利用猪牛胸腺提取胸腺肽工艺技术的研究
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摘要
胸腺肽是胸腺直接分泌的多肽类激素,主要用于治疗自身免疫缺陷疾病以及抑制肿瘤放化疗引起的白血球减少等症。现今市场上所销售的胸腺肽大多是从幼牛胸腺或猪胸腺肽中提取,幼牛胸腺供应量十分不足,导致产品价格居高不下,目前国内外采用的提取方法多数是沿袭Goldstein研究的提取方法,但其纯化步骤繁琐,得到终产物在含量和活性上都存在一定缺陷,国内市场上所销售的胸腺肽中胸腺肽α_1含量很低(一般在1%以下),导致纯度和活性较低、临床疗效差。鉴于以上几种情况,实验力求筛选出一条高效低成本、纯度高、活力强的制备工艺路线。针对以上状况作如下研究:
(1)超声波法提取胸腺肽工艺研究及其技术参数的优化。通过胸腺肽提取工艺方法的研究,确定出高效、低成本的提取工艺技术。以牛胸腺、猪胸腺为原料,采用反复冻融法测定胸腺肽的含量;通过复合旋转实验,确定提取胸腺肽的最佳工艺参数;通过对中性盐的盐析实验,选择盐析方法。研究结果表明:牛胸腺中胸腺肽含量较高(2.0968mg·g~(-1));超声波法提取胸腺肽最佳工艺参数为:浸提液为0.15M NaCl溶液,浸提液液料比为8﹕1,浸提液pH值为4.12±0.01,超声波输出功率320W,超声波处理时间14min,离心转速为8300rpm;硫酸铵在30%～65%饱和度时分离胸腺肽效果最佳;验证实验胸腺肽的提取率可达到4.3961mg·g~(-1),较国内平均提取率3 mg·g~(-1)有明显提高,从成本上每千克节省3000～8000元。
(2)超声波法与常规法提取胸腺肽效果的比较分析。通过对超声波法与反复冻融法这两种方法在活性、提取率及提取效率上的比较,确定超声波法的可行性。采用E-玫瑰花环节法测定胸腺肽的生物活性,福林酚法测定胸腺肽的含量,并确定含量与活性之间的相关性。实验结果表明,超声波法与反复冻融法比较,经T-Test分析,超声波法在提取率方面显著提高(α为0.01),生物活性方面相当,其中超声波法制备的胸腺肽花环结增加值均高于17%,符合国家对药厂出厂的规定(10%),提取效率方面,超声波法节省近200倍时间,超声波法制备的胸腺肽其活性与含量呈不完全正相关关系,r为0.8304,二者之间的回归方程为:Y∧=9. 5393+1.1399X,以上各方面说明,超声波法与反复冻融法相比,提取效率高,工艺过程简单。
(3)胸腺肽α_1分离纯化工艺研究及其技术参数的优化。通过对胸腺肽α_1的分离纯化,提高胸腺肽α_1的纯度,确定简单可行的纯化工艺技术。将QAE Sephadex A-25和D208、D301、717等阴离子交换介质进行静态吸附实验,确定最适离子交换介质;对洗脱液及洗脱方式、流速、填料高度的最佳实验条件进行摸索,选择L9(33)正交实验对流速、填料高度及进样浓度这三个因素进行优化,确定最佳分离条件;采用Sephadex G-25凝胶过滤色谱对纯化的胸腺肽α_1脱盐;选用HPLC和SDS-PAGE对纯化的胸腺肽α_1进行鉴定。结果表明,将QAE Sephadex A-25作为填料介质吸附胸腺肽α_1效果最好,在流速60cm·h~(-1),填料高度50cm,进样浓度8mg·mL~(-1)时,胸腺肽α_1纯化效果理想,经HPLC测定,得到纯度达24.5966mg·mL~(-1)的胸腺肽α_1,回收率达9.7%,质量分数达12.03%,较之市场上所销售的胸腺肽中Tα_1含量(质量分数0.6%～1%)高出近10倍。
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 thymosinα_1 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 thymosinα_1. Through purifying thymosinα_1 and improve the purity of thymosinα_1 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 thymosinα_1.The study chooses HPLC and SDS-PAGE to identifying the thymosinα_1. The results shows that the QAE Sephadex A-25 as a filler media adsorbing thymosinα_1 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 thymosinα_1 is 24.5966 mg·mL~(-1), the Tα_1 recovery is 9.7%, the mass percent is 12.03%,which is 10 times higher than the thymosinα_1 on sale(the mass percent is 0.6% to 1.0%).
