| This thesis aimed to investigate the flavonoids from the leaves of Diospyros kaki based on the studies of Ginkgetin injection made in German (Jinnaduo): Manufactured by Schwabe Germany and "nao xin qing" tablets Manufactured by Guangzhou Baiyunshan Pharmaceutical Co., Ltd, and pertinent literatures. Crude extract of the flavonoids from the leaves of Diospyros kaki were purified, and made it as an intermediates suit for injection. The sterile powder of Shiyehuangtong for injection was successfully developed with better safety, efficiency and stability using the lyophilization technique from the intermediates and maintain the qualities of the product, the formula and the and drying processes were optimized, a quality control methods of freezing-dry products were developed. An accurate and sensitive quality specification of freezing-dry products and the intermediates and the preparation were established. Common safety was investigated, which can provide valuable experimental data for clinical application.Method of identifying total flavonoids was established by chemical reaction. HPLC and UV methods are developed to determine total flavonoids contents in the intermediates for injection and preparation . All of these methods are accurate and sensitive.Based on purity of active substances, the purifying technics of total flavonoids was optimized by using factor experiment and orthogonal design, the types of macroporous resin, temperature, concentration, pH, rate of flow, elutriant were investigated. Finally AB-8 macroporou resin was adopted , and the concentration was 0.06 g·L-1, pH=5.0, temperature was 25℃. Five times column volume of 30% ethanol solution was used to decrease impurity, then following with 50% ethanol solution at flow rate of 2～3 mL·min-1, eluting five times column volume, the eluate were concentrated and dried in vacuum. The purity of total flavonoids in the three batches of samples of the intermediate should be more than 70%, and the transfer rate of total flavonoids more than 60%.Based on the intermediate, the freeze-drying technology was studied, including the screen of the prescription, the selection of the support agent and activated carbon, the appearance, moisture, re-solubility and loss rate of total flavonoids as indicators, to optimize the prescription of freeze-drying preparation. The eutectic point of the solution was -1.2℃determined by thermal analysis system. The curve of freeze-drying was drawn, and the amount of activated carbon is 0.1% (w/v) was established, and Mannitol (250mg/bottle) was used as filler. Research shows that the compatibility were good with saline injection and 5.0% glucose injection (24 h).The quality of preparation was studied, and the quality specification was built up to agree with the quality requirements of Chinese herbal freeze-drying preparation for injection. The quality could be controlled by the specification established considerably and the clinical safety could be ensured.HPLC method was employed to determined kaempferol and quercetin in the hydrolysate of the sample, and the total flavonoids was calculated by the empirical formula: total flavonoids = quercetin×2.55+kaempferol×2.59. Content of total flavonoids is 4.90～5.10mg/bottle, meet requirements. Product quality evaluation system was established, including traits, chemical reaction differential (flavonoids), general inspection (with volume differences, particulate matter, pH, water, tannin, protein, etc.). Toxicity, in vitro hemolysis and vascular irritation were investigated, the results show that it reach the requirements of injection safety. The LD50 could not be determined using high allowable concentration and maximum allowable capacity in Acute toxicity tests in mice, therefore for further study the preparation safety, the maximum tolerable dose (MTD) was determined, results showed that the high dose for the administration is 0.25 g / kg, and the MTD> 0.25g/kg, using injection mice with solution of the preparation by intraperitoneal injection. |
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