【作者】 张友胜；

【导师】 杨伟丽；

【作者基本信息】 湖南农业大学 ， 茶学， 2001， 博士

【摘要】 随着现代科学技术的飞速发展及社会经济和人们生活水平的不断提高，“健康”便成了当今世界研究的主题。开发为人类健康造福的天然野生植物已被认为是世界上最有前途，最有生气的行业之一。其中，类茶植物的研究与开发尤为特别，类茶植物的产品日益受到医药行业、消费者青睐，成为全球需求的热点。 作为一种典型的类茶植物——显齿蛇葡萄(Ampelopsis grossedentata)近年来已引起了人们极大的关注。显齿蛇葡萄系葡萄科蛇葡萄属中的一种野生藤本植物，为粤蛇葡萄的变种A.cantoniesis(H．et A．)Pl．var．grossedentata Hand-Mazz．，全株药用，味甘、淡、性凉，具清热解毒、祛风湿、强筋骨等功效。自古以来，壮族和瑶族人民将其嫩幼茎叶制成保健茶，用于治疗感冒发热、咽喉肿痛、黄疽型肝炎、疮疖等症，夏天泡茶数日不馊，有“神茶”之称。该植物在湖南、湖北、江西、广东、广西、四川、云南、贵州等省份大量分布，同时易于人工栽培，在短时内扩大生产量，且在我们的系列研究中证实栽培种与野生种的主要内含成分相差不大，实属可以直接开发利用的天然野生植物资源。前人对此进行过初步的研究，目前已开发了“茅岩莓茶”、“藤茶”、“甘露茶”等初级类茶产品，但纵观所有实验，研究主要停留在对显齿蛇葡萄粗提物的药理功能研究上，既缺乏对其化学成分的系统分离、鉴定，又缺乏对其主要化学成分的提取、分离、纯化技术等方面的研究，致使该植物至今未能得到迅速、有效、合量、全面的开发与利用。 故此，本课题直接针对目前显齿蛇葡萄研究中亟待解决的几个问题，周密设计、利用薄层层析(TLC)、系列柱层析、HPLC、DSCCC等国内外研究天然野生植物活性成分中最先进的方法，并在实验中首次提出并成功采用“加温溶解保温过柱温水解吸”提制天然野生植物活性成分的新方法，同时运用UV、IR、’HNMR、‘CNMR和MS、LC-MS、GC-MS等国内外鉴定化合物结构中最先进仪器，就植物中主要黄酮类、多酚类以及挥发性化学成分的提取、分离、鉴定；DMY的RP-HPLC检测方法的建立以及植物中DMY含量的测定；提取、分离、纯化DMY的条件优选等内容进行了研究。数据可靠、结论准确。既为研究天然野生植物活性成分提供了新的方法和研究思路，又为全面、系统、合理地开发显齿蛇葡萄植物提供了大量有益的理论和实用参数。实验研究结果表明：1、用丙酮：水(1：1)溶剂回流浸提显齿蛇葡萄植物幼嫩茎叶，提取液用无水乙醚、乙酸乙酯、正丁醇依次萃取、在滤渣和乙酸乙酯浸膏部分，鉴定其中存在二氢杨梅树皮素一 （简称DW人杨梅素（简称W人懈皮素、芦丁四种对人体的生理功能起着非常重要作用的黄酮类化合物，其中懈皮素和芦丁存在于本植物为首次报到。在水相部分首次鉴定其中存在没食子酸、没食子酸乙酯、没食子阶卜卜葡萄糖三种对人体的生理功能具一定作用的多酚类化合物。2、首次建立Nova－PakCl。不锈钢柱（3．9。X 150。）作色谱柱、甲醇／水（36：64、0．1％磷酸）作流动相、二．oml／min流速、进样量为 10 ul、柱温为 30‘C、UV－250urn作检测波长的色谱分析条作下，准确快速地同时测定显齿蛇葡萄中DMY和y含量的HPLC方法，其中RSD分别为5．2 l队3．23％。结果显示：该植物系自然界中黄酮类化合物分布的一种特例，其中Dy含量特高，占幼嫩叶干重的27WeZWh，占茎干重11ng右；y含量占幼嫩叶干重的二．n＊．现占茎干重的1．2％左右，不同季节、不同产地的样品中Dy和y含量有一定差别。Dy系黄酮类化合物中重要的一元，具祛痰、消炎、止咳、降脂、保肝护肝等等非常重要的生理活性作用，该结果进一步证实，该植物是一种开发前景非常广泛的天然野生植物。 3、Dgy提制条件研究结果显示： （1）用水或乙醇浸提显齿蛇葡萄春、夏季样，一次浸提后干燥物中DMY含量为40％ 左右，秋季自然落叶样中不含Dr，结果预示植物中化学成分的转变非常活跃，从现 有的研究结果看，植物原料以春、夏两季为有用原料。 （2）应用目前工业化生产中提制天然植物活性成分的最先进的大孔吸附树脂法提制Dy。在水溶液状态下，物理化学性质各有特点的m、NKA｛、ABS等9种大孔吸附树脂对杂质的吸附比率要大于对Dgr的吸附比率；提纯、精制DMY宜利用大孔吸附树脂吸附杂质的能力优于吸附DMY的能力的特点而实现；首次注意水和温度的综合作用在提纯、精制Dy时起重要影响。 （3）本实验首次提出并采用“增温溶解保温过柱温水洗脱”方法纯化Dy，且获得成功，为快速、有效地提取天然野生植物中不同活性成分增加了新的方法。“增温溶解保温过柱温水洗脱”方法即人为地提高水温，以此增加e在水中的溶解能力；趁热保温过大孔吸附树脂，利用大孔吸附树脂吸附杂质的能力优于吸附Dy的能力，以此提纯DWi；最后，用一定温度的水洗脱被吸附在树脂上的Dr和杂质，既可减少Dy的损失，又可保证树脂的重复使用。结果显示：该方法能快速、有效地提Dgr。提制Dgy时，溶解、过柱温度宜为50－70℃，最好为60℃；用水解吸时，其温度可在60t以上。 N）提纯Dny时，“增温溶解?更多还原

