Anti-inflammatory activity of aronia berry extracts in murine splenocytes

journal of functional foods 8C (2014) 68–75

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Anti-inflammatory activity of aronia berry extracts in murine splenocytes Derek A. Martin a, Rod Taheri a, Mark H. Brand b, Andrew Draghi II c, Francisco A. Sylvester c,d, Bradley W. Bolling a,* a

Department of Nutritional Sciences, University of Connecticut, Storrs, CT, USA Department of Plant Science and Landscape Architecture, University of Connecticut, Storrs, CT, USA c Department of Pediatrics, University of Connecticut Health Center, Farmington, CT, USA d Department of Immunology, University of Connecticut Health Center, Farmington, CT, USA b

A R T I C L E

I N F O

A B S T R A C T

Article history:

Aronia berries are a rich source of dietary polyphenols, with diverse polyphenol profiles among

Received 13 January 2014

its genotypes. The objective of this work was to characterize the anti-inflammatory effects

Received in revised form 28

of underutilized aronia berries and their polyphenols using primary C57/BL6 mouse

February 2014

splenocytes. At 125 μg gallic acid equivalents/mL, the commercial ‘Viking’ aronia berry and

Accepted 5 March 2014

underutilized aronia extracts inhibited LPS-stimulated IL-6 to a similar extent. ‘Viking’

Available online 28 March 2014

extracts inhibited IL-6 predominately in CD4− lymphocytes. The primary polyphenol constituents of extracts were subsequently evaluated for inhibition of LPS-stimulated IL-6.

Keywords:

Cyanidin-3-arabinoside, but not the primary aronia anthocyanin cyanidin-3-galactoside, in-

Aronia

hibited IL-6 at 10 μg/mL. Quercetin, but not its 3-galactoside or glucoside, inhibited LPS-

Chokeberry

stimulated IL-6. Quercetin also inhibited LPS-stimulated IL-10, whereas ‘Viking’ extract

Anti-inflammatory

increased splenocyte IL-10 in the absence of LPS. Thus, the capacity of aronia extracts to

Immune

modulate LPS-stimulated splenocyte IL-6 and IL-10 in vitro was not attributed to its prin-

Polyphenol

cipal polyphenols.

Anthocyanin

1.

Introduction

Inflammation is central to the etiology of many chronic diseases including cancer, cardiovascular disease, and diabetes (Coussens, Zitvogel, & Palucka, 2013; Patel, Buras, & Balasubramanyam, 2013; Pearson et al., 2003). Obesity and autoimmune diseases such as multiple sclerosis, arthritis, and inflammatory bowel diseases are characterized by unresolved inflammation of adipose tissue, nervous tissue, joints, and the intestines, respectively (Abraham & Cho, 2009; Gregor & Hotamisligil, 2011; Park et al., 2011; Reynolds et al., 2011).

* Corresponding author. Tel.: 860-486-2180; fax: 860-486-3674. E-mail address: [email protected] (B.W. Bolling). http://dx.doi.org/10.1016/j.jff.2014.03.004 1756-4646/© 2014 Elsevier Ltd. All rights reserved.

© 2014 Elsevier Ltd. All rights reserved.

Therefore, there is increasing interest in identifying dietary components that could prevent or mitigate chronic inflammation. Berries are promising candidates for dietary interventions targeting chronic inflammation. A diet enriched in blackberries (10% w/w) had antiobesity and anti-inflammatory effects in ovariectomized rats (Kaume, Gilbert, Brownmiller, Howard, & Devareddy, 2012). Spontaneously hypertensive rats fed cranberry and lingonberry had reduced mRNA expression of aortic angiotensin-converting enzyme 1, cyclooxygenase 2, and monocyte chemoattractant protein 1 (Kivimaki et al., 2012). A polyphenol-rich wild blueberry extract inhibited

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lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) protein-DNA binding activity in BV2 murine microglial cells (Lau, Joseph, McDonald, & Kalt, 2009). Aronia berries, juices, and extracts exhibit anticancer, cardioprotective, antihypertensive, antidiabetic, antiinflammatory, and immunomodulatory activities in cellular and animal models (Hellstrom et al., 2010; Kim et al., 2013a; Kim, Park, Wegner, Bolling, & Lee, 2013b; Kokotkiewicz, Jaremicz, & Luczkiewicz, 2010; Sharif et al., 2013). Short-term human intervention studies have also demonstrated reduction in inflammatory biomarkers following aronia consumption. A randomized, placebo-controlled trial of 44 older adults that survived a myocardial infarction demonstrated that 6 week supplementation of 255 mg aronia extract reduced serum interleukin-6 (IL-6), C-reactive protein (CRP), soluble intercellular adhesion molecule, vascular cell adhesion molecule, and monocyte chemoattractant protein-1 compared to the placebo (Naruszewicz, Laniewska, Millo, & Dluzniewski, 2007). Consumption of citrus juice fortified with aronia extract reduced CRP, oxidized LDL, and 8-hydroxydeoxyguanosine and improved glutathione status in adults with metabolic syndrome at 4 and 6 months relative to a placebo drink (Bernabe et al., 2013; Mulero et al., 2012). Despite the promising antiinflammatory effects of aronia consumption, little is known about the bioactivity of underutilized aronia genotypes. Aronia berries belong to the Rosaceae family and have 4 species: Aronia arbutifolia which produce red berries; Aronia melanocarpa and Aronia mitschurinii which produce black berries; and Aronia prunifolia which produce purple berries (Brand, 2010). Aronia berries have varying content of phenolic acids, anthocyanins, flavonoids, and proanthocyanidins based on genotype and sample origin (Taheri, Connolly, Brand, & Bolling, 2013). Despite their enrichment and diversity of polyphenols among aronia berries, little is known about how their polyphenol profiles impact their ability to modulate inflammatory cytokines. Therefore, the aim of this investigation was to examine the effects of aronia berries and their representative phytochemicals on IL-6 and IL-10 production in murine splenocytes in vitro.

