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Article
Synergism between Soluble and Dietary Fiber Bound Antioxidants Ecem Evrim Çelik, Vural Gökmen, and Leif H. Skibsted J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 18 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 27
Journal of Agricultural and Food Chemistry
Synergism between Soluble and Dietary Fiber Bound Antioxidants †
†*
Ecem Evrim Çelik , Vural Gökmen , Leif H. Skibsted § †
Department of Food Engineering, Hacettepe University, 06800 Beytepe, Ankara,
Turkey § Faculty
of Life Sciences, Department of Food Science, Food Chemistry, University
of Copenhagen, Rolighedsvej 30, 1958 Frederiksberg C, Denmark
* Corresponding Author: Prof. Dr. Vural Gökmen Tel: +90 312 2977108; Fax: +90 312 2992123; E-mail: [email protected]
ACS Paragon Plus Environment
1
Journal of Agricultural and Food Chemistry
Page 2 of 27
1
ABSTRACT
2
This study investigates the synergism between antioxidants bound to dietary
3
fibers (DF) of grains and soluble antioxidants of high-consumed beverages or their
4
pure antioxidants. The interaction between insoluble fractions of grains containing
5
bound antioxidants and soluble antioxidants was investigated using; (i) liposome
6
based system by measuring the lag phase before the onset of oxidation, (ii) ESR
7
based system by measuring the reduction percentage of Fremy’s salt radical. In
8
both procedures, antioxidant capacities of DF-bound and soluble antioxidants
9
were measured as well as their combinations, which were prepared for different
10
ratios. The simple addition effects of DF bound and soluble antioxidants were
11
compared with measured values. The results revealed a clear synergism for almost
12
all combinations in both liposome and ESR based systems. The synergism
13
observed in DF bound-soluble antioxidant system paints a promising picture
14
considering their role in human gastrointestinal (GI) tract health.
15
Keywords: Synergism, free antioxidants, bound antioxidants, dietary fibers, cereal
16
grains
17
ACS Paragon Plus Environment
2
Page 3 of 27
Journal of Agricultural and Food Chemistry
18
INTRODUCTION
19
Grains such as wheat, oat and rye are the staple elements of the human being’s
20
daily diet, contributing about 50% of dietary fiber (DF) intake
21
carrier of dietary antioxidants 3. Epidemiological studies report that there is an
22
inverse relationship between whole grain consumption and the incidence of
23
cardiovascular diseases (CVD) 4, type 2 diabetes 5, obesity
24
cancers 7. In the meantime, studies focused on the origin of the health benefits of
25
whole grains in terms of its constituents, namely germ, bran and endosperm
26
showed that bran is the key factor
27
insoluble DF is found as associated with a substantial amount of dietary
28
antioxidants, which can be named as DF-antioxidants 12.
29
DF-antioxidants can contribute to the health effects attributed to DF as well as to
30
dietary antioxidants 3; like protection against CVD
31
diabetes, neurodegenerative diseases, hypertension 14,15,16,17 and regulation of the
32
insulin level in blood after food ingestion
33
significant portion of dietary antioxidants and should not be counted as the minor
34
constituents of DF 3.
35
Since dietary fiber is non-digestible in the upper intestine, it reaches to colon
36
almost in intact form with the antioxidants still bound to it. These antioxidants are
37
released from the fiber matrix slowly in a continuous manner during the
38
considerable survival period of insoluble DF in GI tract by the help of the colon
39
microbiota
40
peak in plasma antioxidant level immediately after ingestion and disappear in few
12.
8,9,10,11.
6
1,2
besides being
and certain types of
Cereal bran, including most of the
18.
13,
certain cancer types,
Furthermore, they constitute a
Thus, in comparison to the soluble antioxidants, which constitute a
ACS Paragon Plus Environment
3
Journal of Agricultural and Food Chemistry
19,
Page 4 of 27
41
hours
antioxidants bound to DF can create a continuous and healthier
42
antioxidant environment in GI tract 12.
