Food Chemistry 134 (2012) 1767–1774

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Food Chemistry
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Antioxidant mechanism of grape procyanidins in muscle tissues: Redox

interactions with endogenous ascorbic acid and a-tocopherol
Jacobo Iglesias a,⇑, Manuel Pazos a, Josep Lluís Torres b, Isabel Medina a
a
Instituto de Investigaciones Marinas (IIM-CSIC), Eduardo Cabello 6, E-36208 Vigo, Spain
b
Instituto de Química Avanzada de Catalunya (IQAC-CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The present study investigates the antioxidant mechanism of grape procyanidins and, in particular, their
Received 28 October 2011 aptitude to establish redox interactions with two important components of the endogenous antioxidant
Received in revised form 25 February 2012 system of muscle tissues, a-tocopherol (a-TOH) and ascorbic acid (AA). To this end, the progress of lipid
Accepted 19 March 2012
oxidation was monitored in fish muscle supplemented with grape procyanidins at the concentrations
Available online 28 March 2012
usually employed in antioxidant food applications, and then related to the redox stability of the endog-
enous a-TOH and AA. In addition to the lipid oxidation protective effect, the incorporation of procyani-
Keywords:
dins also provided an improvement of the redox stability of the endogenous components in a straight
Lipid oxidation
Ascorbic acid
procyanidinic concentration-dependent manner. Results showed the capacity of procyanidins to repair
a-Tocopherol oxidised a-TOH at medium-long term, and to delay the AA depletion. Therefore, such cooperative redox
Fish muscle interaction of exogenous procyanidins adequately complements the natural a-TOH regenerative system
Grape supplied by AA that is efficient at the early post mortem stages.
Procyanidins Ó 2012 Elsevier Ltd. All rights reserved.
Antioxidant

1. Introduction unsaturated lipids. However, this endogenous antioxidant barrier

Lipid oxidation is an important cause of quality deterioration in
muscle foods (Erickson, 2002). Among muscle foods, pelagic fish
muscle is particularly susceptible to lipid oxidation because of
the coexistence of highly oxidisable long chain polyunsaturated
fatty acids (PUFAs) and pro-oxidant substances with the ability
to catalyse lipid oxidation, such as redox-active metals and heme-
proteins (Erickson, 2002; Hultin, 1992). The levels of haemoglobin
typically found in pelagic flesh, which range from 3 to 12 lmol per
kg of muscle (Larsson, Almgren, & Undeland, 2007; Maestre, Pazos,
& Medina, 2011; Richards & Hultin, 2002), have demonstrated an
extensive pro-oxidant activity to catalyse lipid oxidation (Richards
& Hultin, 2002). The oxidative degradation of lipids provokes gen-
eration of volatile compounds responsible for rancid odours and
flavours, the reduction of PUFA and vitamins, and formation of
toxic compounds (Frankel, 1998). The negative repercussion of li-
pid oxidation on sensory analysis is nowadays limiting the com-
mercialisation period of pelagic fish products, causing large
economic costs to the industry and marketers, and its underutilisa-
tion for human food purposes.
Fish muscle is endowed with an efficient endogenous antioxi-
dant defence that stabilises (in vivo) its high content of
⇑ Corresponding author. Tel.: +34 986 231930; fax: +34 986 292762.
E-mail address: jacobo@iim.csic.es (J. Iglesias).
is continuously losing efficiency once fish is sacrificed, resulting
in the onset of lipid deterioration. Antioxidant components of fish
muscle, that include both lipophilic and hydrophilic substances,
are sequentially consumed in post mortem conditions. Several
authors have related the post mortem depletion of endogenous
antioxidants from fish muscle with the so called ‘‘pecking order’’
(Pazos, González, Gallardo, Torres, & Medina, 2005; Petillo, Hultin,
Krzynowek, & Autio, 1998). The ‘‘pecking order’’ postulates a fas-
ter consumption of the antioxidants with higher facility to donate
one-electron, that is, with lower standard one-electron reduction
potentials (Buettner, 1993). Among the antioxidant components
of muscle tissues, a-tocopherol (a-TOH) is suggested to play a
central function since it is the major endogenous lipid-soluble
chain breaking antioxidant and it has been reported as one of
the last protecting barriers of fish muscle against lipid oxidation
(Pazos, Sánchez, & Medina, 2005). The role of a-TOH, as the last
antioxidant defence of muscle tissues, is also supported by the
capacity of ascorbic acid (AA) and other reducing components
of muscle to regenerate a-TOH from the oxidised form of the toc-
opheroxyl radical (Buettner, 1993). This redox cooperation is sug-
gested to explain the antioxidant synergism occurring when a-
TOH and AA are jointly present. The hydrophilic AA is also an
important antioxidant component of muscle tissues due to its
inhibitory activity on lipid oxidation via a free radical-scavenging
mechanism (Frankel, 1998).

