基于活性指数和液质联用技术的桂枝加芍药汤抗炎活性成分快速发现研究

发布时间：2018-06-20 01:02

本文选题：桂枝加芍药汤 + 抗炎活性成分　；参考：《浙江大学》2017年硕士论文

【摘要】：中药方剂活性成分发现是中药现代化研究的重要内容。目前,中药活性成分发现的主要方法有系统分离法、活性导向法、生物色谱法等,前两种方法研究周期长、耗时耗力、命中率低,生物色谱法可以从混合物中快速发现含量较高的潜在活性成分,但可能会忽略某些微量活性成分。为克服以上研究方法的不足之处,本论文通过采用活性指数方法评价中药及其组分中各成分对其活性的贡献,从而预测其中潜在活性成分,并将该研究思路用于仲景方桂枝加芍药汤中抗炎活性成分的快速发现。桂枝加芍药汤出自于《伤寒论》,由桂枝、白芍、生姜、大枣和甘草组成,具有温脾和中,缓急止痛的功效。临床上主要用于治疗腹满引起的胀痛,还可以治疗顽固性菌痢,十二指肠球部溃疡,结核性腹膜炎等疾病。目前该方的药效物质基础尚不清楚。本论文以桂枝加芍药汤为研究对象,以方剂中抗炎活性成分的快速发现为研究目标,具体研究如下：采用液相色谱-四级杆飞行时间质谱(LC-Q-TOF-MS)和液相色谱-离子阱质谱(LC-IT-MS)联用方法对桂枝加芍药汤总提物中化学成分进行了分析。共检测到72个化合物,鉴定和推测了其中54个化合物结构,主要类型包括单萜苷类、三萜皂苷类、黄酮及其苷类和有机酸及其苷类。采用大孔树脂和制备液相色谱方法将桂枝加芍药汤总提物分段制备成64个组分。利用LC-IT-MS对各个组分的化学成分进行表征,一共检测到903个化合物。采用细菌脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7炎症模型,对桂枝加芍汤汤组分的抗炎活性进行评价。最后整合组分的化学信息和药理信息,计算各成分的活性指数,发现61个化合物有较高的活性指数值,推测这些化合物可能是潜在的抗炎活性成分。为了验证预测结果,对其中8个化合物进行了LC-MS导向的半制备液相色谱分离制备,采用NMR谱鉴定其结构,利用LPS诱导小鼠巨噬细胞RAW 264.7的炎症模型进行抗炎活性验证。研究发现,8个化合物均有良好的抗炎活性,其中6个化合物的抗炎活性明显强于阳性药吲哚美辛。此外,3个化合物的抗炎活性为首次报道。本研究结果表明活性指数方法能快速有效地发现中药方剂中的抗炎活性成分。
[Abstract]:The discovery of active components of traditional Chinese medicine prescription is an important content in the modernization of traditional Chinese medicine. At present, the main methods for the discovery of active components of traditional Chinese medicine include systematic separation method, active guidance method, biochromatographic method, etc. The first two methods have long research period, time-consuming and labor-consuming, low hit rate, etc. Biochromatographic method can quickly find the potential active components with high content in the mixture, but some trace active components may be neglected. In order to overcome the shortcomings of the above research methods, this paper uses the activity index method to evaluate the contribution of each component of traditional Chinese medicine and its components to its activity, so as to predict the potential active components. The research idea was applied to the rapid discovery of anti-inflammatory active components in Zhongjing Fang Guizhi Jiaoxiao decoction. Guizhi + Shaoyao decoction, composed of Cinnamon, Radix Paeoniae Alba, Ginger, Jujube and Glycyrrhiza uralensis, has the effect of warming the spleen and relieving pain. It is mainly used to treat distending pain caused by abdominal fullness, obstinate bacillary dysentery, duodenal bulbar ulcer, tuberculous peritonitis and so on. At present, the substance basis of the prescription is not clear. In this paper, Guizhijiaoxiao decoction was taken as the research object, and the rapid discovery of anti-inflammatory active components in the prescription was taken as the research goal. The main results are as follows: LC-Q-TOF-MS) and LC-IT-MS) were used to analyze the chemical constituents in the total extract of Guizhijiaoxiao decoction. A total of 72 compounds were detected and 54 of them were identified and speculated, including monoterpene glycosides, triterpenoid saponins, flavonoids and their glycosides, organic acids and their glycosides. The total extract of Guizhijiaoxiao decoction was prepared into 64 components by macroporous resin and preparative liquid chromatography. The chemical constituents of each component were characterized by LC-IT-MS and 903 compounds were detected. The inflammation model of mouse macrophage RAW264.7 was induced by lipopolysaccharide (LPS), and the anti-inflammatory activity of Guizhijiazhao decoction was evaluated. Finally, the chemical information and pharmacological information of the components were integrated to calculate the activity index of each component. It was found that 61 compounds had higher activity index values, which suggested that these compounds might be potential anti-inflammatory components. In order to verify the predicted results, eight of the compounds were separated and prepared by LC-MS directed semi-preparative liquid chromatography. Their structures were identified by NMR. The anti-inflammatory activity of LPS-induced mouse macrophage RAW 264.7 inflammatory model was verified. It was found that all of the eight compounds had good anti-inflammatory activity, and six of them had stronger anti-inflammatory activity than indomethacin. In addition, the anti-inflammatory activities of three compounds were reported for the first time. The results show that the active index method can quickly and effectively find the anti-inflammatory components in traditional Chinese medicine prescriptions.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284

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