Food Research International 44 (2011) 269–275

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Food Research International

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Efficiency and capacity of antioxidant rich foods in trapping peroxyl radicals: A full
evaluation of radical scavenging activity
Paola Vanzani a,b, Monica Rossetto a,b, Veronica De Marco a, Adelio Rigo a,b,⁎, Marina Scarpa c
a
Department of Biological Chemistry, University of Padova, v. G. Colombo, 3, 35131 Padova, Italy
b
Consortium INBB, section of Padova, v. Medaglie d'Oro, 305, 00136, Roma, Italy
c
Department of Physics, University of Trento, v. Sommarive, 14, 38050, Povo, Trento, Italy

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

Article history: For the first time a variety of foods, characterized by high antioxidant activity, have been tested for the reactivity
Received 30 July 2010 by which the system of antioxidants present in these foods competes for peroxyl radicals with poly-unsaturated
Accepted 14 October 2010 fatty acids. The oxygraphic method we have used, on the basis of a rigorous kinetic model, permits to obtain the
reactivity that is the Peroxyl Radical Trapping Efficiency (PRTE), beyond the Peroxyl Radical Trapping Capacity
Keywords:
(PRTC) and to assign to each food characteristic values of these parameters, so facilitating their inter-comparison.
Beverage
Fruit
In the analyzed foods the PRTE/PRTC ratio spans more than one order of magnitude, so reflecting the quality of
Vegetable antioxidants present in foods. According to the PRTE values and on the basis of the serving size the ranking of
Polyphenols antioxidant food efficiency in trapping peroxyl radicals was blueberryN red chicoryN coffee N pineapple≈ red
Lipid peroxidation wine ≥ orange≈ dark chocolate ≈ apple ≥ tea N pomegranate.
Peroxyl radical scavenging © 2010 Elsevier Ltd. All rights reserved.

1. Introduction parameter depends on the stoichiometry of the trapping process

“Healthy lifestyle” is a dictate of the incoming years and, as a
consequence, the nutraceutical potentiality of food is of primary
interest for consumers and care providers. For instance polyphenols
present in foods may act as antioxidants in the inhibition of lipid
peroxidation process, that may occur very easily under the conditions
present in the gastrointestinal tract (GI) (Gorelik et al., 2005; Lapidot,
Granit, & Kanner, 2005a,b; Ursini et al., 1998). In this context, the
correct ranking of the antioxidant properties of food appears
important. To this regards we should consider two parameters: the
capacity, that is the amount of peroxyl free radicals scavenged by a
given amount of food, and the efficiency, that is the reactivity of foods
in trapping these radicals (Roginsky, 2003) before they damage
critical biological molecules (Frankel & Meyer, 2000). The first
Abbreviations: ABIP, 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride;
DCA, deoxycholic acid sodium salt; DPPH, diphenylpicrylhydrazyl; GI, gastrointestinal;
IC50, concentration of the antioxidant which provides 50% inhibition; IH, antioxidant;
LG, rac1-lauroylglycerol; LH, linoleic acid; LO•2, peroxyl radical; LT, lag-time; PRTE,
Peroxyl Radical Trapping Efficiency; ePRTE, equivalent Peroxyl Radical Trapping
Efficiency; ePRTEss, equivalent Peroxyl Radical Trapping Efficiency per serving size;
PRTC, Peroxyl Radical Trapping Capacity; PRTCss, Peroxyl Radical Trapping Capacity per
serving size; PUFA, poly-unsaturated fatty acids; R0, rate of oxygen consummation due
to azo-compound; RIN, rate of initiation of peroxidation; ss, serving size; TP, total
phenol content; TPss, total phenols per serving size.
⁎ Corresponding author. Department of Biological Chemistry, University of Padova, v.
G. Colombo, 3, 35131 Padova, Italy. Tel.: +39 049 8276107; fax: +39 049 8073310.
E-mail address: adelio.rigo@unipd.it (A. Rigo).
while the second parameter is related to its kinetic rate constant.
However, all the methods until now proposed measure only the
capacity of foods (Roginsky & Lissi, 2005), since also the tests that
report IC50 of foods are static methods being the electron-donating or
H-donating capacity of an antioxidant (i.e. the amount of diphenylpi-
crylhydrazyl, DPPH, scavenged) expressed as IC50. In other words they
measure in some way the stoichiometry of the inhibition process.
To evaluate the capacity and efficiency of antioxidants we set up a
simple oxygraphic method that, based on a rigorous treatment of the
kinetic data relative to the free radical peroxidation of fatty acids,
permits to calculate the Peroxyl Radical Trapping Capacity (PRTC),
that is the amount of peroxyl radicals trapped by a given amount of
antioxidant (usually micromoles of peroxyl radicals trapped by one
gram of antioxidant), and Peroxyl Radical Trapping Efficiency (PRTE)
that is the reciprocal of IC50 (Zennaro et al., 2007), where IC50 is the
concentration of antioxidant which provides 50% inhibition. These
two parameters can be calculated simultaneously from a single
experiment. In this article we have demonstrated that the method can
be applied to a variety of foods with well-recognized antioxidant
properties assigning them typical values of PRTE and PRTC so
facilitating the comparison of their scavenging properties. To obtain
significant and reproducible measurements, which are fundamental
for data comparison, the PRTE and PRTC values were measured in a
simplified micellar system, which is easy to reproduce and mimics
physiological conditions occurring in the upper part of small intestine
during lipid assimilation (Hernell, Staggers, & Carey, 1990; Staggers,
Hernell, Stafford, & Carey, 1990), where the peroxidation of poly-

