Food Chemistry 197 (2016) 466–473

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

Influence of heat treatment on antioxidant capacity and (poly)phenolic

compounds of selected vegetables
Isabel Juániz a, Iziar A. Ludwig b, Estibaliz Huarte a, Gema Pereira-Caro b, Jose Manuel Moreno-Rojas b,
Concepción Cid a, María-Paz De Peña a,⇑
a
Department of Nutrition, Food Science and Physiology, School of Pharmacy, University of Navarra, IdiSNA, Navarra Institute for Health Research, E-31080 Pamplona, Spain
b
Postharvest Technology and Agrifood Industry Area, Andalusian Institute of Agricultural and Fisheries Research and Training (IFAPA), Alameda del Obispo Centre, Córdoba, Spain

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

Article history: The impact of cooking heat treatments (frying in olive oil, frying in sunflower oil and griddled) on the
Received 6 August 2015 antioxidant capacity and (poly)phenolic compounds of onion, green pepper and cardoon, was evaluated.
Received in revised form 13 October 2015 The main compounds were quercetin and isorhamnetin derivates in onion, quercetin and luteolin deri-
Accepted 28 October 2015
vates in green pepper samples, and chlorogenic acids in cardoon. All heat treatments tended to increase
Available online 29 October 2015
the concentration of phenolic compounds in vegetables suggesting a thermal destruction of cell walls and
sub cellular compartments during the cooking process that favor the release of these compounds. This
Keywords:
increase, specially that observed for chlorogenic acids, was significantly correlated with an increase in
Phenolics
Flavonoids
the antioxidant capacity measured by DPPH (r = 0.70). Griddled vegetables, because of the higher temper-
Chlorogenic acids ature applied during treatment in comparison with frying processes, showed the highest amounts of phe-
Antioxidants nolic compounds with increments of 57.35%, 25.55% and 203.06% compared to raw onion, pepper and
Vegetables cardoon, respectively.
Heat treatment Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction wide range of biological effects such as protective effects against

cardiovascular diseases, neurodegenerative diseases and cancer,
The Mediterranean diet is characterized by the high consump- probably due to their ability to protect against oxidative damage
tion of fruit and vegetables. The European Union produces a broad in cells (Del Rio et al., 2013; Rodríguez-Mateos et al., 2014).
range of fruits and vegetables thanks to its varied climatic and Many dietary vegetables are usually eaten both crude or after
topographic conditions and it is one of the main global producers cooking in different ways. Culinary processes induce significant
of some vegetables such as tomatoes, carrots and onions. Specifi- changes in foods such as water loss, changes in the total fat content
cally, 5.7 million tonnes of onions were produced in 2013 in and in the fatty acid profile, degradation of thermolabile com-
Europe, and Spain was one of the main producer countries with pounds, and formation of others due to heat-induced chemical
around 21% of total onion production (Eurostat, 2014). This high reactions (Miglio, Chiavaro, Visconti, Fogliano, & Pellegrini, 2008;
vegetables production favors their high consumption. Recent data Miranda et al., 2010). (Poly)phenolic compounds can also be
indicate that the consumption of fresh vegetables in Spain in affected by thermal processes and, consequently the antioxidant
2014 was 260.96 g/capita/day (MAGRAMA, 2014). This consump- capacity of consumed vegetables too (Ramírez-Anaya,
tion was increased around 31% since 2011 when the intake of fresh Samaniego-Sánchez, Castañeda-Saucedo, Villalón-Mir, & de la
vegetables was 179.17 g/capita/day (AECOSAN, 2011). Onion and Serrana, 2015). There are some studies that report the effect of heat
pepper are two of the most consumed vegetables in Spain, how- treatment on antioxidant activity and (poly)phenolic compounds
ever there are lots of local vegetables as cardoon, chard or borage in vegetables. While boiling is the most investigated cooking
which have also a high acceptability among population depending method, few studies are about frying process, both deep frying
on the region (AECOSAN, 2011). In any case, plant foods are the and pan frying (Palermo, Pellegrini, & Fogliano, 2014) and as far
main source of dietary antioxidants, including phenolic com- as we know, only one study was found about the effect of griddling
pounds. (Poly)phenols rich foods have been reported to exhibit a on the antioxidant capacity of vegetables, (Jiménez-Monreal,
García-Diz, Martínez-Tomé, Mariscal, & Murcia, 2009), but none
⇑ Corresponding author. on the (poly)phenols profile. However, results reported on the
E-mail address: [email protected] (M.-P. De Peña). effect of heat treatment on the (poly)phenolic compounds are