2.

Materials and methods

2.1.

Chemicals and reagents

HPLC grade acetone, acetic acid, and methanol; ammonium chloride, potassium carbonate, and sodium azide were purchased from Fisher Scientific (Pittsburg, PA, USA). Dulbecco’s phosphate buffered saline (DPBS), minimum essential medium (MEM), and fetal calf serum (FCS) were purchased from Thermo Scientific Hyclone (Waltham, MA, USA). Antibiotic/antimycotic, L-glutamine, amino acids, and sodium pyruvate were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Lipopolysaccharide (LPS), 2-mercaptoethanol, >95% purity chlorogenic acid (Cga), >98% purity neochlorogenic acid (nCga), >95% purity quercetin, >90% purity quercetin-3-glucoside (Q3Glu), >97% purity quercetin-3-galactoside (Q3Gal), and analytical standard grade proanthocyanidin B2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cyanidin-3-arabinoside (Cy3A) chloride and cyanidin-3-galactoside (Cy3Gal) chloride of >98% purity were obtained from WuXi Apptec (Shanghai,

69

China). Phorbol myristate acetate (PMA) was from Calbiochem (Gibbstown, NJ, USA). Ionomycin was from Invitrogen (Carlsbad, CA, USA). LIVE/DEAD® Fixable Far Red Dead Cell Stain Kit, for 633 or 635 nm Excitation antibody was purchased from Life Technologies (Carlsbad, CA, USA). Brefeldin A (Golgi PlugTM) and anti-CD16/CD32 were both from BD Biosciences (San Jose, CA, USA). Antibodies for flow cytometry were obtained from eBioscience (San Diego, CA, USA). FITC conjugated anti-CD4 (clone: GK 1.6) and PE conjugated anti-IL-6 (clone: MP5-20F3). Isotype controls were FITC conjugated rat anti-IgG2b κ (clone: eB149/10HS) and PE-conjugated rat anti-IgG1 (clone: eBRG1).

2.2.

Mice

Three-week-old male C57BL6/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were kept at 20–26 °C under a 12 h light/dark cycle and were fed standard chow diet upon arrival to the animal facility. The protocol was approved by the University of Connecticut Institutional Animal Care and Use Committee.

2.3.

Aronia berry extracts

Extracts from aronia berries were prepared as previously described (Taheri et al., 2013). Briefly, fresh A. mitschurinii ‘Viking’, A. arbutifolia (UC021 and UC057 accessions), and A. prunifolia (UC011 and UC047 accessions) berries were harvested at apparent ripeness, frozen at −80 °C, and then lyophilized. Lyophilized berries were powdered and stored at −80 °C until extraction. Extracts were prepared by suspending berry powder in 1:20 (w/v) of 70% acetone, 29.5% ultrapure water, and 0.5% acetic acid. The suspensions were sonicated for 5 min and supernatants collected, and the procedure was repeated twice. Then, the extract was agitated in fresh extract solution for 12 h at room temperature. The supernatants were recombined and dried at 40 °C by rotary evaporation. Aliquots were resuspended in methanol, dried, and stored at −80 °C until use. The polyphenol content of aronia berry extracts was previously reported (Taheri et al., 2013). Total phenols of extracts were determined using the Folin–Ciocalteu method as previously described, and expressed as gallic acid equivalents (GAE) (Kim et al., 2013a; Singleton, Orthofer, & Lamuela-Ravent, 1999). Doses of extracts used in subsequent experiments were normalized to polyphenol content based on GAE.

2.4.

