Food Research International 73 (2015) 97–106

Onion skin — Raw material for the production of supplement that enhances the
health-beneficial properties of wheat bread
Urszula Gawlik-Dziki a,⁎, Kinga Kaszuba b, Katarzyna Piwowarczyk b, Michał Świeca a, Dariusz Dziki
c, Jarosław Czyż b
a Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str. 8, 20-704 Lublin, Poland b
Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7,
30-387 Kraków, Poland c Department of Thermal Technology, University of Life Sciences, Doświadczalna Str. 44, 20-280
Lublin, Poland
a r t i c l e i n f o a b s t r a c t Article history: Received 30 November 2014 Received in revised form 7 February 2015 Accepted
28 February 2015 Available online 7 March 2015
Keywords: Onion skin Bread supplementation Stomach cancer Phenolic compounds Bioavailability Antiradical potential
⁎ Corresponding author. Tel.:+48 81 4623327; fax: +48 81 4623324.
E-mail address: urszula.gawlik@up.lublin.pl (U. Gawlik-Dziki).
Onion skin (OS) is an environmentally problematic waste that is extensively produced during onion bulb processing. Noteworthy,
it contains high amounts of bioaccessible and bioavailable compounds with well docu- mented biological activity. Here, we
estimated the effect of OS-supplementation and gastrointestinal processing of wheat bread on its potential anticancer activity and
ability to modify activity of lipoxygenase (LOX), xanthine oxidase (XO), catalase (CAT) and superoxide dismutase (SOD).
Gastrointestinal processing releases XO and LOX inhibitors and CAT activators from supplemented bread. Most importantly,
synergism between these compounds derived from bread and OS was found. Gastrointestinally adsorbed 3% OS-supplemented
bread extract exerted a considerably stronger cytostatic and anti-invasive effect on gastric cancer AGS cells than its
gastrointestinally digested and buffer masticated counterparts. These effects only partly correlated with the phenolic content of
the extracts and their antioxidative activities. Gastrointestinal digestion and adsorption slightly influence the bioactivity of
non-supplemented bread extracts. However, the activity of gastrointestinally adsorbed, OS-supplemented wheat bread extract
switched from cytostatic and anti-invasive to cytoprotective upon DMSO extraction. These data directly show a complex network
of synergetic and antagonistic effects of single phenolic compounds from onion skin and wheat bread. They indicate that the
bioactivity of phenolic compounds and potentially beneficial effects of wheat bread fortification with onion skin may be
regulated by subtle inter- actions between OS and bread phenolic compounds, and food matrix. They are modulated by
gastrointestinal processing. These findings may open perspectives for the introduction of onion skin into wheat bread supple-
mentation which would increase its pro-healthy properties.
© 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Fruits, cereal grains and vegetables contain large amounts of antiox- idative phenolic compounds that are suggested to account
for beneficial effects of diet rich in plants (Mateo Anson, Havenaar, Bast, & Haenen, 2010; Roldan, Sanchez-Moreno, De Ancos,
& Cano, 2008). Nowadays, the processing of agricultural products generates substantial quantities of by-products, which are
wasted during the food production process. However, these by-products could potentially be employed as food supplements
because they often contain high quantities of bioactive phytochemicals, including phenolic compounds. This postulate con- cerns
in particular the onion skin (OS), in which “production” was increasing over recent years in line with the growing demand for
onion bulbs. More than 500,000 tons of this waste is generated in the
European Union each year, above all in Spain, Holland and the United Kingdom, where it has become an environmental problem.
Noteworthy, OS contains a number of bioaccessible and bioavailable compounds with documented high functional value
(Benítez et al., 2011).
A multidirectional antioxidant activity of potentially bioaccessible and bioavailable phenolic phytochemicals from powdered
OS has previ- ously been demonstrated. It was illustrated by their relatively high anti- radical potential, metal ion chelating
activity, reducing power and the ability to prevent the oxidation of lipids (Gawlik-Dziki et al., 2013a). Antioxidant activity of OS
phenolics, which may determine their postu- lated cancer-preventing abilities, opens perspectives for OS application as a diet
supplement to reduce the risk of cancer formation. OS may be used as a functional additive of daily food without detriment to its
sensory properties and significantly enhance its health-promoting ac- tivity (Gawlik-Dziki et al., 2013b; Hassan et al., 2014). In
particular, bread and bakery products, due to their widespread consumption are considered to be the best vehicle for functional
supplements. Generally, wheat bread made with white flour is considered to be a good source of
http://dx.doi.org/10.1016/j.foodres.2015.02.008 0963-9969/© 2015 Elsevier Ltd. All rights reserved.
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energy and irreplaceable nutrients for the human body, but is character- ized by a relatively low pro-healthy potential (Dziki,
Różyło, Gawlik- Dziki, & Świeca, 2014).
We have previously shown that OS supplementation significantly increases antioxidant activity of wheat bread, without the
loss of its sen- sory properties. This confirms the usefulness of onion skin for bread fortification (ranging up to 3%; Gawlik-Dziki