0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2010.10.022
270 P. Vanzani et al. / Food Research International 44 (2011) 269–275
unsaturated fatty acids (PUFA) present in foods may occur with high
rates. The measured PRTE and PRTC values were correlated with the
polyphenol content emphasizing the deep diversities of the free
radical scavenging properties of foods resulting from the different
type and content of these compounds. On these bases, diverse ranking
of foods was evidenced when their ability to compete for peroxyl
radicals rather than their capacity is taken into account.
2. Materials and methods
2.1. Chemicals
Rac1-lauroylglycerol (LG) was obtained from Sigma-Aldrich
(Milano, Italy). Trolox for HPLC, linoleic acid (LH) and deoxycholic
acid sodium salt monohydrate (DCA) were purchased from Fluka
(Buchs, Switzerland). 2,2′-Azobis[2-(2-imidazolin-2-yl)propane]
dihydrochloride (ABIP) was a kind gift of Wako Chemicals (Germany).
Folin-Ciocalteu phenol reagent was obtained from Sigma-Aldrich
(Milano, Italy).
Quartz distilled water was used to prepare all the aqueous
solutions. Buffers were equilibrated in batch with Chelex-100 (Bio-
Rad, Richmond, CA) to minimize the concentration of heavy metal
ions. Vegetables, fruits, wine, coffee, tea, and chocolate were
purchased from local markets.
2.2. Sample preparation
The extracts of fresh foods were prepared selecting 50 g of edible
part of vegetables or fruits: only leaves in the case of chicory, pulp and
skin in the case of apple, the whole fruit in the case of berries, pulp in
the case of pineapple and orange, arils in the case of pomegranate. The
randomly sampled raw material was homogenized in 200 mL of
ethanol/water solution (85:15 v/v) containing 0.12 M HCl (Rossetto
et al., 2005). The homogenates were centrifuged and the solutions
were stored at −80 °C until activity measurements.
Dry food extracts were prepared as follows: black coffee was
prepared by an Italian Moka coffee machine (5 g of Arabic coffee
powder with 25 mL of distilled water). American coffee was prepared
by an automatic percolator using 40 g of coffee powder in 800 mL of
water. Commercial dark chocolate (2 g), 100% cocoa blend, was
dissolved in 200 mL of distilled water at 100 °C. Green and black teas
were prepared by infusion of 2 g of commercial dry leaves in 200 mL
of distilled water at 100 °C for 2 min (according to the manufacturer's
instructions).
For the various foods, on the basis of the indications of some
authors (Halvorsen et al., 2006; Lee, Kim, Lee, & Lee, 2003; Water-
house, Shirley, & Donovan, 1996), we considered the following
serving size: 1 cup of coffee (5 g of powder), 1 cup of tea (2 g of
powder), 1 glass of red wine (140 g), 1 cup of dark chocolate (7.3 g of
cocoa), 1 bowl of red chicory (100 g), 1 cup of blueberry (100 g), 1
apple (140 g), 1 orange (140 g), 1 cup of pomegranate arils (100 g),
and 1 slice of pineapple (140 g).
2.3. Total phenol content
The total phenol content (TP) was measured according to the
Folin-Ciocalteu method after incubation of the reaction mixture for
30 min at room temperature (Singleton, Orthofer, & Lamuela-
Raventós, 1999). The absorbance measurements were carried out by
a Varian Cary 50 spectrophotometer at 770 nm. The total phenol
content of extracts was expressed in milligrams of gallic acid
equivalent per gram of food. All measurements were performed in
triplicate.
2.4. Measurement of the peroxyl radical scavenging activity
