03
0.06
0.03
0.04
0.02
0.01
0.01

2.47
2.33
2.88
3.32
3.14
3.31
3.27
1.83
2.91

0.72
0.52
1.26
0.88
0.59
0.48
1.31
0.46
1.39

0.93
0.88
0.61
0.92
0.49
0.98
0.58
0.73
0.89

0.08
0.04
0.04
0.01
0.02
0.11
0.01
0.06
0.03

1378
2360
2335
2092
1764
1861
2389
2195
2304
1662
1874
1357
1313
1561
1852
1742

4920
8283
6260
12706
5465
7659

1169
564
442
986
511
610

7951
7821
6766
7169
4732
4181
10800
4885
8097

2267
953
883
142
418
78
175
684
1239

6.33
5.11
4.26
6.14
4.57
3.72
7.85
4.52
3.59
3.96
5.00
5.29
2.33
6.66
6.50
4.31

1.13
0.50
0.15
0.66
0.34
0.32
1.61
0.70
0.22
1.08
0.26
0.62
0.23
0.80
1.09
0.68

11.15
6.32
5.36
7.94
6.06
4.69
11.06
5.95
4.56
5.28
6.53
8.15
3.06
10.37
8.75
5.72

3.65
0.38
0.52
0.67
0.52
0.47
3.41
0.99
0.60
1.18
0.34
1.48
0.35
1.83
1.29
1.00

6.97
13.39
11.39
12.16
9.04
12.67
16.53
20.72
19.08
10.68
16.68
10.27
8.24
14.34
11.11
11.75

1.81
8.10
4.36
3.14
1.14
3.25
5.96
6.78
7.04
2.44
8.58
1.85
1.62
2.21
2.57
4.47

6.96
10.42
9.72
17.00
7.41
15.28
17.41
10.73
9.45
5.22
8.76
6.10
4.65
6.95
8.55
6.37

0.37
1.02
1.07
1.80
0.64
3.12
4.98
1.50
0.40
0.36
1.81
0.50
0.07
0.30
0.98
0.39

R1
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R13
R14
R15
R16

W, white wine; S, sherry wine; R, red wine.


a Expressed as gallic acid equivalents.
b Expressed as M.
c Expressed as TROLOX equivalent antioxidant capacity (TEAC) (mM).

Table 2
Correlation coefficients between total phenol index (TPI) and antioxidant
activity of wines

White wines
(n = 17)
Sherry wines
(n = 9)
Red wines
(n=16)
Total of wines
(n = 42)

ORAC

ABTS2 min

ABTS15 min

DPPH

DMPD

0.7186

0.7219

0.6930

0.5547

0.6656

0.9474

0.0103

0.0213

0.6755

0.0660

0.7519

0.0675

0.1670

0.8237

0.6588

0.9125

0.9012

0.8462

0.9769

0.7771

It seems more reasonable to attribute the remaining antioxidant activity to polyphenolics retained in the cartridge
which are estimated to be around 30-35% from the original content of red wine on the basis of TPI. Indeed, the
values obtained for TPI of the mixture comprising the
three fractions are coherent with their determined antioxidant activity values. The antioxidant behaviour of retained
polyphenolic compounds and non-retained polyphenolics
agrees with other antioxidant features reported in the literature. For instance, it has been published that red wine
polyphenolic monomers fraction had the same activity as
polymeric polyphenolic fraction regarding induced DNA