http://dx.doi.org/10.1016/j.foodchem.2015.10.139
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
 I. Juániz et al. / Food Chemistry 197 (2016) 466–473 467

not clear cut. Onion is one of the most studied vegetables, 2.4. Antioxidant capacity by ABTS assay
nevertheless both losses and gains in (poly)phenolic compounds
after heat treatment are reported in the literature (Crozier, The ABTS antioxidant capacity was performed according to the
Lean, McDonald, & Black, 1997; Ewald, Fjelkner-Modig, method of Re et al. (1999). The radicals ABTS
+ were generated by
Johansson, Sjöholm, & Åkesson, 1999; Harris, Brunton, Tiwari, & the addition of 0.36 mM potassium persulfate to a 0.9 mM ABTS
Cummins, 2015; Lombard, Peffley, Geoffriau, Thompson, & solution prepared in phosphate buffered saline (PBS) (pH 7.4),
Herring, 2005; Price, Bacon, & Rhodes, 1997; Rodrigues, Pérez- and the ABTS
+ solution was stored in darkness for 12 h. The ABTS+
Gregorio, García-Falcón, & Simal-Gándara, 2009; Rohn, Buchner, solution was adjusted with PBS to an absorbance of 0.700 (±0.020)
Driemel, Rauser, & Kroh, 2007). The studies found in literature at 734 nm in a 3 mL capacity cuvette (1 cm length) at 25 °C
about effect of heat treatment on green pepper are focused on (Lambda 25 UV–VIS spectrophotometer, Perkin-Elmer Instru-
antioxidant activity, but not on (poly)phenolic compound profiles ments, Madrid, Spain). An aliquot of 100 lL of each vegetable
changes (Jiménez-Monreal et al., 2009) and up to our knowledge, extract sample properly diluted in demineralized water, was added
this is the first time where the influence of heat treatment on to 2 mL of ABTS
+ solution. The absorbance was measured spec-
antioxidant capacity and (poly)phenol compounds of cardoon trophotometrically at 734 nm after exactly 18 min. Calibration
stalks (Cynara cardunculus L.) has been studied. Therefore, the was performed with Trolox solution (a water-soluble vitamin E
aim of this work was to study the impact of three cooking analog), and the antioxidant capacity was expressed as micromoles
heat treatments (frying in olive oil, frying in sunflower oil and of Trolox equivalent per gram of dry matter sample (lmol Trolox/
griddled) on the antioxidant capacity and (poly)phenolic g dm).
compound profiles of onion, green pepper and cardoon, commonly
consumed as crude in salads and cooked in several ways in the 2.5. Antioxidant capacity by DPPH assay
mediterranean diet.
The antioxidant capacity was also measured using 2,2-
diphenyl-1-picrylhydrazyl (DPPH
) decolorization assay (Brand-
2. Material and methods Williams, Cuvelier, & Berset, 1995) with some modifications. A
6.1  105 M DPPH
methanolic solution was prepared immedi-
2.1. Chemical and reagents ately before use. The DPPH
solution was adjusted with methanol
to an absorbance of 0.700 (±0.020) at 515 nm in a 3 mL capacity
Yellow onion (Allium cepa), sweet Italian green pepper cuvette (1 cm length) at 25 °C (Lambda 25 UV–VIS spectropho-
(Capsicum annuum), cardoon stalks (C. cardunculus L), olive oil tometer, Perkin-Elmer Instruments, Madrid, Spain). Vegetable
and sunflower oil were obtained from local stores. extracts were properly diluted in demineralized water prior to
The methanol and ethanol were of analytical grade from analysis. Samples (50 lL) were added to 1.95 mL of the DPPH
solu-
Panreac (Barcelona, Spain). The methanol (HPLC grade) was tion. After mixing, the absorbance was measured at 515 nm after
purchased from Panreac (Barcelona, Spain). Trolox (6-hydroxy-2, exactly 18 min. Calibration was performed with Trolox solution
5,7,8-tetramethylchroman-2-carboxylic acid), 2,20 -azinobis (3-eth (a water-soluble vitamin E analog). The antioxidant capacity was
ylbenzothiazonile-6-sulfonic acid) diammonium salt (ABTS), expressed as micromoles of Trolox equivalent per gram of dry mat-
2,2-diphenyl-1-picrylhydrazyl (DPPH), as well as the standards ter sample (lmol Trolox/g dm).
used for identification and quantification of phenolic compounds
(quercetin, luteolin, isorhamnetin, 5-caffeoylquinic acid and 2.6. (Poly)phenolic compounds by UHPLC–PDA–HR–MS
caffeic acid), were purchased from Sigma–Aldrich (Steinheim,
Germany). (Poly)phenolic compounds were analysed using an UPLC with a
PDA detector scanning from 200–600 nm, equipped with an
autosampler cooled at 4 °C (Dionex Ultimate 3000 RS, Thermo Cor-
2.2. Samples preparation
poration) and an ExactiveTM Orbitrap mass spectrometer fitted with
a heated electrospray ionization probe (HESI) (Thermo Fisher Sci-
Chopped vegetables (yellow onion, green pepper, and
entific, San José, USA). Chromatographic separation was performed
cardoon stalks) (300 g) were fried with olive or sunflower oils
at 40 °C on a Kinetex 5 lm RP 250  4.6 mm reversed phase col-
(30 mL) at 115 °C for 10 min in a non-stick frying pan. Then,
umn (Phenomenex, Macclesfield, UK). Ten microliter of each
temperature was decreased to 108 °C for 5 min. Chopped
ethanolic extract was analysed using an 80 min 5–50% gradient
vegetables were also submitted to heating at 150 °C for
of acetonitrile in 0.1% aqueous formic acid at a constant flow rate
10 min and then at 110 °C for 5 min in a non-stick griddle
of 1 mL/min. After passing the PDA flow cell, the eluate was split
without oil addition. Then, raw and cooked vegetables were
and 0.2 mL/min was directed to the mass spectrometer with the
lyophilized in a freeze dryer Cryodos-80 (Telstar, Terrasa, Spain),
HESI operating in negative ionization mode. Analysis was carried
and stored at 18 °C until analysis.
out in full-scan (100–800 m/z) and full-scan with In-Source
Collision-induced dissociation (CID) (100–800 m/z; CID 25.0 eV).
2.3. Vegetables extracts Capillary temperature was 300 °C; sheath gas and auxiliary gas
were 60 and 20 units/min, respectively; source voltage was
Vegetables extracts were prepared according to Siddiq, 4.0 kV. Identification was achieved by comparing the exact mass
Roidoung, Sogi, and Dolan (2013) with some modifications. Briefly, and retention time with pure reference standards. In absence of
thirty mL of ethanol/water (80/20) was added to 2 g of lyophilized standards, compounds were tentatively identified by comparing
vegetables. The content was mixed on a mechanical shaker for 1 h the theoretical exact mass of the molecular ion with the experi-
at room temperature and then centrifuged at 4000 rpm for 10 min. mentally measured accurate mass of the molecular ion. In addition
Supernatant was collected and residues were re-extracted twice identification was confirmed by the appearance of typical frag-
using 10 mL of ethanol 80% by vortexing (1 min) and centrifuged ments produced from the molecular ion. Quantification was per-
at 4000 rpm for 5 min. All three supernatants were combined formed by PDA at 325 nm for caffeic acid glucosides and
and frozen at 18 °C for antioxidant capacity and UHPLC–PDA–H chlorogenic acids, and at 360 nm for quercetin-, isorhamnetin-,
R–MS analysis. luteolin-, and apigenin derivatives. Typical UPLC–PDA chro-
468 I. Juániz et al. / Food Chemistry 197 (2016) 466–473

