Pullulan standards

U. conglobata Kjellman was collected from the coast of Yantai, China, on May 2016. The raw material was thoroughly washed with tap water, air-dried, milled using a blender, and then stored at room temperature in a dry environment. Q Sepharose Fast Flow and Sephacryl S-400/HR column were from GE Healthcare Life Sciences (Piscataway, NJ, USA). Dialysis membranes (flat width 44 mm, molecular weight cut-off 3500; flat width 31 mm, molecular weight cut-off 1000) were from Lvniao (Yantai, China). Pullulan standards (Mw: 9.6, 21.1, 47.1, 107, 200, 344, and 708 kDa) were from Showa Denko K.K. (Tokyo, Japan). Penicillin, streptomycin, ConA, LPS, l-rhamnose, d-rhamnose, d-xylose, l-xylose, d-glucuronic acid, and l-glucuronic acid were from Sigma (St. Louis, MO, USA). LH was from Aladdin (Shanghai, China). Enzyme linked immunosorbent assay (ELISA) kits for IgG, IgM, and IgE were from Beyotime (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and Roswell Park Memorial Institute (RPMI) 1640 were from Lonza (Walkersville, MD, USA).

M. angicava was collected from the coastal area of the Yellow Sea in Qingdao, China on June 2013, which is in the growth mature period of the seaweed. The sample is green and 4–9 cm in size. After washing thoroughly in sea water, the sample was dried. Pullulan standards (Mw: 5.9 kDa, 9.6 kDa, 21.1 kDa, 47.1 kDa, 107 kDa, 200 kDa, 344 kDa, and 708 kDa) were purchased from Showa Denko K.K. (Tokyo, Japan). Sephacryl S-400/HR and Sephacryl S-300/HR were purchased from GE Health care Life Sciences (Piscataway, NJ, USA). Dialysis membrane (molecular weight cut-off 1000, 3500) was purchased from Lvniao (Yantai, China). l-rhamnose, l-arabinose, d-xylose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, and heparin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). APTT, TT and PT assay reagents were from MD Pacific (Tianjin, China). An FDP kit was purchased from BIOLINKS CO., LTD. (Tokyo, Japan). PAI-1 and D-dimer kits were purchased from Simens Healthcare Diagnostics Products (Marburg, Germany).

The molecular weight profiles of the starches were analyzed using high-performance size-exclusion chromatography (HPSEC) with refractive index (RI) detector (UFLC, Shimadzu, Kyoto, Japan). The method was according to Nguyen Vu and Lumdubwong [30 (link)]. Six pullulan standards (1 mg/mL) (Showa Denko, Tokyo, Japan) were used to create a standard curve. Weight–average molecular weight ( $\overline{M_w})$ and number–average molecular weight ( $\overline{M_n}$ ) were calculated based on the standard curve. The weight–average degree of polymerization ( $\overline{{\mathrm{DP}}_{\mathrm{w}}}$ ) was obtained by dividing $\overline{M_w}$ by 162. The amylose content of starch was calculated using the ratio of the peak area of amylose and amylopectin.
The maximum absorbance (λ_max) of starch was determined by the modified method of Chrastil [28 (link)]: 30 mg starch was dissolved in 2 M NaOH (1 mL) and distilled water (2 mL); 100 µL of the starch solution was mixed with 5% TCA (5 ml) and 0.01 N I₂-KI solution (I₂ 0.127 g and KI 0.3 mg/ 100 mL) (200 µL). The sample was analyzed by spectrophotometer (GENESYS-10S, Thermo Scientific, Waltham, MA, USA) with a wavelength ranging between 400–900 nm.

p-Nitrophenyl-α-d-glucopyranoside, α-glucosidase (EC 3.2.1.20, from Saccharomyces cerevisiae), 1-phenyl-3-methyl-5-pyrazolone and monosaccharide standards (d-mannose, l-rhamnose, d-glucose, d-glucuronic acid, d-galacturonic acid, N-acetyl-β-d-glucosamine, d-glucose, d-galactose, d-xylose, l-arabinose, and l-fucose) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Pullulan standards (Mw: 5.9, 9.6, 21.1, 47.1, and 107 kDa) were obtained from Showa Denko K.K. (Tokyo, Japan). Q Sepharose Fast Flow and Sephacryl S-100/HR were obtained from GE Healthcare Life Sciences (Piscataway, NJ, USA). Bicinchoninic acid (BCA) protein assay kit and glucose assay kit with o-toluidine were obtained from Beyotime Biotechnology (Shanghai, China). STZ and rosiglitazone were obtained from Aladdin Chemical Co., Ltd. (Shanghai, China). Mouse insulin enzyme-linked immunosorbent assay (ELISA) kit was obtained from Solarbio Biotechnology (Beijing, China). The assay kits for TC, TG, HDL-C and LDL-C were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

The molecular weight distribution of polysaccharide extracts was determined via high-performance gel filtration chromatography (HP-GFC) using a Flexar high-performance liquid chromatography system equipped with a reflective index detector (PerkinElmer, Waltham, MA, USA) [17 (link)–19 (link)]. The analytical column was a serial combination of a PL Aquagel-OH 30 column (300 × 7.5 mm, 8 μm particle size; PL1120-6830; Agilent Technologies, Santa Clara, CA, USA) and a PL Aquagel-OH 40 column (300 × 7.5 mm, 8 μm particle size; PL1149-6840; Agilent). The mobile phase was 0.2 M NaCl with a flow rate of 1 mL/min, and the column and detector temperatures were 25 °C and 40 °C, respectively. Pullulan standards (47.3–788 kDa; Showa Denko, Tokyo, Japan) were used as molecular weight markers. Solutions of the standards and polysaccharide extracts were prepared by dissolving them in distilled water at a concentration of 1 mg/mL. Each solution was filtered, and 30 μL was injected into the HP-GFC system. Data analysis including the determination of peak molecular weight (PMwt), weight average molecular weight (Mw), and number average molecular weight (Mn) was performed using TurboSEC software ver. 6.3.2 (PerkinElmer).

M. nitidum was collected from the coast of Yantai, China on May 2016. The raw material was thoroughly washed with tap water, air-dried, milled using a blender, and then stored at room temperature in a dry environment. Q Sepharose Fast Flow and Sephacryl S-400/HR were from GE Healthcare Life Sciences (Piscataway, NJ, USA). Dialysis membranes (flat width 44 mm, molecular weight cut-off 3500; flat width 31 mm, molecular weight cut-off 1000) were from Lvniao (Yantai, China). Pullulan standards (Mw: 9.6, 21.1, 47.1, 107, 200, 344 and 708 kDa) were from Showa Denko K.K. (Tokyo, Japan). l-rhamnose, l-arabinose, d-xylose, d-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, d-galacturonic acid, and d-glucosamine were from Sigma (St. Louis, MO, USA). APTT, TT, and PT kits were from MD Pacific (Tianjin, China). Heparin (196 U/mg) was obtained from Sigma (St. Louis, MO, USA). Clopidogrel was from Sanofi (Hangzhou, China). Urokinase was obtained from Ndpharm (Nanjing, China). d-Dimer kit was from Siemens Healthcare Diagnostics Products (Marburg, Germany). Factor Xa and thrombin were from Boatman Biotech CO., LTD. (Shanghai, China). AT-III was from Chromogenix (Milan, Italy). HC-II was from Hyphen Biomed (Neuville, France). S-2238 and S-2765 were from Asnail (Beijing, China). FDP kit was from BIOLINKS CO., LTD. (Tokyo, Japan). PAI-1 kit was from Cloud-Clone Corp (Wuhan, China).