桑叶多组分辅助降血糖作用及其对β细胞反馈调节PI-3K/Akt信号通路影响

The mulberry leaf has been used in auxiliary treatment of diabetes since ancient times. Alkaloids, flavonoids and polysaccharides are the main bioactives of mulberry leaves in hypoglycemic activities. However, the synergistic effect and the best proportion of three bioactives remain unclear as well as the underlying mechanisms. Xinjiang province possesses rich and unique germplasm resources of mulberry. While researches on mulberry leaves in Xinjiang province were limited to nutrients analysis, and purification and hypoglycemic effect of bioactives from mulberry leaves still need to be further studied. We optimized the extraction process of total alkaloids and polysaccharides extracted from Xinjiang mulberry leaves. We also revealed bioactives of mulberry leaves are different from each other due to various species and seasons, and mulberry leaves extracts showed stronger anti-oxidative activities. Based on this, total alkaloids, total flavonoids, and polysaccharides extracted from mulberry leaves will be purified and determined by the combination of solvent extraction, chromatographic separation and modern spectroscopic methods, and three bioactives will be quantitative analyzed from different species and seasons. On the other hand, the diabetic rat model will be established to investigate the synergistic hypoglycemic effect and the best proportion of three bioactives from mulberry leaves, so as to confirm the hypoglycemic effect and material basis of mulberry leaves. Moreover, the key genes and key enzymes of PI-3K/Akt insulin signaling pathway participating in the beta cell feedback regulation on glucose homeostasis regulated by mulberry leaf multi-components will be confirmed by the combination of molecular biological methods and statistical correlation analysis method. Therefore, the mechanism of auxiliary hypoglycemic effect exerted by mulberry leaves will be clarified, which will promote the development and utilization of mulberry leaves from Xinjiang province.

桑叶自古便用于“消渴病”即糖尿病辅助治疗，生物碱、黄酮及多糖是桑叶主要降血糖活性成分，三者协同作用最佳配比尚不明确，相关机制研究不够深入。新疆桑种质资源丰富独特，当地桑叶研究仅限于营养成分分析，桑叶生物活性成分分离纯化及其降血糖作用机制有待深入研究。前期已完成新疆桑叶总生物碱及多糖提取工艺优化，发现桑叶生物活性成分因品种季节而异，桑叶提取物具有较强抗氧化活性。本项目拟在此基础上，将溶剂浸提、色谱分离与波谱学方法相结合，分离纯化并定性定量桑叶总生物碱、总黄酮、多糖，系统分析其种属季节差异性。另一方面建立糖尿病大鼠模型，探讨桑叶多组分协同降血糖作用及最佳配比，明确桑叶降血糖功效及其物质基础。将分子生物学手段与统计学相关分析法相结合，确定糖尿病大鼠胰腺β细胞反馈调节PI-3K/Akt胰岛素信号通路中受桑叶多组分调控的关键基因与关键酶，明确其辅助治疗糖尿病作用机制，为新疆桑叶资源开发利用提供依据。

桑叶自古便用于糖尿病的辅助治疗。DNJ、黄酮及多糖是桑叶主要降血糖活性成分，三者协同作用最佳配比尚不明确，相关机制研究亦不够深入。新疆桑种质资源丰富独特，当地桑叶研究仅限于营养成分分析，桑叶生物活性成分分离纯化及其降血糖作用机制有待深入研究。本项目将溶剂浸提与大孔树脂等结合，分离纯化桑叶DNJ、多糖及总黄酮，并对黄酮进行组分分析；以高脂饲料联合腹腔注射链脲佐菌素建立糖尿病大鼠模型，将桑叶多组分（多糖：黄酮：DNJ=8:6:1）喂饲糖尿病大鼠，探讨桑叶多组分协同降血糖作用，明确其降血糖功效及其物质基础。确定大鼠体内PI-3K/Akt胰岛素信号通路中受桑叶多组分调控的关键基因与关键酶，明确其辅助治疗糖尿病作用机制。.研究结果发现，糖尿病大鼠体内出现明显氧化应激损伤，脂肪细胞因子及炎症因子异常高表达，而胰岛素增敏剂脂联素则表达受抑，引发胰岛素信号通路受损及下游肝脏糖代谢紊乱，表现为糖尿病大鼠胰岛素抵抗、血糖持续升高及血脂代谢紊乱。桑叶多组分干预6周后，明显减轻大鼠体内氧化应激损伤，抑制游离脂肪酸等脂肪细胞因子及炎症因子高表达，减轻体内炎症反应，降低外周效应组织的胰岛素抵抗状态；同时激活脂联素，增强胰岛素敏感性，综合发挥降血糖作用。其机制可能是：（1）桑叶上调PI-3K/Akt胰岛素信号通路关键基因PI-3K、Akt、GLUT4及PDX-1基因在mRNA和蛋白水平表达，引发下游糖代谢酶活性恢复正常：肝糖原合成酶及葡萄糖激酶表达增强，糖原合成及效应细胞对葡萄糖摄取、利用增加；糖原磷酸化酶及糖异生作用酶活性受抑，葡萄糖生成减少，大鼠血糖水平下降并接近正常水平。（2）桑叶上调PI-3K、Akt基因表达，恢复二者对下游GSK-3β基因抑制作用，解除后者对下游糖原合成酶抑制作用，促进糖原合成，减少糖原分解，发挥降血糖效应。研究结果明确了桑叶多组分降血糖作用机制，不仅对丰富祖国传统中药研究具有重要学术价值，而且将促进桑叶作为辅助降血糖功能性食品或膳食补充剂深度开发，推动桑叶产业化进程。

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