【作者】 王琴飞；

【导师】 黄绵佳； 李景明；

【作者基本信息】 海南大学 ， 植物学， 2008， 硕士

【摘要】 白藜芦醇（Resrevatrol,Res）做为一种天然的多酚物质,在植物抗病、人类保健与抗病等方面有极其重要的作用。随着白藜芦醇生物活性的深入认识和实验技术的不断更新,人们在葡萄等多种植物中发现了白藜芦醇衍生物及其类似物,这些衍生物和类似物被认为是白藜芦醇的代谢产物。在植物逆境生理、植物病理和植物育种等研究领域被广泛关注。但是由于白藜芦醇代谢在苯丙氨酸代谢途径中至今还是一个空白,因而与其衍生物和类似物的关系至今仍不够清晰。本文在此背景下,以赤霞珠葡萄扦插苗叶片为试材,建立了白藜芦醇及其衍生物的高效液相色谱检测方法和提取方法,研究了紫外诱导后葡萄叶片中白藜芦醇及其部分重要衍生物的积累规律;确定了两种代谢白藜芦醇的酶分别是多酚氧化酶（PPO）和过氧化物酶（POD）;对白藜芦醇代谢酶的代谢机制进行了初步探讨和研究。主要研究结果如下:（1）紫外诱导、避光处理后,白藜芦醇、白藜芦醇糖苷、ε-viniferin,δ-viniferin、紫檀芪五种芪化物均大量积累,白藜芦醇在72h内出现两次合成高峰。除白藜芦醇糖苷以外,ε-viniferin,δ-viniferin、紫檀芪三种芪化物的合成高峰滞后于白藜芦醇,出现在白藜芦醇的第二个合成高峰;（2）实验发现PPO代谢白藜芦醇的主要产物为ε-viniferin,而POD代谢白藜芦醇的产物为δ-viniferin。在两种酶的粗酶反应体系中还存在白藜芦醇代谢的中间产物或其他产物。因此,本实验证明,白藜芦醇至少存在两种以上的代谢途径;（3）通过离子交换柱（Q-Sepharose FF）层析和疏水亲和柱（Butyl-Sepharose FF）层析得到的酶蛋白,电泳结果表明:纯化后酶液中一种代谢白藜芦醇的PPO,分子量约为41.5kD,该酶不存在多亚基现象,其代谢产物为ε-viniferin;（4）虽然紫外诱导后葡萄叶片中δ-viniferin大量积累,但是电泳结果显示,没有新的POD同功酶合成。通过H₂O₂抑制实验,验证了POD在白藜芦醇代谢中的作用;同时表明紫外处理、H₂O₂和POD三者是白藜芦醇代谢合成δ-viniferin的充分必要条件。以上实验结果初步确定了白藜芦醇两条代谢途径,填补了苯丙氨酸代谢途径中白藜芦醇代谢的空白,为进一步探讨白藜芦醇及其衍生物在逆境生理中的作用及调控机制的研究奠定了必要的基础。更多还原

【Abstract】 Resveratrol,as a kind of natural polyphenol, which plays an important role in plant disease resistance, human healthy care and so forth. With the development of experimental technology and people’s further knowledge to explore the biological activity of resveratrol, resveratrol derivatives and their analogues were found in grape and many other plants, which were thought to be the metabolizable products of resveratrol. and it was taken attention to research realms such as plant adversity physiology, plant pathology and plant breeding as well. However, it is still a vacancy for the metabolizability of resveratrol in the phenylalanine metabolism pathway. Therefore, it is needed to know more about the relationship between resveratrol and its derivatives and analogues.In this paper, we took the grapevine leaves of Cabernet Sauvignon as the experimental material ,to establish the methods of high performance liquid chromatography for resveratrol and its derivatives. We wanted to find out the accumulation rules of resveratrol and its important derivatives after treating the leaves with ultraviolet radiation; two enzymes were comfirmed to be the metabolizable enzyme of resveratrol, which were polyphenol oxidase and peroxidase.In addition, the metabolizing mechanism of the metabolizable enzymes of resveratrol were discussed in the research . The main results are given as follows:(1)The resveratrol, piceid,ε-viniferin,δ-viniferin, pterostilene five stilbenes had a significantly accumulated in grape leaves after UV-C irradiation, there are two peaks of resveratrol synthesis in seventy-two hours, the synthesis peak ofε-viniferin,δ-viniferin, pterostilbene three stilbenes delay to resveratrol and appeared in second peak of resveretrol synthesis besides piceid.(2)The study found that main metabolites of resveratrol isε-viniferin andδ-viniferin through polyphenol oxidase and peroxidase,respectively.But there are many other indirect and ultimate metabolites in the two crud enzymes reaction system. the research have proved that resveratrol have two metabolic pathways at least.(3) The enzyme which uses the resveratrol as substract, was preliminary purification through the chromatograghy of ion exchange fiber (Q-Sepharose FF) and hydrophobic affinity chromatography (Butyl-Sepharose FF), the result of activitive electrophoresis showed that there is a polyphenol oxidase ,it is not multipie and molecular weight about 41.5kD, the metabolites isε-viniferin.(4) Although theδ-viniferin had a significantly accumulated in grape leaves after UV-C irradiation, the result of activitive electrophoresis showed that not new peroxidase produced and induced. The result showed that peroxidase involved in oxidation of resveratrol through inhibition of hydrogen peroxide formation we demonstrated ultraviolet radiation , peroxidase and hydrogen peroxide are indispensable in resveratrol metabolize at the same time .The result above ensured that there were two metabolize pathways of resveratrol.it was fill up blank of the resveratol metabolize in the phenylalanine metabolism pathway. These results settle the important foundation for the further reseach on the stress physiology and regulation mechanism of resveratrol and its derivatives.更多还原