matograms of griddled onion, griddled pepper and griddled car- sauteing increased concentrations of predominant quercetin deri-
doon are shown in Fig. 1. Caffeic acid glucoside was expressed as vates in 25% and 7%, respectively, compared to raw onions as in
caffeic acid equivalents by reference to a 0.2–20 lg/mL caffeic acid the present study, while boiling decreased total flavonoid concen-
calibration curve; chlorogenic acids were quantified as 5- tration in 18%. However, other authors reported that frying process
caffeoylquinic acid equivalents by reference to a 0.2–20 lg/mL 5- induced 21–39% losses of flavonoids in onion (Crozier et al., 1997;
CQA calibration curve; and quercetin-, isorhamnetin-, luteolin-, Ewald et al. 1999) while others reported no significantly changes in
and apigenin derivatives were quantified by reference to 0.2– the total levels of quercetin diglucoside and monoglucoside
20 lg/mL calibration curves of their respective aglycones. (Rodrigues et al., 2009). In the present study, all compounds
tended to increase after cooking process, but molar ratio of querce-
tin diglucoside/quercetin glucoside decreases with the frying pro-
2.7. Statistical analysis
cess. In onion fried in olive oil and in sunflower oil, quercetin
diglucoside/glucoside had ratios of 0.74 and 0.72, respectively,
Each parameter was analysed in triplicate. Results are shown as
lower than that of the raw onion (0.83). These results suggest that
the mean ± standard deviation (SD). One-way analysis of variance
during frying process, glucosides of the quercetin diglucosides are
(ANOVA) was applied for each parameter. A Tukey test was applied
thermohydrolyzed producing the corresponding monoglucoside as
as a posteriori test with a level of significance of 95%. Principal
discussed by several authors (Rodrigues et al., 2009; Rohn et al.,
Component Analysis (PCA), based on Pearson’s correlation matrix,
2007). However in griddled onion, total onion quercetin digluco-
was applied in order to study the effect of heat treatment. All sta-
sides significantly increased, probably due to the degradation of
tistical analyses were performed using the STATA v.12.0 software
other quercetin derivates such as quercetin triglucoside, that may
package.

