Preparation,Cellular Uptake And Effect Of Hyperlipidemia Of Mixed Zingerone-loaded Micelles

As an important treasure house for the discovery of natural active ingredients,Chinese medicinal materials with the same food and medicine have been widely studied in recent years.Ginger is a common medicinal and edible Chinese medicinal material,which contains a variety of natural active ingredients,such as curcumin,gingerol,zingerone and so on.Among them,Zingerone（ZO）has a variety of pharmacological activities,such as resistance to radiation-induced stress,antioxidant activity,and anti-tumor activity.The low solubility of zingerone in water reduces its bioavailability and severely limits its clinical application.This subject aims to prepare zingerone nano-mixed micelles to improve its bioavailability and improve its efficacy in lowering blood lipids.In this project,zingerone was loaded onto nano-mixed micelles by the thin film dispersion method,and zingerone nano-mixed micelles（ZNMs）were successfully prepared,and its in vitro release characteristics and rat bioavailability were studied.The cellular uptake of the nano-mixed micellar carrier was evaluated by the cell uptake experiment.This project further verified the blood lipid-lowering effect of ZNMs through the in vitro free fatty acid induced Hep G2 lipid accumulation model and the in vivo high-fat diet induced hyperlipidemia model in mice.The research in this article provides basic research for the development and utilization of zingerone,with a view to expanding its clinical application.Chapter 1 ReviewThis chapter first briefly describes the research progress of hyperlipidemia,including pathogenesis,therapeutic drugs,etc.This chapter continues to review the research progress of zingerone pharmacological activity and related preparations,including physical and chemical properties,pharmacological activity and the preparation of zingerone nano preparations.It focuses on the formation mechanism,mode of action and classification characteristics of the drug-loaded nanomicelle system.This section provides a theoretical basis and direction guidance for the preparation of zingerone nano-mixed micelles.Chapter 2 Pre-prescription study of ZNMsThis chapter focuses on the pre-prescription research of zingerone.An in vitro HPLC analysis method for zingerone was established and the methodology was investigated.The methodological investigation results show that the established HPLC analysis method has strong specificity,good linear relationship（R₂=0.996）,and the precision and accuracy（RSD<2%）meet the methodological requirements.By measuring the equilibrium solubility of zingerone in different p H media（p H=6.8 PBS solution,p H=1.2 HCl solution,double-distilled water solution）,the results show that the equilibrium solubility of zingerone in water is 40.78±2.31μg/m L,and the equilibrium solubility of zingerone in water is 40.78±2.31μg/m L.The equilibrium solubility of zingerone is the smallest,which is 32.78±0.98μg/m L.The results show that the solubility of zingerone is poor.It is a poorly soluble drug and lays the foundation for the method of improving the solubility of zingerone through the micellar delivery system.Chapter 3 Preparation and in vitro evaluation of zingerone nano-mixed micellesThis chapter focuses on the preparation and in vitro evaluation of ZNMs.The zingerone drug-loaded phospholipid-sodium cholate-polyvinylpyrrolidone K30-vitamin E polyethylene glycol succinate quaternary nano-mixed micelles were prepared by the film dispersion method.The optimal prescription of nano-mixed micelles was screened by single factor and orthogonal design method,and the optimal prescription was obtained as follows:zingerone was used as the dosage of 40 mg,the total mass of fixed phospholipid and sodium cholate was 150 mg,m_phospholipid:m_{cholic acid}=2:3,the dosage of PVP K-30 is 30 mg,and the dosage of TPGS is 22 mg.The particle size of the ZNMs prepared by the best prescription is 50.62±0.25 nm,the polydispersity index is 0.168±0.006,the zeta potential is-28.07±0.33 mv,the encapsulation efficiency is94.71±2.02%,and the drug loading is 4.58±0.06%,and the stability is good.In vitro release experiments show that the cumulative release rate of ZNMs is significantly higher than that of zingerone raw materials,and the solubility of zingerone is improved.Chapter 4 Cell uptake of nano-mixed micellesThis chapter focuses on the study of the uptake efficiency and mechanism of nano-mixed micelles.Using coumarin 6 as a fluorescent probe and human hepatocarcinoma cell line（Hep G2）as a cell model,through qualitative and quantitative analysis,the uptake efficiency of Hep G2 cells to nano-mixed micelles was studied,and