Fisheries Science

, Volume 82, Issue 2, pp 357–367 | Cite as

Conjugation with alginate oligosaccharide via the controlled Maillard reaction in a dry state is an effective method for the preparation of salmon myofibrillar protein with excellent anti-inflammatory activity

Original Article Chemistry and Biochemistry

Abstract

The efficacy of the controlled Maillard reaction with alginate oligosaccharide (AO) in a dry state was examined for development of an anti-inflammatory compound from fish myofibrillar protein (Mf). Lyophilized Mf from spawned-out chum salmon was mixed with AO (half of the total protein weight) and incubated at 60 °C and 35 % relative humidity for 0–6 h, followed by digestion with pepsin and trypsin. The anti-inflammatory activity of the digested peptide was improved with the progress of the AO conjugation, and dMSA4 (prepared from Mf–AO conjugate by reaction for 4 h, with 49.6 μg/mg protein of AO attached) was most effective in inhibiting secretions of inflammation-related compounds in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Likewise, dMSA4 suppressed the LPS-induced gene and protein expression of inducible nitric oxide synthase, tumor necrosis factor-α, interleukin-6, and cyclooxygenase (COX)-2, while COX-1 expression was unaffected. Furthermore, oral administration of dMSA4 and the Mf–AO conjugate prior to proteolysis inhibited carrageenan-induced paw volume expansion in mice. These results indicate that AO conjugation using the controlled Maillard reaction in a dry state is a useful approach for enhancing the anti-inflammatory activity of salmon Mf as a nutraceutical food material.

Keywords

Alginate oligosaccharide Anti-inflammation Maillard reaction Muscle protein Salmon 

Introduction

Inflammation is an important host defense reaction by the immune system, which protects the body from harmful stimuli such as invasive pathogens. Immune cells including macrophages, natural killer cells, and dendritic cells eliminate foreign substances such as viruses and bacteria via inflammatory signaling cascades [1]. However, recent reports have indicated that excess or chronic inflammation is associated with diseases such as inflammatory bowel disease, diabetes, cardiovascular disease, and neurodegenerative disease [2, 3, 4, 5], suggesting that the regulation of excess and chronic inflammation is important for health maintenance.

It has also recently been recognized that lifestyle, and diet in particular, is an important factor for mitigating excess inflammation [6], with increasing interest in the use of food components to regulate inflammation. A number of small organic molecules in food components have been shown to possess anti-inflammatory properties [7, 8, 9, 10, 11]. Likewise, some food proteins [12, 13] have been found to exhibit anti-inflammatory activity. These findings suggest that anti-inflammatory effects may be an additional health benefit of foods.

Global consumption of seafood has increased steadily over the past half-century [14], and fish as an important protein source is widely utilized as a raw material for processed seafood. Various processed foods are manufactured using fish myofibrillar proteins (Mf), given their excellent functional properties, including their emulsifying and gel-forming ability and water-holding capacity. There is also increasing interest in the health benefits of seafood, as many epidemiologic studies have provided evidence of the beneficial effects of fish consumption, and fish Mf and Mf-derived peptides have been shown to exert angiotensin-converting enzyme (ACE) inhibitory effects [15] and antioxidant activity [16], and to prevent skeletal muscle insulin resistance [17]. Indeed, ACE-inhibitory peptides from fish Mf are marketed as functional materials defined as Food for Specified Health Uses (FOSHU) approved by the Japanese governmental Consumer Affairs Agency [18]. However, few studies have been reported on the anti-inflammatory activity of Mf and Mf-derived peptides.

The functional properties of food proteins can be improved by conjugation with glycosyl units using the Maillard reaction [19], which commonly takes place between food components during food processing and preservation. This non-enzymatic reaction occurs between the reducing terminus of sugars and the amino groups of proteins, and is suitable for application in the modification of food proteins, as no chemical reagents are required. For example, the antioxidant activity of phosvitin [20], the radical-scavenging activity of soy protein [21], the antimicrobial activity of lysozyme [22], and the immunoreactivity of hazelnut allergen [23] were shown to be enhanced by conjugation with reducing glycosyl units including glucose, galactomannan, curdlan, and allose. Likewise, Isono et al. [24]. reported that conjugation of chicken breast Mf with maltose increased its antioxidant activity.

The protein glycosylation described above proceeded via the Maillard reaction under conditions of elevated temperature and humidity (e.g. >50 °C and/or >65 % relative humidity). In our previous study [25], we demonstrated that peptides derived from fish Mf lyophilized with alginate oligosaccharide (AO) possessed anti-inflammatory activity, which was conferred by attaching a small amount of AO to the protein via the Maillard reaction during lyophilization. However, further study showed that the degree of AO modification in lyophilized Mf often varied even under identical lyophilization conditions, suggesting that the Maillard reaction under lyophilization was not well controlled. Thus it appeared that a controlled AO conjugation system should be adopted for the production of anti-inflammatory compounds from fish Mf.

The objective of this study was to elucidate the efficacy of the controlled Maillard reaction with alginate oligosaccharide (AO) in a dry state, with controlled temperature and humidity, in developing fish protein material with excellent anti-inflammatory properties. Here, Mf was prepared from spawned-out chum salmon Oncorhynchus keta, which is an underutilized protein resource because of its unacceptable soft texture and unpleasant taste and smell. AO was selected as the glycosyl unit to attach to the target protein as it has been found to be superior in enhancing the food functionalities of fish Mf [26, 27]. The chum salmon Mf was mixed with AO and sorbitol (protein denaturant), and the mixture was incubated under controlled temperature and humidity conditions to prepare the Mf–AO conjugates containing different quantities of AO. The effect of the AO conjugation on enhancing the anti-inflammatory activity of Mf was evaluated using lipopolysaccharide (LPS)-stimulated macrophages and carrageenan-induced paw edema in mice as a model of acute inflammation. The effect on gene and protein expression related to secretion of inflammation-related compounds in macrophages was also examined.

