Study On Extraction And Separation Of Nuciferine And The Hypolipidemic Effect Of Lotus Leaf Extract

The lotus leaf is the leaf of the perennial herb of the water lilies of the genus Nelumbo nucifera.The biologically active substances such as nuciferine contained in the lotus leaf have various beneficial effects.The extraction,separation and purification of the nuciferine in the lotus leaf were discussed in this paper.The hypolipidemic function of lotus leaf extract had been deeply explored,and a lotus leaf functional beverage had been developed,which has certain significance and value for the development and utilization of lotus leaf.This subject firstly compared the effect of ultrasonic-assisted extraction,Soxhlet extraction and reflux extraction on the extraction percentage of the nuciferine in the lotus leaf,determined the optimal extraction method,and further optimized the solvent ethanol concentration,material-liquid ratio on the basis of above-mentioned extraction method.The extraction time and extraction temperature were also optimized to determine the optimum extraction conditions of lotus leaf.Under this condition,the nuciferine was extracted,and the D101 macroporous adsorption resin was used to the separation and purification of nuciferine for the extract of lotus leaf.The lotus leaf extract was used as an animal experimental agent to conduct in vivo experiments in mice by the method of intragastric administration.The experimental mice were divided into 7 groups:(1)blank control group,(2)blank administration group,(3)high-fat Model group,(4)Xuezhikang group,(5)low-dose lotus leaf extract group,(6)middle-dose lotus leaf extract group,(7)high-dose lotus leaf extract group.Group(1)and group(2)were fed with common diets,while group(3)-(7)high-fat diets.Blood was taken two weeks after the establishment of model,two weeks after dosing,and four weeks after dosing.TC,TG,LDL-C and HDL-C levels of serum of mice were measured in each group.The mice were sacrificed four weeks after dosing and their liver cells were taken and subjected to real-time fluorescence quantitative PCR assay to determine the expression of metabolic genes in relation to cholesterol and fatty acid in liver tissues.Finally,according to the results of animal experiments,the amount of lotus leaf extract in the lotus leaf beverage was confirmed to determine the formulation of lotus leaf beverage.The experimental result is:(1)The ultrasonic-assisted extraction was 20% higher than that of Soxhlet extraction and 74% higher than that of reflux extraction under optimal extract conditions.Therefore,ultrasonic-assisted extraction was selected and the optimal conditions for it were determined as follows: The ethanol concentration was 70%,the solid-liquid ratio was 1:25,the extraction time was 2.0 h,and the extraction temperature was 85°C.(2)The conditions for separation and purification of lotus leaf extract were determined.For the separation and purification of nuciferine by D101 macroporous adsorption resin,ethanol was used as an eluent gradient elution,and 50% to 70% volume fraction of ethanol eluate was collected and concentrated.The lotus leaf extract with 2% nuciferine could be obtained.(3)The results of animal experiments showed that the lotus leaf extract can reduce the serum levels of TC,TG,and LDL-C in mice.But there is no significant effect on levels of serum HDL-C in mice.The high-dose extract of lotus leaf had similar levels of improvement in serum TC,TG,and LDL-C levels in mice compared with Xuezhikang capsules.The lotus leaf extract had the effect of lowering blood lipids.(4)The results of real-time fluorescence quantitative PCR showed that the lotus leaf extract can significantly down-regulate the expression of protein HMGCR and protein SREBP-2 genes that affect cholesterol anabolism in mouse liver cells,and can significantly up-regulate the expression of protein LXRα and protein CYP7a1 genes that affect cholesterol catabolism to reduce serum TC levels in mice.The lotus leaf extract can strikingly down-regulate the expression of protein ACC and protein SREBP-1c genes affecting fatty acid anabolism and up-regulate the expression of protein CPT-1 gene that affects the catabolism of fatty acids to decrease serum TG levels in mice.(5)The formulas for lotus leaf beverages were determined as lotus leaf,cassia seed,hawthorn,and licorice in a ratio of 5:3:2:0.5,diluted 1:100 and added to 90℃ water.The water ought to have been kept at 90℃ for 25 min,filtered,taken filtered serum and added 5 mL/L honey,5 g/L lotus leaf extract,0.08 g/L citric acid,0.04 g/L malic acid,0.02 g/L sucralose,and 0.03 g/L stevioside.