Analytica Chimica Acta 563 (2006) 101–108

Determination of the phenolic composition of sherry and

table white wines by liquid chromatography and their
relation with antioxidant activity
M.S. Fernández-Pachón, D. Villaño, A.M. Troncoso, M.C. Garcı́a-Parrilla ∗
Área de Nutrición y Bromatologı́a, Facultad de Farmacia, Universidad de Sevilla, C/P. Garcı́a González no. 2, Sevilla E-41012, Spain
Received 22 July 2005; received in revised form 22 September 2005; accepted 26 September 2005
Available online 2 November 2005

Abstract
The purpose of this work is to study possible relationships between the antioxidant activity (AA) of 13 table white and 9 sherry wines and
their phenolic composition. Twenty phenolic compounds were determined by liquid chromathography (LC). AA has been determined by different
methods, including 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and Oxygen Radical
Absorbance Capacity (ORAC). The AA of pure phenolic standard compounds has also been measured. Statistical study has been performed
using multiple regression analysis and artificial neural networks to ascertain if a specific phenolic composition accounts for a higher AA. Certain
phenolic compounds exhibit a high linear correlation with the overall AA of wines. For example, gallic acid, glucoside of cutaric acid and tyrosol
as determined by the ORAC method. If we study AA values obtained by ABTS and DPPH methods, a good correlation is obtained with (−)-
epigallocatechin gallate and ethyl caffeate. The contribution of each phenolic compound to the AA was also examined. Gallic acid, protocatechuic
acid, caffeic acid and tyrosol are the main contributors to the ORAC value. If we use ABTS and DPPH methods, the main contributors are gallic,
caffeic and caftaric acids and (−)-epigallocatechin gallate.
An equation calculated by Multiple Regression Analysis correlates (r = 0.9028) ORAC values of wines with concentration of certain phenolic
compounds (gallic acid, protocatechuic acid, tyrosol, caffeic acid, (−)-epigallocatechin gallate, coumaric acid and 2-furaldehyde).
© 2005 Elsevier B.V. All rights reserved.

Keywords: Antioxidant activity; Wine; Phenolic compounds; Oxygen Radical Absorbance Capacity; 2,2 -Azinobis(3-ethylbenzothiazoline-6-sulfonic acid); 1,1-
Diphenyl-2-picrylhydrazyl

1. Introduction platelet aggregation, the peroxidation of lipids, the oxidation of

Generally, it is established that an oxidation process is
involved in the initial development steps of cancer and cardio-
vascular disease [1,2]. It is accepted that the intake of antioxi-
dant substances reinforces the defences against reactive oxygen
species (ROS). Therefore, the role of antioxidant nutrients as
vitamins (A, E, C) and other food components has been raised
[3]. Phenolic compounds, which are ubiquitous in foods of plant
origin, can contribute to the dietary intake due to their antioxi-
dant nature [4,5]. Thus, they are believed to reinforce antioxidant
system against ROS. Moreover, phenolic compounds are con-
sidered to slow the natural processes of thrombosis by inhibiting
∗ Corresponding author. Tel.: +34 954 55 67 60; fax: +34 954 23 37 65.
E-mail address: mcparrilla@us.es (M.C. Garcı́a-Parrilla).
low-density lipoproteins, etc. [6,7,5].
Among other food commodities such as herbs [8], vegetables,
fruits [9] and tea [10,11], wines are considered to be an excep-
tional dietary source of various classes of phenols, including
benzoic and cinnamic acid derivatives, flavan-3-ols, flavonols
and anthocyanins [12].
Over the last decade health effects of wine consumption have
been studied in depth. Epidemiological studies evidence that
moderate wine consumption protects against degenerative pro-
cesses, such as cardiovascular diseases and cancer, due to its
phenolic content [13].
In vitro antioxidant activity (AA) of wine has been assessed
thoroughly employing different methods [14,15]. As the infor-
mation they provide varies, several methods of assessing AA are
needed [16]. Oxygen Radical Absorbance Capacity (ORAC),
2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)

