Study On The Hypoglycemic Activity And Its Mechanism Of Polyphenol From Agaricus Blazei Murrill' Fruiting Bodies

Agaricus blazei Murrill?ABM?,is one of the most important edible and culinary-medicinal mushrooms,has beneficial effects on various human metabolic diseases.ABM is mostly used for the prevention or treatment of diabetes and cancer.ABM also can be used to enhance the immune system.This study optimized the extraction conditions of polyphenol from ABM by response surface methodology.The objective of this research was to investigate the protective role of polyphenol from ABM against streptozotocin?STZ?-induced diabetic rats,and further investigated its mechanism by metabolomics and genomics.This research provided possible evidence for the hypoglycemic activities of ABM,which might be better used as functional foods or a supplementary diet for diabetic patients.It also might open up new ideas for the prevention and treatment of diabetes.The main research contents and results of this research were as following:1.The extraction conditions of polyphenol from ABM were optimized by response surface methodology and Box-Behnken design.The results showed that the optimal conditions for ABM polyphenols were an extraction temperature of 72�C,extraction time of 2.5 h,and solid to liquid ratio of 1:20.Under the optimal conditions,the total polyphenols content of ABM was 2.92 mg/g,which was close to the predicted value?3.04 mg/g?.The antioxidant activities of polyphenol from ABM were analyzed using 1,1-diphenyl-2-picrylhydrazyl?DPPH?,hydroxyl radical,2,2?-azino-bis?3-ethylbenzothiazoline-6-sulfonic acid??ABTS?and scavenging assays and the reducing power using K₃Fe?CN?₆in vitro.The antioxidant activity of polyphenol from ABM indicated that the polyphenol of ABM could effectively scavenge the radical of DPPH,ABTS and hydroxyl.The EC₅₀values for polyphenol of ABM on scavenging DPPH,ABTS and hydroxyl radicals were 440?g/m L,300?g/m L and 2.84 mg/m L,respectively.And polyphenol of ABM also showed strong reducing power.The reducing power of polyphenol from ABM was 1.49 at 1.25 mg/m L.2.The effects of polyphenol from ABM on?-glucosidase inhibitory activity and improving glucose uptake by liver hepatocellular carcinoma?Hep G2?cells were investigated in vitro.The polyphenol of ABM showed inhibitory activity against the enzyme of?-glucosidase.For the?-glucosidase inhibition,the EC₅₀of ABM ethanol extract was0.95 mg/m L,and EC₅₀of ABM ethyl acetate extract was 0.27 mg/m L.ABM ethyl acetate extract showed higher inhibitory activity on?-glucosidase than ABM ethanol extract.At the concentration of 200?g/m L,ABM extracts would not damage Hep G2 cell integrity during the period of incubation.The analysis of glucose uptake by insulin resistance Hep G2 cells showed that the polyphenol of ABM had significant glucose-lowering activity in vitro?P<0.05?.3.The protective role of polyphenol from ABM against STZ-induced diabetic rats was conducted in vivo.The results showed supplementation with the polyphenol of ABM?250 mg/kg;500 mg/kg?could significantly ameliorate hypoglycemic by controlling fasting blood glucose?FBG?,insulin,insulin resistance index?HOMA-IR?level,glycated hemoglobin?Hb A1c?and glucose tolerance in diabetic rats.The evaluation of the hypolipidemic activity of polyphenol from ABM on diabetic rats showed that the polyphenol of ABM could cause a reduction of the level of aspartate transaminase?AST?,alanine transaminase?ALT?,triglycerides?TG?,total cholesterol?TC?and malondialdehyde?MDA?.In the meantime,the polyphenol of ABM also exhibited strong antioxidant activity on diabetic rats by increasing the level of superoxide dismutase?SOD?,catalase?CAT?and glutathione?GSH?,which could improve the disruption of liver lipid metabolism.In the present study,histopathology analysis results showed that the polyphenol of ABM had beneficial effects on restoration of the damaged liver and pancreatic tissues,improved pancreatic?-cell recovery,decreased glucagon level,increased insulin secretion and protection of islet cells in diabetic rats.4.Western blot and RT-PCR methods were used to investigate the molecular mechanism action of the polyphenol from ABM on diabetic rats.The polyphenol of ABM could up-regulate the gene expression level of Akt,adenine monophosphate activated protein kinase?AMPK?,insulin receptor substrate 1?IRS1?,phosphatidylinositol 3-kinase?PI3K?,glucokinase?GK?and glucose transporter-4?GLUT4?,while down-regulating the expression of glycogen synthase kinase 3??GSK3??,glucose-6-phosphatase?G6Pase?and c-jun N-terminal kinase 1 and 2?JNK1/2?.The polyphenol of ABM also showed a beneficial effect on up-regulating the protein expression level of protein kinase B?Akt?,phospho-protein kinase B?p-Akt?,glutamine synthetase?GS?and PI3K while inhibiting the expression of GSK3?.These findings demonstrated that ABM played a positive role in ameliorating hypoglycemic through activation of the PI3K and AMPK signaling pathways in STZ-induced diabetic rats.5.The high throughput sequencing technology was used to evaluate the difference of gut microbiota after treated with the polyphenol from ABM on diabetic rats.The analysis of gut microbiota indicated that at the phylum level,Firmicutes,Bacteroidetes,Spirochaetes,Proteobacteria,and Actinobacteria were the dominant bacteria in rats.Specifically,compared to diabetic rats,the population of the Bacteroidetes significantly increased and the population of Firmicutes significantly decreased in the polyphenol from ABM treated groups.Therefore,the polyphenol from ABM showed the ability to improving the abundance of gut microbiota.6.Liver samples from different treatment rats?normal rats,diabetic rats and diabetic rats fed with the polyphenol from ABM?were collected to investigate comprehensive metabolic profiling through UPLC-MS based on a metabolomics analysis method.The result showed that the level of NAD was upregulated in the oxidative phosphorylation metabolic pathway.Therefore,the energy metabolism of the organ was enhanced.The level of Glyceraldehyde-3P,2-Hydroxy-ethyl-Th PP and cis-Aconitate were up-regulated in the glucose metabolism pathway.The differential liver metabolites from diabetic rats fed basal and ABM diets related pathways including the glycolysis pathway,pentose phosphate pathway,tricarboxylic acid cycle,and oxidative phosphorylation pathway were obtained.In summary,the results showed that ABM had the activity of hyperglycemic and the underlying mechanisms were as follow:1.The polyphenol from ABM could up-regulate the gene expression level of Akt,AMPK,IRS1,PI3K,GK,and GLUT4,down-regulating the gene express level of GSK3?,G6Pase and JNK1/2.The polyphenol from ABM could up-regulate the protein expression level of Akt,p-Akt,GS,and PI3K,down-regulating the protein expression level of GSK3?;2.The polyphenol from ABM could improve the abundance of gut microbiota and effectively maintain the balance of gut microbiota in diabetic rats;3.The polyphenol from ABM could significantly interfere with the metabolism of the glycolysis pathway,pentose phosphate pathway,tricarboxylic acid cycle and oxidative phosphorylation pathway in diabetic rats;4.The polyphenol from ABM could improve the antioxidant activities in vitro and in vivo,increase the content of glycogen in the liver,and activate the PI3K and AMPK signaling pathways.