0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.03.072
1768 J. Iglesias et al. / Food Chemistry 134 (2012) 1767–1774
In recent decades, many efforts have been focused on the iden-
tification of phenolic compounds with antioxidant properties
which inhibit lipid oxidation in muscle-based foods. Green tea cat-
echins (He & Shahidi, 1997; Medina, Gallardo, Gonzalez, Lois, &
Hedges, 2007), grape procyanidins (Pazos, Iglesias, Maestre, &
Medina, 2010), hydroxycinnamic acids (Iglesias, Pazos, Andersen,
Skibsted, & Medina, 2009; Maqsood & Benjakul, 2010), berry ex-
tracts (Lee, Krueger, Reed, & Richards, 2006; Sampels, Asli, Vogt,
& Morkore, 2010) and olive oil phenolics (Pazos, Alonso, Sanchez,
& Medina, 2008) have successfully been applied to retard lipid oxi-
dation in fish muscle. The antioxidant function of polyphenols has
traditionally been ascribed to their scavenging of free radicals, che-
lating of redox-active metals and inactivating of hemeproteins via
reduction of the highly pro-oxidant ferryl states (Frankel, 1998;
Laranjinha, Almeida, & Madeira, 1995). In addition to these antiox-
idant mechanisms, antioxidant synergisms can arise from cooper-
ative redox interactions between exogenous phenolics and
endogenous antioxidants of muscle (Pedrielli & Skibsted, 2002).
Redox interactions with a-TOH have been thoroughly investigated
in vitro for benzoic- and cinnamic-derived acids, catechin mono-
mers and oligomers (procyanidins) and other flavonoids (Jia, Zhou,
Yang, Wu, & Liu, 1998; Mukai, Mitani, Ohara, & Nagaoka, 2005; Pa-
zos, Andersen, Medina, & Skibsted, 2007; Zhu, Huang, Tsang, &
Chen, 2000). These experiments have evidenced the potential apti-
tude of phenolics, such as caffeic acid, gallic acid, green tea cate-
chins, and procyanidins, to regenerate a-TOH via one-electron
reduction of a-tocopheroxyl radicals. AA has also been able to re-
cycle caffeic acid in LDL (Laranjinha & Lester, 2001). In a recent
study, we have confirmed cooperative redox cycles between exog-
enous caffeic acid and the endogenous a-TOH and AA in fish mus-
cle (Iglesias et al., 2009). However, the contribution of these
interactions to the antioxidant activity in muscle-based foods is
still unknown for most polyphenols.
The present study supplements the knowledge of antioxidant
mechanisms that govern the capacity of exogenous polyphenols
to prevent lipid oxidation in muscle food systems. In particular,
we investigated the interaction of grape procyanidins with two
important components of the endogenous antioxidant system of
muscle tissues, a-TOH and AA. To this end, antioxidant activity of
procyanidins was tested in fish muscle at different concentrations
(10–100 ppm). The efficacy to delay the lipid deterioration was
evaluated by their capacity to inhibit the formation of lipid oxida-
tion products (lipid hydroperoxides, aldehydes and rancid odours)
and their ability to avoid the consumption of PUFAs arising from
the oxidative process. This antioxidant behaviour was then related
to their effects on the decay kinetics of endogenous a-TOH and AA,
and on the formation kinetics of tocopherolquinone (TQ) and dehy-
droascorbic acid (DHAA). TQ and DHAA are main oxidation prod-
ucts derived, respectively, from the antioxidant action of a-TOH
and AA.
The present paper is a continuation of previous studies on the
anti-oxidant activity of procyanidinic fractions and the direct
effects on several pro- and antioxidant endogenous components
of the fish muscle. These fractions have previously shown impor-
tant in vitro properties and ability to regenerate a-TOH (Pazos,
Torres, Andersen, Skibsted, & Medina, 2009) as well as an optimal
activity to inhibit lipid oxidation in fish muscle (Pazos, Gallardo,
Torres, & Medina, 2005). In order to clarify their antioxidant mech-
anisms, this study was focused on the procyanidinic influence on
the well-documented synergistic redox cycle, mediated by a-TOH
and AA (Buettner, 1993), to prevent lipid deterioration in fish mus-
cle. The identification of this redox cooperation in real foods pro-
vides useful information to help develop a new generation of
antioxidant treatments based on enhancing synergisms involving
endogenous components of foods.
2. Materials and methods
2.1. Materials
Thiobarbituric acid, trichloroacetic acid, 1,1,3,3-tetraethoxy
propane (TEP), FeCl24H2O, FeCl36H2O, ascorbic acid, DL-all-
rac-a-tocopherol, sodium dodecyl sulphate (SDS), 4,5-dimethyl-
o-phenylenediamine (DMPD) and streptomycin sulfate were
purchased from Sigma (Steinheim, Germany). The mix of fatty acid
methyl esters (FAME), used as standard, was provided by Supelco
(St. Louis, MO). All chemicals and solvents used were of either ana-
lytical or HPLC grade (Merck, Darmstadt, Germany).
2.2. Grape procyanidins
A polyphenolic fraction rich in procyanidins was extracted from
grape pomace (Vitis vinifera) according to the procedure previously
described by Torres et al. (2002). Briefly, a total extract was first
obtained by application of solid-phase extraction on Parellada
grape ( V. vinifera) pomace with 70% ethanol, followed by a li-
quid–liquid extraction of the ethanolic medium by ethyl acetate.
Grape pomace was the byproduct from pressing destemmed
grapes, and consisted principally of seeds and skins. Size exclusion
chromatography on Toyopearl resin was then performed for the
polypyenols soluble in both ethyl acetate and water, and eluted
with a mixture of acetone:water (3:2). The polyphenolic fraction
obtained was mainly composed of oligomers and polymers of
flavanols. Fig. 1 shows the molecular structures of procyanidins ex-
tracted from grape pomace. HPLC analysis, after depolymerisation
with cysteamine, was used to determine the size of oligomers
(average molecular weight, mean degree of polymerisation) and
the content of galloyl moieties (galloylation) (Torres & Selga,
2003). Mean molecular weight, polymerisation degree, and galloy-
lation percentage were found to be 880 g/mol, 2.7 units and 25%,
respectively (Torres et al., 2002). Fraction labelled as IV was used
in this work since they exhibited the highest capacity to prevent
the formation of lipid oxidation products in fish muscle (Pazos,
Gallardo, et al., 2005).
2.3. Chilled fish muscle experiment
Fresh Atlantic horse mackerel (Trauchurus trauchurus) was ac-
quired in a local market within the first 24 h of capture. Fish pre-
sented an extra quality of freshness (Howgate, Johnston, &
Whittle, 1992). For each experiment, 8 kg of fish (20–24 different
fish) were deboned and eviscerated. The white muscle was sepa-
rated and passed through a mincer (Kenwood Mfg. Co. Ltd., Wok-
ing, UK) fitted with an 8 mm diameter hole size mincing screen
and was supplemented with streptomycin sulfate (200 lg/g) for
inhibiting microbial growth. Procyanidinic fraction was added as
a powder at different concentrations, 10, 25, 50 and 100 lg/g. Con-
trol fish muscle, without the incorporation of polyphenolic frac-
tion, was also prepared. Portions of 8 g of fish muscle were
placed in 50 ml Erlenmeyer flasks and stored at 4 °C on ice during
13 days. Triplicate samples were taken and analysed at different
sampling times. The development of lipid oxidation was monitored
by following the consumption of PUFAs, lipid substrate of lipid oxi-
dation, and the generation of primary and secondary lipid oxida-
tion products measured by the peroxide value (PV),
thiobarbituric acid-reactive substances (TBARS) index and sensory
analysis of rancid odours. Depletion kinetics of the endogenous a-
TOH and AA were determined in the course of storage, together
with the formation kinetics of their oxidative products, TQ and
DHAA.
 J. Iglesias et al. / Food Chemistry 134 (2012) 1767–1774 1769

