| Objective: 1.Optimize the extraction process conditions of stevia root polysaccharide(SRP).2.Study the antioxidant activity of SRP in vitro.3.To study the improvement effect of SRP on non-alcoholic fatty liver rats.Methods: 1.Through the orthogonal experiment of the three factors of solid-liquid ratio,extraction temperature and extraction time,investigate the effects of the above three factors on the yield of Stevia rebaudiana root polysaccharide(SRP),optimize the SRP extraction process conditions,and thus improve the efficiency of SRP extraction process.2.Evaluate the antioxidant activity of stevia root polysaccharides to scavenge DPPH free radicals and hydroxyl free radicals,and the photodecolorization fluorescence recovery(FRAP)value of polysaccharides.3.After one week of adaptive feeding,62 SPF male SD rats were randomly divided into 10 normal control groups and 52 high-fat model groups.The normal group was given normal feed,and the high-fat model group was given high-fat feed.After feeding for 8 weekends,2 rats were randomly selected from the high-fat model group.After sacrifice,livers were taken and stained with oil red O sections to confirm the formation of NAFLD lesions.After successful modeling,the model group was weighed and randomly divided into 5 groups of 10 rats.Intervene for 8 weeks using the following methods:(1)normal group fed normal feed,0.9% saline(Control)gavage daily;(2)model group fed high fat feed,0.9% saline gavage daily(Model);(3)Rats are fed high-fat feed and simvastatin(1 mg/kg/d,Simvastatin)is given by gavage daily;(4)SRP low-dose group rats are fed high-fat feed and low-dose SRP by gavage daily Solution(50mg/kg/d,SRP-L);(5)SRP medium-dose group rats were fed with high-fat diet,and daily oral gavage of medium-dose SRP solution(100mg/kg/d,SRP-M);(6)The rats in the SRP high-dose group were fed with high-fat diet,and the high-dose SRP solution(200 mg/kg/d,SRP-H)was intragastrically administered daily.4.At week 17,rats were anesthetized with 10% chloral hydrate and sacrificed for anatomy.5.Observe the degree of steatosis of rat liver tissue under HE pathological section microscope.6.Take samples of serum and liver tissues,and measure TC,TG,LDL-C,HDL-C,SOD,GSH and other indicators.7.Use enzyme-linked immunosorbent assay(ELISA)to detect the changes of INS,TNF-α and IL-6 in serum.8.Western blot and Immunohistochemistry were used to determine the expression of PPAR-α,AMPK-α and SREBP-1C protein in rat liver tissue.Results: 1.Single factor test and orthogonal test results show that the best extraction conditions of SRP are solid-liquid ratio 1:30,extraction temperature 80℃,extraction time 150 min.2.The in vitro antioxidant activity of SRP showed that when the SRP concentration was 5 mg/mL,the scavenging rates of DPPH radicals and hydroxyl radicals reached 73.30% and 90.51%,respectively.When the SRP concentration is 6 mg/mL,the FRAP value is(33.172±0.184)mmol/mL.3.The observation results under HE pathological microscopy show that the treatment group can significantly improve the degree of liver tissue lesions.4.The results of animal biochemical indicators show that compared with the model group,the three SRP treatment groups can significantly reduce the levels of ALT,AST,TG,TC,LDL-C,FPG,and INS in the serum of NAFLD rats(P<0.05),significantly Increase the levels of SOD,CAT and GSH(P<0.05)in liver tissue.5.ELISA results showed that SRP also inhibited the increase of TNF-α and IL-6(P<0.05)in rats,and relieved the inflammatory environment of NAFLD rats.6.The results of Western blot and immunohistochemistry showed that the protein expression of PPAR-α and AMPK-α in the model group was significantly reduced compared with the normal group(P <0.05),and the protein expression of SREBP-1C in the model group was significant compared with the normal group Rise(P <0.05).Compared with the model group,the protein expressions of PPAR-α and AMPK-α in the three polysaccharide treatment groups increased significantly(P <0.05).Compared with the model group,the protein expression of SREBP-1C in the three polysaccharide treatment groups decreased significantly.(P <0.05).Among them,the SRP-H dose group had the most significant improvement effect on the above indicators(P <0.05).Conclusion: 1.By optimizing the extraction process conditions of SRP,the best extraction conditions of SRP are obtained,and the yield of the sugar is improved.2.SRP has better antioxidant capacity,which may improve NAFLD through antioxidant.3.SRP may improve NAFLD by up-regulating the expression of PPAR-α and AMPK-α proteins and down-regulating the expression of SREBP-1C protein. |
