磁珠固定化凝血酶的制备及其在槐米活性化合物筛选中的应用  

Preparation of Immobilized Thrombin and Its Application in Screening and Analyzing Bioactive Compounds from Flos Sophorae Immaturus

作　　者：张波 胡瑾洁 白小琳 廖循[2] 侯梅芳 ZHANG Bo;HU Jin-jie;BAI Xiao-lin;LIAO Xun;HOU Mei-fang(The Ecological Technique and Engineering College,Shanghai Institute of Technology,Shanghai 201418,China;Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,China)

机构地区：[1]上海应用技术大学生态技术与工程学院,上海201418 [2]中国科学院成都生物研究所,成都610041

出　　处：《天然产物研究与开发》2018年第10期1798-1804,共7页Natural Product Research and Development

基　　金：中国科学院STS项目(ZSTH-007)

摘　　要：槐米是药食两用植物,有清肝明目和凉血止血之功效;其主要化学成分为黄酮类物质,具有抗氧化、抗凝血、降血压等多种生物活性。本文将凝血酶(THR)固定于氨基修饰的磁珠表面,以其为亲和固相萃取吸附剂,开发出一种快速筛选中药中凝血酶抑制剂的方法。首先合成了表面修饰氨基的磁珠,然后以戊二醛作为交联剂将凝血酶共价固定于磁珠表面得到磁珠固定化的凝血酶(MNP@THR)。利用TEM,VSM,FT-IR等对合成的固定化酶进行了表征,同时获得了优化的合成条件为:THR浓度200μg/mg,pH值7. 0,时间60 min,温度37℃为最佳合成条件,此时酶的固定化量为65μg/mg。利用MNP@THR从槐米提取物中特异性地吸附得到两个活性化合物,并通过磁分离进行前处理后,由HPLC-ESI-MS鉴定其分别为槲皮素和芦丁。通过体外酶活抑制实验测出,槲皮素具有较好的抑制活性,其半数抑制浓度IC_(50)为16. 6±0. 68μM;芦丁的凝血酶抑制活性较弱,IC_(50)值为1. 5±0. 14 m M。Flos Sophorae Immaturus is an edible and medicinal plant,which has long been used in traditional Chinese Medicines for cleaning the liver and brightening the eye and cooling the blood.It contains a large number of flavonoids which possess various biological activities such as antioxidant,anticoagulation and hypotensive.In this work,a method for fat screening of thrombin ligands from traditional Chinese medicine by using immobilized thrombin as solid phase extraction adsorbent was developed.Thrombin(THR)was immobilized on the surface of amino-modified meagnetic nanoparticles(MNP@THR)using glutaraldehyde as cross-linking agent.The immobilized enzyme were characterized by TEM,VSM,FTIR.And the optimal synthetic conditions were THR 200μg/mg,pH 7.0,35℃and 60 min,on which the thrombin immobilization is 65μg/mg.The prepared MNPs@THR could bind specifically the bioactive compounds existed in the Flos Sophorae Immaturus extracts.After magnetic separation,two active compounds extracted with MNP@THR were identified as quercetin and rutin by means of HPLC-ESI-MS.According to the enzymatic activity assay,quercertin was found to possess better inhibitory activity than rutin,with IC 50 of 16.6±0.68μM,while the one for rutin is 1.5±0.14 mM.

关 键 词：固定化凝血酶 槐米 抑制剂筛选 亲和吸附 

分 类 号：R917[医药卫生—药物分析学]