The rate of LH peroxidation was measured from the rate of O2
disappearance by a Metrohm 663 VA stand equipped with a Yellow
Spring Oxygen electrode, inserted into a thermostated oxygraphic
cell. The current was recorded by a personal computer equipped with
a data acquisition board (DAQ PCI-6221, M series, National Instru-
ments, Austin, TX). The working electrode was poised at −800 mV vs
Ag/AgCl.
The reaction mixture, as described by Rossetto et al. (2008), was
prepared by drying a solution of LG in dichloromethane and by
dissolving the obtained film in 20 mM phosphate buffer containing
5 mM DCA, pH 7.4. Linoleic acid (2 mM final concentration) was then
added and the solution vigorously vortexed. The micelle containing
solution was equilibrated with atmospheric oxygen by continuous
stirring in the oxygraphic cell, thermostated at 37 ± 0.1 °C. After
thermal equilibrium, ABIP, used as a constant source of peroxyl
radicals, was added (4 mM final concentration) and the rate of oxygen
consumption due to the uninhibited peroxidation of linoleic acid was
recorded for some minutes. At the end of this period the food extract
to be examined was injected into the test solution and the rate of the
oxygen consumption was recorded until the oxygen disappearance,
see Fig. 1. Each food extract was analyzed at four different
concentrations.
2.4.1. Computational procedure to calculate PRTC and PRTE values
The experimental oxygraphic traces were automatically processed
by means of a computational procedure to obtain oxygen consump-
tion rates and PRTC and PRTE values on the basis of a rigorous
treatment of the kinetic data, see Zennaro et al. (2007) and Rossetto
et al. (2008).
2.4.2. A simplified procedure to calculate PRTC and PRTE values
Alternatively to the computational procedure the following
procedure can be followed to obtain the PRTC and PRTE values from
an oxygraphic plot like that reported in Fig. 1:
i) Measure the slopes of the plot at the times t0, tj, t1, t2, t3,…ti… that
is α0, αj, α1, α2, α3,…αi where α0, αj, are the slopes before and just
Fig. 1. Representative oxygraphic records of linoleic acid peroxidation in a micelle
system in the presence and in the absence of an antioxidant. The peroxidation of 2 mM
LH was carried out at 37 °C by decomposition of 4 mM ABIP in a micelle system
containing 5 mM DCA and 0.5 mM LG, in the absence of antioxidant (trace a) and after
injection (arrow) of a green tea extract (0.58 mL/L, see MM) (solid trace b). The slopes
αi at the times ti were measured. Inset: plot of F(xi) versus (ti − tj). The trace c)
represents the oxygraphic trace obtained during the decomposition of 4 mM ABIP in the
micelle system 5 mM DCA and 0.5 mM LG, in the absence of LH.
 P. Vanzani et al. / Food Research International 44 (2011) 269–275
after the injection of the extract at the time tj, respectively, see
Fig. 1. The measure of these slopes can be done graphically.
ii) Calculate the function:
      cross-reactions among the radical species generated by the reaction
−1 −1
F ðxi Þ = xj −xi + ln 1−xj = 1 + xj
+ lnð1 + xi Þ = ð1 − xi Þ
where xj = (αj − R0) / (α0 − R0 ), and xi = (αi − R0 / (α0 − R0 ),
R0 being the rate of O2 consumption due to the formation of
the peroxyl radicals of ABIP, see Fig. 1 (trace c).
iii) Plot the values of F(xi) vs (ti − tj).
iii) Calculate the slope C of this plot. Usually a linear plot
characterized by R2 values N0.99, is obtained.
iv) The PRTC value is calculated from the slope C and the xj value
according to the following equation
 
