Food Chemistry 188 (2015) 430438

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Phenols and the antioxidant capacity of Mediterranean vegetables

prepared with extra virgin olive oil using different domestic cooking
techniques
Jessica del Pilar Ramrez-Anaya a,, Cristina Samaniego-Snchez b, Ma. Claudia Castaeda-Saucedo c,
Marina Villaln-Mir b, Herminia Lpez-Garca de la Serrana b
a
Departamento de Desarrollo Regional, Centro Universitario del Sur (UdeG), Av. Enrique Arreola Silva 883, C.P. 49000 Ciudad Guzmn, Jalisco, Mexico
b
Departamento de Nutricin y Bromatologa, Facultad de Farmacia UGR, Campus Cartuja s/n, C.P. 10871 Granada, Spain
c
Departamento de Sociedad y Economa, Centro Universitario del Sur (UdeG), Av. Enrique Arreola Silva 883, C.P. 49000 Ciudad Guzmn, Jalisco, Mexico

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

Article history: Potato, tomato, eggplant and pumpkin were deep fried, sauted and boiled in Mediterranean extra virgin
Received 26 December 2014 olive oil (EVOO), water, and a water/oil mixture (W/O). We determined the contents of fat, moisture, total
Received in revised form 30 March 2015 phenols (TPC) and eighteen phenolic compounds, as well as antioxidant capacity in the raw vegetables
Accepted 2 April 2015
and compared these with contents measured after cooking. Deep frying and sauting led to increased
Available online 28 April 2015
fat contents and TPC, whereas both types of boiling (in water and W/O) reduced the same. The presence
of EVOO in cooking increased the phenolics identied in the raw foods as oleuropein, pinoresinol, hydrox-
Keywords:
ytyrosol and tyrosol, and the contents of vegetable phenolics such as chlorogenic acid and rutin. All the
Phenolics
Chlorogenic acid
cooking methods conserved or increased the antioxidant capacity measured by DPPH, FRAP and ABTS.
Extra virgin olive oil Multivariate analyses showed that each cooked vegetable developed specic phenolic and antioxidant
Vegetable activity proles resulting from the characteristics of the raw vegetables and the cooking techniques.
Antioxidant capacity 2015 Elsevier Ltd. All rights reserved.
Domestic cooking

1. Introduction
The Mediterranean diet of the Spanish population is character-
ized by a high intake of vegetables and extra virgin olive oil
(EVOO). These are both important sources of biofunctional com-
pounds that have been linked to the prevention of chronic degen-
erative pathologies (Hernndez-Garca, Wood, Castro-Obregn, &
Covarrubias, 2010), not only because they contain nutritive antiox-
idants such as vitamins C, E, and -carotenes, but also important
amounts of non-nutritive antioxidants such as phenolic com-
pounds (Poljsak, 2011). When the supply of these alimentary com-
pounds is guaranteed in the diet, they complement the activity of
endogenous antioxidant defenses and counter the excess of free
radicals caused by pathological conditions (Finley et al., 2011).
Phenols are one of the most signicant types of antioxidants.
They are found in EVOO and vegetables typically consumed in
Spain, such as eggplant, potato, tomato and pumpkin, with qualita-
tive and quantitative compositions specic to each type.
Corresponding author.
E-mail addresses: jessica@cusur.udg.mx (J.P. Ramrez-Anaya), csama@ugr.es
(C. Samaniego-Snchez), claudia.saucedo@cusur.udg.mx (M.C. Castaeda-Saucedo),
marinavi@ugr.es (M. Villaln-Mir), herminia@ugr.es (H.L.-G. de la Serrana).
Measurements by ABTS, DPPH, FRAP and ORAC have found a close
positive correlation between the phenolic contents in extracts of
various vegetables and their antioxidant capacity, with the highest
contribution made by some phenolic acids such as chlorogenic acid
(Kalogeropoulos, Mylona, Chiou, Ioannou, & Andrikopoulos, 2007;
Dini, Tenore, & Dini, 2013; Luthria, 2012; Martnez-Hernndez,
Arts-Hernndez, Gmez, & Arts, 2013). The prole of the phenols
and antioxidant capacity are altered during the domestic proce-
dures foodstuffs are subjected to before consumption, and it is
generally thought that the effects are destructive. In some case,
however, the parameters actually rise in comparison with the
raw foodstuffs (Bunea et al., 2008; Bellail, Omayma, Mohammed,
Ahmed, & El-Gamal, 2012; Chiou, Kalogeropoulos, Salta,
Efstathiou, & Andikopoulos, 2009). Cooking techniques have differ-
ent effects depending on whether they are carried out in polar or
apolar media. Hydrothermal processes have a drastic effect on
the contents of hydrosoluble antioxidants, such as phenols, but,
on the contrary, when apolar media such as deep or surface frying
are used, the phenol concentrations in the vegetable matrix are less
affected (Miglio, Chiavaro, Visconti, Fogliano, & Pellegrini, 2008). It
has been suggested that losses during culinary treatment are
related to other factors such as the vegetable/water proportion,
cooking time, and the surface area exposed to leaching (Volden,