et al., 2013b) and suggests a relatively high contribution of bread phenolic compounds to pro- healthy properties of
OS-supplemented bread. However, OS supple- mentation of bread has not yet been directly considered in terms of its effects on
cancer cell properties, nor was the impact of the interactions between OS and bread components on their bioavailability and
biologi- cal activity estimated. Due to the localization of gastric cancers and their predominantly dietary etiology, bread
supplementation with OS may be especially efficient in the prevention of these tumors. Gastric cancer represents the 2nd most
common cancer in the world, representing a very important health problem with about 900,000 new cases diag- nosed every year
(Kang et al., 2015; Kumar et al., 2013; Mao et al., 2015; Parkin, Bray, & Devesa, 2001). Despite advances in diagnosis and
treatment, the 5-year survival rate of gastric cancer remains at a rela- tively low level of 25% (Karimi, Islami, Anandasabapathy,
Freedman, & Kamangar, 2014; Subapriya & Nagini, 2003).
Accumulating evidence suggests that medicinal plants and phyto- chemicals can offer chemoprotection against toxic,
mutagenic and carcinogenic chemicals that facilitate gastric cancer development (Bhattacharjee & Sengupta, 2009). These facts
confirm the relevance of the models based on gastric cancer cells for the analyses of OS supple- ments on the pro-healthy activity
of wheat bread.
An experimental approach based on gastrointestinal digestion and absorption of plant material, followed by the analyses of
their chemical signature and anti-cancer activity in vitro, has previously enabled us to demonstrate the chemopreventive activity
of broccoli sprout- supplemented bread against gastric cancer (Gawlik-Dziki et al., 2014). Using this approach, we also showed
that the bioavailability of phenolic compounds determines the chemopreventive potential of quinoa leaves (Gawlik-Dziki et al.,
2013b) and broccoli sprouts (Gawlik-Dziki et al., 2012) in a prostate cancer model. Here, we used gastric cancer AGS cell model
to evaluate the effect of gastrointestinal processing on the cytostatic and anti-invasive activities of OS-supplemented bread in the
context of its interference with the activity of pro- and antioxida- tive enzymes. Next, we estimated the contribution of phenolic
com- pounds from bread and OS to the overall activity of OS-supplemented bread. Our observations enabled us to address the
role of the interac- tions between OS and bread phenolic compounds and food matrix in determining the potential anti-cancer
activity of OS-supplemented wheat bread.
2. Materials and methods
2.1. Dry onion skin (OS)-based supplement preparation
Food supplement was prepared as follows: dry onion (Allium cepa, var. Wolska) skin was washed twice with deionized water
and dried in an oven at 50 °C. The detailed OS composition was previously de- scribed (Gawlik-Dziki et al., 2013b).
2.2. Bread making
The flour used in the formula of the control bread was wheat bread flour (600 g), type 750 (average 0.75% ash content, water
content 14% wet basis, wb). The flour was replaced with OS at the 1, 2, 3, 4, and 5% levels, bread OS1–OS5, respectively.
Breads were made according to a procedure described previously (Gawlik-Dziki et al., 2014). The bread slices about 12 mm thick
were dried at 40 °C for about 1 h using a purpose-designed laboratory dryer with an air flow rate of 1.0 m/s until the moisture
content reached ≈10% (wb). Dried bread samples
98 U. Gawlik-Dziki et al. / Food Research International 73 (2015) 97–106
and onion skin were then powdered using a laboratory mill and sieved (60 mesh).
2.3. Extract preparation
Saline buffer (SB), gastrointestinally digested (GD) and absorbed (GDA) extracts of non-supplemented and 1–5%
OS-supplemented wheat bread were prepared as follows. For phosphate buffer saline (PBS) extraction, control (not
supplemented)-, OS-supplemented bread- and OS samples (5 g of dry weight) were homogenized for 1 min with a laboratory
blender, extracted in 25 mL PBS (pH 7.4) for 1 h at room tem- perature (RT), centrifuged (15 min, 3000 g, RT), followed by
supernatant recovery. This procedure was repeated to obtain 50 mL of combined buffer extract (SB). Extracts were stored in
darkness at −20 °C. Simulated mastication, gastrointestinal digestion (GD) and simulated absorption (GDA) of the OS and bread
samples were made according to a procedure described previously (Gawlik-Dziki et al., 2014). To obtain the GD ex- tracts, the
samples were homogenized in the presence of 15 mL of simu- lated salivary fluid (prepared by (1) dissolving 2.38 g Na
2
HPO
4
, 0.19 g KH
2
PO
4
, and 8 g NaCl, 100 mg of mucin in 1 L of distilled water; (2) adjust- ment to pH = 6.75; and (3) addition of α-amylase
(E.C. 3.2.1.1) to obtain the final activity of 200 U/mL) with a stomacher laboratory blender for 1 min to simulate mastication.
For the gastric digestion, the samples were adjusted to pH = 1.2 using 5 mol/L HCl, and subsequently 15 mL of simulated
gastric fluid was added (300 U/mL of pepsin (from porcine stomach mucosa, pepsin A, E.C. 3.4.23.1) in 0.03 mol/L NaCl, pH =
1.2). The samples were shaken for 120 min at 37 °C. After digestion with the gastric fluid, the samples were adjusted to pH = 6
with 0.1 mol/L of NaHCO
3
and then 15 mL of a mixture of bile extract and pancreatin was added (0.05 g
of pancreatin (activity equivalent 4× USP) and 0.3 g of bile extract in 35 mL 0.1 mol/L NaHCO
3
