Protective effects of a blueberry extract in acute inflammation and collagen-induced arthritis in the rat

Biomedicine & Pharmacotherapy 83 (2016) 1191–1202

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Protective effects of a blueberry extract in acute inflammation and collagen-induced arthritis in the rat Maria-Eduardo Figueiraa , Mónica Oliveiraa , Rosa Direitoa , João Rochaa , Paula Alvesb , Ana-Teresa Serrac, Catarina Duartec , Rosário Bronzec , Adelaide Fernandesa , Dora Britesa , Marisa Freitasd , Eduarda Fernandesd , Bruno Sepodesa,* a

iMED.ULisboa and Faculdade de Farmácia – Universidade de Lisboa, Avenida Prof. Gama Pinto, 1649-003 Lisboa, Portugal Faculdade de Medicina, Universidade de Coimbra, 3004-504 Coimbra, Portugal c ITQB/IBET, Avenida da República, Quinta do Marquês, Estação Agronómica Nacional, 2781-901 Oeiras, Portugal d REQUIMTE, Laboratório de Química Aplicada, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal b

A R T I C L E I N F O

Article history: Received 8 April 2016 Received in revised form 28 July 2016 Accepted 15 August 2016 Keywords: Collagen induced arthritis Blueberry fruit Vaccinium corymbosum L. Oxidative burst Anti-inflammatory

A B S T R A C T

Here we investigated the anti-inflammatory effect of a blueberry extract in the carrageenan-induced paw edema model and collagen-induced arthritis model, both in rats. Along with the chemical characterization of the phenolic content of the fruits and extract, the antioxidant potential of the extract, the cellular antioxidant activity and the effects over neutrophils’ oxidative burst, were studied in order to provide a mechanistic insight for the anti-inflammatory effects observed. The extract significantly inhibited paw edema formation in an acute model the rat. Our results also demonstrate that the standardized extract had pharmacological activity when administered orally in the collagen-induced arthritis model in the rat and was able to significantly reduce the development of clinical signs of arthritis and the degree of bone resorption, soft tissue swelling and osteophyte formation, consequently improving articular function in treated animals. ã 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease [1], characterized by inflammation of the synovia, synovial hyperplasia with increased cell density, and infiltration of inflammatory cells leading to pannus formation and irreversible cartilage and bone destruction [2]. The growing knowledge of risk factors for Rheumatoid arthritis (RA) calls for preventive strategies [3] to prevent disability in many patients. Collagen-induced arthritis (CIA) is a model of experimental that is initiated by the administration of type II collagen (CII), a component of the extracellular matrix of articular cartilage located in diarthrodial joints [4]. The similarities between the joint pathology (including erosion) in CIA and RA have been the main driver for the use of this model in drug development targeting RA [5]. The main objective of this work is to study the effects of a blueberry extract in CIA in the rat.

* Corresponding author. E-mail address: [email protected] (B. Sepodes). http://dx.doi.org/10.1016/j.biopha.2016.08.040 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.

Blueberry is any of several Eurasian species of low-growing shrubs in the genus Vaccinium (family Ericaceae, subfamily Vaccinioideae), bearing edible, nearly black berries [6]. There are more than 450 different species of blueberries. Here we studied Vaccinium corymbosum L., originally from North America but now widely distributed around the globe [7]. Blueberries are fruits with high biological activity and known health benefits due to the high content in phenolic compounds. The interest in such compounds arises from the fact that several studies clearly suggest that a diet rich in fruits, vegetables and whole-grain cereals, containing polyphenols, could be related to health benefits and reduce the risk of chronic degenerative diseases [8]. From all the compounds found in blueberries, phenolic compounds are considered of special interest due to their antioxidant activity [9]. The individual contribution of each of these compounds to the total antioxidant capacity of blueberries is dependent on the structure and concentration of each compound in the fruits [10]. The antioxidant capacity of blueberries seems to relate more directly to the total concentration of phenols than to the concentration of anthocyanins, albeit anthocynins also contribute to the antioxidant properties of the fruit [10–12]. Nevertheless, some authors defend that the combination of