Materials and methods

Materials

Spawned-out chum salmon (average weight 3 kg) was obtained from the Hekirichi River hatchery of Hokkaido Prefecture (Hokuto, Hokkaido, Japan). They were immediately chilled, gutted, dressed, and frozen at −25 °C until use. AO (mean degree of polymerization = 6) prepared by alginate-lyase degradation was supplied from Hokkaido Mitsui Chemical Industry Co., Ltd. (Sunagawa, Hokkaido, Japan). The RAW 264.7 murine macrophage cell line was purchased from American Type Culture Collection via Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan). Male ICR mice (age 7 weeks) were obtained from Charles River Japan, Inc. (Yokohama, Japan). Pepsin from porcine mucosa, trypsin from bovine pancreas, and lipopolysaccharide (LPS) from Salmonella typhimurium were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and non-essential amino acids were purchased from Life Technologies (now Thermo Fisher Scientific, Carlsbad, CA, USA). Anti-murine tumor necrosis factor-α (TNF-α), anti-murine interleukin-6 (IL-6), biotinylated anti-murine TNF-α, biotinylated anti-murine IL-6, horseradish peroxidase (HRP)-conjugated streptavidin, and 3,3',5,5'-tetramethylbenzidine (TMB) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The recombinant TNF-α and IL-6 were purchased from BioVision, Inc. (Milpitas, CA, USA). Anti-murine β-actin rabbit IgG, anti-murine iNOS rabbit IgG, and peroxidase-conjugated goat anti-rabbit IgG antibody were purchased from MBL International Corporation (Nagoya, Japan), Cell Signaling Technology Japan, K.K. (Tokyo, Japan), and Bio-Rad Laboratories (Hercules, CA, USA), respectively. Anti-murine COX-1 rabbit IgG and anti-murine COX-2 rabbit IgG were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Immobilon Western Chemiluminescent HRP Substrate was purchased from EMD Millipore (Billerica, MA, USA). Indomethacin and carrageenan were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan), with the exception of the following: bovine serum albumin fraction V (BSA; Merck KGaA, Darmstadt, Germany), Bio-Safe Coomassie G-250 stain (Bio-Rad Laboratories, Hercules, A, USA), interferon-γ (IFN-γ; PeproTech, Inc., Rocky Hill, NJ, USA).

Preparation of salmon Mf

The salmon Mf was prepared as previously described [25]. Briefly, the thawed salmon muscle was minced and washed, and the protein suspension was homogenized. The homogenate was centrifuged to collect the Mf pellet, which was mixed with a cryoprotectant (sorbitol) or AO as follows: sorbitol and AO were added at amounts equal to or half of the total protein weight, respectively, and the protein–sugar mixtures were lyophilized by freeze-drying. In this study, two types of lyophilized samples were prepared, Mf mixed with sorbitol (MS) and Mf mixed with sorbitol and AO (MSA), and samples were stored at −30 °C until use.

Conjugation of Mf with AO

Since denaturation of salmon Mf and the loss of its salt solubility occurs in the dry state under conditions of high humidity (>65 % relative humidity) [27], a low-humidity condition was selected for this study. Thus AO conjugation of MSA was performed in a controlled dry state at 60 °C and 35 % relative humidity in a humidity cabinet (model PR-1G; Tabai ESPEC Corp., Tokyo, Japan) for 0, 2, 4, and 6 h (referred as to MSA0, MSA2, MSA4, and MSA6, respectively).

Enzymatic digestion of MS and MSA

Salmon Mf samples (MS and MSAs) were suspended in 50 mM NaCl and then precipitated in 60 % saturated ammonium sulfate at pH 7.5 to remove sorbitol and unbound AO. After dialysis against 50 mM NaCl, the protein suspensions were digested by pepsin (pH2.0) and trypsin (pH8.0) at 37 °C for 3 h each (enzyme Mf = 1:100 [w/w]). The digested MS and MSA samples were boiled for 15 min, lyophilized, and stored at −30 °C until use. The corresponding enzymatically digested samples as described above are denoted as dMS and dMSA0, dMSA2, dMSA4, and dMSA6, respectively. Before use in cell culture experiments, the digested Mf samples were dissolved in phosphate buffered saline (pH 6.8) and filtered through a 0.22-µm membrane.

Electrophoretic analysis of AO-conjugated protein

The conjugation of MSA with AO was visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), according to the method described by Laemmli [28], with 4.0 and 7.5 % acrylamide slab gels used as the stacking and resolving gels, respectively. Samples were prepared as follows: 0.1 ml of the protein solution was added to 0.2 ml of 20 mM Tris–HCl (pH 8.0) containing 2 % SDS, 8 M urea, and 2 % β-mercaptoethanol and was heated for 2 min, after which 10 μl was loaded onto the gel. Coomassie Brilliant Blue R-250 and Lillie's Cold Schiff’s Reagent were used for protein and carbohydrate staining [29], respectively.

Measurement of protein concentration and amount of AO bound to Mf

The protein concentration of each sample was determined by the Biuret method [30] using bovine serum albumin as a standard, and the AO concentration was determined by the phenol–sulfuric acid method [31]. The amount of AO bound to the protein was expressed as µg/mg of the protein. The assays were carried out after removal of sorbitol and unreacted AO as described above.

Measurement of remaining lysine content

Following reaction with AO, the protein was precipitated with 15 % trichloroacetic acid (TCA) by incubation at room temperature for 30 min to remove the unbound AO. After removal of TCA and AO by centrifugation at 2,100×g for 30 min, the precipitated protein was resuspended in 50 mM phosphate buffer (pH 9.5) containing 2 % SDS. The available lysine content was determined by spectrophotometric analysis using o-phthalaldehyde and N-acetyl-l-cysteine [32]. The available lysine content was calculated using the following equation: available lysine content (g/g protein) = 146.19 × absorbance at 335 nm/(6830 × protein concentration [mg/ml]), where 146.19 is the molecular weight of lysine and 6830 is the molar absorptivity of the OPA-lysine derivative.

