Journal of Food Composition and Analysis 24 (2011) 265–269

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Journal of Food Composition and Analysis

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Short communication

The influence of two different pH levels on the antioxidant properties

of flavonols, flavan-3-ols, phenolic acids and aldehyde compounds
analysed in synthetic wine and in a phosphate buffer
Danila Di Majo a,*, Laura La Neve a, Maurizio La Guardia a, Alessandra Casuccio b,
Marco Giammanco a
a
Division of Physiology and Human Nutrition, Department of Medicine, Pneumology, Physiology and Human Nutrition, University of Palermo, Via A. Elia 3, 90127 Palermo, Italy
b
Department of Clinical Neuroscience, University of Palermo, Via La Loggia 1, 90100 Palermo, Italy

A R T I C L E I N F O A B S T R A C T

Article history: The aim of this study is evaluate either the antioxidant or pro-oxidant behaviour of some typical
Received 1 October 2009 polyphenolic compounds of red wine, to investigate the influence of two different pH levels on the
Received in revised form 2 September 2010 antioxidant properties and to clarify their activity–structure relationship. The antioxidant activity of
Accepted 6 September 2010
compounds in hydrophilic solutions at pH 3.5 and pH 7.4 were measured by a competition kinetic test,
Available online 7 December 2010
based on the crocin bleaching. The position and the number of substitution groups influence the
magnitude of the antioxidant activity of the polyphenolic compounds, but their antioxidant properties
Keywords:
are also strongly influenced by the pH conditions. Increasing the pH, a considerable increase in
Antioxidant activity
antioxidant capacity was observed in all compounds analysed. This study produces information about
Polyphenols
pH the antioxidant properties of the studied compounds at pH 3.5. It is important to clarify that the specific
Crocin action measured in vitro does not necessarily reflect in vivo antioxidant properties.
Red wine ß 2010 Elsevier Inc. All rights reserved.
Food composition

1. Introduction influence of pH on the antioxidant activity is seldom taken into

Some epidemiological evidence suggests beneficial effects on
human health from a diet rich in fruit, vegetables and certain types
of beverages such as tea and wine (Lotito and Frei, 2006;
Fernández-Pachoń et al., 2005; Nigdikar et al., 1998; Wollgast
and Anklam, 2000). The protective effects against cardiovascular
diseases (Serafini et al., 1998, 2000; Chatterjee et al., 2005), certain
types of cancer (Caprino and Russo, 2006), inflammatory
(Fernández-Pachoń et al., 2005; Ghiselli et al., 2000) and age-
related diseases have been partly attributed to antioxidant
constituents (Fernández-Pachoń et al., 2004).
These compounds do not have nutritive properties but they
have a functional role in the human organism (Di Majo et al.,
2008). They trap free radicals through hydrogen atom donation
(Tyrakowska et al., 1999) or electron donation (Lemanska et al.,
2001).
Their antioxidant efficiency has been related to the number of
hydroxyl groups in the molecule, but the mechanism of their action
has not yet been described in full detail. Furthermore, the possible
account. Some studies have investigated the effect of pH on the
antioxidant properties of polyphenols (Tyrakowska et al., 1999;
Lemanska et al., 2001) and have reported that the pH-dependent
behaviour is related to hydroxyl deprotonation. However, these
studies did not compare the antioxidant activity of various classes
of polyphenols and did not take into account the acidic pH.
Furthermore, these authors did not use the crocin bleaching assay.
The aim of this study was to evaluate either the antioxidant or pro-
oxidant behaviours of various polyphenol compounds by the
crocin bleaching assay (CBA) in order to investigate the influence of
two different pH levels. The behaviour of polyphenol compounds
was analysed at pH 3.5 to simulate the food source; on the other
hand, the same compounds were evaluated in a reaction mixture at
pH 7.4 to simulate the in vivo condition. In addition, the effect of
number and position of hydroxyl, methoxy and carboxyl sub-
stituents was explored.
2. Materials and methods
2.1. Standards preparation

* Corresponding author. Tel.: +390916236405, fax: +390916236407. Thirteen polyphenols were analysed: (+)-catechin, ()-epica-
E-mail address: daniladimajo@unipa.it (D. Di Majo). techin, myricetin, quercetin, kaempferol, gallic acid, caffeic acid,

