Study On The Efficacy Of Tanshinone ⅡA Cyclodextrin Inclusion Complex On Hyperlipidemic Zebrafis

Purpose:First,determine the optimal preparation process for the inclusion complex,and then apply the prepared tanshinone ⅡA-β-cyclodextrin inclusion complex to the zebrafish with hyperlipidemia,and evaluate and analyze the hypolipidemic effect of the drug.The purpose is to provide some reference for the prevention and treatment of diseases related to hyperlipidemia,the research of pathogenesis,and the screening and evaluation of high-throughput hypolipidemic drugs.Methods:1.The inclusion complexes of tanshinone ⅡA-β-cyclodextrin were prepared by saturated aqueous solution method,freeze-drying method,ultrasonic method and grinding method,respectively.The optimal method was determined according to the yield.The single factor influence experiments were carried out from the aspects of inclusion time,temperature and mass ratio.The orthogonal experimental method was used to optimize the experimental conditions and determine the optimal inclusion conditions for the inclusion complex.The inclusion complex was characterized by UV spectroscopy,infrared spectroscopy,and fluorescence spectroscopy.Finally,the solubility of the inclusion complex was determined.2.Healthy embryos were selected 6 hours after fertilization(6hpf)and treated with different concentrations of tanshinone ⅡA(2μM,4μM,8μM,16μM)and tanshinone ⅡA-β-cyclodextrin inclusion complex(2μM,4μM,8μM,16μM)for the study,and the drug exposure lasted until 96 hpf.The morphological and developmental changes of zebrafish embryos were observed using a biological microscope at 24 hpf,48hpf,72 hpf,and 96 hpf,respectively.The toxicity of tanshinone ⅡA and tanshinone ⅡA-β-cyclodextrin inclusion complex was compared by using hatching rate,survival rate and malformation rate as evaluation indicators.3.Establish a hyperlipidemia zebrafish model and apply the tanshinone ⅡA-β-cyclodextrin inclusion complex to the hyperlipidemic zebrafish.The hypolipidemic effect of the tanshinone ⅡA-β-cyclodextrin inclusion complex is evaluated according to the changes in body length,body width,and body weight,and the variations in the contents of four biochemical indicators TC,TG,LDL-C and HDL-C of the zebrafish.Results:1.The inclusion complex prepared by the saturated aqueous solution method had the highest yield,and the optimal inclusion conditions were 3 hours of inclusion time,55 ℃ of inclusion temperature,and a mass ratio of 1:4 between tanshinone ⅡA andβ-cyclodextrin.The solubility of the inclusion complex was 24.5 times higher than that of tanshinone ⅡA alone.2.Tanshinone ⅡA and its inclusion complex had almost no effect on the hatching rate and survival rate of zebrafish embryos at concentrations of 2-16 μM.However,the tanshinone ⅡA alone had a certain effect on the teratogenicity of embryos with increasing drug concentration,and there was a potential toxic reaction which affected the normal development of embryos.At the same concentration,the inclusion complex has greater safety than tanshinone ⅡA.3.A zebrafish hyperlipidemia model can be established by feeding 6dpf juvenile fish with a high-fat diet containing 5% cholesterol for two consecutive weeks.The body weight and body width of hyperlipidemic zebrafish were significantly increased(P<0.01)and extremely significantly increased(P<0.001)compared with the control group,respectively.Compared with the control group,the levels of TC,TG,and LDL-C in the juvenile fish were significantly increased(P<0.001),while the levels of HDL-C were significantly reduced(P<0.01).After exposure to different concentrations of inclusion complex solutions(2.5 μM,6.5 μM,10.5 μM),the body width and body weight of the hyperlipidemic model group were improved to some extent.Additionally,there were significant differences in the four indicators of TC,TG,LDL-C,and HDL-C,showing a dose-dependent pattern.Conclusion:The tanshinone ⅡA-β-cyclodextrin inclusion complex prepared by the saturated aqueous solution method has better water solubility that is significantly improved compared with tanshinone ⅡA.More importantly,the inclusion complex is a compound with lower toxicity than tanshinone ⅡA,and it has a certain lipid-lowering effect on the hyperlipidemic zebrafish.