Analysis of molecular weight distribution of digested Mf

The digested and lyophilized Mf samples were dissolved in 50 mM NaCl at a concentration of 10 mg/ml, filtered through a 0.45-µm membrane, and analyzed by gel permeation chromatography (Superdex Peptide 10/300 GL; GE Healthcare Life Sciences, Little Chalfont, UK). Samples were eluted using a flow rate of 0.5 ml/min, and eluents were monitored at 220 nm.

Cell culture

RAW264.7 cells were cultured in DMEM containing 10 % heat-inactivated FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM non-essential amino acids at 37 °C in 5 % CO2.

Analysis of NO production

RAW264.7 cells (200 µl, 2 × 105 cells/well) were seeded in 96-well plates, allowed to adhere for 2 h, and washed twice with phosphate-buffered saline prior to treatment. Cells were cultured in 200 µl of fresh 10 % FBS-DMEM (without phenol red) containing dMSAs (0–500 μg/ml), IFN-γ (0.5 ng/ml), and LPS (2.5 ng/ml) for 24 h. The secretion of nitric oxide (NO) was directly measured in the culture supernatants using the Griess method [33] with sodium nitrite as a standard.

Analysis of TNF-α and IL-6 production by ELISA

The dMSAs (0–500 μg/ml) and LPS (2.5 ng/ml) were added to RAW264.7 cells seeded in 96-well plates and cultured in fresh 10 % FBS-DMEM as described above. After cultivation for 24 h, the culture supernatants were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) to measure TNF-α and IL-6 concentrations, as previously described [25]. Briefly, the culture supernatants (50 µl) were added to 96-well ELISA plates (AGC Techno Glass Co., Ltd., Tokyo, Japan) coated with anti-murine TNF-α or anti-murine IL-6 as primary antibodies, and biotinylated anti-murine TNF-α or biotinylated anti-murine IL-6 as secondary antibodies. The concentrations of TNF-α and IL-6 were calculated by standard curves using recombinant cytokines.

Analysis of iNOS and COX protein expression by immunoblotting

RAW264.7 cells (3.0 ml, 3 × 106 cells/well) were seeded in 6-well plates for 2 h, and then cultured in 2.7 ml of fresh 10 % FBS-DMEM containing dMSAs (0–500 μg/ml), IFN-γ (0.1 ng/ml), and LPS (5.0 ng/ml) for 24 h. Total protein was extracted from the cells using lysis buffer (62.5 mM Tris–HCl, 2 % SDS, 10 % glycerol, 50 mM DTT, 0.01 % bromophenol blue), and the samples were heated at 95 °C for 5 min. Samples were subjected to Laemmli SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; EMD Millipore, Billerica, MA, USA) using a semi-dry blotting system (ATTO Corporation, Tokyo, Japan). The membrane was incubated for 1 h at room temperature in blocking buffer (5 % nonfat dry milk dissolved in 20 mM Tris–HCl, pH 7.6, containing 0.1 % Tween and 140 mM NaCl [TTBS]) and then incubated with the primary antibody at 4 °C overnight. The four primary antibodies were diluted with blocking buffer as follows: anti-murine β-actin rabbit IgG (1:10,000), anti-murine iNOS rabbit IgG (1:1000), anti-murine COX-1 rabbit IgG (1:500), and anti-murine COX-2 rabbit IgG (1:10,000). After washing three times with TTBS, the membrane was incubated with peroxidase-conjugated goat anti-rabbit IgG antibody (1:20,000) at 37 °C for 3 h. After three washes with TTBS, bound antibodies were detected with Immobilon Western Chemiluminescent HRP Substrate and visualized using a CCD camera (ATTO Corp., Japan). The intensity of each band was measured using an image analyzer (CS Analyzer; ATTO Corp., Japan) and was normalized with respect to β-actin (internal control). The resulting iNOS and COX-1 and -2 protein expression is presented as a percentage relative to control (untreated) cells.

Analysis of gene expression of inflammatory mediators

RAW264.7 cells (12.0 ml, 1.0 × 106 cells/ml) were seeded into plastic dishes (diameter: 100 mm) and allowed to adhere for 2 h. Cells were then cultured in fresh 10 % FBS-DMEM containing dMSAs (0–500 µg/ml), IFN-γ (0.1 ng/ml), and LPS (5.0 ng/ml) for 24 h. Extraction of total RNA from the culture cells, reverse transcriptase polymerase chain reaction (RT-PCR), and the primers used in the experiment were described in the previous work [25]. The PCR products were electrophoresed on 2.0 % agarose gel. After staining with ethidium bromide, the DNA bands were detected at 312 nm, and the reverse images are presented. The intensity of each DNA band was measured using the image analyzer and was normalized with respect to β-actin (internal control). The resulting mRNA expression is presented as a percentage relative to control (untreated) cells.

Carrageenan-induced paw edema in mice

In vivo anti-inflammatory activity was measured using a carrageenan-induced paw edema model in mice. Male ICR mice (7 weeks, 8 mice/group) were housed in a temperature- and humidity-controlled environment, and provided ad libitum access to food and water. Mice were then subjected to the experiment following 16 h of fasting. All chemicals and samples used in the animal experiment were dissolved or suspended in sterile saline. Mice in the test group received oral administration of MSA4 or dMSA4 (300 mg/kg of body weight). Negative-control mice received sterile saline only and positive-control mice received indomethacin (10 mg/kg of body weight), both via an oral route. After a period of resting for 1 h, edema was induced in the right hind paw by an intraplantar injection of 40 µl of 1 % (w/v) carrageenan solution, and the paw volume was measured and the increasing rate of paw edema (%) calculated by the method described in the previous work [25]. The experimental protocol was approved by the Committee for Animal Care and Use of Hokkaido University, and the animal experiment was performed in accordance with the Hokkaido University guidelines for the care and use of laboratory animals.

Statistics

Data are presented as mean ± SEM. Statistically significant differences were assessed by the Tukey–Kramer method, at p < 0.05.