http://dx.doi.org/10.1016/j.foodchem.2015.04.124
0308-8146/ 2015 Elsevier Ltd. All rights reserved.
 J.P. Ramrez-Anaya et al. / Food Chemistry 188 (2015) 430438
Borge, Hansen, Wicklund, & Bengtsson, 2009). As a result of all the
transformational processes prior to consumption, the eventual
heterogeneity of the foodstuffs means that the databases on
antioxidant contents are still far from being exhaustive, and even
tables as extensive as Phenol-Explorer have limitations, such as
the lack of data on cooked foodstuffs (Tarascou, Souquet, &
Mazauric, 2010). The aim of the present research is to study the
effect of different domestic cooking techniques on the qualitative
and quantitative phenolic composition of representative
Mediterranean foods.
2. Materials and methods
2.1. Chemicals
All chemicals used were analytical reagent grade, unless other-
wise stated, and water was obtained from a Milli-Q purication
system (Millipore, Bedford, MA). Gallic, p-hydroxybenzoic, p-hy-
droxyphenylacetic and vanillic acids and pinoresinol, quercetin,
luteolin, apigenin and 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-
carboxylic acid (Trolox) standards were supplied by Sigma
Aldrich (Milan, Italy). 3,4-dihydroxybenzoic, chlorogenic, caffeic,
syringic, p-coumaric, o-coumaric and ferulic acids, tyrosol and o-
vanillin standards were supplied by Fluka Chemicals (Buchs,
Switzerland), oleuropein by Extrasynthse (Lyon, France), and
rutin by HWI Analytik GMBH pharma solutions (Ruelzheim,
Germany). Hydroxytyrosol standard was synthesized at the
Organic Chemistry laboratory in the Faculty of Pharmacy of
Granada University. FolinCiocalteu phenol reagent by Merck
(Darmstadt, Germany), 2,2-azinobis-(3-ethylbensothiazoline)-6-
sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH)
were supplied by SigmaAldrich (Milan, Italy). Sodium acetate 3-
hydrate, anhydrous sodium carbonate, methanol, ethanol, acetone,
acetic acid glacial, hydrochloric acid (37%), ferric chloride 6-hy-
drate, orthophosphoric acid were supplied by Panreac (Barcelona,
Spain). The 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) for the FRAP
method was also from Fluka Chemicals (Buchs, Switzerland).
2.2. Foodstuffs and cooking conditions
Fresh potato (Solanum tuberosum), pumpkin (Cucurbita
moschata), tomato (Licopersicum esculentum) and eggplant
(Solanum melongena) were acquired in local commercial establish-
ments in Granada (Spain). The vegetables were washed and dried,
the potatoes and pumpkins peeled, and the seed removed from the
pumpkins and the tomatoes. The pieces obtained from three units
of each vegetable was mixed to avoid unnecessary variations and
cut into one-centimeter cubes. The EVOO from Andalusia (Seville,
Spain) was bought in 5 L plastic bottles and stored at 4 C before
use in 500 mL amber glass containers sealed under nitrogen ow.
Four frequently used domestic cooking techniques were chosen
to study the changes in the foodstuffs: deep frying and sauting,
both using EVOO as medium for transfer of apolar heat, boiling
in water as polar medium, and boiling in a water/EVOO mixture
(W/O). The fresh vegetables were used as control. Each test used
120 g of vegetable. The proportion (w/w) of heat transfer medium
and foodstuff, as well as the temperatures during the process were
dened for each technique according to those described in tradi-
tional recipes. Deep-frying was carried out in a 5:1 proportion at
180 C, sauting in a 0.5:1 proportion at 80100 C, while the
two modes of boiling had a 5:1 proportion at 100 C. In the W/O
boiling technique the proportions of water, oil and foodstuff were
4.5:0.5:1. In all cases the vegetable cubes were held at the estab-
lished temperature for 10 min. Time was controlled by introducing
the foodstuff into the heat transfer medium for 10 min and then
431
draining for 5 min. The vegetable samples were then immediately
refrigerated for later homogenization. Each sample was henceforth
stored under conditions of darkness and cold. Tests on the four
cooking techniques with each vegetable were carried out in
triplicate.
2.3. Moisture, dry matter, and fat content
The moisture, fat, and dry matter (DM) contents of the pro-
cessed vegetables were determined using AOAC methods (2007)
and are expressed as percentages (%).
2.4. Extraction conditions
The extracts were obtained following an adaptation of the
method described by Saura-Calixto (1998), in which 5 g of homog-
enized vegetable were combined (1:2 w/v) with acidied methanol
(pH 2 with HCl 2 N) and shaken for 60 min at 200 rpm. The mixture
was then centrifuged at 6800g for 15 min (Universal 320 Hettich,
Tuttlingen Germany). After recovery of the supernatant the proce-
dure was repeated, this time using 70% acetone. Volume was
adjusted to 25 mL with acidied methanol and the mixture stored
as 7 mL aliquots at 21 C for no longer than 2 months.
2.5. Phenol contents
2.5.1. Total phenolic content (TPC)
Total phenolic content was determined using the Folin
Ciocalteu colorimetric method described by Singleton and Rossi
(1965) and modied in our laboratory (Samaniego-Snchez,
Stagno, Quesada-Granados, Blanca-Herrera, & Brandolini, 2013).
We added 2.5 mL of deionized water and 500 lL of Folin
Ciocalteu reagent to 500 lL of methanolic pulp extract. The mix-
ture was allowed to stand for 5 min, and then 2 mL of a 10% aque-
ous Na2CO3 solution was added. The nal volume was adjusted to
10 mL. Samples were allowed to stand for 90 min at room temper-
ature before measurement at 700 nm versus the blank using a
Boeco S-22 UVVIS spectrophotometer (Hamburg, Germany). The
amount of total phenolics is expressed as gallic acid equivalents
per gram of fresh weight (mg GAE/g FW) through the calibration
curve of gallic acid. The calibration curve range was 0.57.5 lg/
mL (r = 0.999).
2.5.2. Individual phenol content by Liquid chromatography (HPLC)
HPLC was used in the determination of phenolic compounds.
HPLC separation was performed with a Varian Pro Star system
(Madrid, Spain) equipped with an autosampler, binary pump and
UV detector. System Control-Konik software was used. The column
was a Tracer Extrasil ODS25 lm, 15  0.4 cm (Teknokroma,
Barcelona, Spain). Phenolics were evaluated following the modied
method described by Escarpa and Gonzlez (2001). An extract
solution of vegetable in methanol was analyzed. The elution sol-
vents were classied as A (aqueous 0.01 M phosphoric acid) and
B (100% methanol) respectively. The samples were eluted accord-
ing to the following binary gradient: 5% in B as initial conditions,
40% in B for 25 min, 50% in B for 15 min, 70% in B for 5 min: 80%
in B for 10 min and nally 100% in B for 5 min. The ow rate was
1.0 mL/min, maintained for 60 min. Measurements were taken at
280 nm, and quantied by the external standard method. The
mobile phases and samples were previously ltered through a
0.45 lm membrane and ultrasonically degassed prior to use.
After this, 5 lL was injected. Peaks were identied by comparing
the retention times with those obtained with a standard solution
of phenols under study. As external standard we used individual
stock standard solutions prepared daily of around 0.5250 lg/mL
for each phenol. The stock standard was added to several problem
432 J.P. Ramrez-Anaya et al. / Food Chemistry 188 (2015) 430438
samples to verify the increase in area of the chromatographic peak.
Standard solutions were stored in the dark at 20 C. Results were
expressed as lg per gram of fresh vegetable (lg/g FW).
