Study Of Technology For Extracting Thymosin From Bovine Thymus And Porcine Thymus

Thymosin is polypeptide hormones of thymus secreting directly. It's mainly used for healing autoimmune deficiency disorder and inhibit the leukocytopenia of tumor chemotherapy. The thymosin is mostly extracted from the calf thymus and porcine thymus. The flow of calf thymus is in short supply in order to keep prices up. At home and abroad, the extraction of thymosin are mostly based on research of Goldstein. This method has complicated purification steps, meanwhile, the end products have some defects both on the contents and activity. The content of thymosinα₁ of thymosin in domestic market is low (≤1%) so the lower purity quotient and activity induce the medicine may not be very effective. In view of the conditions above, the study tries to pick out a extracting technology which is high performance with low cost, high purity and activity. Based on the above situation, the research is as follows:(1) The optimization of technology and parameters for Ultrasonic Wave extracting thymosin. Through the research of the extraction process for thymosin, high performance and low cost extracting technology is found. Using the bovine thymus and porcine thymus as raw materials, mensurate the content of thymosin by Freezing Thawing Method; The composite rotatable experiment design is used for screen out the optimal extracting parameters; For choosing the salting out method, the neutral salt is studied. The result indicates: The content of thymosin in bovine thymus is higher (2.0968 mg·g^-1); The optimum condition for ultrasonic wave extraction of thymosin were that leaching liquor is 0.15M NaCl, liquid-to-solid ratio of leaching liquor is 8:1, pH of leaching liquor is 4.12±0.01, ultrasonic power is 320W, ultrasonic extraction time is 14min, centrifuge velocity is 8300rpm, the saturation of sulfate ammonium which between 30% and 65% makes the isolation of thymosin better, the extraction ratio of thymosin is 4.3961mg·g^-1, which compared with the average extraction ratio is rasied observably,it could save 3000～8000 yuan/kg on costs.(2) Comparative analysis the Ultrasonic Wave extraction with the conventional extraction. Through the comparison between the Ultrasonic Wave extraction and Freeze Thaw method in the activity and the extraction rate in order to determine the feasibility of ultrasonic Wave extraction. The E-rosettes is used for determining the biological activity of thymosin, and the Lowry is used for determining the extraction rate, meanwhile, the study calculates the relationship between the content and the activity. The results show that Ultrasonic Wave method is improved significantly on the extraction rate(α=0.01), the rosettes of thymosin which is extracted by Ultrasonic Wave method is equal higher than 17%,which comsistents with the Encoding Standard(10%), Ultrasonic Wave method could save almost 140 times on extraction efficiency, the relationship between the content and the activity of thymosin prepared by the ultrasonic Wave method is incomplete positive correlation (r=0.8304) , the regression equation is: Y∧=9. 5393+1.1399X.According above stated, the Ultrasonic Wave extraction method has a higher extraction efficiency and simple procedure.(3) The optimization of technology and parameters for purification thymosinα₁. Through purifying thymosinα₁ and improve the purity of thymosinα₁ in order to research a simple and feasible purification technology. The study chooses QAE Sephadex A-25 and D208, D301, 717 ect. for static adsorption experiment through the anion exchange chromatography in order to determine the optimum ion exchange media; The optimal experiment conditions of the eluent, eluting style, flow rate, height of media are studied and choose L9 (33) orthogonal experiment on optimizing the conditions of the velocity, height and the concentration. The Sephadex G-25 gel filtration chromatography is used for desalting of thymosinα₁.The study chooses HPLC and SDS-PAGE to identifying the thymosinα₁. The results shows that the QAE Sephadex A-25 as a filler media adsorbing thymosinα₁ makes the best effect, the velocity is 60 cm·h^-1, packing height is 50 cm, the concentration is 8 mg·mL^-1, then the purification results is satisfactory. Determined by the HPLC, the purity of thymosinα₁ is 24.5966 mg·mL^-1, the Tα₁ recovery is 9.7%, the mass percent is 12.03%,which is 10 times higher than the thymosinα₁ on sale(the mass percent is 0.6% to 1.0%).