Results

Characterization of Mf after conjugation with AO and molecular mass distribution of the digested peptides

Table 1 shows the changes in the available lysine and the amount of AO bound to Mf with the increasing duration of the Maillard reaction. The reduction in the available lysine content occurred simultaneously with the increase in AO bound to Mf, indicating the progress of AO conjugation through the Maillard reaction in the dry state. Figure 1 shows the change in the migration pattern of Mf during conjugation with AO as visualized by SDS-PAGE. Although no difference was observed between MS and MSA0 by SDS-PAGE (Fig. 1), a decrease in available lysine content of 15.9 % and an increase in bound AO of 8.9 µg/mg protein was detected in MSA0 (Table 1). Thus, it is apparent that a small amount of AO was attached to Mf via the Maillard reaction during lyophilization, as previously reported [25]. On the other hand, after conjugation for 2 h at 60 °C and 35 % relative humidity, the tropomyosin band (TM; 33 kDa) was no longer visible by protein staining, and the mobility of the myosin heavy chain (MHC; 200 kDa) decreased with increasing AO conjugation duration. The carbohydrate staining intensity of the MHC band also increased after AO conjugation. The results shown in Fig. 1 and Table 1 indicate that utilization of the controlled Maillard reaction in a dry state is an effective procedure for preparing neoglycoprotein from salmon Mf.
Table 1

Available lysine content and AO bound to MS and MSA

 

MS

MSA0

MSA2

MSA4

MSA6

Available lysine content (%)

100.0

84.1

68.1

68.4

59.0

AO bound to protein (µg/mg)

0.0

8.9

38.5

49.6

74.8

Salmon myofibrillar protein (Mf) was mixed with sorbitol (MS) or with sorbitol and AO (MSA), as described in the “Materials and methods” section. Conjugation of AO to Mf (MSA) was carried out by incubation at 60 °C and 35 % relative humidity for 0 h (MSA0), 2 h (MSA2), 4 h (MSA4), or 6 h (MSA6)

Fig. 1

Change in SDS-PAGE migration pattern of MSA during AO conjugation. MSA was incubated at 60 °C and 35 % relative humidity for 0–6 h to produce Mf-AO conjugates. MS (Mf-sorbitol mixture) was also examined as a control. After electrophoresis, proteins were subjected to protein and carbohydrate staining. MHC myosin heavy chain, AC actin, TM tropomyosin

The molecular mass distribution of the digested Mf samples (dMS and a series of dMSAs) was investigated by gel permeation chromatography. As shown in Fig. 2, MS and MSA were easily digested into peptides with a molecular mass of <20 kDa, indicating that the major components of Mf (MHC, AC, and TM) were degraded by the artificial digestive system used here, regardless of conjugation with AO. In contrast, the molecular mass distribution of Mf was altered by lyophilization and the progress of AO conjugation via the controlled Maillard reaction at 60 °C and 35 % relative humidity, indicating that AO conjugation affected the digestive pattern of Mf.
Fig. 2

Digested peptide distribution of dMS and dMSAs. MS was digested with pepsin and trypsin for 3 h each. MSAs were incubated at 60 °C and 35 % relative humidity for 0, 2, 4, and 6 h, digested with pepsin and trypsin for 3 h each (referred as to dMSA0, dMSA2, dMSA4, and dMSA6, respectively), and analyzed by gel permeation chromatography

Suppressive effect of digested Mf–AO conjugates on NO and pro-inflammatory cytokine secretion in macrophages

To investigate the effect of AO conjugation via controlled Maillard reaction on the anti-inflammatory activity of Mf, digested Mf–AO conjugates were added to LPS-stimulated RAW264.7 cells, and the secretion of inflammatory mediators after culture for 24 h was measured. The concentrations of NO, TNF-α, and IL-6 in the culture supernatant of LPS-stimulated RAW 264.7 cells without digested Mf (control) was 30.7 μM, 9.4 ng/ml, and 10.3 ng/ml, respectively. Treatment with dMS, on the other hand, significantly reduced the production of NO and TNF-α, and dMSA0 also inhibited the secretion of NO, TNF-α, and IL-6, as shown in Fig. 3a. Furthermore, dMSA2, dMSA4, and dMSA6 significantly reduced the production of all three inflammation-related compounds, and this effect was more pronounced with the AO conjugation. This result, which clearly indicates the progress of AO conjugation in the controlled dry state, confirms that this is an effective process for improving the anti-inflammatory activity of Mf. dMSA4 showed a marked suppressive effect on the secretion of inflammation-related compounds, in particular NO, and thus was chosen for further study.
Fig. 3

Suppressive effects of digested Mfs on secretion of inflammation-related compounds in LPS-stimulated RAW 264.7 cells. adMS and digested Mf–AO conjugates (dMSA0, dMSA2, dMSA4, and dMSA6, described in Fig. 2) at a final concentration of 500 µg/ml were added to the culture medium, along with 2.5 ng/ml LPS and 0.5 ng/ml IFN-γ (no IFN-γ added for cytokine secretion), and the concentrations of NO, TNF-α, and IL-6 in the culture supernatant of LPS-stimulated RAW 264.7 cells were measured after cultivation for 24 h. The control indicates culture supernatant of cells without the addition of digested Mf. b The concentration-dependent effects of dMSA4 on the secretion of NO, TNF-α, and IL-6 were measured using the same conditions as in (a). Data are expressed as mean ± SD (n = 5). Different letters indicate a significant difference (p < 0.05)

As shown in Fig. 3b, dMSA4 suppressed the secretion of NO, TNF-α, and IL-6 in a dose-dependent manner, and a significant reduction in all three inflammatory mediators was observed even at the lowest dose of 300 µg/ml dMSA4. Additionally, AO alone, when used at concentrations of 50–200 μg/ml, higher than the amount attached to Mf, showed no inhibition of NO or cytokine production in LPS-stimulated RAW 264.7 cells (Fig. 4). Therefore, the result shown in Fig. 3 would indicate that conjugation of Mf with AO enhanced the anti-inflammatory activity of digested salmon Mf peptides.
Fig. 4