R1

OH OH OH Fraction IV
R3
OH O
OH R1 OH Polymerization: 2.7
R2
R2
R1 % of esterified galloyl groups (R 2): 25
HO O O OH
OH Averaged molecular weight (Da): 880
OH
OH OH O
OH
R2
R1
OH OH R2
OH

Monomeric catechins Polymeric catechins (arr ows indicated possible polymerization positions)

R1=H, R 2=OH, R 3=H (+)-Catechin

R1=H (Procyanidins) or OH (prodelphinidins)
R1=OH, R 2=H, R 3=H (-)-Epicatechin
R1=H, R 2=OH, R 3=OH (+)-Gallocatechin
OH
R1=OH, R 2=H, R 3=OH (-)-Epigallocatechin
O
OH
R2=OH or Gallate group O
OH

Fig. 1. Chemical structures of the grape procyanidins.

2.4. Extraction and quantification of lipids 2.9. Fatty acid composition

Lipids were extracted from fish muscle according to the method
of Bligh and Dyer (1959) and quantified gravimetrically.
2.5. Peroxide value index (PV)
Changes in the fatty acid profile of lipids extracted from horse
mackerel muscle were monitored during the whole storage exper-
iment by converting fatty acids to methyl esters (Lepage & Roy,
1986) and later analysis by gas chromatography and flame ionisa-
tion detector (GC–FID) (Christie, 1982).

Lipid hydroperoxides were determined in the organic Bligh and
Dyer extract by the ferric thiocyanate method (Chapman & Mac-
kay, 1949). Results were expressed as milliequivalents of oxygen
per kg lipid.
2.6. Thiobarbituric acid-reactive substances (TBARS) index
TBARS index was measured according to Vyncke (1970). Stan-
dard curves were constructed using 1,1,3,3-tetraethoxypropane
as standard and results were expressed as mg malonaldehyde/kg
muscle.
2.7. Determination of a-tocopherol (a-TOH) and tocopherolquinone
(TQ)
a-TOH and its oxidative product TQ were extracted by adapta-
tion of the procedure by Burton, Webb, and Ingold (1985). The
analysis of a-TOH and TQ was performed by HPLC–DAD according
to the method by Cabrini, Landi, Stefanelli, Barzanti, and Sechi
(1992). a-TOH was quantified by using standard curves with com-
mercial a-TOH, and its concentrations expressed in terms of lg per
g of lipid.
2.8. Determination of ascorbic acid (AA) and dehydroascorbic acid
(DHAA)
AA and its oxidative product DHAA were acidically extracted
from fish muscle, derivatised with 4,5-dimethyl-o-phenylenedi-
amine (DMPD), and analysed by HPLC, coupled to a fluorescence
detector, according to the protocol of Iglesias, González and
Medina (2006). The concentrations of AA and DHAA were
expressed as lg per g of muscle.
2.10. Evaluation of antioxidant activity
The antioxidant effectiveness of grape procyanidins was evalu-
ated by comparing induction periods and inhibition percentages
for the formation of oxidation products: lipid peroxides and car-
bonyl compounds by thiobarbituric acid-reactive substances.
Induction periods were calculated as the time in days required
for a sudden formation of the lipid oxidation product by the meth-
od of tangents to the two parts of the kinetic curve (Frankel, 1998).
Inhibition percentages of lipid oxidation were calculated during
the propagation phase of oxidation in controls by the formula pro-
posed by Frankel (1998):
CS
Inhibition ð%Þ ¼  100 ð1Þ
C
where C represents the value of the oxidation product in non-sup-
plemented control muscle and S the value of the oxidation index
in procyanidin-supplemented samples.
2.11. Sensory analysis
Sensory analysis was performed to evaluate the capacity of
procyanidin-supplementation to retard the generation of rancid
odours. The sensory panel was composed of five trained specialists
in the descriptive analysis of fishy off-flavours. Approximately 8 g
of each sample were placed on ice in separate sterile polystyrene
Petri dishes. Panellists concentrated on detecting rancidity/painty
odours using the following structured scale: fresh fish odour, no
odour, incipient rancid odour, rancid odour, putrid odour.
2.12. Statistical analysis
Experiments were repeated twice and each sample was ana-
lysed in triplicate. Means and standard deviations were calculated
1770 J. Iglesias et al. / Food Chemistry 134 (2012) 1767–1774