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anthocyanins in the blueberry fruits might be responsible for 56% of the total value of their antioxidant capacity [9]. Amongst other health benefits attributed to blueberries, evidence has been generated to show that some of the compounds existing in blueberry fruits are potent inhibitors of the inflammatory response, through mechanisms that include the inhibition of the synthesis of cyclooxygenases, lipoxygenase and myeloperoxidase [13–19]. Although some studies have started to unveil the antiinflammatory effects of blueberry extracts in vitro and in vivo, these studies are preliminary and require further elaboration so that a translational approach for clinical practice is a realistic goal when considering an adjunctive therapy for chronic inflammatory conditions such as arthritis. Here we investigated the effects of a chemically characterized blueberry extract on the arthritic inflammatory response caused by the injection of CII in the rat. The amount of components in the extract with expected biological activity could reasonably be achieved in humans, with the consumption of a regular daily quantity of fresh blueberry fruits. Along with the chemical characterization of the phenolic content of the fruits and extract, the antioxidant potential of the extract, the cellular antioxidant activity and the effects over neutrophils’ oxidative burst, we studied the effect of the blueberry extract in two inflammatory response animal models in order to provide a fresh insight into the anti-inflammatory effects observed. We theorize that fresh blueberry fruits, acting as a functional food, or an extract of blueberry fruits, could both have an important role in delaying the progression of some inflammatory chronic diseases such as rheumatoid arthritis. 2. Material and methods 2.1. Materials, solvents and reagents Gallic acid (98%), sulphuric acid (95–97%) and Luminol were purchased from Fluka (Seelze, Germany). Sodium hydroxide (98%), calcium chloride dihydrate, magnesium sulfate and sodium hydrogencarbonate were purchased from Merck (Darmstadt, Germany). Chloride acid (0.1 M), absolute ethanol (99.9%), methanol (99.9%), hydrochloric acid, glacial acetic acid, 99%, and sodium acetate anhydrous 99%, were purchased from Carlo Erba Reagents (Rodano, Italy). Cyanidin-3-O-glucoside was purchased from Extrasynthese (Lyon Nord, France). Tannic acid was purchased from BDH Laboratory reagents (Poole, England). Phosphoric acid p. a (85%) and ascorbic acid were purchased from Panreac Química (Barcelona, Spain). Acetonitrile HPLC gradient grade was purchased from VWR1 (Leuven, Belgium). Milli-Q1 water (18.2 MV. cm) obtained in a Millipore – Direct Q3 UV System equipment (Molsheim, France). Potassium chloride was obtained from Pronalab (Abrunheira, Portugal), sodium chloride and Sodium salt of 2,6-dichlorophenol indophenols, 90%, from Riedel-de Haën (Hanover, Germany). All other reagents were purchased from Sigma-Aldrich (St. Louis, USA). 2.2. Preparation of the blueberry (Vaccinium corymbosum L.) fruit extract For the present study, blueberries from unknown cultivar produced in Portuguese territory were used, and preserved at
20  C until samples are processed. The blueberry fruit extract was prepared according to the method described by You et al. [20], with some modifications. Liquid nitrogen was added to 100 g of fresh fruit, and smashed until a powder was obtained. To this powder, 250 mL of an ethanol (60%, v/v) solution was added (extractant solution). The resulting suspension was agitated during 1 min using a vortex machine, followed by 30 min in an ultrasounds bath

at 30  C. The suspension was centrifuged at 6000 rpm at room temperature for 15 min, and the supernatant was removed. Another 250 mL of extractant solution were added to the sediment and the procedure was repeated until the recovery of the second supernatant that was added to the first collected. The solution was then evaporated until a volume of 100 mL was obtained. In the end, the extract was filtered using 0.45 mm filters, and freezed to
20  C until used. Before administering to animals, the extract was diluted (1:2) with deionized water to simulate the desired fresh fruit daily dose ingestion and, simultaneously, the maximum daily dose to be studied. The volume administered in vivo was 2.1 mL of diluted extract per kg. 2.3. Spectrophotometric assays 2.3.1. Total phenolic content This determination was performed according to Stamatakis et al. [21], with some modifications as previously described by Figueira et al. [5]. Results were expressed as milligrams of gallic acid equivalents (mg GAE) per 100 g of fresh fruit and per mL of extract. Samples were analysed in triplicate. 2.3.2. Total flavonoid content Total flavonoid content was determined in the diluted extract (1:250) as described by Çam M et al. [22]. Results were expressed in mg of catechin equivalents (mg CE) per 100 g of fresh fruit and per mL of extract. Samples were analysed in triplicate. 2.3.3. Total anthocyanins content Total anthocyanins content was determined in the diluted extract (1:20) as described previously [5,21]. For each anthocyanin, a correction factor of molecular weight (CFMW) was used, as described [23]. Results were expressed as milligrams of cyanidin3-O-glucoside equivalents (mg CGE) per 100 g of fresh fruit and per mL of extract. Samples were analyzed in triplicate. 2.3.4. Total hydrolysable tannins content Determined according to the method previously described [22]. Results were expressed as milligrams of tannic acid equivalents (mg TAE) per 100 g of fresh fruit. Samples were analyzed in triplicate. 2.3.5. Total procyanidins content Determined according to the method described by Sun et al. [24]. Results were expressed in mg of catechin equivalents (mg CE) per 100 g of fresh fruit and per mL of extract. Samples were analyzed in triplicate. 2.4. Oxygen radical absorbance capacity (ORAC) The method of Huang et al. [25] modified for the FL800 microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT, USA) [5]. All data were expressed as micromoles of trolox equivalents antioxidant capacity (TEAC) per 100 g of fresh blueberry weight. 2.5. Hydroxyl radical adverting capacity (HORAC) The method developed by Ou et al. [26] modified for the FL800 microplate fluorescence reader as previously described [27] was used. Caffeic acid was used as a standard as it provides a wider linear range as compared to gallic acid. Data was expressed as mmol or mmol of caffeic acid equivalents antioxidant capacity (CAEAC) per 100 g of fresh blueberry weight. Results are a mean of six replicates.

