Ellagitannins from Strawberries with Different Degrees of

Nov 16, 2017 - The present paper describes a comparative study of the metabolism of (1) ellagic acid, (2) monomeric ellagitannins and (3) dimeric ella...
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Ellagitannins from Strawberry with Different Degree of Polymerization Showed Different Metabolism Through Gastrointestinal Tract of Rats Joanna Milala, Monika Kosmala, El#bieta Karli#ska, Jerzy Juskiewicz, Zenon Zdunczyk, and Bartosz Fotschki J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04120 • Publication Date (Web): 16 Nov 2017 Downloaded from http://pubs.acs.org on November 19, 2017

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Journal of Agricultural and Food Chemistry

Ellagitannins

from

Polymerization

Strawberry

Showed

with

Different

Different Metabolism

Degree

of

Through

Gastrointestinal Tract of Rats Joanna Milala,a* Monika Kosmala,a Elżbieta Karlińska,a , Jerzy Juśkiewicz,b Zenon Zduńczyk,b Bartosz Fotschkib a

Institute of Food Technology and Analysis, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Lodz, Poland

b

Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Division of Food Science, Tuwima 10, 10-748 Olsztyn, Poland

*

Corresponding

author,

(Tel:

+48

426312780;

Fax:

+48

426367488:

E-mail:

[email protected])

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Abstract

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The present paper describes a comparative study of the metabolism of 1) ellagic acid, 2)

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monomeric ellagitannins – a mixture of α- and β-bis-hexahydroxydiphenoyl-D-glucose, and

4

3) dimeric ellagitannins – mainly agrimoniin with both glucose residues being esterified with

5

hexahydroxydiphenoyl (HHDP), in rats fed polyphenol-rich diets. Their metabolites were

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identified and quantified in selected parts of the gastrointestinal tract i.e., the stomach, small

7

intestine, and cecum on the 2nd, 4th, and 7th day of the experiment, as well as in the feces,

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blood serum, and urine of rats. Significant differences between the metabolites of strawberry

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ellagitannins and ellagic acid were observed in all parts of the gastrointestinal tract. Urolithin

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A was the predominant polyphenolic metabolite of rats fed a diet supplemented with ellagic

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acid. On the other hands, in rats fed low DP ellagitannins, the main metabolite was nasutin

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followed by urolithin A, while ellagitannins with a higher DP led to nasutin only.

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KEYWORDS: ellagitannins, nasutin, urolithin, rat, agrimoniin, strawberry

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Introduction

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Epidemiological studies have shown that the consumption of fruits and vegetables reduces the

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risk of lifestyle diseases such as atherosclerosis, cancer, and neurodegenerative disorders. 1,2,3

18

1

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cholesterol, low-density lipoprotein cholesterol, and triglyceride levels4, which is largely

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attributable to the activity of polyphenols. Special attention has been attracted by ellagitannins

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(ETs), which are the most abundant strawberry polyphenols, along with anthocyanins and

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flavanols 5 In the study of Juśkiewicz et al .6 strawberry ET preparations were shown to lower

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lipemia and glycaemia.

Berry consumption beneficially modifies the lipid profile by significantly decreasing total

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Ellagitannins can be defined as hexahydroxydifenoyl esters of carbohydrates of

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cylitols as well as compounds derived from further oxidative transformations including

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oligomerization processes.

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differentiated in terms of the degree of polymerization (DP) and susceptibility to hydrolysis.

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The elementary building blocks such as ellagic acid and HHDP may exist as free compounds

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or may be generated as a result of hydrolysis of larger molecules. Ellagic acid is a product of

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spontaneous lactonization within HHDP acid molecule.