different inhibitors were used to Explore the cellular uptake of coumarin 6 nanometer mixed micelles.The results showed that the fluorescence intensity in the C6-NMs group was significantly higher than that of the bulk drug C6 during the same intake time,indicating that the cell uptake efficiency of C6-CNMs was significantly higher than that of the bulk drug.In addition,CPZ has a significant inhibitory effect on the cellular uptake of nano-mixed micelles,which is preliminarily determined to be uptake through clathrin-dependent cellular uptake.Chapter 5 Pharmacokinetics of zingerone and zingerone nano-mixed micelles in ratsThis chapter studies the pharmacokinetic properties of zingerone and its nano-mixed micelles in rats.First,a high-performance liquid chromatography method was established to determine the gingeron content and the method was verified.The in vivo analysis method of zingerone is highly specific,with good precision（RSD<5%）,method recovery（80%～95%）and stability（RSD<5%）.Furthermore,in order to verify that the prepared zingerone nano-mixed micelles can improve their absorption and bioavailability in vivo,this paper uses a non-compartmental kinetic model to conduct pharmacokinetic analysis in rats.The results showed that ZNMs significantly prolonged the half-life T_1/2（3.22±0.24 h vs 1.76±0.09 h）and prolonged the systemic circulation time MRT（6.26±1.04 vs 2.59±1.16）,which was about 1.83 and 2.42 times of the bulk drug,respectively.It shows that ZNMs has a sustained-release effect.The AUC_{0-24 h} of zingerone and its nano-mixed micelles were 8.79±0.21μg/m L and 44.80±0.78μg/m L,respectively.The AUC_{0-24 h}of ZNMs was significantly higher than that of zingerone.The relative oral bioavailability of ZNMs is 5.10 times higher than that of zingerone raw materials,which significantly improves the oral bioavailability of zingerone.Chapter 6 Research on the effect of zingerone and nanomicelles of zingerone on hyperlipidemiaThe research in this chapter focuses on the effects of zingerone and zingerone nano-mixed micelles on blood lipid metabolism in hyperlipidemia C57BL/6 mice and oleic acid-induced lipid accumulation in Hep G2 cells.Free fatty acids induced human liver cancer cell line Hep G2 cells for 24 hours to establish a cell steatosis model to observe the effects of different concentrations of zingerone and its nano-mixed micelles on the lipid accumulation of Hep G2 cells induced by oleic acid.Hep G2 cells were divided into blank group,model group,low-dose zingerone group,high-dose zingerone group,low-dose zingerone nano-mixed micelle group,and high-dose zingerone nano-mixed micelle group.After 24 hours of drug action,the ELISA kit measures the contents of TG,TC,LDL-C,high HDL-C,AST and ALT in the cells,and oil red O staining and Nile red staining to observe the intracellular lipid droplet deposition.Thirty-five C57BL/6 mice were randomly divided into 7 groups,5 in each group,namely blank group,model group,positive control group,low-dose zingerone group,high-dose zingerone group,zingerone nano-mixed micelle low and high dose group..The blank group was fed with basic feed,and the other groups were fed with high-fat feed for 8 weeks to establish a hyperlipidemia mouse model.At the same time,the model was given drug intervention and intragastric administration.It was administered once a day for 8 weeks,and the mice were weighed weekly.After 8 weeks of administration,blood was taken from the eyeball,and the serum was obtained by centrifugation.ELISA kit was used to detect the content of TG,TC,HDL-C,LDL-C,AST,and ALT in the plasma;the liver tissue was separated,the liver mass was weighed,and the liver was calculated.Index;HE staining to observe histopathological changes,oil red O staining to observe lipid accumulation in liver tissue.The lipid droplets of each group with different concentrations of zingerone and its nano-mixed micelles were significantly reduced in the cells,and the administration of each group could reduce the content of TG,TC,and LDL-C in Hep G2 cells,and showed a certain degree.The concentration dependence.The body weight of all mice increased,the weight gain of the model group was the largest,and the weight gain of the administration group was slower than that of the model group.Compared with the model group,the liver index,plasma AST,ALT,TG,TC and LDL-C contents of the simvastatin control group and HLF administration groups decreased,and the lipid droplets of liver cells decreased.The effect of zingerone nano-mixed micelles is more significant than that of zingerone raw materials,which shows that mixed micelle preparations not only have good in vitro properties,but also can effectively play a role in the body.