【作者】 陈玉琼；

【导师】 吴谋成；

【作者基本信息】 华中农业大学 ， 农产品加工与贮藏工程， 2007， 博士

【摘要】 藤茶为我国特有茶用、药用植物，富含二氢杨梅素等黄酮类物质。有关藤茶的研究主要集中在藤茶活性成分的分离鉴定及黄酮抗菌、抗氧化、降血糖作用等方面。本文在对藤茶饮用的安全性进行评价的基础上，以活性为跟踪，采用二次正交旋转组合设计法研究了藤茶主要活性成分黄酮及二氢杨梅素的提取条件，比较了不同藤茶原料黄酮含量及其活性的变化，筛选了利用大孔树脂纯化藤茶黄酮的分离条件，对藤茶水提物、黄酮及二氢杨梅素的降血脂作用及其机理进行系统研究，以期为藤茶的开发应用提供理论基础。1藤茶黄酮提取条件的研究在单因素实验的基础上，采用二次回归正交旋转组合设计方法研究了温度、时间、料液比、乙醇浓度对藤茶黄酮和二氢杨梅素（DHM）提取率、黄酮纯度及其对O₂^·-自由基清除率的影响。结果表明，温度、时间、料液比极显著影响藤茶黄酮及二氢杨梅素的提取率，建立的回归模型显著性检验达极显著水平，回归模型的预测值与实测值能较好地拟和。结合黄酮的纯度和对O₂^·-自由基清除率活性分析，藤茶黄酮的最佳浸提条件：乙醇浓度在-0.8～1水平（67％～85％），浸提温度在0～1水平（65℃～77.5℃），浸提时间在-1～0.3水平（60min～100min），料液比在0～1水平（1∶30～40）。根据二氢杨梅素的提取率和含量分析，其最佳提取条件为：提取温度在-0.8～-0.03水平（55℃～65℃），时间在-0.1～0.1水平（86min～93min），料液比在0～0.5水平（1∶30～35），乙醇浓度在0.5～-0.1水平（74％～80％）。2不同藤茶原料黄酮含量及活性的研究2．1生育期的影响。对藤茶不同嫩度以及新梢不同部位原料的黄酮、二氢杨梅素含量及黄酮清除O₂^·-自由基的活性进行分析，表明随着原料的老化，黄酮含量及其清除O₂^·-自由基的能力呈下降趋势。二氢杨梅素的变化趋势与黄酮基本相同。与叶片相比，藤茶梗中黄酮和二氢杨梅素的含量最低，但活性却相对较高。提示一方面藤茶中的活性黄酮主要集中在叶片，尤其是生育旺盛的部位积累最多，另一方面茎梗与叶片黄酮的组成可能存在较大差异。2．2栽培条件的影响。分别对栽培型、野生型、仿野生型藤茶的黄酮和二氢杨梅素含量以及清除O₂^·-自由基的活性进行分析。结果表明，黄酮和二氢杨梅素含量以及清除O₂^·-自由基的活性大小依次为野生型＞仿野生型＞栽培型。提示藤茶生长条件的改变已对体内生理生化变化产生了较大影响。2．3产地的影响。分析了全国主要藤茶产地湖南、湖北、广东、广西、江西、福建等地所产藤茶原料中黄酮、二氢杨梅素含量及其活性变化。结果表明，以广东连南、福建武平、江西定南所产藤茶的黄酮及二氢杨梅素含量最高，湖南桑植、茶陵、恩施来风所产藤茶的黄酮及二氢杨梅素含量相对较低。各地藤茶黄酮清除O₂^·-自由基的能力与其含量的相关性不明显（r=0.2769），但与二氢杨梅素的含量呈极显著的正相关（r=0.8330）。由此说明，藤茶黄酮的活性主要与其组成有关，而二氢杨梅素是黄酮活性的主体成分。2．4叶型的影响。对大、中、小三种不同叶型的藤茶黄酮进行分析，表明黄酮和二氢杨梅素含量高低依次为中叶＞小叶＞大叶，黄酮清除O₂^·-自由基的活性大小依次为小叶＞中叶＞大叶，但小叶和中叶型藤茶黄酮的活性变化差异不显著。说明在进行品种选育时应以中、小叶型藤茶为主。3藤茶中黄酮的分离纯化及结构鉴定选用11种大孔树脂对藤茶黄酮进行了静态吸附与解吸附实验。结果表明，1号树脂适宜于藤茶黄酮的分离纯化，对黄酮的吸附量可达41.41mg／g，解析率达到89.2％。通过动态吸附实验对1号树脂有关参数进行了优化。结果表明，黄酮上样浓度6-7mg／ml，上样体积为柱体积的1／3，流速1.8ml／min，用95％的乙醇进行洗脱，可达到较好的洗脱与纯化效果，黄酮的洗脱率可达90％左右，洗脱出的黄酮纯度达65％左右。进一步利用重结晶方法得到纯度为98.7％的纯品，经紫外可见光谱，红外光谱、LC-Ms分析表明，黄酮中的主要成分为二氢杨梅素。4藤茶安全性毒理学实验研究根据保健食品检验与评价技术规范对藤茶安全性进行了评价。急性毒性试验结果表明，受试藤茶属无毒级；3项致突变试验结果均为阴性。大鼠90天喂养试验中，藤茶各剂量组动物生长发育良好，对体重、食物利用率无不良影响。各剂量组血常规、白细胞分类结果与对照组无显著差异。大鼠血清ALT、AST、BUN、TC、TG、CRE、GLU、ALB、TP测定结果与对照组比较无显著差异。脏器系数与对照差异不明显。肝、心、脾、肾、胃肠、睾丸、卵巢等器官外观和组织切片与对照相比均未发现实质性病理改变。说明饮用藤茶安全无毒。5藤茶提取物降大鼠及人体血脂作用研究用大鼠脂质代谢紊乱模型法研究藤茶提取液对大鼠的降血脂作用。结果表明，与高脂模型组比较，藤茶高剂量组（4.5g／Kg.bw）大鼠血清TC显著降低，高、中剂量组（3.0g／Kg.bw）血清TC下降＞10％；高、中剂量组TG显著降低，其中，高剂量组TG下降＞15％；高、中、低剂量组（1.5g／Kg.bw）HDL-C显著升高，其中，高剂量组HDL-C升高值＞0.12mmol／L。人体临床试验表明，服用藤茶组降TC有效率为42.0％，降TG有效率为72.0％，降血脂有效率为28.0％，极显著高于对照组。服用藤茶组血清TC、TG明显降低，与试用前及对照组比较差异极显著。试验组实验前后血液学指标及血生化指标检验结果无显著变化。提示藤茶提取物对大鼠及人体都有辅助降血脂的作用。6藤茶中黄酮及二氢杨梅素降血脂作用研究对藤茶中主要活性成分黄酮及二氢杨梅素对高血脂症小鼠的降血脂效果进行了研究。结果表明，与高脂模型组比较，0.5g／kg.bw灌胃（ig）剂量的二氢杨梅素能明显减轻小鼠体重；各剂量黄酮都能显著降低小鼠血清TG含量；0.3g／kg.bw ig剂量二氢杨梅素能显著降低小鼠血清TC、TG、LDL-C含量，0.5g／kg.bw ig剂量二氢杨梅素能极显著提高血清HDL-C含量；黄酮及二氢杨梅素都能明显提高小鼠血清SOD活性，降低血清MDA含量及肝系数。肝脏病理学观察表明，藤茶黄酮及二氢杨梅素能降低肝细胞变性、肿胀的程度。说明藤茶黄酮及二氢杨梅素能降低小鼠血脂，增强机体抗氧化能力，减轻高脂对肝细胞的损伤作用。7二氢杨梅素发挥生理活性的机理研究结合药代动力学原理，应用紫外吸收光谱、荧光光谱等方法对二氢杨梅素与牛血清白蛋白（BSA）相互作用机理进行了研究。结果表明，在弱碱性条件下，二氢杨梅素易解离成为阴离子，紫外吸收峰发生明显红移；在生理pH（7.4）下，BSA中加入不同浓度的二氢杨梅素后，吸收峰发生了红移。二氢杨梅素对BSA的内源荧光强度有较强的猝灭作用，属静态猝灭类型，猝灭速率常数Kq=3.97×10¹²；二氢杨梅素与BSA之间结合位点数n=1.97，结合常数K_A=5.04×10⁹。Ca²⁺、Zn²⁺、Mn²⁺、Cu²⁺金属离子的存在会使二氢杨梅素与BSA的速率猝灭常数增大，结合常数和结合位点数降低，减小了二氢杨梅素与BSA的结合，而Fe³⁺使二氢杨梅素与BSA的结合位点数和结合常数都增加，提高了二氢杨梅素与BSA的结合。说明二氢杨梅素在机体内易与生物大分子结合而发挥其活性功能，金属离子的存在直接影响二者的结合，从而影响其活性的发挥。更多还原

