The Study Of The Preparation Of Chitosan-Egcg-Caseinophosphopeptide Nanocomplexes And Their Anti-oxidant And Anti-inflammatory Activities

Dietary polyphenols plays an important role in health care and treatment of various diseases.Epigallocatechin gallate(EGCG),the main catechin in tea,possesses many therapeutic effects such as antioxidant,anti-inflammatory,anti-tumor,hypolipidemic and so on.However,EGCG is prone to polymerization and degradation in moderately alkaline environments,resulting in instability and low bioavailability,which seriously limit the application of EGCG Numerous studies have shown that encapsulation of EGCG with nanomaterials can enhance its stability under alkaline conditions and enhance its absorption irt the gastrointestinal tract.As a food grade polymer material,chitosan has favorable biodegradability and biocompatibility.Toxicology experiments have demonstrated that chitosan has non-toxic effects to the body.These perfect properties make it the most widely used nano-carrier material for nutrients.In our previous study,EGCG is encapsulated successfully within chitosan(CS)-caseinophosphopeptides(CPP)nanocomplexes by a self-assembly method.The nanocomplexes exhibited a good sustained release effect and the CS-CPP nanocomplexes improved the intestinal permeability and absorption of EGCG Therefore,the bioavailability of EGCG was enhanced through encapsulation by CS-CPP nanocomplexes.However,the biological activity of CS-EGCG-CPP nanocomplexes have not been studied yet.In this study,small angle X-ray scattering(SAXS)was applied to investigate the interaction between CPP and CS.And stability of CS-EGCG-CPP nanocomplexes was investigated in the alkaline environment.In addition,we have studied the anti-oxidant and anti-inflammatory activity of CS-EGCG-CPP nanocomplexes.1.Preparation,characterization and stability of CS-EGCG-CPP nanocomplexesSAXS is effective in the characterization of the nanocomplexation of the digestied peptides and polysaccharide under the varying pH environments from stomach to small intestine.The change of pH value from 2.0 in stomach to 6.2 in small intestine induced the interaction between CPP and CS,forming the nanocomplexes.The structure parameters changed with the mass ratio between CS and CPP.Fractal dimensions(Df)value of domains inside CPP-CS nanocomplexes is more than 3,indicating the formation of denser particle/aggregate.Df value generally increased with the elevation of CS/CPP mass ratios.The particle size of CS-EGCG-CPP nanocomplexes increases with the elevation of CS conceltration and EGCG concentration,however decreases with the elevation of CS/CPP mass ratio.The encapsulation efficiency of EGCG in the nanocomplexes decreases with the increase of CS/CPP mass ratio and EGCG concentration.As CS concentration increases,the encapsulation efficiency of EGCG demonstrates a trend of firstly increasing and then decreasing.The CS-EGCG-CPP nanocomplexes are spherical shape and dispersed uniformly.The average particle size of the nanocomplexes is around 208 nm and the zeta potential of the surface charge(Zeta potential)of the CS-EGCG-CPP nanocomplexes is around+25.6 mV.In addition,CS-EGCG-CPP nanocomplexes can significantly(p<0.01)enhance the stability of EGCG in alkaline environment.2.Study on the anti-oxidant activity of CS-EGCG-CPP nanocomplexesIn the chemical anti-oxidant evaluation system,CS-EGCG-CPP nanocomplexes exhibited similar ability with free EGCG in reduction power,the ferric reducing anti-oxidant power(FRAP),scavenging ABTS radicals and scavenging superoxide anion radicals.Especially,they exhibit much stronger capability than free EGCG in chelating metal ions.At the cellular level,CS-EGCG-CPP nanocomplexes showed the stronger protection against oxidative damage induced by H2O2 in RAW264.7 cells than EGCG3.Study on the anti-inflammatory activity of CS-EGCG-CPP nanocomplexesRAW264.7 and Caco-2 cells were induced by LPS to establish the in vitro inflammatory models.The CS-EGCG-CPP nanocomplexes significantly inhibit LPS-induced production of nitric oxide(NO),TNF-?,IL-1? and IL-6 in the RAW264.7 cells.As for the Caco-2 cells model,CS-EGCG-CPP nanocomplexes also significantly inhibit the secretion of TNF-? and IL-8.And the CS-EGCG-CPP nanocomplexes showed stronger inhibition effect on inflammatory cytokines than the free EGCG We studied the anti-inflammatory mechanism of the CS-EGCG-CPP nanocomplexes through the western blot experiment.The CS-EGCG-CPP nanocomplexes show stronger inhibition effect on the excessive expression of iNOS protein level caused by LPS in RAW264.7 cells than free EGCG Furthermore,they are showed stronger capacity to reduce expression of phosphorylation and degradation of I?B and translocation of NF-?B p65 than free EGCG,which are mediated by NF-?B signaling pathway.Compared with the free EGCG,the anti-inflammatory activity of CS-EGCG-CPP nanocomplexes was significantly improved.