Sephacryl s 100 hr

The lyophilized powders of A. camphorata mycelium were obtained from Dr. Min Ye in the School of Pharmaceutical Sciences, Peking University, Beijing, China. Diethylaminoethyl (DEAE) sepharose Fast Flow and Sephacryl S-100HR were commercially purchased from GE Healthcare Life Science. T-series Dextrans were obtained from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). Monosaccharide standards were all purchased from Fluka, Switzerland. Dimethyl sulfoxide (DMSO) was obtained from Merck (Darmstadt, Germany), whereas 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) came from Sigma-Aldrich, St. Louis, MO, USA. Sodium borohydride (NaBH₄), iodomethane (CH₃I), and trifluoroacetic acid (TFA) were all from Sinopharm Chemical Reagent Co. Ltd. Other reagents were analytical grade unless otherwise stated.

Accelrys Discovery Studio (DS, version 3.1) was used for homology modeling (MODELER) [16 (link)] and docking simulation (ZDOCK) [17 (link),18 (link)]. Pichia pastoris cells carrying the RGD-hirudin gene (Mut⁺) and pPIC9k-RGD-hirudin plasmid were stored in our lab. Briefly, the RGD-hirudin gene was synthesized in the Key Laboratory of Molecular Medicine at Fudan University. cDNA encoding RGD-hirudin was cloned into the plasmid pPIC9K, and this expression vector was transformed into Pichia pastoris GS115. Vector integration into the Pichia pastoris chromosome was confirmed by PCR [14 (link),19 ]. DNA primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The Site-Directed Mutagenesis Kit was purchased from SBS Genetech Co., Ltd. Yeast nitrogen base was obtained from Sigma Aldrich Co., Ltd. Blood plasma was obtained from the Shanghai Blood Center. Sephacryl S-100 HR, Sephadex-G50, and Q-Sepharose-FF were purchased from GE Healthcare Co., Ltd. The Biacore T100 instrument and research grade CM5 chips were purchased from Biacore (GE Healthcare) Co., Ltd. Other reagents were of analytical purity.

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).

GY785 DR was obtained by a free-radical depolymerization process, as previously described [43 ,44 (link)]. Briefly, 2.5 g of the native EPS were solubilized in water (350 mL) and an aqueous solution of hydrogen peroxide was added dropwise to depolymerize the EPS in the presence of copper (II) acetate used as catalyst. After overnight reduction using sodium borohydride and purification on Chelex^® 20 resin, the solution containing EPS was ultrafiltrated on a 10 kDa cut-off membrane and freeze-dried. To obtain homogeneous fractions of GY785 DR with low polydispersity, a predominant population of polysaccharide chains was selected by a gel filtration chromatography on either Superdex^® 30 (GY785 DR of ~20,000 g/mol) or Sephacryl S-100 HR (GY785 DR of ~200,000 g/mol) (GE Healthcare Life Sciences), using an AKTA FPLC system coupled with a refractometric detector (Gilson^®). Samples eluted with water were pooled and freeze-dried.
Highly sulphated GY785 DRS was obtained by a chemical sulphation of GY785 DR of ~20,000 g/mol, as described earlier [43 ,44 (link)]. GY785 DR (50 mg) in its pyridinium salt form was firstly solubilized in anhydrous DMF (100 mL) for 2 h at 45 °C under continuous stirring and then sulphated for the next 2 h at 45 °C in the presence of SO₃∙Py (250 mg). The final aqueous solution (pH 7) was dialyzed against water for 3 days before being freeze-dried.