Effect of AO on secretion of inflammation-related compounds in RAW264.7 cells. AO at a final concentration of 0–200 µg/ml was added to the culture medium, along with 2.5 ng/ml LPS and 0.5 ng/ml IFN-γ (no IFN-γ added for cytokine secretion), and the concentrations of NO, TNF-α, and IL-6 in the culture supernatant of LPS-stimulated RAW 264.7 cells were measured after cultivation for 24 h. Data are presented as mean ± SEM. Statistically significant differences were assessed by the Tukey–Kramer method, at p < 0.05

Effect of dMSA on the protein and gene expression of inflammation-related compounds and COXs

The effect of dMSA4 on the expression of inflammation-related proteins and genes in LPS-stimulated RAW264.7 cells was examined using dMSA0 and dMSA4 to examine the effect of AO conjugation in comparison to control cells receiving no peptide treatment. Before analysis, we confirmed that 500 µg/ml of dMSA had no effect on the expression of the control gene β-actin in the cells. As shown in Fig. 5, dMSA0 showed a weak suppressive effect on the protein expression of iNOS and IL-6. TNF-α and cyclooxygenase (COX)-2 production and their gene expression were significantly reduced by dMSA0 (Fig. 5d), whereas dMSA4 significantly reduced the expression of iNOS, TNF-α, IL-6, and COX-2 at both the gene and protein levels (Fig. 5a–d). In addition, no change was observed in COX-1 expression in the presence of dMSA0 or dMSA4, regardless of the COX-2 expression (Fig. 5d, e). These results indicate that suppressive effects of inflammation-related compounds of dMSA4 occur as a consequence of the reduced inflammation-related gene expression and protein production, without suppression of the constitutively expressed enzyme (COX-1).
Fig. 5

Effect of dMSA4 on protein and gene expression of inflammation-related compounds in LPS-stimulated RAW264.7 cells. RAW 264.7 cells were cultured with 500 µg/ml of dMSA0 or dMSA4 in the presence of 5.0 ng/ml LPS for 24 h, and the protein and gene expression of inflammation-related compounds iNOS (a), TNF-α (b), and IL-6 (c), and cyclooxygenases (COX-1 (e) and COX-2 (d)) were examined by immunoblotting and RT-PCR. The intensity of each band was measured using an image analyzer, and the mRNA and protein expression measurements are normalized to β-actin (internal control), with the exception of the protein expression of TNF-α and IL-6, which are quoted from the data in Fig. 3 measured by ELISA. These data are expressed as a percentage relative to the control (no addition of dMSA). Data are expressed as mean ± SD (n = 3). Different letters indicate a significant difference (p < 0.05)

Anti-inflammatory effects of dMSA4 in a model of acute inflammation in mice

To examine the possible use of dMSA as an anti-inflammatory food material, dMSA4 was evaluated using a model of carrageenan-induced edema in mice. Saline (negative control) and dMSA4 at 300 mg/kg of body weight, and indomethacin at 10 mg/kg of body weight (as a positive control), was orally administered to ICR mice. As shown in Fig. 6a, the increasing rate of paw edema volume in control mice reached 51.7 % 3 h after the carrageenan-injection. In contrast, the exacerbation of the paw edema was significantly inhibited by orally administered dMSA4, and the suppressive effect of dMSA4 was similar to that of the positive control indomethacin, as seen in the results of AUC (Fig. 6a, inset). Additionally, MSA4, AO-conjugated Mf before digestion, also showed an anti-inflammatory effect (Fig. 6b). These results indicate the efficacy of AO conjugation for developing an anti-inflammatory product from fish meat.
Fig. 6

Effect of dMSA4 on progression of carrageenan-induced edema in mice. a Saline (negative control, open circle), dMSA4 at 300 mg/kg (open triangle) or indomethacin at 10 mg/kg (open square) were orally administered to mice (8 mice/group). After resting for 1 h, edema was induced in the right hind paw by an intraplantar injection of 40 µl of 1 % (w/v) carrageenan solution. The paw volume was measured with a plethysmometer before injection and after carrageenan injection at 1 h intervals up to 5 h. The bar chart indicates area under the response–time curve (p < 0.05). b Saline (negative control, closed circle) and MSA4 at 300 mg/kg (closed triangle) were administered orally to mice (8 mice/group). After 1 h, edema was induced in the right hind paw by 1 % (w/v) carrageenan solution. The paw volume was measured with a plethysmometer before and after carrageenan injection at 1-h intervals up to 5 h. The bar chart indicates the area under the curve (p < 0.05). The AUC in the inset is the area under the increasing rate of paw edema volume–time curve. Data are presented as mean ± SEM. Statistically significant differences were assessed by the Tukey–Kramer method at p < 0.05

Discussion

As presented in Table 1, the amount of AO attached to Mf increased with the incubation of the lyophilized Mf–AO mixture under controlled dry conditions at 60 °C and 35 % relative humidity, and the enhanced anti-inflammatory activity of Mf was observed with the progress of the AO conjugation. The results of this work clearly indicate that the Maillard reaction under a controlled dry state is useful for the production of anti-inflammatory fish Mf.

AO conjugation during lyophilization was observed in MSA0, which contained 8.9 µg/mg of AO, whereas 19 µg/mg of AO was conjugated to Mf during lyophilization in the previous work [25]. The variance of the degree of AO conjugation suggests that the Maillard reaction under low-temperature conditions was not precisely controlled. In addition, the anti-inflammatory effect of dMSA0 (Fig. 3a) was reduced in comparison with that observed in the previous work [25], indicating that the level of AO conjugation in the lyophilization process affected the functional improvement of Mf. Therefore, utilization of AO conjugation with lyophilization is unsuitable for developing an anti-inflammatory compound from fish Mf.

