Study On Effect Of Alkaloid Salt From Lotus Leaf On Preventing Of Diet-induced Obesity And Modulation Of Gut Microbiota In Mice

Obesity is a difficult problem which seriously affects the quality of human life and makes the society bear a huge burden.Most of the drugs for obesity have adverse side effects on human body.Dietary intervention with natural products from plants is a relatively healthy and safe way.In recent years,obesity has been proved to be closely related to gut microbiota,which provides a new idea for the prevention and treatment of obesity.Lotus leaf is a kind of medicinal and edible plants with abundant yield,it's rich in alkaloids,flavonoids and other active substances,and has a variety of biological effects.The extraction and purification methods of alkaloid salts from lotus leaves were studied.Then the effect of LLAS on preventing high fat diet induced obesity(DIO)in mice and its effect on gut microbiota were explored.(1)The extraction scheme of alkaloid salts from lotus leaves was optimized by single factor experiment and response surface analysis.The optimized scheme is as follows: heating temperature is 60?,ultrasonic power is 500 W,hydrochloric acid mass fraction is 0.3%,ultrasound time is 41 min,liquid-solid ratio is 27:1.Under this condition,the yield of lotus leaf alkali is(4.12±0.05)mg/g,and the error between actual value and predicted value is less than 1%.(2)Macroporous resin was used to purify crude-extracted alkaloid salts from lotus leaves.The adsorption and desorption properties of four macroporous resins,the maximum sample loading and the maximum sample flow rate were investigated.Finally,D101 macroporous resin was selected for purification,with 2BV/h as the maximum flow rate,6BV as the maximum sample loading,30% ethanol as the impurity removal solution and 70% ethanol as the eluent.The rate of purity and recovery of the purified alkaloids were 40.80% and 81.71%,respectively.(3)High-fat diet was used to induce obesity in mice.High(80mg/kg/d)and low(40mg/kg/d)doses of LLAS were used for dietary intervention in mice.The results showed that high and low dose LLAS could significantly(p<0.001)reduce the body weight of high fat diet mice(MC mice),significantly(p<0.05)reduce the fat accumulation of organs and tissues such as epididymis fat and liver,significantly(p<0.05)reduce the food intake of mice,significantly(p<0.05)reduce the content of triglyceride(TG)and low density lipoprotein cholesterol(LDL-C)in serum,significantly(p<0.05)increase the content of High density lipoprotein cholesterol(HDL-C),the content of total cholesterol(T-CHO)did not be affected,significantly(p<0.05)increased pancreatic lipase's activity;low-dose LLAS significantly(p<0.05)improved the insulin sensitivity of MC mice;high-dose LLAS significantly(p<0.05)increased liver glutathione peroxidase(GSH-Px)content,significantly(p<0.005)reduced liver malondialdehyde(MDA)content;High dose of LLAS significantly(p<0.005)decreased the levels of TNF-?,IL-6 and MCP-1 in epididymal adipose tissue of MC mice.(4)Illumina PE250 high throughput sequencing technique was used to analyze the effects of lotus leaf alkaloid salts(LLAS)on gut microbiota's abundance,diversity and composition of MC mice.The results showed that high-fat diet could decrease the richness and diversity of gut microbiota,but LLAS had no significant effect on the richness and diversity of gut microbiota in MC mice;the ratio of Firmicutes to Bacteroidetes increased significantly(p<0.005)with high-fat diet;and the ratio decreased significantly(p<0.05)with high-dose LLAS.High-dose LLAS can decrease the relative abundance of Proteobacteria significantly(p<0.05),increasing the relative abundance of Akkermania muciniphila significantly(p<0.01),decreasing the relative abundance of Desulfovibrio,and LLAS had no significant effect on the relative abundance of Lactobacillus and Bifidobacterium.LLAS can increase the relative abundance of Phascolarctobacterium,Faecalibacterium,Roseburia and Odoribacter in gut microbiota of mice fed with high-fat diet,their metabolites are short-chain fatty acids,which can indirectly act on central nervous system to regulate appetite.In conclusion,after extraction and purification,LLAS has achieved good results in the dietary intervention of obese mice induced by high-fat diet,which can effectively inhibit the excessive weight growth of MC mice,improve their sensitivity to insulin,improve the antioxidant level of their liver,and reduce the level of inflammatory factors in their adipose tissue.In addition,the dietary intervention of LLAS on MC mice can regulate the structure of gut microbiota and improve intestinal microecology.Therefore,it is speculated that the mechanism of LLAS in preventing obesity may be to inhibit pancreatic lipase,regulate the structure of gut microbiota,thereby alleviating chronic inflammatory response and inhibiting appetite.