Study On Screening Of Supramolecular Nano-carriers From Birch Bark And Curcumin Encapsulation Application

Pentacyclic triterpenoids,which is important functional component in birch bark,have various physiological activities such as hypoglycemic,anti-cancer,and liver protection.But existing studies have ignored the unique amphiphilic structure of pentacyclic triterpenoids,which endowed pentacyclic triterpenoids self-assembly characteristics and potential carrier functions.This project aims to excavate the components with carrier function from the birch bark and provide a new application direction for pentacyclic triterpenoids.First,by means of "ultrasonic emulsification-magnetic assisted separation",micro-nano particles with carrier function were caught from the birch bark extract,and the nanosupramolecular carrier component with self-assembly characteristics was isolated and identified as betulinic acid(BA).Then,the preparation process of BA nanoparticles was optimized,and curcuminloaded BA nanoparticles(Cur-BA NPs)were prepared;furthermore,the stability of curcumin loaded in Cur-BA NPs under different storage conditions were studied and the alcohol-induced liver cell injury model were used to evaluate the biological activity of BA nanoparticles at the cell level.The main results are as follows:(1)Using superparamagnetic iron oxide nanoparticles(SPIO)as probes,with the method of "ultrasonic emulsification-magnetic assisted separation" to catch the nano-supramolecular carrier component from birch bark extract,it is the self-assembly characteristics make it able to load SPIO.Through separation and purification,the main component was obtained,and the structure was determined by thin layer chromatography,high performance liquid chromatography,nuclear magnetic resonance hydrogen spectroscopy and carbon spectroscopy.Finally,nano-supermolecules BA with self-assembly properties were discovered.(2)The preparation process of BA NPs through single factor and orthogonal test with the variables of organic solvent,ultrasonic emulsification time,BA concentration and polyvinyl alcohol(PVA)concentration.With BA retention rate and Zeta potential as indicators,the optimization was carried out,and the best emulsification process for BA NPs was determined: dissolving BA in ethyl acetate,the optimal concentration was 10 mg/m L,the ultrasonic emulsification time was 30 s,and the PVA concentration was 2.5%.Nanoparticles prepared under this process condition have low aggregation,regular and uniform shape,average particle size was 331.942 nm,PDI = 0.281,yield of 75.033 ± 0.83%,and Zeta potential value of 24.3 ± 0.3 m V.(3)Cur-BA NPs were prepared for optimizing the process of encapsulating Cur with BA NPs.The thermal stability,storage stability,ultraviolet light stability,and metal ion stability were studied.The data showed that the best preparation conditions for Cur-BA NPs were BA concentration 10 mg/m L,ultrasonic emulsification time 30 s,and the volume ratio of Cur to BA was 30%.At this time,Cur loading in CurBA NPs was 80.10%,and the average retention rate of BA was 71.84%.When the heat treatment temperature was lower than 80?,the Cur retention rate was at least 64.98 ± 0.17%;after 6 days storage at 4?,the Cur retention rate in Cur-BA NPs was 81.12 ± 0.36%,and the free Cur retention rate was 69.20% ± 0.02%;Cur retention rate in Cur-BA NPs was 83.39 ± 0.88%,while free Cur retention rate was 18.20 ± 1.67%;after 6 h of ultraviolet light;in the range of Na+,Ca2+ ? 800 mmo L/L,200 mmo L/L ? Fe3+ ? 800 mmo L/L,Cur retention rate in Cur-BA NPs was significantly higher than that of free(p < 0.01).BA NPs significantly improved the retention rate of Cur,which could improve Cur stability to a certain extent.(4)An alcohol-induced Hep G2 human liver cancer cell damage model was constructed,high content analysis system was applied analyzing and comparing the cell DNA fluorescence intensity,Ca2+ concentration,reactive oxygen species(ROS)content levels after the action of free BA,BA NPs,and Cur-BA NPs.The protective effects of the three drugs on alcoholic damaged liver cell model were preliminarily evaluated.The results showed that Cur-BA NPs reduced the DNA fluorescence intensity,ROS level and Ca2+ concentration,significantly increased the survival rate of alcohol-induced Hep G2 cells,and its effect was better than free BA and BA NPs group,indicating that Cur-BA NPs were significantly inhibited alcohol-induced apoptosis of Hep G2 cells.