). The extracts were adjusted to pH = 7 with 1 mol/L NaOH and finally 5 mL of 120 mmol/L NaCl and 5 mL of mmol/L
KCl were added to each sample. The prepared samples were subjected to in vitro digestion for 60 min, at 37 °C in the darkness.
Afterwards, the samples were centrifuged and supernatants (extracts after simu- lated digestion, GD) were used for further
analysis. Considering that antioxidant absorption takes place mainly at the intestinal digestion stage, the resulting mixture (fluids
obtained after in vitro digestion) was transferred to the dialysis sacks (D9777-100FT, Sigma-Aldrich), placed in an Erlenmeyer
flask containing 50 mL of PBS buffer and incu- bated in a rotary shaker (2 times per 2 h, 37 °C). The PBS buffer together with
the compounds that passed through the membrane (dialysate, GDA) was treated as an equivalent of the raw material absorbed in
the intestine after digestion.
For the analyses of the bioactivity of OS-phenolics, liophilizated OS extracts were dissolved in DMSO at a concentration of
100 mg/mL, administered to the media at concentrations of 0.1 and 1 mg/mL (cor- responding to 0.1% and 1.0% DMSO;
non-cytotoxic for AGS cells; not shown).
2.4. Biochemical analyses
Total phenolic (TPC) content was estimated according to Singleton and Rossi (1965) and calculated as a gallic acid equivalent
(GAE) (mg/g dry weight, DW). Ability to inhibit xanthine oxidase (XOi) and lipoxygenase (LOXi) was determined based on
Sweeney, Wyllie, Shalliker, and Markham's (2001) and Axelroad, Cheesbrough, and Laakso's (1981) method, respectively.
Inhibitory activity was expressed as EC
50
(Efficient Concentration): the amount of sample (mg DW) needed to obtain 50% activity per 1.0 mL of the initial
solution. Influence on catalase activity (CATa) was based on the Claiborne (1985) method, influence on superoxide dismutase
activity (SODa) was based on Marklund and Marklund (1974) with some modification. Influence of enzyme activity was
expressed as percentage of control (pure enzyme without tested sample) activity.
2.5. Cytostatic and anti-invasive activity of the extracts
Analyses of the biological activity of SB, GD and GDA extracts from control (not supplemented)- and 3% OS-supplemented
breads and of DMSO-extracted fraction of GDA OS extracts were carried out on human stomach cancer AGS cells. For the
proliferation assay, cells were seeded into 6-well flasks (Nunclon) at an initial density of 7.5 × 103 cells/cm2 and cultivated for
24 h. Then, the culture medium (RPMI supplemented with 10% fetal bovine serum, all from Sigma) was exchanged or replaced
with the medium containing the extracts (administered from stock solutions to reach the final concentrations of 1 and 0.1 μg/mL
of culture medium). Cells were cultured for the next 72 h, fixed with 3.7% formaldehyde, and stained with 0.5 μg/mL bis-
benzimide for 20 min. Twenty randomly chosen microphotographs of Hoechst-stained nuclei were taken with a
computer-assisted data ac- quisition system (Leica DM IRE2) for each condition to calculate the av- erage number of cells per
dish (Gawlik-Dziki et al., 2014). Cytoskeleton architecture was analyzed in formaldehyde-fixed, Triton-solubilized cells, stained
with rabbit anti-vinculin IgG (number V9131, Sigma) and counterstained with Alexa 488-conjugated goat anti-rabbit IgG
(number A11008, Invitrogen), TRITC-conjugated phalloidin (number 77418, Sigma), and Hoechst 33258. Image acquisition was
performed with a Leica DMI6000B microscope (Leica Microsystems, Wetzlar, Germany) equipped with the Total Internal
Reflection Fluorescence (TIRF) and Interference Modulation Contrast (IMC) modules (Daniel- Wójcik et al., 2008). Time-lapse
videomicroscopy studies of AGS cell motility were performed on cells plated onto culture flasks (Corning, 25 cm2) at initial cell
densities chosen to compensate for the inhibitory action of the extracts on cell proliferation (200 to 400 cells/mm2). The
movement of individual cells was recorded immediately or 72 h after the extract administration along with the culture medium
(RPMI, Sigma; see above) with the computer-assisted Leica DMI6000B system (recording time: 4 h, with 5-minute time
intervals at 37 °C). Cell trajec- tories (N50 cells, three independent experiments) were pooled and sta- tistically analyzed. The
following parameters were estimated: (i) the total length of cell displacement (TLCD; μm), that is, the distance from the starting
point directly to the cell's final position and (ii) the total length of cell movement (TLCM; μm), that is, the total length of cell tra-
jectory (4 h) (Daniel-Wójcik et al., 2008).
2.6. Calculations
For better evaluation of bioaccessibility in vitro the following factors were determined (Gawlik-Dziki et al., 2013b):
– the antioxidant bioaccessibility index (BAC), which is an indication
of the potential bioaccessibility of antioxidative compounds:
BAC = A
SB
/A
GD
– the antioxidant bioavailability index (BAV):
BAV = A
GD
/A
GDA
– the antioxidant bioefficiency index (BEF), which is an indication of
the bioactivity of bioavailable antioxidant compounds:
BEF = A
SB
/A
GDA
where, A
SB
= EC
50
of buffer extract, A
GDA
= EC
50
of extract after simulated intestinal absorption, and A
GD
= EC
50
of extract after simulated gastrointestinal digestion; – The interaction factor
(IF), which provides an explanation of the
mode of interaction (Gawlik-Dziki, 2012)
IF = A
M
/A
T
99 U. Gawlik-Dziki et al. / Food Research International 73 (2015) 97–106
where, A
M
= the measured activity of a mixture of samples, and A
T