To investigate the enhanced anti-inflammatory activity with the progress of AO conjugation, digested Mf conjugated with different amounts of AO (Table 1) was subjected to in vitro macrophage assay. When the digested Mf samples were subjected to anti-inflammatory assays in LPS-stimulated RAW264.7 cells (Figs. 3 and 4), results clearly showed that conjugation of Mf with AO enhanced the suppressive effects on inflammation-related compounds (NO, TNF-α, and IL-6) of digested salmon Mf peptides. Some studies have reported cytotoxicity of the Maillard reaction products, such as glyceraldehyde-related products and methylglyoxal 3-deoxyglucosone [18, 34], but 1,000 µg/ml of the digested Mf samples in this work had no negative effect on the viability of the RAW 264.7 cells after cultivation for 48 h (data not shown). Additionally, AO that is not attached to Mf showed no inhibition of inflammation-related compound production in LPS-stimulated RAW 264.7 cells (Fig. 4). Therefore, the result of Fig. 3 would indicate that conjugation of Mf with AO enhanced the anti-inflammatory activity of digested salmon Mf peptides. The results described above raise the possibility that the anti-inflammatory compounds contained in the digested Mf samples are digested and/or AO-conjugated peptides. As shown in Fig. 3a, dMS, which was not conjugated with AO, showed anti-inflammatory activity, suggesting that the pepsin-trypsin digestion produced anti-inflammatory peptides from salmon Mf. Additionally, a marked decrease in the production of inflammation-related compounds upon treatment with dMSA2, dMSA4, or dMSA6 suggests the involvement of AO-conjugated peptides in enhancing anti-inflammatory activity. Indeed, the existence of multiple anti-inflammatory peptides in dMSA0 has been suggested using isoelectric focusing chromatography [25]. Structural changes in AO-conjugated protein occurring with the progression of the Maillard reaction may also be related to the anti-inflammatory effect, as intermediate products of the Maillard reaction, such as 5-hydroxymethyl-2-furfural and 5-hydroxymethyl-2-furoic acid, have anti-inflammatory properties [35]. The identification of the compounds responsible for the anti-inflammatory activity observed here is the next step in revealing the relationship between AO conjugation and increased anti-inflammatory activity.

We have examined the molecular mechanism of anti-inflammatory activity of dMSA, and found that dMSA4 exerted its anti-inflammatory activity by suppressing LPS-induced gene and protein expression. Interestingly, dMSA4 had no effect on COX-1 expression as opposed to suppression of COX-2 (Fig. 5). COX enzymes (COXs) are responsible for converting arachidonic acid to prostaglandins [36]. COX-1 is constitutively expressed in most tissues, whereas COX-2 is induced by stimulation with mitogens such as LPS or pro-inflammatory cytokines, resulting in the production of inflammation-enhancing prostaglandins [37]. Concurrent inhibition of COX gene expression is often caused by non-steroidal anti-inflammatory drugs, causing adverse effects such as gastric ulceration and renal toxicity [38]. Hence, selective inhibition of COX-2 is required in anti-inflammatory compounds expected to avoid gastrointestinal and renal damage. dMSA4 may be a potential COX-2 selective inhibitor.

The expression of inflammation-related genes is regulated by signaling cascades in cells. For example, NF-κB, one of the transcriptional factors controlling the expression of inflammatory mediators, is activated by stimuli such as LPS, pro-inflammatory mediators, and oxidative stress [39]. Most anti-inflammatory compounds exert their anti-inflammatory effects by inhibiting NF-κB signaling cascades [8, 40, 41, 42]. dMSA may also inhibit the activation of NF-κB signaling such as phosphorylation of NF-κB p65 or IκBα, as dMSA simultaneously reduced the expression of several NF-κB activated genes including TNF-α, IL-6, iNOS, and COX-2.

Finally, we carried out a test of carrageenan-induced edema in mice to examine the possible use of dMSA as an anti-inflammatory food material. Administration of dMSA4 showed an anti-inflammatory effect, regardless of digestion (Fig. 6), suggesting that anti-inflammatory peptides included in MSA4 could be released by enzymatic digestion (pepsin and trypsin) in vivo in the gastrointestinal tract. The fact that the oral administration of AO-conjugated salmon Mf reduced localized acute inflammation indicates its potential as a nutraceutical. The result shown in Fig. 6 indicates that the digestion process of Mf–AO conjugates is not essential for the exertion of anti-inflammatory effects. However, ingestion of the Mf–AO conjugate after enzymatic digestion is desirable for attaining effective anti-inflammatory activity, as gastrointestinal digestion tends to be affected by the health condition of organisms.

The anti-inflammatory mechanisms of dMSA4 in mice may be related to the inhibition of inflammation-related compounds, as the excess expression of pro-inflammatory cytokines iNOS and COX-2 are involved in the development of carrageenan-induced edema [43, 44, 45]. The pepsin-trypsin digestion may be important for the stable production of anti-inflammatory compounds, as protein digestion is often altered by protein denaturation during the storage process.

In conclusion, conjugation with AO using the Maillard reaction under controlled temperature and humidity conditions is a useful method for producing salmon Mf with excellent anti-inflammatory properties. Since conjugation with AO also improves the solubility, thermal stability, and emulsion-forming ability of fish Mf [27, 46, 47, 48], this work could contribute to the development of an edible anti-inflammatory fish product.

Notes

Acknowledgments

We deeply appreciate the technical assistance of Mr. Yutaka Shimizu, Ph.D., Hokkaido University, for instruction on immunoblotting strategy. We deeply appreciate Hideki Kishimura, Associate Professor, Hokkaido University, for instruction on PCR strategy. We thank Hokkaido Mitsui Chemical Industry Co., Ltd., for supplying alginate oligosaccharide. Part of this work was supported by the Japan Science and Technology Agency, A-STEP Program (FS stage), 2010.

