Extraction And Content Determination Of Active Constituents And Related Substances For Two Medicines

Saffron, obtained from the flower stigma of Crocus sativus L., is the most valuable spice in the world. The major bioactive ingredients of saffron are crocins which show great potential in curing tumor, hypoglycemic and hypotensive. Crocin-Ⅰ and crocin-Ⅱ were recorded in the ChP(2010version) for the quality control of saffron (Crocus sativus L.). In the study, the response surface method was applied to optimize the extraction process of crocin-Ⅰ and crocin-Ⅱ based on single factor tests (ethanol concentration, extraction time and temperature). The optimum parameters were as follows:ethanol concentration of50%, the extraction time of30min and the temperature of45℃. Crocin-Ⅰ and crocin-Ⅱ in saffron collected from Shanghai, Haimen, Jinyun, et al. were extracted under the optimum condition and were analyzed by RP-HPLC. The analytical column was Agilent ZORBAX SB-C18(4.6×250mm,5μm), the mobile phase was methanol-water (45:55) at a flow rate of1.0mL. min-1with column temperature at30℃and the detection wavelength was440nm. This research supplied a theoretical basis for the quantitive analysis of the active ingredients in saffron, and also offered a reference for the crop farming of saffron.Marbofloxacin is a third-generation fluoroquinolone antibacterial drug featuring strong tissue infiltration, long elimination half-life, high bioavailability and a wide safe dose. It is clinically used for the treatment of urinary tract and soft tissue infections of cats and dogs. The regularity of the domestic veterinary market, along with the further development of pets and livestock industry, ensures a promising prospect for marbofloxacin. However, impurities that were introduced in the synthesis and storage process may result in decrease in the quality of marbofloxacin. The related substances of marbofloxacin have neither been sold nor reported in domestic and foreign countries, it’s hence necessary to establish a method to obtain impurities with high purity. In this research, the mother liquor of marbofloxacin provided by Apeloa Biopharmaceutical Company of Zhejiang province was investigated, and four impurities were separated by recrystallization, column chromatography and semi-preparative HPLC. The chemical structures of impurities were successfully determined by spectroscopic methods. Three of the four impurities were identified as impurity C, D and E as prescribed in the European pharmacopoeia (EP7.0), and was postulated to be introduced during the synthesis process of marbofloxacin. Another one turned to be a photodegradation product of marbofloxacin. The method in European Pharmacopoeia (EP7.0) was optimized for better determination of related substance in marbofloxacin production. The optimized method was proved to be simple, feasible and applicable by a series of method validation. The study established an important basis for the purity and safety control, and was also of great significance to the optimization of production process and to the improvement of the quality of marbofloxacin.