0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2005.09.057
102 M.S. Fernández-Pachón et al. / Analytica Chimica Acta 563 (2006) 101–108
and 1,1-diphenyl-2-picrylhydrazyl (DPPH) have been applied
to wine samples [17].
Phenolic compounds are believed to be responsible for the
healthy effects of moderate wine consumption [17,18]. Pheno-
lic compounds are considered free-radical scavengers, their AA
depends on their chemical structure. Specifically, it depends on
their ability to donate hydrogen or electron and their ability to
delocalise the unpaired electron within the aromatic structure.
Several structure–activity relationships have been described
[19,20].
Antioxidant properties of wine can be better understood taken
into account its phenolic composition. It seems interesting to
submit isolated phenols to the above antioxidant tests [21]. There
is not a consensus if the antioxidant properties of wines are
linked with the total phenol concentration, rather than individual
phenols [22]. Therefore, studies on the relationship between phe-
nolic composition and antioxidant properties of sherry wines, as
far as it is known, are scarce. We have selected sherry wines for
our study because they have been less studied and because their
phenolic composition can be described more exactly than red
wines.
This work has been designed as follows: first, we evaluated
the phenolic composition of selected table white and sherry
wines by liquid chromathography (LC) method; second, we
examined the correlation of the phenolic compounds and the
AA of wines; third, we calculated the theorical contribution of
each phenolic compound to the AA of wine samples; and fourth,
we looked for a equation which define AA of wines through their
principal constituents.
2. Experimental
2.1. Samples
A total of 22 wine samples from the market were analysed
including 13 table 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 and reagents
A total of 30 phenolic compounds were studied. Standards
of phenolic compounds were purchased from Merck (vanillic
aldehyde, p-hydroxybenzoic aldehyde, 5-hydroxymethyl-
furfural), Sigma (protocatechuic acid, 2-furaldehyde,
(−)-epigallocatechin, vanillic acid, syringic acid, (−)-epigallo-
catechin gallate, syringic aldehyde, tryptophol, ferulic acid, (−)-
epicatechin gallate, myricetin, trans-resveratrol, kaempferol),
Fluka (gallic acid, tyrosol, 5-methylfurfural, caffeic acid,
(−)-epicatechin, (+)-catechin, p-coumaric acid, quercetin) and
Chromadex (procyanidin B1, procyanidin B2, caftaric acid). The
phenolic compounds were selected for their usual occurrence
in wines and their availability as commercial standards.
Glacial acetic acid and acetonitrile were both LC grade and
were obtained from Fluka and Merck, respectively.
ABTS in the crystallized diammonium salt form, horseradish
peroxidase type VI-A, hydrogen peroxide (30%, v/v) and DPPH
as free radical were obtained from Sigma. Methanol, glycine,
ethanol and hydrochloric acid (32%) were provided by Merck.
␤-Phycoerithrin (␤-PE) from Porphydium cruentum was
obtained from Sigma. Before using this compound, we tested its
suitability for the assay by verifying the loss of more than 90%
fluorescence within 30 min in the presence of 160 mM of 2,2 -
azobis (2-amidino-propane) dihydrochloride (AAPH). AAPH
and 6-hydroxy-2,5,7,8 tetramethylchroman-2-carboxylic acid
(Trolox) were purchased from Wako Chemicals and Aldrich,
respectively. All reagents were of analytical grade. Double
distilled water (Millipore Co.) was used throughout.
2.3. Instrumental
Absorbance measurements were recorded on a UV–VIS U-
2800 Hitachi spectrophotometer. Operating conditions were set
at 25 ◦ C. Fluorimetric measurements were recorded in a F-2500
Hitachi Fluorometer connected to a device which maintained
temperature at 37 ◦ C. The liquid chromatographic equipment
was a Waters 600E System Controller connected in series to a
Waters 996 Photodiode Array Detector.
3. Methods
3.1. ABTS
The method described by Cano [23], with some modifica-
tions, was used. Free radicals are generated by an enzymatic
system consisting of the horseradish peroxidase enzyme, hydro-
gen peroxide and the ABTS chromophore. The radical is gen-
erated by a reaction between 1.5 mM ABTS, 15 ␮M hydrogen
peroxide and 0.25 ␮M peroxidase in 50 mM glycine–HCl buffer
(pH 4.5). The final volume is 60 mL, which yields 30 ␮M of the
ABTS•+ radical cation (final concentration). These concentra-
tions must be checked by measuring their absorbances and using
their molar extinction coefficients. The blank reference cuvette
contained glycine–HCl buffer.
Once the radical was formed, 2 mL of ABTS•+ are mixed
with 100 ␮L of appropriately diluted wine and the decrease in
absorbance is monitored at 414 nm for 15 min.
Standard Trolox solutions (40–200 ␮M) were also evaluated
against the radical. 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 give
a linear response.
3.1.1. DPPH
It used the procedure described by Sánchez-Moreno et
al. [24]. Briefly, 100 ␮L of appropriately diluted wines were
added to 3.9 mL of DPPH•+ methanolic solution (25 mg L−1 ).
Absorbance at 515 nm was measured at different time inter-
vals until the reaction reached an equilibrium. The blank ref-
erence cuvette contained methanol. Standard Trolox solutions
(40–200 ␮M) were also evaluated against the radical. 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 give a linear response.
 M.S. Fernández-Pachón et al. / Analytica Chimica Acta 563 (2006) 101–108 103

