微量富集及模糊衍生技术筛选抗结核新活性成分

发布时间：2018-09-18 19:16

【摘要】：目的:通过微量富集和模糊衍生方法初步建立抗结核新型药物筛选平台,并从已知抗结核药物中筛选新的活性成分。方法:将异烟肼(INH)、利福平(RFP)、乙胺丁醇(EMB)、链霉素(SM)、卡那霉素(KM)、阿米卡星(AMK)、氧氟沙星(Ofx)、左氧氟沙星(Lfx)、加替沙星(Gfx)、莫西沙星(Mfx)、对氨基水杨酸钠(PAS)和丙硫异烟胺(Pto)共12种抗结核药物进行模糊衍生,并对衍生后产生的新成分进行微量富集筛选抗结核新成分。将上述药品分别配制成0.5%的甲醇溶液,每种药品溶液以线条状上样在一张薄层板上,并在薄层板上进行模糊衍生。将衍生的混合物进行二维薄层色谱(2D-TLC)展开,在第一个维度将药品衍生物中的成分根据不同极性进行分离,形成条状谱带,然后将薄层板平行旋转90°进行二维展开,把条状谱带推至薄层板边缘并聚集为点状谱带,达到微量成分富集的作用。将点状谱带逐一分割,分别置于铺满结核分枝杆菌(MTB)的培养基上,37°C恒温培养箱培养4-8周,观察各谱带的抑菌作用。结果:INH和EMB两组中存在相应谱带区域有抑制MTB生长的作用,其中INH组的抑菌实验的灵敏度和特异度较高,进一步分割INH(INH对照组)和INH I(INH衍生物组)的抑菌区域逐一谱带进行培养,从而得出INH I(4号谱带)具有显著抑制敏感结核分枝杆菌和耐药结核分枝杆菌生长的作用,INH I组中可能产生了新成分。结论:初步建立了微量富集方法和模糊衍生技术筛选抗结核新药的平台,使数个实验室工作在同一张薄层板上进行,从而具有高效、低成本和省时省力等优点。本技术可把已知抗结核药物中的微量成分分离出来,通过体外抑菌实验,从已知的药物中发现抗结核新成分。在此平台基础上,初步从INH的衍生物中筛选出抗结核新成分,为药物的分离、筛选及新药的研发奠定了基础。
[Abstract]:Objective: to establish a new anti-tuberculosis drug screening platform by microenrichment and fuzzy derivation, and to screen new active components from known anti-tuberculosis drugs. Methods: twelve species of isoniazid (INH), rifampicin (RFP), (RFP), rifampicin (RFP), (EMB), streptomycin (SM), (KM), (AMK), ofloxacin (Ofx), levofloxacin (Lfx), gatifloxacin (Gfx), moxifloxacin (PAS) and (Pto) were prepared. Antituberculotic drugs are derived by fuzzy methods, New anti-tuberculosis components were obtained by microenrichment and screening. The above drugs were prepared into 0.5% methanol solution, and each drug solution was taken as a linear strip on a thin plate, and fuzzy derivation was carried out on the thin layer plate. The derivative mixture was expanded by two-dimensional thin-layer chromatography (2D-TLC), the components of the derivative were separated according to different polarities in the first dimension to form a strip, and then the thin plate was rotated 90 掳parallel for two-dimensional expansion. The strip band was pushed to the edge of the thin plate and aggregated into a dot band, which resulted in the enrichment of trace components. The dot bands were divided one by one and cultured in a 37 掳C constant temperature incubator for 4-8 weeks on a culture medium covered with Mycobacterium tuberculosis (MTB). The bacteriostatic effects of each band were observed. Results in the two groups, the corresponding band region could inhibit the growth of MTB. The sensitivity and specificity of the bacteriostatic test in the INH group were higher than those in the control group. Further partitioning the INH (INH control group and the INH I (INH derivative group), the inhibitory regions were cultured one by one, It is concluded that INH I (4) has a significant inhibitory effect on the growth of sensitive Mycobacterium tuberculosis and drug-resistant Mycobacterium tuberculosis. Conclusion: a platform for screening new anti-tuberculosis drugs by microenrichment method and fuzzy derivation technique has been established preliminarily, which makes several laboratories work on the same thin-layer plate, which has the advantages of high efficiency, low cost and time saving and labor saving. This technique can isolate the trace components from known antituberculous drugs and find new antituberculotic components from known antituberculous drugs by in vitro bacteriostasis test. On the basis of this platform, a new anti-tuberculosis component was screened from the derivatives of INH, which laid a foundation for the separation and screening of drugs and the research and development of new drugs.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R52

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