2.6. Antioxidant capacity
The antioxidant capacity of the samples was measured in a BMG
Labtech FLUOstar Omega plate reader (Offenburg, Germany) using
the Omega Control program and MARS Data Analysis Software. The
spectrophotometric techniques used were those by Brand-
Williams, Cuvellier, and Berset (1995) with N,N0 -dimethyl-p-
phenylenediamine (DPPH), that of Benzie and Strain (1996) based
on the reducing ability of ferric iron (FRAP), and the antioxidant
equivalent capacity (ABTS) of Pellegrini, Re, Yang, and Rice-Evans
(1999). ABTS and DPPH methods are based on the formation of a
colored radical. Post-addition tests were used, with formation of
the radical in the absence of the sample until a stable signal was
reached. In the FRAP assay, excess FeIII was used, and the rate-lim-
iting factor of FeII-TPTZ, and hence color formation is the reducing
ability of the sample. We then added the sample and measured the
resulting change in absorbance (discoloration of the radical in
DPPH and ABTS, or blue color developing in the FRAP test), which
was proportional to the concentration and antioxidant activity of
the substance analyzed. In all the methods applied, we determined
the dilution of potato, pumpkin, tomato and eggplant methanolic
extracts that gave a linear response. The absorbance signal was
translated into antioxidant activity using Trolox as standard
antioxidant. Different calibration curve ranges were used depend-
ing on the method. Results were expressed as lm of Trolox equiv-
alents per gram of fresh vegetable (lmol TE/g FW). All the
parameters were determined in triplicate and the values expressed
as averages standard deviation.
2.7. Statistical analysis
For each vegetable, the values obtained by the different treat-
ments were compared using ANOVA and the multiple comparisons
of means test (p < 0.05). We also used multivariate analysis tech-
niques: Wards cluster analysis and discriminant analysis. The for-
mer included the results of all the variables, using the city block
algorithm to measure the distance between conglomerates, and,
once grouped according to the conglomerates obtained, they were
compared by applying ANOVA (p < 0.05). We used discriminant
analysis to determine the degree of differentiation between the
vegetables and their different methods of preparation. Three cate-
gories were established by grouping the variables as follows: by
total and individual phenol concentrations, by the antioxidant
capacity measured by the three different methods, and by their
moisture, dry matter and fat contents. Models were obtained that
included one or a combination of the three groups of variables. The
discriminatory functions obtained in each case were evaluated by
their statistical signicance (p < 0.05), the canonic correlation
(r P 0.90), and the percentage of correct classication (100%), tak-
ing the ideal model to be one including the least number of vari-
ables. The analyses were carried out using Statgraphics Plus 4.1.
3. Results
3.1. Moisture, dry matter and fat content
In Tables 14 we can see that the highest loss of moisture and
highest fat gain in the vegetables occurred during deep frying,
whereas sauting (at <100 C) maintained percentages of moisture
similar to those of the raw vegetables and the vegetables boiled by
both methods, but with lower percentages of fats. With boiling, the
trend was to increase (p < 0.05) or not signicantly change
(p > 0.05) the moisture content, while the percentages of dry
extract and fat fell or were unchanged. The trend in the W/O
method was only different to boiling in water in a higher fat con-
tent due to the absorption of EVOO. The variation in the moisture
content of the vegetables affected the proportions of fat and DM in
the food after the different cooking processes. It is known that fry-
ing is a dehydration process in which the water in the surface lay-
ers evaporates giving way to oil within the foodstuffs pores (Chiou
et al., 2009).
3.2. Total phenolic content
Tables 14 show that, in comparison to the raw vegetables,
there were increases in TPC in the four deep fried vegetables, in
the sauted pumpkin and in the eggplant boiled by both methods.
There was also a non-signicant (p > 0.05) increase in the sauted
eggplant and tomato. In contrast, TPC fell in both the potato and
the pumpkin boiled by both methods, while the decrease was
not signicant in the cases of the sauted potato, the potato boiled
in W/O and the tomato boiled by both methods. We may infer from
the foregoing that when EVOO was used as heat transfer medium,
an increase in TPC was more frequent, whereas the use of water
tended to make it fall. Three of the vegetables analyzed (potato,
tomato and pumpkin) showed the following decreasing order of
TPC for the cooking techniques: deep frying > sauting > boil-
ing > boiling W/O. Deep frying caused the highest TPC values, par-
ticularly in the case of eggplant, although this was lower than the
values reported by Kalogeropoulos et al. (2007). The increase in
phenol concentrations observed in the vegetables processed with
EVOO is the result of the simultaneous action of several mecha-
nisms. Some authors have described the transfer to the foodstuff
of the phenols present in the absorbed EVOO, the effect of concen-
tration in the food matrix after partial evaporation of moisture
(Provesi, Dias, & Amante, 2011), and the lack of diffusion to the
EVOO, because migration of hydrosoluble substances toward polar
media does not occur spontaneously (Fillion & Henry, 1998). It has
been shown that there is an increase in the availability of phenols
physically and chemically linked to the microstructure of the pro-
cessed vegetables in comparison to the raw (Martnez-Hernndez
et al., 2013), whether because of the breakage or softening of the
rigid cell walls and other components of the vegetable cells (vac-
uoles and apoplasts), or because of the decomposition of phenolic
compounds linked to the ber (cellulose and pectin) (Gkmen,
Serpena, & Fogliano, 2009). The breaking of phenol-sugar glyco-
sidic links giving rise to aglycons also contributes to the increase
in phenol concentration (Singleton, Orthofer, & Lamuela-
Raventos, 1999). This last mechanism is perhaps the main one con-
cerned in the increase of phytonutrient concentrations, which has
been suggested to explain the variations during not only frying, but
also oven baking, microwave cooking, boiling and the culinary
preparation of various green-leaf vegetables, among others
(Bunea et al., 2008). In addition, the causes of the changes mea-
sured in the vegetables prepared in water included both the
increase of availability by the same causes described for the oil
treatments, and also the decrease of phenol concentrations by
leaching from the vegetable into the boiling water. In these cases,
the destruction of cell walls and subcellular compartments during
boiling facilitated the migration of hydrosoluble substances toward
the extracellular space and from there to the processing water,
thus causing a reduction of TPC in the vegetables with the corre-
sponding enrichment in the cooking water (Miglio et al., 2008).
Similar results were reported for cooking cauliower (Volden
et al., 2009). The reactions of thermal breakdown, of covalent link-
age formation between oxidated molecules and other compounds,
or polymerization and hydrolisis also lead to the reduction of TPC
 J.P. Ramrez-Anaya et al. / Food Chemistry 188 (2015) 430438 433