43
At this point, providing the continuity and improving the quality of the created
44
antioxidant environment becomes an attractive issue. As known, for some
45
combinations of antioxidants, a larger overall effect is observed compared to the
46
simple addition of the individual effects, which is termed as synergism 20. Several
47
studies have shown that plant polyphenols exert a synergistic behavior with other
48
antioxidants present in plant material
49
bound antioxidants of grains and the free soluble antioxidants that are frequently
50
consumed together in the daily human diet; it will help to improve the quality of
51
the healthy antioxidant environment in the GI tract, partly by a mechanism
52
depending on regeneration of the DF antioxidants by the soluble antioxidants.
53
Accordingly, the objective of the present study was to determine if the
54
combination of insoluble antioxidants of grains and soluble antioxidants from
55
different sources (frequently consumed antioxidant rich beverages, pure
56
antioxidant compounds) are synergistically or antagonistically affecting the
57
antioxidant capacity using two different well-established experimental designs; (i)
58
a liposome based system by measuring the lag phase before the onset of oxidation
59
spectrophotometrically, (ii) an ESR based system by measuring the percentage of
60
reduction in a solution of Fremy’s salt as a semi-stable radical by antioxidant
61
compound. In these two different designs, oxidation is determined at two different
62
stages: ESR functions at the radical scavenging stage, while liposomes at the
63
formation of lipid oxidation products i.e. the aldehydes, which create the
21,22,23.
If such interaction exist among DF
ACS Paragon Plus Environment
4
Page 5 of 27
Journal of Agricultural and Food Chemistry
64
fluorescence.
65
MATERIALS AND METHODS
66
Chemicals
67
Potassium
68
phosphatidylcholine from soybean (99%), dipotassium phosphate, sodium
69
phosphate, (-) epicatechin, (-) – epigallocatechin, (-) – epicatechin gallate, (-) –
70
epigallocatechin
71
tetramethylchroman-2 carboxylic acid (Trolox), iron(III) chloride, adenosine 5’ –
72
diphosphate sodium salt (ADP), methanol, chloroform, hexane and ethanol were
73
purchased from Sigma-Aldrich Chemie (Steinheim, Germany). L (+) ascorbic acid,
74
monopotassium phosphate and glycine were purchased from Merck (Darmstadt,
75
Germany). All solvents used were of analytical grade, unless otherwise stated.
76
Water was purified through a Millipore Q-plus purification train (Millipore Corp.,
77
Bedford, MA, USA).
78
Grains and Beverages
79
All grain varieties used were purchased from a local market in Turkey, Ankara.
80
Green tea, pomegranate juice, roasted coffee beans and red wine were of food
81
grade and purchased in Copenhagen, Denmark.
82
brewing 3 g of green tea with 100 mL of hot water for 15 min and espresso was
83
prepared in a kitchen type coffee-machine, Titanium Gaggia, at the 7th level of the
84
dose control knob.
85
Preparation of the insoluble fractions of grains
nitrosodisulfonate
gallate,
(Fremy’s
chlorogenic
salt),
acid,
sodium
resveratrol,
carbonate,
L-α-
6-hydroxy-2,5,7,8
Green tea was prepared by
ACS Paragon Plus Environment
5
Journal of Agricultural and Food Chemistry
Page 6 of 27
86
Ground whole grains were washed according to the procedure described by Çelik
87
et al. (2013)
88
Washed grains were freeze-dried, ground with a ceramic mortar to obtain fine
89
powder form and passed through a sieve (Endecotts Test Sieve, London, UK) of 40
90
mesh size (425 μm). The final insoluble powder was tested and found to be free of
91
soluble antioxidant compounds. It was kept under 4°C prior to experiments.
92
Preparation of liposomes
93
Liposomes were prepared with minor modifications
94
described by Tirmenstein
95
chloroform
96
phosphatidylcholine residue was subsequently re-hydrated with 5 mL of 50 mM
97
sodium phosphate buffer (PB) (pH=7.4) and vortexed. The suspension was
98
sonicated at 100W with 30 sec intervals 10 times to yield a white homogenous
99
suspension of multilamellar liposomes. Between the intervals, the suspension was
100
left to cool down in an ice bath for at least 2 min. Sonicated suspension was
101
centrifuged at 16000 g for 30 min and the supernatant containing the liposomes
102
was collected. The liposomes were stored under +4°C for 1 week before
103
experimental studies to increase the level of lipid hydroperoxides (about 5-10% of
104
the phosphatidyl choline). These will react with Fe3+ to produce free radicals.