3.1.2. ORAC
This procedure is based on a previously reported method
of Cao and Prior [25]. Briefly, it can be described as fol-
lows: 150 ␮L of diluted wine sample is mixed with 150 ␮L
␤-PE (68 mg L−1 ) and 75 ␮L AAPH (160 mM). Fluorescence
is recorded for 60 min until the final value is less than 5%
of the initial value (excitation wavelength is set at 540 nm;
emission wavelength is set at 565 nm). Fluorimetric cuvettes
are kept at 37 ◦ C. 150 ␮L of phosphate buffer (PBS) (75 mM,
pH 7) is used as blank. Trolox solution (20 ␮M) is used as
standard. A triplicate 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 sam-
ple and are expressed as Trolox Equivalents. Trolox fluores-
cence decay curves are registered for every new solution of
␤-PE.
For ORAC assay, wine samples are dealcoholised under vac-
uum at 38 ◦ C in order to avoid ethanol interference in the values
as our previous experience shows that ethanol content stabilises
␤-PE fluorescence avoiding normal decay [21]. PBS is added
to reconstitute an evaporated wine sample to reach the initial
volume.
We have tested that for ABTS and DPPH assays ethanol
does not affect the results. Previous studies reported no reac-
tion between ABTS and ethanol [27].
3.1.3. Chromatographic conditions
The column was a Merck LiChroCART (250 mm × 4 mm)
Superspher 100 RP-18 (5 ␮m), protected by a Merck LiChro-
CART 4-4 guard cartridge. The syringe filters were Millex® -
GV13 (0.22 ␮m) SLGV T13 NL.
The chromatographic conditions have been previously
described [26]. The method uses a binary gradient: A (glacial
acetic acid/water pH 2.65), B (20% A + 80% acetonitrile) pro-
grammed in a gradient as follows: 0 min (100% A); 5 min (98%
A + 2% B); 10 min (96% A + 4% B); 15 min (90% A + 10% B);
30 min (80% A + 20% B); 35 min (70% A + 30% B); 40 min
(100% B); 45 min (100% A); 60 min (100% A).
The flow rate was 1.5 mL min−1 and the temperature was set
at 40 ◦ C.
The photodiode array was programmed to record data from
240 to 450 nm. Unknown spectra were identified by compar-
ing their spectra with those of pure standards and they were
quantified by external calibration. Analyses were carried out in
triplicate.
3.1.4. Statistical analyses
Analyses of linear correlation and multiple regression were
performed with Statistica software.
4. Results and discussion
Tables 1 and 2 present the concentration of phenolic com-
pounds identified in analysed wine samples. Gallic acid accounts
for the largest concentration among the benzoic acids (mean
values are 3.27 and 6.93 mg L−1 in table white and sherry
wines, respectively). These values are very similar in Italian
[28] and Californian wines [29]. Caftaric acid showed the high-

Table 1
Concentration (mg L−1 ) of benzoic and cinnamic acids and derivatives detected in table wine and sherry wines
Wine Benzoic acids (mg L−1 ) Cinnamic acids (mg L−1 )

Gallic Protocatechuic Syringic Total Caftaric Glucoside Cutaric Caffeic p-Coumaric Ethyl Total
acid acid acid acid cutaric acid acid acid acid caffeate