9

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agrimoniin,

two

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hexahydroxydiphenoyl-D-glucose units linked by a C-O-C bond between two gallic acid

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residues.7,10 Raspberries and blackberries primarily contain sanguiin H-6 and lambertianin C,

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the monomeric units of which are linked with a sanguisorboyl group.7,11 These structural

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differences may affect the physiological activity of polyphenols from Rosaceae fruits. 12

a

7,8

GOG-type

ETs constitute a heterogeneous group of compounds

dimer

composed

of

The main strawberry ET is α-1-O-galloyl-2,3:4,6-bis-

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Polyphenols are known as antioxidants, but have a much wider spectrum of activity,

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especially considering their low bioavailability as compared to endogenous antioxidants1 like

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glutathione, alpha-lipoic acid, coenzyme Q, ferritin, uric acid, bilirubin. First of all, it must be

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remembered that large polyphenolic molecules cannot enter the bloodstream, in contrast to

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some simple polyphenols which are absorbed directly. The majority of ETs are hydrolyzed by

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colonic microorganisms, first into ellagic acid, and then into even smaller molecules, such as

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urolithins. The pattern of urolithin formation seems to be as follows: after HHDP acid is

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released form ETs and converted into poly-hydroxylated dibenzopyranone, and then

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3,4,8,9,10-pentahydroxy-6H-dibenzo[b,d]pyran-6-one (Uro-M5). Subsequently, sequential

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dehydroxylation leads to tetrahydroxy- (Uro-M6 and Uro-D), trihydroxy- (Uro-M7 and Uro-

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C), dihydroxy- (Uro-A and IsoUro-A) and monohydroxy- (Uro-B) dibenzopyranones. These

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metabolites may be absorbed into the bloodstream and eventually accumulate in the urine in

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the form of glucuronides and sulfate conjugates; they can be also excreted in the

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feces.13,14,15,16,17,18 A fully validated methodology for urolithin determination in terms of

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linearity, sensitivity, precision, recovery, matrix effect, selectivity, stability in different

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biological samples and quantification was developed by García-Villalba et al.18, even though

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not all metabolites are commercially available.

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Depending on the host’s species and individual characteristics, the resulting

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metabolites may differ.19 20 The significance of polyphenolic metabolites, such as urolithins, is

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not fully understood. It is known that they have antioxidant properties. Urolithins (especially

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A and C) were very active towards O2 •− direct scavenging, significantly inhibited neutrophil

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oxidative burst (stronger than ascorbic acid) 21.and/or activate phase II enzymes, which are

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involved in antioxidant functions or detoxification (thioredoxin reductase-1 or glutathione

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peroxidases)1, show anti-inflammatory activity, anti-estrogenic/estrogenic capacity due to

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structural analogy to estrogens, and anticancer effects.21,22,23,24 On the other hand, non-

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absorbable large compounds may influence the colonic microbiota with the result of

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stimulating the activity of some strains while inhibiting others.22 In our previous study, a

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strawberry polyphenolic extract improved the prebiotic effects of a fructooligosaccharide

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(FOS) diet, beneficially lowering cecal digesta pH and reducing putrefactive short-chain fatty

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acid production. On the other hand, the addition of dietary FOS enhanced the metabolism of

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the examined strawberry extracts in the cecum, thereby increasing the concentrations of

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polyphenolic metabolites in the cecal digesta and urine.25 Our subsequent study26 showed that

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the DP influenced the effectiveness of blood glucose reduction by polyphenolics, with

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monomers more readily mitigating sugar-induced postprandial glycemic loads. Furthermore,

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it was found that ETs contained in a monomeric ET-rich extract were more prone to intestinal

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breakdown in the cecum than those present in a dimeric ET-rich extract, and absorption of

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their metabolites could be increased by dietary FOS; together, they elicited strong

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antibacterial activity. 27 Interestingly, in previous investigations, nasutin was identified as one

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of ET metabolites in rats.12,27,28 This compound was previously found in the feces of some

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wild rodents, including beavers, and termites.13,19,29

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As molecular structure is important to the physiological function of ETs, the aim of

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this study was to identify and quantify the metabolites of ellagic acid and strawberry

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polyphenol preparations with monomeric (EM) and dimeric (ED) ellagitannins (Figure 1) in

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selected parts of the gastrointestinal tract of rats, i.e., the stomach, small intestine, and cecum,

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as well as in rat feces, blood serum, and urine.