0889-1575/$ – see front matter ß 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2010.09.013
266 D. Di Majo et al. / Journal of Food Composition and Analysis 24 (2011) 265–269
Table 1
Composition of the synthetic wine solution.
Synthetic wine Amount to a final volume of
1 L of distilled water
Tartaric acid 4.5 g
Malic acid 1.5 g
Acetic acid 0.11 mg
MgSO4 26 mg
K2SO4 0.1 g
Ethanol 135 mL
syringic acid, syringaldehyde, sinapic acid, ferulic acid, vanillic acid
and vanillin. All the polyphenols were purchased from Sigma–
Aldrich.
They were used at 1 mmol L1 in a phosphate buffer (pH 7.4)
and in synthetic wine (pH 3.5). The myricetin, quercetin and
kaempferol were prepared at concentrations of 0.5 mmol L1 in
both pH, because with an increasing concentration of these
compounds, a decreased sensitivity of the method was observed.
The phosphate buffer was prepared by mixing 7.1 g of KH2PO4 (J.T.
Baker chemicals) and 14.4 g of Na2HPO42H2O (J.T. Baker
chemicals) in distilled water to a final volume of 1 L. The synthetic
wine was prepared according to Table 1 (Barrera Garcia et al.,
2007) and the solution pH was adjusted to 3.5 using NaOH
1 mol L1. Tartaric acid, malic acid, MgSO4, acetic acid and ethanol
were purchased from Fluka.
2.2. Antioxidant activity by crocin bleaching assay (CBA)
The crocin bleaching assay (Ordoudi and Tsimidou, 2006b; Di
Majo et al., 2005; Tubaro et al., 1998) was used to determine the
antioxidant or pro-oxidant activity of all compounds.
The method is based on the crocin bleaching as result of its
oxidation by peroxyl radicals produced from 2,20 -azobis-(2-amidi-
nopropano-dihydrochloride) (AAPH) (Wako Chemicals). Crocin was
extracted twice with methanol, from commercial saffron according
to the validated procedure (Ordoudi and Tsimidou, 2006a) and its
concentration was estimated using the extinction coefficient
reported in the Literature, eMeOH = 1.33  105 M1 cm1. Crocin
working solution (Abs443  1) and AAPH solution (10 mg/mL) were
prepared with phosphate buffer (pH 7.4) or synthetic wine (pH 3.5).
The reaction started with the addition of AAPH (150 mL) to the
reaction mixture containing 150 mL of diluted crocin solution and
increasing volumes (20–150 mL) of standard solutions and solvents
(phosphate buffer or synthetic wine) to a total volume of 1 mL. The
rate of crocin bleaching is followed as decrease of absorbance at
443 nm (Tubaro et al., 1996), using a Beckman 640 UV–vis
spectrophotometer, against a blank composed of the same reagents
without crocin. This bleaching rate, at 443 nm in the absence (V0)
and in presence (Va) of the antioxidant sample, was monitored at
37 8C for 10 min. Various levels of standard compounds (150–
20 mmol L1) were tested so that linear regression curves of relative
rates V0/Va vs. the [antioxidant]/[crocin] rates can be built and the
value Ka/Kc from the slope of the linear regression can be calculated
(Di Majo et al., 2008). Ka/Kc indicates the relative capacity
(antioxidant activity) of different molecules to interact with peroxyl
radicals:
V0 K a ½A
¼1þ  OH
Va K c ½C
The antioxidant activity of analysed compounds was expressed as
mean value  standard deviation of three replications and the Ka/Kc
obtained was multiplied by a thousand. Moreover, the results were
expressed as a Trolox equivalent (TRE), dividing the Ka/Kc value of
each analysed compound by the Ka/Kc of Trolox solution
(1 mmol L1); the Ka/Kc values of Trolox are 6.68  0.57 (pH 7.4)
and 3.83  0.92 (pH 3.5).
2.3. Statistical analysis
Each experiment was repeated three times and the results
reported are the means of the three trials. Data were analysed by
the SPSS Software version 14.0 (SPSS, Inc., Chicago, IL, US) by the
one-way analysis of variance (ANOVA), using the Duncan’s
multiple range test. In all the performed analyses, differences
were considered statistically significant at p  0.05.
3. Results and discussion
3.1. Influence of pH on antioxidant activity of flavonols
The antioxidant properties of three flavonols (Fig. 1) were
measured. Employing CBA at pH 3.5, no difference in the
calculated Ka/Kc values was observed. Kaempferol, quercetin
and myricetin have the same antioxidant activity (p > 0.05). The
analysis of the same compounds at pH 7.4 showed that the
kaempferol (single OH group in the B-ring) has a lower
antioxidant activity (Ka/Kc = 1.96  0.02) than both quercetin and
myricetin (two and three OH groups, respectively) which have an
antioxidant power of 4.60  0.55 and 10.74  1.16, respectively.
These results are consistent with those reported by Lemanska
(Lemanska et al., 2001) and show the importance of OH groups in the
B-ring on the antiradical activity of flavonols. The dihydroxy and
trihydroxy structures are more active than kaempferol because the
30 ,40 -catechol moiety is able to stabilize the corresponding radical
through the formation of an intramolecular hydrogen bond.
Furthermore, according to Lemanska the relative high antioxidant
activity of quercetin may be related to the fact that in this compound
two rather than one active antioxidant structural elements are
present. Both the 30 ,40 -catechol moiety but also 3,5,7-triOH may act
as radical scavenging units. In addition, as shown in Table 2, a
considerable increasing in antioxidant activity was observed with an
increase in the pH. An explanation for this could be that at
physiological pH, the antioxidant value of the polyhydroxyflavones
is a combination of the activity of the neutral as well as of the
deprotonated form in different molar ratios, whereas at pH 3.5 the
neutral form is prevalent. In the neutral form the mechanism of
antioxidant action, i.e. hydrogen atom or electron donation may vary
with the hydroxyl substituent pattern of the hydroxyflavone. It is
generally assumed that the mechanism for antioxidant action of
[()TD$FIG]
OH OH
OH OH
HO O HO O
OH OH
OH OH
(+)-catechin (-)-epicatechin
R'3
HO O R’3 R’5
R'5 H H Kaempferol
OH
OH H Quercetin
OH OH Myricetin
OH O
Fig. 1. Structure of the flavan-3-ols and flavonols.
 D. Di Majo et al. / Journal of Food Composition and Analysis 24 (2011) 265–269 267

