Strawberry Ellagitannins Thwarted the Positive Effects of Dietary

Jun 4, 2014 - Institute of Chemical Technology of Food, Technical University of Łódź, Łódź, Poland. J. Agric. Food Chem. , 2014, 62 (25), ... El...
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Strawberry Ellagitannins Thwarted the Positive Effects of Dietary Fructooligosaccharides in Rat Cecum Bartosz Fotschki,*,† Joanna Milala,‡ Adam Jurgoński,† Elzḃ ieta Karlińska,‡ Zenon Zduńczyk,† and Jerzy Juśkiewicz† †

Division of Food Science, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland Institute of Chemical Technology of Food, Technical University of Łódź, Łódź, Poland



S Supporting Information *

ABSTRACT: Forty-eight male Wistar rats were fed diets containing low (0.051% of diet) or high (0.153% of diet) levels of an ellagitannin-rich (ET) strawberry extract with dietary fructooligosaccharides (FOS) or cellulose (CEL) for 4 weeks. The in vivo study demonstrated that some positive changes in the cecal metabolism resulting from the ingestion of a diet enriched only with FOS were completely or slightly suppressed by the dietary ET. In particular, the pH value (7.21 vs 7.36), short-chain fatty acid production (41.2 vs 30.0 μmol/100g BW), and β-glucuronidase activity (20.2 vs 15.7 μmol/h/g) in the cecum of rats fed with FOS were affected upon the addition of the ET extract. Dietary FOS caused higher metabolism of the tested ET strawberry extract in the gastrointestinal tract of rats. Moreover, the systemic effect of the supplements when consumed together showed undesired serum HDL-cholesterol decrease (0.78 vs 1.02 mmol/L in the treatment with FOS only). KEYWORDS: strawberry extract, fructooligosaccharides, gastrointestinal tract, nasutin A, Wistar rats



transitional forms. Some of the first forms are nasutin A and isonasutin derivatives, which are formed after removing the specific hydroxyls in the 3- and 3′-positions or in the 4- and 3′positions of EA. Another form is urolithin, which is produced after opening the lactone rings in EA.14,15 Urolithins easily pass into intestine−liver circulation, where they are glucuronidated and sulfated. Derivatives of urolithins that are transported in the blood may have a favorable effect on the levels and proportions of cholesterol fractions, the blood level of lipids, and vascular inflammation. In addition, some of the derivatives have proven biological activity, including as analogues of hormones.9,14,16,17 Urolithins can remain in the body up to 4 days after the consumption of ET-containing foods, perhaps due to enterohepatic recirculation and slow microbial metabolism in the colon.18 The metabolism of ETs depends on the activity of individual colonic microflora.18 Therefore, it can be assumed that high activity of the gastrointestinal microbiota will increase metabolism and absorption of the ETs derived from the diet. Well-known dietary ingredients that modulate microbiota are classified as nondigestible saccharides as part of dietary fiber, such as CEL and FOS. CEL has significantly lower susceptibility to fermentation processes in the hindgut than FOS, which has been shown to increase the production of short-chain fatty acids (SCFA) and to have a prebiotic effect.5 In our earlier studies performed with a polyphenol concentrate, the addition of dietary FOS significantly enhanced the metabolic activity of quercetin.6

INTRODUCTION “Fructan” is a general term used for naturally occurring plant oligo- and polysaccharides. As a type of fructan, FOS are plantderived carbohydrates with well-established benefits for the modulation of gastrointestinal functions.1 Currently, a number of studies have shown beneficial effects of the consumption of colorful fruits and vegetables that are rich in FOS and biologically active phytochemicals.2,3 Dietary fiber including polyphenol-rich fiber complexes is resistant to endogenous enzymes of the upper part of the gut in humans as well as monogastric animals. Therefore, the compounds are transported in large quantities to the large intestine, where they undergo fermentation process.1,4 In recent years, scientists have studied the impact of dietary fiber on the metabolism and absorption of phytochemicals.5,6 However, there is little information regarding how phytochemicals may modulate the beneficial effects of FOS in the gastrointestinal tract. An interesting source of phytochemicals, especially ellagic acid (EA) and anthocyanins, is the strawberry.7 EA in this fruit occurs mainly in the form of macromolecular ET, whereas the free form of EA represents only a small proportion of the total content of its derivatives.8 Numerous studies have demonstrated positive effects of ETs and EA on human health, primarily due to the antioxidant, anticarcinogenic, antineurodegenerative, and anti-inflammatory effects.9,10 However, an increased content of polyphenols in the diet may inhibit the growth and reduce the abundance of gastrointestinal microflora.11 ET polymers in the gastrointestinal tract can be absorbed after breaking the ester bonds, which leads to the formation of EA. EA absorption is very low, and the unabsorbed compounds released into the jejunum and colon are extensively metabolized to urolithins by specific microbiota.12,13 The EA metabolism pathway is composed of many © 2014 American Chemical Society

Received: Revised: Accepted: Published: 5871

December 23, 2013 May 5, 2014 June 4, 2014 June 4, 2014 dx.doi.org/10.1021/jf405612a | J. Agric. Food Chem. 2014, 62, 5871−5880