= the theoretically calculated mixture activity (based on the dose response of single components at various concentrations).
IF value b1 indicates synergistic interaction; IF N 1 indicates antago- nism and IF ≈ 1 indicates additional interactions.
2.7. Statistical analysis
Experimental data were shown as mean ± S.D. for the biochemical and mean ± SEM for the anticancer activity assays. In the
biochemical analyses, statistical significances were estimated through Tukey's test for the data obtained from three independent
samples of each extract in three parallel experiments (n = 9). For the estimation of the effect on cell proliferation and motility, the
results from three independent experiments (n = 3) were subjected to statistical analyses using the paired Student's t-test and the
nonparametric Mann–Whitney test, re- spectively (n = 3). Unless stated otherwise, the statistical tests were carried out at a
significance level of α = 0.05. Statistical tests were per- formed using Statistica 6.0 software (StatSoft, Inc., Tulsa, USA).
3. Results
To estimate the effect of wheat bread supplementation with OS on the product's bioactivity, we first analyzed the impact of
saline buffer (SB), gastrointestinally digested (GD) and gastrointestinally digested and absorbed (GDA) extracts on AGS cell
proliferation. For this purpose, we incubated the cells in the presence of 3% OS-supplemented bread ex- tracts administered at the
concentrations of 0.1 and 1 mg/mL (Fig. 1). They correspond to ca. 15 and 150 g fresh weight/75 kg of body mass, respectively.
All analyzed extracts exerted an inhibitory effect of on the proliferation of AGS cells. The GDA extract displayed the highest cy-
tostatic activity at both concentrations tested. However, the GD extract was more active than its SB counterpart at 0.1 mg/mL,
whereas the SB extract was slightly more active than the GD extract at 1 mg/mL. In gen- eral, these observations show the
cytostatic activity of the analyzed extracts, which may be affected by the interactions between their com- pounds. Analyses of the
effects of OS-supplemented bread extracts on the cell motility cells showed that the GDA extract had also the strongest inhibitory
effect on AGS motility at 1 mg/mL (Fig. 2B), in the absence of considerable effects on the morphology and cytoskeletal ar-
chitecture of AGS cells (Fig. 2A). A less pronounced inhibition of AGS motility was seen in the presence of GD extract, whereas
the SB extract slightly increased AGS motility. Thus, gastrointestinal processing in- creases the bioavailability of
OS-supplemented bread compounds that are characterized by inhibitory activity on cell parameters crucial for cancer promotion
and progression.
Whereas phenolic compounds conceivably account for the biolog- ical effects of OS, the cytostatic and anti-invasive effects of
3% OS- supplemented bread extracts did not correlate with their total phenolic content (TPC; Fig. 3). For instance, the more
efficient TPC (estimated as gallic acid equivalent at a concentration of 1 mg DW/mL) of GD (38 μM) than that of the SB (10
μM) extract was accompanied by its more pro- nounced anti-invasive activity and cytostatic effects on AGS cells at low but not
at high concentration (cf. Fig. 1). GDA extract exerted the strongest inhibitory effect on AGS cell proliferation and motility but
its TPC (23 μM) was lower than the TPC of the GD extract. These data support the notion on the role of subtle interactions
between OS- supplemented bread phenolic compounds in the determination of the bioactivity of analyzed extracts.
The role of such interactions was further confirmed by the data on the effect of OS-bread supplementation on the activity of
enzymes in- volved in the cellular redox control. Non-supplemented wheat bread contains bioaccessible and bioavailable
inhibitors of xanthine oxidase (XO), as demonstrated by the inhibitory effect of bread SB, GD and GDA extracts on the activity
of this enzyme (Table 1). This activity
was only slightly increased in SB extracts from OS-supplemented breads. Gastrointestinal processing released XO inhibitors
from bread samples supplemented with OS (in particular 3 and 5%). This effect was not observed in non-supplemented bread.
Strong synergism be- tween bread XO inhibitors and highly bioaccessible, bioavailable and bioefficient XO inhibitors derived
from OS was also observed.
Gastrointestinal digestion reduced LOX inhibitory activity of non- supplemented bread but increased this activity in OS
supplemented bread extracts. Interestingly, the after GDA processing LOXi was reduced in the case of the OS1 and OS2 breads
whereas it remained the same for the OS3–OS4 breads. Probably, in the case of the OS1 and OS2 breads, LOX inhibitors derived
from OS were complexed with proteins/bile salt and these connections were not able to permeate the dialysis mem- brane. In the
cases of more enriched breads, low-molecular LOX inhibi- tors derived from OS may permeate the dialysis membrane and their
activity balanced the negative effect of complex formation. Also interac- tion factor (IF) values suggest the low efficiency of the
synergism be- tween LOX inhibitors from OS and bread.
Gastrointestinal processing of non-supplemented bread slightly de- creased the inhibitory effect of the extract on the activity
of catalase (CAT; Table 1), but OS supplement considerably enriched wheat bread with potentially bioaccessible activators of
this enzyme. Noteworthy,
100 U. Gawlik-Dziki et al. / Food Research International 73 (2015) 97–106
SB GD GDA