【Abstract】 Tengcha is one of the special plants of China for beverage or medicine use, which enriches of flavones. Much attention about the Tengcha focused on the identification of active components and its’ antioxidant, antibacterial, hypoglycemic effects, et al. The studies of this article were as follow: Conditions and parameters for extracting of flavones, dihydromyricetin（DHM） of Ampelopsis grossedentata and its activities; The contents of flavones, DHM, activities from different materials; The conditions of purifying flavones using macroporous adsorbing resins; The safety and Lipid-lowering effects of drinking Tengcha; The Lipid-lowering effects and mechanism of flavones and dihydromyricetin from tengcha. We expect these studies were beneficial to tengcha’s use.1 Extracting conditions of flavones of TengchaThe effects of extracting temperature, time, the ratio of Tengcha and water, ethanol consistency on the extracting rates of flavones, DHM, the purity of material flavones and its activities were studied. The results showed that the factors of temperature, time, the ratio of Tengcha and water had significant effects on extractions of flavones and DHM. The model of regression of flavones extracting rate was statistically significant at 1% level and was appropriate to express response variables. Considering high extracting rates, purity and activity of the flavones, the better variable levels were -0.8～1 ethanol consistency（67%-85 %）, 0～1 temperature（65℃～77.5℃）, -1～0.3 heating time （60min-100min）, 0-1 ratio of Tengcha and water（1:30-40）. The optimum conditions of extracting DHM were -0.8～-0.03 temperature （55℃-65℃）, -0.1～0.1 heating time（86min-93min）, 0-0.5 ratios of Tengcha and water（1:30-35）, 0.5-0.1 ethanol consistency（74%-80%）, according to the extracting rate of DHM and its purity.2 Contents of flavones and its’ activities of different Tengcha materialsThe contents of flavones and DHM of different ripening Tengcha materials and the abilities of scavenging the O₂^·- radicals of the flavones have been studied. The results showed that the contents of flavones and DHM, the abilities of flavones scavenging O₂^·- radicals decreased with the ripening of materials. The contents of flavones in stems were less than that of flavones in leaves, but the scavenging O₂^·- radicals effect of flavones from stems was better than that of flavones from leaves. It was revealed that bioactive