Preparation Of Oleanolic Acid And Construction Of Nanoparticles And Its Protrctive Effect Against CCl₄-induecd Acute Liver Injury In Mice

The liver is the main organ in the body for detoxification and metabolism,which performs important functions,such as detoxification,metabolism,secretion of proteins and retinoid storage.Acute liver injury is mainly caused by the imbalance of the production and metabolism of reactive oxygen species?ROS?,which induces oxidative stress and the disorder of redox status of cells,thus causing toxicity.Oral delivery systems can increase the bioavailability of hydrophobic substances,prepare slow-release agents to protect encapsulated drugs from the effects of p H environment,enzymatic degradation and drug efflux pumps,and target-release drugs.In order to reduce the development of physiological abnormalities such as chemical liver injury induced by oxidative stress,it is particularly important to explore new nanoparticles of traditional natural products as antioxidants to protect the liver.In this paper,the hepatoprotective active substance oleanolic acid?OA?was isolated and purified from Chaenomeles speciose,a traditional Chinese medicine,to prepare oral oleanolic acid nanoparticles?DCA NPs?to study its protective effect against CCl₄-induced acute liver injury and reveal its potential mechanism,thus providing a new strategy for the application of Chaenomeles speciose in the field of biomedicine.The method of extracting and separating oleanolic acid from Chaenomeles speciose and its technological parameters were optimized.The extraction of oleanolic acid from Chaenomeles speciose was optimized by single factor test,Plackett-Burman test and response surface orthogonal test,the best yield of oleanolic acid was 4.16 %;the separation and purification process parameters of oleanolic acid were optimized by the adsorption chromatography of fixed phase medium and mobile phase.The purity of oleanolic acid reached 54.69 % after the secondary purification by silica gel,and the purity of oleanolic acid was 90.68 % through the preparation of performance liquid phase.Preparation and structural characterization of oleanolic acid nanoparticles.The amphiphilic nanocarriers?DCS?was synthesizd by deoxycholic acid modified chito-oligosaccharide,self-assembly encapsulated oleanolic acid to form oleanolic acid nanoparticles?DCA NPs?to improve the hydrophobicity of oleanolic acid.The synthesis process of DCA NPs was optimized,and the structural characterization of DCS and DCA NPs were performed by FT-IR,1H-NMR,DLS and TEM.The results indicated that DCA NPs prepared by 3000 Da COS and 1.4mg /m L DCS by dialysis method had the best drug loading?53.61 %?and encapsulation rate?80.05 %?,COS and DA were successfully conjugated via amide bond,the particle size of DCA NPs were 239.50 nm,stable and obvious core-shell structure were observed.The hepatoprotection of DCA NPs on CCl₄ induced liver injury.The cytotoxicity of DCA NPs was studied in vitro to simulate the release behavior in gastrointestinal environment.In vivo experiment,CCl₄ acute liver injury in mice model wasestablished,through evaluating organ injury,oxidative stress regulation,liver m RNA level and western blotting analysis in 7 groups of mice,the protective effect of oleanolic acid nanoparticles?DCA NPs?on CCl₄ acute liver injury was studied.The results showed that DCA NPs had no toxic effect on Hep G2 cell line at 24 h.Meanwhile,DCA NPs played a sustained-release role in the simulated solution of gastrointestinal tract by reducing the release of OA in an acidic environment.DCS shell had no significant improvement effect on liver injury,and DCA NPs enhanced the protective effect of OA agent on liver.DCA NPs alleviated inflammatory symptoms of CCl₄ acute liver injury by inhibiting NF-?B expression and activated theNrF2-Keap1 pathway to resist oxidative stress,thus ultimately achieving a protective effect on CCl₄ acute liver injuryIn summary,this study revealed the protective effect and mechanism of DCA NPs against CCl₄-induced liver injury,and and laid a theoretical foundation for the development of a new oral delivery system for hydrophobic natural products.