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2.6. Analysis of the blueberry extract by HPLC-DAD HPLC analysis was carried out using a HPLC system from Thermo Finnigan (Surveyor model) equipped with a diode-array detector (DAD) and an electrochemical detector (Dionex, ED40), as previously described [5]. Identification of compounds was done by comparing retention time, spectra and spiking samples with known amounts of pure standards, whenever available. 2.7. In vitro experiments 2.7.1. Cellular antioxidant activity (CAA) Human colon carcinoma Caco-2 cells were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) and seeded at a density of 2  104 cell/well in a 96-well plate in 100 mL of growth medium. The medium was changed every 48 h and the experiments were performed when cells reach confluence (72 h). After that, medium was removed and cells were washed twice with PBS. Triplicate wells were treated for 1 h with 100 mL of different concentrations of blueberry extract plus 25 mM DCFH-DA diluted in PBS. Then, medium was removed and replaced by PBS containing 600 mM AAPH. The 96-well microplate was placed into a fluorescence reader (FL800, Bio-Tek Instruments, Winooski, VT, USA) at 37  C. Emission at 530  25 nm was measured after excitation at 485  20 nm every 5 min for 1 h. Each plate included triplicate control wells (cells treated with DCFH-DA and oxidant, namely AAPH) and blank wells (cells treated with DCFH-DA without oxidant). Quercetin was used as a standard. Cellular antioxidant activity (CAA) of extracts was quantified according to Wolfe et al. [28]. The EC50 values were stated as mean  SD for triplicate sets of data obtained from the same experiment. EC50 were converted to CAA values, expressed as micromoles of quercetin per g of blueberry, using the mean EC50 value for quercetin from three independent experiments. 2.7.2. Isolation of human neutrophils Isolation of human neutrophils was performed by gradient density, as previously reported by [29]. The obtained cell suspensions contained more than 99% of neutrophils and the control of their viability showed more than 95% of the cells excluding trypan blue solution 0.4%. Isolated neutrophils were kept in ice until use. Tris glucose (25 mM Tris, 1.26 mM CaCl22H2O, 5.37 mM KCl, 0.81 mM MgSO4, 140 mM NaCl, and 5.55 mM DGlucose) was the incubation media used, as previously recommended by [30]. 2.7.3. Evaluation of neutrophils’ oxidative burst The chemiluminescent probe luminol has been thoroughly studied and used for monitoring the production of reactive species

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by neutrophils, namely the superoxide anion radical (O2
), hydrogen peroxide (H2O2), hydroxyl radical (HO), hypochlorous acid (HOCl), nitric oxide (NO) and peroxynitrite anion (ONOO
) [31]. The measurement of neutrophils’ oxidative burst was undertaken by chemiluminescence, by monitoring ROS-induced oxidation of luminol, according to a previously described procedure [32]. The reaction mixtures contained neutrophils (1 106cells/ml) and the following reagents at the indicated final concentrations (in a final volume of 250 mL): tested compounds at various concentrations, luminol (500 mM) and phorbol myristate acetate (PMA; 160 nM). Cells were pre-incubated with luminol and the tested compounds for 5 min before the addition of PMA and the measurements were carried out at 37  C, under continuous soft shaking. Kinetic readings were initiated immediately after cell stimulation. Measurements were taken at the peak of the curve. This peak was observed at around 10 min. Effects are expressed as the percent inhibition of luminol oxidation. Each study corresponds to, at least, four individual experiments, performed in triplicate in each experiment. 2.7.4. Statistics Statistic treatments were done using GraphPad PrismTM (version 5.0; GraphPad Software). Results are expressed as mean  standard error of the mean (SEM) Statistical comparison between groups was estimated using the one-way analysis of variance (ANOVA), followed by the Bonferroni’s post hoc test. In all cases, p-values lower than 0.05 were considered as statistically significant. 2.8. In vivo experiments 2.8.1. Animal care and maintenance Experiments were conducted according to the Home Office Guidance in the Operation of Animals (Scientific Procedures) Act 1986, published by Her Majesty’s Stationary Office, London, UK and the Institutional Animal Research Committee Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), as well as to the currently adopted EC regulations. Finally, the studies are in compliance with the ARRIVE Guidelines for Reporting Animal Research’ summarized at www.nc3rs.org.uk. Hence, the ethics committee endorsed the animal study protocol, considering also that the Portuguese General Directorate of Alimentation and Veterinary licensed authors Sepodes and Rocha to coordinate and conduct independent animal research. All studies were carried out using male Wistar rats with 5 weeks of age weighing 100–150 g (Harlan Iberica, Barcelona, Spain). All animals received a standard diet and water ad libitum.

Table 1 Phenolic composition of the fresh blueberry fruit extract and relation to fresh fruit and a comparison to values previously published in the literature (values are expressed as mean of three replicates  standard deviation). Parameter

/100 g of Fresh Blueberry Fruit /mL of Fresh Blueberry Extract Literature reference values (/100 g Fresh Blueberry Fruit)

Total phenolic content (mg of GAE)

236.90  3.40

2.37  0.03

Total flavonoids content (mg of CE)

47.35  2.24

0.47  0.02

Total hydrolysable tannin content (mg of TAE) 276  0.04

2.76  0.04

Total procyanidin content (mg of CE)

116.67  2.53

1.17  0.02

Total anthocyanin content (mg of C3GE)

58.61  1.99

0.58  0.02

GAE – Galic acid equivalents; CE – Catechin equivalents; C3GE – equivalents; TAE – tannic acid equivalents.

289.5 (Giovanelli & Buratti, 2009) 112.0 (Sinelli et al., 2008) 266.0 (Pertuazatti et al., 2007) 179.8 (Gonçalves et al., 2007) 112.7 (Giovanelli & Buratti, 2009)

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2.8.2. Carrageenan-induced paw edema The carrageenan-induced paw edema of the rat hind paw is a suitable model to study acute local inflammation and widely considered to be one of the most useful models in the evaluation of anti-inflammatory activity [33,34]. Paw edema was induced by a single sub-plantar injection into the rat left hind paw of 0.1 mL of a 1% l-carrageenan sterile saline solution. Paw volume was measured by means of a volume displacement method using a plethysmometer (Digital Plethysmometer LE7500; Letica Scientific Instruments, Letica, Spain). Paw volume was measured immediately after the injection of carrageenan (V0 or basal volume) and 6 h later (V6h). Paw edema was induced by sub-plantar injection into the rat left hind paw of 0.1 mL sterile saline containing 1% l-carrageenan. The volume of the paw was measured immediately after the injection and subsequent readings of the same paw were measured at 6 h and compared to the initial readings. The increase in paw volume was taken as edema volume. Animals were randomly allocated into the following groups as described: (i) Control group: animals were subjected to sub-plantar injection into the rat left hind paw of 0.1 mL sterile saline and administered with saline (1 mL/kg, i.p.) (n = 6); (ii) Carrageenan group: animals subjected to paw edema induction and administered with saline (1 mL/kg, i.p.) (n = 6); (iii) Blueberry group: animals subjected to paw edema induction and pre-treated with blueberry fruit extract