*# *# *
*$
Fig. 1. Cytostatic effect of raw (SB), gastrointestinally digested (GD) and gastrointestinally absorbed (GDA) extracts from bread
supplemented with onion skins (3%) on the proliferation of AGS cells. Cells were treated with medium-dissolved extracts (0.1
and 1 mg/mL) and their numbers were compared with the relevant bread control after 72 h (Hoechst 33258 staining; lower
panel). Bars represent mean ± SEM. *P b 0.05 (paired Student's t-test; N = 3; vs. the relevant bread control). #P b 0.05 (vs. SB
sample). $P b 0.05 (vs. GD sample). Scale bar = 100 μm.
140
120
100
80
60
40
20

*
0.1 mg/ml 1 mg/ml
SB GD GDA
these compounds were generally absent in the buffer extracts (illus- trated by only a slight activation of CAT found only in 2 out
of 6 sam- ples). A comparison of the CAT activating effect of SB, GD and GDA extracts demonstrates potential bioavailability of
CAT activators in OS-enriched breads. Most importantly, bioaccessible and bioavailable CAT activators acted synergistically,
whereas additive interaction was observed in the case of the buffer extract. Finally, small amounts of hy- drophilic compounds
capable of activating the superoxide dismutase (SOD) were observed in the tested breads (regardless of composition).
Gastrointestinal processing, however, reduced their activity. It indicates that gastrointestinal processing may release the
compounds that act antagonistically towards the hydrophilic phenolics present in the SB ex- tracts, as also demonstrated by IF
values (Table 1). These data illustrate a very complex network of interactions between phenolic compounds in OS-supplemented
bread extracts, which may disturb the regulation of reactive oxygen species (ROS)-dependent signaling pathways crucial for
AGS cell proliferation and motility. Differences in their bioaccessibi- lity and bioavailability contribute to this complexity.
Together with a conceivable complexity of synergic (agonistic and/or antagonistic) in- teractions between ROS-dependent
intracellular signaling systems, a lack of the correlation between the cytostatic and anti-invasive activi- ties of the extracts and
their estimated TPC is hardly surprising.
Effects of non-supplemented bread extracts on the proliferation and motility of AGS cells (Fig. 4) further confirmed that wheat
bread phenolic compounds might participate in the potentially pro-healthy activities of OS-supplemented bread. For example,
GDA extract from the non-supplemented bread slightly induced AGS motility (Fig. 4), in contrast to its counterpart from 3%
OS-supplemented bread (see Fig. 2). Similar differences were seen between SB and GD extracts from non-supplemented and
OS-supplemented breads. Differences in the activity of 3% OS-supplemented and non-supplemented breads can be interpreted in
terms of the synergy between bread and OS phe- nolic activity. This notion was positively verified by the comparison of
anti-cancer activities of GDA extracts from non-supplemented bread, OS-supplemented bread and “pure” DMSO-extracted
phenolic fraction of the GDA OS extract. When applied at a concentration equivalent to
101 U. Gawlik-Dziki et al. / Food Research International 73 (2015) 97–106