−1
PRTC = xj −xj × RIN = ðzj d CÞ
where zj is the concentration of the food (g L−1) in the oxygraphic
cell just after the injection, and RIN is the rate of initiation of
peroxidation. RIN was calculated according to equation
RIN = 2½α−Tocopherol = LT
where LT is the lag-time, that is the period of time when LH
peroxidation is fully inhibited by addition to the peroxidation
system of α-tocopherol at concentrations in the micromolar range.
v) The PRTE values are calculated according to the following
equation
−1
PRTE = PRTC⋅C = ð1:5⋅RIN Þ = IC50
The PRTE value, which according to the kinetic equations
corresponds to IC−1
50 , is directly proportional to the kinetic rate
constant of the reaction between peroxyl radicals and the
system of antioxidants, and is calculated as liters of testing
system/gram of food.
This procedure may be adopted when the perturbation of the
inhibited oxygraphic trace, following the injection of the food extract
lasts less than 1 min. This perturbation may be due to the different
oxygen concentration between the injected solution and the reaction
system and to the kinetic of distribution of the antioxidants present in
the extract between the aqueous and micelle phases. As a conse-
quence of this simplified procedure there is a slight increase of the
standard deviations of PRTC and PRTE values.
3. Results and discussion
Foods, which have been a matter of debate in the last years for
their interesting antioxidant properties, have been tested according to
the oxygraphic method mentioned in the previous sections. By this
method it was possible to assign typical PRTC and PRTE values to each
food we have tested like those assigned to antioxidant compounds
(Zennaro et al., 2007), so facilitating food inter-comparison. The
calculated PRTC and ePRTE values have been reported in Table 1. Fig. 2
shows the oxygraphic plots obtained in the cases of pineapple and
dark chocolate, which are characterized by high efficiency but low
capacity and vice versa, respectively. The value of R2 higher than 0.99
calculated from the fit of oxygraphic data, see insets of Fig. 2, indicates
a very good agreement between the experimental data and the
theory, Zennaro et al. (2007).
The good matching between the experimental data and the kinetic
model developed for a single antioxidant appears a distinguishing
behaviour of foods we have tested, despite that these foods contain
several antioxidants. This behaviour may trivially be due to the
271
overwhelming activity of one antioxidant with respect to the others or
to the close values of IC50 of the antioxidants present in food. However
the main mechanism leading to the observed behaviour should be the
between the highly reactive peroxyl radicals and the antioxidants
present in foods (Rossetto et al., 2002). This is in accord to literature
data, which demonstrate that, during lipid peroxidation, one-electron
transfer reactions involving couple of antioxidants occur very easily
according to their equilibrium constants (as uric acid, ascorbate,
Trolox, methoxy- and dimethoxy-phenol) (Jovanovic & Simic, 1989;
Simic & Jovanovic, 1989).
The kinetic behaviour of the system of antioxidants present in
foods as a single antioxidant is quite general since it was observed for
most of the foods independently of the system where we have carried
out the lipid peroxidation process (for example independently of the
micelle nature and composition).
The total phenol content (TP) of various foods, measured according
to the Folin-Ciocalteu method, is also reported in Table 1. The TP values
range from about 1 mg of TP/g of fresh weight food in the case of fruits
and vegetables to about tens of milligrams of TP/g in the case of dry
foods such as tea and coffee. Also in the case of ePRTE and PRTC
particularly high values were obtained for dry foods and, as a
consequence, the ratios between the maximum and the minimum
value of the efficiency and of the capacity, that is (ePRTE)max/(ePRTE)min
and (PRTC)max/(PRTC)min, are very high, that is 240 and 600,
respectively. These large fluctuations of the reactivity and capacity,
due in part to the water content of foods, decrease if we consider the
ratios ePRTE/TP and PRTC/TP, which measure the efficiency and the
capacity of foods, respectively, normalised to the polyphenol content,
calculated according to the Folin-Ciocalteu method. In this case the
variations of the ratios ePRTE/TP (about 9 times) and of PRTC/TP (about
14 times) are much smaller, as shown in Table 2, but enough high to
highlight the different quality of the polyphenols present in the foods
we have examined. In fact, while the ratios PRTC/TP are particularly high
in the case of red wine, coffee, red chicory and apple (about 30–45), we
must observe that the polyphenols present in red chicory and blueberry
are characterized by higher efficiencies (ratios ePRTE/TP about 5–6 mg
Trolox/mg of TP) than those present in blackberries, in some red wines
and apple cultivars (ratios ePRTE/TP about 1–2). In the case of red wine,
chocolate and blackberry the low efficiency with respect to the
polyphenol content may be due to the presence in these foods of
polymerized polyphenols. In fact in these foods most of the polyphenols
are present as condensation polymers, where the polyphenol reactive
structures are still present (Afaq, Saleem, Krueger, Reed, & Mukhtar,
2005; Gonzalez-Manzano, Santos-Buelga, Prez-Alonso, Rivas-Gon-
zalo, & Escribano-Bailn, 2006; Hager, Howard, Rohana, Lay, & Prior,
2008; Natsume et al., 2000; Vrhovsek, Rigo, Tonon, & Mattivi, 2004).
As expected the presence of polymerized polyphenols does not
modify essentially the capacity of these foods in trapping reactive
radicals, since the experimental PRTC values result close to those
calculated on the basis of the molarity of the monomer units present
in the polymers. On the contrary we found that the experimental
ePRTE values of the polymer are lower than those expected on the
monomer molarity basis. In fact the kinetic rate constants of
polymerized polyphenols towards peroxyl radical should drop due
to stereo hindrance factors and to the lower diffusion coefficients of
these polymers. This finding is in accord with the literature data
(Arteel & Sies, 1999) from which it appears that some oligomers are
less effective in protecting against oxidation reactions than mono-
mers, when the sum of these repeating units in oligomers is
considered.
No correlation appears to exist between the ePRTE and PRTC
values since the ratio ePRTE/PRTC ranges from a minimum value of
about 0.02 in the case of dark chocolate to values higher than 0.69 in
the case of pineapple, see last column of Table 2, where the reactivity
of various foods is compared on the basis of equal trapping capacity.
272 P. Vanzani et al. / Food Research International 44 (2011) 269–275