Table 2
Antioxidant activity of the flavan-3-ol and flavonol compounds at different pH of the reaction environment.

Compound OH substituent C ring Family Antioxidant activitya TRE valueb

pH 3.5 pH 7.4 pH 3.5 pH 7.4

Catechin 30 ,40 Flavan-3-ol 0.17  0.08c 4.34  0.18d 0.04 0.64

Epicatechin 30 ,40 Flavan-3-ol 0.25  0.11 3.58  0.23d 0.06 0.53
Kaempferol 40 Flavonol 0.48  0.14 1.96  0.02e 0.12 0.29
Quercetin 30 ,40 Flavonol 0.46  0.12c 4.60  0.55e 0.12 0.68
Myricetin 30 ,40 ,50 Flavonol 0.24  0.09 10.74  1.16e 0.06 1.58
a
The results were expressed as Ka/Kc, as mean value  standard deviation of three experiments and after multiplied by thousand.
b
Antioxidant activity expressed as Trolox equivalents.
c
p < 0.01, catechin vs. quercetin.
d
p < 0.01, catechin vs. epicatechin.
e
p < 0.001, myricetin and quercetin vs. kaempferol.

polyphenols in their neutral form may be hydrogen atom donation
(Jovanovic et al., 1994; Sauerwald et al., 1998).
3.2. Influence of pH on antioxidant activity of flavan-3-ol
The antioxidant properties of (+)-catechin and ()-epicatechin
(Fig. 1) were measured. With increasing medium pH the
antioxidant activity of flavan-3-ols increase significantly on its
turn. Flavan-3-ols showed the same trend of flavonols. These
molecules have the same antioxidant activity at pH 3.5 (p = 0.286),
but different antioxidant activities were observed at pH 7.4. In fact
the antioxidant activity of (+)-catechin is higher than ()-
epicatechin (Table 2). This result is the principal effect attributed
to the stereochemistry of hydroxyl groups in the C-ring and this
confirms the results of other Authors (Mendoz-Wilson and
Glossman-Mitnik, 2006). S-configuration of OH group in the C-
ring seems to be a more favourable a structural feature than R-
configuration. ()-Epicatechin has a more twisted structure, and
the molecules with more B-ring twisting are regularly weaker or
inactive (Rice-Evans et al., 1995). Therefore, an unfavourable
conformation of the heterocyclic ring contributes to a lower
activity of the ()-epicatechin. (+)-Catechin and ()-epicatechin
have an identical number of hydroxyl groups in the same position
as quercetin, but they do not contain the 2,3-double bond and the
4-oxo group in the C ring; these structural differences do not confer
an enhancement or reduction of the antioxidant activity. Quercetin
has approximately the same Ka/Kc value at pH 7.4, in comparison
with (+)-catechin and ()-epicatechin (p = 0.472). At physiological
pH the antioxidant values of the two compounds are a combination
of the activity of the neutral as well as of the deprotonated form. At
this pH level the mechanism for the antioxidant action can be
either hydrogen atom and/or electron donation (Lemanska et al.,
2001). These results by crocin bleaching assay reveal that the 4-oxo
group in conjugation with the 2,3-double bond on the A ring
contribute only a little to the hydrogen-donating ability. On the
other hand at pH 3.5 the antioxidant activity of quercetin is higher
than (+)-catechin (p = 0.010). An explanation of these results could
be related to the mechanism of hydrogen atom donation.
3.3. Influence of pH on the antioxidant activity of phenolic acids and
their aldehyde derivatives
Changing the pH, an increased antioxidant activity in phenolic
acids was also observed (Table 3). The pH effect on the antioxidant
activity could be attributed to different degree of dissociation of
COOH and OH groups. A considerable number of dissociated
carboxyl groups are present and ionization of OH groups is also
favoured, under CBA conditions at pH 7.4 (Ordoudi and Tsimidou,
2006b).
For phenolic acids, both pH 3.5 and 7.4 determine a diversifi-
cation in antioxidant potential in relationship to their chemical
structure (Fig. 2). Data produced from CBA (Table 3) indicate that
cinnamic acids are more active than the respective benzoic acids
under both pH conditions. Table 3 shows that the ferulic acid is
more active than the vanillic acid and the sinapic acid is more
active than the syringic, at pH 7.4. A similar behaviour was also
evidenced at pH 3.5, the sinapic acid is more efficient than syringic
acid but no difference in the antioxidant activity of ferulic and