(12.5 mg crude extract/kg/day by gavage) 30 min before l-carrageenan injection (n = 5); (iv) Indomethacin group: animals subjected to paw edema induction and pre-treated with indomethacin (10 mg/kg, i.p.) 30 min before l-carrageenan injection (n = 6); (v) Trolox group: animals subjected to paw edema induction and pretreated with trolox (30 mg/kg p.o.) 30 min before l-carrageenan injection (n = 6). 2.8.3. Collagen-induced arthritis Bovine CII was dissolved in 0.01 M acetic acid at a concentration of 2 mg/mL by stirring overnight at 4  C. Dissolved CII was frozen at
70  C until used. Complete Freund’s adjuvant (FCA) was prepared by the addition of Mycobacterium tuberculosis H37Ra at a concentration of 2 mg/mL. Before injection, CII was emulsified with an equal volume of FCA. CIA was induced as previously described [4,34,35]. On day 1, rats were administered intradermally at the base of the tail with 100 mL of the emulsion (containing 100 mg of CII). On day 21, a second injection of CII in FCA was administered. In another set of experiments, animals (n = 5 per group) were treated with blueberry fruit extract (12.5 mg crude extract/kg/day by gavage) every 24 h, starting on day 23. Rats were evaluated daily for arthritis [35]. Clinical severity was also determined by quantitating the change in the paw volume, as measured by plethysmometry on day 35. At the end of the

Table 2 HPLC-DAD analysis: main phenolic content of the fresh blueberry fruit and extract (values are expressed as means of three replicates  standard deviation). Phenolic compound

mg/100 g of Fresh Blueberry Fruit

mg/mL of Fresh Blueberry Extract

Quinic acid Malic acid Caffeic acid-4-glucoside Caffeic acid-3-glucoside Chlorogenic acid Caffeic acid Isorhamnetin-3-galactoside Isorhamnetin-3-glucoside Quercetin-3-rutinoside Quercetin-3-galactoside Quercetin-3-glucoside Myricetin-3-galactoside Myricetin-3-glucoside Kaempferol-3-rutinoside Quercetin-3-xyloside Isorhamnetin
3- rutinoside Quercetin-3-rhamnoside Kaempferol
3- galactoside Kaempferol
3-glucoside Laricitrin-3-O- galactoside Laricitrin-3-O-glucoside Myricitin-3-O-pentoside Laricitrina-3-rhamnoside Quercetin(acetyl) galactoside Quercetin(acetyl) glucoside Quercetin-3-glucuronide Siringetin-3-O-galactoside Siringetin-3-O-glucoside Siringetin-3-O- rhamnoside Delphinidin
3-O-galactoside Delphinidin
3-O-glucoside Cyanidin-3-O-galactoside Cyanidin-3-O-glucoside Delphinidin-3-O-arabinoside Petunidin-3-O-galactoside Petunidin-3-O-glucoside Cyanidin-3-O-arabinoside Peonidin-3-O-galactoside Peonidin-3-O-glucoside Petunidin-3-O-arabinoside Malvidin-3-O-galactoside Malvidin-3-O-glucoside Peonidin-3-O-arabinoside Malvidin-3-O-arabinoside

1.10  0.20 0.40  0.03 4.70  0.4 4.60  0.2 101.9  0.2 1.70  0.02 2.91  0.03

0.011  0.005 0.004  0.0001 0.047  0.02 0.046  0.01 1.019  0.01 0.017  0.001 0.029  0.001

0.86  0.08 2.88  0.05 4.63 (mg Eq Myricetin-3-glucoside/100 g)

0.008  0.0002 0.028  0.003 0.046  0.005 (mg Eq Myricetin-3-glucoside/mL)

0,91  0.01 (mg Eq Quercetin-3-xyloside/100 g) 6.60  0.1 (mg Eq Kaempferol-3-glucoside/100 g)

0.009  0.001 (mg Eq Quercetin-3-xyloside/mL) 0.066  0.005 (mg Eq Kaempferol-3-glucoside/mL)

3.66  0.01 (mg Eq Quercetin-3-glucuronide/100 g)

0.036  0.001 (mg Eq Quercetin-3-glucuronide/mL)

0.839  0.05 27.6  0.02

0.008  0.003 0.275  0.001

4.98 0.05 22.7  0.3 (mg Cyanidin-3-glucoside/100 g) 20.6  0.01

0.049  0.003 0.226  0.02 (mg Cyanidin-3-glucoside/mL) 0.206  0.005

6.64  0.05 14.14  0.1 (mg Peonidin-3-glucoside/100 g)

0.066  0.02 0.141  0.05 (mg Peonidin-3-glucoside/mL)

36.24  0.6

0.362 0.03

24.65  0.5 (mg Malvidin-3-arabinoside/100 g)

0.246  0.02 (mg Malvidin-3-arabinoside/mL)