Table 1
Total phenol content, ePRTE and PRTC of some antioxidant rich foods.

Food TP (mg/g)a ePRTE (mgTrolox/g)b PRTC (μmolLO2•/g)c

Value Average value Rank Value Average value Rank Value Average value Rank
d
Coffee Coffee (Columbia) 44.8 46.8 1 145 177.8 1 1721 1755 1
Coffee (Kenya)d 45.0 191 1704
Coffee (Brazil)d 49.2 189 2003
American coffee 48.0 186 1592
Tea Black tea 39.4 40.7 3 70.3 69.9 2 726 725 3
Green tea 42.0 69.4 724
Red Wine Pinot noir 1.27 1.46 7 1.38 2.43 7 39.4 59.0 6
Cabernet sauvignon 1.84 3.88 82.3
Merlot 1.27 2.03 55.2
Chocolate Dark chocolate (brand A)e 36.5 40.9 2 30.8 34.0 3 1050 1278 2
Dark chocolate (brand B)e 45.2 37.1 1506
Chicory Verona red chicory 2.51 2.07 5 15.9 11.7 5 110 82.4 4
Treviso red chicory 1.40 9.7 50.0
Wild chicory 2.30 9.48 87.3
Berry Blueberryf (Vaccinium myrtillus) 3.29 3.30 4 17.5 12.6 4 48.5 61.4 5
Blueberry f (Vaccinium corymbosum) 3.62 17.2 70.2
Blackberry 3.00 3.23 65.6
Apple Golden delicious 0.85 0.95 9 1.12 1.26 9 33.7 33.8 7
Morgenduft 1.23 1.85 46.0
Pink lady 0.78 0.80 21.6
Citrus Navel orange 0.84 1.14 8 1.57 1.63 8 3.78 4.45 10
Tarocco orange 1.54 2.11 6.26
Clementine 1.04 1.21 3.31
Pomegranate Pomegranatef (Israel) 1.77 1.60 6 1.29 1.24 10 22.6 17.8 8
Pomegranatef (Greece) 1.43 1.19 12.9
Pineapple Pineapple (Costa Rica, brand A) 0.64 0.76 10 3.66 2.64 6 5.33 5.39 9
Pineapple (Costa Rica, brand B) 0.93 2.16 5.62
Pineapple (Brazil) 0.72 2.11 5.22
a
Total phenols from the Folin-Ciocalteu method expressed as milligrams of gallic acid per gram of food. The values reported are the average of 3 measurements (standard deviation
b 5%).
b
ePRTE is value of equivalent Peroxyl Radical Trapping Efficiency of each food in terms of milligrams of Trolox in 1 g of food. The values reported are the average of 4 measurements at
different concentrations (standard deviation b7%).
c
PRTC is Peroxyl Radical Trapping Capacity expressed as micromoles of peroxyl radicals trapped by 1 g of food. The values reported are the average of 4 measurements at different
concentrations (standard deviation b7%).
d
Coffee prepared using an Italian Moka coffee machine.
e
Chocolate 100% cocoa blend of different commercial brands.
f
Foods which fitting is characterized by a value of R2 ≈ 0.98.

In the case of the ePRTE and PRTC values calculated on the basis of
serving size, that is ePRTESS and PRTCSS, the differences between the
maximum and minimum values are lower than those reported in
Table 1. These differences are still large (see Fig. 3), in particular if we
consider that we are comparing foods with high antioxidant
characteristics. In fact from Fig. 3 it appears the high values of PRTESS
of red chicory and blueberry (about 1300–1700 mg Trolox/serving
size) and the low values of this parameter in the case of apple, tea and
pomegranate (about 200 mg Trolox/serving size). As regards the
PRTCSS, the highest values were measured in the case of dark
chocolate, coffee, red wine, and red chicory (8–10 mmol/serving
size), while the lowest values were obtained in the case of pineapple,
