Development of a Sequential Fractionation-and-Recovery Method for Multiple Anti-Inflammatory Components Contained in the Dried Red Alga Dulse (Palmaria palmata).

A separation process was established to sequentially fractionate and recover three anti-inflammatory components derived from sugars, phycobiliprotein, and chlorophyll from the hot-air-dried thalli of the red alga dulse (Palmaria palmata). The developed process consisted of three steps, without the use of organic solvents. In Step I, the sugars were separated by disrupting the cell wall of the dried thalli with a polysaccharide-degrading enzyme, and a sugar-rich extract (E1) was obtained by precipitating the other components, which were simultaneously eluted by acid precipitation. In Step II, the residue suspension from Step I was digested with thermolysin to obtain phycobiliprotein-derived peptides (PPs), and a PP-rich extract (E2) was obtained by separating the other extracts using acid precipitation. In Step III, solubilized chlorophyll was obtained by heating the residue, which was acid-precipitated, neutralized, and re-dissolved to concentrate the chlorophyll-related components (Chls)-rich extract (E3). These three extracts suppressed inflammatory-cytokine secretion by lipopolysaccharide (LPS)-stimulated macrophages, confirming that the sequential procedure had no negative effects on the activities of any of the extracts. The E1, E2, and E3 were rich in sugars, PPs, and Chls, respectively, indicating that the anti-inflammatory components were effectively fractionated and recovered through the separation protocol.

Glucose-conjugated chicken myofibrillar proteins derived from random-centroid optimization present potent hydroxyl radical scavenging activity.

The optimal conditions for the preparation of glucose-conjugated chicken myofibrillar proteins (Mfs) via the Maillard reaction, presenting strong antioxidant activity against hydroxyl radicals (ï½¥OH) and high solubility in low ionic strength medium, were sought using random-centroid optimization (RCO). Four parameters of temperature, relative humidity (RH), reaction time, and glucose-to-Mfs mixing ratio, were examined, resulting in a total of 24 vertices. Evaluations were carried out relatively to each individual vertex, and the optimal preparatory conditions to obtain the highest antioxidant activity were determined as follows: temperature of 52 °C, RH of 38%, reaction time of 6.79 h, and a glucose to Mfs mixing ratio of 11.7 (w/w). The resulting glucose-conjugated chicken Mfs gained thermal gel-forming ability and its ï½¥OH averting capacity reached 9.7 ± 0.7 µmol of gallic acid equivalent/g of protein.Abbreviations: GA: gallic acid; HORAC: hydroxyl radical averting capacity; IC50: half-maximal inhibitory concentration; 2-ME, 2-mercaptoethanol; Mfs: myofibrillar proteins; MHC: myosin heavy chain; ï½¥OH: hydroxyl radical; PAGE: polyacrylamide gel electrophoresis; RCO: random-centroid optimization; RH: relative humidity; RLU: relative light units; SDS: sodium dodecyl sulfate; SEM: scanning electron microscope; SS: disulfide.

Molecular and immunological characterization of ß'-component (Onc k 5), a major IgE-binding protein in chum salmon roe.

Salmon roe has a high allergic potency and often causes anaphylaxis in Japan. The major allergic protein of salmon roe is ß'-component, which is a 35kDa vitellogenin fragment consisting of two subunits. To elucidate structural information and immunological characteristics, ß'-component and the subunit components were purified from chum salmon (Onchorhincus keta) roe and vitellogenin-encoding mRNA was used to prepare ß'-component subunit-encoding cDNA. This was PCR-amplified, cloned and sequenced and the deduced amino acid sequence compared with partial sequences of ß'-component obtained by peptide mapping. The recombinant ß'-component subunit was produced by bacterial expression in Escherichia coli and its IgE-binding ability was measured by ELISA using the sera of a patient allergic to salmon roe. This was then compared with that of the native ß'-component with and without carboxymethylation. Following successful cloning of the cDNA encoding the ß'-component subunit, 170 amino acid residues were deduced and matched with the amino acid sequences of 121 and 88 residues in the 16kDa and 18kDa subunits, respectively. The sequences of both ß'-component subunits were almost identical, and the predicted secondary structure of the ß'-component showed a high content of ß-pleated sheets and no α-helices. There was no difference in IgE-binding ability between the native and recombinant ß'-component subunits at the same protein concentration, regardless of carboxymethylation. In conclusion, ß'-component is a homodimer protein composed of two isoform subunits having the same level of IgE-binding ability and, therefore, allergenic identity.