A
Control GDA bread contol GDA OS-suppl. bread B

control

*
*
Fig. 2. The immediate effect of raw (SB), gastrointestinally digested (GD), and gastrointestinally absorbed (GDA) extracts from
bread supplemented with onion skins (3%) on the cytoskel- eton architecture (actin/vinculin; A) and motility (B) of AGS cells.
Circular diagrams (axis scale inμm) are drawn with the initial point of each trajectory placed at the origin of the plot. Data are
summarized as % of the relevant (SB, GD, GDA, respectively) bread control (see Fig. 3). Bars represent mean ± SEM. *P b
0.001 (Mann–Whitney test; N = 3; vs. the relevant bread control). #P b 0.05 (Student's t-test; N = 3; vs. SB sample). $P b 0.05
(Student's t-test; N = 3; vs. GD sample). Scale bar = 20 μm (A) and 100 μm (B).

OS OS SB OS GD GDA 20
Velocity
140
120

Displacement
100
80
60
40
SB GD GDA
1.0 mg of extract/mL, phenolic fractions of SB and GD extracts from OS inhibited AGS proliferation and motility. Thus, they
acted in a fashion similar to OS-supplemented bread extracts. However, the phenolic fraction of GDA OS-extracts exerted a
cytoprotective activity similar to that seen upon AGS cell treatment with non-supplemented bread extracts and opposite to that
observed for GDA extracts from OS- supplemented breads (Fig. 5). This observation may point to the role of high molecular
mass protein and polysaccharide compounds in the determination of the OS-supplemented bread extract activity.
4. Discussion
Previously, we have demonstrated that the bioavailability and anti- oxidative activity of phenolic compounds of onion skin is
affected by
SB GD GDA
Fig. 3. Total phenolic contents in control and enriched bread. SB — raw, GD — gastrointestinally digested, GDA —
gastrointestinally absorbed extracts.
Table 1 Influence of control and enriched bread phytochemical compounds on xanthine oxidase (OX), lipoxygenase (LOX),
catalase (CAT), superoxide dismutase (SOD) activity and their inter- action, bioaccessibility, bioavailability and bioefficiency in
vitro factors.
Activity Sample Raw (SB) Digested (GD) Absorbed (GDA) Interaction factor BAC BAV BEF
A
M
50
45
g 40
35
30
25
20
15
10
5
0
C OS1 OS2 OS3 OS4 OS5
its gastrointestinal processing in vitro and by phenolic interactions with food matrix proteins (Świeca, Gawlik-Dziki, Dziki,
Baraniak, & Czyż, 2013). Here, we show that OS-supplementation and gastrointestinal processing in vitroincreases the inhibitory
effect of wheat bread extracts on gastric cancer cell proliferation and motility. This effect was accom- panied by the augmenting
effects of gastrointestinal processing on the bioavailability and bioaccessibility of phenolic compounds that
Absorbed (GDA)
XOi
[EC
50
A
T
A
M
A
T
A
M
A
T
Raw (SB) Digested
(GD)
C 11.84 ± 0.38aA* – 11.86 ± 0.25aA – 12.48 ± 0.62aA – – – – 1.00 0.95 0.95 OS1 10.37 ± 0.52bA 43.94 6.03 ± 0.48bB 25.57
6.34 ± 0.21bB 25.30 0.23 ± 0.001 0.23 ± 0.002 0.25 ± 0.002 1.72 0.95 1.63 OS2 10.24 ± 0.12bA 43.54 6.56 ± 0.51bB 28.36 6.91
± 0.35bB 27.46 1.56 0.95 1.48 OS3 10.09 ± 0.09bA 43.04 5.86 ± 0.23bcB 25.13 6.17 ± 0.41bB 24.42 1.72 0.95 1.64 OS4 10.31 ±
0.13bA 44.13 5.98 ± 0.18cB 25.79 6.30 ± 0.24bC 24.83 1.72 0.95 1.64 OS5 10.03 ± 0.42bA 43.08 5.84 ± 0.037bcB 25.32 6.14 ±
0.26bB 24.10 1.72 0.95 1.63
LOXi
[EC
50
mg DW/mL]
C 27.20 ± 1.12aA – 30.58 ± 1.38aB – 33.24 ± 1.24aC – – – – 0.89 0.92 0.82 mg
OS1 25.09 ± 1.42bA 46.25 21.35 ± 1.42bB 35.47 27.40 ± 1.13bC 41.35 0.54 ± 0.002 0.59 ± 0.011 0.66 ± 0.003 1.18
0.78 0.92 DW/mL]
OS2 23.79 ± 1.38cA 43.97 21.43 ± 1.25bB 35.41 26.10 ± 1.25cC 39.51 1.11 0.82 0.91 OS3 21.80 ± 1.22dA 40.40 21.14 ±
1.09bA 36.66 21.59 ± 1.48dA 32.78 1.03 1.00 1.00 OS4 24.28 ± 0.91cA 45.11 21.63 ± 1.11bB 36.67 21.77 ± 1.22dB 33.16 1.12
0.99 1.12 OS5 25.05 ± 1.21bA 46.67 21.39 ± 1.05bB 37.10 21.55 ± 1.18dB 32.93 1.17 0.99 1.16
CATa* [% of control]
C 99.94 ± 2.00aA – 89.92 ± 2.49aB – 78.82 ± 2.11aC – – – – 0.90 0.88 0.79 OS1 102.20 ± 3.00bA 100.17 111.95 ± 3.28bB 89.24
105.38 ± 4.48bC 79.08 1.00 ± 0.049 0.76 ± 0.028 0.73 ± 0.012 1.10 0.94 1.03 OS2 107.61 ± 3.09cA 100.34 121.49 ± 3.33cB
89.47 110.32 ± 4.32cC 79.34 1.13 0.91 1.03 OS3 98.82 ± 3.00aA 100.51 117.07 ± 4.11dB 89.71 109.65 ± 3.98cC 79.60 1.18
0.94 1.11 OS4 98.49 ± 2.45aA 100.68 116.06 ± 5.07dB 89.94 108.98 ± 5.46cC 79.86 1.18 0.94 1.11 OS5 94.75 ± 2.11dA 100.85
124.10 ± 5.32cB 90.18 110.87 ± 5.11cC 80.12 1.31 0.89 1.17
SODa** [% of control]
C 106.59 ± 3.85aA – 103.97 ± 3.27aB – 91.18 ± 3.34aC – – – – 0.98 0.88 0.86 OS1 104.20 ± 2.48bA 106.70 87.89 ± 2.85bB
104.06 70.31 ± 2.71bC 91.33 1.01 ± 0.011 1.28 ± 0.163 1.32 ± 0.231 0.84 0.80 0.67 OS2 106.72 ± 5.22aA 106.82 77.25 ±
2.17cB 104.15 80.20 ± 2.09cC 91.47 0.72 1.04 0.75 OS3 106.07 ± 3.45aA 106.93 69.57 ± 2.85dB 104.25 55.66 ± 2.02dC 91.62
0.66 0.80 0.52 OS4 107.88 ± 4.11cA 107.05 80.35 ± 3.01eB 104.34 64.28 ± 1.42eC 91.77 0.74 0.80 0.60 OS5 106.65 ± 3.98aA
107.16 97.72 ± 4.04fB 104.43 86.18 ± 2.33fC 91.91 0.92 0.88 0.81
*Sample concentration 10 mg/mL. **The values ± SD designated by the different small letters (in the columns, concerning the
individual activity) and by the different capital letters (in the rows, concerning individual extracts) are significantly different (α =
0.05). XOi — xanthine oxidase inhibitory activity, LOXi — lipoxygenase inhibitory activity, CATa — catalase activity, SODa
— superoxide dismutase activity, A
M
—
theoretically calculated activity, IF — interaction factor, BAC — bioaccessibility factor, BAV — bioavailability factor, BEF —
bioefficiency factor.
— measured activity, A
T
102 U. Gawlik-Dziki et al. / Food Research International 73 (2015) 97–106
ab
h
j
i
i
ee
fe
e
b
modulate the activity of enzymes involved in intracellular oxidative stress and ROS-dependent signaling. Mechanistic studies
indicated the role of the interactions between phenolic compounds of wheat bread and OS, and of phenolic-food matrix
interactions in determining OS-supplemented wheat bread bioactivity.
Cytostatic and anti-invasive effects of OS-supplemented bread ex- tracts suggest the possible interference of
OS-supplemented bread
c
j
c
j
d
with gastric cancer promotion and progression. They remain in agree- ment with the findings on the effect of vegetables on the
function of cancer cells (Clere, Faure, Martinez, & Andriantsitohaina, 2011). Exper- imental models based on the analyses of
basic cellular traits crucial for cancer development, such as cancer cell proliferation and motility in vitro, are commonly
employed to estimate the potential of phyto- chemicals for cancer prophylactics and treatment. They can be used to estimate the
bioactivity of purified factors (Czernik, Sroka, Madeja, & Czyz, 2008; Wójcik et al., 2013) and of complex plant extracts
(Gawlik-Dziki et al., 2013a, 2013b; Gawlik-Dziki et al., 2014). Using this approach, we have previously shown that wheat bread
supplemen- tation with broccoli sprouts augments their anti-cancer potential but the simulated gastrointestinal processing
decreases their bioactivity (Gawlik-Dziki et al., 2014). Our current observations confirm pro- healthy potential of OS phenolic
compounds (Świeca et al., 2013). Comparison of AGS behavior in the presence of non-supplemented and OS-supplemented
bread extracts shows that the activity of OS phe- nolics may at least partly account for their cytostatic and anti-invasive effects.
They also show that the bioactivity of OS compounds is retained in wheat bread and increases upon gastrointestinal processing.
103 U. Gawlik-Dziki et al. / Food Research International 73 (2015) 97–106

