Tracking Of The Active Ingredients Of Arisaema Heterophyllum Blume Against Mycobacterium Tuberculosis

Tuberculosis(TB)is an infectious disease caused by Mycobacterium tuberculosis(Mtb),which is one of the top ten causes of death in the world.BCG vaccination only has a certain effect on preventing meningeal tuberculosis in children,and the main treatment for tuberculosis still is drug treatment.Under the stress of drug selection,drug-resistant Mycobacterium tuberculosis strains continue to increase,leading to the number of cases "Multidrug-resistant Tuberculosis(MDR-TB)" and "Extensively drug resistant Tuberculosis(XDR-TB)" is increasing.Therefore,it is necessary to research and develop new anti-tuberculosis drugs.Arisaema heterophyllum Blume,also known as Pinellia pedatisecta Schott,Arisaema clavatum Buchet,and Arisaema erubescens(Wall.)Schott,Which is a tuber of Arisaema heterophyllum Blume,Arisaema amurense Maxim or Pinellia pedatisecta Schott.It has the effects of removing dampness and reducing phlegm,expelling wind and relieving spasm,and dispersing nodules and swelling.It is used to treat lymph in folk tuberculosis.The purpose of this study is to track and isolate molecules with anti-TB activity from Arisaema heterophyllum Blume,and to preliminarily explore the mechanism of action of the active molecules against tuberculosis.The activity tracking separation method was used to track and isolate the compound SF with anti-tuberculosis activity from araceae.According to the spectral data combined with the literature,the compound was identified as silibinin.Using resazurin color method,the MIC value of silibinin against mycobacterium tuberculosis was measured to be 160 μg/ml,and the MBC value was also 160 μg/ml measured by colony counting.Using a checkerboard test,it was found that silibinin,pyrazinamide and isoniazid had a combined antibacterial effect,which showed an additive effect and had no antagonistic effect with rifampicin.In addition,silibinin has an inhibitory effect on the biofilm of Mycobacterium tuberculosis.After the Mycobacterium tuberculosis is treated with silibinin,it can be seen that the Mycobacterium tuberculosis bacteria grow longer under an optical microscope.We judged that the antibacterial effect of silibinin may be related to the inhibition of the division of Mycobacterium tuberculosis.First,perform virtual molecular docking.The results show that silibinin has a strong affinity with the FTSZ protein and SSD protein of Mycobacterium tuberculosis.Then the fluorescence quantitative PCR technology was used to determine the relative transcription level of the ssd gene of Mycobacterium tuberculosis after being treated with silibinin.The results showed that when the concentration of silibinin was 80μg/ml,the transcription of ssd gene was down-regulated,and the concentration of silibinin was 160μg /ml,ssd gene transcription is up-regulated.The SSD protein is a key regulatory protein that determines the formation of the diaphragm during the division of Mycobacterium tuberculosis.Increased expression of the SSD protein will result in the loss of the diaphragm during the division of Mycobacterium tuberculosis and make the bacteria longer.This suggests that silibinin interferes with the division of Mycobacterium tuberculosis.Finally,scanning electron microscopy was used to observe that the low concentration of silibinin made Mycobacterium tuberculosis elongated and swelled locally.After increasing the concentration of silibinin,the tuberculosis became spherical until it disintegrated.We speculate that silibinin blocks the formation of the diaphragm when Mycobacterium tuberculosis divides and becomes a diploid cell,which increases the demand for nutrients.The unique cell wall structure of Mycobacterium tuberculosis restricts the entry of more nutrients into the cell.Disorders of metabolic regulation are caused,the mycolic acid layer of the cell wall becomes thinner,and a large amount of water molecules enter the cells with high osmotic pressure,which eventually causes the cells to expand and rupture.In summary,silibinin has good compatibility with first-line anti-tuberculosis drugs and has a relatively unique antibacterial mechanism.When designing a new anti-tuberculosis combination regimen,silibinin can be considered.This study provides an in vitro test basis for the use of araceae to treat tuberculosis,and provides a new idea for the development of anti-tuberculosis drugs with new mechanisms of action.