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subjected to freeze-drying in a TG 5 Lyophilizer (VEB Hochvakuum Dresden, Germany). The yield of the dry weight of the preparation that was rich in polyphenols was 2.6% of the weight of the dried industrial, seedless strawberry pomace. Chemical Analyses of the Polyphenolic Extract. The chemical composition of the preparations was determined by AOAC methods (2005)19 using the following procedure: dry matter and ash content, 940.26; protein content, 920.152; crude fat, 930.09; total dietary fiber, 985.29. Sucrose, fructose, glucose, and other saccharides were measured after desalination of the strawberry extract solution in water (5 mg/mL) on an anion exchange column (two parts of Amberlite IRA 400 anion exchanger purchased from Sigma-Aldrich (Germany), one part of Amberlite IR 120 cation exchanger also purchased from Sigma-Aldrich (Germany)). The analysis was performed using an HPLC system (Knauer, Berlin, Germany) with an RI detector and a BIO-RAD Aminex HPX-87C column, 300 × 7.8 mm (Phenomenex, Torrance, CA, USA), with a flow rate of 0.5 mL/ min at 85 °C. Sucrose (limit detection at 0.08 mg/mL), glucose (limit detection at 0.08 mg/mL), and fructose (limit detection at 0.04 mg/ mL) were used as standards. Determination of Polyphenols. The concentrations of EA and total ETs, such as agrimoniin, including agrimoniin alone and other polyphenols (anthocyanins and flavonols), were determined in 50% methanol solutions prepared by resolving 5−25 mg of the freeze-dried polyphenolic extract in 5 mL of methanol diluted in water to 10 mL according to Sójka et al. (2013).20 The solution was then subjected to HPLC analysis. A Smartline (Knauer, Berlin, Germany) chromatograph equipped with degasser Smartline Manager 5000 unit, two pumps Smartline Pump 1000, mixing chamber, autosampler Smartline Autosampler 3950, column oven Jetstream 2 Plus, and detector Smartline PDA 2800 was used for the chromatographic analysis. The separation was conducted on a Phenomenex Gemini 5u C18 110A column (250 × 4.60 mm; 5 μm) (Phenomenex Torrance, CA, USA). The column was maintained at 35 °C. Eluent A contained 0.05% phosphoric acid in water, and eluent B contained 0.05% phosphoric acid in acetonitrile. The flow rate was 1.25 mL/min. The gradient program was as follows: 0−5 min, 4% eluent B; 5−12.5 min, 4−15% eluent B; 12.5−42.5 min, 15−40% eluent B; 42.5−51.8 min, 40−50% eluent B; 51.8−53.4 min, 50−55% eluent B; and 53.4−55 min, 4% eluent B. The volume of the injected sample was 20 μL. The detection conditions were as follows: 280 nm [p-coumaric acid, kaempferol-3-Oβ-D-(6″-E-p-coumaroyl)-glucopyranoside, (+)-catechin agrimoniin], 360 nm (EA, quercetin, and kaempferol glycosides, quercetin, kaempferol), and 520 nm (anthocyanins). The data were registered by ClarityChrom (Knauer, Berlin, Germany) chromatography software. The following standards were used: EA, p-coumaric acid, and (+)-catechin (Sigma-Aldrich, St. Louis, USA) and quercetin-3-Oglucopyranoside, kaempferol-3-O-glucoside, quercetin, pelargonidin-3O-glucoside, and kaempferol (Extrasynthese, Genay, France). The agrimoniin standard was obtained from strawberry press cake and was purified by preparative chromatography. The agrimoniin with limit detection at 0.55 mg/L was used as the basic standard for the ET content determination. Analysis of Proanthocyanidins and Free Catechins. A proanthocyanidin degradation method in an acidic environment with an overdose of phloroglucinol was employed. The method was described by Kennedy and Jones (2001).21 Approximately 20 mg of sample was weighed in a 2 mL Eppendorf tube, and 800 μL of a methanol solution containing phloroglucinol (75 g/L) and ascorbic acid (15 g/L) was added to the sample. Phloroglucinolysis was started by adding 400 μL of 0.2 mol/L hydrochloric acid to methanol. The incubation was conducted for 30 min at 50 °C. Next, the samples were instantly cooled in an ice bath, and the reaction was stopped by adding 600 μL of a 40 mmol/L sodium acetate solution. The samples were centrifuged for 5 min at 3600g and then diluted with a 40 mmol/L sodium acetate solution. The products of the acidic degradation of proanthocyanidins were separated with the Knauer Smartline chromatograph (Berlin, Germany) equipped with a UV−vis P2800 detector (Knauer, Berlin, Germany) and a fluorescent detector (FD) RF-10AXL (Schimadzu, Tokyo, Japan). The separation was conducted

The environment of the large intestine is of paramount importance to host health. Our previous experiments showed beneficial action of dietary FOS5 or strawberry polyphenols17 in the rat’s cecum. Therefore, in the reported experiment it was hypothesized that a dietary combination of both supplements, FOS and polyphenols, would increase the desired beneficial effect in the lower gut environment. Moreover, intensified metabolism of strawberry polyphenolic compounds when combined with dietary FOS was expected.