105
Peroxidation of liposomes
106
Liposomes prepared 1 week prior to measurement were pipetted into a 96 well
107
transparent plate as 10 µL for each well and mixed with 50 µL of sample dilutions
108
or standard solution (Trolox), while MilliQ water was used as zero-sample. Sample
24
in order to remove water, alcohol and lipid soluble fractions.
and
26.
evaporated
25
according to the method
40 mg of phosphatidylcholine was dissolved in into
dryness
under
ACS Paragon Plus Environment
nitrogen
flow.
The
6
Page 7 of 27
Journal of Agricultural and Food Chemistry
109
dilutions were prepared as; 500, 100, 50 and 10 µM for pure antioxidant
110
compounds, 1/500, 1/1000, 1/5000, 1/10000 dilutions with water for beverages
111
and 10, 5, 1 and 0,5 mg per mL aqueous solutions for insoluble and whole grains.
112
Mixtures of insoluble grains with beverages/pure antioxidant solutions were
113
prepared by combining two different concentrations of grains (10 and 5 mg/mL)
114
with two different concentrations of beverages (1/500, 1/1000) or pure solutions
115
(500 and 100µM) at a ratio of 1:1 to give 50 µL sample. Since aldehydes generated
116
by lipid peroxidation can react with amines to produce fluorescent products,
117
glycine was added to the reaction medium as glycine ascorbate buffer (GAB),
118
which was prepared as 50 mM potassium phosphate buffer (PPB) with 100 mM
119
glycine and 450 µM ascorbate at pH 7.4, for 120 µL per well. Background wells
120
were made by pipetting GAB buffer instead of liposome and samples. Lipid
121
peroxidation was initiated by introducing 20 µL of an oxidizing agent comprised of
122
25 µM FeCl3 in 50 mM PPB (pH=7.4) with 1 mM ADP.
123
The plate was inserted into the plate reader of Tecan fluorometer immediately
124
after addition of oxidizing agent. The lag phase before the onset of oxidation was
125
measured for 4 h at 37° C (excitation at 360 nm, emission at 460 nm) in 10 min
126
intervals and samples were analyzed in triplicate.
127
Inhibition of lipid oxidation was calculated by the following formula:
128
Inhibition of lipid oxidation (%) = 100 – (AUCsample/AUCcontrol) x 100
129
where AUCsample is the area under the curve of sample containing antioxidant as
130
measured in well, and AUCcontrol is the area under the curve measured for the
131
control well. In order to avoid the differences originating from different starting
ACS Paragon Plus Environment
7
Journal of Agricultural and Food Chemistry
Page 8 of 27
132
levels of oxidation, all fluorescent values were subtracted the lowest value,
133
considering the lowest point as zero and the AUC was calculated by the trapezoid
134
rule.
135
Scavenging of Fremy’s salt radical
136
The scavenging of Fremy’s salt radical was determined with minor modifications
137
according to the method described by Rødjter et al. (2006)
138
whole grains, 10, 15 and 20 mg of sample was mixed with 1 mL of MilliQ water and
139
200 µL of a solution of Fremy’s salt (1.64 mM) in 25% saturated sodium carbonate
140
solution. For pure antioxidant solutions (10 µM) and beverages (1:1000 diluted)
141
100, 150 and 200 µL of sample was used instead of 10, 15 and 20 mg.
142
Combinations of insoluble grains with beverages/pure antioxidant solutions were
143
made in the ratio of 10 mg: 100 µL and then 100, 150 and 200 µL of sample from
144
the prepared mixture was added into the reaction medium as described for
145
beverages and pure solutions above. 6 min after the mixing, ESR spectra was
146
recorded with a MiniScope MS200 ESR spectrometer (Magnettech GmbH, Berlin,
147
Germany). The measurements were carried out with the following settings: BO-
148
field, 3366.90 G; sweep width, 49.82 G; sweep time, 60 sec; smooth, 0; steps, 4096;
149
number, 1 pass; modulation, 1000 mG; Mw atten, 10 dB; gain, 2; E,1.
