The purification and molecular mass analysis of polysaccharides
The extraction of polysaccharide was performed according to a previously described method by a previously reported procedure . Briefly, 200 mg of powdered seaweed was incubated in 20 ml of 85% ethanol at 80°C for 4 h (three times) to remove lipids. Polysaccharides in the residue were extracted with 30 ml of cold water, dialyzed against distilled water, and then freeze-dried (32.0 mg). The total sugar content was determined by phenol-sulfuric acid method using galactose as a standard . The sulfate content was determined by the BaCl2-gelatin method  using κ-carrageenan (TCI, Tokyo, Japan) as a positive control. The relative molecular weight of the polysaccharides was determined by high-performance size exclusion chromatography with Shodex OHpak SB-807G (Guard), SB-807 HQ, and SB-806M HQ (8.0 mm ID × 300 mm length; Showa Denko KK, Tokyo, Japan) at 40°C and estimated using dextran standards (150, 270, and 670 kDa from Sigma Aldrich Corp., 3,755 kDa from American Polymer Standards Corp., Mentor, OH, USA). Sample (injected volume: 20 μl) was eluted using 0.3 M NaNO3 at a flow rate of 1 ml/min and was detected using a refractive index (RI) detector RID10 (Shimadzu Corp., Kyoto, Japan).
Preparation of polyphenol extract and measurement of polyphenols
Powdered C. hypnaeoides were extracted with 80% ethanol at room temperature for 48 h. The ethanol extract was filtered and concentrated using a rotary evaporator. Total polyphenols were determined by a previously reported method with slight modifications . Phenolic extract (10 ml) was placed in triplicate in a 96-well plate, and mixed with 25 ml of 1 N Folin-Ciocalteu reagent and 20% sodium bicarbonate, followed by 150 ml distilled water. The mixture was incubated for 30 min at room temperature before recording the absorbance at 630 nm. Total polyphenol was calculated according to the gallic acid standard curve. The total polyphenol content in ethanol extract was 9.65 mg gallic acid equivalent/g.
Animals and dietary treatment
Male C57BL/6J mice (6-week-old) were purchased from CLEA Japan Inc. (Tokyo, Japan) and housed at a constant temperature of 22±1°C with a 12 h light/dark cycle. After 1 week of acclimation, the animals were randomly divided into 4 groups of 12-13 animals, as follows: 1) normal diet (normal) group, high-fat (HF) diet group, HF diet supplemented with 2% C. hypnaeoides(HF+ChL) group, and HF supplemented with 6% C. hypnaeoides (HF+ChH) group. The doses of C. hypnaeoides were decided based on the previous studies . The C. hypnaeoides sample was harvested on the coast of Niigata Prefecture and was washed with water. It was dried, reduced to a fine powder using a food mixer, and mixed into HF chow. The composition of the experimental diets was adjusted by considering the nutritional components of C. hypnaeoides as described previously . The normal diet provided 354 kcal/100 g of energy (14.4% calories from protein, 11.1% calories from fat and 74.4% calories from carbohydrate), while the HF diet provided 493 kcal/100 g of energy (17.9% calories from protein, 60.7% calories from fat and 21.4% calories from carbohydrate). All experimental diets were based on the AIN-76 diet (Oriental Yeast Co. Ltd., Tokyo, Japan). Animals were allowed ad libitum access to food and drinking water. The food, including the HF diet was exchanged with new food every day. The body weight and food intake were monitored twice a week. After 13 weeks of feeding, the mice were deprived of food overnight, and blood samples were withdrawn under mixed anesthetic agent (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol; Fujifilm Wako Pure Chemical Co., Osaka, Japan). Liver tissue and epididymal, peritoneal, and mesenteric white adipose tissues were removed, weighed, and stored at -80°C. Some mice were used for histological examinations. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Fukui Prefectural University (approval No. 19-14).
Serum biochemical analyses
Serum was obtained by centrifugation at 1,500×g for 15 min at 4°C. The serum levels of total cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed using a Hitachi 7060 Automatic Analyzer (Hitachi, Tokyo, Japan) with commercial kits (Fujifilm Wako Pure Chemical Co., Osaka, Japan). The non-HDL cholesterol levels were calculated by subtracting the HDL-cholesterol from the total cholesterol level. The serum insulin (Morinaga Institute of Biological Science, Yokohama, Japan), adiponectin (Otsuka Pharmaceutical Co. Ltd. Tokyo, Japan), tumor necrosis factor-a (TNF-a; Fujifilm Wako Pure Chemical Co., Osaka, Japan), and monocyte chemoattractant protein-1 (MCP-1; Proteintech, Rosemont, IL, USA) levels were also determined using a commercial ELISA kit. Serum malondialdehyde (MDA) levels were determined using a commercial kit (Japan Institute for the Control of Aging Co. Ltd., Shizuoka, Japan).