3. Results and discussion

Total of seven flavonoids were identified and quantified in

onion samples. All of them were glucosides of quercetin and
isorhamnetin. Aglycones of these two flavonoids were not
detected. Other minor flavonoids such as kaempferol, luteolin
and myricetin derivates, or phenolic acids identified in onion by
other authors were not detected in our samples, probably due to
differences in onion variety, as well as other factors like cultivar
(Lanzotti, 2006; Lu, Ross, Powers, & Rasco, 2011; Rodríguez
Galdón, Rodríguez Rodríguez, & Díaz Romero, 2008; Sellappan &
Akoh, 2002; Simin et al., 2013). The most abundant compounds
in onion were three quercetin derivates, one quercetin glucoside
and two quercetin diglucosides, which accounted approximately
for 90% of total flavonoids in all samples (Table 1). This is similar
to the percentage of quercetin diglucosides and monoglucosides
(83–93% of the total flavonols content) reported by Lombard
et al. (2005) in different raw onions varieties. The total flavonoid
content in raw onion obtained by addition of every identified flavo-
noids by HPLC, was 12.43 mg/100 g fresh weight. This concentra-
tion is higher than that reported by Rodríguez Galdón et al.
(2008) (7.48–9.92 mg/100 g fresh weight) but in the lower values
of the majority data range described in the literature (12.21–
62.1 mg/100 g fresh weight) (Bonaccorsi, Caristi, Gargiulli, &
Leuzzi, 2005; Lombard et al., 2005; Sellappan & Akoh, 2002),
maybe due to the onion variety taking into account that red onion
tends to have higher amounts of flavonoids than yellow ones
(Lombard et al., 2005; Nuutila, Puupponen-Pimiä, Aarni, &
Oksman-Caldentey, 2003). In all studies found, quercetin and quer-
cetin derivates were also the main compounds in raw onion.
Griddled onion showed the highest amount of flavonoids with
an increment of 57.35% compared to raw. Additionally, the use of
olive oil for frying resulted in a higher increase of flavonoids
(34.55%) than the use of sunflower oil (15.44%). Quercetin deri-
vates present a higher thermal stability than isorhamnetin deri-
vates, since all phenolic compounds increased with the exception
of isorhamnetin glucoside, that decreased around 90%, remaining
quercetin derivates the most abundant after cooking process. How-
ever, in literature, both losses and gains in quercetin derivates con-
tent after cooking process of onions have been reported depending
on the heat treatment conditions. Quercetin derivates did not sig-
nificantly change when brown-skinned onion was fried during
5 min, whereas at higher frying times (15 min) decreased signifi- Fig. 1. UPLC–PDA chromatograms of (A) griddled onion, k = 360 nm; (B) griddled
cantly (Price et al., 1997). Lombard et al. (2005) also showed how pepper, k = 360 nm and (C) griddled cardoon, k = 325 nm. For peak assignments, see
different heat treatments could affect phenolic content; baking or Tables 1–3.
 I. Juániz et al. / Food Chemistry 197 (2016) 466–473 469

Table 1
(Poly)phenolic compounds in raw and cooked onions (fried in olive oil, fried in sunflower oil and griddled). Results are expressed as mean ± standard deviation (mg/g sample dm).
Rows in bold indicate the total values of each (poly)phenol category.