In vitro splenocyte cultures

Mice were anesthetized under isofluorane and sacrificed by cervical dislocation. Splenocytes were obtained as previously described, with modifications (McAleer, Saris, & Vella, 2011). Spleens were removed and strained through a 70 μm cell strainer. Cells were pelleted in MEM by centrifuging at 400 × g for 5 min at 4 °C, and resuspended in 2 mL MEM. Red blood cells were lysed by adding 5 mL 0.15 M ammonium chloride with 10 mM potassium carbonate. After 5 min, 30 mL DPBS was added, cells were pelleted, and washed twice with MEM. Cells were counted in a Bio-Rad (Hercules, CA, USA) TC-10 cell counter or by a hemacytometer following Trypan blue staining. Cells were then plated at 106 cells/well with 250 μL of MEM containing 10% FCS and supplemented with amino acids,

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dextrose, sodium pyruvate, antibiotics, antimycotic, and 2-mercaptoethanol (McAleer et al., 2011). For LPS-induction experiments, 10 μg LPS/mL and aronia extracts or polyphenol standards (in <1% methanol) were dissolved in media and incubated with cells for 12 h at 37 °C with 5% CO2. Following incubation, the cells were pelleted as above, and the supernatants were stored at −80 °C until analysis. Concentrations of the phytochemicals were chosen to reflect the ranges found in the aronia berry extracts effective at inhibiting LPS-induced IL-6 in splenocytes. Cga, nCga, Cy3Gal, and Cy3A were tested at 10, 5, and 1 μg/mL (28.22, 14.11, and 2.822 μM for Cga and nCga; 20.63, 10.31, 2.063 μM for Cy3Gal and Cy3A). Q3Gal and Q3Glu were tested at 10, 1, and 0.1 μg/ mL (21.53, 2.153, and 0.5153 μM). Quercetin was tested at an equimolar basis of the quercetin glycones at 6.5, 0.65, and 0.0658 μg/mL. Proanthocyanidin B2 was tested at 5, 2.5, and 1 μg/ mL (8.643, 4.321, and 1.729 μM).

2.5.

Determination of cell viability

Following incubation and pelleting (Section 2.3), the cells were washed with MEM and then DPBS. Splenocytes were pelleted and then resuspended in 100 μL DPBS with 1 μL LIVE/DEAD® Fixable Far Red Dead Cell Stain Kit antibody. The cells were incubated for 30 min at 4 °C in darkness. Cells were pelleted, the supernatant was decanted, and then washed twice in DPBS with 3% fetal bovine serum and 0.1% sodium azide and resuspended in 1.2 mL plastic tubes in 500 μL of the same buffer. Data were acquired on a BD FACSCalibur flow cytometer (Franklin Lakes, NJ, USA) and analyzed with FlowJo Version 7.6.5 analysis software (Treestar, Ashland, OR, USA).

2.6.

Determination of cytokines by ELISA

IL-6 and IL-10 contents of cell supernatants were determined by enzyme-linked immunosorbent assay (ELISA) kits from eBioscience (San Diego, CA, USA) and were performed according to the manufacturer’s instructions.

2.7.

Determination of IL-6 by flow cytometry

Splenocytes were cultured as described in Section 2.3 with the following modifications: phorbol myristate acetate (50 ng/ mL) and ionomycin (1 μg/mL) (PI) were used in lieu of LPS and 5 μg/mL Brefeldin A was added to incubates (McAleer et al., 2011). Cells were incubated for 5 h and washed twice with MEM following incubation. Nonspecific binding was blocked using anti-CD16/CD32 and cells were surface stained with LIVE/ DEAD® Fixable Far Red Dead Cell Stain Kit and FITC conjugated anti-CD4 or the corresponding isotype controls. Following permeabilization and fixation (BD Biosciences, Cytofix/Cytoperm – Plus) according to manufacturer’s instructions, cells were stained intracellularly with PE conjugated anti-IL-6. Cells were resuspended in stain/wash buffer and data were acquired on a BD FACSCalibur flow cytometer and analyzed with FlowJo Version 7.6.5 analysis software.

2.8.

Statistical analysis

Data are expressed as the mean ± standard error. Statistical analyses were performed in GraphPad Prism 5 (La Jolla, CA,

Fig. 1 – Dose–response inhibition of LPS-stimulated IL-6 release from primary mouse splenocytes. C57/BL6 primary mouse splenocytes were incubated at 106 cells/well for 12 h with 10 μg/mL LPS and ‘Viking’ aronia berry extract at varying concentrations. Cell supernatant IL-6 was determined by ELISA. Data are mean ± SE, n = 3/treatment. P < 0.001 for all aronia treatments versus LPS control by one way ANOVA with Tukey’s post-test.

USA). One way ANOVA followed by Tukey’s HSD test was performed to test significance (P < 0.05). Principal component analysis was performed using Minitab 16 (State College, PA, USA).

3.

Results and discussion

3.1. Effects of aronia extracts on IL-6 production in murine splenocytes The addition of 10 μg LPS/mL to primary C57/BL6J mouse splenocytes induced extracellular IL-6 to 585 ± 36 pg/mL, while unstimulated splenocytes did not produce detectable IL-6 (Fig. 1). ‘Viking’ aronia berry extract dose dependently inhibited LPSstimulated splenocyte IL-6 (P < 0.001). A 125 μg GAE/mL extract dose inhibited IL-6 by 58%, whereas at 500 μg GAE/mL, IL-6 inhibition was ~96%. Subsequent experiments showed that viability at 125 μg GAE/mL was not different from LPS control cells. We sought to determine the immunomodulatory properties of aronia extract by examining the effects of the extract on splenic cells and specifically the CD4+ T helper cells. The IL-6 inhibition of ‘Viking’ extract was characterized in PMA and ionomycin-stimulated splenocytes using flow cytometry. ‘Viking’ extracts inhibited intracellular IL-6 in both CD4− and CD4+ lymphocytes (Fig. 2). IL-6+ CD4− lymphocytes decreased from 22.3 ± 1.4 to 10.6 ± 1.0% upon treatment with 2 mg GAE/mL Viking aronia extract (P = 0.02). Similarly, IL-6+ CD4+ lymphocytes decreased from 6.4 ± 0.4 to 4.1 ± 0.3% (P = 0.049). Another study utilizing primary murine splenocytes (Lin, Li, & Hwang, 2008) has assessed the effects of plant extracts on IL-6 production, but were not examined by flow cytometry. Here, we show that IL-6 inhibition occurs in both CD4− and CD4+ lymphocytes.