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Fig. 1. Inhibitory effect of blueberry extract (1.1-35 mg/mL) on human neutrophils’ oxidative burst. Neutrophils’ were stimulated with phorbol myristate acetate, as measured by luminol-amplified chemiluminescence. ***p<0.001 compared with the control assay (PMA alone). The values are given as the mean  SEM (n 4).

experimental protocol, blood samples were collected into a BD Vacutainer SST II Advance gel and clot activator tube [35 mL (BD Diagnostics – Preanalytical Systems, Oxford, UK)] and were centrifuged (3000 rpm for 10 min at room temperature) to separate serum, to be analyzed by a Cobas Analyzer Roche1 Automated system. Alanine aminotransferase (ALT, a specific marker for hepatic parenchymal injury) and aspartate aminotransferase (AST, a non-specific marker for hepatic injury) were measured to evaluate the liver function. Creatine Kinase (CK) was measured to evaluate the neuromuscular injury. Urea and creatinine were measured to evaluate the renal function. Measurements of serum levels of pro-inflammatory cytokines (TNF-a, IL1b, IL-6) were also performed by means of ELISA assays (Quantikine1 Rat TNFa/TNFSF1A Immunoassay, Quantikine1 Rat IL-1b/IL-1F2 Immunoassay, Quantikine1 IL-6 Immunoassay from R&D Systems Inc. (Minneapolis, EUA). 2.8.3.1. Histologic (light microscopy) assessment of arthritis. On day 35, animals were sacrificed while under anesthesia, and the paws and knee joints were removed and fixed in formalin for light

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microscopy examination. Knee joint tissue samples were obtained at the end of the experimental period and were fixated in a solution of paraformaldehyde in PBS (10% v/v), at room temperature. Before dehydration and inclusion in paraffin blocks, samples were decalcified with formic acid (10%) for a minimum of 8 days. Samples were dehydrated using ethanol gradients and included on paraffin blocks, following sectioning at 3 mM. All sections were deparaffinized with xylol and hydrated in progressively less concentrated solutions of ethanol, ending with distilled water. Afterwards, they were stained with hematoxilin/eosin, dehydrated, diphanized and placed on a mount fluid. All sections were observed with an optical microscope with the 10 x and 40 x lens and photographed. For the light microscopy analysis, an optical microscope Zeiss, a photographic camera Leica DFC490, and an images capture software Leica IM50 were used. An investigator who was blinded to the treatment regimen performed the histologic examination, scoring the morphologic features on a scale of 0–3, where 0 = no damage, 1 = edema, 2 = inflammatory cells, and 3 = bone resorption. 2.8.3.2. Immunohistochemistry for iNOS and COX-2. After sacrifice, animals were perfused with 0.1 M phosphate buffer saline (PBS) (pH 7.4) followed by the same buffer containing 4% paraformaldehyde (PFA). Fixed tissues were post-fixed in 4% PFA in PBS for 72 h at room temperature (RT), decalcified, dehydrated through a graded ethanol series and embedded in paraffin. Histopathologic features (inflammation, pannus formation, cartilage damage, and bone resorption) in knee joint specimens were observed following Hematoxylin-eosin staining. For immunostaining, 6 mm sections were submitted to antigen retrieval in 20 mM citrate buffer with 1.5% H2O2 for 15 min at RT in the dark, incubated for 10 min in Tris/EDTA buffer at 84  C and blocked for 1 h at RT in 1% bovine serum albumin (BSA) in PBS. Primary antibodies, rabbit anti-COX-2 (Cell Signaling #4842, 1:100) and mouse anti-iNOS (BD Transduction Laboratories #610328, 1:100) were used in 0.5% BSA in PBS overnight at 4  C. After washing in PBS, sections were incubated for 1 h at RT with antibodies anti-rabbit and anti-mouse coupled with horseradish

Fig. 2. Effects of blueberry extract on carrageenan-induced paw volume increase. Effect of a single administration of blueberry extract (12.5 mg crude extract/kg/day by gavage; n = 5) on rat paw edema development elicited by carrageenan 6 h after induction and comparison with the effect of indomethacin (10 mg/kg, i.p.; n = 6) and trolox (30 mg/kg, i.p.; n = 6). The administration of the extract significantly inhibited rat paw edema formation. The data are presented as mean  SEM. *P<0.001 vs. control group; z P < 0.005 vs. control group; #P<0.001 vs. carrageenan group; x P < 0.005 vs. carrageenan group.

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Fig. 3. Effects of blueberry extract on CIA associated paw volume increase. Effect of treatment with blueberry extract (12.5 mg crude extract/kg/day) on rat paw edema development associated to CIA (as seen on RA group). Treatment with the extract significantly reduced paw edema. Results are presented as mean  SEM. *P < 0.001 vs. RA group.

peroxidase (Santa Cruz Biotechnology, 1:5000) in 0.5% BSA in PBS, incubated for 10 min in SIGMAFASTTM DAB with Metal Enhancer (SIGMA) and mounted with EntellanÒ (Merck). Tissue sections were visualized in an Axioskop bigthfield microscope and images were acquired with a camera DFC 490 (Leica). Densitometric analysis of immunocytochemistry photographs from paw section was performed using Optilab Graftek software.