MATERIALS AND METHODS

Chemicals. Acetone (99.5%), pure p.a., was purchased from Basic POCH S.A. (Gliwice, Poland). Ethanol (96.2%), rectified, was purchased from “NORD CLAS” Sp. z o.o. (Łódz, Poland). Phloroglucinol for HPLC (≥99%), methanol for HPLC (99.9%), gradient grade, and sucrose, glucose, and fructose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid for HPLC (99− 100%) and phosphoric acid (85−87%), ACS reagent grade, were purchased from J.T. Baker (Phillipsburg, NJ, USA). Ascorbic acid and hydrochloric acid (35−38%), pure p.a., were purchased from ́ s̨ kie, Poland). Acetonitrile (>99.9%), CHROChemPur (Piekary Sla MASOLV gradient grade for HPLC, was purchased from SigmaAldrich (Darmstadt, Germany). FOS Preparation. A commercial FOS preparation (Raftilose P95) was kindly provided by Orafti (Oreye, Belgium). According to the producer’s declaration, it contains 93.2−95.8% oligofructose, as 2−8 degree of polymerization (DP) of fructose polymers and no more than 7.0% of free fructose. The content of oligo-, di-, and monosaccharides in the preparation was characterized using an HPLC system (Knauer, Berlin, Germany) equipped with a EuroChrom 2000 data control system, RI K-2301 Knauer detector (Germany), and a Shodex NH2P (250 × 4 mm) column (Japan), using an acetonitrile−water mixture (67−33% v/v) as a mobile phase at a flow rate of 0.8 mL/min at 20 °C. FOS preparation (0.5 g) was weighed in a 50 mL volumetric flask and dissolved in water (concentration 10 mg/mL) and was injected to the chromatograph for separation and registration of signals by RI detector. The composition of the individual di- to heptamers was determined on the basis of registered areas of the signals. The results were as follows: fructose, 5.1%; DP 2, 2.7%; DP 3, 25.6%; DP 4, 31.2%; DP 5, 18.7%; DP 6, 12.3%; and DP 7, 4.4% (94.4% of oligofructose). Limit of detection for fructose as a standard was 0.06 mg/mL. Strawberry Polyphenolic Extract. The strawberry press cake (750 kg) was taken from the concentrated juice production line of the Alpex Company (Łec̨ zeszyce, Poland) and dried in an industrial vacuum dryer (Polfarmex Company, Kutno, Poland) at 70 ± 2 °C. After drying, the pomace (400 kg) was separated into a seed fraction (diameter 0.5−1 mm) and a seedless fraction (diameter 1−3 mm) using a vibration sieving machine with sieves of the sizes 0.5 mm, 1 mm, and 3 mm (each was designed by the company Polfarmex S.A. Kutno, Poland) equipped with a vibration device necessary to move the material on the sieve and to renew the surface of sieving. The material with particle sizes between 1 and 3 mm, constituting part of pomace devoid of seeds (achenes), was used for successive production of a strawberry polyphenolic extract applied in the present study. The water−acetone ET strawberry concentrate was prepared at laboratory scale using the seedless fraction and a 60% solution v/v of acetone (5 L per 1 kg of pomace). Extraction was conducted in two stages for a minimum of 12 h. After partial removal of the solvent by distillation, approximately 5% of the resulting solution was subjected to adsorption using 500 g of adsorbent Amberlite XAD 16 in a column with a diameter Φ of 40 mm and a height of 1000 mm. After washing away the saccharide dirt with water, the desorption of polyphenols was performed using 10%, 40%, and 60% solutions of ethanol successively in amounts of 0.5, 1, and 2 L per 1 kg of adsorbent, respectively. Solutions containing desorbed polyphenols from the Amberlite XAD were gathered and divided into fractions, of which only the eluates rich in ET were concentrated to approximately 15% of the dry weight and 5872

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on Gemini 5u C18 110A (250 mm × 4.6 mm; 5 μm) column (Phenomenex Torrance, CA, USA) using gradient elution with 2.5% water solution (v/v) of acetic acid (phase A) and 80% (v/v) acetonitrile in water (phase B). The following gradient was used: 0−10 min, 4−7% B; 10−27 min, 7−30% B; 27−29 min, 30−70% B; 29−34 min, 70% B; 34−35 min, 70−4% B; and 35−40 min, 4% B. The flow rate was 1 mL/min, the temperature was 25 °C, and the injection volume was 20 μL. The identification of components was conducted by comparing the retention times and the UV−vis spectra of standards of (−)-epicatechin, (+)-catechin, (−)-epicatechin-phloroglucinol, and (+)-catechin-phloroglucinol. Quantitative analyses of the released flavonols, i.e., (+)-catechin and (−)-epicatechin, were conducted based on chromatograms recorded with the FD detector set at an excitation wavelength of 278 nm and an emission wavelength of 360 nm. Quantities were calculated by standard curves of (−)-epicatechin (0.625−50 mg/L; detection limit at 0.03 mg/L) for terminal units, and (−)-epicatechin-phloroglucinol adduct for extender units (0.6−50 mg/L; detection limit at 0.3 mg/L). Phloroglucinol adducts were determined based on chromatograms registered with the PDA detector set at a 280 nm wavelength. Extraction of Nasutin A (4,9-Dehydroxy-ellagic Acid) and Polyphenols. Polyphenol compounds, including nasutin A, were extracted with a 70% solution of acetone. A sample containing 0.5 g of the ground frozen digesta was placed in a 7 mL test tube; next, 5 mL of a solvent was added, mixed using a vortex (IKA Yellowline Test Tube Shaker TTS2, Wilmington, NC, USA) and sonicated (Inter Sonic IS-4, Inter Sonic s.c., Olsztyn, Poland) for 10 min. Following the sonication, the solution was left in the dark for 15 min for extraction. The solution was centrifuged for 5 min (4800g) and poured into a 20 mL tube with a cup. The above procedure was repeated three more times; the combined extracts were poured into a 20 mL tube with a cup. The obtained extracts were stored at 20 °C for a maximum of 3 days before further analysis. Next, the combined extracts were poured into a vacuum distillatory flask, and the solvent was removed. Next, the dry residue was dissolved in 2 mL of 70% methanol. The solution was centrifuged for 5 min (4800g) and was submitted to HPLC analysis. A Smartline (Knauer, Berlin, Germany) chromatograph equipped with the same elements as described above was applied for the chromatographic analysis. The separation was conducted on a Phenomenex Gemini 5u C18 110A (250 × 4.60 mm; 5 μm) column (Phenomenex, Torrance, CA, USA). The conditions of separation were the same as for the determination of polyphenols described above. The urolithin A (3,8-dihydroxy-6H-dibenzo-[β-D]-pyran-6-one) was used as a standard for the determination of nasutin A. To date, commercial nasutin is not available. Urolithin A was prepared as described by Cerdá et al. (2004).18 The raw material for the isolation of urolithin A was two liters of urine from three healthy volunteers (25−45 years old) who consumed 400 g of strawberry per day. The urine was acidified to pH 4.8 by acetic acid. Then, 1.2 mg of glucuronidase bovine liver (Sigma-Aldrich, St. Louis, MO, USA) was added, maintained at 37 °C for 60 min and passed through Amberlite XAd-7 (Sigma-Aldrich, St. Louis, MO, USA) resin for the absorption and purification of polyphenols. In the next step, the resin was washed, and the adsorbed polyphenols were desorbed by methanol. The methanol fractions rich in urolithin A were concentrated and subjected to purification on a Strata SPE 1 g column (Strata - x 33u Polymeric Reversed phase 1 g/20 mL, Giga Tubes Phenomenex, Torrance, CA, USA) and were separated on a preparative HPLC (Knauer, Berlin, Germany) chromatograph, composed of two pumps (Knauer K-501) forming gradient, Phenomenex Luna 10u C18 100A column (250 × 21, 20 mm; 10 μm) (Torrance, CA, USA), UV−vis detector, Foxy R1 Teledyne ISCO (Lincoln, USA) fraction collector, and Eurochrom chromatographic software. Two eluents were used for separation: eluent A, 0.1% water solution of formic acid; eluent B, 80% methanol. The flow rate was 15 mL/min. The following gradient was used: 0−15 min 30% eluent B; 15−50 min 30−75% eluent B; 50−60 min 75% eluent B; 60−63 min 75−30% eluent B; 63−66 min 30% eluent B; and 55−60 min 20% eluent B. The volume of injected sample was 500 μL. Selected fractions containing urolithin A were subjected to freeze-