W01 8.37 ± 0.0 n.d. 0.47 ± 0.0 8.84 11.3 ± 0.2 n.d. n.d. 1.57 ± 0.0 0.97 ± 0.0 n.d. 13.8
W02 2.60 ± 0.2 n.d. n.d. 2.60 14.6 ± 1.0 6.07 ± 0.7 n.d. 1.77 ± 0.0 0.84 ± 0.0 1.71 ± 0.0 24.9
W03 n.d. 4.74 ± 0.2 n.d. 4.74 19,1 ± 0.2 7.72 ± 0.8 12.4 ± 0.0 2.71 ± 0.3 3.04 ± 0.2 2.40 ± 0.3 47.4
W04 2.46 ± 1.1 n.d. n.d. 2.46 13.5 ± 0.1 n.d. n.d. n.d. n.d. 2.47 ± 0.0 16.0
W05 3.28 ± 0.4 2.45 ± 0.1 n.d. 5.73 27.4 ± 0.1 9.80 ± 6.4 n.d. 2.23 ± 0.0 1.15 ± 0.1 n.d. 40.5
W06 3.54 ± 0.0 n.d. n.d. 3.54 11.2 ± 0.5 n.d. 6.97 ± 0.0 1.63 ± 0.2 0.47 ± 0.1 n.d. 20.3
W07 7.17 ± 1.2 n.d. n.d. 7.17 14.6 ± 0.4 n.d. n.d. 0.99 ± 0.0 0.58 ± 0.0 3.47 ± 0.0 19.6
W08 3.72 ± 0.7 2.86 ± 1.4 n.d. 6.58 9.80 ± 0.5 3.70 ± 2.1 8.12 ± 3.3 0.99 ± 0.1 0.70 ± 0.1 1.54 ± 0.2 24.8
W09 0.69 ± 0.1 n.d. n.d. 0.69 53.5 ± 2.9 5.10 ± 0.0 14.4 ± 0.4 1.99 ± 0.1 n.d. 1.65 ± 0.4 76.5
W10 1.78 ± 0.1 n.d. n.d. 1.78 24.6 ± 1.9 2.85 ± 0.1 8.03 ± 0.1 1.14 ± 0.0 n.d. 1.63 ± 0.1 38.2
W11 2.28 ± 0.0 n.d. n.d. 2.28 3.86 ± 0.1 n.d. n.d. n.d. n.d. 1.39 ± 0.0 5.25
W12 n.d. 2.90 ± 0.5 n.d. 2.90 13.0 ± 0.3 5.01 ± 0.7 7.85 ± 0.0 1.65 ± 0.1 1.60 ± 0.4 1.42 ± 0.0 30.5
W13 6.68 ± 0.4 3.21 ± 0.2 n.d. 9.89 15.5 ± 0.3 n.d. n.d. n.d. 0.91 ± 0.0 n.d. 16.4
S01 4.42 ± 0.4 n.d. n.d. 4.42 37.6 ± 0.5 4.80 ± 0.4 9.87 ± 1.5 n.d. n.d. 1.08 ± 0.0 54.1
S02 9.71 ± 0.5 1.27 ± 0.6 n.d. 11.0 n.d. n.d. n.d. n.d. n.d. n.d. 0.00
S03 7.82 ± 2.1 n.d. n.d. 7.82 29.7 ± 1.9 10.8 ± 0.9 9.04 ± 1.7 3.33 ± 0.5 1.32 ± 0.3 1.90 ± 0.0 56.1
S04 10.7 ± 0.8 n.d. 1.30 ± 0.0 12.0 42.9 ± 0.9 n.d. 10.7 ± 4.8 n.d. n.d. 1.91 ± 0.4 55.5
S05 n.d. 14.1 ± 0.0 1.09 ± 0.0 15.2 13.1 ± 0.1 n.d. n.d. 4.24 ± 0.0 4.06 ± 0.2 n.d. 21.4
S06 9.20 ± 0.1 n.d. n.d. 9.20 19.4 ± 0.4 8.24 ± 0.2 5.96 ± 0.1 n.d. n.d. 1.06 ± 0.1 34.6
S07 4.45 ± 0.9 n.d. n.d. 4.45 13.5 ± 0.4 n.d. 4.31 ± 1.1 2.36 ± 0.1 n.d. n.d. 20.1
S08 5.91 ± 3.4 n.d. 1.56 ± 0.0 7.47 6.29 ± 0.3 3.92 ± 0.0 n.d. n.d. n.d. n.d. 10.2
S09 10.2 ± 0.3 1.37 ± 0.0 2.12 ± 0.8 13.7 10.5 ± 0.3 n.d. n.d. n.d. 2.04 ± 0.1 n.d. 12.5

Values are means of triplicate determination (n = 3) ±standard deviation; n.d.: not detected.
 104
Table 2
Concentration (mg L−1 ) of flavanols and other no phenolic compounds detected in the white and sherry wines
Wine Flavanols (mg L−1 ) Other no phenolic compounds (mg L−1 )

M.S. Fernández-Pachón et al. / Analytica Chimica Acta 563 (2006) 101–108

Catechin Procyanidin Epigallocatechin Epicatechin Total 5-Hydroxy 2-Fural- Tyrosol Tryptophol Aldehyde Aldehyde p-Hydroxy benzoic
B1 gallate methylfurfural dehyde syringic vanillic aldehyde

