关键词: 固定化凝血酶, 槐米, 抑制剂筛选, 亲和吸附

Abstract: 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 200 μg/mg,pH 7.0,35 ℃ and 60 min,on which the thrombin immobilization is 65 μg/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 IC₅₀ of 16.6 ± 0.68 μM,while the one for rutin is 1.5 ± 0.14 mM.

Key words: immobilized thrombin, Flos Sophorae Immaturus, inhibitor screening, affinity adsorption