Preparation, Recognition Mechanism And Application Of Molecularly Imprinted Polymers For Bioactive Compounds Of Traditional Chinese Medicines

Molecularly imprinted polymers (MIPs) are novel functional materials with molecular recognition capability for target molecules. Since MIPs have the advantages of predetermination, specificity and practicability, steady physical and chemical properties, easy preparation, they have been extensively used in chromatogramphic separation, solid-phase extraction, biosensors, membrane separation and antibody mimic.Due to the advantages of MIPs, this dissertation is focused on the preparation and recognition mechanism of MIPs, and separation of bioactive compounds from traditional Chinese medicine by molecularly imprinted solid-phase extraction (MISPE). The detailed content is described as follows.(1) A novel method was reported for preparation of monodisperseβ-cyclo -dextrin(β-CD)-based surface molecularly imprinted microspheres (MIMs). Firstly, monodisperse crossing-linked poly(glycidyl methacrylate) microspheres (PGMA) were prepared by one-step swelling and polymerization method using monodisperse linear polystyrene seed particles as the shape template. Secondly, monodisperse functionalized poly(glycidyl methacrylate) microspheres (F-PGMA) were prepared throughβ-CD and (/or) hydroxyethyl methacrylate reaction with epoxy groups of PGMA. Finally, three kinds of monodisperse surface MIMs were prepared using ursolic acid (UA) as the template molecule, F-PGMA as the support matrix by a surface molecular imprinting technique. The pre-organization process of the monomer and the template was evaluated by UV and FT-IR. Molecular simulation was applied to optimize the conformations of UA, acrylamide (AA),β-CD and the complexes. The binding energy between the template and the monomer was calculated according to the single energies of UA, AA,β-CD and the complexes. The results of the adsorption dynamics study showed that the adsorption equilibrium was achieved quickly because a thin layer of MIPs containing recognition sites was grafted on the surface of the multiporous F-PGMA and the abtained MIMs showed good site accessibility for UA. The results of static adsorption experiments showed that these MIMs, prepared using bondedβ-CD and AA as the functional group/monomer, either separately or in combination, have shown various recognition properties. The adsorption capacities and imprinting factors of these MIMs were in the order of MIMs-1 (β-CD and AA) >MIMs-2 (β-CD) >MIMs-3 (AA), which was consistent with the order of the binding energies of the three kinds of imprinting systems. Scatchard analysis indicated there were two classes of affinity binding sites in MIMs-1 and one class of affinity binding site in NIMs-1. The adsorption curve of MIMs-1 was in good agreement with the two-site binding model. The feasibility of employing the MIMs-1 as the stationary phase of liquid chromatography to separate UA and OA was investigated. The results showed baseline separation was achieved on the imprinted column, but the non-imprinted column and C18 column could not achieve.(2) The adsorption thermodynamics of UA on MIMs-1 were investgated. The adsorption isotherms were fitted with Freundlich isotherms. The enthalpy change, entropy change and free energy change were estimated. The results indicated that the adsorption was a favourable, physical and radiative adsorption process. The adsorption capacity decreased with the increase of the solution temperature.(3) Separation of UA from Chinese traditional medicine, Ilex kudingcha C. J. Tseng, using MIMs-1 as the adsorption materials was investigated detailedly. The conditions of solid-phase extraction were optimized using sample solution, and relatively pure UA was obtained. The recoveries of UA on MIMs-1and NIMs-1 were 96.8% and 15.2%, respectively. Compared with NIMs-1, MIMs-1 had higher affinity and selectivity to UA. It shows the possibility for application of MIMs-1 as a selective adsorbent for enrichment and isolation of UA from herbs.(4) Two kinds of MIPs (PM, PO) were prepared using matrine (MT) and its structural analogue oxymatrine (OMT) as the template, respectively. The pre-organization process of the monomer and the template was evaluated by IR and 1H NMR. The results indicated that hydrogen bonds formed between the template and the monomer of the two MIPs, and the stoichiometric mole ratio of the template-monomer complexe was 1:2. The conformations of the template, the monomer and the complex were optimized by density functional theory, and the binding energy between the monomer and the template molecule was calculated. The affinity and selectivity of the two MIPs were also predicted by molecular simulation. Recongition properties of two MIPs were investigated by static adsorption experiments. The results indicated they showed good recongition properties for their own template, and relative poor for the analogues. The affinity and selectivity performances of the two kinds of MIPs consisted with the binding energies calculated by molecular simulation, which indicated molecule simulation had important meanings for understanding the recongnition mechanism of MIPs. Scatchard analysis was applied to study the recongnition mechanism of MIPs. The results of scatchard analysis indicated that there were two kinds of binding sites in each MIPs. The equilibrium dissociation constants of the lower and higher affinity binding sites of PM were more than those of PO, and the apparent maximum numbers of binding sites of the lower and higher affinity binding sites of PM were lower than those of PO for the corresponding binding sites. It indicated the adsorption capability of PM for MT was lower than that of PO for OMT.(5) Separation of MT and OMT from Sophora flavescens Ait using PM and PO as the adsorbents, respectively. The conditions of solid-phase extraction were optimized using sample solution. Relatively pure MT and OMT were obtained, respectively.(6) Two-template MIPs (PMO) were prepared using MT and OMT as the mixed template. PMO has shown good recognition ability for MT and OMT simultaneously. The distributions of recognition sites for MT and OMT were 57.71% and 66.15%, respectively, and the distribution of the cross reactivity binding sites was 23.86%. The results of MISPE indicated that MT and OMT could be separated simultaneously from Sophora flavescens Ait using PMO as the adsorbent.