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Methods

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Preparation of ET-rich strawberry extracts

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ET extracts were obtained from strawberry fruit pomace, a by-product in the manufacture of

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concentrated strawberry juice (ALPEX Co., Łęczeszyce, Poland), as described by Jurgoński

86

et al.27 In brief, fresh pomace was dried in an industrial vacuum dryer at 70±2°C and passed

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through sieves. The seedless fraction was subjected to two-step extraction with 60% aqueous

88

solution of acetone. Next, after partial removal of the solvent via distillation, the resultant

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solutions were transferred onto a column packed with polymeric resin (Amberlite XAD 16,

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Sigma-Aldrich, Poznan, Poland). Sugars and other water-soluble compounds present in the

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solutions were eluted from the column with water. Then, fractions rich in monomeric and

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dimeric ETs were desorbed with 10% and 40% aqueous solution of ethanol, respectively,

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concentrated to approx. 15% dry matter, and lyophilized (Table 1).

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Analytical methods

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Basic composition. AOAC methods 30 were used to determine dry matter and ash (940.26),

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protein (920.152), crude fat (930.09), total dietary fiber (985.29), and insoluble dietary fiber

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(991.42).

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Polyphenols: HPLC analysis. The concentration of ETs, ellagic acid, anthocyanins, and

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flavonols was determined in extracts diluted with methanol (1 mg/mL) using a Knauer

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Smartline HPLC system with a photodiode array detector (Berlin, Germany) coupled with a

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Gemini C18 column (110 Å, 250×4.60 mm; 5 µm, Phenomenex, Torrance, USA). The

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detailed separation and detection conditions are described elsewhere Fotschki et al.

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following standards were used for the identification of polyphenols: ellagic acid, flavonols

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(quercetin-3-O-glucoside, kaempferol-3-O-glucoside, quercetin, kaempferol, tiliroside),

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pelargonidin-3-O-glucoside (all from Extrasynthese, Genay, France), p-coumaric acid

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(Sigma-Aldrich), as well as ETs (bis-hexahydroxydiphenoyl-D-glucose and agrimoniin),

107

obtained by semi-preparative HPLC as described by Sójka et al. 10

25

. The

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The concentration of proanthocyanidins in the extracts was determined by HPLC

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following their breakdown in an acidic environment with excess phloroglucinol according to

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Kennedy and Jones (2001). The obtained products were separated using a Knauer Smartline

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chromatograph (Berlin, Germany) equipped with a UV–Vis detector (PDA 280, Knauer,

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Berlin Germany) and a fluorescence detector (Shimadzu RF-10Axl, Kyoto, Japan) coupled

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with a Gemini C18 column (110 Å, 250×4.60 mm; 5 µm, Phenomenex, Torrance, USA). The

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separation conditions were as described by Kosmala et al.12 Identification was done at 280 nm

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using a UV–Vis detector and the following standards: (–)-epicatechin, (+)-catechin, and (–)-

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epigallocatechin, and their respective phloroglucinol adducts. Phloroglucinol adducts were

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previously synthetized and isolated from corresponding standard. Quantification was carried

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out by measuring peak areas recorded by the fluorescence detector (excitation wavelength:

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278 nm; emission wavelength: 360 nm). The breakdown products were quantified using

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standard

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(–)-epicatechin-phloroglucinol adduct for extender units.

curves

of

(–)-epicatechin

and

(+)-catechin

for

terminal

units

and

122 123

High Performance Liquid Chromatography – Electrospray Ionization – Mass Spectrometry

124

(HPLC-ESI-MS).

