Study On The Pharmacokinetic Characteristics Of Nuciferine And Its Mechanism Of Hypolipidemic Effect

Objective: Nuciferine from lotus leaf(Nelumbo nucifera Gaertn)can decompose fat tissues in the body.With diuretic and laxative effect and ameliorating hyperlipidemia,nuciferine was looked as an excellent product for regulating lipid metabolism and losing weight.As early as in ancient China,it was recorded in the literature that "taking lotus leaf makes people thin ".And the effect of nuciferine on reducing fat and losing weight has also been proved by modern medicine.Lotus leaf,as a natural plant,has the advantages of extensive sources and natural characteristics.However,the detection results of nuciferine bioavailability are significantly inconsistent,which seriously affects the clinical use of nuciferine.The purpose of our study was to clarify the reasons on the bioavailability of nuciferine and deeply reveal the mechanism of nuciferine on ameliorating hypolipidemia,and to provide scientific basis for the clinical application of nuciferine.Method: 1)To establish the quantitative detection method of nuciferine in cell culture medium,and the transmembrane transport mode and influencing factors of nuciferine in Caco-2 cells were clarified.2)Three breeds of experimental animals were given intraperitoneal injection of nuciferine at a dose of 50 mg/kg and blood concentration was detected by HPLC.The pharmacokinetics parameters were calculated by DAS 3.0 software.Meanwhile,liver microsomes from them were prepared to study the metabolism of nuciferine in vitro.3)The excretion and metabolism of nuciferine were studied by HPLC and LC/MS-IT-TOF methods,respectively.And some in vitro experiments were also carried out to explain the elimination characteristics of nuciferine.4)Hyperlipidemia models in rat were established,and q RT-PCR and Western blotting experiments were performed to detect the relative gene expression levels for lipid metabolism in rat liver and small intestine.Then,rat serum exosomes were prepared and characterized,the concentrations of miRNAs in the exosomes were also quantitatively determined by BCA kits,and interactions between miRNAs and target m RNAs were studied with the double luciferase reporter gene assay.Lastly,miRNA antagonists were used to evaluate the effect of miRNA in serum exosomes on the dyslipidemia.Results: 1)The transmembrane transport of nuciferine was capable of being markedly affected by the concentration of nuciferine and p H value of medium.Chlorogenic acid and caffeic acid significantly promoted the transport of nuciferine,while ouabain had no significant effect on it,while tetraethylamine significantly inhibited the transport of nuciferine across membranes.2)The pharmacokinetic results showed that nuciferine fitted two-compartment model in rabbits and rats,while one-compartment model in mice.The Km value of nuciferine was the lowest in mouse liver microsomes.3)The results showed that the relative excretion rate(RE,%)of nuciferine in feces after oral administration was highest,and the value in the urine was highest after intravenous injection.Then,the relative excretion rate of nuciferine in bile is also higher.And the significant metabolism/degradation existed in liver microsome and stimulated gastric fluid(SGF).4)Nuciferine was able to significantly ameliorate the blood lipid level in rats.q RT-PCR showed that nuciferine significantly induced the expression levels of PPARα,ABCA1,CYP7A1,LXRα in liver and ABCG5,CD36 in intestine,and significantly inhibited the expression of NPC1L1 in intestine.In vitro double luciferase reporter assay results showed that miRNA-764-5p,miRNA-758-3p and miRNA-126a-5p in serum exosomes had significant interaction with target gene m RNA of PPARα,ABCA1 and CYP7A1,respectively.Conclusion: 1)The transmembrane transport of nuciferine was mainly fitted with passive transport.Drug transporters(OCT)have significant influence on nuciferine transmembrane transport.2)The elimination of nuciferine was the fastest in mice and the slowest in rabbits,which was in line with the in vitro metabolism trends by liver microsome from different species.3)The oral bioavailability of nuciferine was poor and inconsistent,which was capable of being associated with poor uptake in intestine,higher intake and metabolism in hepatocyte and massive degradation in stomach.4)The effect of nuciferine on lipid metabolism in rats was related to miRNAs in the serum exosomes.These miRNAs were able to reduce lipid levels by targeting lipid metabolism genes and promoting PPARα-LXRα-ABCA1 pathway and CYP7A1 expression.It was also related to absorption and transport of cholesterol in small intestine.