(n = 3) and data were compared by one-way analysis of variance Control 10 µg/g

(ANOVA). Means were compared by a least squares difference A 140
25 µg/g 50 µg/g
method, with the Statistica 6.0 programme (Statsoft, Tulsa, Okla- 120
homa). The level of significance was set at p < 0.05. 100 µg/g
100

PV (meq. O2/Kg lip)

80
3. Results and discussion
60
3.1. Grape procyanidins as inhibitor of lipid oxidation in pelagic fish
40
muscle
20
The antioxidant behaviour of grape procyanidins (in retarding
lipid oxidation) was investigated in chilled fish muscle within the 0
0 2 4 6 8 10 12 14
antioxidant concentration range commonly employed for antioxi- -20 Time (days)
dant food applications (10–100 ppm). The implementation of anti-
oxidant treatments under chilling conditions has current
relevance, due to the development of novel refrigerated surimi Control 10 µg/g
B 25 µg/g 50 µg/g
and minced muscle products. Moreover, the development of lipid
10 100 µg/g

TBARS (mmol MDA/Kg muscle)

oxidation presents similarities, from a mechanistic point of view,
in both refrigerated and frozen environments, and accordingly,
refrigerated experiments can be employed as faster model systems
to elucidate antioxidant efficiencies during frozen storage (Medina,
González, Iglesias, & Hedges, 2009).
Panellists were not able to detect organoleptic differences
among the samples with and without added procyanidinic frac-
tions at the beginning of the experiment, including samples with
the highest antioxidant concentration (100 lg/g), which is a key is-
sue for the commercial application of new antioxidants in foods.
Non-supplemented samples showed induction periods of 2 days
for the formation of lipid hydroperoxides (Fig. 2A). Supplementing
fish muscle with polyphenolic concentrations as low as 10 or
25 ppm resulted in a delay in the induction periods of peroxides
up to 4 days. The incorporation of procyanidins at 25 ppm was also
effective in reducing the formation rate of peroxides during the
propagation phase of oxidation (Fig. 2A). At higher concentrations,
procyanidins were found to be more efficient in preventing the for-
mation of peroxides. The induction period of peroxides was 5 days
in samples supplemented with 50 ppm, whereas a concentration of
100 ppm provided a complete protection against the formation of
peroxides over the entire experiment (13 days). Accordingly, the
inhibition percentages of the formation of peroxides were main-
tained above 95% during the entire chilling storage by the highest
polyphenolic concentration (100 ppm) (Table 1). This elevated effi-
ciency of procyanidins, to avoid lipid oxidation in fish muscle, is in
agreement with their stability and antioxidant properties in the
typical pH range of post mortem fish muscle (pH 5–7) (Wood, Sent-
hilmohan, & Peskin, 2002; Hu, McClements, & Decker, 2004). The
inhibition of peroxides was concentration-dependent, showing a
direct relationship between the polyphenolic concentration and
the inhibitory efficiency: 100 ppm > 50 ppm > 25 ppm > 10 ppm.
The effect of grape procyanidins on the formation of secondary
lipid oxidation products, such as aldehydes, showed tendencies
similar to those above indicated for the inhibition of the primary
oxidation product peroxides. High polyphenolic concentrations
9
8
7
6
5
4
3
2
1
0
0 2 4 6 8 10 12 14
Time (days)
Fig. 2. Kinetics of formation of lipid hydroperoxides (A) and aldehydes (B) during
the chilled storage of pelagic fish muscle supplemented with different concentra-
tions of grape procyanidins. (PV, peroxide value index; TBARS, thiobarbituric acid-
reactive substances index). Error bars correspond to one experimental standard
deviation.
provided the longest induction periods (Fig. 2B) and the strongest
inhibition for the formation of TBARS (Table 1). The supplementa-
tion of fish muscle with 100 ppm of grape procyanidins was able to
retard the induction period for TBARS until 13 days and to main-
tain inhibitions of 80% for the formation of TBARS throughout the
experiment. Sensory analysis was correlated with these results
since rancidity was detected after 2 days in muscle supplemented
with 10 and 25 ppm; samples with 50 lg/g exhibited rancid off-
odours after 7 days of storage and no rancidity was detected in
the system supplemented with the highest concentration of the
antioxidant (100 ppm). These results highlighted the elevated sus-
ceptibility of pelagic fish muscle to undergo lipid oxidation during
chilled storage, and the ability of grape procyanidins to avoid such
deteriorative change of lipids in a concentration-dependent man-
ner. The antioxidant behaviour of procyanidins was found to be
similar to that of caffeic acid which achieves maximum activity
in pelagic fish muscle at the highest phenolic concentration,

