Activity Tracking And Structural Modification Of Anti-tuberculosis Components Of Prunus Mume

Tuberculosis（TB）is an infectious disease caused by Mycobacterium tuberculosis（MTB）.Mycobacterium tuberculosis is usually spread from the respiratory tract,mainly invading the lungs,and can also infect other tissues and organs.In recent years,due to frequent population movements,cross-infection of Mycobacterium tuberculosis and HIV and other pathogens,and irregular use of anti-tuberculosis drugs,tuberculosis is difficult to cure.China not only has a large base of tuberculosis patients,but also has a relatively large proportion of drug-resistant strains.The situation is very serious.Therefore,the development of new anti-TB drugs is of great significance.The Dark Plum Fruit is the dry and nearly mature fruit of the Rosaceae Dark Plum Fruit,which has the effects of restraining the lungs,astringent intestines,producing body fluid and soothing roundworms.It was found in the early stage that the ethanol extract of Dark Plum Fruit has anti-TB activity,and the active part is ethyl acetate extract.This project intends to use activity tracking separation methods to isolate and identify active molecules,and carry out structural modifications,laying a foundation for the development of new anti-TB drugs basis.Ethanol extract of Dark Plum Fruit（434.4 g）was obtained by ethanol heat reflux method,and extracted with ethyl acetate to obtain ethyl acetate fraction（117.3 g）.The ethyl acetate fraction was separated by silica gel column chromatography to obtain FrA（11.23 g）,FrB（13.71 g）,FrC（17.24 g）,FrD（21.63 g）,FrE（15.39 g）,FrF（25.47g）six components,after the activity tracking,it shows that the FrE part has the best inhibitory activity against Mycobacterium tuberculosis,The MIC value is 200μg/mL,and the remaining MIC values are all greater than 200μg/mL.The FrE part was tracked and separated by silica gel column chromatography,and the active monomer compound 1 was obtained,which was identified as 5-hydroxymethyl-furfural,and the MIC value for inhibiting Mycobacterium tuberculosis is 120μg/mL.The reaction of 5-hydroxymethyl-furfural with O-（tetrahydro-2H-pyran-2-yl）hydroxylamine yields compound 2（5-hydroxymethyl-furfural oxime）with an MIC of60μg/mL,The activity of compound 2 is double that of compound 1.Based on the experimental basis that compound 1 enhances anti-TB activity through oximation reaction,we believe that oxime compounds may generally have anti-TB activity.The oximationreactionofcinnamaldehyde,indole-3-carboxaldehyde,pyridine-2-carboxaldehyde,salicylaldehyde,4-cyanobenzaldehyde,3,4,5-trimethoxybenzaldehyde and hydroxylamine compounds respectively,and the Schiff base reaction of indole-3-carbaldehyde and N-Aminothiourea resulted in 8compounds.The results of the anti-tuberculosis test show that the MIC of compounds3,4,and 5 are 10μg/mL,the MIC values of compounds 6,7 are 20μg/mL,and the MIC values of compounds 8,9,and 10 are all greater than 20μg/mL.KatG enzyme is the key enzyme for Mycobacterium tuberculosis to decompose intracellular oxygen free radicals.Inhibition of KatG will cause the accumulation of H₂O₂and cause the death of Mycobacterium tuberculosis.In order to explore the mechanism of action of the above compounds,firstly,molecular virtual docking was performed.The results show that compound 5 has a strong affinity with KatG enzyme,and the docking score is 107.98.The activity of KatG enzyme was determined by UV spectrophotometry.The inhibitory rate of compound 5 on KatG enzyme is 11%,which is the highest among the 10 compounds.The MIC value of compound 5inhibiting Mycobacterium tuberculosis is 10μg/mL.The experimental results show that the measured value is consistent with the score of the virtual docking.The MIC value of compound 10 inhibiting Mycobacterium tuberculosis is greater than 20μg/mL,but the score of virtual docking with KatG enzyme is higher（90.58）.The actual measured inhibition rate of compound 10 on KatG enzyme is 7%.We believe that the three methoxy groups on the benzene ring of the compound limit the transport of the molecule across the membrane,resulting in a decrease in its actual antibacterial effect.Generally speaking,the anti-tuberculosis activity of the 10 compounds is significantly correlated with the KatG enzyme inhibition rate.To sum up,this topic uses oximation reaction to modify the aldehyde group on the compound into an oxime group,and the compound’s anti-Mycobacterium activity is significantly improved,providing a new idea for the development of anti-Mycobacterium tuberculosis molecules with a new mechanism of action.