| Catechins are the major active components in green tea with a good application prospect for their good resistance to oxidation and inhibition activity against tumor cells.Catechins are prone to oxidation and polymerization to reduce their biological activity and limit their application in various fields due to the molecular structure with polyhydroxyl groups.Beta-lactoglobulin??-LG?is the main component of whey protein in milk and can be used as a good embedding material for bioactive substances.Applying ?-LG to embed catechins to prepare the nano-formulation of catechins can effectively improve the stability of the catechins and bioavailability.In present study,four different catechin-?-LG nanoparticles including EGCG-NPs,ECG-NPs,EGC-NPs and EC-NPs have been prepared,and the structure-activity relationship between the nanoparticles and the difference of proliferation inhibitory activity against different tumor cell lines,the proliferation inhibition mechanism of EGCG-NPs against A375 human melanoma cells,and the inhibition effect of EGCG-NPs on the tumors born by mice have been investigated.The main results were listed as follows:1.With different molar ratio of catehins/?-LG,four nanoparticles EGCG-NPs,ECG-NPs,EGC-NPs and EC-NPs were prepared at pH 7.0 under temperature 85?.With the increasing of molar ratio,the size and zeta potential of nanoparticles increased,the embedding rate and load capacity of catechins decreased;with a molar ratio of catechins/?-LG of 32:1,four nanoparticles with the zeta potential-30?-34 mV and mean diameter 30-35 nm were prepared.The nanoparticles exhibited satisfied spherical shape.2.The basic parameters of the catechin-nanoparticles closely associated with the type of catechins,i.e.the size of EGCG-NPs and ECG-NPs was smaller than EGC-NPs' and EC-NPs';the zeta potential of them were bigger than EGC-NPs' and EC-NPs';and the embedding rate and loading amount of EGC-NPs and EC-NPs were more than those of EGCG-NPs and ECG-NPs.The nano-formulation of catechins could improve the antioxidant ability of catechins.3.EGCG,ECG and EC combined to ?-LG produced fluorescence quenching effect,and the quenching belong to a static process with thermal release.The thermodynamic parameters and molecular docking showed that the ?S values of the process of all catechins combined to P-LG were positive,while the ? H values were negative,which suggested that the major forces were hydrophobic effect,hydrogen bond and electrostatic interactions between catechin molecules and ?-LG,the binding energy of EGCG and ECG to ?-LG is stronger than that of EC.4.The proliferation inhibition rate of nanoparticles on tumor cells for different type of catechins were difference,but dosage-effect and time-effect were observed.Nano-formulation of the catechins improved the proliferation inhibition rate.EGCG-NPs obviously improved the proliferation inhibition rate against A375,MSTO-211H,Hela,TE-1,SK-OV-3,C-33A and CACO-2 cells compared with native EGCG;ECG-NPs obviously improved the proliferation inhibition rate against A375,MSTO-211H,NCI-N87,Hela,TE-1,SPC-A-1,B16,SK-OV-3 and C-33A cells compared with to native ECG;EGC-NPs obviously improved the proliferation inhibition rate against MSTO-211H,B16,SK-OV-3,and C-33A cells compared with native EGC;EC-NPs improved the proliferation inhibition rate against MSTO-211H,SK-OV-3,NCI-N87 and C-33A cells compared with native EC.The improvement of proliferation inhibition rate did not display for 769-P,22RV1,4T1 and BxPC-3 cells.5.A375 human melanoma cells appeared typical apoptosis morphology after the treatment of EGCG-NPs with a dose of 40 ?g·mL-1 equivalent EGCG,though the necrosis of A375 cells was observed.The cell apoptosis was the major effect which led to the death of the tumor cells based on the detection of flow-cytometry analysis.6.Spectrophotometry detection with the corresponding reagent kits showed that EGCG-NPs increased the activity of Caspase-3 and Caspase-9 significantly.The expression of Cleaved-Caspase-3,Cleaved-Caspase-9 and Bax increased,and Bcl-2 decreased after the treatment of EGCG-NPs based the assay by Western bloting.The results demonstrated that EGCG-NPs affected the permeability of mitochondrial membrane in the cells,and prompted the release of cytochrome C and apoptosis signal,which suggested that the release of endogenous mitochondrial cytochrome C is the mechanism that EGCG-NPs induced apoptosis of A375 cells.7.The in vivo inhibition rate of EGCG-NPs against A375 tumor born by mice with dose of 10 mg EGCG·kg-1 day was 39.74%,which was 1.6 times that of EGCG. |
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