Table 1
Inhibition percentages on the generation of lipid peroxides (PV) and aldehydes (TBARS) in chilled fish muscle supplemented with 10, 25, 50 and 100 lg/g grape procyanidins.
Inhibitions were calculated at initiation (day 3), propagation (day 5) and termination (day 13) phase of lipid oxidation in control non-supplemented muscle.A

PV (%) TBARS (%)

Day 3 Day 5 Day 13 Day 3 Day 5 Day 13
10 lg/g 84.2 ± 3.5a 2.9 ± 18.2a 5.7 ± 6.0a 54.8 ± 1.2a 4.2 ± 0.1a 12.4 ± 1.18a
25 lg/g 64.7 ± 12.5a 75.3 ± 2.7b 28.3 ± 9.6b 56.7 ± 7.2a 27.9 ± 11.3b 20.5 ± 0.3b
50 lg/g 98.6 ± 2.8b 99.3 ± 0.0c 72.9 ± 7.3c 75.2 ± 5.5b 78.7 ± 3.3c 48.3 ± 7.2b
100 lg/g 100.1 ± 2.8b 99.4 ± 0.6c 96.6 ± 6.2d 88.4 ± 0.7c 83.0 ± 4.0c 79.8 ± 0.8c
A
Values with different letters in the same columns indicate significant differences among means (p < 0.05).
 J. Iglesias et al. / Food Chemistry 134 (2012) 1767–1774 1771

100 ppm (Iglesias et al., 2009). This antioxidant conduct of procy- 0.30
anidins differs from that of hydroxytyrosol which exhibited the
Control Procyanidins
most elevated antioxidant activity for the intermediate phenolic
concentrations of 50 ppm (Pazos et al., 2008). This behaviour of

a
hydroxytyrosol is explained by the activation of metal-reducing

mg DHA/ mg lipid
0.20
capacity and other pro-oxidant mechanisms at high phenolic
concentration.