Peak no. Compound Raw Fried in olive oil Fried in sunflower oil Griddled
1 Quercetin triglucoside 0.01 ± 0.00a 0.01 ± 0.00a 0.01 ± 0.00a 0.01 ± 0.00a
2 Quercetin diglucoside I 0.22 ± 0.00a 0.30 ± 0.01a 0.24 ± 0.03a 0.35 ± 0.06a
3 Quercetin diglucoside II 0.35 ± 0.00a 0.42 ± 0.01b 0.38 ± 0.02ab 0.56 ± 0.03c
5 Quercetin glucoside I 0.04 ± 0.00b 0.02 ± 0.00a 0.02 ± 0.00a 0.03 ± 0.00ab
6 Quercetin glucoside II 0.64 ± 0.01a 0.95 ± 0.01c 0.83 ± 0.00b 1.02 ± 0.04c
Total Quercetin derivates 1.26 ± 0.01a 1.71 ± 0.03bc 1.48 ± 0.05ab 1.96 ± 0.12c

4 Isorhamnetin diglucoside 0.02 ± 0.00a 0.12 ± 0.02bc 0.09 ± 0.02b 0.17 ± 0.02c
7 Isorhamnetin glucoside 0.08 ± 0.00a 0.01 ± 0.00a 0.01 ± 0.00a 0.01 ± 0.00a
Total Isorhamnetin derivates 0.10 ± 0.01 0.13 ± 0.02ab 0.10 ± 0.02a 0.18 ± 0.01b

Total Flavonoids 1.36 ± 0.01a 1.83 ± 0.05bc 1.57 ± 0.07ab 2.14 ± 0.14c

Different letters for each row indicate significant differences (p 6 0.05) among samples.

be strongly linked to other structures in the food matrix and thus well as in onion samples, no aglycone flavonoids were detected.
have not been fully extracted. The softening effect of the cooking Quercetin rhamnoside and luteolin 7-O-(2-apiosyl-6-malonyl) glu-
process of vegetables, in this case mainly griddling in onion, due coside were the most abundant in all green pepper samples,
to heat-induced wall and cells ruptures can also affect (poly)phe- accounting for around 80% of total phenolic compounds. These
nolic extractability (Harris et al., 2015; Palermo et al., 2014). results are in agreement with those reported in previous studies
Although some authors reported that quercetin monoglucoside in raw green pepper (Marín, Ferreres, Tomás-Barberán, & Gil,
can be also deglycosilated by thermohydrolysis to the correspond- 2004). In green pepper, also an increase of the total (poly)phenolic
ing aglycon (Rodrigues et al., 2009; Rohn et al., 2007), no quercetin compounds after heat treatment was observed. Griddled pepper
or isorhamnetin aglycons were detected after cooking process in showed the highest amount of phenolic compounds (0.96 mg
the present work in agreement with Harris et al. (2015), probably (poly)phenolic compounds/g sample dm). Frying also induced an
due to both heat degradation and contribution of the formation of increase of (poly)phenolic compounds, but the use of olive oil for
Maillard reaction products, like melanoidins (Pérez-Jiménez, Díaz- frying resulted in a lower increase in flavonoids, and therefore in
Rubio, Mesías, Morales, & Saura-Calixto, 2014). total (poly)phenolic compounds (48.14%) than with sunflower oil
The increment of phenolic compounds corresponded with an (103.70%). Caffeic acid derivates, as well as luteolin hexoside–pen-
increase in antioxidant capacity measured by DPPH assay (Fig. 2A). tosides and luteolin glucosides, were only found after heat
DPPH tended to be higher in cooked onions, specially in griddled treatment.
onion, however this increase was not significant. No data of DPPH As well as in onion samples, the increase in the identified (poly)
in cooked onion were found in literature. On the contrary, raw phenolic compounds by HPLC also agrees with a high antioxidant
onion showed the highest antioxidant capacity measured by ABTS activity measured by DPPH, with the exception of green pepper
(162.10 ± 1.33 lmol Trolox/g sample dm) (Fig. 2B) and it decreased fried with olive oil (Fig. 2A). By contrast, some authors indicated
with the heat treatment, maybe due to the degradation of non phe- that radical-scavenging activity (DPPH) differences between raw
nolic antioxidants. These results disagree with those reported by and stir frying in different pepper samples, including green ones,
some authors where no losses or increases in the ABTS antioxidant were not significant (Chuah et al., 2008) or even losses were
activity of onion after frying and griddling were found (Jiménez- observed in red pepper (Hwang, Shin, Lee, Lee, & Yoo, 2012). These
Monreal et al., 2009; Pellegrini et al., 2009). differences, as well as in onion, could be due to different pepper
A total of twelve flavonoids and three phenolic acids were iden- variety and cultivars. On the other hand, and as well as in onion
tified in green pepper (Table 2). Flavonoid compounds (quercetin samples, antioxidant activity measured by ABTS assay decreased
and luteolin derivates) were the main (poly)phenols, accounting after heat treatment (Fig. 2B) probably due to degradation of ther-
for more than 90% of total phenolic compounds in all samples. As molabile non-phenolic antioxidants. This decrease was lower in