Table 1
Moisture, fat, dry matter, phenolic concentration and antioxidant capacity in potatoes prepared by four cooking techniques.

Domestic cooking technique

Raw Deep fried Sauted Boiled Boiled (W/O)
Moisture, fat and dry matter content (%)A
Moisture 81.12 0.2c 44.39 3.2a 73.63 0b 83.44 0.5d 80.20 1.4c
Fat 0.05 0.05a 13.10 0.4c 3.10 0.4b 0.06 0a 3.07 0.9b
Dry matter 18.33 0.14b 42.5 2.58d 23.27 9.29c 15.89 0.35a 16.65 1.63a
Total phenol content (mg GAE/g FW)A
Total phenols 0.35 0.03b 0.55 0.05c 0.32 0.01b 0.25 0.03a 0.19 0.01b
Individual phenol content (lg/g FW)A
Gallic acid 0.017 0a 0.067 0c 0.066 0c 0.059 0b 0.064 0c
Hydroxytyrosol nd 1.329 0.04c 0.878 0.06b nd 0.558 0.01a
3,4-Dihydroxybenzoic acid 0.142 0ab 0.195 0.01c 0.151 0.01b 0.1467 0.01ab 0.139 0a
Tyrosol nd 0.177 0.01b 0.147 0.01a nd nd
p-Hydroxyphenylacetic acid 0.261 0.01a 0.277 0.01a 0.273 0.02a nd nd
p-Hydroxybenzoic acid 0.047 0a 0.072 0b 0.069 0.01b 0.054 0a 0.053 0a
Chlorogenic acid 0.191 0a 0.439 0.03b 0.413 0b 0.173 0a 0.177 0a
Vanillic acid 0.131 0a 0.152 0a 0.146 0.01a 0.129 0.01a 0.139 0a
Caffeic acid 0.019 0cd 0.019 0bc 0.021 0d 0.017 0ab 0.016 0a
Syringic acid 0.023 0ab 0.022 0 a 0.025 0c nd 0.024 0bc
p-Coumaric acid 0.067 0a 0.214 0d 0.138 0.01c nd 0.089 0.01a
o-Vanillin 1.012 0.05c 0.142 0a 0.137 0a 0.679 0.06b 0.127 0.01a
o-Coumaric acid 0.083 0b 0.182 0.01e 0.123 0.01d 0.044 0a 0.094 0c
Oleuropein nd 1.196 0.02e 0.679 0.03a nd 0.797 0.07b
Pinoresinol nd 0.31 0.01b 0.247 0.02a nd 0.269 0.02ab
Luteolin nd 0.138 0c 0.127 0b nd 0.119 0.01a
Apigenin 0.519 0.03c 0.204 0.01b 0.262 0.02a nd 0.202 0.01b
Rutin nd nd nd nd nd
Antioxidant capacity (lmol TE/g FW)A
DPPH 0.53 0.04a 3.45 0.39e 2.44 0.02d 1.19 0.22c 0.89 0.13b
FRAP 0.68 0.0a 1.78 0d 1.54 0.17c 0.88 0.02b 0.93 0.03b
ABTS 3.66 0.19b 10.13 0.13e 4.91 0.30c 2.57 0.03a 5.67 0.16d

nd: not detected, FW: fresh weight, W/O: water/oil mixture.

ae
Means with different letters within the same line are signicantly different (p < 0.05).
A
Values are the means SD (n = 3).

