Synergism between Soluble and Dietary Fiber Bound Antioxidants

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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

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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

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ABSTRACT

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This study investigates the synergism between antioxidants bound to dietary

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fibers (DF) of grains and soluble antioxidants of high-consumed beverages or their

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pure antioxidants. The interaction between insoluble fractions of grains containing

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bound antioxidants and soluble antioxidants was investigated using; (i) liposome

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based system by measuring the lag phase before the onset of oxidation, (ii) ESR

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based system by measuring the reduction percentage of Fremy’s salt radical. In

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both procedures, antioxidant capacities of DF-bound and soluble antioxidants

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were measured as well as their combinations, which were prepared for different

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ratios. The simple addition effects of DF bound and soluble antioxidants were

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compared with measured values. The results revealed a clear synergism for almost

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all combinations in both liposome and ESR based systems. The synergism

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observed in DF bound-soluble antioxidant system paints a promising picture

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considering their role in human gastrointestinal (GI) tract health.

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Keywords: Synergism, free antioxidants, bound antioxidants, dietary fibers, cereal

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grains

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INTRODUCTION

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Grains such as wheat, oat and rye are the staple elements of the human being’s

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daily diet, contributing about 50% of dietary fiber (DF) intake

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carrier of dietary antioxidants 3. Epidemiological studies report that there is an

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inverse relationship between whole grain consumption and the incidence of

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cardiovascular diseases (CVD) 4, type 2 diabetes 5, obesity

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cancers 7. In the meantime, studies focused on the origin of the health benefits of

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whole grains in terms of its constituents, namely germ, bran and endosperm

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showed that bran is the key factor

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insoluble DF is found as associated with a substantial amount of dietary

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antioxidants, which can be named as DF-antioxidants 12.

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DF-antioxidants can contribute to the health effects attributed to DF as well as to

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dietary antioxidants 3; like protection against CVD

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diabetes, neurodegenerative diseases, hypertension 14,15,16,17 and regulation of the

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insulin level in blood after food ingestion

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significant portion of dietary antioxidants and should not be counted as the minor

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constituents of DF 3.

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Since dietary fiber is non-digestible in the upper intestine, it reaches to colon

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almost in intact form with the antioxidants still bound to it. These antioxidants are

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released from the fiber matrix slowly in a continuous manner during the

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considerable survival period of insoluble DF in GI tract by the help of the colon

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microbiota

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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


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19,

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hours

antioxidants bound to DF can create a continuous and healthier

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antioxidant environment in GI tract 12.

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At this point, providing the continuity and improving the quality of the created

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antioxidant environment becomes an attractive issue. As known, for some

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combinations of antioxidants, a larger overall effect is observed compared to the

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simple addition of the individual effects, which is termed as synergism 20. Several

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studies have shown that plant polyphenols exert a synergistic behavior with other

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antioxidants present in plant material

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bound antioxidants of grains and the free soluble antioxidants that are frequently

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consumed together in the daily human diet; it will help to improve the quality of

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the healthy antioxidant environment in the GI tract, partly by a mechanism

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depending on regeneration of the DF antioxidants by the soluble antioxidants.

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Accordingly, the objective of the present study was to determine if the

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combination of insoluble antioxidants of grains and soluble antioxidants from

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different sources (frequently consumed antioxidant rich beverages, pure

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antioxidant compounds) are synergistically or antagonistically affecting the

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antioxidant capacity using two different well-established experimental designs; (i)

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a liposome based system by measuring the lag phase before the onset of oxidation

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spectrophotometrically, (ii) an ESR based system by measuring the percentage of

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reduction in a solution of Fremy’s salt as a semi-stable radical by antioxidant

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compound. In these two different designs, oxidation is determined at two different

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stages: ESR functions at the radical scavenging stage, while liposomes at the

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formation of lipid oxidation products i.e. the aldehydes, which create the

21,22,23.

If such interaction exist among DF


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fluorescence.

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MATERIALS AND METHODS

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Chemicals

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Potassium

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phosphatidylcholine from soybean (99%), dipotassium phosphate, sodium

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phosphate, (-) epicatechin, (-) – epigallocatechin, (-) – epicatechin gallate, (-) –

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epigallocatechin

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tetramethylchroman-2 carboxylic acid (Trolox), iron(III) chloride, adenosine 5’ –

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diphosphate sodium salt (ADP), methanol, chloroform, hexane and ethanol were

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purchased from Sigma-Aldrich Chemie (Steinheim, Germany). L (+) ascorbic acid,

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monopotassium phosphate and glycine were purchased from Merck (Darmstadt,

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Germany). All solvents used were of analytical grade, unless otherwise stated.

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Water was purified through a Millipore Q-plus purification train (Millipore Corp.,

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Bedford, MA, USA).

