Research On Construction And Adsorption Mechanism Of Cyanidin Anthocyanin Molecular Imprinting Recognition Receptor

Excessive free radical production or reduced scavenging ability in human body are the potential cause of human disease and aging.Anthocyanin is a kind of natural pigment similar to flavonoid and widely existing in plants,which is rich in phenolic hydroxyl and can effectively remove free radicals in human body.It has become the most promising natural antioxidant in the field of food and medicine.However,the traditional separation process of anthocyanin is complicated and its structure is easy to be destroyed,which leads to the decrease or even loss of its biological activity,consequently becoming a bottleneck problem restricting its further research and development.In this work,anthocyanin as the research object,based on the integration of the advantages of magnetic nanoparticles（rapid separation）,covalent organic frameworks（high adsorption capacity）and molecularly imprinted polymers（selective recognition）,novel nano separation materials for rapid separation,efficient adsorption and specific identification of anthocyanin are developed,aiming to promote the in-depth study of anthocyanin structure-activity relationship and its industrialization.The main research contents are as follows:In view of the disadvantages of the traditional separation methods,such as unsatisfactory separation effect,long separation period and extremely low product yield,the surface molecular imprinting technology combined with magnetic separation technology was used to construct the adsorption medium with specific recognition and high separation efficiency for cyanidin-3-O-rutinoside（C3R）for the first time.Double bonded functional magnetic carriers were prepared by introducing double bonded groups on the surface of silanized magnetic nanoparticles,and then the imprinted layers were coated on the surface of magnetic carriers by double-bonded polymerization.Rutin with similar structure to C3R was selected as the dummy template to solve the problem of instable anthocyanin.Non-molecularly imprinted polymer library screening method was adopted to screen suitable polymerization systems with high throughput.The adsorption capacity and selectivity coefficient to quercetin of prepared dummy template magnetic surface molecularly imprinted polymers（DMMIPs）were 15.69 mg/g and15.49,respectively.The adsorption kinetic time was 80 min,and the magnetic responsiveness was good.Through shape selectivity,π-πinteraction and hydrogen bonding,DMMIPs realized the specific recognition and enrichment of C3R in water phase.The purity of C3R in crude extract of Lonicera caerulea was increased to 89.16%.The recovery was 82.34%.The whole process only needed 3 h.This method simplifies the complicated separation and purification steps of anthocyanin and provides a new idea for the separation of anthocyanin.Aiming to overcome the shortcoming of low adsorption capacity of surface molecularly imprinted polymers,magnetic covalent organic framework molecularly imprinted polymers（MCMIPs）linked byβ-ketoenamine were synthesized by fusion of molecularly imprinted polymers（MIPs）and covalent organic frameworks（COFs）.Primarily,the amino-functional magnetic carrier was prepared by doping 1,6-hexamethylenediamine in magnetic nanoparticles,and then the COFs with imprinted holes was formed on the magnetic carrier through Schiff-base reaction.The introduction of COFs with porous structure and largeπelectron system provided more recognition sites and enhancedπ-πinteraction,which significantly increased the adsorption capacity of MIPs（126.71 mg/g）by about 7 times compared to DMMIPs.It was much higher than that of macroporous resin XAD-2（0.67 mg/g）reported in the paper.The rigid skeleton structure of COFs can effectively prevent the collapse of molecularly imprinted cavities and ensure the accuracy of spatial configuration of recognition sites.The novelβ-ketoenamine-linked MCMIPs can be synthesized under mild conditions to ensure the structural integrity of C3G to a large extent,which can replace the dummy template molecularly imprinted strategy and provide a new strategy for imprinting of thermosensitive/photosensitive substance.The experimental results showed that MCMIPs had excellent selectivity（the coefficient selectivity for quercetin of 5.95）,adsorption reversibility（elution rate of 98.68%）and good stability,which could be reused.With the help of MCMIPs,C3G with a purity of more than 88%was obtained from two kinds of berries of black chokeberry and bilberry after one separation,which has a great application prospect in the field of separation and purification of anthocyanin.In order to solve the problem of low affinity and selectivity between biomimetic receptor MIPs compared with natural recognition system,hydrophilic multifunctional monomer covalent organic framework molecularly imprinted polymers（HMCMIPs）were prepared.p-aminobenzene sulfonic acid,2,6-diaminoanthraquinone and calcium ion were applied as functional monomers to form electrostatic interaction,π-πinteraction,hydrogen bond and chelation with different parts of the template molecule C3G.Various types of interaction synergically enhanced the interaction force between the template molecule and the functional monomer,thus improving selectivity and affinity.The binding constant of HMCMIPs to C3G was as high as 3.33×10⁶ M^-1,which is four orders of magnitude higher than the reported binding constant of MIPs(4.71×10²M^-1),and even two or three orders of magnitude higher than the binding constant of natural receptor recognition lectin(10³～10⁴ M^-1).The selectivity of HMCMIPs was about 6 times higher than that of MCMIPs（the selection factor for quercetin of 36.67）,and the adsorption capacity of HMCMIPs was up to 1566.11 mg/g,making a breakthrough.In addition,a new functional modification method“functional module embedding method”was proposed in this study,which realized the integration of synthesis and modification,simple operation and extended the diversity of COFs.The functional module in this method acts as“terminator”to truncate the growth of material.By adjusting the termination rate and elongation rate,the material morphology is precisely controlled,and the problem of slow mass transfer rate is overcome.It only took 2 h to obtain 93.72%purity C3G with a recovery of 97.09%.The high selectivity of HMCMIPs can effectively improve the separation efficiency,shorten the separation time,avoid the loss caused by irreversible adsorption of stationary in multiple separations,and improve the recovery rate,which has great application potential.This study will lay a theoretical foundation for further research and industrialization of anthocyanin.