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A Gastrointestinally-digested Ribes nigrum L. Fruit Extract Inhibits Inflammatory Response in a Co-culture Model of Intestinal Caco-2 Cells and RAW264.7 Macrophages Anna Olejnik, Katarzyna Kowalska, Mariola Olkowicz, Wojciech Juzwa, Rados#aw Dembczy#ski, and Marcin Schmidt J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02776 • Publication Date (Web): 26 Sep 2016 Downloaded from http://pubs.acs.org on September 27, 2016
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Journal of Agricultural and Food Chemistry
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A Gastrointestinally-digested Ribes nigrum L. Fruit Extract Inhibits Inflammatory Response
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in a Co-culture Model of Intestinal Caco-2 Cells and RAW264.7 Macrophages
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Short title: Anti-inflammatory effects of blackcurrant fruit extract
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Anna Olejnik*, Katarzyna Kowalska, Mariola Olkowicz, Wojciech Juzwa, Radosław Dembczyński,
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Marcin Schmidt
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Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Wojska Polskiego 48, 60-627 Poznań, Poland
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*Corresponding author contact information:
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Anna Olejnik, PhD
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Address: Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences,
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Wojska Polskiego 48, PL 60-627 Poznań, Poland;
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Tel.: +48 618466008. Fax: +48 618466003. E-mail:
[email protected] 17 18
Authors’ contribution
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Anna Olejnik: concept of work, study design, “in vitro” digestion, anti-inflammatory experiment, DNA
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damage detection, and manuscript preparation; Katarzyna Kowalska: quantitative analysis of
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inflammatory gene expression (real time PCR analysis); Mariola Olkowicz: analysis of the
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blackcurrant extract composition by HPLC-DAD coupled with ESI/MS; Wojciech Juzwa:
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measurement of intracellular ROS production (flow cytometry analysis); Radosław Dembczyński:
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blackcurrant extract preparation; Marcin Schmidt: design of primers for real time PCR.
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ABSTRACT
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Blackcurrant fruits are a rich source of polyphenolic compounds with high antioxidant capacity and
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potent anti-inflammatory properties. In this study, blackcurrant extract digested in an artificial
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gastrointestinal tract and its intestinal permeable fraction were investigated for their ability to suppress
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inflammatory responses induced in a two-component cell culture system of intestinal epithelial cells
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and macrophages. The obtained results showed the capacity of the extract at a concentration of 1 mg of
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freeze-dried blackcurrant powder per ml to down-regulate the expression of inflammatory mediators,
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such as IL-8 (54 ± 7%) and COX-2 (17 ± 6%), in intestinal cells and IL-1α (76 ± 4%), IL-1β (91 ±
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2%) and IL-6 (61 ± 5%) in macrophages stimulated with lipopolysaccharides. Inhibited COX-2 (44 ±
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6%) and iNOS (15 ± 7%) expression played a role in reducing the production of prostaglandin E2 (40 ±
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20%) and NO (31 ± 9%), respectively. Decreased TNF-α secretion (24 ± 5%) by activated
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macrophages was also observed after treatment with blackcurrant extract. Moreover, the
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gastrointestinal-digested extract (0.01−1 mg/ml) dose-dependently decreased the enhanced ROS
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generation (14−54%) and oxidative DNA damage (16−37%) induced in intestinal cells. The increased
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intestinal permeability caused by proinflammatory mediators, as assessed by transepithelial electrical
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resistance, was completely counteracted.
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KEYWORDS: blackcurrant fruits, “in vitro” digestion, intestinal permeability, antioxidant activity,
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anti-inflammatory effects
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Blackcurrant (BC) Ribes nigrum L. is considered as a valuable source of bioactive compounds
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with therapeutic potential for the prevention and treatment of many human diseases, such as obesity
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and metabolic disorders, cardiovascular diseases and cancer.1,2 Although BC fruits contain macro- and
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micro-nutrients, such fiber, minerals (K, Ca, Mg, Fe) and vitamins (with a high content of vitamin C),
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many phytochemicals, including polyphenolic compounds (such as anthocyanins, phenolic acid
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derivatives, flavan-3-ols, flavonols, hydrolyzable tannins, stilbenoids), present in these fruits have been
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found to exert health-promoting and disease preventive effects. Biochemical analyses also revealed a
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high content of polyunsaturated fatty acids (mainly γ-linolenic acid and stearidonic acid) in the BC
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seeds.1 The favorable health effects may be related to the different biological activities of BC
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components, including their high antioxidant capacity and potent anti-inflammatory properties.1,3 Few
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“in vitro” experiments have shown the ability of BC fruits to regulate the cellular response of
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inflammation-related cells. The preparations of BC fruits have been found to exhibit significant
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immune effects by controlling the levels of inflammatory cytokines and mediators, including
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interleukin 6 (IL-6),4 interleukin 1β (IL-1β),5,6 tumor necrosis factor α (TNF-α)4-6 and inducible nitric
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oxide synthase (iNOS)5,7. However, to date, anti-inflammatory research has been focused solely on the
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pure chemical constituents of BC fruits or crude BC extracts, without considering their bioavailability,
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which largely depends on the digestive stability and the efficiency of intestinal absorption and affects
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the health-beneficial properties.