3.2. Protection of PUFAs from pelagic fish muscle by grape 0.10

procyanidins

Fatty acids from fish muscle were monitored during the entire
chilling experiment. The initial content of polyunsaturated fatty
acids (PUFAs) in horse mackerel muscle was found to be about 0.00
0.29 ± 0.01 mg per mg of lipid, representing approximately 0 1 2 3 4 5 6 7 8 9 10 13
42.1 ± 1.2% of total fatty acids (Table 2). Among PUFAs, docosahex- Days at 4ºC
aenoic acid (DHA, 22:6 x-3) was clearly the most abundant PUFA
Fig. 3. Protection of endogenous docosahexaenoic acid (DHA, 22:6 x-3) by
in fresh fish muscle, with a content of 0.20 ± 0.01 mg per mg of li-
supplementing pelagic fish muscle with 100 lg/g of grape procyanidins. Error bars
pid and a proportion of about 30% of total fatty acids. DHA also correspond to one experimental standard deviation.
exhibited the greater abundance than did saturated fatty acids
(SFAs) or monounsaturated fatty acids (MUFAs). Eicosapentaenoic
acid (EPA, 20:5 x3) was the second most abundant PUFA, with a The consumption of DHA between days 2 and 4 is in agreement
content of 0.045 ± 0.001 mg per mg of lipid (Table 2). Palmitic acid with the kinetics obtained for the formation of lipid peroxides and
(16:0) was the SFA present in highest concentration aldehydes (Figs. 2 and 3). Both lipid oxidation products were
(0.125 ± 0.005 mg per mg of lipid), whereas oleic acid (18:1 x-9) essentially produced from day 2 of storage and, at day 4, reached
was the most abundant MUFA in fresh horse mackerel muscle their maximum accumulation in chilled fish muscle. Moreover,
(0.123 ± 0.004 mg per mg of lipid). The contents of fatty acids DHA is comprehensively expected to be the largest lipid substrate
showed similarities to previous results found for other fish species. of peroxides and aldehydes, considering its large abundance and its
DHA and palmitic acid have been reported, respectively, as the elevated susceptibility to develop oxidations. It is well-docu-
most abundant PUFA and SFA in cod, herring and salmon (Larsson mented that the vulnerability of fatty acids to oxidation is directly
et al., 2007). Moreover, oleic acid has also been described as the related to the carbon-chain length and the number of double bonds
most important MUFA in cod and salmon, in contrast to herring (Frankel, 1998). The supplementation of fish muscle with increas-
muscle, in which erucic acid (22:1 x-9) has been reported as the ing procyanidin concentrations was found to retard the consump-
more abundant MUFA. tion of DHA in a straight concentration-dependent manner. Fig. 3
Chilled storage provoked a decrease in the amount of the most shows the evolution of endogenous DHA levels in fish muscle sup-
abundant PUFA, DHA, in control non-supplemented muscle. DHA plemented with 100 ppm of grape procyanidins, which was able to
was drastically depleted between days 2 and 4 by reducing over keep the initial amount of DHA intact (0.20 mg per mg of lipid)
24% of its content compared to initial levels (Fig. 3). From day 4 during the 13 days of chilled storage (Fig. 3). This observation is
up to day 13, the amount of DHA was maintained stable at approx- concordant with the above results that showed the ability of
imately 0.16 mg per mg of lipid. The results are in agreement with 100 ppm of grape procyanidins to completely avoid the generation
previous studies that reported loss of DHA in chilled and frozen pe- of lipid oxidation products, peroxides and aldehydes in chilled fish
lagic fish (Pazos et al., 2005; Pazos et al., 2008). Chilled storage did muscle.
not provoke significant changes in the amounts of SFAs and MUFAs
in absolute terms (data not shown). 3.3. Interaction between exogenous grape procyanidins and
endogenous a-TOH and AA from fish muscle
Table 2
Fatty acid profile of fresh horse mackerel muscle. Control non-supplemented fish muscle developed a rapid
reduction of a-TOH and AA levels (Figs. 4 and 5). After 2 days of
mg FA/mg lipid % FA
chilled storage, both antioxidant components of muscle were com-
14:0 0.020 ± 0.002 3.1 ± 0.2
pletely exhausted. This observation is comprehensible, considering
16:0 0.125 ± 0.005 18.2 ± 0.2
16:1x7 0.025 ± 0.001 3.7 ± 0.1
the antioxidant role of a-TOH and AA in fish muscle, the rapid
18: 0.044 ± 0.002 6.5 ± 0.1 development of lipid oxidation observed in chilled fish muscle,
18:1x9 0.123 ± 0.004 18.0 ± 0.4 and the post mortem conditions by which the principal supply of
18:1x7 0.016 ± 0.001 2.3 ± 0.1 these fish muscle components is aborted. Procyanidin-supple-
18:2x6 0.007 ± 0.000 1.1 ± 0.0
mented muscle samples showed a delay in the a-TOH depletion
18:3x3 0.002 ± 0.000 0.3 ± 0.0
18:4x3 0.003 ± 0.000 0.5 ± 0.0 directly related to the procyanidin concentration (Fig. 4A). After
20:1x9 0.020 ± 0.000 2.9 ± 0.2 2 days, muscle enriched with the lowest procyanidin level
20:4x6 0.007 ± 0.000 1.1 ± 0.1 (10 ppm) preserved 32% of the initial levels of a-TOH, in contrast
20:4x3 0.003 ± 0.000 0.5 ± 0.0 to the entire loss of this component in control non-supplemented
20:5x3 0.045 ± 0.001 6.6 ± 0.2
22:1x11 0.021 ± 0.003 3.1 ± 0.4
muscle. The supplementation with higher polyphenolic levels
22:5x3 0.018 ± 0.000 2.6 ± 0.1 (25–100 ppm) was able to conserve (intact) the initial a-TOH con-
22:6x3
P
0.202 ± 0.007 29.5 ± 0.7 tent after 2 days of chilling. Fish supplemented with 25 ppm exhib-
SFAs 0.190 ± 0.008 27.8 ± 0.5 ited a significant loss of a-TOH after 3 days. Samples supplemented
P
MUFAs 0.205 ± 0.011 30.0 ± 1.2
P with 50 and 100 lg/g were able to maintain the original concentra-
PUFAs 0.288 ± 0.010 42.2 ± 1.2
tions of a-TOH for 5 and 13 days, respectively. The analysis of TQ,
1772 J. Iglesias et al. / Food Chemistry 134 (2012) 1767–1774

A Control 10 µg/g A 2.5 Control

300 25 µg/g 50 µg/g 10 µg/g

100 µg/g 2.0
25 µg/g

AA (µg/g muscle)
250
1.5 50 µg/g
α-TOH (µg/g lip)

200
100 µg/g
150 1.0

100
0.5
50
0.0
0 0 1 2 3 4 5 6 7
0 2 4 6 8 10 12 14
-50 -0.5 Time (days)
Time (days)
B 2.5 Control
B Control 10 µg/g 10 µg/g
25 µg/g 50 µg/g 2.0
4.5 25 µg/g
100 µg/g

DHAA (µg/g muscle)

4.0 50 µg/g
1.5
α-TQ (Counts/g lip)

3.5
100 µg/g
3.0
2.5 1.0

2.0
1.5 0.5

1.0
0.5 0.0
0 2 4 6 8
0.0
0 2 4 6 8 10 12 14 Time (days)

