双水相萃取-凝胶色谱联用法提取菲牛蛭中的水蛭素

Extraction of Hirudin by Double Aqueous Phase Combined with Gel Chromatography

目的建立水蛭素的双水相萃取-凝胶色谱联用提取法。方法以抗凝血酶活性单位(ATU)为指标,在优化双水相萃取工艺条件基础上,建立以广西菲牛蛭消化液为原料的聚乙二醇/硫酸铵双水相萃取-凝胶色谱联用提取水蛭素新工艺。结果在所考察的实验范围内,水蛭素对照品双水相萃取最佳工艺条件是:PEG 2000、(NH4)2SO4、NaCl质量分数依次为22%、20%、0.04%,萃取液温度35℃、pH 6.0;萃取物经凝胶色谱除杂以后,得到纯度较高的产品。实验建立的提取工艺处理水蛭素对照品ATU回收率达到81.92%,粗提液分离纯化及小规模工艺放大实验ATU回收率分别为80.34%、80.36%。所获产品比活性达到3 210.27 ATU.mg-1、不连续聚丙烯酰胺凝胶电泳及光谱扫描结果与水蛭素对照品相同。结论双水相萃取-凝胶色谱联用提取水蛭素效果较好。

OBJECTIVE To establish an extraction method of hirudin by double aqueous phase combined with gel chromatography. METHODS Anti-thrombin activity units （ATU） was adopted as the index. On the basis of optimizing the double aqueous phase extraction conditions, a new extraction technology of hirudin was established by employing double aqueous phase system combined with gel chromatography. The double aqueous phase was composed of polyethylene glycol and ammonium sulfate and took the di- gestive juice of Poecilobdella manillensis from Guangxi Province as the raw material. RESULTS In the studied experimental range, the optimum technological conditions of double aqueous phase extraction of hirudin reference substantce were as follows ： the contents of PEG 2000, （NH4）2SO4 and NaC1 were 22%, 20% and 0. 04% , respectively. The extraction temperature was 35 %, and pH was 6. 0. The obtained extracts were further purified by gel chromatography. The recoveries of ATU reached 81.92%, 80. 34% and 80. 36% for hirudin reference substance, hirudin crude extracts and scale-up experiment, respectively. The specific activity of obtained products reached 3 210. 27 ATU ·mg-1. The results of discontinuous polyacrylate gel electrophoresis and spectral scan for the extract were the same with for the reference substance. CONCLUSION The double aqueous phase extraction combined with gel chromatography is good for hirudin.