| Propylene glycol alginate sodium sulfate (PSS), the first marineheparinoid-active sulfated polysaccharide developed and producted by Chinaindependently, and has been commonly used as a clinical anti-cardiovascular diseasedrug for nearly30years. Some problems were exposed during the clinical use of PSS,such as too many manufacturers, unconsistent production process and some adversereactions. The current quality standard which cannot meet the requirements of thequality control of PSS, need to be standardized and perfected. A new highperformance liquid chromatography with pre-column derivatization method wasdeveloped for determination of the M/G ratio and the content of PSS. The factorsaffecting determination results of molecular weight of PSS by gel permeationchromatography method were investigated. PSS polysaccharide fragments withdifferent molecular weight was prepared by gel column chromatography and PSSoligosaccharides were further prepared by a solid phase acid (732#resin) degradationmethod. Their anticoagulation and the binding activity for FGF-2were evaluated andthe active fragments of PSS oligosaccharides were preliminarily determined. Therelated works provided useful reference for the quality control of PSS and itssecondary development.1) A reliable high performance liquid chromatography method by pre-columnderivatization with1-phenyl-3-methjl-5-pyrazolone (PMP) was developed fordetermination of the M/G ratio of PSS. The best hydrolysis method of PSS wasdetermined after the comparison four degradation methods based on the degree ofdestruction of M and G, with the best degradation condition, temperature of120℃,time of4h, sulfuric acid concentration of0.1mol/L and the chromatographicseparation conditions were also optimized. A satisfactory resolution of M and G was achieved on a KP-C18column using0.1mol/L phosphate buffer (pH7.0)-acetonitrile(83/17, v/v) as a mobile phase. The M/G ratio of PSS determined by the method wasin good accordance with that obtained by1H-NMR method with a desulfurizationstrategy.2) The high performance liquid chromatography with pre-column derivatizationmethod was successfully used for determining the content of PSS based on themolecular skeleton characteristic of PSS (composed of M and G). Reference to thecontent determination method of sodium chondroitin sulfate, glucuronic acid (GlcUA)was chosed as internal standard, and the standard curve is drawn with PSS referencesubstance, then the content of PSS was calculated by the peaks area of M and G, theinfluence of responsivity correction factor for content determination was alsoinvestigated. The results indicated that the calibration curve was linear in the range of0.3~15mg/mL with correlation coefficient of0.9966. The intra-day average RSD forPSS in three PSS concentrations (high, medium and low) was0.78%(n=6) and theinter-day average RSD was3.08%(n=5). The RSD for repeatability is3.24%(n=5)and for stability is2.93%.The average recovery was115.77%and the low dection limitwas0.25μg/mL. This method had a good specificity, high sensitivity, goodrepeatability and can reflect more the immanent quality properties of PSS than thecurrent quality standard with a sulfur content conversion method. This method wassuitable for quality control of PSS. The contents of ten PSS sample from differentmanufacturer in China were determined and compared, and obvious qualitydifferences were discovered in these samples, which showed that the productiontechnology and quality control of PSS need to be strengthened.3) The factors affecting determination results of molecular weight of PSS by gelpermeation chromatography method were investigated. The sample were separated ondifferent chromatography column, and detected by refractive index detectors (IR).Retention time (tR) and the distribution coefficient (Kav) were used in the calculationof Mp. The testing results were compared with the absolute molecular weight detectedby DAWN HELEOS II. The molecular weight of PSS sample from differentmanufacturer were determined and compared by this method. This work provided good reference for the molecular weight limiting and determination methodimprovement.4) PSS was classified to different fragments by gel column chromatographymethod. A kind of environmental friendly solid acid degradation was established(solid acid at a concentration of200mg/mL, temperature of100℃, time of6h).Thirteen PSS oligosaccharide fractions were obtained by Bio-Gel P6separation andtheir structural compositions were characterized by ESI-MS.5) The anticoagulation and FGF-2binding activity of PSS fragmentswithdifferent molecular weight or PSS oligosaccharide were evaluated. The resultsindicated that the anticoagulant activity of PSS would decreased when its molecularweight decreased, but there are no relationship between the molecular weight of PSSand its FGF-2binding activity. The G monosaccharide would improve anticoagulantactivity of polysaccharide, and decrease their FGF-2binding activity. In addition,sulfate groups are key groups for the above two activities. Some PSS oligosaccharideswith high degree of polymerization showed significant FGF-2binding activity but noanticoagulant activity. Particularly, fraction F8showed higher activity and was to bethe research object of oligosaccharide drug for binding to FGF-2with noanticoagulant activity.The detection methods for M/G ratio and content established by this paper andthe research results for the factors influencing the determination of molecular weightcontribute to the revision of PSS quality standard and quality control of PSS. Theactivities investigation in this paper was used for our further understanding of thestructure-activity relationship of PSS and the development of PSS oligosaccharidedrug. |
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