Time (days)
C 2.0 Control
Fig. 4. Effects of different concentrations of grape procyanidins on the depletion of 1.8 10 µg/g
endogenous a-tocopherol (a-TOH) (A) and on the accumulation of tocopherolqui-
none (TQ) (B), the main oxidative degradation byproduct of a-tocopherol in pelagic 1.6 25 µg/g
Ratio AA/DHAA

fish muscle. Error bars correspond to one experimental standard deviation.
the principal oxidation product of the antioxidant action of a-TOH
in fish (Pazos, Sánchez, et al., 2005) and beef (Faustman, Liebler, &
Burr, 1999) muscle, indicated a similar tendency. Non-supple-
mented muscle exhibited a rapid generation of TQ after 1 day of
chilled storage (Fig. 4B). The incorporation of procyanidins at 10
or 25 ppm was effective in delaying, to some extent, the formation
of TQ, and increasing polyphenolic concentrations to 100 ppm
reached stronger inhibitory against accumulation of TQ. TQ levels
remained low during 5 days of chilling by supplementing procy-
anidins at 50 ppm; after that period, a drastic increment of TQ
was observed (Fig. 4B). Finally, the formation of TQ was intensely
retarded in muscle supplemented with procyanidins at 100 ppm
during the entire experiment. These results indicate the significant
protective ability of grape procyanidins to avoid the oxidative deg-
radation of a-TOH.
Procyanidin-supplemented muscle exhibited slower depletion
kinetics for AA than did control samples (non-supplemented)
(Fig. 5A). The ability of procyanidins to protect endogenous AA
showed a straight concentration-dependence. Endogenous AA
was totally consumed in controls after 2 days of experiment,
whereas systems with 10 or 25 ppm of polyphenols contained
75% of the original AA. The depletion of AA in samples with higher
polyphenolic concentration (50 and 100 ppm) started after 2 days
of storage, but these samples still conserved approximately 50%
of the initial AA after 5 days of storage. Fish muscle, enriched with
100 ppm of procyanidins, exhibited the highest AA amounts after
7 days. However, the kinetics for the formation DHAA, the primary
oxidative product of AA, did not show a polyphenolic concentra-
tion dependency (Fig. 5B). The levels of DHAA were found initially
to be 1.5–2.0 ppm, and started to decrease after 4 days of chilled
1.4 50 µg/g
1.2 100 µg/g
1.0
0.8
0.6
0.4
0.2
0.0
0 1 2 3 4 5 6 7
Time (days)
Fig. 5. Effects of different concentrations of grape procyanidins on the depletion of
endogenous ascorbic acid (AA) (A) and on the accumulation of dehydroascorbic acid
(DHAA) (B), the oxidation by-product of ascorbic acid. Oxidative stability of
different samples is also shown by the AA/DHAA ratio (C). Error bars correspond to
one experimental standard deviation.
storage in either procyanidin-supplemented or non-supplemented
muscle, without significant differences. This observation may be
attributed to the elevated electrophilic character of DHAA, as the
1,2,3-tricarbonyl form, and its fast and irreversible conversion to
diketogulonate and other breakdown products, through oxidation
or hydrolysis (John, 2000). Conversely, the AA/DHAA ratio
displayed evident differences among samples (Fig. 5C). Non-
supplemented muscle showed a faster reduction of the AA/
DHAA ratio, followed (in increasing order of stability) by
10 ppm  25 ppm > 50 ppm and 100 ppm, which provided a higher
ratio for a longer period of time. The AA/DHAA ratio has been
associated with the oxidative stability of fish muscle, I.E. lower ra-
tios for muscle from pelagic fish species compared to those from
species less liable to develop oxidative degradation, e.g. lean fish
 J. Iglesias et al. / Food Chemistry 134 (2012) 1767–1774
(Iglesias et al., 2006). These results confirmed the ability of procy-
anidins to protect endogenous AA from muscle tissues against oxi-
dative decomposition.
The redox interaction between the endogenous components,
a-TOH and AA, is a well-documented example of synergistic anti-
oxidant action (Buettner, 1993). a-TOH acts principally by neutral-
ising lipid radicals by electron/hydrogen atom donation, and that
radical-scavenging activity leads, in first instance, to the formation
of tocopheroxyl radicals (a-TO). AA can donate one electron/
hydrogen atom to a-TO to regenerate a-TOH, giving rise to the for-
mation of the semihydroascorbyl radical (AA) and then, DHAA.
Earlier spin resonance spectroscopy (ESR) experiments have dem-
onstrated the ability of the grape procyanidins evaluated here to
reduce (in vitro) a-TO in a phospholipid-like system, based on so-
dium dodecyl sulfate (SDS) micelles (Pazos et al., 2009). The con-
tent of galloyl residues (0.25 gallate units/procyanidin molecule)
was found to contribute positively in this redox regenerative
mechanism of procyanidins on a-TOH. The present investigation
confirms a protective role of grape procyanidins on the endoge-
nous a-TOH from fish muscle. Lourenço, Gago, Barbosa, De Freitas,
and Laranjinha (2008) have also proved the protecting action of
grape procyanidins on a-TOH in isolated LDL. From a thermody-
namic point of view, the presence of galloyl structures favour the
reducing reaction of a-TO by procyanidins since the one-electron
reduction potentials of galloylated polyphenols are significantly
lower than are non-galloylated or a-TOH. As an example, epicate-
chin and its galloylated derivatives epigallocatechingallate (EGCG)
