Study On The Chemical Components Of Total Flavonoids Extract From Pueraria Lobate(Willd.) Ohwi And Its Intervention On Obesity

Flavonoids are a kind of natural plant nutrients with important biological activities.Their strong antioxidant biological activities are related to their biological functions such as lowering blood pressure and blood lipid.Obesity is a kind of metabolic disease,which is affected by many factors such as heredity,metabolism,environment,physiology and so on.It is closely related to more than 20 kinds of medical diseases,such as type 2 diabetes,hypertension,cardiovascular disease and so on.Most methods of weight loss have the disadvantages of large side effects and high recurrence rate.It is a feasible method to find the natural products from the natural active products to prevent the occurrence and development of obesity safely and effectively.There are a lot of flavonoids in Pueraria lobate(Willd.)Ohwi,a traditional Chinese medicine with the same source of medicine and food.Therefore,it is of great significance to extract flavonoids from Pueraria lobate(Willd.)Ohwi,and study its intervention on obesity.The flavonoids in Pueraria lobate(Willd.)Ohwi were extracted and purified.The intervention effect of total flavonoids extract from Pueraria lobate(Willd.)Ohwi,TFERP(TFERP)on obese mice was evaluated by establishing fat mice model induced by high fat diet.HepG2 cells were incubated with oleic acid(OA)to establish ahepatocyte steatosis cell model to evaluate the effect of TFERP on lipid deposition of hepatocytes,and the effect of autophagy on lipid deposition of TFERP was studied.The main research contents and results are as follows:1.Ultrasonic assisted extraction was used to extract flavonoids from Pueraria lobate(Willd.)Ohwi.TFERP was purified by D101 macroporous adsorption resin.The contents of puerarin,daidzein,3’-hydroxypuerarin and 3’-methoxypuerarin in TFERP were determined by HPLC.Finally,the antioxidant capacity of TFERP was determined by DPPH,ABTS and total reduction force experiment.The results showed that the content of total flavonoids in Pueraria lobate(Willd.)Ohwi was 9.54 ± 0.26%,the total flavonoids purity of TFERP was 89.46±0.67%,The contents of,3’-hydroxypuerarin,puerarin,3’-methoxypuerarin,daidzein of TFERP were 16.18±0.19,182.20±0.96,61.31±0.38 and 69.18±0.53mg/g.TFERP scavenged DPPH and ABTS in antioxidant experiment the IC50 values of free radicals were 0.18mg/ml and 0.16mg/ml.In the concentration range of 0.01-0.40mg/ml,the reduction capacity of Fe3+ by TFERP raised with the increase of concentration.The antioxidant results showed that TFERP had favourable antioxidant capacity.2.A model of obesity mice induced by high fat diet was established.Ten weeks of TFERP intervention with high(800mg/kg),medium(400mg/kg)and low(200mg/kg)dose groups,the effects of TFERP on body weight,Lee’s index,serum lipid index,liver lipid content,histopathological sections and liver related antioxidant indexes were studied.After 10 weeks of TFERP intervention,it was found that TFERP could significantly decrease the food utilization rate,weight gain rate,Lee’s index,fat coefficient,the size of fat cells,reduce the content of TG,TC,LDL-C in serum,the accumulation of TG,TC in liver improving the level of AST and ALT in liver.In addition,TFERP can restore the activities of GSH,SOD and CAT in mice liver,reducing the content of MDA,ameliorating the oxidative stress state in mice and the fatty degeneration of liver induced by high fat diet,800mg/kg TFERP works best.3.MTT method was used to select the appropriate OA concentration to establish the HepG2 cell lipid deposition model,screening the safe concentration range of TFERP and puerarin(Pur).Oil red O staining was used to observe the effect of TFERP and Pur on the lipid accumulation in HepG2 cells induced by OA,and to further detect the content of TC and TG in HepG2 cells.Finally,Western blot was used to detect the expression levels of three autophagy related proteins LC3-II,LC-I and P62,and to study the relationship between the TFERP and Pur regulate function of liver cell lipid deposition and autophagy.Screening out the OA molding concentration was 1 mM,and the safe concentrations of TFERP and Pur were 10-500μg/mL and 5-40μg/mL.After intervented with the TFERP and Pur,the number of lipid droplets in HepG2 cells reduced,the color became lighter,and the content of TC and TG in HepG2 cells decreased significantly(P<0.01),and the 165pg/mL TFERP and 30μg/mL Pur work best.TFERP and Pur can significantly increase the LC3-Ⅱ/Ⅰ ratio(P<0.01),decreasing the P62 protein content(P<0.01),increasing the autophagy level to improve the oleic acid-induced lipid deposition of hepatocytes,and TFERP works better than Pur.In conclusion,TFERP plays a role in obesity intervention by improving the oxidative stress state of mouse liver and up regulating autophagy level,and it can be used as a new potential drug for the prevention of obesity and non-alcoholic fatty liver.