Isolation and Characterization of Blueberry Polyphenolic Components

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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Isolation and Characterization of Blueberry Polyphenolic Components and Their Effects on Gut Barrier Dysfunction Michael A. Polewski,* Daniel Esquivel-Alvarado, Nicholas S. Wedde, Christian G. Kruger, and Jess D. Reed

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Reed Research Group, Department of Animal Sciences, University of WisconsinMadison, 1675 Observatory Drive, Madison, Wisconsin 53706, United States ABSTRACT: Highbush blueberries contain anthocyanins and proanthocyanidins that have antimicrobial and antiinflammatory bioactivities. We isolated and characterized three polyphenolic fractions, a total polyphenol fraction (TPF), an anthocyanin-enriched fraction (AEF), and a proanthocyanidin-enriched fraction (PEF), from freeze-dried blueberry powder and evaluated their effects on an in vitro model of gut barrier dysfunction. High-performance liquid chromatography chromatograms illustrate successful fractionation of the blueberry powder into TPF, AEF, and PEF. AEF contained 21 anthocyanins, and PEF contained proanthocyanidin oligomers of (epi)catechin with primarily B-type interflavan bonds. The model uses a strain of Escherichia coli to disrupt a Caco-2 cell monolayer on Transwell inserts. Barrier function was measured by transepithelial electrical resistance (TEER), a marker of membrane permeability. All fractions were able to restore TEER values after an E. coli challenge when compared to the control, while AEF was able to attenuate the E. coli-induced decrease in TEER in a dosedependent manner. KEYWORDS: blueberry, anthocyanins, proanthocyanidins, E. coli, gut barrier dysfunction, transepithelial electrical resistance

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required to have effects in cell culture and ex vivo studies.11 Given that polyphenols have low bioavailability, they may have several effects within the gut lumen and on the gut mucosa as previous research in mice has demonstrated.12,13 Intestinal epithelial cells grown on Transwell inserts are used to determine the pharmacological and toxicological effects of substances on GBF.14 Such models can adequately reproduce both healthy and inflamed intestinal tissue that provide a useful tool for elucidating the mechanisms of GBF, intestinal inflammation, and absorption of therapeutic compounds.15 In Transwells, the function of the epithelial cell barrier is determined by measuring transepithelial electrical resistance (TEER), which is a surrogate marker of membrane permeability. TEER is widely accepted as a quantitative technique to measure the integrity of tight junction dynamics between epithelial cells in studies of drug transport and barrier disruption.16 In this study, we isolated and characterized three chromatographic fractions, a total polyphenolic fraction (TPF), an anthocyanin-enriched fraction (AEF), and a proanthocyanidinenriched fraction (PEF), from freeze-dried whole highbush blueberry powder. We then evaluated each fraction for its ability to attenuate E. coli-induced reduction in TEER. Our hypothesis was that the presence of blueberry polyphenols would attenuate the E. coli-induced reduction of TEER and

INTRODUCTION The gut epithelium is the largest mucosal surface of the body and is continuously exposed to potentially pathogenic microorganisms, toxins, and antigens. The gut barrier protects the body from these potentially harmful luminal components while allowing for absorption of nutrients and minerals. Dysfunction of the gut barrier allows for passage of harmful luminal substances, which results in an excessive immune response that contributes to systemic inflammation and disease.1,2 High-fat and high-sugar (HF/HS) diets increase dysbiosis and susceptibility to invasion by pathogenic bacteria, particularly Escherichia coli.3 In addition, HF/HS diets are predisposing factors that lead to increased gut permeability and subsequent gut barrier dysfunction.4,5 The negative effects of a HF/HS diet on gut barrier function (GBF), gut inflammation, and circulating lipopolysaccharide can be attenuated by a cranberry extract that was enriched in cranberry polyphenolic components as recent research has demonstrated.4 Increased exposure of the gut lumen to significant amounts of polyphenols from foods may protect the intestinal epithelium against different luminal challenges, thus helping to promote intestinal health and mitigate chronic intestinal and metabolic diseases.6 In particular, highbush blueberries (Vaccinium corymbosum) contain anthocyanins and proanthocyanidins that have antimicrobial and anti-inflammatory effects; however, there has been little research on how these polyphenols affect GBF.7−9 Much of the health research on polyphenolic compounds in foods has focused on post-absorptive effects of native compounds and their metabolites in peripheral tissues.10 However, polyphenolic compounds and their metabolites do not reach high concentrations in systemic circulation and peripheral tissues and are often below a concentration that is © XXXX American Chemical Society

Special Issue: Advances in Polyphenol Chemistry: Implications for Nutrition, Health, and the Environment Received: March 15, 2019 Revised: May 31, 2019 Accepted: June 4, 2019

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DOI: 10.1021/acs.jafc.9b01689 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry that the three fractions of polyphenols would differ in their ability to attenuate the reduction.