and epigallocatechin (EGC) have, respectively, reducing potentials
of 0.57, 0.43 and 0.42 V, and a-TOH a reducing potential of
0.48 V (Jovanovic, Hara, Steenken, & Simic, 1995). The nature of
galloyl structures facilitates the physical concentration of polyphe-
nols in the phospholipid membranes, where the endogenous
a-TOH is located, and that fact should enhance their redox interac-
tion with a-TOH. Moreover, grape procyanidins are incorporated in
large excess, 50–100 ppm, compared to the endogenous content of
a-TOH in muscle tissue, 2–3 ppm, kinetically enhancing the reduc-
tion of a-TO by procyanidins. All these factors indicate a central
role of the regenerative action of grape procyanidins, via redox
reduction of a-TO, in the observed protection of endogenous
a-TOH in procyanidin-supplemented muscle tissues. The identifi-
cation of this cooperative interaction, between grape procyanidins
Proc-O· Proc-OH
I
Proc-OH Proc-O· AA A·
Cytosol III
α-TO· α-TO· α-TOH
α-TOH
R· RH
R· RH
RH
II
Phospholipid Proc-OH Proc-O·
membrane
Fig. 6. Proposed redox interactions of grape procyanidins with endogenous a-TOH
and AA in pelagic fish muscle.
1773
and endogenous a-TOH in muscle tissues, is suggested to make a
great contribution to the antioxidant efficiency since similar inter-
actions between galloylatedcatechins/a-TOH provide antioxidant
synergisms (Zhou, Wu, Yang, & Liu, 2005).
The consumption of endogenous AA from fish muscle was also
effectively retarded by the incorporation of procyanidins. This ef-
fect of procyanidins on endogenous AA differs from that reported
previously for caffeic acid (E° = 0.54 V), which significantly dimin-
ishes the redox stability of AA (Iglesias et al., 2009). This behaviour
of caffeic acid has been explained by the repairing action of AA on
caffeic o-semiquinone radicals, according with the lower reduction
potential for AA (E = 0.28 V). Mechanistically, three facts may ex-
plain the protective effect of grape procyanidins on endogenous
AA, as indicated in Fig. 6: (i) a regenerative pathway via donation
of electrons or hydrogen atoms to AA or DHAA by procyanidins,
(ii) an indirect slow-down of AA consumption, attributable to the
free radical-scavenging activity of procyanidins, and (iii) an indi-
rect reduced consumption of AA, caused by the strong redox stabil-
ity of a-TOH in the presence of procyanidins, since AA is involved
in the repairing mechanism of a-TOH. The lower standard reduc-
tion potential of AA (E = 0.28 V) compared to that of galloylated
catechin (monomeric unit of grape procyanidins) does not make
the regeneration of AA by procyanidins thermodynamically feasi-
ble. However, a regenerative action of AA by procyanidins cannot
be totally rejected since the reduction potentials are not measured
under realistic conditions in fish muscle, and the reduction reac-
tion of AA by procyanidins should be kinetically driven by the ex-
tremely high stability of AA and the relatively higher
concentration of procyanidins (10–100 ppm) compared to endoge-
nous AA found in fish muscle (1.5–2.0 ppm). This hypothesis is in
agreement with an in vitro experiment that described the regener-
ative activity of a procyanidin-rich extract on AA (Packer, Rimbach,
& Virgili, 1999), but is contrary to an investigation with linoleic
acid SDS micelles that reported regenerative action of AA on green
tea catechins under equimolar conditions (Dai, Chen, & Zhou,
2008). The ability of procyanidins to sink free radicals and to pro-
tect a-TOH from oxidation is proposed to indirectly extend the re-
dox stability of AA in procyanidin-supplemented muscle pending
confirmation of the extent of the interaction between procyanidins
and AA in muscle tissues.
In summary, we have investigated the aptitude of exogenous
grape procyanidins to establish redox interactions with two key-
components of the endogenous antioxidant system of fish muscle,
a-TOH and AA. The results underline the strong ability of grape
procyanidins to protect a-TOH against oxidative degradation via
one-electron reduction of a-tocopheroxyl radicals, thus reinforc-
ing, the natural regenerative system of a-TOH provided by AA in
muscle tissues (Fig. 6). The endogenous reducing system of a-
TOH, based on AA, is extremely active at the early post mortem
stage, but it rapidly loses efficiency due to the strong reducing
power of AA that provokes its faster oxidation by molecular oxygen
and reactive oxygen species. Grape procyanidins are endowed with
an intermediate reducing power, and so, they are able to strength-
en the repairing system of a-TOH in muscle tissue at medium-long
term, when AA is not active in regenerating a-TOH. The present
study adds valuable information about new antioxidant treatments
based on the protection of a-TOH, considered one of the last anti-
oxidant barriers against oxidation in muscle tissues, and the estab-
lishment of synergistic antioxidant actions with endogenous
components of muscle tissues.
Acknowledgements
This work was financed by the Spanish Ministry of Science and
Innovation (Grant AGL2009-12374-C03-01). Consejo Superior de
Investigaciones Científicas (CSIC) and Xunta de Galicia are
1774 J. Iglesias et al. / Food Chemistry 134 (2012) 1767–1774
gratefully acknowledged for a PhD Grant to J.I. and postdoctoral
contract ‘‘Isidro Parga Pondal’’ for M.P.
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