【作者】 龚受基；

【导师】 刘仲华；

【作者基本信息】 湖南农业大学 ， 药用植物资源工程， 2012， 博士

【摘要】 六堡茶始于广西梧州市苍梧县六堡乡,属于中国特有的后发酵茶----黑茶家族成员,传统消费区域为华南一带及东南亚各国。黑茶具有多种特殊保健功效,其独特的减肥降脂功能得到了消费者的认可,降血糖功效也引起了科学工作者的关注。茉莉花窨制花茶剩余的花渣,有可能作为废弃物造成环境二次污染,目前没有综合利用的报道,没有实现综合开发,其最大价值没有得到体现。六堡茶和茉莉花基础研究和应用研究都已经滞后于市场脚步,它们能够降低糖尿病小鼠血糖,挖掘六堡茶和茉莉化降血糖有效成分及其降糖机制将有助于植物资源的综合开发。本研究的主要总结如下：1、六堡茶和茉莉花化学成分分析以红外光谱法表征六堡茶,测定六堡茶化学成分红外图谱总叠加,根据化学成分各基团的红外指纹特征可以对六堡茶的陈化时间先后作出判断。六堡茶粉末和提取物的红外光谱和二阶红外光谱分析结果表明,六堡茶红外光谱有类似的光谱特征,两者光谱的相同波数峰强度相似系数均大于0.96,系统进化树图表明相近陈化时间六堡茶有较近的关系。茶叶粉末和提取物红外光谱有较大差异,它们的红外光谱和二阶红外光谱随陈化时间延长都出现了变化。六堡茶和茉莉花化学成分测定发现,六堡茶与其他黑茶成分存在共性,随陈化时间延长,多酚类、总糖含量呈减少趋势,茶黄素、茶红素、茶褐素含量呈增加趋势,茶褐素含量大幅度增加；与未发酵茶比较,化学成分含量变化较大,化学成分变化特点可能是六堡茶保健功能优异的物质基础。不同陈化时间六堡茶茶黄素含量为0.19-0.42%,茶红素含量为1.26-3.24%,茶褐素含量为9.49-16.29%。茉莉花蛋白质含量0.68%,与六堡茶相当,多酚含量高达6.84%,比六堡茶高,总糖含量为8.6%,稍低于六堡茶。2、软脂酸对HepG2细胞糖脂代谢的影响软脂酸0.4mmol/L剂量刺激HepG2细胞16h可以有效建立胰岛素抵抗模型,HepG2细胞胰岛素抵抗模型葡萄糖消耗减少,细胞中TC、TG堆积量增加。GLUT2、HL和AMPKa2基因表达降低,PEPCK和ACC2基因表达升高。二甲双胍改善HepG2细胞胰岛素抵抗模型的胰岛素抵抗症状,葡萄糖消耗量增加,细胞中TC、TG堆积量减少,糖原堆积量增加。胰岛素抵抗模型经治疗后,GLUT2、HL和AMPKa2等糖脂代谢相关基因表达上调。辛伐他汀能够改善HepG2细胞胰岛素抵抗模型胰岛素抵抗症状,细胞中TC、 TG堆积量减少,糖原堆积量增加。胰岛素抵抗模型经治疗后,GLUT2和HL基因表达上调,PEPCK和ACC2表达下调。3、六堡茶和茉莉花对高脂饮食大鼠胰岛素抵抗的影响给大鼠喂养猪油、蔗糖和蛋黄含量高的饲料8周,大鼠体重增加很快,形成肥胖大鼠模型。六堡茶和茉莉花能够降低高脂饮食大鼠体重、脂肪含量、谷草转氨酶、甘油三酯水平,升高HDL水平,降低大鼠空腹血糖和空腹胰岛素浓度,提高胰岛素敏感指数,降低葡萄糖耐量试验AUC,对高脂饮食导致的高血糖和胰岛素抵抗有改善作用。4、六堡茶和茉莉花对糖尿病大鼠胰岛素抵抗的影响高脂喂养加小剂量STZ腹腔注射大鼠可以成功建立糖尿病大鼠模型。糖尿病大鼠模型经六堡茶给药干预,大鼠食量减少,但幅度不大。实验结束,六堡茶高剂量组体重比模型组高,但幅度不大,能减少糖耐量AUC,降低糖尿病大鼠空腹血糖和空腹胰岛素水平。茉莉花各剂量组体重都减少,随着剂量增加,体重减少变慢,能够减少糖耐量AUC,降低糖尿病大鼠空腹血糖,升高空腹胰岛素水平。5、六堡茶和茉莉花对糖脂代谢机制的影响六堡茶水提物可以促进胰岛素抵抗状态HepG2细胞的葡萄糖摄取,中剂量、高剂量六堡茶促进葡萄糖吸收,改善细胞内总胆固醇含量。这种作用可能是通过增加AMPKα2和HL mRNA表达、减少ACC1、ACC2mRNA表达,调控能量代谢和加速脂肪氧化来实现。在蛋白水平上能够促进Akt ser473位点磷酸化,改善胰岛素抵抗导致的葡萄糖转运和肝糖原合成紊乱。茉莉花水提物促进葡萄糖吸收,显著降低总胆固醇含量,在基因水平上增加HL mRNA表达,降低AMPKα2、 ACC1mRNA表达,茉莉花不同剂量对ACC2mRNA表达影响不同。6、六堡茶和茉莉花改善胰岛素抵抗成分及其机制六堡茶化学成分中与减肥降脂降血糖相关成分有没食子酸、儿茶素、茶黄素、他汀类、茶多糖、茶多酚和茶褐素。渥堆过程中,儿茶素、茶多酚含量减少,茶黄素、茶褐素、他汀类含量增加,改善胰岛素抵抗化学成分很可能与茶黄素、茶褐素增加有关,也可能与各成分含量比例改变有关。六堡茶对胰岛素抵抗的改善作用是各化学成分对不同靶点共同作用的结果,通过上调AMPK表达,提高Akt活性,增加葡萄糖摄取,促进脂肪氧化来实现的。茉莉花中富含胡萝卜素、齐墩果酸、槲皮素、芦丁等黄酮及多酚类物质,对降低脂肪堆积有很好的作用,多糖对降低血糖也有很好的作用,其作用机制可能是通过增加Akt蛋白磷酸化,提高Akt活性,增加HL表达,减少脂肪合成增加脂肪分解的途径实现对糖脂代谢的调整。更多还原

【Abstract】 Liupao tea is one of Chinese post-fermented teas with unipue flavour, originating in Liubao town, Cangwu County, Guangxi, which has traditionally been drunk in Southern part of China and Southeast Asia. Dark tea, so called post-fermented tea, was proved to possess unipue healthy efficacy, especially anti-obesity, hypolipidemia, hypoglycemia effects attracting more scientists’ attention. Jasmine flower residue is left after Jasmine tea scented, which is usually regarded pollutional garbage. Comprehensive ultilization of Jasmine flower residue has not been found currently and the essential value is fully ignored. Basic and applied research in Liupao tea and Jasmine have been