150
Statistical Analysis
151
The analytical data were reported as the mean ± standard deviation of duplicate
152
independent measurements. The significance of mean differences was determined
153
by Independent Samples t-test using SPSS version 17.0.
ACS Paragon Plus Environment
27.
For insoluble and
8
Page 9 of 27
Journal of Agricultural and Food Chemistry
154
RESULTS AND DISCUSSION
155
Antioxidant activity measurement by ESR spectroscopy assay
156
Table 1 gives relative decrease (%) in the intensity of measured and estimated
157
electron spin resonance (ESR) signal values for the mixtures of insoluble grains
158
and beverages rich in antioxidants. The antioxidant activities of samples were
159
determined as their ability to reduce the stable Fremy’s salt radical as measured by
160
ESR spectrometer. Diamagnetic reduction products are formed, which are ESR-
161
invisible, when antioxidant compounds scavenge Fremy’s salt radicals, leading to a
162
decrease in the ESR-signal
163
signal was determined for insoluble grains, beverages and pure antioxidant
164
compounds separately, and the decrease in the ESR signal for combinations of
165
grains with beverages/pure antioxidant solutions were calculated by using these
166
data. At the same time, the experiments were performed for combinations of
167
grains with beverages/pure antioxidant solutions. Estimated data were obtained
168
by summing up the halves of the percentage decreases (%) in the intensities of ESR
169
signals separately measured for the concentrations/dilutions of insoluble grains
170
and beverages/pure antioxidant compounds, which were used to make 1:1
171
combination.
172
In general, an almost linear relationship was observed between the mixture
173
volume added to the reaction medium and estimated/measured values of decrease
174
in the ESR signals. The measured values of decrease in the intensity of ESR signals
175
were significantly higher than the estimated ones for combinations of almost all
176
insoluble grains with all beverages used, pointing toward a clear synergistic effect
27.
The percentage decrease in the intensity of ESR
ACS Paragon Plus Environment
9
Journal of Agricultural and Food Chemistry
Page 10 of 27
177
(p
Article
Synergism between Soluble and Dietary Fiber Bound Antioxidants Ecem Evrim Çelik, Vural Gökmen, and Leif H. Skibsted J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 18 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 27
Journal of Agricultural and Food Chemistry
Synergism between Soluble and Dietary Fiber Bound Antioxidants †
†*
Ecem Evrim Çelik , Vural Gökmen , Leif H. Skibsted § †
Department of Food Engineering, Hacettepe University, 06800 Beytepe, Ankara,
Turkey § Faculty
of Life Sciences, Department of Food Science, Food Chemistry, University
of Copenhagen, Rolighedsvej 30, 1958 Frederiksberg C, Denmark
* Corresponding Author: Prof. Dr. Vural Gökmen Tel: +90 312 2977108; Fax: +90 312 2992123; E-mail: [email protected]
ACS Paragon Plus Environment
1
Journal of Agricultural and Food Chemistry
Page 2 of 27
1
ABSTRACT
2
This study investigates the synergism between antioxidants bound to dietary
3
fibers (DF) of grains and soluble antioxidants of high-consumed beverages or their
4
pure antioxidants. The interaction between insoluble fractions of grains containing
5
bound antioxidants and soluble antioxidants was investigated using; (i) liposome
6
based system by measuring the lag phase before the onset of oxidation, (ii) ESR
7
based system by measuring the reduction percentage of Fremy’s salt radical. In
8
both procedures, antioxidant capacities of DF-bound and soluble antioxidants
9
were measured as well as their combinations, which were prepared for different
10
ratios. The simple addition effects of DF bound and soluble antioxidants were
11
compared with measured values. The results revealed a clear synergism for almost
12
all combinations in both liposome and ESR based systems. The synergism
13
observed in DF bound-soluble antioxidant system paints a promising picture
14
considering their role in human gastrointestinal (GI) tract health.
