苦地丁消除细菌耐药性的活性成分及作用机制

In recent years, the problem of drug resistance of bacteria has been the grave concern of the international community and the medical profession and the traditional Chinese medicine must be come into notice because of its characteristics with multiple ways, levels and effect targets. On the basis of identification of the effective fraction of Corydalis Bengeana Tures for eliminating bacteria drug resistance and its mainly pharmacological activity, this project planned to separate the effective fraction by chromatographic separation strategy such as ODS and Sephadex. The alkaloids and flavonoids from the separation were prepared for systematic chemical separation. The chemical structure of monomeric compound from the separation was elucidated by the means of spectroscopy, assisted by the pharmacodynamical research to identify active constituent and effective components of Corydalis Bengeana Tures. And on this basis, we researched on how effective substance of effective fraction, active constituent and effective components affect the plasmids, biofilm formation and beta-lactamase of drug-resistance bacteria, investigated the relationship of effective fraction/active constituent/effective components, pharmacological activity and mechanism, elucidated each level of effective substance and mechanism of elimination bacteria drug resistance of Corydalis Bengeana Tures. This project would deepen the cognition of traditional effects scientific connotation, provide significant scientific basis for developing good curative effect, with independent intellectual property rights of the antagonist bacteria resistant new drugs, provide new ideas of researching other antimicrobial and anti-inflammation traditional Chinese medicine.

细菌耐药性是医药界面临的巨大的科学难题，而中药作为新的具有多途径、多层次、多靶点的特点备受瞩目。在已确定苦地丁消除细菌耐药性有效部位及其主要药理活性的基础上，本项目拟采用ODS及Sephadex等色谱分离策略对有效部位进行拆分，将拆分得到的生物碱类组分和黄酮类组分进行系统的化学分离，采用光谱学测试技术确定单体化合物的化学结构，并结合药效学研究确定苦地丁的有效组分和有效成分。在此基础上，研究有效部位、有效组分、有效成分三个层面的药效物质基础对耐药细菌的质粒、生物膜形成、主动外排泵系统、β-内酰胺酶的产生的变化，探讨有效部位/有效组分/有效成分?功效(药理作用)?作用机制的相关性，阐明苦地丁消除细菌耐药性的各层次药效物质基础及其作用机制。本项目可深化对苦地丁传统功效科学内涵的认识，为研制出疗效好、具有自主知识产权的拮抗细菌耐药性的新药提供重要科学依据，并可为其他抗菌消炎类中药提供新的思路。

本项目在构建体外消除大肠杆菌耐药性和体外抑菌、杀菌药理模型基础上，发现了罂粟科植物苦地丁的消除细菌耐药性的有效部位和有效组分，并采用大孔吸附树脂和聚酰胺等柱色谱实现了有效组分的拆分。发现苦地丁90%乙醇提取物经大孔吸附树脂梯度洗脱后的90%洗脱层为消除细菌耐药性的有效部位；其中生物碱类组分为有效组分；采用自制的LH-3大肠杆菌为研究对象，以消除剂SDS为阳性对照药，对苦地丁有效部位和有效组分给药前后菌株的质粒进行提取，对菌株质粒凝胶糖电泳结果进行分析，发现有效部位和有效组分的作用机制，可能是通过消除多重耐药性菌株的质粒而起到作用。对有效部位进行系统细致的化学分离，分离鉴定了30个生物碱类成分、8个黄酮类成分、1个脂肪酸类成分和2个甾体类成分。并采用RP-HPLC法建立了6个活性生物碱的质量控制方法，建立的方法简单灵敏、重现性好，并用该方法对不同产地的苦地丁药材进行了含量测定。在此基础上，对活性成分生物碱的大鼠体内药代动力学进行了研究。首先采用UHPLC-MS/MS法，对紫堇灵、乙酰紫堇灵以及苦地丁乙醇提取物给药后的紫堇灵、乙酰紫堇灵的药物动力学进行了研究。然后采用UPLC-ESI-MS/MS法对给予苦地丁提取物后大鼠体内5种生物碱的多组分药动学进行了研究。这些结果都可以为苦地丁在体内的药理学，毒理学，药物代谢研究奠定基础。