In order to develop a direct and reliable method for discovering lead compounds from traditional Chinese medicines (TCMs), a comparative online ligand fishing platform was developed using immobilized capillary enzyme reactors (ICERs) in combination with liquid chromatography-mass spectrometry (LC-MS). Methacrylate-based monolithic capillaries (400 μm I.D. × 10 cm) containing epoxy reactive groups were used as support to immobilize the target enzyme acetylcholinesterase (AChE). The activity and kinetic parameters of the AChE-ICER were investigated using micro-LC-UV. Subsequently, ligand fishing and identification from mixtures was carried out using the complete AChE-ICER-LC-MS platform. For efficient distinction of true actives from false positives, highly automated comparative analyses were run alternatingly using AChE-ICERs and negative control-ICERs, both online installed in the system. After washing unbound compounds to the waste, bound ligands were eluted from the AChE-ICER to a trapping loop using a denaturing solution. The trapped ligands were further separated and identified using LC-MS. Non-specific binding to the monolith support or non-functional sites of the immobilized enzyme was investigated by exposing analytes to the negative control-ICER. The specificity of the proposed approach was verified by analyzing a known AChE inhibitor in the presence of an inactive compound. The platform was applied to screen for AChE inhibitors in extracts of Corydalis yanhusuo. Eight compounds (columbamine, jatrorrhizine, coptisine, palmatine, berberine, dehydrocorydaline, tetrahydropalmatine and corydaline) with AChE binding affinity were detected and identified, and their AChE inhibitory activities were further verified by an in vitro enzymatic inhibition assay. Experimental results show that the proposed comparative online ligand fishing platform is suitable for rapid screening and mass-selective detection of AChE inhibitors in complex mixtures.

Neuraminidase (NA) is a glycoside hydrolase that has been proposed as a potential therapeutic target for influenza. Thus, the identification of compounds that modulate NA activity could be of great therapeutic importance. The aim of this study is to develop a drug discovery tool for the identification of novel modulators of NA from both compound libraries and natural plant extracts. NA was immobilized onto the surface of magnetic beads and the inherent catalytic activity of NA-functionalized magnetic beads was characterized. Based on the enzymatic activity (hydrolysis ratio), the inhibitory activities of 12 compounds from plant secondary metabolites were screened, and the desired anti-NA activities of flavonoids were certified. Ligand fishing with the immobilized enzyme was optimized using an artificial test mixture consisting of oseltamivir, lycorine and matrine prior to carrying out the proof-of-concept experiment with the crude extract of Flos Lonicerae. The combination of ligand fishing and HPLC-MS/MS identified luteolin-7-O-β-D-glucoside, luteolin, 3,5-di-O-caffeoylquinic acid and 3,4-di-O-caffeoylquinic acid as neuraminidase inhibitory ligands in Flos Lonicerae. This is the first report on the use of neuraminidase functionalized magnetic beads for the identification of active ligands from a botanical matrix, and it sets the basis for the de novo identification of NA modulators from complex biological mixtures.

Abstract:The content of toxic trace elements is an important index to measure the quality of plastic products. For a long time, the determination of toxic trace elements is the focus of third party detection and so on. It is very important to evaluate the accuracy of detection. In this paper, a mathematical model of the uncertainty of trace elements based on regression equation, constant volume and weighing weight is established. The results show that the content of Boron and Lead in the plastic samples is (66.3 ± 5.4) mg/kg and (500 ±30) mg/kg, respectively.

The content of Chromium in plastic products is (276±6) mg/kg, which is determinated by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) using Microwave digestion. A mathematical model of the uncertainty containing variables such as regression equation, constant volume , weighing weight and so on is established.