concentrations in the vegetables after boiling (Friedman, 1996) In
the case of the vegetables boiled in W/O, the TPC fell despite the
enrichment of the prole with the phenols of the EVOO. This ten-
dency can be explained by the simultaneous action of the transfer
phenomena observed during frying and leaching during boiling.
3.3. Individual phenolic content
In the proles of the vegetables prepared with EVOO and the W/
O mixture, we detected additional phenols to those identied in
the raw vegetables and those prepared without oil. The ve most
abundant phenols in the raw vegetables and those cooked without
EVOO (Tables 14) were o-vanillin, hydroxyphenylacetic, chloro-
genic and vanillic acids, and rutin, the last only in tomatoes. We
also measured dihydroxybenzoic, gallic, o-coumaric acids, and api-
genin. However, in addition to these, in the vegetables processed
with EVOO we detected and quantied oleuropein, hydroxytyrosol,
tyrosol, pinoresinol, and p-coumaric and hydroxybenzoic acids. We
conrmed the transfer of phenols from the EVOO to the vegetables,
as they were enriched by compounds exclusively found in the oil
(oleuropein, pinoresinol, tyrosol and hydroxytyrosol) (Boskou,
2009), whereas there were no initial contents of these compounds
in the raw vegetables (Tables 14). Similar dynamics have been
reported in other studies on frying potatoes and eggplant, with
retention percentages of 25% (oil), 38% (fried potato) and 70% (fried
eggplant) (Kalogeropoulos et al., 2007). We likewise conrmed the
increase in availability of phenols in the vegetables after thermal
processing by analyzing the phenols absent from EVOO such as
chlorogenic acid and rutin. The data show an increase in the con-
centrations of these compounds in the vegetables even though
the oil did not provide them, for these phenols were not detected
in the fresh EVOO of our study (data not shown) and, moreover,
they have not been reported as part of the typical phenolic prole
of EVOO (Boskou, 2009). Similarly to other authors, we quantied
chlorogenic acid in tomato and pumpkin, and found it to be the
majority phenol in potato and eggplant (Tables 14) (Das et al.,
2011; Luthria, 2012). The variation in this phenolic acid during
deep frying and sauting coincided with the rising trend of the
total phenol concentrations in deep fried potato, tomato, eggplant
and pumpkin. Deep frying was the technique with highest associ-
ated increases and can therefore be considered an improvement in
the cooking process, although it also increases the calorie density
of the food because of the amount of oil absorbed. The increases
or conservation we found agree with those reported for sauted
bamboo shoots (Zhang, Ji, Hu, Chen, & Ye, 2011), and sauted pota-
toes, peppers and zucchini (Kalogeropoulos et al., 2007). However,
a decrease in TPC, in particular of chlorogenic acid, was also
detected in sauted eggplant, for which a possible explanation
might be oxidation caused by higher exposure to air, as the food
is not totally submerged in the oil (Kalogeropoulos et al., 2007).
This is also supported by the results of Chiou, Kalogeropoulos,
Efstathiou, Papoutsi, and Andrikopoulos (2013), who found higher
concentrations of oleuropein in the oil recovered after deep-frying
potatoes, in comparison with the oil used for pan-fried potatoes.
During the assays of frying techniques, leaching was not consid-
ered a cause of the variations in phenol contents, as migration of
hydrosoluble substances toward polar media does not occur spon-
taneously (Fillion & Henry, 1998). In the cases of boiling in water
and in W/O, where water is used as the heat transfer medium,
the concentrations of chlorogenic acid in potato, pumpkin and
tomato remained practically the same as in the raw vegetables.
The only case of signicant increase (p < 0.05) was eggplant boiled
434 J.P. Ramrez-Anaya et al. / Food Chemistry 188 (2015) 430438

Table 2
Moisture, fat, dry matter, phenolic concentration and antioxidant capacity in eggplant prepared by four cooking techniques.

Domestic cooking technique

Raw Deep fried Sauted Boiled Boiled (W/O)
Moisture, fat and dry matter content (%)A
Moisture 94.23 0.75d 31.81 3.4a 66.55 1.03b 95.94 0.27d 83.49 1.2c
Fat 0.01 0.01a 56.63 4.5d 27.37 0.2c 0.07 0.08a 12.70 1.5b
Drymatter 5.76 0.76b 11.56 1.1c 6.08 1.03b 3.99 0.29a 3.81 0.54a
Total phenol content (mg GAE/g FW)A
Total phenols 0.27 0.02a 2.80 0.25c 0.38 0.02a 0.69 0b 0.65 0.02b
Individual phenol content (lg/g FW)A
Gallic acid Trazas nd 0.049 0a Trazas nd
Hydroxytyrosol nd 0.408 0.01c 0.170 0b nd 0.165 0ab
3,4-Dihydroxybenzoic acid 0.134 0.01a 0.310 0.01c nd 0.139 0a 0.183 0.01b
Tyrosol nd 0.234 0.01b 0.134 0a nd 0.135 0a
p-Hydroxyphenylacetic acid 0.260 0.02a 0.325 0.01b 0.322 0b 0.285 0a 0.281 0a
p-Hydroxybenzoic acid 0.054 0a 0.336 0d 0.052 0a 0.126 0b 0.147 0.01c
Chlorogenic acid 1.315 0.07a 10.901 0.57d 2.528 0.07b 3.851 0.13c 3.738 0.11c
Vanillic acid 0.133 0a 0.264 0.02d 0.160 0.01b 0.165 0b 0.232 0c
Caffeic acid 0.014 0a 0.054 0c 0.051 0c 0.022 0b 0.014 0a
Syringic acid 0.023 0a 0.029 0b 0.055 0d 0.047 0c 0.028 0b
p-Coumaric acid 0.071 0a 2.15 0.10d 0.207 0b 0.359 0.01c 0.370 0c
o-Vanillin 0.132 0a 0.197 0c 0.174 0.01b 0.126 0.01a nd
o-Coumaric acid 0.023 0a 0.023 0a 0.027 0b 0.023 0a 0.022 0a
Oleuropein nd 0.263 0.01b 0.169 0a nd nd
Pinoresinol nd 0.288 0.01a 0.212 0a nd 0.207 0a
Luteolin nd nd nd nd nd
Apigenin nd 0.206 0.01a nd nd 0.220 0b
Rutin nd nd nd nd nd
Antioxidant capacity (lmol TE/g FW)A
DPPH 0.57 0.06ab 13.39 0.39d 1.19 0.07c 3.68 0.33bc 2.66 0.12abc
FRAP 1.41 0.08ab 38. 88 1.02d 2.69 0.28b 10.42 0.82c 10.13 0.49c
ABTS 1.34 0.08a 18.19 1.46b 3.43 0.1d 4.10 0.06e 2.43 0.14c

nd: not detected, FW: fresh weight, W/O: water/oil mixture.

ae
Means with different letters within the same line are signicantly different (p < 0.05).
A
Values are the means SD (n = 3).