2.8.3.3. Radiographic analysis. Animals were anesthetized with sodium pentobarbital (45 mg/kg, intraperitoneally) and placed on a radiographic box at a distance of 90 cm from the X-ray source. Radiography of normal and arthritic rat hind paws was performed using an X-ray machine, with a 40-kW exposure for 0.01 s. An investigator who was blinded to the treatment regimen performed the radiographic scoring, using a scale of 0–3, where 0 = no bone

Fig. 4. Radiographic progression of CIA in the tibiotarsal joints. (A) There is no evidence of pathological alterations in the tibiotarsal joints of control (sham) animals. (B) The hind paws of rats with CIA at day 35 demonstrated bone resorption and quite significant joint erosion. Treatment with blueberry extract (C, D) significantly suppressed the joint pathology and the soft tissue edema in the hind paws. The X-ray images are representative of at least 3 experiments performed on different experimental days.

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Fig. 5. Histopathology findings regarding the effects of blueberry extract in a rat CIA model. Rats were treated with the extract (12.5 mg crude extract/kg/day). Specimens from vehicle-treated CIA rat exhibit severe synovitis, cartilage damage (large arrow), and marked infiltration and pannus (small arrow) and bone resorption (arrow head). Specimens from rats treated with blueberry extract exhibit normal synovium, small cartilage damage (large arrow), and reduced marginal zone pannus (small arrow) or bone resorption (arrow head). (original magnification x100).

damage, 1 = tissue swelling and edema, 2 = joint erosion, and 3 = bone erosion and osteophyte formation. 3. Results and discussion Spectrophotometric analysis allowed us to determine the total phenolic compounds, total flavonoids, total anthocyanins, total hydrolysable tannins, total condensed tannins and total procyanidins content of the blueberry extract to be used in the in vivo experiments (Table 1). The concentration of these compounds was expressed per 100 g of fresh fruit and per mL of extract of fresh blueberries, allowing for a comparison with the data available in the literature. We were able to conclude that the extract content is directly comparable to what is described in the literature for blueberry samples. Using chromatographic techniques, phenolic compounds were separated, identified and quantified. The main phenolic compounds identified are listed in Table 2. It is clear that blueberries exhibit a wide range of phenolic compounds. Chlorogenic acid is the most abundant phenolic acid, followed by caffeic acid. The most abundant flavonol is quercetin, followed by myricetin, siringetin, laricitrin, isorhamnetin and kaempferol, with these last two representing less then 5% of total flavonoids. With the exception of quercetin, all other flavonols are more dependent on

the species studied [36–39]. In Table 2 we can also find the identified anthocyanins. Amounts vary between species due to several factors. In Vaccinium corymbosum fruits, malvidin and delphinidin glucosides are present in high quantities, followed by petunidin and cyanidin, while peonidin glucosides are present in reduced amounts [40–42]. The antioxidant capacity was evaluated by the ORAC and HORAC assays. For this extract, the ORAC assay value was 22869 mmol of equivalents of trolox/L of extract and the HORAC assay value was 5998 mmol of equivalents of caffeic acid/L of extract. This means that the blueberry extract administered to rats has a high scavenger capacity for reactive oxygen species, hence the blueberry fruit has a significant high antioxidant capacity when compared to other fruits such as raspberry [5], blackberry [12,28,43] and gooseberry [43]. Although the antioxidant activity of this blueberry extract is considered high, there is no consensus on the compounds that predominantly contribute for this effect. The intracellular antioxidant activity assay has the advantage of providing information on what is happening in the intracellular environment, since the ORAC and HORAC methods only target chemical reactions that cannot be interpreted as occurring in vivo. The intracellular antioxidant activity assay takes into account the complexity of biologic systems namely bioavailability, stability, tissue binding and/or retention, and the reactivity of the

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Fig. 6. Effect of blueberry extract treatment on the histologic damage score (A) and the radiographic score (B) in animals with collagen-induced arthritis (CIA). The values presented are the mean and standard error of mean. *p<0.001 versus sham; #p<0.001 versus vehicle-treated (CIA).

compounds under physiologic conditions [44]. Results of the inhibition of neutrophils oxidative burst by blueberry extract are represented in Fig. 1. Pre-incubation of neutrophils with blueberry extract resulted in a significant increase of the oxidation of luminol with ascending concentrations of the extract. The oxidation of luminol results from the activation of neutrophils with PMA. Obtained results are all statistically significant, demonstrating dose-dependent antioxidant effect even at low concentrations (1.1 mg/mL) of the extract. At 18 mg/mL, inhibition of luminol oxidation reached 90%. The dosedependent effect should be related to the increasing phenolic content of the extract. To assess the anti-inflammatory capacity of the extract we tested the extract in the carrageenan paw edema model and Collagen type II induced rheumatoid arthritis. As expected, in the carrageenan paw edema model in rats, there is a significant increase of the hind paw volume, attributed to the inflammatory reaction, in animals administered solely with carrageenan (Fig. 2). The administration of blueberry extract significantly reduced edema formation, by 30%, suggesting a direct interference with the inflammatory response elicited by

carrageenan. Results relate to edema after 6 h of the administration of the flogistic agent. This time point is associated to the late inflammatory response phase, mainly mediated by prostaglandins and interleukins, produced after the migration of polymorphonuclear cells [45]. Results obtained suggest that the blueberry extract had an anti-inflammatory effect that could be justified not only by the antioxidant properties but also by other mechanisms, that could account not only for the effects seen even over 6 h postadministration. This is also in line with the comparison to other drugs tested in the same experimental model, with different known mechanisms of action. Trolox is a hydro soluble analogue of vitamin E and reduced edema by 50% when compared to the carrageenan control; tempol is a superoxide dismutase mimetic with low molecular weight able to reduce edema by 50% when compared to carrageenan control; indomethacin is a non-selective cyclooxygenase inhibitor, able to reduce edema in this experimental model by 50% when compared to carrageenan control. These results demonstrate that the blueberry extract showed a significant anti-inflammatory, nevertheless inferior to the effect observed when known anti-inflammatory drugs acting via different mechanisms were used. Interestingly, when the same