In vitro and in vivo anti-inflammatory activity of digested peptides derived from salmon myofibrillar protein conjugated with a small quantity of alginate oligosaccharide.

Salmon myofibrillar protein (Mf) was investigated as a source of edible anti-inflammatory products. Peptides produced by stepwise digestion of Mf (without carbohydrate) with pepsin and trypsin had little effect on the secretion of inflammation-related compounds from lipopolysaccharide-stimulated RAW 264.7 macrophage cells. However, peptides prepared from Mf conjugated with alginate oligosaccharide (AO; 19 µg/mg protein) (dMSA) through the Maillard reaction in the presence of sorbitol significantly reduced the secretion of the pro-inflammatory mediators nitric oxide, tumor necrosis factor (TNF)-α and interleukin (IL)-6, as well as mRNA expression of TNF-α, IL-6, inducible nitric oxide synthase and cyclooxygenase-2. Additionally, dMSA inhibited acute inflammation in a carrageenan-induced model of paw edema in mice, but had no effect on natural killer cell cytotoxic activity or macrophage phagocytosis. These results suggest that fish Mf conjugated with AO may be a potential food material with anti-inflammatory function.

Enhanced anti-inflammatory effect of fish myofibrillar protein by introducing pectin oligosaccharide and its molecular mechanisms.

This study investigated the efficacy of glycation with edible uronic acid-containing oligosaccharides via the Maillard reaction to enhance the anti-inflammatory effect of fish myofibrillar protein (Mf). Lyophilized Mf was reacted with pectin oligosaccharide (PO, half of the total protein weight) at 60 °C and 35 % relative humidity for up to 12 h to produce glycated Mf (Mf-PO). After pepsin and trypsin digestion, the anti-inflammatory effect was assessed by measuring the secretions of proinflammatory cytokines in LPS-stimulated RAW 264.7 macrophages, and the anti-inflammatory effect of Mf was enhanced by PO-glycation without marked lysine loss and browning. The effects on the expressions of genes related to the LPS-stimulated signaling pathway in macrophages were also examined. PO-glycation suppressed LPS-stimulated inflammation by suppressing expression of cd14 and enhancing suppressive effect of Mf on the TLR4-MyD88-dependent inflammatory signaling pathway. Therefore, as an edible reducing sugar, PO could be an effective bioindustrial material for developing anti-inflammatory Mf.

Maillard-type glycated collagen with alginate oligosaccharide suppresses inflammation and oxidative stress by attenuating the expression of LPS receptors Tlr4 and Cd14 in macrophages.

Inflammation and oxidative stress contribute to noncommunicable diseases (NCDs), with macrophages playing pivotal roles. Glycated collagen through Maillard-type glycation holds promise for enhancing anti-inflammatory properties, but its mechanism remains unclear. This study investigates the cellular mechanism and aims to contribute to expanding collagen utilization. Collagen was glycated with alginate oligosaccharide (AO) and glucose (Glc: as a comparative case) at 60 °C and 35% relative humidity for up to 24 h (C-AO and C-Glc, respectively). The anti-inflammatory activities of both C-AO and C-Glc were evaluated using an LPS-stimulated macrophage model. 18 h AO-glycated collagen (C-AO18 h) was found to significantly reduce the production of nitric oxide and proinflammatory cytokines (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß). In contrast, C-Glc did not exhibit enhanced anti-inflammatory activity during any of the glycation periods. The enhanced anti-inflammatory activity of C-AO18 h was attributed to its downregulating effect on LPS receptors (toll-like receptor 4, Tlr4; cluster of differentiation 14, Cd14) and myeloid differentiation primary response 88 (Myd88) mRNA expression, with suppression in receptor expression resulting in decreased phagocytic ability of macrophages against E. coli. In addition, compared with intact collagen, C-AO18 h exhibited improved antioxidant activity in the LPS-stimulated macrophage model, as it significantly upregulated superoxide dismutase (SOD) and catalase (CAT) activities while reducing malondialdehyde (MDA) levels. Overall, this study contributes to the development of collagen-based functional foods for mitigating inflammation and oxidative stress in NCDs.