in water and in W/O. The increase detected in the frying techniques
means that these increased the availability of chlorogenic acid,
whereas with boiling techniques leaching is the factor that most
contributes to the fall in concentration of this phenol. Francisco,
Velasco, Moreno, Garca-Viguera, and Cartea (2010) attributed this
reduction in Brassica rapa to the decomposition of the phenol dur-
ing boiling at 100 C, and Mulinacci et al. (2004) associated it with
isomerization and hydrolysis, which caused redistribution of phe-
nolic acids by transesterication during boiling.
3.4. Antioxidant capacity
The antioxidant capacity of the vegetables was determined with
the DPPH, FRAP and ABTS methods and the results are summarized
in Tables 14. All the domestic processing techniques increased the
antioxidant capacity of the four vegetables. A fall in capacity or
absence of signicant change (p > 0.05) were only found after
hydrothermal cooking, the most frequent being reductions
detected by ABTS in pumpkin prepared by all methods, and in
boiled potato, as well as in the FRAP determinations of tomato
boiled in water and W/O and in pumpkin boiled in W/O. Lack of
change was only detected in DPPH determination in sauted and
boiled (both types) eggplant. The cooking techniques can be
ranked in decreasing order of antioxidant capacities determined
for the four vegetables as follows: deep frying > sauting > boil-
ing > raw. This pattern was found to be most consistent in the
results of the DPPH and FRAP tests, occurring in all four vegetables
in the case of the former and in three (potato, tomato and pump-
kin) in the latter. The results of the ABTS test did not, however,
show a common trend among the vegetables. It can be seen that
the antioxidant capacity of the foods processed only in oil
increased more than when processed in water or W/O. The two
types of hydrothermal cooking remained grouped together in the
ranking, because there was no consistency to the effects of adding
oil to the cooking water on the antioxidant capacity of the vegeta-
bles, as there were almost as many cases of increase as of decrease.
We only observed a consistent additional effect in the ABTS results
of eggplant, tomato and pumpkin and the DPPH results of eggplant
and potato. We even found higher antioxidant capacity in the veg-
etables boiled in water than in W/O in the cases of potato (ABTS),
tomato (DPPH) and pumpkin (DPPH and FRAP), and no additional
improvement was detected in the potato, tomato and eggplant
tested by FRAP. Other authors results depend on the vegetable
and the cooking method. As in this study, signicant increases
were found by Bellail et al. (2012) using DPPH to test boiled and
microwave cooked, oven baked and deep-fried potatoes, with high-
est results shown by the last technique. The same was true for
Miglio et al. (2008) using ABTS on boiled, sauted and steam
cooked carrots and zucchini. Increases were also found using the
FRAP method by Martnez-Hernndez et al. (2013) in boiled and
grilled broccoli, by Dini et al. (2013) in boiled, baked and fried
pumpkin, and by Pellegrini et al. (2009) in boiled and deep-fried
brassicaceae, potatoes, zucchini and carrots. The increase in the
antioxidant capacity of the vegetables processed as studied can
be attributed to the increase in phenol concentration due to both
higher availability and also to the increase associated with EVOO
absorption as mentioned in Section 3.1. This is supported by the
fact that the total phenol concentration and the antioxidant capac-
ity measured by DPPH, FRAP and ABTS showed signicant correla-
tion (Section 3.3). On the contrary, reduction of antioxidant
capacity has been measured by DPPH in boiled mushrooms
(Nguyen, Nagasaka, & Ohshima, 2012), by ABTS in boiled tomatoes
 J.P. Ramrez-Anaya et al. / Food Chemistry 188 (2015) 430438 435

Table 3
Moisture, fat, dry matter, phenolic concentration and antioxidant capacity in tomatoes prepared by four cooking techniques.

Domestic cooking technique

Raw Deep fried Sauted Boiled Boiled (W/O)
Moisture, fat and dry matter content (%)A
Moisture 94.54 0.13c 32.22 1a 88.42 2.17b 96.67 0.45d 94.10 0.63c
Fat 0.07 0.03a 13.53 1.3d 7.20 0.1c 0.07 0a 1.19 1.02b
Drymatter 5.39 0.11a 51.48 5.59b 4.38 1.46a 3.25 0.32a 4.72 0.99a
Total phenol content (mg GAE/g FW)A
Total phenols 0.21 0.01a 5.34 0.52b 0.33 0.02a 0.13 0.01a 0.13 0a
Individual phenol content (lg/g FW)A
Gallic acid 0.039 0a 0.064 0c 0.050 0b nd nd
Hydroxytyrosol nd 4.715 0.14b 0.183 0a nd nd
3,4-Dihydroxybenzoic acid nd 0.354 0.01b 0.153 0a nd nd
Tyrosol nd 0.145 0b 0.143 0b nd 0.085 0a
p-Hydroxyphenylacetic acid 0.330 0b 0.639 0.01c 0.325 0b 0.278 0.01a nd
p-Hydroxybenzoic acid 0.050 0a 0.075 0b 0.073 0b nd nd
Chlorogenic acid 0.077 0a 0.439 0.01c 0.108 0b nd nd
Vanillic acid 0.132 0a nd 0.132 0a 0.124 0.01b 0.063 0a
Caffeic acid 0.025 0c 0.052 0d 0.019 0b nd 0.010 0a
Syringic acid nd 0.071 0c 0.024 0b nd 0.019 0a
p-Coumaric acid 0.091 0b 0.204 0c 0.074 0b 0.075 0b 0.034 0a
o-Vanillin 0.151 0.01abc 0.355 0.01cd 0.244 0.01bc 0.148 0abc 0.075 0ab
o-Coumaric acid 0.032 0c 0.027 0b 0.022 0b 0.022 0b 0.011 0a
Oleuropein nd 0.487 0.01c 0.314 0.02b nd 0.124 0.01a
Pinoresinol nd 0.364 0.03c 0.236 0.01b nd 0.186 0.01a
Luteolin 0.125 0c 0.175 0d 0.124 0b nd 0.072 0a
Apigenin 0.231 0.01b nd nd 0.245 0.01b 0.115 0a
Rutin 0.179 0.01b 0.354 0.02c 0.197 0.01b 0.148 0.012a nd
Antioxidant capacity (lmol TE/g FW)A
DPPH 2.08 0.07c 13.60 0.24e 8.28 0.28d 0.98 0.09a 1.56 0.11b
FRAP 2.02 0c 16.97 1.05e 2.43 0.03d 0.57 0.04a 1.09 0.04b
ABTS 0.52 0.02a 11.06 0.14e 0.81 0.02b 2.94 0.16d 1.75 0.05c

nd: not detected, FW: fresh weight, W/O: water/oil mixture.

ae
Means with different letters within the same line are signicantly different (p < 0.05).
A
Values are the means SD (n = 3).

