Preparation Of Molecular Imprinting Absorbents For Oleanane Triterpenoidal Saponins Separation In Chinese Herbs

Molecular imprinted technology(MIT)is a process to prepare the polymer which exhibits extraordinary affinity to the target molecule,a technology which is visually named as "an artificial lock" making the molecular key.The remarkable selectivity and recognition property of MIP derived from the molecular imprinted polymers(MIPs),a kind of synthetic polymers which are matched well with the target molecule by the spatial structure and binding sites.MIP has high selectivity,pre-schedvuled,recognition property,stable performance and other characteristics,and was widely used in biomimetic sensors,enzyme catalysis simulation,solid phase extraction,drug discovery and delivery and other areas.Molecularly Imprinted-Solid Phase Extraction(MISPE)is a new separation technology,which used the MIP as SPE sorbents for separation,enrichment and analysis of a specific target molecule in complex samples.MISPE not only has the advantages of SPE,but also give the MISPE better function,such as simple manipulation,selectively enriched target molecules in samples,effective removed interference components and impurities.MISPE is a powerful tool for separation in Chinese herbs.As the development of computer and the molecular mechanics,the computer molecular simulation technology has been used in molecular imprinted area and could provide a speedy and efficient guidance to the screening of functional monomer in molecular imprinted system.It could not only analysis mechanism of molecular recognition between MIP and target molecule,but also guide the rational design and preparation of MIP to reduce experimental fumble and tihe waste of material and reagents.In this paper,oleanolic acid was used as the target molecule(template).The functional monomer was screened by the computer molecular simulation technology.Then OA-MIPs was synthesized by bulk polymerization,and was characterized and evaluated by some analytical methods.Used OA-MIP as SPE adsorbents,the special affinity and high selectivity of MISPE to separate the pentacyclic triterpenoids(OA)in Chinese Herbs(Ligustrum LucidumAit)was investigated.1.Objectives and ContentsThe functional monomer was predicted by the computer molecular simulation.OA-MIPs were prepared by bulk polymerization with OA as the template and its characteristics are analysed by some analytical methods.Used OA-MIP as SPE sorbents to prepare OA-MISPE column for the separation and enrichment of OA from Ligustrum Lucidum Ait in order to established a separation methods for effective constituent in Chinese Herbs.(1)Screening functional monomer by computer molecular simulation(2)The preparation of OA-MIPs(3)The structure characterization and property evaluation of OA-MIPs(4)Application of OA-MISPE to the extraction from ligustrum lucidum ait for separation and enrichment of OA2.Methods2.1 Screening functional monomer by computer molecular simulationA computational approach using Hyperchem(?)7.5 molecular modelling software was firstly applied to predict the non-covalent interactions between OA and functional monomers.The minimum energy conformations of three commonly used functional monomers,methacrylic acid(MAA)as acidic,Acryla mide(AM)as neutral and 4-Vinyl pyridine(4-VP)as basic functional monomers,were simulated by molecular rmechanics(MM)and then refined using PM3 semi-empirical mechanic methods.Afterward,the Amber MM method was used to analyse possible interactions between template and functional monomers and to calculate their single point energies(Epolymer,Efunction,Etemplate)and binding energies(?E),so as to select the suitable monomer and thus to design for template-MIP development.2.2 The preparation,structure characterization and property evaluation of OA-MIP(1)Three kinds of MIPs were synthesized by bulk polymerization using methacrylic acid(MAA)as acidic,Acrylamide(AM)as neutral,and 4-Vinyl pyridine(4-VP)as basic functional monomers and ethylene glycol dimethacrylate(EGDMA)as a cross-linking agent,initiated thermally with azobisisobutyronitrile(AIBN).The adsorption property and imprinted mechanism of three kinds of OA-MIPs were evaluated by adsorption isotherm and adsorption kinetics test.(2)The best OA-MIP was characterized by SEM,Brunauer-Emmett-Teller(BET)and IR spectroscopy with aperture surface,surface proportion,aperture distribution,and characteristic peak as indexs.2.3 Application of OA-MISPE to the extraction for separation and enrichment of OA from ligustrum lucidum ait(1)Use OA-MIP as SPE sorbents to prepare OA-MISPE.After changing loading,washing and eluting steps,the recovery of OA in loading,washing,and eluting solution was tested by HPLC to determine the optimal condition.