| Aronia melanocarpa(Michx.)Elliott(A.melanocarpa)is a perennial deciduous shrub from the Rosaceae family.A.melanocarpa berry is rich in anthocyanins and have a variety of biological activities,such as lowering blood sugar and lipid,improving alzheimer's disease,protecting the liver,preventing cardiovascular and cerebrovascular diseases.Modest supplement of A.melanocarpa berry have an benefit effect on promoting health.Type 2diabetes mellitus(T2DM)is a chronic metabolic disease.A variety of complications are cause by the chronic high glucose,which bring a great threat to human health.Liver is one of the major target tissues of insulin,and plays a vital role in stabilizing plasma glucose levels via regulation of hepatic glucose utilization or production.Hepatic insulin resistance contributes to hyperglycaemia and glucose intolerance,and becomes a principal component in the pathogenesis of type 2 diabetes.Therefore,ameliorating hepatic insulin resistance may constitute an effective therapeutic strategy for regulating glucose homeostasis and T2DM.In this study,the extraction and purification process of anthocyanins from A.melanocarpa were optimized and the main composition of A.melanocarpa anthocyanins were also identified.The effects of A.melanocarpa anthocyanin extracts(AMAE)on hepatic insulin resistance and its mechanism at the molecular level in high fat diet(HFD)and streptozotocin(STZ)-induced T2DM rats were studies in this paper.The main results are as following:(1)Ultrasonic assisted extraction method was used to extract anthocyanins from A.melanocarpa.According to the results of single factor experiment,the Box-Behnken design in response surface analysis method was used to optimize the extraction process conditions of anthocyanins from A.melanocarpa The optimal parameters were obtained:ethanol concentration was 63%,extraction time was 32min,extraction temperature was 42?,solid-liquid ratio was 1:20,and the extraction yield of anthocyanins reached 5.61 mg/g under the optimal conditions.Comparing and analyzing the effect of four different types of macroporous resin on the purification of A.melanocarpa anthocyanins,D101 macroporous resin was selected as the purified resin.The optimal conditions was determined as follows:sample concentration was 1.2 mg/mL,adsorption flow rate was 1 mL/min,maximum sample amount is 375 mL,desorption solution was 60%ethanol,desorption flow rate was 1 mL/min and desorption solution amount was 250 mL.The purified A.melanocarpa anthocyanin extracts under these conditions was black purple powder,and the anthocyanins content was254.73 mg/g.Four anthocyanins,five flavonoids,and three phenolic acids were identified in A.melanocarpa anthocyanin extracts by HPLC-DAD-ESI-MS~2.Anthocyanins were predominant among the detected compounds,with cyanidin-3-galactoside exhibiting the highest content(55.09%).The flavonoids in the extract were mainly consisted of quercetin derivative,although the content was much lower than that of anthocyanins.The content of phenolic acid was second to that of anthocyanin,which was mainly composed of neochlorogenic acid,chlorogenic acid and caffeic acid.(2)Rats were fed with HFD and intraperitoneally injected with 35 mg/kg STZ to induce type2 diabetic rat model.The rats were supplemented with AMAE at doses of 100 and 400 mg/kg body weight(bw)daily for 8 weeks.AMAE improved the increase of food intake,water intake and excretion and the decrease of body weight gain;effectively reduced blood glucose,insulin and lipid content;mitigated oxidative stress and inflammatory response;alleviated insulin resistance;and had a protective effect on the liver in T2DM rats.(3)AMAE significantly increased hepatic glycogen content and key glycolytic enzymes activities,including hexokinase(HK),glucokinase(GK),pyruvate kinase(PK),decreased key gluconeogenic enzymes activities(phosphoenolpyruvate carboxykinase(PEPCK)and glucose-6-phosphatase(G6Pase)),reduced triglyceride(TG),total cholesterol(TC)and Free fatty acids(FFA)levels,decreased reactive oxygen species(ROS)generation,improve glutathione(GSH)content,reduced the damage of lipid,protein and DNA induced by free radicals and inhibited the expression of interleukin-1?(IL-1?),IL-6 and tumor necrosis factor-?(TNF-?)in the liver of T2DM rats.Moreover,AMAE markedly increased the phosphorylation levels of insulin receptor substrate-2(IRS-2),phosphoinositide 3-kinase(PI3K),protein kinase B(Akt),glycogen synthase kinase-3?(GSK-3?),elevated the mRNA and protein expression of glucose transporter 2(GLUT2)and peroxisome proliferators-activated receptors?(PPAR?)and promoted the phosphorylation of AMP-activated protein kinase?(AMPK?).These results indicated that AMBE exhibited a hyperglycemic effect in HFD/STZ-induced T2DM rats,as evidenced by improving glucose utilization,promoting hepatic glycogen synthesis and inhibiting gluconeogenesis.Moreover,AMAE reduced lipid accumulation and oxidative stress along with inflammation in the hepatic tissues of T2DM rats,and activated the PI3K/Akt,GLUT2,PPAR?and AMPK signaling pathway,which might contribute to improved hepatic insulin resistance. |
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