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Fig. 2 – Modulation of PMA/ionomycin (PI) stimulated IL-6 production in murine splenocytes. C57/BL6 primary mouse splenocytes were incubated at 106 cells/well for 5 h with PMA and ionomycin with 2 mg/mL Viking aronia extract. Cells were surface stained with FITC-anti-CD4 and intracellularly stained with PE-anti-IL-6 and examined by flow cytometry. (A, B) Representative dot plots. Quadrants established based on isotype control staining. (C, D) t-tests were performed in CD4− and CD4+ populations. Data are mean ± SE, n = 2. P = 0.02 for % of total cells IL-6+ in CD4− population; P = 0.049 for % of total cells IL-6+ in CD4+ population.

We further tested the capacity of underutilized aronia extracts to inhibit LPS-stimulated splenocyte IL-6 relative to the ‘Viking’ extract. The aronia accessions analyzed were chosen to represent the varying enrichment of polyphenol classes among aronia extracts. The red aronia UC021 was chosen because of its high proanthocyanidin content, while the red aronia UC057 contained high flavonoid and hydroxycinnamic acids (Taheri et al., 2013). The purple aronia UC047 was chosen because of its increased hydroxycinnamic acid content, whereas the purple UC011 had mid-range distribution of all polyphenol classes (Taheri et al., 2013). Relative to the untreated, LPS-stimulated controls, all aronia berry extracts inhibited the production of IL-6 in murine splenocytes, from 29 to 45% at 125 μg GAE/mL (P < 0.001) (Fig. 3A). Despite the varying enrichment of polyphenol classes, there were no significant differences in inhibition capacity between the accessions. Likewise, cell viability was unaffected by aronia treatments (Fig. 3B). Principal component analysis of these data suggested that the inhibition of IL-6 was most closely associated with the flavonoid and phenolic acid content of aronia extracts (Fig. 4). Polyphenols present in aronia extracts could inhibit IL-6 by several mechanisms. LPS-stimulation induces lymphocyte immunostimulatory cytokines and chemokines by initiating the

toll-like receptor 4 (TLR4) pathway and activating NF-κB, activator protein 1 (AP-1), or interferon response factors (Ospelt & Gay, 2010). Flavonoids have been shown to inhibit NF-κB activation through p38, extracellular signal-regulated kinases ERK, c-Jun N-terminal kinases (JNK), mitogen-activated protein (MAP) kinases and by inhibiting IKK-β and IκB-α in a variety of cell types (González et al., 2011). The flavonol quercetin also inhibits lipid raft formation, a first step in TLR4 signaling, and induces the negative TLR regulator Toll-interacting protein in LPS-stimulated macrophages (Byun et al., 2013; Kaneko et al., 2008). Polyphenol-rich red cabbage juice also increased IL-10 and decreased IL-6 in murine splenocytes (Lin et al., 2008). Consequently, polyphenols also inhibit pro-inflammatory signaling in T cells. Proanthocyanidin-rich grape seed extract decreased arthritis symptoms and induced T regulatory cells in an arthritis mouse model (Park et al., 2011) and also decreased TNF-α and IL-17 producing cells in the synovial tissue (Cho et al., 2009).

3.2. Inhibition of IL-6 from LPS-stimulated splenocytes by polyphenols As principal component analysis suggested that aronia phenolic acids and flavonoids were better correlated with IL-6

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Fig. 3 – (A) Inhibition of LPS-stimulated splenocyte IL-6 by aronia berry extracts. C57/BL6 primary mouse splenocytes were incubated at 106 cells/well for 12 h with 10 μg/mL LPS, and 125 μg GAE aronia berry extract/mL. IL-6 in supernatant was determined by ELISA. Data are the mean of n = 3 experiments ± SE, expressed as pg IL-6, as the % of control. One way ANOVA revealed no significant differences between aronia accessions. (B) Viability of splenocytes incubated with LPS and aronia extracts determined by flow cytometry. Data are mean ± SE of duplicate experiments, as % live cells. One way ANOVA revealed no significant differences between aronia accessions.