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Grains and Beverages

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All grain varieties used were purchased from a local market in Turkey, Ankara.

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Green tea, pomegranate juice, roasted coffee beans and red wine were of food

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grade and purchased in Copenhagen, Denmark.

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brewing 3 g of green tea with 100 mL of hot water for 15 min and espresso was

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prepared in a kitchen type coffee-machine, Titanium Gaggia, at the 7th level of the

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dose control knob.

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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


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Ground whole grains were washed according to the procedure described by Çelik

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et al. (2013)

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Washed grains were freeze-dried, ground with a ceramic mortar to obtain fine

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powder form and passed through a sieve (Endecotts Test Sieve, London, UK) of 40

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mesh size (425 μm). The final insoluble powder was tested and found to be free of

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soluble antioxidant compounds. It was kept under 4°C prior to experiments.

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Preparation of liposomes

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Liposomes were prepared with minor modifications

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described by Tirmenstein

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chloroform

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phosphatidylcholine residue was subsequently re-hydrated with 5 mL of 50 mM

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sodium phosphate buffer (PB) (pH=7.4) and vortexed. The suspension was

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sonicated at 100W with 30 sec intervals 10 times to yield a white homogenous

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suspension of multilamellar liposomes. Between the intervals, the suspension was

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left to cool down in an ice bath for at least 2 min. Sonicated suspension was

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centrifuged at 16000 g for 30 min and the supernatant containing the liposomes

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was collected. The liposomes were stored under +4°C for 1 week before

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experimental studies to increase the level of lipid hydroperoxides (about 5-10% of

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the phosphatidyl choline). These will react with Fe3+ to produce free radicals.

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Peroxidation of liposomes

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Liposomes prepared 1 week prior to measurement were pipetted into a 96 well

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transparent plate as 10 µL for each well and mixed with 50 µL of sample dilutions

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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


nitrogen

flow.

The

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dilutions were prepared as; 500, 100, 50 and 10 µM for pure antioxidant

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compounds, 1/500, 1/1000, 1/5000, 1/10000 dilutions with water for beverages

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and 10, 5, 1 and 0,5 mg per mL aqueous solutions for insoluble and whole grains.

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Mixtures of insoluble grains with beverages/pure antioxidant solutions were

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prepared by combining two different concentrations of grains (10 and 5 mg/mL)

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with two different concentrations of beverages (1/500, 1/1000) or pure solutions

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(500 and 100µM) at a ratio of 1:1 to give 50 µL sample. Since aldehydes generated

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by lipid peroxidation can react with amines to produce fluorescent products,

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glycine was added to the reaction medium as glycine ascorbate buffer (GAB),

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which was prepared as 50 mM potassium phosphate buffer (PPB) with 100 mM

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glycine and 450 µM ascorbate at pH 7.4, for 120 µL per well. Background wells

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were made by pipetting GAB buffer instead of liposome and samples. Lipid

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peroxidation was initiated by introducing 20 µL of an oxidizing agent comprised of

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25 µM FeCl3 in 50 mM PPB (pH=7.4) with 1 mM ADP.

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The plate was inserted into the plate reader of Tecan fluorometer immediately

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after addition of oxidizing agent. The lag phase before the onset of oxidation was

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measured for 4 h at 37° C (excitation at 360 nm, emission at 460 nm) in 10 min

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intervals and samples were analyzed in triplicate.

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Inhibition of lipid oxidation was calculated by the following formula:

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Inhibition of lipid oxidation (%) = 100 – (AUCsample/AUCcontrol) x 100

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where AUCsample is the area under the curve of sample containing antioxidant as

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measured in well, and AUCcontrol is the area under the curve measured for the

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control well. In order to avoid the differences originating from different starting


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levels of oxidation, all fluorescent values were subtracted the lowest value,

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considering the lowest point as zero and the AUC was calculated by the trapezoid

134

rule.

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Scavenging of Fremy’s salt radical

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The scavenging of Fremy’s salt radical was determined with minor modifications

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according to the method described by Rødjter et al. (2006)

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whole grains, 10, 15 and 20 mg of sample was mixed with 1 mL of MilliQ water and

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200 µL of a solution of Fremy’s salt (1.64 mM) in 25% saturated sodium carbonate

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solution. For pure antioxidant solutions (10 µM) and beverages (1:1000 diluted)

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100, 150 and 200 µL of sample was used instead of 10, 15 and 20 mg.

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Combinations of insoluble grains with beverages/pure antioxidant solutions were

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made in the ratio of 10 mg: 100 µL and then 100, 150 and 200 µL of sample from

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the prepared mixture was added into the reaction medium as described for

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beverages and pure solutions above. 6 min after the mixing, ESR spectra was

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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.


27.

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

Synergism between Soluble and Dietary Fiber Bound Antioxidants

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