| Tuberculosis(TB)remains one of the leading causes of death from infectious diseases worldwide.Chemotherapy is the dominant means of controlling TB.Due to antibiotic abuse and irregular treatment,the number of patients with drug-resistant TB is increasing,and the cure rate for multidrug-resistant tuberculosis(MDR-TB)and extensively drug-resistant tuberculosis(XDR-TB)is less than 50%,posing a serious threat to human health.Therefore,there is an urgent need to develop anti-TB drugs with new mechanisms of action.Guizhou Mengzi Shu is a species of Guizhou Miao People medicine with the scientific name of Xylosma racemosa(Sieb.& Zucc.)Miq.var.caudata(S.S.Lai)S.S.Lai,whose bark is used by Guizhou Miao People folk doctors to treat TB.Plant medicines play an important role in modern drug development,and there are about 8000 species of plants with medicinal value in China.Therefore,the discovery of molecules against Mycobacterium tuberculosis(MTB)from plant secondary metabolites holds great promise.In this study,we first isolated the anti-MTB active components from the X.racemosa,then synthesized seven compounds based on the active molecular characteristics;and finally explored the anti-MTB mechanism of action.The ethanol extract of X.racemosa bark was prepared by hot reflux extraction,and the anti-MTB activity was determined by the Lowenstein-Jensen medium absolute concentration method.The MIC value of the extract was 1000 μg/m L.Extracted sequentially with petroleum ether and ethyl acetate,and the active ingredient was found to be concentrated in the ethyl acetate site(MIC=500 μg/m L).Separated by column chromatography and thin-layer chromatography,the active monomer(MIC=250 μg/m L)was obtained and identified as chlorogenic acid by NMR.Structurally chlorogenic acid contains quinic acid and caffeic acid structural units.Drawing on some successful modifications of natural products,seven Schiff base analogs(compounds X01-X07)were synthesized by molecular collocation with triazoles using analogs of quinic acid and caffeic acid as raw materials.In vitro antibacterial experiments showed that all seven compounds showed good inhibitory effects against MTB(MIC values ranged from 4μg/m L to 64μg/m L.),with compound X04 showing strong inhibitory activity(MIC=4 μg/m L)and a 62-fold increase in antibacterial effect compared with chlorogenic acid.In addition,computer-aided drug design software was used to predict the drug-forming properties and pharmacokinetic characteristics of seven compounds.Compound X04 has the potential to be a lead compound,and the mechanism of action of this compound was investigated.Firstly,the molecular docking method was used to predict its binding targets.20 proteins of MTB were selected from the PDB protein database and docked with X04 respectively,and it was found that X04 has a strong affinity with mvi N and SOD.mvi N is related to MTB peptidoglycan synthesis and energy metabolism,and SOD is a key enzyme for superoxide anion scavenging by MTB.Through experimental verification,it was found that X04 could stimulate MTB to highly express mvi N,could change the structure of MTB peptidoglycan,and could change the shape of MTB,and it was hypothesized that X04 might have an inhibitory effect by inhibiting mvi N.X04 could increase the activity of SOD,and it was hypothesized that when X04 interfered with peptidoglycan synthesis,the sugar chain was in the polymerization-decomposition-polymerization state,which consumed a large amount of ATP from MTB.Superoxide anion increases,causing SOD overexpression through a negative feedback mechanism.Of course,a lot of work needs to be done to determine the target of X04’s action.This study provides evidence for the folk use of the X.racemosa in the treatment of TB.Compound X04 is highly active and has low toxicity to Vero cells,and its structural optimization may lead to anti-TB lead compounds with new mechanisms of action. |
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