inhibition than anthocyanins and proanthocyanidins, we subsequently tested the efficacy of representative polyphenols in aronia extracts. We tested polyphenols at levels approximating concentrations present in the aronia extracts. For example, when standardized to 125 μg GAE /mL, ‘Viking’ aronia berry extract contained ~3 μg Cga/mL, while UCO47 had 10.3 μg Cga/mL. Cy3A, but not Cy3Gal, inhibited LPS-stimulated splenocyte IL-6 at 10 μg/mL (Table 1). Quercetin at 6.5 μg/mL inhibited LPS-stimulated splenocyte IL-6 by 80%. In contrast, Q3Glu and Q3Gal increased IL-6 13–16% at 10 and 0.1 μg/mL, respectively. Notably, quercetin at 6.5 μg/mL reduced cell viability to 69% of control cells. The structure–function relationship of quercetin and its conjugated glycosides and oxidative stress has been well estab-

Fig. 4 – Principal component analysis of aronia extract polyphenol content. (A) The biplot of principal components 1 and 2, whereas ● is black, ■ is purple, and ▲ is red aronia berries; (B) loading plot of inhibition of LPS-stimulated mouse splenocyte IL-6 by 125 μg GAE aronia extract/mL and the aronia polyphenol content of extracts, as reported previously (Taheri et al., 2013).

lished. Conjugation at the 3′ or 4′ position decreases its antioxidant activity (Morand et al., 1998). However, less is known about how conjugation affects anti-inflammatory activity. Although Q3Gal and Q3Glu had reduced efficacy in inhibiting splenocyte IL-6, these compounds possess anti-inflammatory activity in some, but not all, models of inflammation. Quercetin, but not its 3-glucuronide, inhibited LPS-stimulated IL-6 mRNA in RAW264.7 macrophages (Boesch-Saadatmandi et al., 2011). In contrast, a 500 mg/kg oral dose of Q3Gal and Q3Glu inhibited carrageenan-induced hind paw and TPA-induced mouse ear edema (Erdemoglu, Akkol, Yesilada, & Calis, 2008). Quercetin and quercetin-3-glucuronide also inhibited IL-6 and TNF-α production through similar mechanisms in human umbilical vein endothelial cells exposed to palmitate (Guo et al., 2013). Work by Terao, Murota, and Kawai (2011) has suggested hydrolysis of quercetin-3-glucuronide is necessary for anti-inflammatory activity. Therefore, the anti-inflammatory activity of quercetin glycosides appears to be cell-type dependent. Hydroxycinnamic acids also increased IL-6 in LPS-stimulated splenocytes, by 31–34% at 1 μg/mL (Table 1). The immunestimulating activity of hydroxycinnamic may be unique to

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Table 1 – Effect of phytochemical standards on IL-6 production in primary murine splenocytes stimulated with LPS. Flavonoid class

Hydroxycinnamic acid Anthocyanin Proanthocyanidin Flavonol

Compound

IL-6/cell, % of untreated control Dose (μg/mL medium)

Chlorogenic acid Neochlorogenic acid Cyanidin-3-galactoside Cyanidin-3-arabinoside Proanthocyanidin B2 Quercetin-3-galactoside Quercetin-3-glucoside Quercetina

10

5

1

0.1

113.4 ± 8.7 137.9 ± 8.6* 105.5 ± 4.5 79.4 ± 3.4* –b 97.28 ± 9.3 115.7 ± 2.2** 20.2 ± 7.8**

127.7 ± 11.2 130.7 ± 8.3* 98.2 ± 12.5 78.7 ± 7.9 76.9 ± 11.9 – – –

133.7 ± 6.2* 130.7 ± 6.3* 106.9 ± 12.0 113.9 ± 11.0 90.8 ± 10.1 97.4 ± 5.9 100.1 ± 8.0 105.2 ± 14.9

– – – – – 112.5 ± 3.3* 108.5 ± 6.2 108.3 ± 10.9

Data are mean ± SE of duplicate experiments and expressed as % of untreated control, where * P < 0.05 and ** P < 0.01 in two-tailed t-tests versus control (100%). a Quercetin was equimolar with quercetin 3-galactoside and –glucoside, at 6.5, 0.65, and 0.065 μg/mL. b –, not determined.

Table 2 – Effect of representative aronia polyphenols on LPS-stimulated IL-10 from primary mouse splenocytes. Polyphenol

IL-10/cell (% of untreated control)

P valuea

Chlorogenic acid Neochlorogenic acid Cyanidin-3-galactoside Cyanidin-3-arabinoside Quercetin-3-galactoside Quercetin-3-glucoside Quercetinb

89.19 ± 1.659 83.16 ± 2.65 101.46 ± 3.63 98.05 ± 2.69 92.83 ± 7.67 107.35 ± 5.52 80.89 ± 4.25

0.0029 0.0031 0.7080 0.5075 0.3928 0.2406 0.0064

Data are mean ± SE of duplicate experiments, normalized to cell viability, and expressed as % of untreated control. a P values are two-tailed t-tests versus controls (100%). C57/BL6 primary mouse splenocytes were incubated at 106 cells/well for 12 h with 10 μg/mL LPS with 10 μg/mL polyphenol standards. Cell supernatant IL-10 was determined by ELISA. b Quercetin was equimolar (21.5 μM) with quercetin 3-galactoside and 3-glucoside at 6.5 μg/mL.

splenocytes, as prior studies have found chlorogenic acid inhibits LPS-stimulated cytokines in RAW 264.7 macrophages and rat hepatic stellate cells (Hwang, Kim, Park, Lee, & Kim, 2014; Shi et al., 2013). Taken together, the most abundant constituents of aronia extracts, mainly hydroxycinnamic acids and Cy3Gal, did not exhibit potent IL-6 inhibition in splenocytes. Thus, the IL-6 inhibition observed with aronia may be due to minor polyphenols, novel constituents, or synergies among these components.

