Research on Extraction and Purification of Spinosad
|School||Wuhan Polytechnic University|
|Course||Microbial and Biochemical Pharmacy|
|Keywords||Saccharopolyspora spinosa spinosad extraction purification decolorization response surface methodology|
At first, the method of detection and analysis for spinosad by high performance liquid chrpmatography was established.The concentration of spinosad was accurately detected by HPLC. The physico-chemical characteristics of spinosad were reported in this paper. Spinosad was purified by solvent extraction, macroporous adsorbent resin, silica column chromatography and ion-exchange chromatography methods.The extraction, isolation and purification technique of spinosad were obtained.The physic-chemical characteristics of spinosad showed that it was stable in hot and alkaline conditions, but not stable in superacid conditions. It was found that pH 10 was propitious to extract spinosad from fermentation broth. Ethyl acetate was confirmed to the optimum extracting solvent from different solvents. On the basis of which, three times was the optimal extracting times and the equivalent volume of ethyl acetate and fermentation extracting broth was the best extraction proportion.The extraction technique of spinosad from fermentation broth was optimized by central composite rotatable design. On the basis of one-factor experiments, the method of response surface analysis with 3 factors and 5 levels was adopted. A second order equation for extracting spinosad was built. Response surface and contour were graphed with the concentration of spinosad as the response value. Based on the analysis of the response surface plots and their corresponding contour plots, effects of extracting time, the ratio of material to solvent and ultrasonic extracting frequency was explored. The optimal extraction conditions, by using this new method, were determined as follows: extracting frequency 75kHZ, extracting time 74min and the ratio of material to solvent 1:1.5. Under the optimized conditions the average concentration of spinosad extracted from fermentation broth was 100.75μg/mL.Spinosad extract was firstly absorbed by macroporous adsorbent resin, and then most of spinoad was eluted with a mixture of acetone and water(70:30~95:5). Its recovery was up to 80.72%. Further purification of the above concentrated residue was achieved by an adsorption chromatography on silica gel. A linear gradient elution was carried out using petroleum/ethyl acetate/methanol. The effluent solutions in different vials were monitored by thin-layer chromatograph (petroleum ether:ethyl acetate:methanol=10:10:1). The fractions containing spinosad were polled and concentrated in vacuum, which were dissolved. This fraction was in a minimum volume of methanol and further analyzed by analysis HPLC. The result showed that the purity of spinosad was 90%~95%.The decolorization process of spinosad extract by anion exchange resin(201×7) and the effects of adsorption conditions on its decolorization capacity were investigated.The optimal operation conditions were as following: adsorption equilibrium time 3h , resin content 4%( g/V) , rotation speed 150 r/min, and pH 10. Under this conditions, the decolorization rate was above 80% and the loss of spinosad was only 2%. This method is simple and available for the commercial process.