Fig. 2. Antioxidant capacity (DPPH and ABTS) of raw and cooked vegetables. Different letters indicate significant differences (p 6 0.05).
470 I. Juániz et al. / Food Chemistry 197 (2016) 466–473

Table 2
(Poly)phenolic compounds in raw and cooked green pepper (fried in olive oil, fried in sunflower oil and griddled). Results are expressed as mean ± standard deviation (mg/g
sample dm). Rows in bold indicate the total values of each (poly)phenol category.

Peak No. Compound Raw Fried in olive oil Fried in sunflower oil Griddled
4 Quercetin rhamnoside glucoside I nda 0.01 ± 0.00a 0.01 ± 0.00a 0.01 ± 0.00a
11 Quercetin rhamnoside glucoside II nda 0.01 ± 0.00a 0.01 ± 0.00a 0.02 ± 0.00b
6 Quercetin 3-sambubioside-7-rhamnoside 0.01 ± 0.00a 0.01 ± 0.00a 0.01 ± 0.00a 0.02 ± 0.00a
14 Quercetin rhamnoside 0.17 ± 0.00a 0.19 ± 0.02ab 0.24 ± 0.01b 0.42 ± 0.01c
13 Quercetin glucoside 0.01 ± 0.00a 0.02 ± 0.00a 0.04 ± 0.00ab 0.06 ± 0.00b
Total Quercetin derivates 0.18 ± 0.41a 0.23 ± 0.02a 0.32 ± 0.02b 0.53 ± 0.01c

9 Luteolin glucoside I nda tra 0.01 ± 0.00a 0.01 ± 0.00a

10 Luteolin glucoside II nda tra 0.01 ± 0.00a 0.02 ± 0.00a
5 Luteolin hexoside pentoside I nda tra 0.01 ± 0.00a 0.01 ± 0.00a
7 Luteolin hexoside pentoside II nda tra tra tra
8 Luteolin hexoside pentoside III nda tra tra 0.01 ± 0.00a
12 Luteolin 7-O-(2-apiosyl)glucoside nda 0.01 ± 0.00a 0.01 ± 0.00a 0.02 ± 0.00a
15 Luteolin 7-O-(2-apiosyl-6-malonyl)glucoside 0.08 ± 0.01a 0.12 ± 0.02ab 0.16 ± 0.00b 0.29 ± 0.02c
Total Luteolin derivates 0.08 ± 0.01a 0.15 ± 0.02b 0.20 ± 0.00b 0.37 ± 0.02c

Total Flavonoids 0.26 ± 0.02a 0.38 ± 0.04b 0.52 ± 0.02c 0.90 ± 0.03d

1 Caffeic acid glucoside I nda tra tra tra

2 Caffeic acid glucoside II tra tra 0.01 ± 0.00a 0.03 ± 0.00b
Total Caffeic acid derivates 0.00 ± 0.00a 0.00 ± 0.00a 0.01 ± 0.00a 0.03 ± 0.00a

3 CQA 0.01 ± 0.00a 0.02 ± 0.00ab 0.02 ± 0.00ab 0.03 ± 0.00b

a ab ab
Total CQA derivates 0.01 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 0.03 ± 0.00b

Total Phenolic acids 0.01 ± 0.00a 0.02 ± 0.00a 0.03 ± 0.00a 0.06 ± 0.00b

Different letters for each row indicate significant differences (p 6 0.05) among samples.
nd: no detected, tr: traces.