【Abstract】 With the development of modern sciences and technology and the improvement of social economy and human’ s living standards. "Health" is becoming the roost important Topic in the world . It become one of the most hopeful and the mostvigorous business to develop botanical resources for human being’ s happiness, among of which , studying and developing on tea substitute is more important .Products of tea substitute are to be the hottest products in the world , more and more medical industries and more and more patients pay more attention on which.As a typical tea substitute, Awpelopsis grossedentata , was paid more attention in recent years. It belongs to Ampelopsis Michx of Vitaceae and A. cantoniesis(E. et A.) PL var. grossedentata Hand-Mazz. All parts of this plant can cat as medical materials . It is sweet in taste , cool in nature, treating common cold and pyretic fever , pain-swelling of pharynx and larynx as well as jaundice hepatitis ,with a long history of several hundred years among Yao and Zhuang nationality people in China .The tender steins and leaves have been made into a healthy drink which is called "mystic tea" . The water extract can keep fresh for several days in summer season . It grows in Hunan , Guangxi, GuangDong, GuiZhou, Rennan provinces .It is easy to add its materials by cultivation in a short time and the main chemical compounds between cultivated species and wild species keep same according to our series experiments .So , it can be called useful wild plant .Many people have studied the plant , what’ s more , they developed the plant’ s tender stems and leaves into many tea-substitute products , for example , Mao An-mei Cha , Can Lu Cha , Teng Cha and so on. But what they studied is focused on medical functions of water extraction, not involving the systemic isolation and identification of components and purification related for main chemical components . Thus , in this paper, the isolation and identification of flavonids , polyphenols and essential oils in this plant .establishment a HPLC method for DMYrvmeasurement , detecting of the content of DMY , selection of the factors for extraction , especially isolation and purification around DMY were studied. In these experiments, several methods as TLC, Column chromatography , HPLC, HSCCC and "dissolving with heat and flowing with the same temperature , desorption with warm water" and many modern instruments of IV , IR , ’HMNR , I3CNMR , MS , LC-MS , GC-MS were used .All dates and results in this study are true .which not only provided many new methods for developing other botanical resources , but also many theoretical and practical dates for developing A. grossedentata.The results showed that:1. Dihydromyricetin, myricetin , quercetin and rutin which are important to human beings health were identified in the fractions of filter residue and ethyl acetate extraction of A. grossedentata .which was extracted with acetone/water (1:1), ethyl ether, ethyl acetate , n-butyl alcohol in turn .Among of which , quercetin and rutin were firstly reported in this plant. Gallic acid, ethyl gallate , galloyl glucose were firstly detected in water fraction.2.AHRP-HPLC method for the detection and measurement of Dihydromyricetin and myricetin in Ampelopsis grossedentata was developed firstly. Operating conditions were UV-250nm detection, Nova-pak €?columnlSOmraXS. 9mmi. d., and the flow phase of v(methanol)/v(water) = (36:64, plus 0.1% phosphoric acid) , the flow speed of 1. Oml/min , lOul injection volume , 30癈 temperature for column . The coefficient of variation of this method were 5. 21% and 3. 23% for DMY and MY respectively . Dihydromyricetin contents were varied from 27-28% in the yound leaves to 11% in the tender stem .Myricetin contents from 1.8-1.9% to 1. 2% . The contents of Dihydromyricetin and myricetin were varied with seasons and places .All dates above identified that the plant is a very important botanical resource which has wide developing future.3. The results of extraction and purification for DMY showed:(1)更多还原