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The aim of the present study was to investigate whether the anti-inflammatory effects of BC fruits
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can be modulated by metabolic transformations. The investigation was based on the analysis of the
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cellular immune responses in lipopolysaccharide-stimulated RAW264.7 macrophages treated with the
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bioavailable intestinal permeable fraction of gastrointestinal-digested BC fruit extract. In addition,
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anti-inflammatory and antioxidant activities of gastrointestinal-digested BC extract were evaluated in
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the differentiated enterocyte-like Caco-2 cells, which mimic the intestinal barrier.
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MATERIALS AND METHODS
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Chemicals
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Pepsin from porcine gastric mucosa, pancreatin from porcine pancreas and bile extract porcine were
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obtained from Sigma–Aldrich (Steinheim, Germany). Likewise, HPLC-MS analysis chemicals
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(solvents and standards), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid diammonium
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salt), Trolox, Dulbecco’s modified Eagle’s medium (DMEM), non-essential amino acids 100X,
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trypsin–EDTA solution, lipopolysaccharides (LPS), budesonide, TRI–Reagent and Griess reagent were
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purchased from Sigma–Aldrich. Fetal bovine serum (FBS) and gentamycin solution were obtained
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from Gibco BRL (Grand Island, NY, USA). Fluorescein-5-(and-6)-sulfonic acid (FSA), 2',7'-
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dichlorodihydrofluorescein diacetate (DCFH-DA) and SYBR® Select Master Mix were supplied by
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Life Technologies (Carlsbad, CA, USA). RNase-free DNase I recombinant and Transcriptor First
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Strand cDNA Synthesis Kit were provided by Roche Diagnostics GmbH (Mannheim, Germany).
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ELISA kits used for quantification of IL-6, TNF-α and prostaglandin E2 (PGE2) were obtained from R
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& D Systems Inc. (Minneapolis, MN, USA). CytoTox-One™ Homogeneous Membrane Integrity
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Assay kit was provided by Promega GmbH (Mannheim, Germany).
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Blackcurrant extract preparation
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The mature fruits of BC (Ribes nigrum L.) Titania cv. were homogenized with a small amount of
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distilled water. The obtained fruit pulp was frozen at -80 °C and subjected to freeze-drying at a
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vacuum pressure of 0.1 mm Hg and drying at 20 °C for 23 hours using a freeze-dryer (LMC-1, Martin
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Christ Gefriertrocknungsanlagen GmbH, Germany) and then subsequent drying at 23 °C for 3 hours.
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The lyophilized BC fruits were ground into a fine powder, packaged under nitrogen atmosphere and
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stored at -20 °C until analysis.
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Freeze-dried BC powder was suspended in deionized water to obtain a concentration of 0.05 g/ml
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and subjected to a digestion process simulated in an artificial alimentary tract, comprising gastric (1 M
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HCl, pepsin solution, pH 2, 2 h) and intestinal (1 M NaHCO3, pancreatin and bile solution, 106 colony-
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forming units of human fecal bacterial culture, pH 6.0–7.4, 2.5 h) phases. The digested BC mixture 4 ACS Paragon Plus Environment
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was incubated at 37 °C under continuous agitation (150 rpm). In addition, anaerobic conditions were
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maintained during passage through the intestinal tract. The gastrointestinal digestion procedure was
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developed on the basis of previously published data8-11 and was described in detail by Olejnik et al.12
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After gastrointestinal digestion, the BC extract was centrifuged (10 min, 10 000 g), filtered (0.22 µm
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pore size), and stored at −80 °C until analysis.