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MATERIALS AND METHODS

Chemicals and Reagents. Water, methanol, acetone [highperformance liquid chromatography (HPLC) grade], sodium carbonate, sodium chloride, agar, Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin (Pen/Strep) mixture (10 000 units of each antibiotic/mL), gentamicin sulfate (50 mg/ mL), non-essential amino acid (NEAA) solution (100×), GlutaGRO supplement (200 mM L -alanyl- L-glutamine), and Dulbecco’s phosphate-buffered saline solution 10× (D-PBS) with calcium and magnesium (PBS + Ca2+/Mg2+, 0.1 g/L CaCl2 and MgCl2) were purchased from Fisher Scientific (Fair Lawn, NJ, U.S.A.). Ethanol (200 proof) was obtained from Decon Laboratories, Inc. (King of Prussia, PA, U.S.A.). Sterilized water, thyamin hydrochloride, 2,5dihydroxybenzoic acid (DHB), gallic acid, Folin−Ciocalteu reagent, and Triton X -100 were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Sephadex LH-20 was purchased from GE Healthcare (Uppsala, Sweden). Tryptose and dextrose were obtained from BD Biosciences (Sparks, MD, U.S.A.). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Lawrenceville, GA, U.S.A.). HyClone D-PBS without calcium and magnesium and 0.25% HyClone trypsin in 0.1% ethylenediaminetetraacetic acid (EDTA) were obtained from Thermo Scientific (South Logan, UT, U.S.A.). Caco-2 epithelial cells (HTB-37) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, U.S.A.). Samples. The freeze-dried blueberry powder (FDBP) was a 50:50 blend of ‘Tifblue’ and ‘Rubel’ cultivars. The FDBP was supplied by the United States Highbush Blueberry Council (USHBC) in a vacuum-sealed aluminum container to prevent oxidation and ensure maximum shelf life. Fractionation of the FDBP. The FDBP (10 g) was extracted with 70% acetone (v/v, 100 mL) in an ultrasonic bath for 20 min. Previous reports indicate that an optimal total polyphenol extraction from berries can be obtained with 70% acetone.17,18 The extract was centrifuged at 1800g and 20 °C for 15 min, and the supernatant was collected. Acetone was removed by evaporation under vacuum at 35 °C. The aqueous extract was loaded onto a Hypersep C18 cartridge that was previously activated with methanol and equilibrated with water. The aqueous extract was washed with 3 bed volumes of water and eluted with 3 bed volumes of methanol to obtain TPF. An aliquot of the TPF was concentrated to dryness, suspended in water, and loaded onto a glass column (4 cm internal diameter × 15 cm length, Kontes) that was packed with Sephadex LH-20 (18−111 μm, GE Healthcare), which was equilibrated in water. The TPF was eluted with water, 50% (v/v) ethanol, and 80% (v/v) acetone. The 50% (v/ v) ethanol fraction was defined as the AEF, and the 80% (v/v) acetone fraction was defined as the PEF (Figure 1). All three fractions were evaporated to dryness as described above and solubilized in methanol. The total phenolic content of the three fractions was quantified using the Folin−Ciocalteu assay, and concentrations were expressed as gallic acid equivalents (GAE), so that equivalent concentrations of each fraction could be tested in in vitro studies. Reversed-Phase High-Performance Liquid Chromatography with a Diode Array Detector (RP-HPLC−DAD) Analysis. Analysis of the fractions was performed using a HPLC system that consisted of a Rheodyne 7125 manual injector, two Waters 501 HPLC pumps, a Waters 2998 DAD, and a Zorbax SB-C18 column (4.6 mm × 150 mm × 5 μm) with a C18 guard column. Prior to injection, fractions were filtered with a polytetrafluoroethylene (PTFE) membrane (0.45 μm, with a diameter of 30 mm). The injection volume was 20 μL, with a flow rate of 1.5 mL at 30 °C. The mobile phase consisted of two solvents, (A) water and (B) methanol, both with 10% (v/v) formic acid. The gradient elution program was as follows: the first 2 min, isocratic at 5% B; 2−4 min, B increased linearly from 5 to 25%; 4−25 min, B increased linearly from 25 to 40%; 25−30 min, B increased linearly from 40 to 50%; and 30−33 min, B increased linearly from 50 to 99%, followed by reconditioning