drying. The UV spectrum of the obtained substances had maxima at 246, 278, 305, and 356 nm, which was the same pattern as the main metabolite of ET detected in urine.14,22 Identification of Nasutin A. The UV spectra of polyphenols have often been used for their identification by HPLC DAD analysis. UV spectra are generally used for the identification of flavonoids and urolithins. In the present study, the spectra of ET metabolites were studied using HPLC UV-DAD to establish the structure of the microbial degradation of agrimoniin. The maximum UV wavelengths for the detected substance (with retention time 26 min) have been determined to be 250, 285, 373, and 389 nm. Next the solution of the substance, separated by HPLC, was directly injected into an ESI-MS/ MS detector (LCQ DECA, Thermo-Finnigan). The analyses were conducted in the positive ion mode.18 The source parameters were as follows: ion spray voltage 3.00 kV; capillary temperature 325 °C; sheath gas and auxiliary gas 30 and 10 units/min, respectively. To generate MS/MS data, the precursor ions were fragmented by helium gas collision in the ion trap by optimizing the collision energy in order to obtain an intensity of the precursor ion close to 10% of the relative scale of the spectrum. The MS [M − H+] 269 for the substance was determined. Data obtained for UV spectrum and value of [M − H+] indicate that nasutin A was the main metabolite in the experiment.14 In Vivo Experiment, Preparation of the Diets, and the Animal Protocol. The animal protocol used in this study was approved by the Local Institutional Animal Care and Use Committee, and the study was performed in accordance with EU Directive 2010/ 63/EU for animal experiments. The assessment was conducted on 48 male adult Wistar rats aged 130 d and weighing 440.3 g (pooled SEM 0.839). The rats were randomly divided into six groups of eight animals each. All animals were housed individually for 4 weeks in metabolic cages with free access to water and the experimental diets (Table 1). The selection of the animals and their maintenance over the 28 d experiment followed common regulations. The environment was controlled with a 12 h light−dark cycle, a temperature of 21 ± 1 °C, relative humidity of 50 ± 5%, and 20 air changes per h. Diets contained similar levels of protein (from casein supplemented with methionine), fat (soybean oil), and minerals and vitamins (from AIN93 M mixtures)23 and a similar content of fiber. However, the fiber was from different sources, i.e., insoluble α-cellulose (SIGMA, Poznań, Poland) or a prebiotic FOS that was fermentable in the lower gut (Orafti, Oreye, Belgium). In the standard-fiber diets, the content of CEL was 6%. In the prebiotic-supplemented diets, half of the dietary CEL was replaced with FOS. The strawberry polyphenolic preparation was added to each type of diet in an amount of 0.051% or 0.153% of the air-dried feed, which constituted 0.04% and 0.12% of total strawberry polyphenols in the diet, respectively (in regard to ETs, the main polyphenolic component of the extract, 0.03% and 0.09% of ETs, respectively). The individual BW and feed intake of the rats were recorded. At the termination of the experiment, the rats were anesthetized with sodium pentobarbital according to the recommendations for the euthanasia of experimental animals. After a laparotomy, blood samples were taken from the caudal vena cava, and then serum was prepared by solidification and low-speed centrifugation (350g, 10 min, 4 °C). The small intestine, cecum, and colon were removed and weighed. As soon as possible after euthanasia (ca. 10 min), the small intestinal, cecal, and colonic pH values were measured (pH meter model 301, Hanna Instruments, Vila do Conde, Portugal). The dry matter of the cecal digesta was determined at 105 °C for 4 h. In the fresh cecal digesta, ammonia (NH3) was determined by microdiffusion analysis in Conway’s dishes, and the SCFA were analyzed using GC as described previously.24 For SCFA, the samples (0.2 g) were mixed with 0.2 mL of formic acid, diluted with deionized water, and centrifuged at 7211g for 10 min. The supernatant was loaded onto a capillary column (SGE BP21, 30 m × 0.53 mm) using an on-column injector. The initial oven temperature was 85 °C and was raised to 180 °C at a rate of 8 °C/min and held for 3 min. The temperatures of the flame ionization detector and the injection port were 180 and 85 °C, respectively. The sample volume for GC analysis was 1 μL. The cecal putrefactive SCFA (PSCFA) concentration was calculated as the sum of the isobutyrate, isovalerate, and valerate concentrations in the cecal 5873

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statistical analysis was performed. Differences with P < 0.05 were considered to be significant.

