ABSTRACT
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 digestive 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 NaCl were 22%, 20% and 0.04%, respectively. The extraction temperature was 35°C, 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 3210.27 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. Copyright 2012 by the Chinese Pharmaceutical Association.