Figure 1. Separation strategy used to obtain the three polyphenolic fractions. The blueberry TPF was obtained via C18 chromatography. The blueberry AEF and PEF were further separated from TPF using LH-20 chromatography. of the column. Empower Pro software was used for collecting and analyzing three-dimensional chromatograms. Electrospray Ionization Tandem Mass Spectrometry (ESI− MS/MS) Analysis. Characterization of AEF was performed on an Agilent 1200 series system (Agilent Technologies, Santa Clara, CA, U.S.A.), which was equipped with an API 4000QTrap triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, U.S.A.). Prior to injection, each fraction was filtered using a PTFE membrane (0.22 μm, with a diameter of 17 mm). A total of 8 μL was injected into a Zorbax-SB C18 column (4.6 mm × 250 mm × 10 μm) set at 35 °C. The mobile phase consisted of two solvents, (A) water and (B) methanol, both with 5% (v/v) formic acid at a flow rate of 0.4 mL/min. The gradient elution program was as follows: the first 6 min, isocratic at 10% B, 6−17 min, B increased linearly from 10 to 40%; and 17−30 min, B increased linearly from 40 to 70%, followed by reconditioning of the column. The flow generated by the chromatographic system was introduced directly into the ESI source, and the following parameters were employed: positive ionization mode, curtain gas (CUR, 30), ion spray (IS, 4000 V), turbo gas temperature (TEM, 375 °C), nebulizer gas pressure (GS1, 25 psi), and gas turbo (GS2, 18 psi). Identification of individual compounds was based on MS/MS spectral data, which matched those reported in existing literature.19 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI−TOF MS). MALDI−TOF MS methodology was applied to characterize PEF.20 Mass spectra were collected on a Bruker Microflex mass spectrometer (Billerica, MA, U.S.A.). PEF at a concentration of 20 mg/mL was mixed with a 0.64 M solution of 2,5-dihydroxybenzoic acid (DHB) in methanol in a 1:1 volume ratio. An aliquot (0.7 μL) of the mixture was spotted onto a ground stainless-steel MALDI target using the dry droplet method. All analyses were performed in positive reflectron mode with a voltage source of 12 kV, a voltage pulse of 1.75 kV, and a reflectron voltage of 2.5−5.2 kV. Deflection was set at 800 Da. Bradykinin [1060.6 molecular weight (MW)] and glucagon (3483.8 MW) were used as external standards. FlexControl and FlexAnalysis (version 3.0, Bruker Daltonik GmbH, Bremen, Germany) were used for data acquisition and data processing, respectively. Deconvolution Method. A previously developed method for deconvolution of overlapping isotope patterns in MALDI spectra was used to quantify the percentage of A- to B-type interflavan bonds at each degree of polymerization in proanthocyanidins.21 Data were excluded from the analysis when one of the peaks included in the deconvolution of the isotope pattern had a signal/noise ratio of less than 3.0. Bacterial Culture. The E. coli strain (E. coli-5011) used in our studies was a clinical isolate obtained from the University of B