Table 3
Antioxidant activity of the phenolic acids and aldehyde compounds at different pH of the reaction environment.

Compounds Structure Family Antioxidant activitya TRE valueb

pH 3.5 pH 7.4 pH 3.5 pH 7.4

Gallic acid 3,4,5-triOH Benzoic 0.27  0.03c 42.12  1.40c 0.07 6.19
Syringic acid 3,5-diOMe-4-OH Benzoic 0.12  0.03c,d 9.79  1.89c,d,e,f 0.03 1.44
Vanillic acid 3-OMe-4-OH Benzoic 0.05  0.04 0.76  0.02e,g,h 0.01 0.11
Sinapic acid 3,5-diOMe-4-OH Cinnamic 0.37  0.01d,i 17.51  1.68d,i 0.10 2.57
Caffeic acid 3,4-diOH Cinnamic 0.20  0.05j 9.19  1.10j 0.05 1.35
Ferulic acid 3-OMe-4-OH Cinnamic 0.14  0.04i,j 5.38  0.69g,i,j 0.04 0.79
Syringaldehyde 3,5-diOMe-4-OH Aldehyde 0.12  0.01 23.86  6.58f,k 0.03 3.51
Vanillin 3-OMe-4-OH Aldehyde 0.11  0.01 2.37  0.27h,k 0.03 0.35
a
The results were expressed as Ka/Kc, as mean value  standard deviation of three experiments and after multiplied by thousand.
b
Antioxidant activity expressed as Trolox equivalent.
c
p < 0.01, gallic acid vs. syringic acid at both pH.
d
p < 0.01, sinapic acid vs. syringic acid at both pH.
e
p < 0.01, syringic acid vs. vanillic acid.
f
p < 0.01, syringaldehyde vs. syringic acid.
g
p < 0.01, ferulic acid vs. vanillic acid.
h
p < 0.01, vanillin vs. vanillic acid.
i
p < 0.01, sinapic acid vs. ferulic acid at both pH.
j
p < 0.01, caffeic acid vs. ferulic acid at both pH.
k
p < 0.01, syringaldehyde vs. vanillin.
[()TD$FIG]
268 D. Di Majo et al. / Journal of Food Composition and Analysis 24 (2011) 265–269
CH=CHCOOH
MeO OMe MeO
OH
sinapic acid
CH=CHCOOH
OMe
OH
ferulic acid
CH=CHCOOH
OH HO
OH
caffeic acid
Fig. 2. Structure of the cinnamic acids, benzoic acids and aldehyde compounds.
vanillic acid was observed. An explanation of this could be that the
proximity of the COOH substituent to the aromatic ring is not
beneficial for the antioxidant activity of phenol acids and the
insertion of an ethylenic group between the phenyl ring and the
carboxyl group has a favourable effect on the reducing properties
of the OH group. Moreover, the CH5 5CH–COOH group plays a role in
stabilizing the radical by resonance (Rice-Evans et al., 1995),
offering an explanation for the higher activity of cinnamic acids
compared to benzoic acids.
The antioxidant activity of two different aldehydes was
compared to those of the benzoic and cinnamic acids. The
syringaldehyde and the vanillin were found to be far more active
than the correspondent phenolic acids (Table 3). This result
deserves further elaboration, in fact an electron-withdrawing
effect of the aldehyde was foreseen and thus a lesser degree of
antioxidant activity. Moreover the antioxidant activity of the two
aldehydes was compared. Syringaldehyde is more active than
vanillin. This result shows as the presence of two methoxy groups
influences the redox potential of the phenol. The difference in
antioxidant activity is due to the differences in molecular planarity
and so to the differences in the stabilization of the corresponding
phenoxy radical.
3.3.1. Influence of hydroxylation on antioxidant activity of the
phenolic acids
The trihydroxy configuration to the benzoic ring was shown to
significantly enhance the antiradical performance of phenol acids
at both pH levels. Gallic acid presents three available hydroxyl
groups that can donate H to stabilize the free radicals. The
substitution of the hydroxyl group with the methoxy group
causes a decrease in antioxidant activity of phenol acids. In fact
gallic acid is more active than syringic with an increase of 76% in
antioxidant activity (Table 3) and caffeic acid results more active
