Investigation On Some New Methods Of Molecularly Imprinted Solid Phase Extraction For Active Ingredients Of Traditional Chinese Medicines

The complex matrix in traditional Chinese medicines(TCMs)increases the difficulties for the separation of active components.Molecularly imprinted polymers(MIPs)possess the excellent adsorption property and selectivity to template,and can specifically identify target compounds from the complex matrices.Therefore,molecular imprinting technology was used for extraction,separation,blood drug concentration monitoring and metabolism research of active components of TCMs,which could promote the modernization of TCMs.This thesis has carried out the following five parts of work:(1)MIPs based on polydatin were prepared by precipitation polymerization and used for extraction of polydatin from TCMs and biological samples.The prepared MIPs possessed excellent selectivity,good stability and reusability.MIPs were combined with dispersive solid phase extraction technology for the separation of polydatin from the extract of Polygoni Cuspidati Rhizoma et Radix,plasma and urine of SD rats.MIPs can effectively extract polydatin from the complex systems and realize the selective extraction of active ingredients of TCMs.(2)Magnetic molecularly imprinted polymers(MMIPs)were prepared by surface molecular imprinting method with Fe₃O₄ magnetic spheres as the support materials.MMIPs were used for the selective extraction and rapid separation of hesperetin.The best preparation conditions of MMIPs were obtained with the adsorption capacity as the index.Scatchard analysis showed that MMIPs had two kinds of binding sites(high-affinity sites and low-affinity sites),which preliminarily illustrated the existence of specific recognition sites on MMIPs.It was found that MMIPs showed high adsorption capacity and outstanding selectivity to hesperetin.Compared with MIPs,MMIPs had the advantages of easy template elution,fast mass transfer and high adsorption performance.MMIPs were successfully used to separate the hesperetin from the extract of Citrus reticulata Blanco.Meanwhile,MMIPs could be quickly separated from the solution using magnets.The selective extraction and rapid separation of active ingredients of TCMs were simultaneously achieved by MMIPs.(3)MMIPs based on phloridzin were prepared for first time and used for the extraction and blood concentration monitoring of phloridzin.Fe₃O₄ particles were prepared by solvothermal method and modified by tetraethyl orthosilicate and(3-aminopropyl)triethoxysilane.MMIPs with specific adsorption function were prepared using the modified Fe₃O₄ as magnetic materials and phloridzin as template.The appearance,core-shell structure,characteristic chemical bond,magnetization and the mass percentage of coatings of MMIPs were investigated by characterizations.In addition,the adsorption capacity,adsorption efficiency and specific recognition ability of MMIPs were studied.Finally,MMIPs were used as adsorbents for the enrichment of phloridin from the extract Malus doumeri(Bois)A.Chev and plasma of Kunming mice.The results showed that MMIPs had the outstanding specific recognition ability for phlorizin.MMIPs could reach adsorption equilibrium within 60 min and performed a high adsorption efficiency.The recovery of phloridin from the extract Malus doumeri(Bois)A.Chev reached 81.45%-90.27%by MMIPs.MMIPs were used to monitor blood concentration of phloridzin in rat?s plasma.In addition,compared with the traditional organic solvent protein precipitation method,MMIPs could extract more phloridin from plasma and reflect the blood concentration of drugs.Therefore,MMIPs are a kind of promising extraction material.(4)Deep eutectic solvent magnetic molecularly imprinted polymers(DES-MMIPs)based on baicalein were prepared with a ternary DES as the new functional monomer.DES-MMIPs were evaluated and used for extraction of baicalein.DES had a large number of groups that could generate hydrogen bond with baicalein,which was conducive to the specific recognition of baicalein by the polymers.DES usually included the binary DES and ternary DES.Compared with binary DES,the ternary DES had the advantages of low viscosity,high stability,good solubility,and more functional groups that could generate hydrogen bonds.The adsorption capacity of ternary DES-MMIPs for baicalein was higher than that of binary DES-MMIPs and MMIPs,indicating that there were the stable interactions between DES and template molecules.And the binding ability of ternary DES to template was stronger than that of binary DES.Therefore,DES-MMIPs were prepared using a ternary DES as the functional monomer.It was found that DES-MMIPs had a large specific surface area and the uniform mesoporous structures by the specific surface area test,indicating that DES-MMIPs had the ultra-high adsorption capacity and effective imprinting sites.DES-MMIPs possessed a strong specific recognize ability to baicalein and successfully separated baicalein from the extract of Scutellaria baicalensis Georgi.In addition,as a functional monomer,DES can not only improve the binding ability between MIPs and templates,but also promote the development of MIPs in green chemistry.(5)Restricted access molecularly imprinted polymers(RAMIPs)fibers were prepared for the first time and combined with solid-phase microextraction technology for the study of hesperetin in vivo metabolism.Bovine serum albumin is negatively charged and can exclude macromolecules through the electrostatic repulsion.RAMIPs fibers were prepared with bovine serum albumin as a restricted access material modifying on the surface of MIPs.RAMIPs not only have a specific recognition ability to templates,but also can effectively exclude macromolecules in biological systems and maintain a good adsorption performance.The RAMIPs fibers were successfully used to extract hesperetin and its metabolites from the liver.The concentration of hesperetin in the liver was calculated by sampling rate calibration method.After 60 min of administration,the concentration of hesperetin reached the maximum in the liver(4.87±0.43?g/g).Moreover,four metabolites of hesperetin were identified by HPLC-MS/MS,namely hesperetin-7-O-glucuronide,hesperetin-3?-O-glucuronide,eriodictyol and homoeriodictyol,respectively.RAMIPs fibers can continuously detect the changes of drugs in living animals for a long time,realizing the in vivo metabolism study of the active ingredients of TCMs.In summary,various extraction materials with high adsorption performance,high selectivity and high efficiency based on the principle of molecular imprinting technology were prepared in this paper.And extraction materials were systematically investigated and evaluated.The new methods of selective extraction,rapid separation,blood concentration monitoring and in vivo metabolism of active ingredients of TCMs were established step by step through MIPs,magnetic MIPs,new green functional monomer-MIPs and restricted access MIPs.The separation effect of active ingredients in TCMs was improved effectively.The work in this paper provides technical support for the development of medicinal ingredients of TCMs,and provides the ideas for study of mechanism and material basis of pharmacodynamics for TCMs.