Studies On Preparation, Structure And Activities Of Polyguluronate Sulfate And Its Oligosaccharides

Urolithiasis is a common disorder with a high recurrence rate. Urinary macromolecules, especially glycosaminoglycans (GAGs) play important roles as inhibitors of urinary stone formation. GAGs and their analogues have attracted considerable interest as potential and promising drug candidates for the prevention and treatment of urolithiasis. Polyguluronate sulfate (PGS) as an analogue of GAGs, which has high charge density and strong polyanionic property, is prepared by chemical sulfation of polyguluronate (PG) that separated from alginate. The structure of PGS is characterized by modern analytical techniques. Results of antiurolithiatic activity and safety of PGS show a good prospect on the prevention and treatment of urolithiasis. PGS oligosaccharides with different degrees of sulfate substitution and different polymerization degrees are prepared by several methods, and their activities on the incorporation with fibroblast growth factors (FGF) and fibroblast growth factor receptors (FGFR) are assayed also in this paper.PG is obtained by pH-fractionated precipitation from the hydrolysate of alginate. PGS is prepared by chemical sulfation of PG with sulfur trioxide trimethylamine complex (SO3-TMA) method, sulfur trioxide pyridine complex (SO3-Py) method and chlorosulfonic acid-foramide method, respectively. Compared with other methods, the reaction condition of SO3-Py method is mild, and it is simple and suitable to prepare PGS with high degree of sulfate substitution. The structure of PGS is characterized by UV, IR, GC, CD, 1H-NMR, 13C-NMR, 1H-1HCOSY, 1H-13C HMQC and other chemical analyses. Sulfation is demonstrated to occur at the C-2 and C-3 positions of the guluronic acid residues. Molecular conformation simulated by Sybyl software show that PGS is a flexible long chain molecule. PGS molecule is a random coil and can sway freely in water solution. The quality stability of PGS are tested under high temperature, high humidity and strong light, respectively. Results show that PGS exhibits a good quality stability, but it should keep away from high temperature and high humidity.Antiurolithiatic activities of PGS are assayed on two experimental urolithiasis models in rats. The contents of renal oxalate and calcium in ethylene glycol-induced nephrolithiasic rats are decreased significantly, and the renal crystal deposition and pathological changes are improved after administration of PGS at dose-levels of 50 and 100 mg/kg. The formation of zinc disc implant-induced urinary bladder stones in rats are inhibited considerably after administration of PGS at dose-levels of 50 and 100 mg/kg. PGS exhibits good effects both on the prevention and the treatment of urolithiasis. In addition, PGS exhibits considerable anti-inflammatory activity in the cotton pellet-induced granuloma in rats. Acute toxicity results of PGS show that it is a safe drug candidate. The intravenous LD50 in mice is 6.29 g/kg, and the oral maximum tolerance values of LPGS in mice is 25 g/kg.PGS oligosaccharides with different degrees of sulfate substitution and different polymerization degrees are prepared by free radical degradation, solid phase acid degradation and by chemical sulfation of PG oligosaccharides, respectively. 732# resin as a solid phase acid is used for the degradation of sulfated polysaccharides at firstly in this paper. Not only PGS can be degraded effectively by the solid phase acid method, but also there are no salt impurities exist in PGS oligosaccharides. The difficulty of separation between oligosaccharides and salt is avoided by the solid phase acid method, but the PGS oligosaccharides are desulfated obviously. So, the solid phase acid method is suitable to prepare PGS oligosaccharides with low degrees of sulfate substitution. The free radical degradation and chemical sulfation of PG oligosaccharides method are suitable to prepare PGS oligosaccharides with high degrees of sulfate substitution, but it is difficult to separate these PGS oligosaccharides. PGS oligosaccharides prepared by solid phase acid method are separated by Bio-Gel P6 column, and thirteen oligosaccharide fractions (F1-F13) are obtained. The polymerization degree of F1-F13 is one to thirteen, respectively, demonstrated by ESI-MS analysis. Results of PGS oligosaccharides incorporated with FGF-FGFR1c show that the least segments of PGS oligosaccharides to incorporate with FGF1-FGFR1c and FGF19-FGFR1c are PGS4 and PGS12, respectively. The ability of PGS oligosaccharides to incorporate with FGF7-FGFR1c is increased when the polymerization degree is increased.In addition, PGS liposomes are prepared by different methods in order to improve oral absorption and bioavailability of PGS. The encapsulation efficiency of PGS liposomes prepared by reverse-phase evaporation is up to 39.13%. A HPGPC method is established also for the determination of encapsulation efficiency of PGS liposomes.All results on PGS above are important and useful to guide the further clinical research of PGS. Preparation and structure analysis of PGS oligosaccharides with different polymerization degrees have provide a base not only for the research on structure-activity relationship of PGS, but also for further research on the relationship of sulfated oligosaccharides with some diseases, such as cancer, which are relevant to FGF-FGFR.