References

  1. 1.
    Chen GY, Nuñez G (2010) Sterile inflammation: sensing and reacting to damage. Nat Rev Immunol 10:826–837CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Daniel K, Podlsky MD (2002) Inflammatory bowel disease. N Engl J Med 347:417–429CrossRefGoogle Scholar
  3. 3.
    Pearson TA, Mensah GA, Alexander RW, Anderson JL, Cannon RO, Criqui M, Fadl YY, Fortmann SP, Hong Y, Myers GL, Rifai N, Smith SC, Taubert K, Tracy RP, 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 107:499–511CrossRefPubMedGoogle Scholar
  4. 4.
    Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010) Mechanisms underlying inflammation in neurodegeneration. Cell 140:918–934CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Wellen KE, Hotamisligil GS (2005) Inflammation, stress, and diabetes. J Clin Invest 115:1111–1119CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Wu X, Schauss AG (2012) Mitigation of inflammation with foods. J Agric Food Chem 60:6703–6717CrossRefPubMedGoogle Scholar
  7. 7.
    Chan MM, Fong D, Ho CT, Huang HI (1997) Inhibition of inducible nitric oxide synthase gene expression and enzyme activity by epigallocatechin gallate, a natural product from green tea. Biochem Pharmacol 54:1281–1286CrossRefPubMedGoogle Scholar
  8. 8.
    Lin YL, Tsai SH, Lin-Shiau SY, Ho CT, Linet JK (1999) Theaflavin-3,3′-digallate from black tea blocks the nitric oxide synthase by down-regulating the activation of NF-kappaB in macrophages. Eur J Pharmacol 367:379–388CrossRefPubMedGoogle Scholar
  9. 9.
    Ren J, Chung SH (2007) Anti-inflammatory effect of alpha-linolenic acid and its mode of action through the inhibition of nitric oxide production and inducible nitric oxide synthase gene expression via NF-kappaB and mitogen-activated protein kinase pathways. J Agric Food Chem 55:5073–5080CrossRefPubMedGoogle Scholar
  10. 10.
    Kim JA, Kong CS, Pyun SY, Kim SK (2010) Phosphorylated glucosamine inhibits the inflammatory response in LPS-stimulated PMA-differentiated THP-1 cells. Carbohydr Res 345:1851–1855CrossRefPubMedGoogle Scholar
  11. 11.
    Wang J, Mazza G (2002) Inhibitory effects of anthocyanins and other phenolic compounds on nitric oxide production in LPS/IFN-γ-activated. J Agric Food Chem 50:850–857CrossRefPubMedGoogle Scholar
  12. 12.
    Yamaguchi M, Yoshida K, Uchida M (2009) Novel functions of bovine milk-derived alpha-lactalbumin: anti-nociceptive and anti-inflammatory activity caused by inhibiting cyclooxygenase-2 and phospholipase A2. Biol Pharm Bull 32:366–371CrossRefPubMedGoogle Scholar
  13. 13.
    Hartog A, Leenders I, van der Kraan PM, Garssen J (2007) Anti-inflammatory effects of orally ingested lactoferrin and glycine in different zymosan-induced inflammation models: evidence for synergistic activity. Int Immunopharmacol 7:1784–1792CrossRefPubMedGoogle Scholar
  14. 14.
    FAO Fisheries and Aquaculture Department (2012) The state of world fisheries and aquaculture 2012. Food and Agriculture Organization of the United Nations, Rome, pp 3–5Google Scholar
  15. 15.
    Je JY, Park PJ, Kwon JY, Kim SK (2004) A novel angiotensin I converting enzyme inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate. J Agric Food Chem 52:7842–7845CrossRefPubMedGoogle Scholar
  16. 16.
    Ren J, Wang H, Zhao M, Cui C, Hu X (2010) Enzymatic hydrolysis of grass carp myofibrillar protein and antioxidant properties of hydrolysates. Czech J Food Sci 28:475–484Google Scholar
  17. 17.
    Lavigne C, Tremblay F, Asselin G, Jacques H, Marette A (2001) Prevention of skeletal muscle insulin resistance by dietary cod protein in high fat-fed rats. Am J Physiol Endocrinol Metab 281:E62–E71PubMedGoogle Scholar
  18. 18.
    Ryan JT, Ross RP, Bolton D, Fitzgerald GF, Stanton C (2012) Bioactive peptides from muscle sources: meat and fish. Nutrients 3:765–791CrossRefGoogle Scholar
  19. 19.
    Saeki H (2012) Protein–saccharide interaction. In: Hettiarachchy NS et al (eds) Food proteins and peptide, chemistry, functionality, interactions and commercialization. CRC Press, New York, pp 230–261Google Scholar
  20. 20.
    Nakamura S, Ogawa M, Nakai S, Kato A, Kitts DD (1998) Antioxidant activity of a maillard-type phosvitin-galactomannan conjugate with emulsifying properties and heat stability. J Agric Food Chem 46:3958–3963CrossRefGoogle Scholar
  21. 21.
    Junfeng F, Yanyan Z, Szesze T, Fengjuan L, Manyu Z, Saito M, Eizo Tatsumi E, Lite L (2006) Improving functional properties of soy protein hydrolysate by conjugation with curdlan. J Food Sci 71:C285–C291CrossRefGoogle Scholar
  22. 22.
    Nakamura S, Kato A (2000) Multi-functional biopolymer prepared by covalent attachment of galactomannan to egg-white proteins through naturally occurring Maillard reaction. Nahrung 44:201–206CrossRefPubMedGoogle Scholar
  23. 23.
    Iwan M, Vissers YM, Fiedorowicz E, Kostyra H, Kostyra E, Savelkoul HF, Wichers HJ (2011) Impact of Maillard reaction on immunoreactivity and allergenicity of the hazelnut allergen Cor a 11. J Agric Food Chem 59:7163–7171CrossRefPubMedGoogle Scholar
  24. 24.
    Isono M, Saeki H, Nishimura K (2012) Properties of glycated chicken myofibrillar proteins with enhanced antioxidant abilities. J Home Econ Jpn 63:461–468Google Scholar
  25. 25.
    Saigusa M, Nishizawa M, Shimizu Y, Saeki H (2015) In vitro and in vivo anti-inflammatory activity of digested peptides derived from salmon myofibrillar protein conjugated with a small quantity of alginate oligosaccharide. Biosci Biotechnol Biochem 79:1518–1527CrossRefPubMedGoogle Scholar