**
*
Fig. 4. Effect of raw (SB), gastrointestinally digested (GD), and gastrointestinally absorbed (GDA) extracts from control breads
administered at a concentration of 1 mg/mL on the motility of AGS cells. Circular diagrams (axis scale in μm) are drawn with the
initial point of each trajectory placed at the origin of the plot. Data are summarized as % of the control (cells cultivated in the
absence of any extract). Bars represent mean ± SEM. *P b 0.001 (Mann–Whitney test; N = 3); vs. the control cells. #P b 0.05
(Student's t-test; N = 3; vs. SB sample). $P b 0.05 (Student's t-test; N = 3; vs. GD sample). Scale bar = 20 μm.
140

120 control
100
SB GD GDA

bread SB bread GD bread GDA

80
60
40
20
Further analyses revealed the enhancement of antioxidative prop- erties of wheat bread upon OS supplementation illustrated
by the effects of OS-supplemented bread extracts on the activity of XO, LOX, CAT and SOD. Anticancer activity of phenolic
compounds in vivo has long been ascribed to their direct antioxidant effects and their in- terference with the activity of pro- and
anti-oxidative enzymes at the cellular level (Han, Shen, & Lou, 2007). ROS-dependent signaling participates in aberrant cell
behavior, a hallmark of cancer. It is de- termined by a complex network of synergic interactions between sig- naling systems. XO
and LOX are major sources of endogenous ROS. They modify the activity of the signaling pathways, which regulate cancer cell
proliferation, differentiation and apoptosis (Wu, 2006). Thus, they contribute to oxidative stress and are directly associated with
the occurrence of pathological conditions, such as tumor pro- motion and progression (Ferraz-Filha, Vitolo, Fietto, Lombardi, &
Saude-Guimaraes, 2006). Also arachidonic acid metabolism by the LOX-dependent pathway produces reactive oxygen species,
which may play a role in inflammation and tumor promotion. In contrast, CAT and SOD are potent scavengers of free radicals
(Hileman, Achanta, & Huang, 2001).
OS-supplemented bread extracts (3% in particular) had no signifi- cant effects on LOX activity, exerted an inhibitory effect on
SOD and XO activities, and activated CAT. Thus, it is a plausible hypothesis that ROS-mediated signaling is targeted by the OS
compounds of wheat bread, leading to the inhibition of cancer cell proliferation and motility. This notion is substantiated by the
different involvement of these enzymes in the development of various cancers. A decrease of SOD and CAT activity was found
in patients with colon cancer (Kubiak et al., 2011). In contrast, a significant increase in the level of SOD and lowered CAT
activity were observed in breast cancer patients (Krishna Veni, Usha, Muni Kumar, & Raghava Rao, 2011). The inhibition of
ROS formation in cancer cells by SODs resulted in decreased pancreatic tumor growthin vitro (Afanas'ev, 2011). However,
increased SOD serum levels in gastric cancer patients were associated with an increased risk of this cancer (Lin, Kikuchi, Obata,
& Yagyu, 2002). Owing to their antioxidant and detoxification properties, SOD and CAT have been pro- posed as the biomarkers
of chemoprevention (Weisburger, 2001). Also arachidonic acid metabolism is a postulated target in the preven- tion of many
types of cancer, especially cancers of the digestive tracts (Hong, Smith, Ho, August, & Yang, 2001; Sadik, Sies, & Schewe,
2003; Yamamoto et al., 2005).
However, versatile modulating effects of OS-supplemented wheat bread extracts on XO, LOX, CAT and SOD activities
pinpoint just one of the possible mechanisms of their potential pro-health activity. We still have to consider that the interference
of phenolic compounds with signaling systems involved in the regulation of cancer development also results from their inhibitory
effect on other signaling pathways,
104 U. Gawlik-Dziki et al. / Food Research International 73 (2015) 97–106

