金银花干燥过程中酶促褐变机理和酚类物质氧化衰退途径研究

Quality deterioration is the key problem in honeysuckle drying process, the pathways of chlorogenic acid and galuteolin oxidation recession and enzymatic browning metabolism of honeysuckle phenols are lack of system study. On the basis of previous researches, Combination rules of PPO activity center coupled copper and oxygen free radical ,electron and proton transfer situation in enzymatic catalytic oxidation process, and study change rule of Copper oxidation state in activity center and the mechanism of catalytic oxidation were analyzed by electrochemical and spectral techniques. Honeysuckle PPO catalytic oxidation simulation research was implemented, phenols substances enzymatic oxidation law and metabolism of oxidation products were analyzed through high performance liquid chromatography-electrospray ionization mass spectrometry. Using immobilized enzyme technology prepared honeysuckle phenols compounds’o-benzoquinone, and the coupling oxidation pathway of model compounds with other phenols in non-enzymatic were studied, honeysuckle phenols non-enzymatic oxidation law were analyzed. According to the phenols simulator investigation results, oxidation recessionary pathway of main functional component chlorogenic acid and galuteolin were revealed, and the mechanism of deterioration and phenolic oxidation process of honeysuckle In Hot air drying process were clarified. This research will promote the healthy development of honeysuckle industry in Henan province.

品质劣变是金银花干燥过程中的关键问题，而引发劣变的酚类物质酶促褐变机理，以及主要功效成分绿原酸与木犀草苷的氧化衰退途径缺乏深入研究。本课题拟采用光谱技术、电化学技术，分析酶促氧化时PPO活性中心耦合铜与氧自由基的结合规律及其电子与质子的转移情况，探讨PPO酶促氧化机理；模拟金银花PPO和酚类物质的酶促氧化反应，并通过高效液相色谱-电喷雾电离质谱法分析酚类氧化过程及其产物代谢规律；利用固定化酶制备金银花酚类的邻苯醌模型物，并与其他酚类物质进行非酶促耦合氧化途径模拟研究，分析金银花酚类非酶促氧化规律及其产物在酶促褐变中的作用与地位；根据模拟研究结果探讨金银花酶促褐变时酚类代谢过程，揭示主要功效成分绿原酸和木犀草苷氧化衰退途径，阐明热风干燥过程中金银花酚类氧化规律和品质劣变机理。本课题研究成果将促进河南省金银花产业的健康发展。

金银花是一种重要的传统中药材，应用非常广泛。采收后需及时恰当的干燥，否则会造成颜色劣变和功效成分降低等品质劣变问题，直接影响其商品价值。前期研究结果证实，其品质劣变主要是由多酚氧化酶催化氧化酚类物质的酶促褐变造成的。本项目以金银花为研究对象，首先确定功效成分同时检测和提取的方法，为后续研究提供条件；然后通过气调热泵干燥新鲜金银花，研究干燥过程中功效成分的降解动力学，揭示其氧化损失过程，最后通过模拟酚类物质酶促褐变及非酶促褐变，进一步研究其氧化损失机理，为褐变抑制及稳定获得高品质金银花提供理论依据。通过气调（充氮）热泵干燥实验，探讨干燥过程中水分比的变化规律，研究干燥温度、装载量和氧体积分数对绿原酸、木犀草苷含量和L 值的影响，建立干燥模型及绿原酸和木犀草苷降解动力学模型。以新鲜金银花为研究对象，通过匀浆浸提、盐析透析及柱层析，分离得到比活力较高的金银花多酚氧化酶；采用分光光度法和双倒数作图法对金银花多酚氧化酶的酶学性质、理化性质及其抑制剂进行了系统的研究。以绿原酸标品通过体外模拟金银花非酶褐变，通过HPLC分析氧化产物得出，非酶氧化产物共检测到8种。通过体外由粗提PPO催化绿原酸标品模拟金银花酶促氧化反应，由HPLC分析模拟体系的反应产物，检测到一种新生成的物质，并且还发现6种物质的反应途径与绿原酸一致。采用HPLC-MS分析氧化产物的结构，得出绿原酸在非酶氧化过程中不存在聚合反应现象，但是发生同分异构现象。绿原酸在PPO的催化作用下生成羟基绿原酸、绿原酸p-醌衍生物和绿原酸o-醌衍生物。