15
Keywords: Synergism, free antioxidants, bound antioxidants, dietary fibers, cereal
16
grains
17
ACS Paragon Plus Environment
2
Page 3 of 27
Journal of Agricultural and Food Chemistry
18
INTRODUCTION
19
Grains such as wheat, oat and rye are the staple elements of the human being’s
20
daily diet, contributing about 50% of dietary fiber (DF) intake
21
carrier of dietary antioxidants 3. Epidemiological studies report that there is an
22
inverse relationship between whole grain consumption and the incidence of
23
cardiovascular diseases (CVD) 4, type 2 diabetes 5, obesity
24
cancers 7. In the meantime, studies focused on the origin of the health benefits of
25
whole grains in terms of its constituents, namely germ, bran and endosperm
26
showed that bran is the key factor
27
insoluble DF is found as associated with a substantial amount of dietary
28
antioxidants, which can be named as DF-antioxidants 12.
29
DF-antioxidants can contribute to the health effects attributed to DF as well as to
30
dietary antioxidants 3; like protection against CVD
31
diabetes, neurodegenerative diseases, hypertension 14,15,16,17 and regulation of the
32
insulin level in blood after food ingestion
33
significant portion of dietary antioxidants and should not be counted as the minor
34
constituents of DF 3.
35
Since dietary fiber is non-digestible in the upper intestine, it reaches to colon
36
almost in intact form with the antioxidants still bound to it. These antioxidants are
37
released from the fiber matrix slowly in a continuous manner during the
38
considerable survival period of insoluble DF in GI tract by the help of the colon
39
microbiota
40
peak in plasma antioxidant level immediately after ingestion and disappear in few
12.
8,9,10,11.
6
1,2
besides being
and certain types of
Cereal bran, including most of the
18.
13,
certain cancer types,
Furthermore, they constitute a
Thus, in comparison to the soluble antioxidants, which constitute a
ACS Paragon Plus Environment
3
Journal of Agricultural and Food Chemistry
19,
Page 4 of 27
41
hours
antioxidants bound to DF can create a continuous and healthier
42
antioxidant environment in GI tract 12.
43
At this point, providing the continuity and improving the quality of the created
44
antioxidant environment becomes an attractive issue. As known, for some
45
combinations of antioxidants, a larger overall effect is observed compared to the
46
simple addition of the individual effects, which is termed as synergism 20. Several
47
studies have shown that plant polyphenols exert a synergistic behavior with other
48
antioxidants present in plant material
49
bound antioxidants of grains and the free soluble antioxidants that are frequently
50
consumed together in the daily human diet; it will help to improve the quality of
51
the healthy antioxidant environment in the GI tract, partly by a mechanism
52
depending on regeneration of the DF antioxidants by the soluble antioxidants.
53
Accordingly, the objective of the present study was to determine if the
54
combination of insoluble antioxidants of grains and soluble antioxidants from
55
different sources (frequently consumed antioxidant rich beverages, pure
56
antioxidant compounds) are synergistically or antagonistically affecting the
57
antioxidant capacity using two different well-established experimental designs; (i)
58
a liposome based system by measuring the lag phase before the onset of oxidation
59
spectrophotometrically, (ii) an ESR based system by measuring the percentage of
60
reduction in a solution of Fremy’s salt as a semi-stable radical by antioxidant
61
compound. In these two different designs, oxidation is determined at two different
62
stages: ESR functions at the radical scavenging stage, while liposomes at the
63
formation of lipid oxidation products i.e. the aldehydes, which create the
21,22,23.
If such interaction exist among DF
ACS Paragon Plus Environment
4
Page 5 of 27
Journal of Agricultural and Food Chemistry
64
fluorescence.
65
MATERIALS AND METHODS
66
Chemicals
67
Potassium
68
phosphatidylcholine from soybean (99%), dipotassium phosphate, sodium
69
phosphate, (-) epicatechin, (-) – epigallocatechin, (-) – epicatechin gallate, (-) –
70
epigallocatechin
71
tetramethylchroman-2 carboxylic acid (Trolox), iron(III) chloride, adenosine 5’ –
72
diphosphate sodium salt (ADP), methanol, chloroform, hexane and ethanol were
73
purchased from Sigma-Aldrich Chemie (Steinheim, Germany). L (+) ascorbic acid,
74
monopotassium phosphate and glycine were purchased from Merck (Darmstadt,
75
Germany). All solvents used were of analytical grade, unless otherwise stated.