DOI: 10.1021/acs.jafc.9b01689 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Wisconsin Hospital and Clinics. The strain was characterized to be genotypically representative of extra-intestinal pathogenic E. coli (ExPEC) and found to express both P and type 1 fimbriae. These are virulence factors for ExPEC colonization of the gut and are involved in attachment and invasion of E. coli to epithelial cells in the urinary tract, colon, and ileum.8,22 E. coli was cultured from frozen stock under static culture conditions in tryptose broth (10 g of tryptose, 2.5 g of sodium chloride, 0.5 g of dextrose, and 0.0025 g of thyamin hydrochloride in 500 mL of deionized water) at 37 °C and washed twice with 1× PBS + Ca2+/Mg2+ by centrifugation at 1800g for 10 min. The optical density of the inoculum suspension at 450 nm was used to calculate and adjust the bacterial cell density using a previously established bacterial density−absorbance curve.11 Gut Epithelial Cell Culture. Caco-2 (HTB-37) epithelial cells were cultured in complete media (DMEM supplemented with 10% FBS, 1% non-essential amino acids, 1% L-alanyl-L-glutamine, and 1% penicillin (100 units/mL)/streptomycin (100 μg/mL)] at 37 °C in 5% CO2. Caco-2 cells were subcultured following a low-density protocol and seeded for experiments at 6.75 × 104 cells/Transwell into 24-well Transwell inserts (MilliporeSigma).23 The apical and basolateral compartments of the Transwells were maintained in complete media for 10 days, with media in both compartments changed every 2 days. On day 10, the media in the apical compartment was switched to serum-free complete media, while the basolateral media remained in complete media.24 This protocol aided in epithelial cell differentiation and more accurately resembled an in vivo environment.25 In addition, the lack of serum in the apical compartment reduced the variation when E. coli and treatment(s) were added. On day 12, TEER values were recorded to obtain a baseline reading and to ensure that the Caco-2 cells were sufficiently differentiated (TEER > 450 Ω cm2). TEER. Transwell plates were removed from the incubator and allowed to equilibrate to room temperature in a Biological Safety Cabinet (BSC) for 10 min before measurements were recorded. Electrical resistance was measured using a Millicell-ERS resistance system (MilliporeSigma) until similar values were recorded on three consecutive measurements. TEER was calculated as

Figure 2. Experimental timeline of cell seeding, E. coli challenge, addition of blueberry fraction treatment, and TEER measurements of Caco-2 cells on Transwell inserts.

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RESULTS AND DISCUSSION RP-HPLC−DAD Analysis. The fractionation of FDBP with C18 and subsequent LH-20 resins was analyzed using RPHPLC−DAD (Figure 3). λmax of phenolic compounds may be used to classify compounds that are present in chromatographic fractions. λmax of proanthocyanidins is 280 nm, with no absorbance at higher wavelengths. λmax of hydroxycinnamic acids, flavonols, and anthocyanins is approximately 320, 370, and 520 nm, respectively. As seen in Figure 3A, these four wavelengths show multiple peaks, indicating that TPF contains a mixture of these four classes of compounds, including a broad, poorly resolved area of absorbance at 280 nm that is typical of proanthocyanidins. In contrast, the AEF chromatogram shows an increase in anthocyanins (520 nm) and a concurrent reduction in the other classes of compounds (Figure 3B). This reduction in the absorbance wavelengths that characterizes other polyphenols is a good indicator that AEF has a higher proportion of anthocyanins. The PEF chromatogram shows a broad unresolved peak associated with proanthocyanidins and the absence of absorbance at the other wavelengths (Figure 3C). Together, these three chromatograms provide evidence of successful fractionation of blueberry polyphenols into TPF, AEF, and PEF by C18 and LH-20 chromatography. Characterization of AEF by ESI−MS/MS. A total of 21 anthocyanins were detected in AEF (Table 1). The presumed structural assignment was made on the basis of the MS2 spectral data. AEF contained the 6 most common of the 19 naturally occurring anthocyanidins: malvidin, petunidin, delphinidin, peonidin, cyanidin, and pelargonidin.26 Glycosylated (hexosides and pentosides) and acylated (p-coumaryl, malonyl, and acetyl) anthocyanidins were also detected. Free anthocyanidins were not detected in AEF. The relative ion area was calculated by summing up intensities of all mass spectral peaks and dividing this total by the specific compounds. The predominant anthocyanins in AEF were malvidin-3-O-hexosides and malvidin-3-O-pentosides. For both anthocyanins, the MS2 mass fragment was m/z 331, which suggested the loss of one hexose (m/z 162) and one pentose (m/z 132), respectively. Previous reports indicate that the main blueberry anthocyanins are derived from delphinidin, petunidin, and malvidin. Our results are consistent with previously published findings.19 Characterization of PEF by MALDI−TOF MS. The MALDI−TOF MS spectra of PEF acquired in reflectron mode show masses that correspond to an oligomeric series of (epi)catechin units (288 Da) up to the nonamers (Figure 4). MALDI−TOF MS spectra showed structural variation in the nature of the interflavan bond (A and B types, 2 Da). Our results suggest that blueberry proanthocyanidins in PEF contain predominantly “B-type” interflavan bonds. The oligomeric series of (epi)catechin units can be explained

