title: Screening of thrombin inhibitors from phenolic acids using enzyme-immobilized magnetic beads through direct covalent binding by ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.
abstract: Thrombin was immobilized on dynabeads®M-270 epoxy by direct covalent binding method for the first time. The enzyme coated magnetic beads were combined with ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry technique to establish a simple, rapid and reliable approach for screening thrombin inhibitors from Danshen preparation. The conjugation of thrombin to the magnetic beads was characterized using scanning electron microscope, transmission electron microscope and infrared spectroscopy, and the enzyme activity was determined by the analysis of enzyme-bead ratio and peak areas of target compounds. Several factors including amount of magnetic beads, type of elution solvent, incubation temperature and time were optimized. Additionally, two thrombin-bound compounds (protocatechuic aldehyde and salvianolic acid C) in Danshen injection were validated by conventional inhibitory assay and the IC50values were 286.11 and 66.09μg/mL, respectively. Our findings suggested that the proposed method was efficient in screening active compounds from medicinal plants.
authors.affiliation: College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036, China. Electronic address: caojun91@163.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Cao
authors.affiliation: College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036, China.
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Xu
authors.affiliation: College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036, China.
authors.forename: Xun-Gao
authors.initials: XG
authors.lastname: Liu
authors.affiliation: College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036, China.
authors.forename: Shu-Ling
authors.initials: SL
authors.lastname: Wang
authors.affiliation: College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036, China.
authors.forename: Li-Qing
authors.initials: LQ
authors.lastname: Peng
mesh_terms: D000991:Antithrombins / Q000737:chemistry / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D004365:Drugs, Chinese Herbal / Q000737:chemistry*; D062385:Hydroxybenzoates / Q000737:chemistry*; D027561:Salvia miltiorrhiza; D012997:Solvents / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D013917:Thrombin / Q000037:antagonists & inhibitors*
chemical_list: D000991:Antithrombins; D004365:Drugs, Chinese Herbal; D062385:Hydroxybenzoates; D012997:Solvents; C028972:dan-shen root extract; D013917:Thrombin; C017616:phenolic acid
keywords: Danshen injection; Magnetic beads; Phenolic acids; Quadrupole time-of-flight tandem mass spectrometry; Thrombin; Ultra high-performance liquid chromatography