Research On Extraction And Purification And Preliminary Study On Microcapsule Of Doramectin

The study was conducted to investigate the purification processes and optimize a method for the delayed-release preparation of doramectin which was a new antibiotics veterinary medicine. This paper will firstly discuss 4 processes which could conduct and lay foundation for further pilot enlargement tests including establish the method of HPLC, experiments for stabily, decolorization by activated carbon, purification by macroporous resin and silica gel. Secdonly the paper optimized a methods for a delayed-release preparation, ALG-Zein microcapsules, its ability of stability and in vitro releasing performance were tested as well. The main conclusions were as follows:(1) High performance liquid chromatography method have been developed for the detection of doramectin. The component of doramectin was detected by UV. The correlation coefficient exceeded 0.9996. Factors including distribution ratio of doramectin in fermentation broth(hyphae or supernatant), extractants, extraction times, storage time, pH and temperature on extraction results were investigated with one-factor experiments. Meanwhile, using ethyl acetate for extraction solvent, the optimal extraction times and extracting volume were optimized. It was found that the distribution ratio of doramectin in hyphae reached up to 97.79 %. The optimal extraction solvent was methanol and twice extraction were enough. Doramectin in fermentation broth could be stored for 6 days at least within the condition of pH from 3 to 13, and temperature from 20 to 80℃. Using ethyl acetate to extract doramectin, twice was the optimal extracting time and the twice volume of ethyl acetate and fermentation extracting broth was the best extraction proportion. The optimal extraction conditions of doramectin in fermentation broth of Streptomyces avermitilis were obtained. Under the optimal extractions, the concentration of doramectin and its extraction rate were 151.78μg/mL and 98.00 %, respectively. ( 2 ) Response surface methodology was applied to study the decolorization of activated carbon for doramectin in fermentation broth of Streptomyces avermitilis. Based on decoloring rate by the detection of UV and loss of doramectin detected by HPLC, effect of activatied carbon to decoloration was investigated. The optimal technical conditions were as follows: 1.0 % activated carbon, temperature 33℃and decoloration pH of 3. Under the optimal conditions, the decoloring rate was above 68.78 % in further verified experiment and the difference was not significant with predicted value of 70.35 %. The method was effective, simple and reliable, also warranted for the commercial process.(3) Domestic DM11 resign was optimized for separation. Doramecin was absorbed to 100 % in 3 h and its desorption could be fully completed in 0.5 h. Low-purity doramection was obtained by using 1:50 ratio of methanol to concentrate as well. The concentrated extract of doramectin was aborbed by DM11 resign and eluted twice by concentration gradient of methanol and water. Through these processes the purity of doramectin was increased from 4 % to 45 % approximately. The total recovery of doramectin in concentrated extract was about 80 %. Doramectin was purified by silica column chromatography method. Ultimately, doramectin was obtained with a purity 92 % and yield 60 %.(4) The last part of this paper was carried out about the controlled release preparation of doramectin. The processing technology was optimized as followed: mass ratio of doramectin and zein was 1:12, initial concentration of ethanol was 66.6 % with terminal concentration of 40 %, and agitation speed was 1500 r/min. Protein microsphere was obtained with 11 % encapsulation and its particle size reached about to 200 nm. During the second coating process, ALG was adding with mass concentration of 1.5 %, and the ratio of zein solution and ALG solution was 1:4. Under these processing technologies, microcapsule was obtained with 80 % encapsulation and it was morphologically intact and evenly distributed. Experiments were carried out in simulate gastric juice and intestinal liquid to evaluate the releasing property of microcapsules. Microcapsules released well in simulate intestinal liqudid whereas not in simulate gastric juice, the release rate of which was about 25 % in the first 5 hours, but microcapsules released low concentration and had slow-release effect afterwards and released completely in 8 d. To study its high-temperatrue stability, experiments carried out in 60℃for 10 d and the conclusion was that microcapsules performed well in high temperature and their concentration of drug were constant as well as the appearance, their moisture content remained to be 8 % constantly after the high temperature experiments.