Table 3
(Poly)phenolic compounds in raw and cooked cardoon (fried in olive oil, fried in sunflower oil and griddle). Results are expressed as mean ± standard deviation (mg/g sample dm).
Rows in bold indicate the total values of each (poly)phenol category.

Peak No. Compound Raw Fried in olive oil Fried in sunflower oil Griddled
1 CQA I 0.03 ± 0.00a 0.07 ± 0.00a 0.08 ± 0.00a 0.06 ± 0.00a
2 CQA II 0.03 ± 0.01a 0.03 ± 0.00a 0.05 ± 0.00a 0.08 ± 0.00a
3 5-CQA 3.32 ± 0.15a 2.84 ± 0.05a 3.05 ± 0.05a 7.29 ± 0.35b
4 CQA III 0.08 ± 0.00c 0.03 ± 0.00a 0.04 ± 0.01b 0.03 ± 0.00a
Total CQAs acids 3.46 ± 0.02a 2.98 ± 0.05a 3.22 ± 0.05a 7.46 ± 0.35b

6 1,3-diCQA nda nda 0.03 ± 0.00a 0.02 ± 0.00a

7 3,4-diCQA tra 0.07 ± 0.00b 0.10 ± 0.00c 0.12 ± 0.00d
8 1,4-diCQA nda 0.07 ± 0.00b 0.10 ± 0.01c 0.12 ± 0.00d
9 3,5-diCQA 0.90 ± 0.04a 1.61 ± 0.01b 2.01 ± 0.06c 4.42 ± 0.15d
10 1,5-diCQA 0.47 ± 0.02a 1.08 ± 0.06b 1.26 ± 0.02c 2.10 ± 0.02d
11 4,5-diCQA 0.03 ± 0.00a 0.29 ± 0.02c 0.25 ± 0.01b 0.43 ± 0.01d
Total DiCQAs acids 1.41 ± 0.06a 3.12 ± 0.07b 3.76 ± 0.08c 7.21 ± 0.18d

12 SuccinyldiCQA I 0.13 ± 0.01a 0.32 ± 0.01b 0.40 ± 0.00c 0.64 ± 0.00d

13 SuccinyldiCQA II 0.18 ± 0.00b 0.11 ± 0.00a 0.13 ± 0.00a 0.47 ± 0.01c
14 SuccinyldiCQA III 0.01 ± 0.00a tra tra 0.01 ± 0.00a
16 DisuccinyldiCQA 0.02 ± 0.00a 0.01 ± 0.00a 0.02 ± 0.00a 0.02 ± 0.00a
Total SuccinyldiCQAs acids 0.35 ± 0.02a 0.45 ± 0.12b 0.55 ± 0.00c 1.14 ± 0.01d

Total Chlorogenic acids 5.22 ± 0.09a 6.55 ± 0.13b 7.53 ± 0.14b 15.82 ± 0.54c

15 Apigenin glucoside nda nda nda tra

5 Luteolin glucoside tra tra tra 0.02 ± 0.00b
Total Flavonoids tra tra tra 0.02 ± 0.00b

Different letters for each row indicate significant differences (p 6 0.05) among samples.
nd: no detected, tr: traces.

griddled pepper (32.92%) in comparison to green pepper fried with Cardoon is a vegetable from the species of C. cardunculus L.
sunflower oil (62.41%) and with olive oil (80.80%). Previous studies Some parts of the plant can be edible. In Navarra and other Spanish
also reported losses on scavenging capacity (ABTS) in peppers after regions, stalks are consumed fresh in salads (when they are soft) or
frying (30–50% losses) and griddling (5–30% losses), but lower in typical meals applying frying or griddling. Although several
because time is shorter (up to 8 min vs 15 min in the present studies about composition and total phenolic compounds of differ-
study) (Jiménez-Monreal et al. 2009). ent varieties and parts of C. cardunculus L. have been found, up to
 I. Juániz et al. / Food Chemistry 197 (2016) 466–473 471

Fig. 3. Principal Component Analysis (PCA) of the raw and cooked vegetables. (a) Parameter loadings. (b) Sample scores.