Table 1. Diet Composition (%)



experimental diet casein DL-methionine cellulosea FOS soya oil cholesterol mineral mixb vitamin mixc strawberry extract corn starch calcd dietary ellagitannins calcd dietary polyphenols

CCEL

CFOS

ELCEL

ELFOS

EHCEL

EHFOS

14.8 0.2 6 0 8 0.5 3.5 1 0

14.8 0.2 3 3 8 0.5 3.5 1 0

14.8 0.2 6 0 8 0.5 3.5 1 0.051

14.8 0.2 3 3 8 0.5 3.5 1 0.051

14.8 0.2 6 0 8 0.5 3.5 1 0.153

14.8 0.2 3 3 8 0.5 3.5 1 0.153

66 0

66 0

65.949 0.030

65.949 0.030

65.847 0.090

65.847 0.090

0

0

0.040

0.040

0.120

0.120

RESULTS The preparation obtained from strawberry used in this study contained 78.7 g of polyphenols per 100 g of preparation (Table 2). The main phenolic compounds in the preparation Table 2. Basic Chemical and Polyphenolic Composition (%) of the Strawberry Extract composition main components chemical composition dry matter crude protein ether extract crude ash total dietary fiber carbohydratesc, (including F+G < 1%)a polyphenols total polyphenolic compounds (HPLCDAD):b free ellagic acid total ellagitannind (containing 27% agrimoniin) p-coumaric acid and derivatives flavonoidse proanthocyanidins

a α-Cellulose preparation was obtained from Sigma-Aldrich (No. C8002). bAIN-93 M (Reeves 1997),23 g per kg mix: 357 g of anhydrous calcium carbonate (40.04% Ca), 196 g of monobasic potassium phosphate (22.76% P, 28.73% K), 70.78 g of potassium citrate and tripotassium monohydrate (36.16% K), 74 g of sodium chloride (39.34% Na, 60.66% Cl), 46.6 g of potassium sulfate (44.87% K, 18.39% S), 24 g of magnesium oxide (60.32% Mg), 6.06 g of ferric citrate (16.5% Fe), 1.65 g of zinc carbonate (52.14% Zn), 1.45 g of sodium meta-silicate·9H2O (9.88% Si), 0.63 g of manganous carbonate (47.79% Mn), 0.3 g of cupric carbonate (57.47% Cu), 221.026 g of powdered sucrose, and 0.275 g of chromium potassium sulfate·12H2O (10.42% Cr). The following components were added in mg per kg mix quantities: 81.5 mg of boric acid (17.5% B), 63.5 mg of sodium fluoride (45.24% F), 31.8 mg of nickel carbonate (45% Ni), 17.4 mg of lithium chloride (16.38% Li), 10.25 mg of anhydrous sodium selenate (41.79% Se), 10 mg of potassium iodate (59.3% I), 7.95 mg of ammonium paramolybdate·4H2O (54.34% Mo), and 6.6 mg of ammonium vanadate (43.55% V). cAIN-93 M (Reeves 1997),23 g per kg mix: 3.0 g of nicotinic acid, 1.6 g of Ca pantothenate, 0.7 g of pyridoxine−HCl, 0.6 g of thiamin-HCl, 974.655 g of powdered sucrose, 0.6 g of riboflavin, 0.2 g of folic acid, 0.02 g of biotin, 2.5 g of vitamin B12 (cyanocobalamin, 0.1% in mannitol). The following components were added in IU per g quantities: 15.0 IU of vitamin E (all-rac-α-tocopheryl acetate, 500), 0.8 IU of vitamin A (all-transretinyl palmitate, 500000), 0.25 IU of vitamin D3 (cholecalciferol, 400000), and 0.075 IU of vitamin K-1 (phylloquinone).

polyphenols

93.2 2.5 0.0 0.4 0.0 11.6 78.7 78.7 1.3 59.3 0.2 1.0 16.9

a

F+G, content of fructose and glucose all other groups, P < 0.05). Additionally the ELCEL rats had the lowest concentration of this metabolite, and it differed significantly in this respect from group EHCEL. Regardless of the diet type, the dietary application of both strawberry extract dosages significantly decreased the cecal concentration and pool of total SCFA, mainly due to the lower concentration of acetic acid (Table 5). The dietary supplementation with the higher dosage of strawberry extract significantly (P < 0.05) decreased the cecal concentration of putrefactive SCFA compared to the treatment without extract and with a lower extract dietary application. The dietary FOS significantly increased the cecal propionic and butyric acid concentrations and significantly decreased the concentration of total putrefactive SCFA. The cecal SCFA pool was also increased by dietary FOS application (P < 0.05). The analysis of the SCFA C2:C3:C4 profile indicates a lower ratio of acetic acid and an increased ratio of propionic and butyric acids after 5876

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Table 5. Short Chain Fatty Acid (SCFA) Concentration (μmol/g Digesta), Total Pool (μmol/100 g BW), and Profile (%)a SCFA concn group

SCFA profile

C2b

C3

C4i

C4

C5i

C5

42.0 45.0 36.4 35.7 38.7 37.6 0.95

8.36 10.7 7.88 9.36 8.07 9.68 0.27

0.73 0.60 0.74 0.50 0.62 0.43 0.02

6.57 9.23 5.73 6.21 5.91 8.27 0.36

0.61 0.64 0.71 0.52 0.48 0.43 0.03

PSCFA

SCFA

1.06 1.13 1.05 0.93 0.95 0.93 0.03

2.40 2.36 2.49 1.95 2.05 1.80 0.07

61.7 69.7 55.0 55.2 56.7 59.2 1.5

SCFA pool

C2

C3

C4

36.9 45.5 27.7 31.5 25.6 35.2 1.4

68.0 65.0 66.2 65.1 68.1 63.4 0.5

13.6 15.3 14.3 16.8 14.4 16.4 0.3

10.5 13.0 10.5 11.0 10.1 14.1 0.4

c

CCEL CFOS ELCEL ELFOS EHCEL EHFOS SEM extract (E) C EL EH P value fiber (F) CEL FOS P value interaction E × F P value

