目的 从大蓟根中分离抗结核杆菌活性成分，并初步探讨其抗结核杆菌的作用机制。方法 采用活性跟踪分离方法，从大蓟根中分离抗结核杆菌活性成分，运用棋盘试验法研究大蓟活性成分与异烟肼、利福平和吡嗪酰胺的联合抗菌作用，运用分子对接软件初步探讨活性单体成分与KatG酶的亲和力，采用qRT-PCR法考察活性化合物干预结核分枝杆菌后KatG酶基因的相对转录量，采用紫外分光光度法测定KatG酶的活力，采用扫描电镜观察结核杆菌的形态。结果 从大蓟根分离得到具有抗结核杆菌活性的化合物HP01，鉴定为槲皮素。槲皮素抑制结核杆菌的最低抑菌浓度（minimum inhibitory concentration，MIC）值为160 μg/mL。槲皮素与吡嗪酰胺存在相加作用，与异烟肼、利福平无拮抗作用。分子对接显示，槲皮素与KatG酶具有较强的亲和力。酶活力测试结果表明槲皮素对结核杆菌KatG酶具有抑制作用。结核杆菌被槲皮素处理后其KatG基因转录出现明显的补偿上调。扫描电镜结果显示，槲皮素能破坏结核杆菌细胞壁。结论 大蓟抗结核杆菌的活性成分为槲皮素，槲皮素能够抑制结核杆菌KatG酶活性，导致结核杆菌结构破坏，从而杀死结核杆菌。在设计新的抗结核组合用药方案时，可以考虑加入槲皮素。

Objective To isolate the anti-Mycobacterium tuberculosis active components from roots of Cirsium japonicum, and preliminarily explore the anti-M. tuberculosis mechanism. Methods Using bioassay-guided isolation method to isolate active molecules against M. tuberculosis from roots of C. japonicum. The checkerboard test was used to study the synergic effects of active ingredients with isoniazid, rifampicin and pyrazinamide. Using molecular docking software to preliminarily explore the affinity between active monomer and KatG. qRT-PCR was used to study the relative transcription of KatG gene after the active compound interfered with M. tuberculosis. KatG activity was determined by ultraviolet spectrophotometry. Scanning electron microscope was used to observe the morphology of M. tuberculosis. Results Compound HP01 with anti-M. tuberculosis activity was isolated from roots of C. japonicum, and was identified as quercetin. Quercetin inhibited M. tuberculosis with a MIC value of 160 μg/mL. Quercetin had an additive effect with pyrazinamide, but had no antagonistic effect with isoniazid and rifampicin. Molecular docking showed that quercetin had a strong affinity with KatG. The enzyme activity test showed that quercetin had an inhibitory effect on KatG of M. tuberculosis. After M. tuberculosis was treated with quercetin, the transcription of KatG gene showed a compensatory up-regulation. Scanning electron microscopy showed that quercetin could destroy the cell wall of M. tuberculosis. Conclusion The active component of C. japonicum against M. tuberculosis was quercetin. Quercetin could kill M. tuberculosis by inhibiting KatG of M. tuberculosis and causing structural damage. When designing a new anti-tuberculosis combination regimen, quercetin can be considered.

R285.5

国家自然科学基金资助项目（82060635）；国家自然科学基金资助项目（81760629）；贵州省社会发展科技攻关项目（SY字[2013]3058号）；“国重室开放课题”及“黔科合平台人才”（[2017]5101）