than ferulic acid. These results are in agreement with general SAR
criteria concerning the effect of OH and OMe groups on the
COOH CHO
OMe MeO OMe
OH OH
syringic acid syringaldehyde
COOH CHO
OMe OMe
OH OH
vanillic acid vanillin
COOH
OH
OH
gallic acid
antioxidant activity of phenolic compounds (Ordoudi and
Tsimidou, 2006b).
3.3.2. Influence of methoxy groups on the antioxidant activity of the
phenolic acids
It was evidenced that the number and position of methoxy
groups on the phenyl ring affect the antioxidant activity of phenol
acids and aldehyde compounds, at both pH values. At pH 7.4, the
presence of the two methoxy groups in the phenyl ring enhances
the stabilization of the corresponding phenoxy radical formed after
abstraction of the H atom, compared to the molecules with only
one methoxy group adjacent to the OH group. In fact syringic acid
is more active than vanillic acid, as well as sinapic acid is more
active than ferulic acid, and syringaldehyde is more efficient than
vanillin (Table 3). This result depends on the electronic effect of
methoxy groups which help to stabilize the phenoxy radicals
formed after the reaction with free radicals. At pH 3.5, a similar
behaviour was obtained for sinapic acid that has shown a higher
antioxidant activity than ferulic acid, while no differences in the
antioxidant activity of syringic vs. vanillic acid was observed.
4. Conclusion
The data of this study show the influence of two different pH
levels on the antioxidant properties of flavonols, flavan-3-ols,
phenolic acids and aldehyde compounds. These data produce new
information about the antioxidant properties of the studied
compound at pH 3.5. The position and the total number of
hydroxyl and methoxy groups influence the magnitude and
mechanism of the antioxidant activity. This result is consistent
with those reported by other authors (Lemanska et al., 2001). The
flavonols and flavan-3-ols reveal differences in antioxidant
property only at physiological pH, whereas for phenolic acids,
both pH 3.5 and 7.4 determine a diversification in antioxidant
potential in relationship to their chemical structure. Data produced
 D. Di Majo et al. / Journal of Food Composition and Analysis 24 (2011) 265–269
from CBA indicate that cinnamic acids are more active than the
respective benzoic acids at both pH levels. The influence of the
carboxyl group in the phenolic acid is undeniable and the aldehyde
group confers a remarkable antioxidant activity to the aromatic
molecules. This result necessitates further studies. The similar
behaviour of quercetin and catechin at physiological pH and with
the crocin bleaching assay show that the double bond between C2
and C3 and the carbonyl group at C4 of the C-ring of flavonols also
influence antioxidant activity. On the other hand, this study
confirms that at pH 7.4 the 30 ,40 -catechol structure is important for
the antiradical activity of flavonoids, as it has been seen in
myricetin and quercetin vs. kaempferol.
Data produced indicate that the antioxidant properties of
polyphenol compounds are strongly influenced by pH level.
Increasing pH led to a considerable increase in observed
antioxidant activity. The polyphenolic compounds are more
sensitive to pH change than to the number and position of
substitution groups. It is important to clarify that the specific
action measured in vitro does not necessarily reflect the in vivo
antioxidant properties. In a biological system, in addition to
chemical and environmental behaviours, there are many factors
influencing antioxidant activity.
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