greatly lagging behind market demand. Liupao tea and Jasmine flower can ameliorate hyperglycemia in diabetic mice. Insulin resistance is the indispensable stage and outcome of diabetes. Research on chemical ingredients and hypoglycemia mechanism will help to highlight the ultilization of Liupao tea and Jasmine. The main results are as follows:1chemical ingredients’analysis of Liupao tea and JasmineLiupao tea was characterised by infrared spectrum without the process of chemical ingredients’ isolation of Liupao tea. The Liupao tea’s quality is concluded according to IR fingerprint of different perssads in multi-ingredients. IR spectrum and second derivative spectrum of Liupao tea power and extracts were conducted. The results showed that IR spectum of Liupao tea possesses the same characters, in which the similarity factor of peak intensity with same wave number are more than0.96. Cluster Dendrogram shows that similar aging time Liupao teas have close relationship. Difference can be found in IR spectrums of power and extract from a same tea, and also in which of powers or extracts from different aging time teas.Ingredients’analysis results show that Liupao tea shares great same constituents with other dark teas. Polyphenols and total sugar content have a reducing trend and theaflavin, Thearubigin and Theabrownin content increase with aging time extending. Alteration of chemical ingredients may be the key factor on Liupao tea healthy efficacy compared to non-fermented teas. Theaflavins in different aging time Liupao tea extracts range from0.19to0.42%, thearubigin from1.26to3.24%, theabrownin from9.49to16.29%.Jasmine extracts contain0.68%protein,6.84%polyphenol,8.6%total sugar.2the effects of palmitate on glucose and lipid’s metabolism in HepG2cellsInsulin resistant HepG2cell model is established by incubation with0.4mmol/L palmitate for16h, with glucose consumption decreasing, content of total cholesterol and triglyceride accumulation increasing, expression of GLUT2, HL and AMPKa2mRNA decreasing, expression of PEPCK and ACC2mRNA increasing.Metformin can ameliorate insulin resistance in HepG2cell model, with increasing glucose consumption and glycogen content, decreasing content of total cholesterol and triglyceride accumulation, up-regulating the expression of GLUT2, HL and AMPKa2mRNA. Simvastatin also ameliorate insulin resistance in HepG2cell model, with increasing glycogen content, decreasing content of total cholesterol and triglyceride accumulation, up-regulating the expression of GLUT2, HL mRNA, down-regulating the expression of PEPCK and ACC2mRNA.3the effects of Liupao tea and Jasmine on high fat diet-induced insulin resistance in ratsObese rat model is successfully established by feeding high fat chow containing high content of lard, sugar and yolky powder