蜈蚣抗凝血肽的提取分离与分析

【作者】 黄能听；

【导师】 王玉蓉；

【作者基本信息】 北京中医药大学 ， 中药制药学， 2010， 硕士

【摘要】 目的：研究仿生酶解法提取蜈蚣的工艺条件,探索蜈蚣酶解物中具有抗凝血作用的物质基础,进一步分离分析其组分结构,为制剂工艺提供合理依据。方法：(1)提取工艺的研究：采用活化部分凝血活酶时间(APTT)试验及纤维蛋白平板试验,以体外抗凝与纤溶活性为指标,对仿生酶解法、水提醇沉、醇提醇沉和水浸提等方法进行比较,筛选出蜈蚣的提取方法；仍以APTT实验和纤维蛋白平板实验为指标,进一步对胃蛋白酶酶解法、胰蛋白酶酶解法、仿生酶解法和复合酶解法进行比较,筛选最优的酶解方法；以水解度为指标,通过单因素考察,确定蜈蚣仿生酶解法的最佳工艺条件。(2)抗凝血肽的分离与分析：以APTT为考察指标,应用SephadexG-50、SephadexG-25凝胶过滤色谱和C18反相层析法分离蜈蚣胃蛋白酶酶解物中的抗凝血肽,并辅以高效液相色谱,MAILDIA-TOF-MASS质谱,凝胶电泳和薄层色谱等手段进行分析。结果：(1)经过提取方法和酶解方法的筛选,确定仿生酶解法为蜈蚣的提取方法,单因素考察结果确定酶解工艺为：蜈蚣药材细粉加入20倍量人工胃液,密闭,于37℃恒温水浴作用30min后,加入4.0%胃蛋白酶水解4h,将酶解液置于85℃恒温水浴作用15min,冷却至室温,以4200r/min离心15min,取上清液,干燥得蜈蚣胃蛋白酶酶解物；沉淀干燥,继加入20倍量人工肠液,密闭,于37℃恒温水浴作用30min后,加入5%胰蛋白酶水解4h,将酶解液于85℃恒温水浴作用15min,冷却至室温,以4200r/min离心15min,取上清液,干燥得蜈蚣胰蛋白酶酶解物。(2)经Sephadex G-50、SephadexG-25两次凝胶过滤色谱分离后得到一个组分F3-3,显示了很强的抗凝活性,F3-3经过C18反相色谱柱进一步纯化后,得到4个组分,其中第二组F3-3-2具有很强的抗凝效果,但并不是单一组分；(3)分离出第三组分F3-3-3,经HPLC和MAILDIA-TOF-MASS质谱法分析,为较纯物质但抗凝效果不明显。结论：仿生酶解法适于提取蜈蚣抗凝血和溶栓的有效部分,综合应用凝胶和C18反相柱可有效分离蜈蚣胃蛋白酶酶解物中的抗凝血组分,MAILDIA-TOF-MASS质谱显示,蜈蚣中的抗凝物质是小分子肽类,本研究揭示蜈蚣中起抗凝血作用的物质并非某一单一成分而是分子量在某一范围的肽类混合物。更多还原

【Abstract】 Objective:To study the process condition of bionic enzymatic hydrolysis of Centipede,explore the effective parts of anticoagulation of Centipede Hydrolysates. Further separation and analysis of its component structure to provide a reasonable basis for the pharmaceutical preparationsMethod:(1) Extraction Process:Taken activated partial thromboplastin time (APTT) and fibrinogen plate assay as the index, Comparison of bionic enzymatic hydrolysis, water extraction and alcohol precipitation, alcohol extraction and alcohol precipitation and water boiling, selected the extraction method of centipede; taken activated partial thromboplastin time (APTT) and fibrinogen plate assay as the index too, further Comparison of pepsin Pepsin hydrolysis method, trypsin hydrolysis method, Bionic enzymatic hydrolysis method and Combined enzymatic hydrolysis methods, Select the best hydrolysis method. Single factor explored the process condition of Centipede’s extraction with hydrolysis degree as the index,to determine the best conditions of bionic enzymatic hydrolysis. (2) anticoagulant peptide separation and analysis:Taken activated partial thromboplastin time (APTT) as the index, separate the anti-coagulate active components from the pepsin enzymolysis liquid of Centipede by SephadexG-50, SephadexG-25 gel filtration chromatography and C18 reversed phase chromatography, and supplemented by high performance liquid chromatography, MAILDIA-TOF-MASS mass spectrometry, gel electrophoresis and thin layer chromatography methods for analysis.Results:(1) Bionic enzymatic hydrolysis is the best extraction method for Centipede, The best extracting technique of them as follows:Centipede was firstly hydrolyzed for 4hours in an artificial gastric liquid at 37℃which contained 4.0% pepsin (pepsin to substrate). Then the residue was hydrolyzed for 4 hours in artificial intestine liquid at the same temperature and contained 5.0% trypsin (trypsin to substrate) (2) A component F3-3 can separated by SephadexG-50 and SephadexG-25 gel filtration chromatography, showed strong anticoagulant activity. It can further purified by C18 reversed phase chromatography and get four components, the second Fraction F3-3-2 has a strong anticoagulant effect, but it is not a single component; (3) In addition,the third component F3-3-3 is a relatively pure material, but the anticoagulant effect is not obvious.Conclusion:The bionic enzymatic method is suitable for extracting the effective parts of anticoagulation and thrombolysis.Used gel and C18 reversed phase column can effectively separate the anti-coagulate active components from the pepsin enzymolysis liquid;MAILDIA-TOF-MASS MS show, the anticoagulant of centipede is a small molecule peptide,and they are not a single substance,but some Peptide in the context of a certain molecular weight.更多还原