Study On Chinese Herbal Medicine Combined With Pyrazinamide

Tuberculosis(TBerculosis,TB)is caused by the pathogen Mycobacterium tuberculosis(MTB)and is an infectious disease with high prevalence,high infectivity and chronic wasting characteristics.Until last century,the emergence of an anti-tuberculosis drug that named streptomycin,tuberculosis was effectively treated.In recent years,due to the emergence of drug-resistant tuberculosis and cross-infection with AIDS,its mortality rate is second only to that of AIDS,which seriously threatens human's health.Therefore,there is an urgent need to develop new drugs,find new targets and new combinations of treatment methods to cope with the growing trend of tuberculosis.Among the first-line anti-tuberculosis drugs,Pyrazinamide(PZA)can kill tubercle bacilli and retaine bacteria in the semi-dormant period under acidic conditions.And pyrazinamide can shorten the treatment time of tuberculosis from 9-12 months to 6 months,so it plays an important role in shortening the treatment time in short-course chemotherapy of tuberculosis.Although pyrazinamide can completely eradicate tuberculosis,it has obvious disadvantages in clinical application.Compared with other first-line anti-tuberculosis drugs(such as isoniazid in vitro MIC value of 0.061 ?g/m L,rifampicin in vitro MIC Value is 60 ?g/m L),in vitro MIC value of pyrazinamide(50-120 ?g/m L)is high,so it must be administered in large doses to play a therapeutic role,long-term large doses of pyrazinamide will cause significant liver toxicity,if the liver protection treatment is not carried out in time,the patient may die.Therefore,it is of great significance to search for drugs that have a combined effect with pyrazinamide from Chinese herbal medicine,reduce it's dosage and liver toxicity,and provide new treatment plan for tuberculosis.Chinese herbal medicine resources are very rich,with a wide variety and complex chemical composition.It is a huge library of natural compounds and one of the main sources of new anti-tuberculosis drugs.In this experiment,the diversity of Chinese herbal medicine resources was used to establish a sample library of ethanol extracts of Chinese herbal medicine.An in vitro anti-M.Tuberculosis activity screening test was conducted on the combination of 15 Chinese herbal medicine alcohol extracts and pyrazinamide.When pyrazinamide(60 ?g/m L)is added with alcohol extract of traditional Chinese medicine(1000 ?g/m L),the traditional Chinese medicine Ramulus cinnamomi,Fructus mume,Morus alba L.,Common Vladimiria Root,Rheum palmatum L.,Angelica sinensis(Oliv.)Diels,Bupleurum,Glabrous Greenbrier Rhizome,Curcuma phaeocaulis Valeton of alcohol extracts combined with pyrazinamide have in vitro anti-tuberculosis activity,further experimental results show that pyrazinamide(60 ?g/m L)is added with alcohol extract of traditional Chinese medicine(500 ?g/m L),the Chinese medicine Ramulus cinnamomi,Morus alba L.,Fructus mume,Common Vladimiria Root,Rheum palmatum L.and pyrazinamide have a combined antibacterial effect.Therefore,in the follow-up work,an in-depth study of Ramulus cinnamomi and Fructus mume was found that the main component of anti-tuberculosis activity in Ramulus cinnamomi was cinnamaldehyde,and the minimum inhibitory concentration of cinnamaldehyde was 10 ?g/m L.When used in combination,pyrazinamide(60 ?g/m L)is added with cinnamaldehyde(4 ?g/m L)that has inhibitory activity against Mycobacterium tuberculosis.In order to speculate the mechanism of cinnamaldehyde,scanning electron microscope(SEM)and molecular docking experiments were carried out.The preliminary results speculate that cinnamaldehyde may play an anti-tuberculosis role by interacting with the active site of fatty acid synthase I enzyme.It will be verified at the molecular(enzyme)level later.The Fructus mume ethanol extract is extracted in stages to obtain two parts of ethyl acetate part and residue.Further activity tracking showed that the ethyl acetate layer had a weaker bacteriostatic effect when the concentration was 500 ?g/m L;the pyrazinamide(60 ?g/m L)is added with ethyl acetate layer(500 ?g/m L)that had effect stronger.When the concentration of the remaining layer is 500 ?g/m L and 1000 ?g/m L,there is no inhibitory activity against tubercle bacilli.Combined with anti-tuberculosis activity,the active site was determined to be the ethyl acetate layer.The ethyl acetate layer was separated by silica gel column chromatography and silica gel chromatography analysis,activity tracking separation,and the active monomer compound WD-01 with in vitro inhibition of M.tuberculosis was isolated.After a series of physical and chemical properties and standard identification,it was identified as ursolic acid with a MIC of 100 ?g/m L;in order to speculate the possible mechanism of ursolic acid,molecular docking experiments were carried out,and the preliminary results speculated that ursolic acid may play an anti-tuberculous role by interacting with the active sites of mycolic acid cyclopropane synthase I,arabinosyltransferase C,and acyl carrier protein reductase to exert anti-tuberculosis activity,which will be verified at the molecular(enzyme)level later.In summary,this paper found 9 Chinese herbal medicines with anti-tuberculosis activity combined with pyrazinamide,which provides an experimental basis for clinical medicine;it is determined that cinnamaldehyde is the active components of Ramulus cinnamomi,and cinnamaldehyde has a strong antibacterial effect.It has a potential to be developed as an anti-tuberculosis drug;ursolic acid is found to be one of the anti-tuberculosis components of Fructus mume,and ursolic acid and pyrazinamide have a joint effect.The combination of ursolic acidand pyrazinamide can reduce the hepatotoxicity of pyrazinamide.