flavones were mainly existed in leaves especially growing vigorously and varied in flavones compositions between leaves and stems.Conditions of cultivation effected the contents of flavones, DHM and the abilities of scavenging the O₂^·- of flavones. The tendency of the contents of flavones and DHM, the abilities of scavenging the O₂^·- of flavones were uncultivated materials＞half cultivated materials＞cultivated materials. It was revealed that conditions of living environment had great effects on physiology of Tengcha.The contents of flavones and DHM, the abilities of scavenging the O₂^·- of flavones from Hunan, Hubei, Guangdong, Guangxi, Jiangxi, Fujian were analyzed. The results showed that the contents of flavones and DHM from Guangdong Liannan, Fujian Wuping, Jiangxi Dingnan were the highest, but that from Hunan Sangzhi, Chaling, Enshi Laifeng were the lest. The abilities of scavenging the O₂^·- of flavones had litter correlation with the contents of flavones（r=0.2769）, but DMY（r=0.8330）. This revealed that the activity of flavones was related on its’ composition and DHM was the main active component.The contents of flavones, DHM from different shape leaves were different, which the middle shape leaves contained more flavones and DHM than that of large shape leaves significantly. The effects of scavenging the O₂^·- radicals of flavones from small shape leaves were the highest, that of flavones from large shape leaves were the lowest. Breed with middle or small shape leaves might be a good selected.3 Purification and structure identification of flavones from TengchaExperiments of 11 kinds of macroporous adsorbing resins adsorbing and desorbing flavones showed that number 1 was one of the most appropriate resins in all for preliminary purification of flavones, and the adsorbing capacities were 41.41mg/g, desorbing rate were 89.5%. In column of number 1, the optimum conditions were that the concentration of crude flavones was 6-7mg/ml, the volume of sample was 1/3 or 1/4 that of column, flowing velocity was 1.8ml/min, eluting solution was 95% ethanol, which led to 65% purity of the flavones and 90% desorbing rate of flavones. The flavones which was purified by recrystallization was dihydromyricetin, according to UV, IR, and LC-MS analysis.4 Toxicology assessments on safety of TengchaThe safety of water extracting of Tengcha on the animal was investigated according to the Standard of Health Food Test and Assess. Acute toxicity test on mice showed that LD50 was greater than 30g/Kg.bw. Three genetic tests showed negative results. 