(1)超声波法提取胸腺肽工艺研究及其技术参数的优化。通过胸腺肽提取工艺方法的研究,确定出高效、低成本的提取工艺技术。以牛胸腺、猪胸腺为原料,采用反复冻融法测定胸腺肽的含量;通过复合旋转实验,确定提取胸腺肽的最佳工艺参数;通过对中性盐的盐析实验,选择盐析方法。研究结果表明:牛胸腺中胸腺肽含量较高(2.0968mg·g~(-1));超声波法提取胸腺肽最佳工艺参数为:浸提液为0.15M NaCl溶液,浸提液液料比为8﹕1,浸提液pH值为4.12±0.01,超声波输出功率320W,超声波处理时间14min,离心转速为8300rpm;硫酸铵在30%～65%饱和度时分离胸腺肽效果最佳;验证实验胸腺肽的提取率可达到4.3961mg·g~(-1),较国内平均提取率3 mg·g~(-1)有明显提高,从成本上每千克节省3000～8000元。
(2)超声波法与常规法提取胸腺肽效果的比较分析。通过对超声波法与反复冻融法这两种方法在活性、提取率及提取效率上的比较,确定超声波法的可行性。采用E-玫瑰花环节法测定胸腺肽的生物活性,福林酚法测定胸腺肽的含量,并确定含量与活性之间的相关性。实验结果表明,超声波法与反复冻融法比较,经T-Test分析,超声波法在提取率方面显著提高(α为0.01),生物活性方面相当,其中超声波法制备的胸腺肽花环结增加值均高于17%,符合国家对药厂出厂的规定(10%),提取效率方面,超声波法节省近200倍时间,超声波法制备的胸腺肽其活性与含量呈不完全正相关关系,r为0.8304,二者之间的回归方程为:Y∧=9. 5393+1.1399X,以上各方面说明,超声波法与反复冻融法相比,提取效率高,工艺过程简单。
(3)胸腺肽α_1分离纯化工艺研究及其技术参数的优化。通过对胸腺肽α_1的分离纯化,提高胸腺肽α_1的纯度,确定简单可行的纯化工艺技术。将QAE Sephadex A-25和D208、D301、717等阴离子交换介质进行静态吸附实验,确定最适离子交换介质;对洗脱液及洗脱方式、流速、填料高度的最佳实验条件进行摸索,选择L9(33)正交实验对流速、填料高度及进样浓度这三个因素进行优化,确定最佳分离条件;采用Sephadex G-25凝胶过滤色谱对纯化的胸腺肽α_1脱盐;选用HPLC和SDS-PAGE对纯化的胸腺肽α_1进行鉴定。结果表明,将QAE Sephadex A-25作为填料介质吸附胸腺肽α_1效果最好,在流速60cm·h~(-1),填料高度50cm,进样浓度8mg·mL~(-1)时,胸腺肽α_1纯化效果理想,经HPLC测定,得到纯度达24.5966mg·mL~(-1)的胸腺肽α_1,回收率达9.7%,质量分数达12.03%,较之市场上所销售的胸腺肽中Tα_1含量(质量分数0.6%～1%)高出近10倍。
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 thymosinα_1 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 thymosinα_1. Through purifying thymosinα_1 and improve the purity of thymosinα_1 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 thymosinα_1.The study chooses HPLC and SDS-PAGE to identifying the thymosinα_1. The results shows that the QAE Sephadex A-25 as a filler media adsorbing thymosinα_1 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 thymosinα_1 is 24.5966 mg·mL~(-1), the Tα_1 recovery is 9.7%, the mass percent is 12.03%,which is 10 times higher than the thymosinα_1 on sale(the mass percent is 0.6% to 1.0%).
引文
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