Fabrication Of Zein-Based Delivery Systems For Quercetin On Investigating Its Bioactivities And Bioavailability

Recently,an abundant of functional biological active substances have been widely used in food industry,while most of hydrophobic active substances have real low solubility,stability and bioavailability limiting their application.Zein has been used as the matrix material for delivery the bioactive substances based on the capacity of self-assembly and biocompatibility to improve their bioavailability and biological value,while zein nanoparticles tend to aggregation with the poor stability under neutral condition.In this study,polysaccharide,polyphenol,or lecithin were used to regulate the zein particle surface through the interaction with zein to improve the properties of particle based on their synergistic effects,and the mechanism of quercetin biological activity was further investigated based on the characteristics of particles.The Pickering emulsions were fabricated using the prepared nanoparticles,and the digestion behavior,microstructure of delivery systems,absorption and activities change of quercetin were investigated using the in vitro digestion combined cells models to discuss the relationship between the structures of delivery systems and the bioavailability and biological activity of quercetin;further the utilization and metabolism of quercetin in systems were investigated by pharmacokinetics.The main conclusions are as follows:（1）Zein-chitosan composite colloidal particles（ZCPs）were prepared to explore the antioxidant activity mechanisms of quercetin systematically.The relationship between ZCPs and the enhancement of antioxidant activity of quercetin was investigated by inhibiting the AAPH-induced erythrocyte hemolysis and Hep G2 cell antioxidant activity.The results showed that ZCPs significantly improved the solubility and photothermal stability of quercetin and enhanced the protective effect on erythrocyte hemolysis,especially under low concentration.According to RT-PCR and Western Blot assay,free quercetin,ZCPs-Q,and ZCPs could activate the Nrf2 signal pathway combined with ARE to regulate the m RNA and protein of antioxidant enzymes to scavenge the excessive intracellular ROS for improving the antioxidant activity.（2）The effects of particle properties on the bioactive activities were investigated,which used different polysaccharide（chitosan,HTCC,and pectin）to regulate the surface properties of zein particles to obtain the nanoparticles with different particle size and charge.The cell uptake mechanism of particles and its effects on the bioactive activities were investigated used Caco-2 cell,Hep G2 cell,and RAW264.7 cell model.The results showed that different nanoparticles could enter Caco-2 cells through different endocytosis mechanism based on the characteristics of particles.Moreover,the different particles could improve the cell uptake,cell antioxidant activity,and anti-inflammatory activity based on the smaller size and higher charge.（3）The effects of different nanoparticles on the bioavailability and biological value were further investigated from the cell and animal level.A simulated gastrointestinal tract and Caco-2 cell monolayer model were applied to illustrate the relationship between the structures of particles digestion and enhancing the cell uptake,bioavailability,and biological value of quercetin.The results indicated that the structures of particles changed and formed pseudo-micelles after digestion to re-resolve quercetin to improve its bioaccessibility.Then the micelles improved the cell uptake based on the micelle structures,and then the bioavailability was improved.Meanwhile,the particles digesta enhanced the antioxidant activity before and after Caco-2 cell uptake.Besides,the pharmacokinetic study of ZCPs-Q and ZP-Q were investigated in rats using free quercetin as control,indicating that the nanoparticles improved the quercetin AUC_0-∞,and ZCPs and ZP improved for 10.4 and 5.86 times,respectively,which related to the propertied of particles.Meanwhile,the main metabolic pathways were identified by LC-MS/MS.（4）The effects of polyphenols and small molecular surfactants used to improve the properties of zein particles on the bioactive activity of quercetin were investigated to realize multifunctional bioactive substances co-delivery,which used soybean lecithin-zein-chlorogenic acid conjugate（Z-CA-L-CN）or non-covalent mixtures（Z-CA-L-MN）to prepare ternary composite colloidal particles to encapsulate quercetin.The synergy anti-aging activity and stability of quercetin with CA in different nanoparticles were investigated using H₂O₂-induced HSF cells based on the free radical theory of senescence.The results showed that different nanoparticles improved the anti-aging activity based on the synergy effect of quercetin and CA through regulating the intracellular Nrf2 and PI3K/AKT signaling pathways in H₂O₂-induced HSF cells.The presence of lecithin and Z-CA conjugate significantly improved the cellular uptake to enhance the anti-aging activity.Besides,the stability of anti-aging activity of quercetin and CA in Z-CA-L-CN significantly improved under the ultraviolet radiation（UV）and heating conditions,which provided new systems for local delivery of bioactive substances.（5）Pickering emulsions fabricated with ZCPs-Q were used to systematically study the effects of quercetin forms on the bioavailability using the combination of simulated gastrointestinal digestion in vitro and Caco-2 cell uptake and transport model,which linked the types of digesta with improving the cell absorption and oral bioavailability of quercetin.The results showed that the stability of Pickering emulsions was improved based on the formed multi-layer interface film of ZCPs-Q on the surface inhibiting the polymerization between particles and austenitic maturation.After the in vitro gastrointestinal digestion,the structures of ZCPs-Q and Pickering emulsions changed and formed micelles to dissolve quercetin,and then the bioaccessibility of quercetin was significantly improved.The micelles enhanced the cell uptake of quercetin in Caco-2,especially for ZCP-QE,which significantly improved the bioavailability of quercetin due to the combination function of mixed micelles and particles digesta through digestion-passive diffusion,from free quercetin of 2.71%to 41.22%.（6）The sub-micron Pickering emulsions were fabricated with ZP/ZP-Q using high pressure microfluidics to investigate the oil（corn oil and MCT）and quercetin position（shell or core quercetin in emulsions）on the bioavailability and biological value of quercetin using simulated gastrointestinal digestion in vitro and Caco-2 cell uptake and transport model.The results showed that the oils and quercetin position in emulsions affected the formation of mixed micelles,while corn oil enhanced the powerful micellization of quercetin and shell quercetin in Pickering emulsions benefitted the transfer into micelles,and then improved its bioaccessibility.Meanwhile,the emulsions overcame the demulsification of traditional Pickering emulsions during gastric digestion.Moreover,the mixed micelles were benefitted to improve the cell absorption related to the micelle structures and concentration,especially for LCT-ZPQ-E,which increased the bioavailability of quercetin by 69.87%.Besides,the emulsions improved the anti-inflammatory activity of quercetin with different degrees during digestion and cell transport related to the structures,which provided the emulsion-based targeted delivery system for hydrophobic nutraceuticals.In conclusion,this paper provided the ideal delivery system for oral administration and local delivery of functional bioactive substances,which could not only improve the bioavailability and biological value of hydrophobic bioactive substances specifically,but also realize the co-delivery of multiple functional substances.