orange, and tea (≈ 1 mmol/serving size). In other words the ratio
between the highest and lowest values of the PRTC and of the ePRTE is
about one order of magnitude. However this ratio decreases by a
factor of three if polyphenol content is considered. This is due to the
difference in quality of polyphenols present in the foods we have
tested.
We have considered also the influence of the extraction process on
the PRTE and PRTC. In particular a set of experiments was carried out
on some foods, which are representative of the various classes we
have examined, to simulate in a simple way the processes occurring
during the gastrointestinal digestion. In particular in Table 3 we have
compared the results obtained using as extraction medium i) water;
ii) aqueous 0.12 M HCl; iii) aqueous ethanol (15% water) containing
0.12 M HCl and iv) aqueous 0.12 M HCl followed by neutralization of
the homogenate and treatment with an aqueous solution containing
50 g/L of sodium cholate, to aid the extraction of more hydrophobic
molecules and to simulate the processes occurring in the GI tract.
From the results reported in Table 3 it appears the importance of
acidic conditions similar to those occurring in our stomach (0.12 M
HCl in the gastric juice after stimulation), to achieve a high extraction
yield of polyphenols. This is in accord with the results we reported in a
previous article on red chicories (Rossetto et al., 2005), demonstrating
the importance of low pH values to achieve a high extraction yield of
phenolics with antioxidant properties. Furthermore the extraction
experiments carried out by aqueous acidic ethanol that we have used
in this work or by acidic water followed by treatment with sodium
cholate, so reproducing the condition occurring in the GI tract,
produced similar results.
Because of the high relevance of phenolics in small intestine (Kerem,
Chetrit, Shoseyov, & Regev-Shoshani, 2006) the knowledge of PRTC and
PRTE values of foods under the conditions present in this body
compartment permits to evaluate the protection afforded by the
antioxidants before lipids are adsorbed. According to the data reported
in Fig. 3 the rank of foods changes significantly if we consider the
ePRTESS or PRTCSS values. As regards the capacity to trap peroxyl
radicals, the ranking is dark chocolate ≥ coffee≥ red wine≥ red chicor-
y N blueberry ≥ apple N pomegranate ≈ tea N pineapple ≈ orange. This
order agrees broadly with those reported by other authors (Halvorsen
et al., 2002; Lee et al., 2003; Pellegrini et al., 2003), who measured the
antioxidant capacity of foods with various types of assays such as FRAP,
TEAC, and DPPH. In general, with reference to the serving size, the PRTC
values we have obtained are two–three up to ten times higher, as in the
case of apples, with respect to the capacity values reported by some
authors. This difference could be explained by the different reactivities
 P. Vanzani et al. / Food Research International 44 (2011) 269–275 273