TEER = (R m − R i) − A where Rm is transmembrane resistance, Ri is intrinsic resistance of cellfree media, and A is the surface area of the membrane in cm2. E. coli-Induced Reduction in TEER. On day 14, Caco-2 cells seeded on Transwell inserts were challenged with E. coli. On the day of the challenge, E. coli was brought to 5 × 104 colony-forming units (CFU)/mL in serum and antibiotic-free complete media, so that a multiplicity of infection (MOI) of 0.1:1 was achieved. Both compartments of the Transwell were washed with D-PBS. The apical compartment was replaced with 200 μL of E. coli-containing media per Transwell, and the basolateral compartment was replaced with 900 μL of antibiotic-free complete media per Transwell. To evaluate the efficacy of the three blueberry fractions, varying concentrations of each fraction were added simultaneously to the apical compartment concurrently with E. coli. TEER values were measured before and after the E. coli challenge (t = 0 and 24). After 24 h, cells were washed with D-PBS and incubated in complete media with 100 μg/mL gentamycin for 1 h to kill any adhered bacteria. Gentamycin media were then removed, and cells were washed with D-PBS. Serum-free complete media with or without the blueberry fractions were again added to the apical compartment. TEER values were recorded 24 h (t = 48) and 48 h (t = 72) after the E. coli challenge to monitor recovery of the epithelial cell layer (Figure 2). Data and Statistical Analysis. All data are reported as the mean ± standard deviation of at least three replicates. Statistical analysis was performed using SAS (version 9.4, SAS Institute, Inc.), setting α = 0.05. Results were analyzed with two-way analysis of variance (ANOVA) models with interactions between the independent variables “time”, “concentration”, and “fraction” to assess significant differences. The dependent variable was TEER. mMass version 5.5.0 was used for mass spectra analysis. C

DOI: 10.1021/acs.jafc.9b01689 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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hexose (162 Da) to the (epi)catechin units was detected. The ratio of A-/B-type interflavan bonds was calculated using a previously described deconvolution method (Figure 5).21 Approximately 41% of proanthocyanidins in PEF contained one or more “A-type” interflavan bond. E. coli-Induced Reduction in TEER. We developed a model of gut barrier dysfunction that uses a strain of E. coli to induce a reduction in TEER to identify which fractions from FDBP attenuated this response. In this model, E. coli causes a reduction in TEER via attachment and invasion of the Caco-2 cell monolayer, which represents the first step in a mucosal inflammatory response.27 Using a pathogenic microbe is a more physiologically relevant stimulus of gut barrier dysfunction than the use of pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), or inflammatory cytokines, such as interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), or interferon γ (IFNγ).28 Using this model, we demonstrated that when Caco-2 cells are challenged with E. coli at a multiplicity of infection (MOI) of 0.1:1 for 24 h, TEER values decline significantly (p < 0.05; Figure 6). TEER values remain suppressed until 48 h after the E. coli challenge. At 72 h, TEER values started to recover. The ability of this strain of E. coli to illicit a drop in TEER can be attributed to the expression of virulence factors that allows for adhesion, invasion, and colonization of epithelial cells. These processes alter intestinal permeability by inducing the expression of the pore-forming proteins and displacing protein from apical tight junctions, thus leading to decreased transepithelial resistance and loss of barrier function.8,29 The continued decrease in TEER values 24 h after the removal of E. coli may be caused by invaded bacteria that replicate inside the Caco-2 cells and further damage tight junctions of the cell barrier. Alternatively, invaded bacteria could destroy cells or be expelled and then invade new cells. The increase in TEER values at 48 h after the removal of E. coli may occur because of a change in media that removes any expelled bacteria coupled with the presence of antibiotics that kill newly expelled bacteria. In addition, epithelial cells can continue to grow and replenish destroyed areas of the confluent monolayer. Effect of TPF, AEF, and PEF on E. coli-Induced Reduction in TEER. We evaluated all three fractions for their ability to attenuate the E. coli-induced reduction in TEER. Results in Figure 7 illustrate the effect of the three blueberry fractions on E. coli-induced reduction in TEER over a 72 h period. During the 24 h E. coli challenge, TPF (Figure 7A) and AEF (Figure 7B) at all concentrations were neither different from nor better than the (negative) E. coli control, while PEF was neither different from nor worse than the E. coli control. A twoway ANOVA showed significant (p < 0.001) effects caused by fractions, concentrations, and times. When AEF at 50 and 100 μg of GAE/mL was compared to the TPF and PEF at equivalent concentrations, there was a significant effect (p < 0.05) on TEER values. This attenuation in the E. coli-induced reduction in TEER by AEF suggests that anthocyanins are interfering with the mechanism by which E. coli causes a drop in TEER. While not directly tested, we speculate that there could be a direct interaction between E. coli and AEF that results in the attenuation of the E. coli-induced reduction in TEER after 24 h. Previous research has indicated that components of AEF, particularly anthocyanins, have antimicrobial and anti-adhesive effects that inhibit the growth of pathogenic bacteria, including E. coli.30 Furthermore, compo-