M.S. Fernandez-Pachon et al. / Analytica Chimica Acta 513 (2004) 113118

117

Table 3
Antioxidant activity of phenolic fractions from red wines
Wine

ORACb

F1
R3
R4
R6
R8
R9
R10
R11
R13
R15
R16

1085
2700
1956
1526
1653
1201
1660
1349
1539
1036

59
293
195
48
144
33
370
37
210
95

0.60
0.28
0.90
0.74
1.02
0.78
0.80
0.33
0.45
0.89

0.03
0.12
0.11
0.33
0.12
0.00
0.30
0.03
0.02
0.11

0.74
0.62
1.11
0.92
1.30
0.93
1.41
0.44
0.41
0.97

0.00
0.16
0.20
0.55
0.28
0.15
0.54
0.10
0.23
0.39

1.14
4.99
1.00
11.57
8.04
6.61
10.63
1.29
4.78
7.18

0.42
0.00
0.00
0.18
0.39
0.77
1.59
0.01
0.56
1.00

2.80
1.81
0.95
1.83
2.26
2.00
1.72
0.86
0.55
0.82

0.12
0.02
0.06
0.03
0.00
0.74
0.24
0.03
0.00
0.10

F2
R3
R4
R6
R8
R9
R10
R11
R13
R15
R16

3096
1260
3053
4407
3959
2614
2909
2219
4744
2695

655
40
822
547
299
260
335
90
628
370

1.99
0.83
1.89
2.15
1.23
1.29
1.67
0.52
2.43
2.14

0.25
0.51
0.00
0.30
0.54
0.01
0.00
0.01
0.30
0.04

2.66
1.08
2.43
2.79
1.60
1.93
3.79
0.59
3.27
2.96

0.39
0.78
0.00
0.57
0.86
0.13
2.12
0.00
0.45
0.02

0.83
1.77
1.71
8.72
3.38
1.88
3.32
0.71
2.65
2.55

0.43
0.08
1.76
3.19
0.36
0.64
0.48
0.01
0.41
0.61

4.25
1.98
0.98
4.88
3.83
2.55
2.60
4.10
2.98
3.59

0.47
0.27
0.05
0.21
0.21
0.08
0.16
1.36
0.91
0.08

F3
R3
R4
R6
R8
R9
R10
R11
R13
R15
R16

1250
1260
536
473
604
613
460
455
879
902

178
40
138
220
19
115
69
144
2
97

0.06 0.04
0.23 0.03
0.20 0.03
0.14 0.00
0.13 0.00
0.09 0.03
0.22 0.04
0.07 0.00
0.18 0.03
0.11 0.00

0.12
0.32
0.19
0.18
0.17
0.14
0.25
0.09
0.28
0.17

0.00
0.04
0.06
0.00
0.00
0.06
0.18
0.00
0.01
0.01

0.26
0.73
0.33
1.26
1.99
2.29
1.75
0.24
0.54
0.46

0.08
0.07
0.00
0.10
0.22
1.21
0.00
0.02
0.00
0.00

0.40
0.80
0.64
0.39
0.48
0.22
0.42
0.30
0.36
0.19

0.05
0.04
0.02
0.07
0.06
0.01
0.01
0.07
0.01
0.01

ABTS2 min c

ABTS15 min c

DMPDc

DPPHc

a Expressed as gallic acid equivalents.


b Expressed as M.
c Expressed as TROLOX equivalent antioxidant capacity (TEAC) (mM).

oxidative damage [10]. These results must be taken into


account when correlating in vitro and in vivo antioxidant
results as polymeric phenolics are unlikely to be absorbed.
Half of the radical quenching activity (as reflected the sum
of the three fractions) is probably exerted by absorption

through the intestinal barrier. The other half could represent


the antioxidant activity available in the colonic medium.
Three methods (ABTS, DPPH and ORAC) offered the
same reactivity order for the different analysed fractions:
Fraction 2 is the highest followed by Fractions 1 and 3.

Table 4
Antioxidant values analytically determined in the mixture of the three phenolic fractions and the theoretically calculated value obtained by summing the
antioxidant values of the fractions analysed separately
Sample

Fractions

ORACb

ABTS2 min c

ABTS15 min c

DPPHc

TPIa

R3

D
C

3739 213
5431 892

1.75 0.25
2.65 0.32

2.06 0.60
3.52 0.39

5.03 0.89
7.45 0.64

1418
1382

R4

D
C

4084 62
4360 373

0.44 0.02
1.34 0.66

0.63 0.03
2.02 0.98

2.30 0.01
4.59 0.33

1367
1313

R8

D
C

3499 307
6407 815

2.56 0.40
3.03 0.63

3.64 0.54
3.89 1.12

8.39 2.10
7.10 0.31

1468
1403

a
b
c

Expressed as gallic acid equivalents.