A**
B
140
eq. 0.1% DMSO
120
eq. 0.5% DMSO eq. 1.0% DMSO

*
100
80
control SB
60
40

*
20
GD GDA SB GD GDA

C
20
Fig. 5. The effect of DMSO-extractable fraction of raw (SB), gastrointestinally digested (GD), and gastrointestinally absorbed
(GDA) OS extracts on the proliferation (A), cytoskeleton ar- chitecture (actin/vinculin; B) and motility (C) of AGS cells. Data
are summarized as % of a relevant DMSO control (0.1 and 1.0% DMSO for 0.1 and 1 mg/mL, respectively). Bars represent mean
± SEM. *P b 0.05 (paired Student's t-test; N = 3; A) and *P b 0.001 (Mann–Whitney test; N = 3; B); #P b 0.05 (Student's t-test;
vs. SB sample); $P b 0.05 (Student's t-test; vs. GD sample; B). Scale bar = 20 μm.
140
Displacement

*
120
Velocity 100 80 60

* * 40
SB GD GDA
for example those dependent on protein kinases (Clere et al., 2011). Thus, a comprehensive elucidation of the mechanisms of
cytostatic and anti-invasive effects of OS phenolics requires further study. Validity of this postulate is illustrated by comparison
of bioactivity of wheat bread and OS-supplemented bread extracts in the AGS cell system, and the non-linear character of
dose-dependent effects of extracts on cancer cell properties. Increased motility of the cells incubated in the presence of
non-supplemented bread extracts and SB extract of 3% OS-supplemented bread may illustrate “cytoprotective” or “stimulatory”
properties of certain phenolic combinations. They can result from ROS scavenging properties of phenolics or from their effect on
other intracel- lular signaling pathways. This notion is also illustrated by relatively distinct differences between the effects of
OS-supplemented breads differing in supplementation level (1–5%) on the activity of analyzed enzymes. Moreover, the switch
between “cytoprotective/simulatory” and cytostatic effect of OS supplementation and gastrointestinal pro- cessing prompts
speculations on the multidirectional role of wheat bread compounds in the regulation of the bioactivity of OS compounds. In
particular, the differences in bioactivity of GDA extracts from OS- supplemented breads and DMSO-extracted fraction of GDA
extract from OS suggest the role of high molecular mass protein and polysaccharide compounds in the determination of the
OS-supplemented bread extract activity. The presence of indigestible protein–flavonoid complexes with molecular weights close
to the GDA membrane cut-off capability (between 14.5 and 25 kDa) was found in OS-supplemented bread extracts (Świeca et al.,
2013). These phenol–protein interactions can differentially affect the bioavailability of the phenolic compounds via
the effect on the efficiency of their penetration though the membrane. Phenol–protein/polysaccharide interactions can also
differentially affect the quality and quantity of phenolic interactions with cellular effectors. Gastrointestinal absorption may thus
enrich OS-supplemented bread extracts in highly bioactive compounds, whereas DMSO-extraction re- duces anti-cancer
activities of OS phenolic compounds. This interplay may add to the complexity of networks governing agonistic and antag-
onistic effects of phenolic compounds on cancer cell behavior.
5. Conclusion
Considerable attention has recently been directed towards the pleio- tropic effects of plant phenolic compounds on intrinsic
cellular proper- ties crucial for cancer development (Clere et al., 2011; Weng & Yen, 2012). OS contains significantly higher
levels of flavonoids than the edible portion of this vegetable. Therefore, this waste material is a po- tential source of bioactive
compounds for wheat breads. Our data sug- gest that OS-supplementation elevates bioactivity of wheat bread incl. antioxidant
potential, and OS phenolics that interfere with the cellular traits crucial for gastric cancer promotion and progression. These
effects were seen at the physiologically relevant concentrations of the extracts, equivalent to ca. 150 g of bread for 75 kg of body
mass. The phenolic content of the extracts at the concentrations used in this study (10–38 μM) is higher than the reported serum
concentrations of poly- phenols after their dietary uptake, but may be corresponding to their local concentrations in specified
tissues (for instance in tumor mass). The functional links between antioxidant potential of the analyzed extracts and their effects
on cancer cells remain to be elucidated. How- ever, the synergistic effects of bread and OS phenolics and the possible facilitating
effect of the bread matrix on the bioavailability of bioactive OS compounds suggest that wheat bread constituents may signifi-
cantly contribute to the beneficial effects of OS-supplementation of wheat bread. These data indicate that the anti-cancer activity
of OS- supplemented food may depend on the complex network of interac- tions between the phenolics and food matrix. Thus,
onion skins may serve as a valuable food supplement preventing upper gastrointestinal system cancers. Together with satisfactory
overall consumer acceptabil- ity of the product containing up to 3% onion skin powder, these data confirm OS suitability for
bread supplementation (Gawlik-Dziki et al., 2013b). They also justify further studies on the interplay between bio- logical
activity of single polyphenolic compounds, their combinations and flavonoid–matrix interactions, and its contribution to the
cytostatic and anti-invasive activities of the food products.
Acknowledgments
This scientific study was financed by the Polish Ministry of Scientific Research and Higher Education (grant NN312233738).
Faculty of Bio- chemistry, Biophysics and Biotechnology of Jagiellonian University is a partner of the Leading National
Research Center (KNOW) supported by the Ministry of Science and Higher Education.
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