  26. 26.
    Sato R, Katayama S, Sawabe T, Saeki H (2003) Stability and emulsion-forming ability of water-soluble fish myofibrillar protein prepared by conjugation with alginate oligosaccharide. J Agric Food Chem 51:4376–4381CrossRefPubMedGoogle Scholar
  27. 27.
    Takeda H, Iida T, Okada A, Ootsuka H, Ohshita T, Masutani E, Katayama S, Saeki H (2007) Feasibility study on water solubilization of spawned out salmon meat by conjugation with alginate oligosaccharide. Fish Sci 73:924–930CrossRefGoogle Scholar
  28. 28.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  29. 29.
    Zacharius RM, Zell TE, Morrison JH, Woodlock JJ (1969) Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem 30:148–152CrossRefPubMedGoogle Scholar
  30. 30.
    Gornall AG, Bardawill CJ, David MM (1949) Determination of serum proteins by means of the biuret reaction. J Biol Chem 177:751–766PubMedGoogle Scholar
  31. 31.
    Dubois M, Gilles AK, Hamilton KJ, Rebers AP, Smith F (1956) Colorimetric method for determination of sugers and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  32. 32.
    Hernandez MJM, Alvarez-coque MCG (1992) Availble lysine in protein assay using o-phthalaldehyde/N-acetyl l-cysteine spectrophotometric method. J Food Sci 57:503–505CrossRefGoogle Scholar
  33. 33.
    Baer HP, Schmidt K, Mayer B, Kukovetz WR (1995) Pentamidine does not interfere with nitrite formation in activated RAW 264.7 macrophages but inhibits constitutive brain nitric oxide synthase. Life Sci 57:1973–1980CrossRefPubMedGoogle Scholar
  34. 34.
    Suzuki K, Koh YH, Mizuno H, Hamaoka R, Taniguchi N (1998) Overexpression of aldehyde reductase protects PC12 cells from the cytotoxicity of methylglyoxal or 3-deoxyglucosone. J Biochem 123:353–357CrossRefPubMedGoogle Scholar
  35. 35.
    Kitts DD, Xiu-Min C, Hao J (2012) Demonstration of antioxidant and anti-inflammatory bioactivities from sugar-amino acid Maillard reaction products. J Agric Food Chem 60:6718–6727CrossRefPubMedGoogle Scholar
  36. 36.
    Okada N, Hirata A, Murakami Y, Shoji M, Sakagami H, Fujisawa S (2005) Induction of cytotoxicity and apoptosis and inhibition of cyclooxygenase-2 gene expression by eugenol-related compounds. Anticancer Res 25:3263–3269PubMedGoogle Scholar
  37. 37.
    Dubois RN, Abramson SB, Crofford L, Gupta RA, Simon LS, Van De Putte LB, Lipsky PE (1998) Cyclooxygenase in biology and disease. FASEB J 12:1063–1073PubMedGoogle Scholar
  38. 38.
    Jones MK, Wang H, Peskar BM, Levin E, Itani RM, Sarfeh IJ, Tarnawski AS (1999) Inhibition of angiogenesis by nonsteroidal anti-inflammatory drugs: insight into mechanisms and implications for cancer growth and ulcer healing. Nat Med 5:1418–1423CrossRefPubMedGoogle Scholar
  39. 39.
    Egan LJ, Toruner M (2006) NF-kappaB signaling: pros and cons of altering NF-kappaB as a therapeutic approach. Ann N Y Acad Sci 1072:114–122CrossRefPubMedGoogle Scholar
  40. 40.
    Håversen L, Ohlsson BG, Hahn-Zoric M, Hanson LA, Mattsby-Baltzer I (2002) Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NF-kappa B. Cell Immunol 220:83–95CrossRefPubMedGoogle Scholar
  41. 41.
    De Mejia EG, Dia VP (2009) Lunasin and lunasin-like peptides inhibit inflammation through suppression of NF-kappaB pathway in the macrophage. Peptides 30:2388–2398CrossRefPubMedGoogle Scholar
  42. 42.
    Singh S, Aggarwal BB (1995) Activation of transcription factor NF-kappaB is suppressed by curcumin (diferuloylmethane). J Biol Chem 270:24995–25000CrossRefPubMedGoogle Scholar
  43. 43.
    Cuzzocrea S, Sautebin L, De Sarro G, Costantino G, Rombolà L, Mazzon E, Ialenti A, De Sarro A, Ciliberto G, Di Rosa M, Caputi AP, Thiemermann C (1999) Role of IL-6 in the pleurisy and lung injury caused by carrageenan. J Immunol 163:5094–5104PubMedGoogle Scholar
  44. 44.
    Tan-No K, Nakajima T, Shoji T, Nakagawasai O, Niijima F, Ishikawa M, Endo Y, Sato T, Satoh S, Tadano T (2006) Anti-inflammatory effect of propolis through inhibition of nitric oxide production on carrageenin-induced mouse paw edema. Biol Pharm Bull 29:96–99CrossRefPubMedGoogle Scholar
  45. 45.
    Omote K, Hazama K, Kawamata T, Kawamata M, Nakayaka Y, Toriyabe M, Namiki A (2001) Peripheral nitric oxide in carrageenan-incuced inflammation. Brain Res 912:171–175CrossRefPubMedGoogle Scholar
  46. 46.
    Sato R, Sawabe T, Saeki H (2005) Characterization of fish myofibrillar protein by conjugation with alginate oligosaccharide prepared using genetic recombinant alginate lyase. J Food Sci 70:58–62CrossRefGoogle Scholar
  47. 47.
    Sato R, Sawabe T, Kishimura H, Hayashi K, Saeki H (2000) Preparation of neoglycoprotein from carp myofibrillar protein and alginate oligosaccharide: improved solubility in low ionic strength medium. J Agric Food Chem 48:17–21CrossRefPubMedGoogle Scholar
  48. 48.
    Maitena U, Katayama S, Sato R, Saeki H (2004) Improved solubility and stability of carp myosin by conjugation with alginate oligosaccharide. Fish Sci 70:896–902CrossRefGoogle Scholar

Copyright information

© Japanese Society of Fisheries Science 2015

Authors and Affiliations

  • Mizuho Nishizawa
    • 1
  • Musashi Saigusa
    • 1
  • Hiroki Saeki
    • 1
  1. 1.Faculty of Fisheries SciencesHokkaido UniversityHakodateJapan

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