[A case of liver metastasis from gastric cancer responding completely to S-1/docetaxel chemotherapy].

A 68-year-old man was introduced to our hospital with right lower abdominal pain. Endoscopic examination and abdominal CT revealed gastric cancer with liver metastasis. We started chemotherapy using S-1(120 mg/body/day), orally administered for 2 weeks followed by a 2-week rest period, and docetaxel(35 mg/m(2)), administered intravenously on day 1 and 15 as 1 course. After 4 courses of chemotherapy, the liver tumor reduced markedly and no new cancerous region was found by examination; therefore total gastrectomy and partial hepatectomy were performed. Histological examination showed an undifferentiated adenocarcinoma remaining as Grade 1b in the resected stomach. A resected specimen of the liver showed necrotic tissue without any cancer cells. This case suggests that S-1/docetaxel chemotherapy may reduce the stage of unresectable liver metastasis from gastric cancer and make a curative operation possible.

Variation in shrimp tropomyosin allergenicity during the production of Terasi, an Indonesian fermented shrimp paste.

Terasi is a fermented shrimp paste in Indonesia. We examined the effect of the Terasi manufacturing process on the abundance of the allergen tropomyosin (TM) and its IgG/IgE-binding ability. Terasi was produced from three shrimps, Akiami (Acetes japonicus), Okiami (Euphausia pacifica), and Isazaami (Neomysis awatchensis). Protein degradation and TM IgE-binding activity were examined by immunoblotting using anti-TM rabbit IgG and competitive enzyme-linked immunosorbent assays using shrimp-allergic patients' sera. The processing caused TM degradation, and the IgG-specific response in Akiami meat disappeared at the second fermentation step but remained in both Okiami and Isazaami Terasi. In contrast, TM IgE-binding in all meats decreased gradually during manufacturing and nearly completely disappeared in Akiami Terasi. Conclusively, Terasi production is an effective manufacturing process to reduce the IgE-binding ability of TM, and Terasi can be recognized as a low allergenic seafood when produced under an appropriate manufacturing condition.

Glycation with uronic acid-type reducing sugar enhances the anti-inflammatory activity of fish myofibrillar protein via the Maillard reaction.

The role of carboxyl group in uronic acid in enhancing the anti-inflammatory activity of fish myofibrillar protein (Mf) was investigated, when lyophilized Mf was reacted with various reducing sugars at 60 °C and 35% relative humidity through the Maillard reaction. After pepsin and trypsin digestion, the anti-inflammatory activity was evaluated by measuring the secretions of tumor necrosis factor-α, interleukin-6, interleukin-1ß, and nitric oxide in lipopolysaccharide-stimulated RAW 264.7 macrophage. The anti-inflammatory activity of Mf was not affected by glycation with glucose or galactose, whereas strongly enhanced by glycation with uronic acid-type reducing sugars: glucuronic acid, galacturonic acid, and alginate oligosaccharide. These results indicate that the presence of carboxyl group in reducing sugar is important for enhancing the anti-inflammatory activity of Mf. This study also shows that the enhanced effect could depend upon the number of carboxyl group in bound reducing sugar.

Water-soluble protein from walleye pollock (Gadus chalcogrammus) suppresses lipopolysaccharide-induced inflammation by attenuating TLR4-MyD88 expression in macrophages.