3.3. Modulation of splenocyte IL-10 release by aronia and its polyphenols IL-10 is an anti-inflammatory cytokine that plays a role in inflammatory and autoimmune disorders (Iyer & Cheng, 2012). IL-10 is produced by immune cells such as T helper cells, monocytes, macrophages, dendritic cells, B cells, cytotoxic T cells, natural killer cells, mast cells, neutrophils and eosinophils as well as epithelial cells and keratinocytes (Iyer & Cheng, 2012). Therefore, we evaluated the ability of aronia extract and its associated polyphenols to modulate IL-10. In LPS-stimulated splenocytes, quercetin decreased IL-10 production by 19.1 ± 4.3% (Table 2). CGA and nCGA decreased IL-10 production by 10.1 and 16.8%, respectively (Table 2). No other treatment, including ‘Viking’ aronia berry extract, modulated LPS-stimulated splenocyte IL-10 release compared to control (Table 3). In unstimulated cells, IL-10 secretion increased to 140% of the control with 125 μg/mL ‘Viking’ aronia berry extracts (Table 3). Previous studies of polyphenols and IL-10 production differ from the present study. Differences in concentration, polyphenol content and LPS-stimulation times could account for the differences between these studies. Fractions of polyphenolrich cabbage extract induced IL-10 secretion in LPS-stimulated mouse splenocytes, at 500 μg/mL (Lin et al., 2008). A 600-fold dilution of aronia extract also inhibited human monocyte (Mono Mac 6) IL-10 stimulated with 1 μg LPS/mL for 18 h, although a 2000-fold dilution did not (Xu & Mojsoska, 2013). Thus, higher concentrations of aronia extract may inhibit LPS-stimulated IL-10.

Table 3 – Effect of ‘Viking’ aronia berry extract on IL-10 production in primary mouse splenocytes. LPS

Treatment

IL-10 (pg/mL/105 cells)

IL-10 (% control)

0 μg/mL 0 μg/mL 10 μg/mL 10 μg/mL

Control Viking extract Control Viking extract

2.31 ± 0.61 2.73 ± 0.62 34.1 ± 7.6 32.2 ± 5.9

100 140.3 ± 8.8 100 106.1 ± 5.7

P valuea 0.0026 0.9609

Data are mean ± SE of triplicate experiments. a Significance was by two-tailed t-test versus the control value (100%). Aronia extract was incubated at 125 μg GAE/mL for 12 h with concurrent LPS stimulation, as indicated in the first column.

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Further work is needed to understand the mechanism of aronia induction of IL-10 secretion from splenocytes at basal conditions. Many transcriptional regulators are involved in the regulation of IL-10 production including specificity factor, STAT1 and STAT3, c-musculoapneurotic fibrosarcoma, AP-1, CCAAT/ enhancer binding proteins, interferon regulatory factors, cyclic AMP response element binding protein, NF-κB, GATA3; further, IL-10 is also subject to epigenetic and post-translational regulation (Iyer & Cheng, 2012).

4.

Conclusions

Aronia berries represent a rich source of dietary polyphenols and have been used in human and animal intervention studies with promising results. Aronia berry extracts inhibit LPSinduced IL-6 secretion and induce IL-10 excretion in unstimulated splenocytes. These cytokines are implicated in the development of autoimmune inflammatory disorders such as multiple sclerosis, arthritis, and inflammatory bowel diseases. This work provides further insight to the structure– function anti-inflammatory relationships of aronia berry polyphenols, as the most abundant aronia polyphenols are likely not the major effectors of IL-6 inhibition, and underutilized berries with reduced anthocyanin content retain their antiinflammatory potential. Further work is needed to elucidate the molecular basis for the increased IL-6 inhibition of Cy3A relative to Cy3Gal.

Acknowledgements The authors are grateful for the technical assistance of Bryan A. Connolly, Liyang Xie, and Ruisong Pei. This work was supported by the University of Connecticut Diet and Health Initiative and USDA Hatch CONS0080 to Dr. Bolling. The funding sources had no involvement in the research conduct or manuscript preparation.