43.5 a 36.1 b 38.2 b 0.004

9.55 8.62 8.87 0.283

0.66 a 0.62 a 0.53 b 0.015

7.90 5.97 7.09 0.061

0.63 a 0.61 a 0.46 b 0.035

1.09 0.99 0.94 0.185

2.38 a 2.22 a 1.92 b 0.026

65.7 a 55.1 b 58.0 b 0.008

41.2 a 29.6 b 30.4 b 0.000

66.5 65.7 65.7 0.709

14.4 15.5 15.4 0.053

11.6 10.7 12.1 0.307

39.0 39.5 0.802

8.10 b 9.92 a 0.001

0.70 a 0.51 b 0.000

6.07 b 7.90 a 0.007

0.60 0.53 0.234

1.02 1.00 0.753

2.32 a 2.04 b 0.045

57.8 61.4 0.201

30.1 b 37.4 a 0.002

67.4 a 64.5 b 0.002

14.1 b 16.2 a 0.000

10.4 b 12.7 a 0.004

0.583

0.720

0.570

0.342

0.291

0.555

0.320

0.494

0.520

0.281

0.715

0.189

a

Mean values within a column with unlike letters a and b were shown to be significantly different (P < 0.05); differences among groups CCEL, CFOS, ELCEL, ELFOS, EHCEL, and EHFOS were indicated only in the case of a statistically significant interaction E × F (P < 0.05). bC2, acetate; C3, propionate; C4i, isobutyrate; C4, butyrate; C5i, isovalerate; C5, valerate; PSCFA, putrefaction short chain fatty acid (sum of C4i, C5i, and C5). c CCEL, control diet with 6% cellulose (CEL) as the dietary fiber; CFOS, control diet with 3% fructooligosaccharides (FOS) and 3% cellulose as the dietary fiber; ELCEL, diet with a low level of the strawberry extract and CEL as the dietary fiber; ELFOS, diet with a low level of the extract and FOS/ CEL as the dietary fiber; EHCEL, diet with a high level of the strawberry extract and CEL as the dietary fiber, EHFOS, diet with a high level of the extract and FOS/CEL as the dietary fiber.

Table 6. Biochemical Indicators of the Blood Serum of Rats Fed Experimental Dietsa U/L ALTb group

mmol/L

mmol/L

AST

TC

HDL

93.9 93.6 101 99.3 99.2 99.8 2.6

2.08 2.10 2.05 1.84 2.11 1.91 0.03

0.75 1.02 0.87 0.80 0.82 0.75 0.02

HDL profile, % of TC

TG

GL

0.97 1.03 0.99 0.87 1.08 0.90 0.04

8.81 7.59 6.53 6.47 6.98 6.16 0.20

c

CCEL CFOS ELCEL ELFOS EHCEL EHFOS SEM extract (E) C EL EH P value fiber (F) CEL FOS P value interaction E × F P value

33.0 34.3 32.6 27.1 30.7 32.3 1.1

b a b b b b

36.5 48.7 42.6 43.3 38.9 39.5 1.0

b a ab ab b b

33.7 29.8 31.5 0.378

93.7 99.9 99.5 0.593

2.09 1.95 2.01 0.268

0.88 0.83 0.78 0.074

42.6 42.9 39.2 0.172

1.00 0.93 0.99 0.770

8.20 a 6.50 b 6.57 b 0.000

32.1 31.2 0.693

97.9 97.6 0.958

2.08 1.95 0.068

0.81 0.85 0.224

39.3 b 43.9 a 0.014

1.01 0.93 0.330

7.44 a 6.74 b 0.034

0.356

0.990

0.313

0.000

0.014

0.447

0.326

a

Mean values within a column with unlike letters a and b were shown to be significantly different (P < 0.05); differences among the groups CCEL, CFOS, ELCEL, ELFOS, EHCEL, and EHFOS were indicated only in the case of a statistically significant interaction E × F (P < 0.05). bALT, alanine transaminase; AST, aspartate transaminase; TC, total cholesterol; TG, triglycerides; GL, glucose. cCCEL, control diet with 6% cellulose (CEL) as the dietary fiber, CFOS, control diet with 3% fructooligosaccharides (FOS) and 3% cellulose as the dietary fiber, ELCEL, diet with a low level of the strawberry extract and CEL as the dietary fiber, ELFOS, diet with a low level of the extract and FOS/CEL as the dietary fiber, EHCEL, diet with a high level of the strawberry extract and CEL as the dietary fiber, EHFOS, diet with a high level of the extract and FOS/CEL as the dietary fiber.