Table 2
ePRTE/TP, PRTC/TP and ePRTE/PRTC ratios of some foodstuffs.

Food ePRTE/TP PRTC/TP ePRTE/PRTC

(mg Trolox/mg) (μmol LO2•/mg) (mg Trolox/μmol LO2•)

Coffee (Columbia) 3.23 38.4 0.08

Coffee (Kenya) 4.24 37.9 0.11
Coffee (Brazil) 3.84 40.7 0.09
American coffee 3.88 33.2 0.12
Black tea 1.78 18.4 0.10
Green tea 1.65 17.2 0.10
Pinot noir 1.09 31.0 0.04
Cabernet sauvignon 2.11 44.7 0.05
Merlot 1.60 43.5 0.04
Dark chocolate (brand A) 0.84 28.8 0.03
Dark chocolate (brand B) 0.82 33.3 0.02
Verona red chicory 6.33 43.8 0.14
Treviso red chicory 6.93 35.7 0.19
Wild chicory 4.12 38.0 0.11
Blueberry 5.32 14.7 0.36
(Vaccinium myrtillus)
Blueberry 4.75 19.4 0.25
(Vaccinium corymbosum)
Blackberry 1.08 21.9 0.05
Golden delicious 1.32 39.7 0.03
Morgenduft 1.50 37.4 0.04
Pink lady 1.03 27.7 0.04
Navel orange 1.87 4.50 0.42
Tarocco orange 1.37 4.06 0.34
Clementine 1.16 3.18 0.37
Pomegranate (Israel) 0.73 12.8 0.06
Pomegranate (Greece) 0.83 9.02 0.09
Pineapple 5.72 8.33 0.69
(Costa Rica, brand A)
Pineapple 2.32 6.04 0.38
(Costa Rica, brand B)
Pineapple (Brazil) 2.93 7.25 0.40

TP: total phenols from the Folin-Ciocalteu method expressed as milligrams of gallic acid
per gram of food.
Fig. 2. Inhibition of LH peroxidation by pineapple and dark chocolate. Representative
ePRTE: equivalent Peroxyl Radical Trapping Efficiency of each food in terms of milligrams
oxygraphic records of linoleic acid peroxidation in a micelle system in the presence of
of reference compound (Trolox) in 1 g of food.
food extracts. Panel A: pineapple extract was added (arrow) in the oxygraphic cell at
PRTC: Peroxyl Radical Trapping Capacity expressed as micromoles of peroxyl radicals
final concentration of 0.25 g fresh fruit/L. Panel B: dark chocolate extract was added
trapped by 1 g of food.
(arrow) in the oxygraphic cell at final concentration of 0.012 chocolate g/L. Insets: plot
of F(xi) versus time according to the described procedure. The peroxidation of LH was
carried out in a solution containing 5 mM DCA, 0.5 mM LG, 2 mM LH and 4 mM ABIP at
37 °C, pH 7.4.