(2)The extraction solution from ligustrum lucidum ait was loaded on MISPE column xunder the optimal condition of the loading,washing,eluting steps and the recovery of OA in each step was tested by HPLC in order to established a new separation methods on the basis of OA-MISPE for OA from ligustrum lucidum ait.3.Results3.1 Screening functional monomer by computer molecular simulationThe minimum energy conformations of three commonly used functional monomers,methacrylic acid(MAA)as acidic,Acryla mide(AM)as neutral and 4-Vinyl pyridine(4-VP)as basic functional monomers,were simulated by Hyperchern(?)7.5 molecular modelling software.The binding energy of each compound(?E(OA-AM),?E(OA-MAA),?E(OA-4-VP))were-7.42kcal/mol,-3.53kcal/mol and-4.50kcal/mol.Among them,?E(OA-AM)was the highest,followed by AE(OA-4-VP),?E(OA-MAA)was the lowest.3.2 Preparation of OA-MIPMIPs(MIP1(MAA)?MIp(AM)?MIP3(4-VP)were prepared by bulk polymerization.The precise parameters of synthesis:0.1mmol OA,1.6mmol MAA/AM/4-VP,8.0mmol EGDMA,0.17mmol AIBN.3.3 Binding properties of OA-MIP to OAThrough the kinetics curve analysis,the adsorption of MIPI1(MAA),MIP2(AM)and MIP3(4-VP)were 57.2 ?mol/g?157.3 ?mol/g and 44.9 ?mol/g separately in the initial stages.With the increase of time,the adsorption of MIP1(MAA),MIP2(AM)and MIP3(4-VP)reached the peak value(75.4 ?mol/g,273.0 ?mol/g,145.3 ?mol/g)respectively in 20 min,2 h and 8 h.The adsorption of three MIPs begined to be down and leveling off as the adsorption time continue to increase.The adsorption of OA-MIPs to OA was significantly higher than that of NIPs,the adsorption of MIP1(MAA),MIP2(AM)and MIP3(4-VP)basically reached equilibrium at the concentration of 3.0mmol/L,4.0 mmol/L,3.5 mmol/L OA respectively.The adsorption capacity of MIP2(AM)(Q=280.98 ?mol/g)was highest,followed by MIP3(4-VP)(Q=232.18?mol/g),MIP1(MAA)(Q=141.71 ?mol/g)was the weakest.The adsorption capacity of NIPs(Q2=217.17 ?mol/g,Q3=186.64 ?mol/g and Q1=114.20 ?mol/g)were lower than those of MIPs.3.4 Structural characterization of OA-MIPThe best MIP(OA-MIP2)was characterized by SEM,Brunauer-Emmett-Teller(BET)and IR spectroscopy.The results showed that the surface of OA-MIP2 was rough and had a complex aperture structure,while NIP was smooth.It showed that all EGDMA and AM was participated in the polymerization reaction by IR.The obvious stretching vibration of amino group in 3466 cm-1 and carbonyl group in 1732 cm-1 were observed in MIP after elution of the template molecule and disappeared when MIP interacted with OA.The Brunauer-Emmett-Teller(BET)test showed that surface proportion 203.5 m2/g,the volume of total aperture 0.242 cm3/g,bore diameterl7 A-3000 A,the average of bore diameter 47.6 A(belongs to medium aperture).3.5 Application of OA-MISPE to the extraction for separation and enrichment of OA from ligustrum lucidum ait(1)With the OA standard solution as loading solution and the recovery of OA as index,the optimal condition was determined,which were loading with 1.0 mg/mL of OA standard solution,washing with 0.5 mL of toluene,and eluted with 2.0 mL×2 of methanol.(2)The method of determining the content of OA had been established by MISPE.The standard curve equation of OA:y=7944.1x-179.39(r=0.9991),which in the range of 0.2 mg/mL?1.4 mg/mL of OA,linear relationship was satisfactory.The precision of RSD was lower than 0.28%.The relative recovery of OA was 95.9%?84.3%,RSD wasl.47%?2.39%.(3)The extraction solution from ligustrum lucidum ait was loaded on MISPE column under the optimal condition of the loading,washing,eluting steps for the separation and enrichment of OA from Ligustrum Lucidum Ait and the recovery of OA reached 76.0%.4.Conclusions4.1 We combined template(OA)with functional monomers by molecular rmechanics(MM),PM3 semi-empirical mechanic and the Amber MM methods.The results showed that ?E(OA-AM)=-7.42(kcal/mol)??E(OA-MAA)=-3.53(kcal/mol)??E(OA-4-VP)=-4.50(kcal/mol).Compared with OA-4-VP and OA-MAA,which only had one hydrogen bond,the binding energy of OA-AM was the largest and it could form a stable six-member ring structure with double hydrogen bond.Consequently,the molecular imprinted system of OA-AM was the best,followed by OA-4-VP and OA-MAA was in the last place.4.2 MIPs(MIP1(MAA)?MIP2(AM)?MIP3(4-VP)were prepared by bulk polymerization.The adsorption isotherm and adsorption kinetics test showed that the adsorption of OA-MIP2 was 280.98 ?mol/g,which was 2 and 1.2 times larger than those of MIP1 and MIP3.The results of SEM and BET showed that the surface of OA-MIP2 was roughness and had a complex aperture structure,while NIP was smooth.These results show that comparing with MIP1 and MIP3,MIP2 had a special affinity and specific adsorption ability for OA.Satisfactorily,the experimental results of binding properties were in accord with the outcomes from computer simulation.An elementary research system of computer-assisted design for the preparation of MIPs for functional monomers had been established and this method was feasible,simple and effective.4.3 OA-MISPE using OA-MIP as the sorbents had specially separated and enriched pentacyclic triterpenoids(OA)under the optimal condition of the loading,washing,eluting steps from the extraction of ligustrum lucidum ait and the recovery was 76.0%.The method of determining the content of OA had been established by MISPE.Therefore,MISPE could enrich effective constituent from the extraction of Chinese Herbs.