肝X-受体激动剂马尾藻甾醇的体外抗结核活性研究

Anti-tuberculosis activities in vitro of LXR agonist saringosterol

目的评价马尾藻甾醇及其24-差向异构体的抗结核活性。方法运用半制备高效液相色谱法(HPLC)分到马尾藻甾醇异构体,核磁共振氢谱(1H-NMR)鉴定异构体的化学结构;采用倍比稀释法测定化合物对结核分枝杆菌Mycobacterium tuberculosis的最小抑菌浓度(minimal inhibit concentration,MIC)、单独使用及联合用药的杀菌作用;采用对硝基乙酸苯酯(PNPA)法评价化合物对芳胺N-乙酰基转移酶(arylamine N-acetyltransferases,NAT)的抑制作用,并使用ledock_go软件进行分子虚拟对接实验。结果获得24S-和24R-马尾藻甾醇;马尾藻甾醇、24S异构体和24R异构体对M.tuberculosis的MIC值分别是25、12.5μmol/L和>100μmol/L,对NAT蛋白的抑制率分别为45.27%±2.49%、46.95%±8.30%和29.20%±5.30%。马尾藻甾醇和24S异构体还能增强氧氟沙星的杀菌作用。从数值上看,24S异构体活性强于24R异构体,分子对接实验提示24S异构体与NAT蛋白之间的结合比24R异构体更牢固。结论马尾藻甾醇及其24S异构体在体外对结核分枝杆菌具有杀菌作用,可能与细菌胆固醇代谢有关,尚待深入研究。

Objective To investigate the anti-tuberculous activities of saringosterol and its 24-epimers.Methods Semi-preparative HPLC was used to separate saringosterol epimers and 1H-NMR was employed to identify chemical structures.The doubling dilution method was used to determine the minimum inhibitory concentration(MIC)of the compounds on Mycobacterium tuberculosis,and bactericidal effect of single and combined treatment.P-nitrophenylacetate(PNPA)method was used to evaluate the inhibitory effect of compounds on arylamine N-acetyltransferases(NAT),and ledock_go software was used for molecular docking experiments.Results 24 epimers of saringosterol were purified and identified.The MIC values of saringosterol,24S epimer,and 24R epimer against Mycobacterium tuberculosis were 25、12.5μmol/L,and>100μmol/L,respectively,and their inhibition effects on NAT were 45.27%±2.49%、46.95%±8.30%and 29.20%±5.30%,respectively.24S epimer was more active than the 24R epimer.Saringosterol and its 24S epimer enhanced the bactericidal effect of ofloxacin.Molecular docking showed that the binding between 24S epimer and NAT protein was stronger than that of 24R epimer.Conclusion Saringosterol and 24S-saringosterol had a bacteriocidal effect in vitro.The mechanism might be related to bacterial cholesterol metabolism,which needs further studies.