(Danesi & Bordoni, 2008) and by FRAP in various types of sauted
cauliower and zucchini (Pellegrini et al., 2009; Volden et al.,
2009). Zhang et al. (2011) mention that the migration of a large
amount of substances into the cooking medium after boiling bam-
boo shoots caused a fall in antioxidant capacity in the shoots and a
rise in that of the water as measured by DPPH. Observations such
as this lead to the recommendation that the water in which food-
stuffs have been cooked should also be consumed (Danesi &
Bordoni, 2008; Francisco, Velasco, Moreno, Garca-Viguera, &
Cartea, 2010).
3.5. Correlations
The correlations between the antioxidant capacity measured by
DPPH, FRAP and ABTS and the TPC were signicant (p < 0.05) and
positive (r = 0.78, 0.70 and 0.52, respectively). Positive correlations
of antioxidant capacity determined by DPPH with phenol contents
have previously been reported in foodstuffs such as eggplant
(r = 0.53 to r = 0.79) (Martnez-Hernndez et al., 2013) and tomato
(r = 0.98) (Samaniego-Snchez et al., 2013), among others. In this
study, the correlation between ABTS measured antioxidant capac-
ity and total phenol contents were low, whereas in other studies it
has been high, e.g., Rautenbach, Faber, Laurie, and Laurie (2010) in
boiled potatoes (r = 0.93). Also in boiled potatoes the same authors
reported high correlations between FRAP measured antioxidant
capacity and polyphenol contents (r = 0.95). In eggplants the corre-
lation was even found with the contents of chlorogenic acid and its
derivatives (Devanand, 2012). The correlations of the antioxidant
capacity measured by the different methods were signicant
(p < 0.05) between FRAP and DPPH (r = 0.76), and between DPPH
and ABTS (r = 0.29), but not between FRAP and ABTS (p > 0.05).
DPPH and FRAP tend to be used together in the study of the effects
of domestic processing on antioxidant capacity, the rst to evalu-
ate the capacity to capture free radicals and the second to deter-
mine the reductive capacity of a sample (Dini et al., 2013).
Antioxidant compounds, such as polyphenols, can be very efcient
reductive agents of ferric iron, but some are not efcient captors of
DPPH radicals because of steric obstacles (Dini, Tenore, & Dini,
2010). The limitations of the ABTS method in comparison with
FRAP and DPPH such as the higher afnity for hydrosoluble com-
pounds regardless of their true antioxidant potential contribute
to the low correlations of antioxidant capacity with TPC
(Roginsky & Lissi, 2005). Limitations of the method also gave a
lower proportion of signicant differences between the values of
the vegetables prepared using less drastic methods (raw, the two
boiling techniques and sauteeing), because the ABTS method is less
useful for showing more subtle differences in antioxidant capacity
(Gahler, Otto, & Bohm, 2003).
3.6. Multivariate analysis
3.6.1. Cluster analysis
The 20 observations of the various combinations of vegetables
and cooking methods were grouped in 7 clusters (Fig. 1). Clusters
2, 5 and 7 (respectively deep fried pumpkin, tomato and eggplant)
were cases of deep frying separated from the other groups by the
particularly high levels of TPC and antioxidant capacity measured
by FRAP and DPPH. They also presented high concentrations of
dihydroxybenzoic acid, oleuropein, pinoresinol, low moisture con-
tents, and high fat contents. Because of these characteristics, the
deep fried vegetables in these groups could be seen as fatty
antioxidant concentrates. Clusters 1, 3, 4 and 6 tended to form
436 J.P. Ramrez-Anaya et al. / Food Chemistry 188 (2015) 430438

Table 4
Moisture, fat, dry matter, phenolic concentration and antioxidant capacity in pumpkin prepared by four cooking techniques.

Domestic cooking technique

Raw Deep fried Sauted Boiled Boiled (W/O)
Moisture, fat and dry matter content (%)A
Moisture 87.89 0.95c 46.11 2.7a 82.61 1.31b 92.41 0.66d 89.56 1.5c
Fat 0.09 0.03a 17.58 0.8d 4.09 0.27c 0.08 0.01a 2.75 0.27b
Drymatter 12.02 0.03b 36.31 2.43c 13.29 1.32b 7.51 0.65a 6.68 1.23a
Total phenol content (mg GAE/g FW)A
Total phenols 0.38 0.01c 1.69 0.02e 0.5 0d 0.36 0.04b 0.27 0a
Individual phenol content (lg/g FW)A
Gallic acid 0.027 0.00b 0.026 0e 0.044 0c 0.026 0b 0.056 0d
Hydroxytyrosol nd 1.479 0.1c 1.355 0.06b nd 0.786 0a
3,4-Dihydroxybenzoic acid 0.19 0b 0.184 0.13c 0.179 0.00ab 0.184 0.13ab 0.175 0.01a
Tyrosol nd 0.125 0.01c 0.167 0.01ab nd 0.171 0.002b
p-Hydroxyphenylacetic acid 0.468 0.01a 1.049 0.01b 0.46 0.01a 1.049 0.01c 1.647 0.05d
p-Hydroxybenzoic acid 0.057 0a 0.072 0c 0.077 0.003b 0.072 0b 0.061 0.01a
Chlorogenic acid 0.076 0.01a 0.078 0b 0.087 0.01a 0.078 0.001a 0.084 0.003a
Vanillic acid nd 0.122 0.01b 0.134 0a 0.122 0.01a 0.133 0.002a
Caffeic acid 0.018 0b 0.015 0d 0.023 0d 0.015 0a 0.02 0c
Syringic acid 0.022 0a 0.027 0d 0.028 0.002c 0.027 0bc 0.024 0ab
p-Coumaric acid 0.073 0.01a 0.085 0e 0.111 0.004d 0.085 0b 0.092 0c
o-Vanillin 0.135 0.01a 0.14 0.004b 0.134 0.003a 0.14 0.004a 0.133 0a
o-Coumaric acid 0.099 0.01ab 0.103 0.004c 0.111 0.002b 0.103 0.004b 0.086 0.01a
Oleuropein nd nd 0.313 0.023b nd 0.266 0.01a
Pinoresinol nd nd nd nd nd
Luteolin nd nd nd nd nd
Apigenin nd 0.233 0.01a nd 0.233 0.01b nd
Rutin nd nd nd nd nd
Antioxidant capacity (lmol TE/g FW)A
DPPH 0.03 0c 3.96 0.05e 0.75 0.01d 0.06 0a 0.13 0b
FRAP 1.27 0.06b 8.1 0.59d 2.78 0.05c 1.22 0.03b 0.56 0.05a
ABTS 3.51 0.25e 1.71 0.06b 2.96 0.03d 2.81 0.14c 1.35 0.02a

nd: not detected, FW: fresh weight, W/O: water/oil mixture.

ae
Means with different letters within the same line are signicantly different (p < 0.05).
A
Values are the means SD (n = 3).