Expressed as M.
Expressed as TROLOX equivalent antioxidant capacity (TEAC) (mM).

118

M.S. Fernandez-Pachon et al. / Analytica Chimica Acta 513 (2004) 113118

ORAC
DMPD

f1
f2

DPPH

f3

ABTS

15min

ABTS
2min

0%

20%

40%

60%

80%

100%

with the other radical quenching antioxidant tests. Besides


the standard deviations found for DMPD results are very
high. Hence, in our opinion it is not advisable to assess
antioxidant activity of wines with this test.
The time (1 h) needed to perform a single determination
either for the DPPH test or the ORAC assay can be considered a disadvantage of these methods. In addition, ORAC
reagents are expensive. However, the results obtained are
reproducible and coherent. It must be pointed out that despite their methodological differences, results obtained with
ABTS, DPPH or ORAC methods allow similar conclusions.

Fig. 1. Percentage of antioxidant activity achieved by each fraction with


regard total wine antioxidant activity.

4. Conclusions
Fraction 2 achieved values ranging from 32.1 to 40.2% of
the whole wine value except for the DMPD method which
yielded lower values. On the other hand, Fraction 1 accounts
for values ranging from 12.8 to 20.8% of the wine activity
depending on the method used. As has been noticed, Fraction 1 accounts for a non-negligible activity which may not
be rejected. It has been reported that the vasodilation activity
of wines is well correlated with a single phenolic family: total anthocyanins [11]. However, Fraction 1 (phenolic acids)
should not be disregarded concerning free radical quenching activity or protein protection from oxidation. ANOVA
showed that there are no significative differences among this
fraction value and results obtained for white or sherry wine
with ABTS, DMPD or ORAC method.
Some considerations can be drafted from the comparison
of data obtained with different antioxidant methods. ABTS
absorbance measures were recorded at two times (2 and
15 min). Absolute values are higher at longer reaction times.
However, wines keep the same ranking activity, differences
among white and red wines are similar and correlation with
TPI is the same. Different results were expected for the fractions determined at different times since their reactivity towards ABTS and hydrosolubility/liposolubility coefficients
are considerably distinct. Moreover, phenolic fractions behaviour is almost identical (Fig. 1). On the other hand, the
ABTS test has already been applied to evaluate antioxidant
capacity of different wines [12]. As previously mentioned
there are different methodologies involved in the ABTS test:
radicals generation, wavelength of measurement and temperature, which makes it difficult to compare our results with
those already reported. However, the same conclusion is arrived at: white wines are 10 times less powerful than red
wines. Though experimental conditions in the ABTS test influence absolute antioxidant values, in our opinion conclusions extracted from ranking or comparing wines and their
phenolic fractions are rather robust and coherent. Therefore,
measures at 2 min offer the same information as those at
15 min and they can be performed in a shorter time.
The low reactivity of anthocyanins and flavanols towards
DMPD radical can explain the lower correlation with TPI
and the smaller difference found for red and white wines.
Results obtained with the DMPD method are not consistent

ABTS, DPPH and ORAC methods can be recommended


to evaluate antioxidant activities of wines. Red wines shows
higher antioxidant activity than white or sherry wine. Between these two last there are no significative differences
regardless of the antioxidant measurement method used.
Fifty percent of the total red wines scavenging radical activity (ABTS, DPPH methods) is attributed to polymeric
phenolic compounds. Regarding the remaining 50% the reactivity order is as follows: anthocyanins and flavan-3-ol are
the most active followed by phenolic acids and flavonols.
No synergic effects among the three mentioned fractions
could be proved. No significative differences were found
between phenolic acids fraction and white wines.
Acknowledgements
Authors thank Ministerio de Ciencia y Tecnologia for financial support (Research Project 2001-2368).
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