of the titrating molecule (peroxyl radical in our case) that are used to
carry out the various assays, and/or by the acidic conditions of extraction
procedure and/or by the correct calculation procedure we have followed
(Zennaro et al., 2007).
As regards the efficiency of foods that is the reactivity in trapping
reactive radicals, which has never been measured before, the ranking
of foods is blueberry N red chicory N coffee N pineapple ≈ red wine ≥ -
orange ≈ dark chocolate ≈ apple ≥ tea N pomegranate. The efficiency
appears as an important property, for some aspects more informative
than the amount of radicals trapped, since it indicates the possibility
of foods, or better of the system of antioxidants present in foods, to
compete with other molecules such as PUFA, proteins etc. for peroxyl
radicals and therefore to preserve these molecules from oxidative
damages.
An example of the relative importance of PRTE and of PRTC could
be the protection afforded by the foods of Table 2 in the GI tract,
where peroxyl radicals are generated by the reaction between PUFA
and iron containing compounds (Lapidot et al., 2005a,b; Vulcain,
Goupy, Caris-Veyrat, & Dangles, 2005). To verify the possibility that
lipid peroxidation occurs also in the small intestine under the
conditions occurring in this body compartment, PRTC–PRTE experi-
ments, were carried out saturating the LH micelle system with N2–O2
atmosphere, characterized by O2 partial pressures in the range 10–
50 Torr. The addition of 0.1–1 μM bovine Hb, incubated for 1 hour at
37 °C in 50 mM HCl to simulate the passage through the stomach,
leads to a very fast peroxidation process characterized by a constant
rate of oxygen consumption of the order of 2–3 μmol O2 s−1, which
brought in a few seconds the disappearance of the oxygen. This
demonstrates that under the conditions present in small intestine
(low O2 partial pressure and pH values around 7), lipid peroxidation
occurs and is a very fast process.
According to the data of Fig. 3, the amount of peroxyl radicals
which potentially may be trapped after ingestion of a serving size of
these foods is of the order of a millimole or higher. This amount is by
far higher than the amount of peroxyl radicals that can be generated
on the basis of the limited amount of oxygen present in the initial part
of GI tract, that is the stomach and upper part of the small intestine
where the absorption of PUFA occurs (Gorelik, Ligumsky, Kohen, &
Kanner, 2008). As a consequence the foods we have considered
appear equivalent in the protection of the GI tract on a capacity basis
that is on the basis of the PRTC values. However, if we consider the
reactivity, there are large differences in the fraction of peroxyl radicals
escaping the food antioxidants, and consequently the possibility of
radical damages decreases increasing the PRTESS, and therefore also
this parameter should be considered to evaluate the protection
afforded by foods.
4. Conclusions
With the aid of a simple kinetic model, which is rigorously
followed, foods particularly rich of antioxidants have been classified
on the basis of their ability to trap peroxyl radicals according to
274 P. Vanzani et al. / Food Research International 44 (2011) 269–275

Fig. 3. Efficiency, capacity and total phenol content of antioxidant rich foodstuffs on the basis of typical serving sizes. The average values and intervals of ePRTEss as milligrams of
Trolox/serving size (A), PRTCss as mmol of peroxyl radical/serving size (B), and TPss as g gallic acid/serving size of foodstuffs calculated according to the data of Table 1.

stoichiometric (PRTC) or to kinetic parameters (PRTE). This permits a important since only this parameter gives information on the
full and more accurate estimate of their activity as antioxidant in competition for peroxyl radicals between the antioxidants present
protecting the GI tract. In fact important differences in the ranking of in food and biomolecules, preserving them from oxidative damages.
the antioxidant activity of foods exist if the capacity (PRTC) or the
efficiency (PRTE) is considered. The efficiency appears particularly
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