Figure 3. HPLC chromatograms recorded at 280, 320, 370, and 520 nm of the three blueberry fractions: (A) TPF, (B) AEF, and (C) PEF.

according to the equation m/z = 290 + 288d − 2A + b, where 290 represents the molecular weight of the terminal (epi)catechin unit, d is the number of (epi)catechin extension units, A is the number of A-type interflavan bonds, and b is the molecular weight of sodium cations (23 Da). Mass differences of 16 Da from the oligomeric series of (epi)catechin units are also observed and can be explained by an additional hydroxyl substitution in the B ring of the repeating flavan-3-ol unit or the splitting of the signal as a result of sodium and potassium adducts. In addition, an additive mass corresponding to one D

DOI: 10.1021/acs.jafc.9b01689 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Anthocyanin Profiles Obtained for AEF by HPLC−ESI−MS/MS anthocyanina

structure

relative ion areab (%)

molecular weight (m/z)

aglycon (m/z)

anthocyanidin

moiety

malvidin-3-O-hexoside malvidin-3-O-pentoside petunidin-3-O-hexoside delphinidin-3-O-hexoside peonidin-3-O-hexoside petunidin-3-O-pentoside delphinidin-3-O-pentoside cyanidin-3-O-hexoside peonidin-3-O-pentoside cyanidin-3-O-pentoside delphinidin-3-O-(6″-p-coumaryl)hexoside petunidin-3-O-(6″-p-coumary)hexoside delphinidin-3-O-(6″-O-malonyl)hexoside malvidin-3-O-(6″-O-acetyl)hexoside pelargonidin-3-O-hexoside cyanidin-3-O-(6″-coumaryl)hexoside cyanidin-3-O-(6″-O-malonyl)hexoside petunidin-3-O-(6″-O-acetyl)hexoside delphinidin-3-O-(6″-O-acetyl)hexoside pelargonidin-3-O-pentoside malvidin-3-O-(6-O-coumaryl)hexoside

C23H25O12+ C22H23O11+ C22H23O12+ C21H21O12+ C22H23O11+ C21H21O11+ C20H19O11+ C21H21O11+ C21H21O10+ C20H19O10+ C30H27O14+ C31H29O14+ C24H23O15+ C25H27O13+ C21H21O10+ C30H27O13+ C24H23O14+ C24H25O13+ C23H23O13+ C20H19O9+ C32H31O14+

39.43 10.58 9.67 9.52 8.26 5.04 4.61 4.27 4.18 3.45 0.3 0.28 0.18 0.05 0.05 0.04 0.03 0.02 0.01 0.01 0.01

493.43 463.41 479.41 465.38 463.41 449.38 435.35 449.38 433.38 419.35 611.52 625.55 551.42 535.47 433.38 595.52 535.43 521.44 507.42 403.36 639.57

331.3 331.3 317.27 303.24 301.27 317.27 303.24 287.24 301.27 287.24 303.24 317.27 303.24 331.3 271.24 287.24 287.24 317.27 303.24 271.24 331.3

malvidin malvidin petunidin delphinidin peonidin petunidin delphinidin cyanidin peonidin cyanidin delphinidin petunidin delphinidin malvidin pelargonidin cyanidin cyanidin petunidin delphinidin pelargonidin malvidin

hexoside pentoside hexoside hexoside hexoside pentoside pentoside hexoside pentoside pentoside coumaryl-hexoside coumaryl-hexoside malonyl-hexoside acetyl-hexoside hexoside coumaryl-hexoside malonyl-hexoside acetyl-hexoside acetyl-hexoside pentoside coumaryl-hexoside

a Anthocyanins were identified on the basis of the combined information on ultraviolet/visible (UV/vis), molecular ion, and mass fragments. bThe percentage of anthocyanin was based on the relative ion area.