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Ellagitannins were confirmed by HPLC-ESI-MS using a Dionex UltiMate 3000 UHPLC and

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a Thermo Scientific Q Exactive series quadrupole ion trap mass spectrometer (Jurgoński et

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al., 2015). A Phenomenex Luna 5 µm C18 (250 × 4.6 mm) column was used with a binary

128

gradient of 1% formic acid as mobile phase A and water/acetonitrile (1:4 v/v) as mobile phase

129

B at a flow rate of 1.0 mL/min. The gradient program was 5% of mobile phase B for the first

130

6.5 min, 5–15% B from 6.5 to 12.5 min, 15–45% B from 12.5 to 44 min, 45–75% B from 44

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to 45 min, isocratic conditions at 75% B from 45 to 50 min, 75–5% B from 50 to 52 min, and

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column equilibration at 5% B from 52 to 65 min. Mass spectrometry analysis was performed

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in negative ion mode under the following conditions: capillary voltage +4 kV, sheath gas

134

pressure 75 au (arbitrary units), auxiliary gas at 17 au, and scan range from 200 to 2000 m/z.

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In vivo experiments

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Animals and diets. Experiments were conducted on 30 male Wistar rats aged approx. 4 weeks,

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randomly assigned to one of three groups of ten rats each. The animals were maintained

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individually in metabolic cages at a stable temperature (21–22°C), a 12 h light:12 h dark

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cycle, and a ventilation rate of 20 air changes per hour. The rats were used in compliance with

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the European guidelines for the care and use of laboratory animals (EU Directive

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2010/63/EU), and the animal protocol was approved by the Local Institutional Animal Care

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and Use Committee (permission number 32/2012). Individual body weight and food intake

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data were recorded. Samples of intestinal digesta, feces, urine, and blood were collected after

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2, 4 and 7 days of experimental feeding, from 3 rats each time. All urine and feces for 24 h

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were collected to measure how much of ET and ellagic acid metabolites was produced during

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1 day. The rats had free access to tap water and semipurified diets, which were prepared and

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then stored at 4°C in sealed containers until the end of the experiment. The diets were

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modifications of a casein diet for laboratory rodents recommended by the American Institute

150

of Nutrition

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achieve a final concentration 0.1% of ellagitannins or ellagic acid. The composition of

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experimental diets is summarized in Table 3. All diets contained 3% FOS to mitigate any

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potential negative effect of polyphenols on the fermentative function of the large gut

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microbiota 32. Three individuals from each experimental group fed polyphenol-rich diets were

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anesthetized after 2 days, another 3 after 4 days, and the rest after 7 days in order to collect

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intestinal digesta and blood samples. Three rats fed a control diet without polyphenols were

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sacrificed after 7 days of the experiment. Urine was collected after 1, 2, 4, and 7 days, and

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feces were collected after 1, 4, and 7 days. Upon the termination of the experiment, the rats

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were weighed and anesthetized with 50 mg sodium pentobarbital/kg body weight (they were

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not deprived of feed prior to anesthesia). Following laparotomy, blood samples were collected

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from the caudal vein and serum was prepared by solidification and low-speed centrifugation

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(350×g, 10 min, 4°C). Serum samples were kept frozen at -70°C until assayed. Selected

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intestinal segments (stomach, small intestine, cecum) were removed, and samples of digesta

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were collected.

31

.Polyphenolic preparations were added to the diets in such proportions as to

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Free ellagic acid and ET metabolites in stomach, small intestinal, and cecal digesta and

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feces. Extraction of free ellagic acid and ET metabolites was carried out using 100% and 70%

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acetone. To 500 mg of digesta or feces 2 mL of 100% acetone was added, mixed, sonicated

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for 10 min, and centrifuged (800 rad/s). The procedure was repeated twice with 1.5 mL of

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70% acetone and the extracts were combined, evaporated to dryness under vacuum, dissolved

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in 1 mL of 100% methanol and analyzed with HPLC. For HPLC parameters see Polyphenols:

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HPLC analysis. Detection was performed at 360 nm. Standards used were: Ellagic acid,

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Urolithin A, Urolithin B (Sigma Aldrich, Steinheim, Germany). Urolithin A glucuronide was

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prepared as described by Cerdá et al. (2004).18 The raw material for the isolation of urolithin

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A glucuronide was two liters of urine from three healthy volunteers (25−45 years old) who

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consumed 400 g of strawberry per day, as described in Fotschki et al. (2014).Nasutin was

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isolated from rats fed ellagitannin preparation faeces in the same manner. Identification of

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metabolites was confirmed by HPLC-MS and compared with literature.