90 days feeding test investigated that the outer appearance, behavior, weights, organ coefficients, the indexes of hematology and biochemistry in rats have no significant difference comparing with the control group. Significant pathological changes of inherent organs were not discovered. All the results showed that drinking Tengcha was safety on Rats. 5 The Serum Lipid-Lowering Action of Water Extracting from Tengcha on Rats and HumanThe serum Lipid-lowering effects of water extracting of Tengcha was studied. The results showed that the TC level of high group （4.5g/Kg.bw） and the TG levels of both middle （3.0g/Kg.bw） and high group dropped greater than the positive control group significantly. However, HDL-C levels in the three Tengcha treated groups were higher significantly （p＜0.05）. Clinical trial showed the effective rate of lowering TC, TC, and serum lipid of Tengcha on human were 42.0%, 72.0%, 28.0%. The TC, TG level of Tengcha treated groups dropped greater than before or the control group significantly （p＜0.01）. The indexes hematology and biochemistry of the Tengcha treated groups did not change significantly. Tengcha was safe and effective on lowering rats and human serum lipid.6 The Hypolipidemic Effect of Flavones and Dihydromyricetin from TengehaThe effects of Tengcha flavones and dihydromyricetin （DHM） on hypolipidemic mice were investigated. The results showed, compared with the hyperlipidemia control group, 0.5g/kg.bw DHM reduced the weights of mice significantly; Serum TG level distinctly dropped with each dose flavones and 0.3g/kg bw DHM which also decreased serum TC、TG、LDL-C levels. 0.5g/kg.bw DHM increased serum HDL-C. Each dose of flavones and DHM enhanced the activities of SOD and reduced the content of MDA and liver coefficient. The liver tissue pathology observation showed that Tengcha flavones and DHM could lighten liver cell denatured. As results, Tengcha flavones and DHM could prevent hypolipidemia in mice and protect myocardial cells from oxidation and the liver from harm of high lipid.7 Spectroscopic Investigation of the Interaction between Dihydromyricetin and Bovine Serum AlbuminThe mechanism of interaction between dihydromyricetin （DHM） and bovine serum albumin （BSA） has been investigated by UV absorption and fluorescence spectrum. The results showed that DHM existed at anion and caused the UV absorption red shift at alkalescent condition; DHM with different concentration caused the UV absorption spectrum of BSA red shift at physiology pH7.4. DHM showed the florescence quenching on BSA. The quenching rate constant was 3.97×10¹², which suggested as static quenching process. The number of binding sites was 1.97, and the binding constant was 5.04×10⁹. Ca²⁺、Zn²⁺、Mn²⁺、Cu²⁺、Fe³⁺ could increase quenching rate constant and decrease the binding constant and binding sites between DHM and BSA, while Fe³⁺ could increase the binding constant and binding sites.更多还原