Water-soluble protein (WSP) from fish meat is abundant in the waste effluent generated via the surimi manufacturing process. This study investigated the anti-inflammatory effects and mechanisms of fish WSP using primary macrophages (MΦ) and animal ingestion. MΦ were treated with digested-WSP (d-WSP, 500 µg/mL) with or without lipopolysaccharide (LPS) stimulation. For the ingestion study, male ICR mice (5 weeks old) were fed 4% WSP for 14 days following LPS administration (4 mg/kg body weight). d-WSP decreased the expression of Tlr4, an LPS receptor. Additionally, d-WSP significantly suppressed the secretion of inflammatory cytokines, phagocytic ability, and Myd88 and Il1b expressions of LPS-stimulated macrophages. Furthermore, the ingestion of 4% WSP attenuated not only LPS-induced IL-1ß secretion in the blood but also Myd88 and Il1b expressions in the liver. Thus, fish WSP decreases the expressions of the genes involved in the TLR4-MyD88 pathway in MΦ and the liver, thereby suppressing inflammation.

Immunomodulatory Properties of Highly Viscous Polysaccharide Extract from the Gagome Alga (Kjellmaniella crassifolia).

Marine brown algae are rich in sulfated polysaccharides, which have the ability to form gels and viscous solution. Sulfated polysaccharides exhibit many biological activities; however, little is known whether the viscoelastic property in the polysaccharide extract is correlated with biological activities. We examined the immunomodulatory properties of highly viscous polysaccharide extract (HVPE) from Gagome Kjellmaniella crassifolia in a murine model, and the effects were compared with those of a less viscous polysaccharide extract (LVPE). HVPE or LVPE (10, 30, and 100 mg/kg/day) were orally administered to C57BL/6 mice for 14 days. Secretions of cytokine and IgA in Con A-stimulated spleen and Peyer's patch (PP) cells and phagocytic activity of peritoneal macrophages was determined. IFN-γ, IL-12, IL-6, and IgA secretions showed high levels in spleen cell cultures from mice administered HVPE, whereas these effects were diminished in the LVPE-administered mice. The phagocytic activity of peritoneal macrophages was enhanced by the continuous oral administration of HVPE, and these effects were higher than those of LVPE. Furthermore, an increase in IgA secretion by administration of HVPE was observed in Con A-stimulated PP cells. These results suggest that the polysaccharide extract from K. crassifolia has immunomodulatory activities, which depend on the viscosity.

Comparison of Maillard-Type Glycated Collagen with Alginate Oligosaccharide and Glucose: Its Characterization, Antioxidant Activity, and Cytoprotective Activity on H2O2-Induced Cell Oxidative Damage.

To improve the antioxidant activity of collagen molecules using Maillard-type glycation, the relation between antioxidant activity and progress indexes for the Maillard reaction must be understood. In this study, lyophilized tilapia scale collagen was mixed with a half weight of alginate oligosaccharide (AO) or glucose and incubated at 60 °C and 35% relative humidity for up to 18 h to produce the Maillard-type glycated collagen (C-AO and C-Glu, respectively). As glycation progressed, the amount of conjugated sugar coupled with UV-vis absorbance at 294 nm and 420 nm increased more rapidly in C-Glu than in C-AO, and the available lysine decreased rapidly in C-Glu compared with C-AO. The early-to-middle- and late-stage products of the Maillard reaction were involved in enhanced antioxidant activity of digested C-AO and digested C-Glu, respectively. Additionally, C-AO acquired the antioxidant activity without marked available lysine loss. The cytoprotective effect of collagen in H2O2-induced damage was enhanced by glycation, achieved by reducing malondialdehyde content and increasing superoxide dismutase and catalase activities. These results indicate that AO is an excellent reducing sugar that enhances the health benefits of collagen without excessive loss of lysine, which is a nutritional problem of the Maillard-type glycation.

Antioxidative ability of chicken myofibrillar protein developed by glycosylation and changes in the solubility and thermal stability.