W01 n.d. 2.60 ± 0.00 8.40 ± 0.00 n.d. 11.0 n.d. 0.39 ± 0.1 n.d. n.d. n.d. n.d. n.d.
W02 n.d. n.d. n.d. n.d. 0.00 0.99 ± 0.4 n.d. 4.76 ± 4.7 1.06 ± 0.2 n.d. n.d. n.d.
W03 n.d. 4.00 ± 0.60 15.6 ± 0.00 n.d. 19.6 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
W04 n.d. n.d. n.d. n.d. 0.00 n.d. 0.66 ± 0.0 1.51 ± 0.0 n.d. n.d. n.d. n.d.
W05 n.d. n.d. n.d. n.d. 0.00 n.d. n.d. n.d. 0.86 ± 0.1 n.d. n.d. n.d.
W06 n.d. n.d. 2.70 ± 0.00 n.d. 2.70 1.67 ± 0.0 1.60 ± 0.1 n.d. 0.71 ± 0.0 n.d. n.d. n.d.
W07 11.1 ± 1.6 8.60 ± 0.80 n.d. 10.4 ± 2.1 30.1 0.67 ± 0.1 n.d. 8.39 ± 0.0 1.58 ± 0.0 n.d. n.d. n.d.
W08 n.d. n.d. n.d. n.d. 0.00 n.d. 1.17 ± 0.3 n.d. n.d. n.d. n.d. n.d.
W09 n.d. n.d. 5.80 ± 0.40 n.d. 5.80 1.81 ± 0.2 0.77 ± 0.1 11.9 ± 2.6 n.d. n.d. n.d. n.d.
W10 n.d. n.d. n.d. n.d. 0.00 1.20 ± 0.2 0.05 ± 0.0 2.43 ± 1.7 n.d. n.d. n.d. n.d.
W11 n.d. n.d. n.d. n.d. 0.00 1.38 ± 0.0 0.15 ± 0.0 n.d. n.d. n.d. n.d. n.d.
W12 n.d. n.d. n.d. n.d. 0.00 n.d. n.d. 3.60 ± 0.0 n.d. n.d. n.d. n.d.
W13 n.d. n.d. n.d. n.d. 0.00 n.d. n.d. n.d. 3.04 ± 1.1 n.d. n.d. n.d.
S01 n.d. 12.6 ± 4.70 1.80 ± 0.00 n.d. 14.4 n.d. 0.20 ± 0.0 n.d. n.d. n.d. n.d. n.d.
S02 3.55 ± 0.1 32.6 ± 2.20 13.7 ± 5.90 3.36 ± 0.9 53.2 n.d. 0.24 ± 0.1 10.8 ± 2.5 n.d. n.d. n.d. n.d.
S03 n.d. n.d. 6.10 ± 0.00 n.d. 6.10 63.8 ± 3.3 3.04 ± 0.6 n.d. n.d. n.d. n.d. n.d.
S04 n.d. n.d. 6.50 ± 0.00 n.d. 6.50 n.d. 0.34 ± 0.0 45.6 ± 0.0 n.d. n.d. n.d. n.d.
S05 n.d. n.d. n.d. n.d. 0.00 36.2 ± 1.1 8.41 ± 1.3 40.5 ± 0.0 n.d. n.d. n.d. 2.34 ± 0.6
S06 n.d. 28.6 ± 0.00 10.1 ± 0.00 n.d. 38.7 n.d. n.d. 2.33 ± 0.3 n.d. n.d. n.d. n.d.
S07 n.d. 6.90 ± 1.70 n.d. n.d. 6.90 1.52 ± 0.0 0.35 ± 0.0 19.4 ± 0.7 n.d. n.d. n.d. n.d.
S08 n.d. n.d. 3.70 ± 0.40 n.d. 3.70 22.3 ± 0.9 3.29 ± 0.5 25.7 ± 1.9 n.d. n.d. n.d. 0.74 ± 0.1
S09 n.d. n.d. 7.40 ± 0.40 n.d. 7.40 19.9 ± 0.1 8.87 ± 0.3 11.2 ± 1.7 n.d. 4.29 ± 0.1 0.55 ± 0.3 2.14 ± 0.1

Values are means of triplicate determination (n = 3) ±standard deviation; n.d.: not detected.
 M.S. Fernández-Pachón et al. / Analytica Chimica Acta 563 (2006) 101–108 105