76
Water was purified through a Millipore Q-plus purification train (Millipore Corp.,
77
Bedford, MA, USA).
78
Grains and Beverages
79
All grain varieties used were purchased from a local market in Turkey, Ankara.
80
Green tea, pomegranate juice, roasted coffee beans and red wine were of food
81
grade and purchased in Copenhagen, Denmark.
82
brewing 3 g of green tea with 100 mL of hot water for 15 min and espresso was
83
prepared in a kitchen type coffee-machine, Titanium Gaggia, at the 7th level of the
84
dose control knob.
85
Preparation of the insoluble fractions of grains
nitrosodisulfonate
gallate,
(Fremy’s
chlorogenic
salt),
acid,
sodium
resveratrol,
carbonate,
L-α-
6-hydroxy-2,5,7,8
Green tea was prepared by
ACS Paragon Plus Environment
5
Journal of Agricultural and Food Chemistry
Page 6 of 27
86
Ground whole grains were washed according to the procedure described by Çelik
87
et al. (2013)
88
Washed grains were freeze-dried, ground with a ceramic mortar to obtain fine
89
powder form and passed through a sieve (Endecotts Test Sieve, London, UK) of 40
90
mesh size (425 μm). The final insoluble powder was tested and found to be free of
91
soluble antioxidant compounds. It was kept under 4°C prior to experiments.
92
Preparation of liposomes
93
Liposomes were prepared with minor modifications
94
described by Tirmenstein
95
chloroform
96
phosphatidylcholine residue was subsequently re-hydrated with 5 mL of 50 mM
97
sodium phosphate buffer (PB) (pH=7.4) and vortexed. The suspension was
98
sonicated at 100W with 30 sec intervals 10 times to yield a white homogenous
99
suspension of multilamellar liposomes. Between the intervals, the suspension was
100
left to cool down in an ice bath for at least 2 min. Sonicated suspension was
101
centrifuged at 16000 g for 30 min and the supernatant containing the liposomes
102
was collected. The liposomes were stored under +4°C for 1 week before
103
experimental studies to increase the level of lipid hydroperoxides (about 5-10% of
104
the phosphatidyl choline). These will react with Fe3+ to produce free radicals.
105
Peroxidation of liposomes
106
Liposomes prepared 1 week prior to measurement were pipetted into a 96 well
107
transparent plate as 10 µL for each well and mixed with 50 µL of sample dilutions
108
or standard solution (Trolox), while MilliQ water was used as zero-sample. Sample
24
in order to remove water, alcohol and lipid soluble fractions.
and
26.
evaporated
25
according to the method
40 mg of phosphatidylcholine was dissolved in into
dryness
under
ACS Paragon Plus Environment
nitrogen
flow.
The
6
Page 7 of 27
Journal of Agricultural and Food Chemistry
109
dilutions were prepared as; 500, 100, 50 and 10 µM for pure antioxidant
110
compounds, 1/500, 1/1000, 1/5000, 1/10000 dilutions with water for beverages
111
and 10, 5, 1 and 0,5 mg per mL aqueous solutions for insoluble and whole grains.
112
Mixtures of insoluble grains with beverages/pure antioxidant solutions were
113
prepared by combining two different concentrations of grains (10 and 5 mg/mL)
114
with two different concentrations of beverages (1/500, 1/1000) or pure solutions
115
(500 and 100µM) at a ratio of 1:1 to give 50 µL sample. Since aldehydes generated
116
by lipid peroxidation can react with amines to produce fluorescent products,
117
glycine was added to the reaction medium as glycine ascorbate buffer (GAB),
118
which was prepared as 50 mM potassium phosphate buffer (PPB) with 100 mM
119
glycine and 450 µM ascorbate at pH 7.4, for 120 µL per well. Background wells
120
were made by pipetting GAB buffer instead of liposome and samples. Lipid
121
peroxidation was initiated by introducing 20 µL of an oxidizing agent comprised of
122
25 µM FeCl3 in 50 mM PPB (pH=7.4) with 1 mM ADP.