Myofibrillar protein prepared from chicken breast muscle was incubated with several concentrations of glucose or maltose for 6 h at 60 °C and 35% relative humidity in order to obtain glycosylated chicken protein. When the ratio of the weights of the myofibrillar protein and glucose or maltose had respectively reached 1:6 or 1:3-5, the solubility of each type of glycosylated chicken protein in a 0.1 M NaCl solution was exceeded by about 60%, although the myofibrillar protein was insoluble in a low ionic strength solution. Moreover, when the myofibril and maltose reaction (myofibril:maltose = 1:4) was extended to 36 h, the glycosylated protein did not undergo denaturation when held at 50 °C for 2 h, while it also exhibited an antioxidative function against superoxide anion radicals.

Purification and characteristics of trypsin from masu salmon (Oncorhynchus masou) cultured in fresh-water.

Trypsin from the pyloric ceca of masu salmon (Oncorhynchus masou) cultured in fresh water was purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl cellulose to obtain a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The molecular mass of the purified trypsin was estimated to be approximately 24,000 Da by SDS-PAGE. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and N ( alpha )-p-tosyl-L: -lysine chloromethyl ketone. Masu salmon trypsin was stabilized by calcium ion. The optimum pH of the masu salmon trypsin was around pH 8.5, and the trypsin was unstable below pH 5.0. The optimum temperature of the masu salmon trypsin was around 60 degrees C, and the trypsin was stable below 50 degrees C, like temperate-zone and tropical-zone fish trypsins. The N-terminal 20 amino acid sequence of the masu salmon trypsin was IVGGYECKAYSQPHQVSLNS, and its charged amino acid content was lower than those of trypsins from frigid-zone fish and similar to those of trypsins from temperate-zone and tropical-zone fish. In the phylogenetic tree, the masu salmon trypsin was classified into the group of the temperate-zone fish trypsin.

Digestibility in the gastrointestinal tract and migration to blood of ß'-component (Onk k 5), a major salmon roe IgE-binding protein.

The major allergen of chum salmon (Oncorhynchus keta) roe is the ß'-component (Onc k 5, ß'-c), which is a yolk protein and a fragment of vitellogenin. When yolk content containing ß'-c was orally administered to mice, ß'-c passed through the gastrointestinal tract and was excreted in feces without marked degradation. The direct administration of ß'-c to ligated jejunal and ileal loops showed that ß'-c was absorbed through the small intestine and transferred into the blood. Immunohistochemical staining showed that orally administered ß'-c was distributed from the apical side to the basal side of intestinal epithelial cells, suggesting that endocytosis may be involved in the intestinal absorption of ß'-c. In conclusion, ß'-c is absorbed along a large portion of the small intestine and circulates in the blood stream without significant digestion. The resistance of ß'-c to gastrointestinal digestion seems to contribute to its strong allergenicity.

Anti-obesity effects of chondroitin sulfate oligosaccharides from the skate Raja pulchra.

We aimed to investigate the anti-obesity effects of chondroitin sulfate (CS) oligosaccharides obtained from cartilage of the skate Raja pulchra and to compare them with those of CSs of other molecular weights (MWts) (skate CS polysaccharides) and origins (shark CS, bovine CS). CSs suppressed pancreatic lipase activity as well as proliferation and lipid accumulation in mature adipocytes. Higher MWt CS had a greater lipase inhibitory activity than lower MWt CS. CSs of different origin show differing potencies for lipase inhibition and effects on adipocytes. Also, dietary intake of skate CS oligosaccharides could ameliorate obesity in high-fat diet mice model: it prevented gaining in body weight, liver weight and adipose tissue weight, maintained lower food consumption, inhibited intestinal absorption of triglyceride, and adjusted the serum endotoxin level. In conclusion, skate CS oligosaccharides have an anti-obesity activity, and the MWt and origin of the CSs may affect this activity.

Anti-inflammatory effects of dulse (Palmaria palmata) resulting from the simultaneous water-extraction of phycobiliproteins and chlorophyll a.