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Fig. 7. Blueberry extract administration reduces COX2 and iNOS activation in the CIA model – immunohistochemistry qualitative analysis. Rats were treated with the extract (12.5 mg crude extract/kg/day) and samples immunostained for COX2 (A) and iNOS (B). Specimens from non-treated animals exhibit almost no expression of COX-2 and iNOS, while vehicle-treated arthritic rats exhibit marked production of COX-2 and iNOS (brown staining
large arrow) and neutrophils infiltration (large circle). Specimens from animals treated with the blueberry extract exhibit reduced neutrophils infiltration and COX-2 staining and almost no iNOS expression (original magnification x100).

experimental setting was used for a raspberry extract [5], the raspberry extract produced a more powerful anti-inflammatory effect compared to trolox, tempol and indomethacin and, subsequently, when compared to the blueberry extract. We cannot define the precise mechanism behind the anti-inflammatory effect observed with the blueberry extract. Nevertheless, the ability to scavenge oxygen reactive species seems to be undeniably involved in the anti-inflammatory effect of this extract. The phenolic content of the extract can certainly account for the antioxidant capacity, but other compounds could also contribute for the antiinflammatory effect. Wang and colleagues [46–48] compared the action of isolated cyanidin to several non-steroidal anti-inflammatory drugs such as naproxen, ibuprofen and acetylsalicylic acid, confirming the anti-inflammatory action of cyanidin. Rotelli et al. [49] evaluated the action of quercetin and rutin in the carrageenan-induced paw edema experimental model, demonstrating that quercetin had higher anti-inflammatory effect when compared to both rutin and phenylbutazone. A model of rheumatoid arthritis induced by type-II collagen was used to understand the effect of this extract in a more complex inflammatory response, involving edema but also a more robust cellular inflammatory response. In non-treated animals, where rheumatoid arthritis was induced, positive controls of

inflammation were identified (e.g. edema and loss of function of the hind paws). We quantitatively measured edema of the hind paws in all experimental groups. The non-treated group (AR group) shows a significant increase of volume, suggesting an edema formation directly proportional to the inflammatory insult in this group (Fig. 3). In animals treated with the blueberry extract, there is a statistically significant reduction in edema formation (70%) when compared to animals non-treated with the extract; this demonstrates that in a chronic inflammatory response setting, the blueberry extract administered orally had a clear anti-inflammatory effect. When compared to results obtained in the acute inflammatory setting, here we can observe a higher reduction of edema, probably due to multiple anti-inflammatory effects. Subarnas and Wagner [50] demonstrated that an extract with high concentration of anthocyanins had an anti-inflammatory effect by inhibition of cyclooxygenases and inhibition of TNF- a production. Other polyphenols that are part of the extract, such as chlorogenic acid and quercetin, are able to inhibit the activity of cyclooxygenases and lipoxygenase [51,52]. As contributor to this reduction in edema, one cannot disregard the role of the repeated administration of extract in this experimental model (daily for 13 days, p.o.), probably originating higher and stable plasmatic levels of phenolic compounds that could account for the observed

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Fig. 8. Blueberry extract administration reduces COX2 and iNOS activation in the CIA model – immunohistochemistry quantitative analysis. Rats were treated with the extract (12.5 mg crude extract/kg/day) and samples immunostained for COX2 and iNOS. Specimens from animals treated with the blueberry extract exhibit a reduced infiltration and COX2 staining and almost no iNOS expression as seen on Fig. 7. Densitometric analysis of immunocytochemistry photographs from paw section was performed using Optilab Graftek software. *p<0.001 versus Vehicle, #p<0.001 versus CIA + Vehicle.

effects along time. Our results are consistent with the previous work of Torri et al. [53] where a crude hydroalcoholic extract of Vaccinium corymbosum was administered orally at doses of 100, 200 or 300 mg/kg for all the assays. In the carrageenan test, the crude extract reduced rat paw oedema by 9.8, 28.5 and 65.9%, respectively [53]. Considering that the crude extract of blueberry displayed antinociceptive and anti-inflammatory activity, the authors clearly conclude that its consumption may be helpful for the treatment of inflammatory disorders [53]. Radiographic analyses are shown in Fig. 4. Periarticular erythema and edema were observed in the hind paws 21 days post-challenge. A 100% incidence of CIA was observed by day 23 in all CII-immunized rats. No clinical signs of CIA were observed in rat fore paws during the 35-day evaluation period. Hind paw erythema and swelling increased in severity and time-dependent manner (data not shown) with a maximum arthritis index observed till day 35. Treatment with the extract was able to significantly reduce edema formation in the hind paws when compared to untreated animals. On day 35, the histologic evaluation revealed that specimens from a non-treated control rat exhibit normal synovium, no cartilage damage and no marginal zone pannus or bone resorption. Samples from a vehicle-treated CIA rat exhibit severe synovitis, cartilage damage, marked infiltration and pannus, and bone resorption. Specimens obtained from animals treated per os exhibit normal synovium, small cartilage damage, and reduced marginal zone pannus or bone resorption (Figs. 4 C, D and 5 ). When considering the radiographic examination of the hind paws