Figure 4. Mass spectra obtained with MALDI−TOF MS in positive reflectron mode for PEF. “r.int.” and “DP” correspond to relative intensity and degree of polymerization, respectively. (Inset) Enlarged spectrum to assist with visualizing the isotopic distribution of potassium adducts of proanthocyanidin (PAC) tetramers.

and 100 μg of GAE/mL restored TEER values to the same level as the positive control. In addition, these concentrations of AEF were better at restoring TEER values than both TPF and PEF at equivalent concentrations. These results are supported by previous results that indicate that anthocyanins are able to protect epithelial cells from a reduction in TEER when challenged with a cytokine.33 The results in Table 1 indicate that AEF has a high content of malvidin, and we speculate that the chemical structure and conformation of these anthocyanins are responsible for the observed effects. This is in contrast to a previous study that showed that

nents in AEF could have a direct interaction on the expression of E. coli virulence factors that prevent them from adhering to and invading epithelial cells.31,32 Future studies should evaluate the growth kinetics of E. coli in the presence of these fractions to determine any bactericidal or bacteriostatic effects. In addition, future experiments should evaluate the expression of specific virulence factors when E. coli is grown in the presence of these fractions. At 48 h, only TPF and AEF at concentrations greater than or equal to 25 μg of GAE/mL were able to improve TEER values when compared to their respective E. coli controls. AEF at 50 E

DOI: 10.1021/acs.jafc.9b01689 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 5. Deconvolution of MALDI−TOF positive reflectron mode mass spectra of PEF. Relative percentage of proanthocyanidins that contain one or more A-type interflavan bonds at a degree of polymerization between the dimer (2) and heptamer (7).

Figure 7. Effect of E. coli (MOI of 0.1:1) added simultaneously with either (A) TPF, (B) AEF, or (C) PEF on Caco-2 TEER. TEER was measured sequentially over a 72 h experimental period as described in the Materials and Methods. The apical compartment was simultaneously stimulated with E. coli (MOI of 0.1:1) and either TPF, AEF, or PEF (10 25, 50, or 100 μg of GAE/mL) for 24 h. After 24 h, TEER values were measured, cells were washed to remove E. coli, and the media were replenished. TEER values were measured again at 48 and 72 h after the E. coli challenge. Results are the mean ± SD of three replicates per treatment. Results were analyzed by two-way ANOVA with α = 0.05.

Figure 6. Effect of E. coli (MOI of 0.1:1) on Caco-2 TEER was measured sequentially over a 72 h experimental period as described in the Materials and Methods. E. coli causes a time-dependent drop in Caco-2 TEER. Results are the mean ± standard deviation (SD) of two independent experiments. Treatments not connected by the same letter are significantly different (p = 0.05) as analyzed by one-way ANOVA.

improve TEER values when compared to their respective E. coli controls. In contrast to the observed effects at 48 h, TPF at 50 and 100 μg of GAE/mL restored TEER values to the same level as the positive control treatment. In addition, these concentrations of TPF significantly increased TEER values in comparison to AEF and PEF at equivalent concentrations. The decrease in TEER at 72 h by AEF may be caused by the loss of active compounds through metabolism because apical media and treatments were replaced at 24 h but not at 48 h. PEF (Figure 7C) did not prevent the E. coli-induced reduction in TEER during the 24 h E. coli challenge when compared to the control. PEF concentrations of 25, 50, and 100 μg of GAE/mL caused a further reduction in TEER when compared to the E. coli control at 24 h. At 48 and 72 h, only the 50 μg of GAE/mL concentration of PEF restored TEER values when compared to its E. coli control. Furthermore, all PEF concentrations were lower than AEF and TPF at preventing and restoring TEER at 24, 48, and 72 h at equivalent concentrations. The negative effect of PEF on TEER values at 24 h and the delayed ability to restore TEER values at 48 and 72 h suggest an interaction of PEF with the epithelial cell membrane. The ability of proanthocyanidins to interact with membrane lipids and cell surface proteins is an important feature of their biological activity, especially at higher degrees of polymerization. 37 While the direct