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Analysis of the blackcurrant extract composition by HPLC-DAD coupled with ESI/MS
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Analyses of the BC fruit extract composition were performed using an Agilent 1200 series HPLC
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system (Agilent Technologies, Inc., Santa Clara, CA, USA) coupled on-line with a mass spectrometer
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fitted with an electrospray ionization (ESI) source. The LC system consisted of a G1312A binary
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pump, a G1329 autosampler, a G1316A temperature-controlled column compartment, and a G1315D
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photodiode array detector and was connected directly to an Agilent 6224 time-of-flight MS system
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with the use of an ESI interface. Data acquisition and processing were performed using MassHunter
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2.0 software (Agilent Technologies, Inc.).
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Chromatographic separations were carried out on a 150 mm x 2.1 mm, 5 µm ACE (Advanced
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Chromatography Technologies, Aberdeen, Scotland) C18 column. The mobile phase consisted of two
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solvents: 5% (v/v) formic acid in water (A) and methanol (B). A gradient elution procedure was
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performed as follows: 5–25% B, 0–8 min; 25–45% B, 8–30 min; 45–5% B, 30–40 min; 5% B, 40–55
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min. The flow rate was at 0.3 ml/min, and the injection volume was 5 µl. The HPLC chromatograms
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were recorded at 280, 325, 360 and 520 nm, which are the recommendations for the detection of
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flavan-3-ols, hydroxycinnamic acid derivatives,
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Quantification of BC compounds was based on the standards of (-)-catechin (catechin derivatives),
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chlorogenic, p-coumaric, ferulic and sinapic acids (hydroxybenzoic acid derivatives), quercetin,
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kaempferol
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(anthocyanins).
and
myricetin
(flavonols),
flavonols, and anthocyanins, respectively.
cyanidin-3-O-glucoside
and
cyanidin-3-O-rutinoside
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The mass spectrometer was operated with an ESI interface in positive or negative ionization
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mode, depending on the physicochemical properties of the compounds. The major mass spectrometer 5 ACS Paragon Plus Environment
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parameters were as previously described.10 The identities of the phenolic compounds were obtained by
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matching their molecular ion m/z values (obtained by LC-ESI/MS) with published data.13-17
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Measurement of total antioxidant capacity using ABTS assay
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An ABTS assay was performed according to the method of Re et al.,18 adapted to 96-well
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microplates. The results were expressed as µM of Trolox equivalents per 1 ml of BC extract.
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Cell cultures
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Caco-2 (Cat. no: 86010202, passage no: 45) and RAW264.7 (Cat. no: 91062702, passage no: 3)
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cell lines were obtained from the European Collection of Cell Cultures and supplied by Sigma–
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Aldrich. Cells were cultured in DMEM supplemented with 100 ml/l heat-inactivated FBS, 10 ml/l non-
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essential amino acids 100X and 50 mg/l gentamycin. Cell cultures were incubated at 37 °C in a
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humidified atmosphere (5% CO2, 95% air) and subcultured twice a week after reaching ca. 80%
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confluence. A trypsin–EDTA solution (0.25%) was used to harvest the Caco-2 cells.
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Caco-2 cells at passage number 48-53 were seeded at a density of 4.4 · 105 cells/cm2 on
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polycarbonate membranes (Millicell™ Culture Plate Inserts, 12 mm in diameter) with a pore size of
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0.4 µm (Millipore; Merck KGaA, Darmstadt, Germany). The cells were cultured for 21 days with
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growth medium replacement every 2-3 days until the cells were fully differentiated. The integrity of
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the Caco-2 monolayer was determined by measuring the transepithelial electrical resistance (TEER)
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using a Millicell Electrical Resistance System (Millipore, Merck KGaA, Darmstadt, Germany). Caco-2
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cultures with TEER values above 800 Ω · cm2, indicating tightness of the junctions between intestinal
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epithelial cells, were used in the experiments. Moreover, the apical−basolateral permeability of FSA
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was measured as a transport marker. At physiological pH, FSA is a hydrophilic, charged molecule with
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a molecular weight of 478 Da and is considered to be cell impermeable.19 The samples taken from the
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basolateral side of the Caco-2 cultures were analyzed at 495/538 nm using a Tecan M200 Infinite
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microplate reader (Tecan Group Ltd., Männedorf, Switzerland).
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RAW264.7 macrophages at passage number 6−9 were seeded onto 24-well plates at a density of 4 · 105 cells/cm2 and incubated for 24 hours to allow cells to adhere completely to the wells. 6 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
Anti-inflammatory experiment design
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A co-culture model consisting of a differentiated 21-day Caco-2 cell monolayer (placed in the
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apical compartment) and a 24-hour culture of RAW264.7 macrophages (located in the basolateral
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compartment of the bicameral chamber) was used to determine the inflammatory response induced by
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non-digested and gastrointestinal-digested BC extracts.