REFERENCES

Abraham, C., & Cho, J. H. (2009). Inflammatory bowel disease. New England Journal of Medicine, 361(21), 2066–2078. Bernabe, J., Mulero, J., Cerde, B., Garcia-Viguera, C., Moreno, D. A., Parra, S., Aviles, F., Gil-Izquierdo, A., Abellan, J., & Zafrilla, P. (2013). Effects of a citrus based juice on biomarkers of oxidative stress in metabolic syndrome patients. Journal of Functional Foods, 5(3), 1031–1038. Boesch-Saadatmandi, C., Loboda, A., Wagner, A. E., Stachurska, A., Jozkowicz, A., Dulak, J., Doring, F., Wolffram, S., & Rimbach, G. ( 2011). Effect of quercetin and its metabolites isorhamnetin and quercetin-3-glucuronide on inflammatory gene expression: role of miR-155. The Journal of Nutritional Biochemistry, 22(3), 293–299. Brand, M. (2010). Aronia: native shrubs with untapped potential. Arnoldia, 67(3), 14–25. Byun, E. B., Yang, M. S., Choi, H. G., Sung, N. Y., Song, D. S., Sin, S. J., & Byun, E. H. (2013). Quercetin negatively regulates TLR4 signaling induced by lipopolysaccharide through Tollip

expression. Biochemical and Biophysical Research Communications, 431(4), 698–705. Cho, M. L., Heo, Y. J., Park, M. K., Oh, H. J., Park, J. S., Woo, Y. J., Ju, J. H., Park, S. H., Kim, H. Y., & Min, J. K. ( 2009). Grape seed proanthocyanidin extract (GSPE) attenuates collagen-induced arthritis. Immunology Letters, 124(2), 102–110. Coussens, L. M., Zitvogel, L., & Palucka, A. K. (2013). Neutralizing tumor-promoting chronic inflammation: a magic bullet? Science, 339(6117), 286–291. Erdemoglu, N., Akkol, E. K., Yesilada, E., & Calis, I. (2008). Bioassay-guided isolation of anti-inflammatory and antinociceptive principles from a folk remedy, Rhododendron ponticum L. leaves. Journal of Ethnopharmacology, 119(1), 172–178. González, R., Ballester, I., López-Posadas, R., Suárez, M. D., Zarzuelo, A., Martínez-Augustin, O., & Sánchez de Medina, F. (2011). Effects of flavonoids and other polyphenols on inflammation. Critical Reviews in Food Science and Nutrition, 51(4), 331–362. Gregor, M. F., & Hotamisligil, G. S. (2011). Inflammatory mechanisms in obesity. Annual Review of Immunology, 29(1), 415–445. Guo, X. D., Zhang, D. Y., Gao, X. J., Parry, J., Liu, K., Liu, B. L., & Wang, M. (2013). Quercetin and quercetin-3-O-glucuronide are equally effective in ameliorating endothelial insulin resistance through inhibition of reactive oxygen species-associated inflammation. Molecular Nutrition & Food Research, 57(6), 1037–1045. Hellstrom, J. K., Shikov, A. N., Makarova, M. N., Pihlanto, A. M., Pozharitskaya, O. N., Ryhanen, E. L., Kivijarvi, P., Makarov, V. G., & Mattila, P. H. ( 2010). Blood pressure-lowering properties of chokeberry (Aronia mitchurinii, var. Viking). Journal of Functional Foods, 2(2), 163–169. Hwang, S. J., Kim, Y. W., Park, Y., Lee, H. J., & Kim, K. W. (2014). Anti-inflammatory effects of chlorogenic acid in lipopolysaccharide-stimulated RAW 264.7 cells. Inflammation Research, 63(1), 81–90. Iyer, S. S., & Cheng, G. (2012). Role of interleukin 10 transcriptional regulation in inflammation and autoimmune disease. Critical Reviews in Immunology, 32(1), 23–63. Kaneko, M., Takimoto, H., Sugiyama, T., Seki, Y., Kawaguchi, K., & Kumazawa, Y. (2008). Suppressive effects of the flavonoids quercetin and luteolin on the accumulation of lipid rafts after signal transduction via receptors. Immunopharmacology and Immunotoxicology, 30(4), 867–882. Kaume, L., Gilbert, W. C., Brownmiller, C., Howard, L. R., & Devareddy, L. (2012). Cyanidin 3-O-beta-D-glucoside-rich blackberries modulate hepatic gene expression, and anti-obesity effects in ovariectomized rats. Journal of Functional Foods, 4(2), 480–488. Kim, B., Ku, C. S., Pham, T. X., Park, Y., Martin, D. A., Xie, L., Taheri, R., Lee, J., & Bolling, B. W. ( 2013a). Aronia melanocarpa (chokeberry) polyphenol-rich extract improves antioxidant function and reduces total plasma cholesterol in apolipoprotein E knockout mice. Nutrition Research, 33(5), 406–413. Kim, B., Park, Y., Wegner, C. J., Bolling, B. W., & Lee, J. (2013b). Polyphenol-rich black chokeberry (Aronia melanocarpa) extract regulates the expression of genes critical for intestinal cholesterol flux in Caco-2 cells. The Journal of Nutritional Biochemistry, 24(9), 1564–1570. Kivimaki, A. S., Ehlers, P. I., Siltari, A., Turpeinen, A. M., Vapaatalo, H., & Korpela, R. (2012). Lingonberry, cranberry and blackcurrant juices affect mRNA expressions of inflammatory and atherothrombotic markers of SHR in a long-term treatment. Journal of Functional Foods, 4(2), 496–503. Kokotkiewicz, A., Jaremicz, Z., & Luczkiewicz, M. (2010). Aronia plants: a review of traditional use, biological activities, and