Hepatic biochemical analysis
Lipids were extracted from the liver according to a previously described method . In brief, frozen liver tissues (50 mg) were homogenized in 5 volumes of isopropanol. The homogenate was kept at room temperature for 2 days and then centrifuged at 1,000×g for 10 min. Aliquots of supernatant were analyzed for triglyceride content using a commercial kit (Fujifilm Wako Pure Chemical Co., Osaka, Japan). Liver samples for the biochemical analysis were homogenized in 5 volumes of cold 20 mM Tris-HCl buffer (pH 7.4), and centrifuged at 12,000×g for 15 min at 4°C. The supernatant was used for the biochemical analysis. Lipid peroxidation was determined by estimating malondialdehyde (MDA) using a commercial kit (Japan Institute for the Control of Aging Co. Ltd., Shizuoka, Japan). The reduced glutathione (GSH) levels were determined using a commercial kit (Japan Institute for the Control of Aging Co. Ltd., Shizuoka, Japan). The inflammatory cytokine levels in the liver were quantified using enzyme-linked immunosorbent assay (ELISA) kits specific for mouse TNF-a and interleukin-6 (IL-6). (Fujifilm Wako Pure Chemical Co., Osaka, Japan). The protein concentration was determined using a Quick Start Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA)
The mice were anesthetized with an intraperitoneal injection of mixed anesthetic agent (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol; Fujifilm Wako Pure Chemical Co., Osaka, Japan), and transcardially perfused with a fixative containing 4% paraformaldehyde and 1.5% glutaraldehyde in phosphate-buffered saline (PBS). After perfusion, the liver and white adipose tissue were removed and allowed to stand in the same fixative for one day. The tissues were rinsed several times with PBS and embedded in paraffin. Tissues were cut into 5-μm-thick sections, mounted on slides, and stained with hematoxylin eosin (HE).
Glucose tolerance test
A glucose loading test was performed one week before the end of experiment to access glucose intolerance . The mice were fasted overnight and intraperitoneally injected with glucose (2 g/kg body weight). Blood samples were collected from the tail veins of the mice, and glucose levels were measured at 0, 30, 60, 90, and 120 min after injection using a blood glucometer Nipro Stat Strip (Nipro, Osaka, Japan).
During fecal collection, mice were separated, and fecal samples were collected for a 24-h period from each mouse and weighed. These samples were ground into a powder in a mortar, and 50 mg of feces was extracted with 300 ml of distilled water. After centrifugation (16,000×g, 30 min, 4°C), ethanol was added to the supernatant (final concentration of 85%), and polysaccharides were obtained as the precipitate. The resulting residue was washed with 85% ethanol and dried. The residue was then resuspended in distilled water and centrifuged (16,000×g, 10 min, 4°C), and the polysaccharide content in the supernatant was measured using the phenol-sulfuric acid method, which has been described elsewhere, with galactose as the standard . For the measurement of triglycerides, lipids were extracted by adding isopropanol (10 times the weight) to the fecal powder. The sample was then dried and dissolved in isopropanol. The triglyceride concentration was measured using a commercial kit (Fujifilm Wako Pure Chemical Co., Osaka, Japan).
Nitric oxide and cytokine assays
RAW264.7 cells were prepared to a concentration of 2×105 cells/ml using MEM medium (M5650, Sigma Aldrich) supplemented with 10% fetal bovine serum and antibiotics (penicillin 100 U/ml and streptomycin 100 μl/ml) The cell suspension was added to a 96-well plate and cultured in a 5% CO2 incubator for 24 h. After washing each well with phosphate-buffered saline, lipopolysaccharide (LPS; derived from Escherichia coli O111, Fujifilm Wako Pure Chemical Co., Osaka, Japan) with a final concentration of 100 ng/mL and polysaccharide or ethanol extract solution were added to the medium. The cells were cultured in a 5% CO2 incubator for an additional 24 h. The culture supernatant (100 ml) was collected, 100 ml of Griess reagent was added, and the mixture was left for 20 minutes in the dark; the absorbance was then measured by microplate reader at 543 nm. The nitric oxide concentration in the medium was calculated from the standard curve prepared from sodium nitrite. Cell viability was evaluated via a 3- (4,5-dimethylthial-2-yl) -2,5-diphenyltetrazalium bromide (MTT) colorimetric assay.
Results are expressed as the mean ± SEM. Data were analyzed by a one-way analysis of variance (ANOVA) followed by Turkey’s multiple range tests. P values of p<0.05 were considered to indicate statistical significance.