for8weeks. Body weight, content of lipid, level of AST and TG all decrease, level of HDL increases after administration of Liupao tea and Jasimine extracts in high fat diet induced obese rats. Levels of rats’ fasting plasma and insulin, AUC of glucose tolerance test also lower, while the insulin sensitivity index rises. Symptom of hyperglycemia and hyperinsulinemia induced by high fat diet are improved.4the effects Liupao tea and Jasmine on insulin resistance in diabetic ratsDiabetic rat model is successfully established by feeding high fat chow combining with intraperitoneal inject of streptozotocin. Food intake of rats has a little decrease after administration of Liupao tea extracts in the end of the experiment. Body weight of high dose group is a little heavier than which of model group, AUC of glycose tolerance test less, fasting plasma glucose and fasting insulin lower compared with model group. Three group rats of Jasmine extracts has weight loss, AUC of glycose tolerance test less, fasting plasma glucose level lower, fasting insulin level higher compared with model group.5the effect of Liupao tea and Jasmine on metabolism mechanism of glucose and lipidsLiupao tea and Jasmine extracts can improve glucose intake of insulin resistant HepG2cell and lower level of total cholesterol. This may attributes to up-regulate the expression of AMPKα2, HL mRNA, accelerating energy metabolism and lipid oxidation. Akt phosphorylation in ser473may improve glucose transport and liver glycogen synthesis. Jasmine extracts also can improve glucose intake, and lower level of total cholesterol, increase the expression of HL mRNA, reduce the expression of MPKa2,ACC1mRNA.6incredients and mechanism of ameliorating insulin resistance in Liupao tea and JasmineIngredients in Liupao tea related to antiobsity, hypolipidemia and hypoglycemia include gallic acid, catechins, theaflavins, stastins, tea polysaccharides, tea polyphenols and theabrownins. In the fermenting stage, the content of catechins and tea polyphenols reduce while the content of theaflavins, statins and theabrownins increase. The effective ingredients of ameliorating insulin resistance may be related to the increase of theaflavins and theabrownins’content, or to the alteration of ratio among different ingredients. The improvement of insulin resistance may attribute to the synergistic effect of various effective ingredients acting on multiple targets. The curative effect is through up-regulation of AMPK mRNA expression, activation of Akt, increase of glucose intake and promotion of lipid oxidation. Jasmine contains high content of flavonoids and polyphenols such as carotene, oleanolic acid, quercetin and rutin, which help to lower the accumulation of lipids. Polysaccharides in Jasmine can also lower the blood glucose level.更多还原