123
The plate was inserted into the plate reader of Tecan fluorometer immediately
124
after addition of oxidizing agent. The lag phase before the onset of oxidation was
125
measured for 4 h at 37° C (excitation at 360 nm, emission at 460 nm) in 10 min
126
intervals and samples were analyzed in triplicate.
127
Inhibition of lipid oxidation was calculated by the following formula:
128
Inhibition of lipid oxidation (%) = 100 – (AUCsample/AUCcontrol) x 100
129
where AUCsample is the area under the curve of sample containing antioxidant as
130
measured in well, and AUCcontrol is the area under the curve measured for the
131
control well. In order to avoid the differences originating from different starting
ACS Paragon Plus Environment
7
Journal of Agricultural and Food Chemistry
Page 8 of 27
132
levels of oxidation, all fluorescent values were subtracted the lowest value,
133
considering the lowest point as zero and the AUC was calculated by the trapezoid
134
rule.
135
Scavenging of Fremy’s salt radical
136
The scavenging of Fremy’s salt radical was determined with minor modifications
137
according to the method described by Rødjter et al. (2006)
138
whole grains, 10, 15 and 20 mg of sample was mixed with 1 mL of MilliQ water and
139
200 µL of a solution of Fremy’s salt (1.64 mM) in 25% saturated sodium carbonate
140
solution. For pure antioxidant solutions (10 µM) and beverages (1:1000 diluted)
141
100, 150 and 200 µL of sample was used instead of 10, 15 and 20 mg.
142
Combinations of insoluble grains with beverages/pure antioxidant solutions were
143
made in the ratio of 10 mg: 100 µL and then 100, 150 and 200 µL of sample from
144
the prepared mixture was added into the reaction medium as described for
145
beverages and pure solutions above. 6 min after the mixing, ESR spectra was
146
recorded with a MiniScope MS200 ESR spectrometer (Magnettech GmbH, Berlin,
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Germany). The measurements were carried out with the following settings: BO-
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field, 3366.90 G; sweep width, 49.82 G; sweep time, 60 sec; smooth, 0; steps, 4096;
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number, 1 pass; modulation, 1000 mG; Mw atten, 10 dB; gain, 2; E,1.
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Statistical Analysis
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The analytical data were reported as the mean ± standard deviation of duplicate
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independent measurements. The significance of mean differences was determined
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by Independent Samples t-test using SPSS version 17.0.
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For insoluble and
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RESULTS AND DISCUSSION
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Antioxidant activity measurement by ESR spectroscopy assay
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Table 1 gives relative decrease (%) in the intensity of measured and estimated
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electron spin resonance (ESR) signal values for the mixtures of insoluble grains
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and beverages rich in antioxidants. The antioxidant activities of samples were
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determined as their ability to reduce the stable Fremy’s salt radical as measured by
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ESR spectrometer. Diamagnetic reduction products are formed, which are ESR-
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invisible, when antioxidant compounds scavenge Fremy’s salt radicals, leading to a
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decrease in the ESR-signal
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signal was determined for insoluble grains, beverages and pure antioxidant
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compounds separately, and the decrease in the ESR signal for combinations of
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grains with beverages/pure antioxidant solutions were calculated by using these
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data. At the same time, the experiments were performed for combinations of
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grains with beverages/pure antioxidant solutions. Estimated data were obtained
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by summing up the halves of the percentage decreases (%) in the intensities of ESR
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signals separately measured for the concentrations/dilutions of insoluble grains
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and beverages/pure antioxidant compounds, which were used to make 1:1
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combination.
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In general, an almost linear relationship was observed between the mixture
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volume added to the reaction medium and estimated/measured values of decrease
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in the ESR signals. The measured values of decrease in the intensity of ESR signals
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were significantly higher than the estimated ones for combinations of almost all
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insoluble grains with all beverages used, pointing toward a clear synergistic effect
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The percentage decrease in the intensity of ESR
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(p