est content among the cinnamic acids and derivatives (mean Table 4
values are 17.8 mg L−1 in table white wines and 19.2 mg L−1 Antioxidant activity of wines determined by ORAC, ABTS and DPPH methods
in sherry wines). The concentrations of phenolic acids obtained Wines ORAC (␮M) ABTS (mM) DPPH (mM)
are very similar to those described in French wines [30] and W01 425 ± 59 0.54 ± 0.05 0.65 ± 0.02
from Penedés [26]. Between flavanols, procyanidin B1 was the W02 717 ± 51 0.53 ± 0.07 0.58 ± 0.02
major component. Other no phenolic compounds were identi- W03 1096 ± 397 0.69 ± 0.06 0.80 ± 0.03
fied and considered for statistical analysis. Tyrosol is present W04 953 ± 185 0.21 ± 0.02 0.47 ± 0.04
in both types of wine in amount (mean values are 2.51 and W05 1019 ± 156 0.28 ± 0.05 0.60 ± 0.02
W06 1168 ± 136 0.29 ± 0.03 0.62 ± 0.02
17.3 mg L−1 in table white and sherry wines, respectively); 5- W07 1727 ± 592 0.58 ± 0.06 0.95 ± 0.10
hydroxymethylfurfural has a high concentration in sherry wines W08 609 ± 8 0.30 ± 0.02 0.54 ± 0.03
(mean value is 16.0 mg L−1 ). Certain phenolic compounds, such W09 1660 ± 45 0.33 ± 0.03 0.63 ± 0.04
as vanillic acid, gentisic acid, ferulic acid, procyanidin B2, (−)- W10 570 ± 133 0.14 ± 0.01 0.39 ± 0.04
epigallocatechin, (−)-epicatechin gallate, kaempferol, quercetin W11 840 ± 28 0.59 ± 0.06 0.62 ± 0.01
W12 2135 ± 313 0.37 ± 0.03 0.53 ± 0.02
or trans-resveratrol were not present in the samples, although W13 1564 ± 281 1.45 ± 0.29 2.21 ± 0.33
have been described in similar wines [31].
S01 1118 ± 200 0.26 ± 0.03 0.61 ± 0.04
Tables 3 and 4 present the AA of phenolic standard com-
S02 1391 ± 47 0.57 ± 0.04 0.88 ± 0.04
pounds and wines under study. These data have been reported S03 1782 ± 96 0.27 ± 0.03 0.98 ± 0.11
previously [21,15]. S04 1300 ± 150 0.21 ± 0.05 0.93 ± 0.08
We tested if a single phenolic concentration was corre- S05 1291 ± 9 0.08 ± 0.01 0.58 ± 0.01
lated with AA of wines. Correlation coefficients are shown in S06 1336 ± 103 0.57 ± 0.05 0.92 ± 0.01
S07 1158 ± 109 0.19 ± 0.02 0.49 ± 0.02
Table 5. The correlation coefficients of each compound vary
S08 1304 ± 71 0.11 ± 0.01 0.73 ± 0.06
according to the method used. The ORAC assay presents the S09 995 ± 102 0.11 ± 0.02 0.89 ± 0.03
highest number of significant correlation coefficients (p < 0.05).
W: white wine; S: sherry wine.
For both types of wine, gallic acid, glucoside of cutaric acid,
5-hydroxymethylfurfural and tyrosol showed a high linear
correlation with the ORAC values. Ethyl caffeate and (−)-
epigallocatechin gallate are the phenolic compound with larger reported in Table 5. As can be seen, flavanols are the group with
correlation coefficient with all tests under study. A good corre- largest coefficients with all methods for both types of wines. The
lation between (−)-epigallocatechin gallate concentration and correlation is smaller in the case of benzoic and cinnamic acids.
DPPH method has been reported [31,32]. It must be considered AA of wines is not neccesarily correlated with the compounds
that gallic acid, (−)-epigallocatechin gallate and tyrosol showed which present highest concentrations. For instance, caftaric acid
also the highest concentrations in benzoic acids, flavanols and is the quantitative largest of cinnamic acid; nevertheless presents
“other compounds”, respectively. a weak correlation with the AA in both types of wines for all
In order to test if a phenolic class of compounds could achieve the methods involved. Chemical structure determines AA val-
for the AA of wines, we summed the concentrations of pheno- ues. AA of phenolic compounds was analysed previously with
lic compounds with similar chemical structure, that is: benzoic available commercial pure standards [21].
acids, cinnamic acids and flavanols. Correlation coefficients are The contribution to wine AA of each phenolic compound has
been calculated, multiplying its concentration in the wine by its
activity value. Thus, theorical values were obtained. Table 6
Table 3
shows the medium contribution (%) of phenolic compounds to
Antioxidant activity values of standard compounds determined by triplicate
the values of total AA of table white and sherry wines. As can
Phenolic compound ABTS (mM) DPPH (mM) ORAC (␮M) be seen the wine AA is larger than the sum of their phenolic
Gallic acid 1.98 ± 0.01 2.66 ± 0.06 6.97 ± 2.04 compounds (12.53% of total table white wine ORAC value).
Protocatechuic acid 0.32 ± 0.01 1.29 ± 0.09 18.16 ± 2.10 The contribution of gallic and caffeic acids to the AA of table
Syringic acid * 1.26 ± 0.05 8.27 ± 0.63 white wines is relevant whatever the method used. In sherry
Caffeic acid 1.01 ± 0.00 1.11 ± 0.01 15.28 ± 0.39
wines, the single compound contributing in a large extent is
p-Coumaric acid * * 10.16 ± 2.09
Ferulic acid 0.23 ± 0.00 0.60 ± 0.03 8.48 ± 1.10 gallic acid (40% with ABTS AA). The contribution of the rest
Caftaric acid 0.21 ± 0.00 0.76 ± 0.00 n.d. of compounds to the AA of wines is less than 15%. Besides these
(+)-Catechin 0.57 ± 0.01 2.16 ± 0.16 12.22 ± 2.01 two, tyrosol and protocatechuic acid contribute to ORAC value
(−)-Epicatechin 0.99 ± 0.00 3.22 ± 0.19 11.69 ± 1.63 of table white wines (2.14 and 2.07%, respectively). On the other
(−)-Epicatechin gallate 3.00 ± 0.04 3.46 ± 0.14 3.97 ± 1.35
hand, caftaric acid and (−)-epigalocatechin gallate account for
(−)-Epigallocatechin gallate 2.69 ± 0.07 3.58 ± 0.21 5.37 ± 1.64
Procyanidin B1 1.88 ± 0.22 3.92 ± 0.33 8.99 ± 0.54 a remarkable activity measured by methods ABTS and DPPH.
Procyanidin B2 1.83 ± 0.08 4.35 ± 0.01 7.82 ± 0.61 The percentage contribution of (−)-epigallocatechin gallate
Tyrosol * * 10.59 ± 1.61 and procyanidin B1 is higher in sherry wines than in analysed
2-Furaldehyde n.d. n.d. 1.76 ± 0.11 table white wines. These compounds are present in most of
Syringaldehyde n.d. n.d. 5.11 ± 0.05
sherry wine samples than table white ones. Hence, average con-
(*) Does not react at the conditions described; n.d.: no detected. centration is higher.
106 M.S. Fernández-Pachón et al. / Analytica Chimica Acta 563 (2006) 101–108