35 days after immunization with CII revealed bone matrix resorption and osteophyte formation at the joints (Figs. 4 B and 5). There was no evidence of CIA pathology in control (normal) rats. Radiographic examination confirmed that the blueberry extract was able to markedly reduce the degree of bone resorption, soft tissue swelling and osteophyte formation, improving articular function in treated animals. Our results show that macroscopic evidence of CIA appeared firstly as periarticular erythema and edema of the hind paws. The incidence of CIA was 100% by day 23 in the challenged rats, and the severity of CIA progressed over a 35day period. Radiographs revealed focal resorption of bone, with osteophyte formation in the tibiotarsal joint, and soft tissue swelling. Treatment with the extract starting at the onset of arthritis (day 23) delayed the development of the clinical signs on days 24–35 and improved the histologic and radiographic scores of the knee joint and hind paw (Fig. 6). The inflammatory signalling net is significantly attenuated with the proposed treatment, and there is even no significant production or release of proinflammatory cytokines that would induce COX-2 expression (Figs. 7 and 8) and there a down-regulation of iNOS expression in samples collected from animals treated with the blueberry extract (Figs. 7 and 8). Also, the determination of serum biochemical biomarkers to detect liver (AST and ALT), kidney (creatinine and urea) and neuromuscular (CK) injury/dysfunction, in animals subjected to a repeated administration of the extract as seen during the collagen induced arthritis experimental protocol, demonstrated the clear safety profile of this extract with no

Table 3 Serum levels of biochemical markers of liver, kidney and neuromuscular injury at the end of the CIA experimental protocol and repeat-dose administration of the extract (values are expressed as mean  standard error of mean, n = 5 per group). Biochemical parameters

Control + Vehicle

CIA + Vehicle

CIA + Blueberry extract

ALT (IU/L) AST (IU/L) Urea (mg/dL) Creatinine (mg/dL) CK (IU/L)

32  3 120  10 301  8 0.3  0.07 350  83

39  7 129  3 314  15 0.4  0.03 400  84

29  5 114  3 288  20 0.3  0.01 375  70

ALT – Alanine aminotransferase, AST – Aspartate aminotransferase, CK – creatine kinase. Blood was collected on the last day of CIA experimental protocol and centrifuged at 3000 rpm for 10 min to obtain serum. Statistical analysis did not reveal any significant differences between experimental groups.

M.-E. Figueira et al. / Biomedicine & Pharmacotherapy 83 (2016) 1191–1202 Table 4 Serum levels of interleukins at the end of the experimental protocol (values are expressed as mean  standard error of mean, n = 5 per group). *p < 0.001 vs. Vehicle; #p < 0.001 vs. CIA + Vehicle. Experimental Groups

TNF-a (pg/mL)

IL-1b (pg/mL)

IL-6 (pg/mL)

Vehicle CIA + Vehicle CIA + Blueberry Extract p.o.

0.11  0.005 0.46  0.01 * 0.25  0.03 *#

0.05  0.01 0.31  0.03 * 0.08  0.02 #

0.011  0,001 0.180  0,02 * 0.016  0,001 #

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Acknowledgements Financial support should be acknowledged to Fundação para a Ciência e Tecnologia (FCT) through the grant REDE/1518/REM/2005 for the LC–MS/MS equipment. Marisa Freitas acknowledges FCT the financial support for the Pos-doc grant (SFRH/BPD/76909/ 2011), in the scope of “QREN e POPH e Tipologia 4.1 e Formação Avançada”, co-sponsored by FSE and by National Funds of MCTES. References

significant effects on these parameters (Table 3). The serum levels of cytokines (TNF-a, IL-1b and IL-6) in animals treated with the blueberry extract are significantly reduced when compared to nontreated arthritic animals (Table 4), further supporting the concept that the inflammatory signalling net is significantly attenuated with the administration of the blueberry extract, as demonstrated by the reduction in production or release of pro-inflammatory cytokines such as TNF-a, IL-1b and IL-6. This is consistent with the results of iNOS and COX-2 expression, and also with the histological analysis. 4. Conclusions The chemical characterization of the blueberry extract with HPLC-DAD and LC-ESI–MS/MS allowed us to conclude that the blueberries used have a wide range of phenolic compounds. The most common flavonol was identified as being quercetin. A significant portion of the extract is constituted by anthocyanins. We have also demonstrated that this extract has high antioxidant capacity both in vitro and in vivo. In a CIA animal model the blueberry extract, when administered orally for 13 days was able to visibly reduce edema formation but also delayed the development of the clinical signs on days 24–35 and improved the histologic and radiographic scores of the knee joint and hind paw. Taken together, these results are good indicators for the use of the blueberry extract as a therapeutic adjuvant in the treatment of rheumatoid arthritis. This is particularly important in a polymedicated population that will benefit from this adjunctive therapy or supplementation. The effects we observed in the current work can be translated to practice or to a pharmaceutical dosage, when formulated as a supplement, considering the average consumption of blueberries in the population. This is in line with the work of Zhong et al. [54] where fresh juice of mashed blueberries was administered to a group of patients with Juvenile Idiopathic Arthritis and it was demonstrated that the consumption of juice for 6 months improved the therapeutic effect of etanercept in these patients. We hereby provide a mechanistic explanation for these observations and support the idea that a blueberry extract could be used as an adjunctive therapy. This is a clear translational example of how a functional food can improve quality of life when having pharmacological impact on a body function. Conflict of interest None. Financial support Financial support should be acknowledged to Fundação para a Ciência e Tecnologia (FCT) through the grant REDE/1518/REM/2005 for the LC–MS/MS equipment. Marisa Freitas acknowledges FCT the financial support for the Pos-doc grant (SFRH/BPD/76909/ 2011), in the scope of “QREN e POPH e Tipologia 4.1 e Formação Avançada”, co-sponsored by FSE and by National Funds of MCTES..

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