journal of functional foods 8C (2014) 68–75

perspectives for modern medicine. Journal of Medicinal Food, 13(2), 255–269. Lau, F. C., Joseph, J. A., McDonald, J. E., & Kalt, W. (2009). Attenuation of iNOS and COX2 by blueberry polyphenols is mediated through the suppression of NF-kappa B activation. Journal of Functional Foods, 1(3), 274–283. Lin, J. Y., Li, C. Y., & Hwang, I. F. (2008). Characterisation of the pigment components in red cabbage (Brassica oleracea L. var.) juice and their anti-inflammatory effects on LPS-stimulated murine splenocytes. Food Chemistry, 109(4), 771–781. McAleer, J. P., Saris, C. J. M., & Vella, A. T. (2011). The WSX-1 pathway restrains intestinal T-cell immunity. International Immunology, 23(2), 129–137. Morand, C., Crespy, V., Manach, C., Besson, C., Demigné, C., & Rémésy, C. (1998). Plasma metabolites of quercetin and their antioxidant properties. American Journal of Physiology – Regulatory Integrative and Comparative Physiology, 275(1), R212–R219. Mulero, J., Bernabe, J., Cerda, B., Garcia-Viguera, C., Moreno, D. A., Albaladejo, M. D., Aviles, F., Parra, S., Baellan, J., & Zafrilla, P. (2012). Variations on cardiovascular risk factors in metabolic syndrome after consume of a citrus-based juice. Clinical Nutrition, 31(3), 372–377. Naruszewicz, M., Laniewska, I., Millo, B., & Dluzniewski, M. (2007). Combination therapy of statin with flavonoids rich extract from chokeberry fruits enhanced reduction in cardiovascular risk markers in patients after myocardial infraction (MI). Atherosclerosis, 194(2), e179–e184. Ospelt, C., & Gay, S. (2010). TLRs and chronic inflammation. The International Journal of Biochemistry & Cell Biology, 42(4), 495–505. Park, M. K., Park, J. S., Cho, M. L., Oh, H. J., Heo, Y. J., Woo, Y. J., Heo, Y. M., Park, M. J., Park, H. S., Park, S. H., Kim, H. Y., & Min, J. K. (2011). Grape seed proanthocyanidin extract (GSPE) differentially regulates Foxp3 + regulatory and IL-17 + pathogenic T cell in autoimmune arthritis. Immunology Letters, 135(1–2), 50–58. Patel, P. S., Buras, E. D., & Balasubramanyam, A. (2013). The role of the immune system in obesity and insulin resistance. Journal of Obesity, 2013, 1–9.

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Pearson, T. A., Mensah, G. A., Alexander, R. W., Anderson, J. L., Cannon, R. O., Criqui, M., Fadl, Y. Y., Fortmann, S. P., Hong, Y., Myers, G. L., Rifai, N., Smith, S. C. Jr, Taubert, K., Tracy, R. P., & Vinicor, F. ( 2003). Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association. Circulation, 107(3), 499–511. Reynolds, R., Roncaroli, F., Nicholas, R., Radotra, B., Gveric, D., & Howell, O. (2011). The neuropathological basis of clinical progression in multiple sclerosis. Acta Neuropathologica, 122(2), 155–170. Sharif, T., Stambouli, M., Burrus, B., Emhemmed, F., Dandache, I., Auger, C., Etienne-Selloum, N., Schini-Kerth, V. B., & Fuhrmann, G. ( 2013). The polyphenolic-rich Aronia melanocarpa juice kills teratocarcinomal cancer stern-like cells, but not their differentiated counterparts. Journal of Functional Foods, 5(3), 1244–1252. Shi, H., Dong, L., Jiang, J., Zhao, J., Zhao, G., Dang, X., Lu, X., & Jia, M. ( 2013). Chlorogenic acid reduces liver inflammation and fibrosis through inhibition of toll-like receptor 4 signaling pathway. Toxicology, 303C, 107–114. Singleton, V. L., Orthofer, R., & Lamuela-Ravent, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods in Enzymology, 299, 152–178. Taheri, R., Connolly, B. A., Brand, M. H., & Bolling, B. W. (2013). Underutilized chokeberry (Aronia melanocarpa, Aronia arbutifolia, Aronia prunifolia) Accessions are rich sources of anthocyanins, flavonoids, hydroxycinnamic acids, and proanthocyanidins. Journal of Agricultural and Food Chemistry, 61(36), 8581–8588. Terao, J., Murota, K., & Kawai, Y. (2011). Conjugated quercetin glucuronides as bioactive metabolites and precursors of aglycone in vivo. Food and Function, 2(1), 11–17. Xu, J., & Mojsoska, B. (2013). The immunomodulation effect of aronia extract lacks association with its antioxidant anthocyanins. Journal of Medicinal Food, 16(4), 334–342.