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the ellagitannin metabolites was presented in Table 2.

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Urine samples. Urine (2–4 mL) was acidified with 0.25 mL of 1 mol/L H3PO4 and was

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applied to a 60 mg SPE Strata X-33 column conditioned according to the manufacturer’s

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instructions (Phenomenex, Torrance, USA). The column was washed with 3 mL of water, 3

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mL of 0.1 mol/L acetate buffer, and 6 mL of water, and dried. The analyte was eluted with

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100% methanol (2 × 0.5 mL) and analyzed by UHPLC-MS.

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The metabolites were determined by HPLC-ESI-MS using a Dionex UltiMate 3000 UHPLC

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and a Thermo Scientific Q Exactive series quadrupole ion trap mass spectrometer. The

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column was a Kinetex 2.6 µ C 18 100 Å; 150×2.1 mm (Phenomenex, Torrance, CA, USA)

187

kept at 35°C. A binary gradient of 0.1% formic acid as mobile phase A and 0.1% formic acid

188

in acetonitrile as mobile phase B was used at a flow rate of 0.5 mL/min. The gradient program

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was 5% of mobile phase B for the first 1.44 min, 5–15% B from 1.44 to 2.98 min, 15–40% B

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Recovery of

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from 2.98 to 10.1 min, 40–73% B from 10.1 to 11.5 min, isocratic conditions at 73% B from

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11.55 to 12.7 min, 73–5% B from 12.7 to 13.28 min, and column equilibration at 5% B from

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13.28 to 18 min. Mass spectrometry analysis was performed in negative ion mode under the

193

following conditions: capillary voltage +4 kV, sheath gas pressure 60 au (arbitrary units),

194

auxiliary gas 10 au, and scan range from 120 to 1200 m/z.

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Blood serum samples. To 0.5 mL serum 1 mL 100% acetone was added, mixed, sonicated for

196

10 min, and centrifuged (900 rad/s). The procedure was repeated with 1.0 mL of 100%

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acetone and the extracts were combined and evaporated to dryness under vacuum in a

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ScanSpeed 40 low speed centifuge (Labogene, Denmark), lyophilized in an Alpha 1-2 LD

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plus freeze dryer (Germany), solubilized in 0.2 mL of 100% methanol, and then analyzed by

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UHPLC-MS (parameters as for urine).

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Statistical analysis. The results are expressed as means and pooled SEM. Two-way analysis

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of variance (ANOVA) was used to evaluate the effects of polyphenolic supplementation (S;

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extracts rich in monomeric or dimeric ETs, and ellagic acid), feeding duration (D; 2, 4, 7 days

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for gastrointestinal digesta and feces and blood samples and 1, 2, 4, 7 days for urine samples),

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and interactions between these two factors (S × D). If analysis revealed a significant

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interaction (P≤0.05), differences between the respective study groups were determined with

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Duncan’s post hoc test at P≤0.05. Statistical analysis was performed using STATISTICA

208

software, version 10.0 (StatSoft Corp., Cracow, Poland).

209 210

Results

211 212

The preparations used in the experiment were characterized by a high content of polyphenols,

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more than 95%, mostly ellagitannins. Both preparations, EM and ED, contained ellagitannins

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typical for strawberry fruits.5,6,7,10,12 EM was characterized by a high quantity of monomeric

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ellagitannins, 67%, while ED was characterized by a high quantity of dimeric ellagitannins,

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mostly agriimonin. Structures are presented in Figure 1. The remaining polyphenols were

217

proanthocyanidins, 27 and 11% respectively, which are difficult to separate. Both

218

preparations were deprived of other groups of polyphenols like anthocyanins or flavonols

219

(Table 1).

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The highest concentration of ellagic acid in stomach digesta was found in rats fed a diet

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supplemented with ellagic acid (EA treatment) with the difference being statistically

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significant (P