The use of dulse (Palmaria palmata) as a source of edible anti-inflammatory products was evaluated in this study. Phycobiliproteins and chlorophyll a were simultaneously extracted from lyophilized dulse leaves via water-extraction, and subjected to thermolysin digestion to produce thermolysin-digested water-extract (d-DWE). d-DWE significantly reduced tumor necrosis factor-α, interleukin-6, and nitric oxide in LPS-stimulated murine macrophages (RAW 264.7 cells), and orally administered d-DWE mitigated acute inflammation in carrageenan-induced paw edema of mice. Mass spectrometry revealed d-DWE contained peptide LRDGEIILRY (derived from phycoerythrin ß-chain) and chlorophyll a decomposition products, and they individually reduced the secretion of the proinflammatory mediators in LPS-stimulated RAW 264.7 cells. These results indicate the anti-inflammatory activity could be from a combined effect of phycobiliprotein and chlorophyll a decomposition products prepared from the water-extract of dulse. Thus, inexpensive and safe water-extraction method is effective for the extraction of anti-inflammatory components from dulse.

Changes in allergenicity and digestibility of squid tropomyosin during the Maillard reaction with ribose.

The effect of the Maillard reaction on the allergenicity of squid tropomyosin (TM) was investigated. When TM was reacted with ribose (TM-ribose), its human-specific IgE-binding ability decreased markedly and alpha-chymotryptic digestibility of TM was also altered at the early stage of the Maillard reaction. On the other hand, the modification of the lysine residues in TM using 2,4,6-trinitrobenzenesulfonic acid had no effect on the allergenicity and alpha-chymotryptic digestibility of TM. Therefore, the structural change in TM induced by the Maillard reaction would cause the reduction of the allergenicity, rather than the block of lysine residues. Although peptic digestion diminished the specific IgE-binding ability of TM, the reduction of the allergenicity by the Maillard reaction remained after peptic digestion. These results suggest that hypersensitive reaction of TM-ribose in the human body might be lower than that of native TM.

Trypsins from yellowfin tuna (Thunnus albacores) spleen: purification and characterization.

Two anionic trypsins (A and B) were purified to homogeneity from yellowfin tuna (Thunnus albacores) spleen by a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. Purity was increased to 70.6- and 91.5-fold with approximately 2.8% and 15.6% yield for trypsin A and B, respectively. The apparent molecular weight of both trypsins was estimated to be 24 kDa by size exclusion chromatography and SDS-PAGE. Both trypsin A and B appeared as a single band on native-PAGE. Trypsin A and B exhibited the maximal activity at 55 and 65 degrees C, respectively, and had the same optimal pH at 8.5 using TAME as a substrate. Both trypsins were stable to heat treatment up to 50 degrees C and in the pH range of 6.0 to 11.0. Both trypsin A and B were stabilized by calcium ion. The activities were inhibited effectively by soybean trypsin inhibitor, TLCK and partially inhibited by EDTA, but were not inhibited by E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). Apparent Km and Kcat of trypsin A and B for TAME were 0.2-0.33 mM and 66.7-80 S(-1), respectively. The N-terminal amino acid sequences of trypsin A, IVGGYECQAHSQPHQVSLNA, and trypsin B, IVGGYECQAHSQPPQVSLNA, indicated the high homology between both enzymes.

Effect of maillard reaction on allergenicity of scallop tropomyosin.

Scallop tropomyosin (TM), the major allergen of shellfish, was prepared from adductor muscles and reacted with four reducing sugars to investigate the effect of the Maillard reaction on the allergenicity of TM. The IgE-binding ability of TM increased significantly with the progress of the reaction with glucose, ribose, and maltose, but not with maltotriose. The allergenicity was enhanced at the early stage of the Maillard reaction, and the trend of the effect depended on the type of reducing sugar used. 2,4,6-Trinitrobenzenesulfonic acid treatment of the lysine residues in TM showed that the protein surface charge resulting from the Maillard reaction had no effect on the enhancement of the allergenicity. Thus, the change in the allergenicity would be closely related to the structural change caused by the Maillard reaction.