Study On The Construction Of EGCG-Loaded Nanoparticles Based On The Self-Assemblely Of Sodium Caseinate, EGCG And Gum Arabic

(-)-Epigallocatechin-3-gallate(EGCG),the major constituent of tea polyphenols,is also the most active compounds in tea polyphenols.However,poor stability and low bioavailability of EGCG greatly limit its application.Nanotechnology is an effective approach to solve this problem.Sodium caseinate(SC)and gum arabic(GA)are ideal materials for the construction of nanocarriers,and the self-assembled nanocarrier based on their interaction possesses unique advantages for the delivery of bioactive compounds.For this reason,in the present study,on the basis of research on the interaction of SC and GA along with SC,EGCG and GA,SC and GA were employed as wall materials to fabricate EGCG-loaded nanoparticles(EGCG-NP)based on the self-assembly of sodium caseinate,EGCG and gum arabic.The mechanism of EGCG-NP formation was discussed as well.Genipin(GP),a natural crosslinking agent,was introduced to crosslink EGCG-NP.The stability of GP-crosslinked nanoparticles(EGCG-GNP),its antioxidant activity as well as release in vitro were investigated further.The EGCG-GNP obtained above present many characteristics such as high drug loading content,high safety,excellent stability,energy conservation,high added-value activity and so on.The contents and results are as follows:(1)Study on the interaction between SC and GA and the construction and formation mechanism of SC-GA nanoparticles(SC-GA-NP).The results indicated that the stable SC-GA-NP were obtained with the following conditions: the concentration ratio of SC to GA was 1:1,the pH value was 4.2,the total concentration of bioploymers was 3 mg/mL and the concentration of NaCl was 10 mmol/L.The results of dynamic light scattering(DLS)and zeta potential measurements revealed that the particle size,Polydispersity index(PDI)and zeta potential value of nanoparticles formed on above mentioned conditions were about 142 nm,0.093 and-21.43 mV respectively.The resulted nanoparticles remained stable at 4℃ for 30 days and presented excellent thermal stability.The morphologies of these nanoparticles were characterized by transmission electron microscopy,which showed that the nanoparticles were spherical in shape.Differential scanning calorimetry(DSC)results indicated that the combination of GA to SC could impove the thermal stability of SC.What’s more,study also found that pH and ionic strength had a significant influence on the interaction between SC and GA and the formation of nanoparticles,suggesting that the main force of the formation of SC-GA-NP was electrostatic interaction.Futhermore,the results of fourier transform infrared spectrometer(FTIR)and fluorescence spectroscopy demonstrated that the electrostatic interaction between SC and GA to form nanoparticles occurred in the positively charged amino of SC and the negatively charged carboxyl of GA and the combination was low affinity.(2)Research on the interaction between SC,EGCG and GA along with the preparation,characterization and the formation mechanism of EGCG-NP.The results revealed that the conditions for the preparation of EGCG-NP were as follows: the concentration ratio of SC/EGCG/GA was 5:2:5(mg/m L),the pH value was 4.2,the total concentration was 1.8 mg/m L and the concentration of NaCl was 10 mmol/L.The particle size of resulted nanoparticles was about 173 nm,the zeta potential was about-19.94 mV,the polydispersity index was about 0.109,the encapsulation efficiency was about 62.82%,and the drug loading content reached up to 338.49 μg/mg.TEM reaults showed that the nanoparticles were spherical in shape.X-ray diffraction(XRD)results confirmed that the existing state of EGCG in nanoparticles was amorphous state,which was of great significance to improve the bioavailability of EGCG.According to the results of FTIR,fluorescence spectroscopy as well as CD,the mechanism of the formation of EGCG-NP was concluded as follows: Firstly,EGCG bound to EGCG with the driving force of hydrogen bonding.Subsequently,with the addition of GA,electrostatic interaction occurred between the positively charged amino of SC and negatively charged carboxyl of GA,which could inhibit the excessive combination and aggregation between SC and EGCG.The two non-colvant interactions mentioned above induced the self-assembly of SC,EGCG and GA to form EGCG-NP.The separation conditions of the nanoparticles were determined to be as follows: centrifugal speed at 12000 r/min and centrifugal time 30 min.The characterization results of redispersed nanoparticles obtained on above separation condition indicated that both deionized water and 10 mmol/L NaCl could be employed as redispersed solution of EGCG-NP and the redispersed property was excellent.According to stability results,the prepared nanoparticles had good storage stability whereas they were not stable in different pHs and simulated gastrointestinal matrixes,suggesting there existed a tendency for nanoparticles to dissociate.(3)Preparation,characterization,stability and in vitro release of EGCG-GNP.The results showed that the preparation conditions of EGCG-GNP were as follows: the concentration ratio of GP/SC/EGCG/GA was 10:5:2:5(mg/m L),the pH value was 4.2,the total concentration was 3.3 mg/mL,the concentration of NaCl was 10 mmol/L,the crosslinking temperature was 37 ℃ and the crosslinking time was 21 h.The particle size of resulted EGCG-GNP was about 206 nm,the zeta potential was about-21.63 mV,the polydispersity index was about 0.131,the encapsulation efficiency was about 63.71%,and the drug loading content reached up to 340.23 μg/mg.TEM reaults showed that the nanoparticles were spherical in shape and dispersed well.XRD results confirmed that the existing state of EGCG-GNP was amorphous state.FTIR results revealed that GP-crosslinked with the amino groups of SC and GA.The stability results showed that the prepared EGCG-GNP had good storage stability.Futhermore,compared with the non crosslinked nanoparticles,the nanoparticles crosslinked with GP tended to be more stable in different pH matrixes and simulated gastrointestinal environment,suggesting that GP crosslinking could significantly improve the stability of nanoparticles.It was found that the release rate of EGCG was about 27% and 48% in the simulated gastric fluid and intestinal fluid respectively at the first 5 h after GP crosslinking,and then increased to 32% and 56% at 24 h respectively,indicating that EGCG-GNP could achieve sustained and controlled release of EGCG.(4)Research on the antioxidant activity of GP-crosslinked SC-EGCG-GA nanoparticles in vitro.The in vitro antioxidant activity of GP-crosslinked SC-EGCG-GA nanoparticles were evaluated by DPPH free radical scavenging ability,reducing power,ABTS free radical scavenging ability and ferric reducing ability before and after illumination and heat treatment.The results showed that the encapsulation of EGCG by EGCG-GNP could protect EGCG from oxidative browning caused by light and heat,and the antioxidant activity was improved.