decreased the amount of cecal digesta and tended to increase

FOS and ET strawberry extract showed an opposite effect on

the cecal dry matter concentration. It was also noted that the

the measured parameters. 5877

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increase in the concentration of nasutin A in the gastrointestinal tract after the consumption of the diet with FOS and variable doses of the ET strawberry extract. In this study, trace amounts of nasutin A were observed in the stomach; it can be speculated that hydroxyls can be removed from EA under the physiological conditions of the stomach. The acidic environment of the stomach leads to the production of EA.7,14 The highest concentration of nasutin A was observed in the small intestines. Larrosa et al. (2006)43 showed that ETs are mostly hydrolyzed to EA under the physiological conditions of the small intestine. In the cecum, a lower concentration of nasutin A was observed compared to the small intestine. When ETs or EA reach the distal part of the small intestine and the colon, they are largely metabolized by the gut microflora to urolithins;42 we speculated that the main differences in the concentration of nasutin A were associated with specific microflora that are not the same in all parts of the gastrointestinal tract. Determining the causes of such a phenomenon requires further experiments. Numerous studies have shown that the administration of FOS leads to an enhancement of SCFA production, mainly acetate, propionate, and butyrate.3,6 After absorption, SCFAs play important roles in an organism. Propionate has a positive influence on lipid metabolism in liver, whereas butyrate is an energy substrate for colonocytes.37 In this study, the addition of FOS into the diet improved the cecal production of propionate and butyrate acids. Thus, a higher concentration of butyric may explain the significantly higher mass of cecal tissue in the groups consuming FOS. Zduńczyk et al. (2006)11 demonstrated that the presence of polyphenols in inulin-containing diets did not change the total SCFA production in the cecum of rats. In addition, Juśkiewicz et al. (2010)6 reported that the polyphenol concentrate from apple pomace and FOS did not show any serious negative effects on the basic parameters of the cecum and SCFA production. However, the addition of the ET strawberry extract into the diet slightly reduced the beneficial effect of FOS on enhancing the production of SCFA, especially acetic acid in the cecum. The high content of ETs and their metabolites suppressed the activity of the microbiota and thereby decreased the production of SCFA. The results of other studies indicate that an increased content of polyphenols in the diet may inhibit the growth and reduce the abundance of gastrointestinal microflora.11,38 In this study, the group with a high content of ET strawberry extract exhibited significantly reduced production of PSCFA, which may suggest less intensive anaerobic bacterial fermentation of polypeptides and amino acids.24 Based on the results obtained in this study, we assume that the above changes in the cecal concentrations of SCFA were consistent with blood markers of the lipid profile in the rats. Propionate is reported to be involved in the cholesterollowering effect of fiber by impairing acetate utilization, especially when cholesterol synthesis is activated to compensate for enhanced fecal losses of steroids.44 In this study, rats that consumed the diet with FOS had a significantly higher value of HDL-C, a significantly lower concentration of glucose in the blood, and a statistical tendency (P = 0.068) toward a lower serum TC concentration compared to rats that consumed the diet with CEL. These results confirm earlier observations made by Giacco et al. (2004),45 who reported that positive systemic effects of fructans could reduce plasma glucose as well as the total and LDL cholesterol concentrations. The combination of a high level of the ET strawberry extract with FOS in the diet caused a higher reduction of glucose in the blood, and the

The activity of microbiota in the cecum depends on the supplied nutrients. Kosmala et al. (2014)35 have shown that the inclusion of 5% FOS significantly decreased α-galactosidase, βglucosidase, and β-glucuronidase activity, while the activity of α-glucosidase and β-galactosidase did not change statistically when compared to the CEL-enriched diet. In our experiment, the addition of 3% FOS to the diet significantly reduced the activity of β-glucuronidase and increased that of β-galactosidase in the cecal digesta compared to the CEL groups. The addition of ET strawberry extract improved the effect of ingested diet by significantly decreasing the activity of β-glucosidase in the cecal digesta. However, the addition of FOS and a high level of the ET strawberry extract (group EH) together reduced the beneficial effect of FOS by increasing the β-glucuronidase activity in the cecal digesta. These enzymes may exert toxic, carcinogenic, or mutagenic effects in the gastrointestinal tract.36 It can be assumed that the addition of ETs to diet could selectively modulate the composition of the microbiota and thus increase potentially harmful species of bacteria which were responsible for the enhanced activity of β-glucuronidase in the cecum. FOS is known as a prebiotic, which selectively stimulates the growth of bifidobacteria, thus causing significant changes in the composition of the gut microflora by increasing the number of potentially health-promoting bacteria and reducing the number of potentially harmful species, including Escherichia coli and Clostridium.37 Larrosa et al. (2010)38 found that ETs and ET metabolites from pomegranate decreased enterobacteria and increased lactobacilli and bifidobacteria. The ET strawberry extract in addition to ETs is a source of proanthocyanidins, which may reduce the activity of digestive enzymes when present at elevated levels.39 The ET strawberry extract used in this study was also a source of flavonoids; previously, a flavonoid extract applied as a dietary supplement was shown to decrease the activity of bacterial β-glucosidase and β- and α-galactosidases in the cecal digesta of rats.11 Roberton et al. (1982)40 suggested that the most important factor in the modulation of β-glucuronidase activity in the rat large bowel is bile flow. Indeed, dietary fiber is the main factor that intensifies peristalsis and thus the bile flow through the large intestine.41 In this study, the identification and determination of the ET metabolites in the gastrointestinal tract of the rats revealed the presence of nasutin A and EA. However, urolithins, which are considered to be typical reaction products of ET metabolism in the gastrointestinal tract of humans and experimental animals, were not detected.12,13 One of the possible ways in which the intensification or modulation of the ET biodegradation processes may be occurring is through an increase in the population of the lactobacilli bacteria that are capable of producing tannase and changes in the pH of the environment.42 Tannase causes hydrolysis of ester bonds, including the bonds between hexahydroxydiphenic acid (HHDP) and glucose; free HHDP can then undergo spontaneous lactonization to EA. Lactonization of the acid with the hydroxyl group of the benzene ring can be accompanied by decarboxylation of the second acid moiety and the subsequent loss of hydroxyl residues to form urolithins.14,15 A small amount of experimental data14 and the results of computer simulations of chemical reactions12 indicated the formation of nasutin A and isonasutin as byproducts of lactonization with the removal of two hydroxyl residues from EA. Nevertheless, there is no clear explanation for the spontaneity or the conditions of the formation process of nasutin A. The data presented in Figure 1 show a significant 5878