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To evaluate the anti-inflammatory effect, the BC extracts were added at a concentration of 1 mg
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BC powder/ml DMEM to the apical side of the co-culture system and incubated on a plate shaker (120
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rpm) under standard culture conditions. Budesonide (1 µM) with an anti-inflammatory potential was
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applied to the apical side as a positive control, instead of the BC extracts. The cell culture system
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containing only medium (DMEM) in the apical compartment was regarded as a negative control.
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Following the 3-hour incubation, LPS from E. coli O127 were added at a dose of 5 ng/ml to the
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basolateral compartment to stimulate the RAW264.7 macrophages. After 3-hour LPS treatment, the
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basolateral culture medium was centrifuged (1000 g, 5 min) and collected for TNF-α, IL-6, PGE2 and
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nitric oxide (NO) measurements. The Caco-2 and RAW264.7 cells were harvested for total RNA
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isolation and inflammatory gene expression analysis by real-time PCR.
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Cytotoxicity assay
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To detect the cytotoxic effect of the BC extract, LPS and budesonide on the Caco-2 and
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RAW264.7 cells under experimental conditions, the release of lactate dehydrogenase (LDH) was
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determined using a CytoTox-One™ Homogeneous Membrane Integrity Assay, according to the
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manufacturer’s protocol.
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Measurement of intracellular ROS production
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The effect of BC extracts on intracellular reactive oxygen species (ROS) generation was evaluated
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using the DCFH-DA assay described by Bass et al.20 with some modifications.12 The Caco-2 cells were
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treated with the BC extract (0.01; 0.1 and 1 mg BC powder/ml) both in the presence and absence of
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100 µM hydrogen peroxide (H2O2) as an oxidative stress inductor. Intracellular dichlorofluorescein
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(DCF) fluorescence of the Caco-2 cells was analyzed at 488/530 nm by flow cytometry (BD FACS 7 ACS Paragon Plus Environment
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AriaTM III, BD Bioscience, San Jose, CA, USA). The mean fluorescence intensity was quantified by
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FACSDiva™ Version 6.1.3 software (BD Bioscience, San Jose, CA, USA).
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Detection of DNA damage
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Single cell gel electrophoresis (SCGE) was used to analyze the DNA damage induced in the Caco-
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2 cells by treatment with the BC extracts (0.01, 0.1, 1 and 10 mg of BC powder/ml) and/or H2O2 (100
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µM). The cells were incubated with non-digested or gastrointestinal-digested extract and oxidant at 37
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ºC for 30 min. After exposure, the cells were prepared for SCGE and analyzed in terms of DNA
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damage according to the previously described procedure.12 Data on the DNA strand breaks were
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expressed as the mean percentage of DNA in the comet tail using CometScoreTM software (TriTek
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Corp., Sumerduck, VA, USA).
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Quantitative analysis of inflammatory gene expression
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Total RNA was isolated from the Caco-2 cells and RAW264.7 macrophages using TRI-Reagent
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according to the manufacturer’s instructions. The RNA samples were DNase-treated by incubation at
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37 °C for 15 min with the reaction buffer and RNase-free DNase I recombinant. The quantity and
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purity of the RNA were determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher
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Scientific, Waltham, USA), and the ratio of absorbance at 260/280 nm of all the samples was higher
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than 2. The total RNA (0.5 µg) was reverse-transcribed using a Transcriptor First Strand cDNA
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Synthesis kit, following the manufacturer’s protocol. The resulting cDNA was amplified using a real-
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time quantitative PCR system (SmartCycler DX real-time PCR System Cepheid, USA) with SYBR®
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Select Master Mix. PCR was performed using a final volume of 25 µl, including 10 ng sample cDNA,
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25 µM specific forward and reverse primers, and 12.5 µl SYBR® Select Master Mix. The primers
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designed with NCBI/Primer-Blast are shown as supporting information (Table S1). The following
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amplification conditions were used in real-time PCR: 10 min 94 °C, 40 × 40 s 95 °C, 30 s 59 °C, and
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30 s 72 °C. The purity of the PCR products was determined based on the melting curve analysis.
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The levels of transcripts IL-1α, IL-1β, IL-6, TNF-α, prostaglandin-endoperoxide synthase 2
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(Ptgs2, commonly referred to as COX-2), and iNOS (also known as NOS2) in the RAW264.7 cells and 8 ACS Paragon Plus Environment
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the levels of IL-8, TNF-α, and COX-2 in the Caco-2 cells were normalized to that of β-actin and
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glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. The relative amount of each gene
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was calculated using the 2-∆∆CT method.21 The levels of each different mRNA in the cells of the control
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co-culture system (treated only with LPS) were designated as 1, and the relative levels of the gene
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transcripts in the samples were expressed as the fold change.