our knowledge only one included stalks, however it is focused on identified by HPLC increased after heat treatment. Chlorogenic
total phenolic compounds by Folin Ciocalteu method and not in acids can be found free, but also ionically or covalently attached
the (poly)phenolic profile by HPLC (Velez et al., 2012). In contrast to other food structures, like melanoidin. DiCQAs, where the addi-
to the phenolic compounds characteristics of onion and green pep- tion of a caffeic acid moiety increases the hydroxyl groups, can ion-
per, the most abundant (poly)phenols of cardoon were chlorogenic ically interact with other macromolecules (Monente, Ludwig,
acids (four caffeoylquinic acids (CQA), six diccaffeoylquinic acids Irigoyen, De Peña, & Cid, 2015). The softening effect of the cooking
(diCQA), three succinyldicaffeoylquinic acids (succinyldiCQA) and process of vegetable due to heat-induced wall and cells ruptures
one disuccinyldicaffeoylquinic acid (dicuccinyldiCQA) (Table 3). can affect (poly)phenolic extractability (Palermo et al., 2014), so
These last compounds were reported for the first time in raw heat treatment could hydrolyzed and release those compounds
cardoon leaves (C. cardunculus L.) by Pinelli et al. (2007). In the from the food matrix. The increment in total (poly)phenolic com-
cardoon stalks of the present study, 5-CQA, 3,5-diCQA and pounds was higher in griddled cardoon (203.06%) than in cardoon
1,5-diCQA were the most abundant compounds, accounting for fried in sunflower oil (44.25%) and in cardoon fried in olive oil
80–90% of total (poly)phenolic compounds detected by HPLC in (25.47%). This could be due to the higher temperature applied dur-
all samples. Traces of some flavonoids were also detected in car- ing griddled treatment than that applied during frying processes,
doon, mainly in griddled one. Up to our knowledge, this is the first which might favor the release of these compounds from the food
time where the influence of heat treatment on antioxidant capacity matrix, specially 5CQA which increased dramatically after grid-
and (poly)phenol compounds of cardoon stalks (C. cardunculus L.) dling process probably due to the hydrolysis of diCQAs compounds.
has been studied. The total amount of (poly)phenolic compounds On the other hand, the less increment in fried samples may be due
472 I. Juániz et al. / Food Chemistry 197 (2016) 466–473

to the slight decrease of 5-CQA after frying processes both with compounds, caused by heat treatment also increases the bioavail-
olive and sunflower oils, maybe due to isomerization into other ability of these compounds after consumption.
CQAs. These results are in agreement with Ferracane et al. (2008)
which confirmed that cooking practices caused a marked Acknowledgments
intramolecular transesterification of caffeoylquinic acid.
Highly significant correlations were found between 5-CQA, total This research was funded by the Spanish Ministry of Economy
CQAs, total diCQAs and total succinyldiCQAs, and antioxidant and Competitiveness (AGL2014-52636-P) and PIUNA (Plan de
capacity measured by DPPH assay (r = 0.70, p < 0.001), becoming Investigación de la Universidad de Navarra). We thank Ms. Gwe-
griddled cardoon the most antioxidant sample. Contrary to onion naelle Ceniceros for her kind help. I.J wishes to express her grati-
and pepper samples, DPPH values did not increase in cardoon fried tude to the Association of Friends of the University of Navarra for
with sunflower oil, but as well as in pepper samples, a loss of scav- the grant received. GP-C is supported by a postdoctoral research
enging activity (DPPH) took place in samples fried with olive oil contract funded by the Andalusian Institute of Agricultural and
(Fig. 2A). On the other hand, ABTS assay showed higher values in Fisheries Research and Training (IFAPA) and the European Social
cardoon after fried with sunflower oil, and specially after griddled, Funds (ESF).
than raw samples (Fig. 2B).
Principal Component Analysis (PCA) has been applied to evalu- Appendix A. Supplementary data
ate at a glance the influence of heat treatment on the antioxidant
activity and (poly)phenolic compounds of different vegetables. Supplementary data associated with this article can be found, in
Fig. 3 shows the bidimensional representation of all the variables the online version, at http://dx.doi.org/10.1016/j.foodchem.2015.
(Fig. 3A) and vegetables samples (Fig. 3B) according to the two 10.139.
selected Principal Components (PC). PC1 (48.89% of the total vari-
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