| Hippophae rhamnoides L. is a traditional medicine for its high medicinal value in Tibetan and Mongolian. Our previous studies have found that flavonoid-rich alcohol extracts of seabuckthorn seed residues possessed effective regulation on the disorders of glucose and lipids metabolism. However, the mechanisms underlying these effects have remained unclear. Therefore, the present study was designed firstly to obtain higher purity of total flavonoids of seeds in Hippophae rhamnoides L.(TFSH) through optimization the extraction technology of TFSH, and then to investigate the effects and regulating mechanisms of TFSH, quercetin, and chlorogenic acid on glucose metabolism in 3T3-L1 adipocytes, which immediately followed by studying the effects and further mechanisms of TFSH on the regulation of glucose and lipid metabolic disorders in db/db diabetes mice. This research would provide theoretical basis for the resource of depth development and its main contents and conclusions are as follows:(1) Based on the flavonoids extraction technology of ethanol reflux extraction method in the lab, a response surface optimization experiment were designed to investigate the optimum extraction condition of TFSH. The results indicated the following maximum extraction efficiency of TFSH for four extractions:extraction temperature,79.0 ℃; extraction time,2.05 h; solid to liquid ratio,1:27.5; and ethanol concentration,59.37%. The highest yield of TFSH is 2.985% under actual optimum conditions. The purity of TFSH reached 85.03% after purification. Furthermore, results showed that TFSH exhibited obvious scavenging effect against DPPH and hydroxyl free radicals and inhibitory effect on proliferation of lung cancer A549 cells.(2) The fully differentiated 3T3-L1 adipocytes were obtained and the effects of TFSH and its components quercetin and chlorogenic acid on glucose consumption and their possible mechanisms were investigated.The results indicated that 400μg/mL TFSH promoted glucose consumption in 3T3-L1 adipocytes and simultaneously up-regulated the mRNA expressions of CAP, the phosphorylation of AMPK, and the mRNA and protein expressions of GLUT1, and suppressed the mRNA expressions of leptin, resistin, and TNFa. We suspected that TFSH stimulated glucose consumption partly through the activation of AMPK and CAP/Cbl pathways, up-regulted the expression of GLUT1, and suppressed the expression of adipocytokines like leptin, resistin, and TNFa.We evaluated the activities of two monomers in seabuckthorn. Data showed that chlorogenic acid and quercetin could obviously promote the glucose consumption in adipocytes. In addition, both significantly improved the glucose consumption in adipocytes under the condition of insulin stimulating. Mechanisms of experiments shows:100 μmol/L Quercetin significantly improved the mRNA and protein expressions of GLUT1 and mRNA expressions of GLUT4 and adiponectin, while lowered the mRNA expression of TNFa in some degree. We thus suspected that Quercetin stimulated glucose consumption probably through up-regulating the expression of GLUT1, and partly facilitating the expression of adiponectin and suppressing the expression of TNFa.Compared with insulin stimulating alone, the combination of 100 μmol/L quercetin with 100 nmol/L insulin increased the phosphorylation of AKT and the mRNA expressions of CAP, PPARy, adiponectin and GLUT1. We speculated that quercetin prompted glucose consumption in 3T3-L1 adipocytes under the stimulating of insulin partially by activating the CAP/Cbl and PI3K/AKT pathway, at the same time up-regulating the mRNA expressions of PPAR y, adiponectin, and GLUT1.25 μmol/L Chlorogenic acid could increase the phosphorylation of AMPK and the mRNA and protein expressions of PPARy, meanwhile up-regulate the mRNA expressions of CAP and PI3K and down-regulate the mRNA expressions of TNFa. Chlorogenic acid significantly prompted glucose consumption in 3T3-L1 adipocytes partially though activating CAP/Cb1, PI3K and AMPK pathways, increasing the expression of PPARy, and inhibiting the expression of TNFa.Similarly, compared with insulin stimulating alone, the combination of chlorogenic acid with 100 nmol/L insulin increased the phosphorylation of AMPK, the mRNA and protein expressions of PPARy, GLUT4 and GLUT1, and the mRNA expressions of CAP, GLUT4, and adiponectin. Therefore, chlorogenic acid increased glucose consumption in 3T3-L1 adipocytes partly by activating the AMPK and CAP/Cbl pathways and raising the expressions of GLUT1, PPARy, GLUT4, and maybe also associated with the increasing expression of adiponectin and decreasing expression of resistin.(3) The C57BL/6-db/db model was used to explore glucose and lipid metabolism of TFSH in type 2 diabetes. db/db mice of 8 weeks old continuously administrated TFSH(100 mg/kg,300 mg/kg) by drenching for 8 weeks. Compared with the db/db mice, db/db mice had a significant decrease in body weight, showing the effect of TFSH in increasing weight controlling and blood lipid modulating. The reductions in fasting blood glucose and GSP exhibited the function of TFSH in modulating blood glucose. Moreover, the increased insulin secretion and HOMA-ISCI and decreased islet area also indicated that TFSH could improve insulin sensitivity and the function of islet beta cells. In the same time, the increase in adiponectin secretion and reduces in leptin secretion and HOMA-IR possibly indicated that TFSH could increase insulin sensitivity and improve insulin resistance.In summary, TFSH and the two monomers all significantly influenced the important factors’expression in the process of glucose metabolism in 3T3-L1 adipocytes. TFSH also put up remarkable regulation on blood glucose and lipid disorders in type 2 diabetic mice. These results could consequently promote the clinical applications of TFSH, quercetin and chlorogenic acid in a near future. |
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