Table 5
Correlation coefficients (r) found from linear correlation analysis (95% significance level) between antioxidant activity values of wines and concentration of phenolic
compounds
Phenolic compounds W S

ORAC ABTS DPPH ORAC ABTS DPPH

* *
Gallic acid 0.8942 0.5448 0.5094 0.8263 0.2967 0.6751
Protocatechuic acid 0.3937 0.3776 0.2806 0.2681
Syringic acid 0.9634*
Benzoic acids 0.6771 0.6812 0.7173* 0.6984 0.0775 0.4320
Caffeic acid 0.5643 0.5028 0.8600* 0.2200 −0.4986 −0.0490
p-Coumaric acid 0.4774 0.2317 0.7312* −0.3872 −0.6473 −0.1092
Caftaric acid 0.5508 −0.1266 −0.0400 0.2986 0.0547 0.3499
Glucoside of cutaric acid 0.9087* 0.0229 0.3206 0.8508* 0.0431 0.5091
Cutaric acid 0.7218* −0.1657 0.4590 0.2148 −0.3962 0.1123
Ethyl caffeate 0.9309* 0.7765* 0.7566* 0.6309 −0.0221
Cinnamic acids 0.6802 0.1390 −0.0638 0.7028* -0.2423 0.2222
(−)-Epigallocatechin gallate 0.4011 0.9614* 0.9567* 0.7766* 0.7501* 0.6182
Procyanidin B1 0.9901* 0.9769*
Flavanols 0.6313 0.9761* 0.7658* 0.7798* 0.9464* 0.2605
5-Hydroxymethylfurfural 0.8938* −0.3373 0.7295* 0.8587* −0.4403 0.2476
2-Furaldehyde 0.6939 −0.5659 -0.3476 0.2271 −0.6041 −0.0497
p-Hydroxybenzoic aldehyde −0.4306
Tyrosol 0.8458* 0.8327* −0.8416 −0.2906
Tryptophol 0.6206 0.6443 0.7317*

W: table white wines; S: sherry wines.

* Significant linear correlation.

Table 6 (Continued )

Table 6 Phenolic compounds Methods W S

Average contribution (%) of phenolic compounds to the antioxidant activity of Average (n = 13) Average (n = 9)
wines (+)-Catechin ORAC 0.60 ± 2.18 0.35 ± 1.04
Phenolic compounds Methods W S ABTS n.d. n.d.
Average (n = 13) Average (n = 9) DPPH n.d. n.d.
(−)-Epicatechin ORAC 0.54 ± 1.88 0.31 ± 0.94
Gallic acid ORAC 2.83 ± 3.49 3.80 ± 2.08
ABTS 0.35 ± 1.02 n.d.
ABTS 7.36 ± 6.58 40.8 ± 31.7
DPPH 0.76 ± 2.08 n.d.
DPPH 5.90 ± 5.61 14.3 ± 5.95
(−)-Epigallocatechin gallate ORAC 0.92 ± 1.90 1.78 ± 4.13
Protocatechuic acid ORAC 2.07 ± 3.13 2.66 ± 6.49
ABTS 2.94 ± 4.86 13.3 ± 12.2
ABTS 0.54 ± 0.72 0.59 ± 1.58
DPPH 2.79 ± 5.04 4.92 ± 3.99
DPPH 1.38 ± 1.79 1.11 ± 2.81
(−)-Epicatechin gallate ORAC n.d. n.d.
Syringic acid ORAC 0.07 ± 0.25 0.48 ± 0.63
ABTS 0.22 ± 0.78 n.d.
ABTS n.d. n.d.
DPPH 0.23 ± 0.82 n.d.
DPPH n.d. n.d.
Procyanidin B1 ORAC 0.27 ± 0.98 1.46 ± 4.37
Benzoic acids ORAC 4.98 ± 4.49 6.93 ± 5.78
ABTS 0.64 ± 1.41 6.94 ± 8.42
ABTS 7.89 ± 6.29 41.4 ± 31.5
DPPH 0.94 ± 1.93 7.75 ± 10.1
DPPH 7.28 ± 5.01 15.4 ± 7.38
Procyanidin B2 ORAC n.d. n.d.
ABTS n.d. 0.27 ± 0.80
Caffeic acid ORAC 2.16 ± 1.74 1.22 ± 1.92
DPPH n.d. 0.41 ± 1.22
ABTS 2.32 ± 1.55 5.22 ± 9.86
DPPH 1.64 ± 1.34 1.17 ± 1.55 Flavanols ORAC 2.34 ± 4.26 3.90 ± 6.95
ABTS 4.14 ± 5.52 20.5 ± 12.1
Caftaric acid ORAC n.d. n.d.
DPPH 4.72 ± 7.00 13.1 ± 13.7
ABTS 4.08 ± 3.70 6.23 ± 4.57
DPPH 7.96 ± 5.26 5.60 ± 4.16 2-Furaldehyde ORAC 0.08 ± 0.11 0.40 ± 0.57
ABTS n.d. n.d.
p-Coumaric acid ORAC 0.83 ± 0.90 0.67 ± 1.17
DPPH n.d. n.d.
ABTS n.d. n.d.
DPPH n.d. n.d. Tyrosol ORAC 2.14 ± 2.90 14.5 ± 13.9
ABTS n.d. n.d.
Ferulic acid ORAC n.d. 0.07 ± 0.21
DPPH n.d. n.d.
ABTS n.d. 0.40 ± 0.41
DPPH n.d. 0.31 ± 0.30 Syringaldehyde ORAC n.d. 0.24 ± 0.73
ABTS n.d. n.d.
Cinnamic acids ORAC 2.99 ± 2.47 1.96 ± 2.74
DPPH n.d. n.d.
ABTS 6.40 ± 4.53 11.9 ± 13.0
DPPH 9.61 ± 5.72 6.98 ± 4.98 W: table white wines (n = 13); S: sherry wines (n = 9); n.d.: no detected.
 M.S. Fernández-Pachón et al. / Analytica Chimica Acta 563 (2006) 101–108 107