anthocyanin-rich extracts containing a high cyanidin and delphinidin content protected a Caco-2 monolayer from a cytokine-induced decrease of TEER, while extracts high in malvidin content were less effective.34 The difference in results could be explained by the time and type of challenge to the Caco-2 cell monolayer (cytokine versus bacteria) and the duration of treatments (hours versus days). While not directly measured, we also speculate that the positive effect of AEF on TEER recovery is associated with an increase in tight junction protein abundance in the cells. Tight junction proteins, such as zonulins, occludins, and claudins, seal and strengthen the paracellular space between epithelial cells, thus preventing the passage of microorganisms and other antigens through the epithelium.35 Previous work has suggested that anthocyanin-rich extracts prevent the downregulation of tight junction proteins in Caco-2 cells when challenged with a cytokine by inhibiting the nuclear factor κB (NF-κB) inflammatory signaling pathway.9,34,36 We speculate that AEF could be acting through similar mechanisms in our E. coli model, but future studies need to evaluate these blueberry fractions for effects on tight junction protein expression. At 72 h, TPF at concentrations greater than or equal to 25 μg of GAE/mL and all concentrations of the AEF were able to F

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Journal of Agricultural and Food Chemistry interaction of PEF and E. coli was not evaluated, we speculate that the presence of A-type proanthocyanidins in PEF may enhance in vitro bacterial anti-adhesion activities and aid in the observed effects seen with PEF.38 It is important to note that the effect of PEF on membrane permeability was not measured in these experiments and that TEER is a surrogate marker of membrane permeability based on electrical impedance. It is possible that the proanthocyanidins in PEF are artificially lowering TEER values rather than directly affecting membrane permeability by incorporating into the epithelial cell membrane and increasing electrical conductance.39 Future experiments need to correlate TEER measurements with a direct indicator of membrane permeability that measures transmembrane flux across the epithelial cell layer. In summary, our results show that we were able to isolate and characterize three polyphenolic fractions from freeze-dried blueberry powder. A total of 21 anthocyanins were identified in AEF by ESI−MS/MS. Condensed polymeric anthocyanins and proanthocyanidins up to nonamers were determined in PEF by MALDI−TOF MS. A deconvolution method showed that at least 88% of the proanthocyanidins in PEF contain one or more “B-type” interflavan bond. This research provides a useful in vitro model to simultaneously study the interaction of polyphenolic fractions with E. coli and an epithelial cell membrane. Using this model, we evaluated the three fractions for their ability to affect the E. coli-induced reduction of TEER. We show that blueberry polyphenols attenuate the E. coliinduced reduction in TEER and that the three fractions differ in their abilities to modulate this response. While all three fractions were able to recover TEER at 72 h when compared to E. coli alone, only AEF was able to limit the E. coli-induced reduction in TEER at 24 h. These findings further support the paradigm that consumption of berries or supplements containing polyphenol enrichments could afford beneficial health effects in the gastrointestinal tract. These beneficial effects could be applicable in conditions of chronic gut inflammation associated with inflammatory bowel disease and the consumption of simplified liquid diets. Further investigation is necessary to identify which specific components of AEF are responsible for this effect. Future research should also investigate the direct antimicrobial and antibiotic interactions of these blueberry polyphenolic fractions with E. coli. Such studies will aid in the development of innovative strategies for preventing or alleviating chronic inflammatory processes.

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cyanidin-enriched fraction; GBD, gut barrier dysfunction; TEER, transepithelial electrical resistance; PAC, proanthocyanidin

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 608-263-4314. E-mail: [email protected]. ORCID

Michael A. Polewski: 0000-0002-8759-2664 Daniel Esquivel-Alvarado: 0000-0003-3557-2562 Funding

The authors thank the United States Highbush Blueberry Council (USHBC) for providing the funding and the freezedried whole blueberry powder. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED FDBP, freeze-dried blueberry powder; TPF, total polyphenolic fraction; AEF, anthocyanin-enriched fraction; PEF, proanthoG

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