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Quantification of IL-6, TNF-α α and PGE2 production
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The secretion of IL-6, TNF-α and PGE2 into the basolateral culture medium by RAW264.7
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macrophages was quantified using ELISA kits according to the manufacturer’s instructions.
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Measurement of NO production
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The quantity of nitrite, considered as an indicator of NO production, was determined using the
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Griess reagent, as described previously.12
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Statistical analysis
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Physicochemical and biological analyses were conducted in triplicate for each non-digested and
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gastrointestinal-digested BC extract obtained from three independent digestive experiments. Statistical
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analysis was performed using STATISTICA version 12.0 software (Statsoft, Inc., Tulsa, OK, USA).
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All data were expressed as the means ± SD. Two-way analysis of variance (ANOVA) followed by
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Tukey’s post hoc test was used to determine the differences between the mean values of multiple
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groups. Student’s t tests were applied to measure the significance of the difference in the means
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between two distributions. Distributional assumptions were assessed graphically by histograms and by
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Shapiro-Wilk test on the observations as a whole and for each individual. Moreover, the equality of
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variances assumption was verified using Levene's test. Statistical significance was considered at p
0.05) from the untreated cells constituting the control cell culture system. The BC extracts at
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concentrations lower than 1.5 mg/ml and the fraction of transported BC extract did not show any
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cytotoxic effects on differentiated Caco-2 cells and RAW264.7 macrophages, respectively (data not
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shown).
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Effect on transepithelial intestinal permeability
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Differentiated Caco-2 cells co-cultured with LPS-stimulated RAW264.7 macrophages showed a
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significant reduction in TEER (26.8%, p < 0.01). The supplementation of the Caco-2/RAW264.7
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model with non-digested or gastrointestinal-digested BC extracts counteracted this phenomenon.
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Similarly, budesonide (1 µM) completely inhibited the proinflammatory effect of the activated
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RAW264.7 cells on Caco-2 cell monolayer integrity (Figure 4A).
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Effect on inflammatory gene expression and mediator production
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The expression of proinflammatory genes was analyzed in the intestinal Caco-2 cells after
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treatment with non-digested and gastrointestinal-digested extracts with the simultaneous LPS 13 ACS Paragon Plus Environment
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activation of RAW264.7 macrophages. As shown in Figure 4B, the BC extracts were found to suppress
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the expression of transcript copies of the proinflammatory mediators IL-8 and COX-2. The
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gastrointestinal-digested BC extract down-regulated IL-8 and COX-2 mRNA expression levels by
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54% (p < 0.001) and 17% (p < 0.05), respectively. In contrast, the influence of the BC extracts on
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TNF-α mRNA expression in the Caco-2 cells was not observed (Figure 4B).
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The intestinal permeable fractions of the non-digested and gastrointestinal-digested BC extracts
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significantly inhibited LPS-induced NO generation (31 ± 9%, p < 0.05) (Figure 5D) and PGE2
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secretion (40 ± 20%, p < 0.05) (Figure 5C) by RAW264.7 macrophages. They caused a decrease in the
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mRNA levels of iNOS (15 ± 7%, p < 0.05) and COX-2 (44 ± 6%, p < 0.001) (Figure 5B), the
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corresponding enzymes catalyzing the production of NO and PGE2, respectively. In addition, the
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extracts’ permeable fractions down-regulated the excessive expression of proinflammatory cytokines,
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such as IL-1α, IL-1β and IL-6. The bioavailable fraction of gastrointestinal-digested BC extract
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penetrating across the Caco-2 monolayer inhibited the mRNA expression of IL-1β and IL-6 in
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RAW264.7 macrophages stimulated with LPS by 91 ± 2% (p < 0.0001) and 61 ± 5% (p < 0.001),
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respectively (Figure 5A). The significant inhibitory effect was also evident in reduced TNF-α
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secretion. When the Caco-2/RAW264.7 culture system was stimulated with LPS, approx. 343 ng/ml
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TNF-α was produced in the basolateral compartment (Figure 5C). Following the exposure of
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RAW264.7 macrophages to the intestinal permeable fractions of non-digested and gastrointestinal-
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digested BC extracts, the production of TNF-α was decreased by 12 ± 6% (p < 0.05) and 24 ± 5% (p