AA of wines does not rely on one single phenolic compound
but on the sum of them. Indeed, when the contribution of com-
pounds of the same class is summed, the percentages increase.
Multivariate statistical analysis was applied to data. The pur-
pose was to obtain a linear equation that can explain the AA of
the wine of the type:
AAwine = a + b × AAgallic ac + c × AAprotocatechuic
+d × AAsyringic ac + ······
AA values of phenolic compounds (obtained multiplying its
concentration in the wine by its activity value determined with
standard compounds) were taken into account. AA of the wine
is the dependent variable and the theoretical AA of phenolic
compounds are independent variables. The obtained multiple
regression equations are the following:
For ABTS and DPPH methods, the inclusion in the model of
several compounds does not improve the correlation coefficients
obtained with the single concentrations.
However, multiple regression of phenolic compounds with
ORAC values substantially improves the regression coefficients
of individual phenolic concentrations, being wines ORAC values
better related with its phenolic profile than for the other AA
ac methods used.
Acknowledgements
The authors thank the Spanish Government (Ministerio
de Ciencia y Tecnologı́a) for financial support (Research
Projects 2001-2368 and 2001-2396) and the Regional Gov-
ernment (Junta de Andalucı́a) (2003-AGR 167) for a research
grant.

ORAC assay:
ORACwhite wine = −1270.28 + 0.587 × ORACgallic ac + 0.431 × ORACprotocatechuic ac − 0.18 × ORAC2-furaldehyde
+ 0.359 × ORACtyrosol + 0.456 × ORACcaffeic ac + 0.324 × ORACepigallocatechin gallate

−0.68 × ORACcoumaric ac

r = 0.9028
ORACsherry wine = 2693.76 + 0.117 × ORACgallic ac + 0.474 × ORACprotocatechuic ac − 0.30 × ORACtyrosol
+0.256 × ORACcaffeic ac + 0.139 × ORACepigallocatechin gallate − 0.86 × ORACsyringic ac

−1.2 × ORACcoumaric ac

r = 0.9327
ABTS method:

TEACwhite wine = −0.6042 + 0.608 × TEACgallic ac + 0.280 × TEACepigallocatechin gallate + 0.290 × TEACprocyanidin B1

r = 0.6236

TEACsherry wine = 0.0242 − 0.21 × TEACgallic ac + 0.418 × TEACepigallocatechin gallate + 0.488 × TEACprocyanidin B1

r = 0.7404
DPPH method:

TEACwhite wine = −0.7892 + 0.646 × TEACgallic ac + 0.119 × TEACepigallocatechin gallate + 0.401 × TEACprocyanidin B1

r = 0.6195

TEACsherry wine = 0.3245 + 0.280 × TEACgallic ac − 0.19 × TEACepigallocatechin gallate + 0.652 × TEACprocyanidin B1

r = 0.7410
108 M.S. Fernández-Pachón et al. / Analytica Chimica Acta 563 (2006) 101–108
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