240 concentrations of antioxidants, such as eggplant, and when consid-

ered without the addition of oil it makes up, together with pump-
200 kin (raw and boiled in water and W/O) one of the groups with the
lowest concentration of total and single phenols (cluster 1), both
160
Distance

because of the nature of the foodstuff and because of the detrimen-

120 tal effect of boiling (higher moisture content overall with reduction
of dry extract). Cluster 4 (raw and boiled tomato) is a similar case
80 to cluster 1.
40

0 3.6.2. Discriminant analysis

Boiled Eggpl
Boiled Pto

Boiled W/O Eggp

In the analysis by cooking method and by vegetable, we

Boiled W/O Tmt

Dp Fried Eggp
Dp Fried Pto
Boiled Tmt

Raw Eggpl

Dp Fried Tmt
Boiled W/O pmpk

Sauted Eggpl
Raw Pmpk

Boiled W/O Pto

Sauted Pmpk
Boiled Pmpk

Raw Pto

Sauted Pto
Raw Tmt

Sauted Tmt

Dp Fried Pmpk

obtained satisfactory discriminant models based on the proles

11 4 6 2 3 5 7
Fig. 1. Dendrogram plot of distance in cooking technique-vegetable cases.
1
Indicates cluster number; Dp fried: Deep fried; Pto: potato; Eggpl: eggplant; Tmt:
tomato; Pmpk: pumpkin.
according to the vegetable in question, but without attaining com-
plete resolution, so that clusters 1 (pumpkin) and 3 (potato) were
associated, as were clusters 4 (tomato) and 6 (eggplant). Cluster 6
is based on the high concentrations of chlorogenic acid and the
highly antioxidant nature of eggplant (Luthria, 2012), where the
effect of concentration was not determinant. In cluster 3, the effect
of antioxidant concentration through moisture loss and fat supply
increased the properties measured in potato cooked in EVOO. The
nature of potato does not correspond to that of a source of high
of single and total phenols (Fig. 2) that provided good grouping
around the centroids, good differentiation between groups and
the lowest number of variables (nineteen). The model for the cook-
ing methods consisted of 5 equations, and that of the vegetables of
4. The rst equation covered the highest discrimination percentage
in both cases (79.39% and 67.47%, respectively). The ve most
important phenols were dened on the basis of the rst equation,
taking as criterion those that presented the highest coefcients. In
the model of discrimination by cooking method, the phenols with
highest coefcients in the rst discriminant equation were gallic,
chlorogenic and hydroxybenzoic acids, tyrosol and pinoresinol. In
the model discriminating between the four vegetables, the ve
most important phenols in the main equation were chlorogenic,
p-coumaric and dihydroxybenzoic acids, pinoresinol and luteolin.
Cluster analysis showed that the grouping of observations is a
function of both the antioxidant characteristics of each vegetable,
and of the cooking method used, because the clusters tended to
form according to the vegetable, and were not made up of homo-
geneous groups of cooking techniques or specic vegetables.

11
7 Boiled W/O
3 Raw
-1
Second discriminant function
-5
-9
-21 -11 -1 9 19
21
16
11
6 Centroids
1 Chiou, A., Kalogeropoulos, N., Efstathiou, P., Papoutsi, M., & Andrikopoulos, N. K.
-4
-9
-24 -14 -4 6 16
First discriminant function
Fig. 2. Score plots on the rst and second functions of the model by cooking
technique (a) and vegetable (b); W/O: water/oil.
Despite the similarities established by the cluster analysis, the dis-
criminant analysis showed that the differences between cooking
techniques and between vegetables were sufciently clear. The
combination of the variables measured is so specic to each cook-
ing method that the prole produced for each one is the same
regardless of the vegetable cooked. Likewise, each vegetable has
such a specic prole that, regardless of cooking technique, that
combination will be maintained even after cooking. Therefore,
after processing by any of the techniques under the same condi-
tions, any of the vegetables studied here will develop an end-pro-
le of phenols, antioxidant capacity, moisture, fat and dry mater
that will depend on both the vegetables initial characteristics,
and on the proles caused by each cooking technique.
4. Conclusions
Each cooked vegetable developed a specic prole of phenols,
moisture, fat, dry matter and antioxidant activity determined by
the original characteristics of the raw vegetables and by the cook-
ing technique applied. The choice of cooking method depends on
the raw ingredient. When the phenol contents of the raw vegetable
are high, the TPC rises even more if EVOO is included in processing
and the application of hydrothermal treatments does not affect the
nal concentrations. When this is not the case, deep frying and
sauting conserve and enrich the phenolic composition.
Hydrothermal cooking methods can be recommended when the
foodstuff is consumed together with the cooking water, when the
addition of EVOO improves the phenolic prole and compensates
for the deciencies of the raw food.
Conict of interest
The authors declare that there are no conict of interest.
Acknowledgments
This article on the results of the rst authors doctoral thesis.
We wish to express our gratitude to PROMEP/SEP, Mxico
J.P. Ramrez-Anaya et al. / Food Chemistry 188 (2015) 430438 437
Cooking Technique UDG-598, and to Robert Ian MacCandless Carrey for his assistance
(a) Boiled in improving the English language version of the manuscript.
Deep fried
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