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(6) Juśkiewicz, J.; Milala, J.; Jurgoński, A.; Król, B.; Zduńczyk, Z. Consumption of polyphenol concentrate with dietary fructooligosaccharides enhances cecal metabolism of quercetin glycosides in rats. Nutrition 2010, 27, 351−357. (7) Silva Pinto, M.; Lajolo, F. M.; Genovese, M. I. Bioactive compounds and quantification of total ellagic acid in strawberries (Fragaria x ananassa Duch.). Food Chem. 2008, 107, 1629−1635. (8) Seeram, N. P.; Lee, R.; Scheuller, H. S.; Heber, D. Identification of phenolic compounds in strawberries by liquid chromatography electrospray ionization mass spectroscopy. Food Chem. 2006, 97, 1− 11. (9) Giménez-Bastida, J. A.; González-Sarrías, A.; Larrosa, M.; TomásBarberán, F.; Espín, J. C.; García-Conesa, M. T. Ellagitannin metabolites, urolithin A glucuronide and its aglycone urolithin A, ameliorate TNF-α-induced inflammation and associated molecular markers in human aortic endothelial cells. Mol. Nutr. Food Res. 2012, 56, 784−796. (10) Landete, J. M. Ellagitannins, ellagic acid and their derived metabolites: a review about source, metabolism, functions and health. Food Res. Int. 2011, 44, 1150−1160. (11) Zduńczyk, Z.; Juśkiewicz, J.; Estrella, I. Cecal parameters of rats fed diets containing grapefruit polyphenols and inulin as single supplements or in a combination. Nutrition 2006, 22, 898−904. (12) Mertens-Talcott, S. U.; Jilma-Stohlawetz, P.; Rios, J.; Hingorani, L.; Derendorf, H. Absorption, metabolism, and antioxidant effects of pomegranate (Punica granatum L.) polyphenols after ingestion of a standardized extract in healthy human volunteers. J. Agric. Food Chem. 2006, 54, 8956−8961. (13) Tomás-Barberan, F. A.; Garcia-Conesa, M. T.; Larrosa, M.; Cerdá, B.; Gonzalez-Barrio, R.; Bermudez-Soto, M. J.; GonzalezSarrias, A.; Espín, J. C. Bioavailability, metabolism, and bioactivity of food ellagic acid and related polyphenols. Recent Adv. Polyphenol Res. 2008, 1, 263−277. (14) González-Barrio, R.; Truchado, P.; Ito, H.; Espín, J. C.; TomasBarberán, F. A. UV and MS identification of urolithins and nasutins, the bioavailable metabolites of ellagitannins and ellagic acid in different mammals. J. Agric. Food Chem. 2011, 59, 1152−1162. (15) Mazzone, G.; Toscano, M.; Russo, N. Density functional predictions of antioxidant activity and UV spectral features of nasutin A, isonasutin, ellagic acid, and one of its possible derivatives. J. Agric. Food Chem. 2013, 61, 9650−9657. (16) Aviram, M.; Rosenblat, M.; Gaitini, D.; Nitecki, S.; Hoffman, A.; Dornfeld, L.; Volkova, N.; Presser, D.; Attias, J.; Liker, H.; Hayek, T. Pomegranate juice consumption for three years by patients with carotid arterial stenosis reduces common carotid-media thickness, blood pressure and LDL oxidation. Clin. Nutr. 2004, 23, 423−433. (17) Jarosławska, J.; Juśkiewicz, J.; Wróblewska, M.; Jurgoński, A.; Król, B.; Zduńczyk, Z. Polyphenol-rich strawberry pomace reduces serum and liver lipids and alters gastrointestinal metabolite formation in fructose-fed rats. J. Nutr. 2011, 141, 1777−1783. (18) Cerdá, B.; Espín, J. C.; Parra, S.; Martínez, P.; Tomás-Barberán, F. A. The potent in vitro antioxidant ellagitannins from pomegranate juice are metabolised into bioavailable but poor antioxidant hydroxy6H-dibenzopyran-6-one derivatives by the colonic microflora of healthy humans. Eur. J. Nutr. 2004, 43, 205−220. (19) AOAC. Official methods of analysis of AOAC International, 18th ed.; Horwitz, W., Latimer, G. W., Eds.; AOAC International: Gaithersburg, MD, 2005. (20) Sójka, M.; Klimczak, E.; Macierzyński, J.; Kołodziejczyk, K. Nutrient and polyphenolic composition of industrial strawberry press cake. Eur. Food. Res. Technol. 2013, 237, 995−1007. (21) Kennedy, J. A.; Jones, G. P. Analysis of proanthocyanidin cleavage products following acid-catalysis in the presence of excess phloroglucinol. J. Agric. Food Chem. 2001, 49, 1740−1746. (22) Espín, J. C.; Gonzalez-Barrio, R.; Cerdá, B.; Lopez-Bote, C.; Rey, A. I.; Tomás-Barberan, F. A. Iberian pig as a model to clarify obscure points in the bioavailability and metabolism of ellagitannins in humans. J. Agric. Food Chem. 2007, 55, 10476−10485.

HDL-C level in the serum was lower than in the group without the ET strawberry extract in the diet. Other authors have found that ET metabolites, urolithins, are responsible for serum and liver lipid reductions.43,16 Moreover, some studies have shown that polyphenolic compounds may reduce the activity of endogenous enzymes in the small intestine and thereby may influence the regulation of blood glucose levels.22,46 In summary, the present experiment demonstrated that some of the positive changes in the cecal metabolism of rats that resulted from the ingestion of a diet enriched only with FOS were suppressed or slightly suppressed by the addition of an ET strawberry extract. The pH value, SCFA production, and βglucuronidase activity in the cecum of rats fed with FOS were affected upon the addition of the ET strawberry extract. The addition of FOS to the diet resulted in higher metabolism of the tested ET strawberry extract in the gastrointestinal tract of rats. Moreover, the systemic effect of the supplements when consumed together showed undesired decrease in HDL-C.



ASSOCIATED CONTENT

* Supporting Information S

Figure 2: HPLC-DAD chromatogram (λ = 360 nm) of the rat gastric contents after the administration of high doses of ETs (EH) and FOS compared to the control group (C) without ETs and with FOS. Figure 3: HPLC-DAD chromatogram (λ = 360 nm) of the rat small intestine contents after the administration of high doses of ETs (EH) and FOS compared to the control group (C) without ETs and with FOS. Figure 4: HPLC-DAD chromatogram (λ = 360 nm) of the rat cecum contents after the administration of high doses of ETs (EH) and FOS compared to the control group (C) without ETs and with FOS. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Phone: +48-89-523-4600. Fax: +48-89-524-0124. E-mail: b. [email protected]. Funding

This work was supported in part by the National Science Centre Poland (Grant DEC-2012/05/B/NZ9/03402) and by the Polish Ministry of Science and Higher Education as part of the resources allocated for science in 2010-2013 under research project No. NN312360139. Notes

The authors declare no competing financial interest.



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