基于化学生物学方法结合高分辨质谱技术的中药药效物质快速筛选方法研究

发布时间：2018-05-27 17:34

本文选题：中药药效物质 + 化学生物学　；参考：《浙江大学》2017年博士论文

【摘要】：中药药效物质基础研究是中药作用机理探讨、创新药物研制和建立与药效相关的中药质量控制标准的基础,是中药现代化研究中的重中之重。而中药药效物质发现是中药药效物质基础研究的核心内容,建立中药药效物质的快速筛选方法对中药现代化研究具有重要的推动作用。本论文聚焦于中药药效物质发现方法的技术性问题,着重研究基于化学生物学方法结合高分辨质谱技术的中药药效物质快速筛选新方法,其主要研究内容如下:1.建立HPLC-ESI-Q-TOF-MS/MS方法对养心氏片的化学成分进行了系统性分析,共鉴定了其中127个化合物,对其结构进行了分类和质谱裂解规律的解析。采用DPPH-LC-MS分析技术对养心氏片中的抗氧化物质基础作了进一步的研究;从中快速筛选到34个潜在的抗氧化成分。其中丹酚酸A和丹酚酸B为最强的抗氧化物质,其DPPH自由基清除活性高于VC。通过对大鼠口服养心氏片的入血成分进行分析,发现丹酚酸A和丹酚酸B可以入血,可能是养心氏片发挥体内抗氧化作用的重要药效物质。2.创新性地提出了一种血脑屏障人造膜平行性透过试验与DPPH探针识别相结合的PAMPA-BBB-DPPH方法,并将其应用于中药中可透过血脑屏障的抗氧化物质筛选。发现知母乙酸乙酯提取物为活性中药提取物。通过DPPH-HPLC结合高分辨质谱技术,鉴定出知母提取物中的可透过血脑屏障的抗氧化物质为2,6,4'-Trihydroxy-4-methoxybenzophenone。体外研究表明,该物质具有中等强度的抗氧化活性,其DPPH自由基清除能力略弱于VC等抗氧化剂,但是其血脑屏障人造膜透过能力很强,具有深入开发为脑保护药物的价值。3.将亲和超滤LC-MS方法与分子对接技术相结合,首次应用于中药注射剂与人血清白蛋白相互作用的研究。发现丹红注射液中的丹酚酸类成分有很强的人.血清白蛋白结合作用,且与联用药阿司匹林的主要代谢产物水杨酸存在竞争作用。通过竞争性结合试验、分子对接、结合位点探针替代试验等方法解释了其竞争机制。研究内容为丹红注射液与人血清白蛋白的相互作用研究奠定了基础,同时也为中药注射剂与人血清白蛋白的相互作用研究提供了新的思路。4.创新性地提出一种基于固定化融合靶点亲和色谱的亲和选择方法,将其应用于PPARy配体的筛选。通过基因工程技术将靶点受体与亲和标签融合,可以实现靶点蛋白的快速制备、定向固定及纯化,从而应用于天然产物中相应配体的筛选。相比于之前报道的亲和选择筛选方法,可低成本地获得大量靶点蛋白,实现快速、定向的靶点固定,并能提高筛选通量。应用该方法发现野菊花中的异绿原酸A具有较强的PPARγ结合能力。该研究为亲和选择方法的建立提供了参考。
[Abstract]:The basic research on the substance of traditional Chinese medicine (TCM) is the most important part in the study of TCM modernization. It is the basis of innovating the development of Chinese medicine and establishing the quality control standard of Chinese medicine related to its efficacy. The discovery of effective substances of traditional Chinese medicine is the core of the basic research on the substance of traditional Chinese medicine and the establishment of a rapid screening method of Chinese medicine pharmacodynamic substances has an important role in promoting the modernization of traditional Chinese medicine. In this paper, we focus on the technical problems of the discovery methods of Chinese traditional medicine pharmacodynamic substances, and focus on the study of a new method for rapid screening of Chinese traditional medicine pharmacodynamic substances based on chemical biological method and high resolution mass spectrometry. The main research contents are as follows: 1. A HPLC-ESI-Q-TOF-MS/MS method was established for the systematic analysis of the chemical constituents of Yangxin tablets. 127 of them were identified and their structures were classified and analyzed by mass spectrometry. The basis of antioxidant substances in Yangxin tablets was further studied by DPPH-LC-MS analysis, and 34 potential antioxidants were screened from them. Salvianolic acid A and Salvianolic acid B were the strongest antioxidants and the scavenging activity of DPPH radical was higher than that of VC. By analyzing the components of Yangxin tablet in rats, it was found that Salvianolic acid A and Salvianolic acid B could enter the blood, which may be an important antioxidation substance of Yangxin tablet. A novel PAMPA-BBB-DPPH method combining parallel permeability test of blood brain barrier (BBB) with DPPH probe identification was proposed and applied to the screening of antioxidants in Chinese medicine. It was found that the ethyl acetate extract of Anemarrhena emarginosa was active Chinese medicine extract. By means of DPPH-HPLC and high resolution mass spectrometry, the antioxidants in the extracts of Anemarrhena emarginosa through the blood-brain barrier were identified to be 2x6O4- Trihydroxy-4-methoxybenzophenone. In vitro studies showed that the compound had moderate antioxidant activity, and its scavenging ability of DPPH free radical was slightly weaker than that of VC, but its blood-brain barrier artificial membrane was highly permeable, and had the value of further development as a brain protection drug. The affinity ultrafiltration (LC-MS) method was combined with molecular docking technique to study the interaction between traditional Chinese medicine injection and human serum albumin (HSA) for the first time. It was found that Salvianolic acid in Danhong injection had strong human. Serum albumin binds and competes with salicylic acid, the main metabolite of aspirin. The competitive mechanism was explained by competitive binding test, molecular docking and site probe substitution test. The study laid a foundation for the study of the interaction between Danhong injection and human serum albumin, and also provided a new idea for the study of the interaction between traditional Chinese medicine injection and human serum albumin. A novel affinity selection method based on immobilized fusion target affinity chromatography was proposed and applied to the screening of PPARy ligands. Through the fusion of target receptor and affinity label by genetic engineering technology, the target protein can be quickly prepared, immobilized and purified, and applied to the screening of corresponding ligands in natural products. Compared with the previously reported affinity selection method, a large number of target proteins can be obtained at low cost, which can achieve fast, targeted target fixation and improve screening flux. By using this method, it was found that Isochrysanthemum Lv Yuan A had strong binding ability to PPAR 纬. This study provides a reference for the establishment of affinity selection method.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R284

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