Research Of Mechanism On The Antituberculosis Action Of Baicalin

ObjectiveThe epidemic situation of tuberculosis has been aggravated again since1980’s, and TB has been the most important infectious diseases in recent years. The presence of drug-resistant bacterial strains is the major cause of tuberculosis epidemic and becomes a serious challenge to anti-tuberculosis drugs. The presence of drug-resistant bacterial strains is related to HIV infection, improper use of anti-TB drugs, inappropriate treatment, less patients compliance. Recent hot-spot researches on herbal extracts make good progress and develop effective drug varieties which have low drug resistance TB and side effect. Many experiments show that Baicalin has broad antimicrobial spectrum, but there is little research on its inhibitory effect on Mycobacterium tuberculosis in vitro. Focusing on a large number of side effects of chemotherapy, increasing number of drug resistance TB and other hot issues, in this study antibacterial tests on baicalin in vitro are researched and cell morphological change after inhibitory effects of baicalin on Mycobacterium tuberculosis observed with electronic microscope, in order to define baicalin inhibitory mechanism and effect on Mycobacterium tuberculosis and provide a theory basis for development of new clinical antituberculosis drugs.MethodsBy preparation of modified L-J drug-containing medium, with various baicalin concentration of48mg/ml,12mg/ml,6mg/ml,3mg/ml,1.5mg/ml,0.75mg/ml,0.375mg/ml, and inoculation of clinical TB strains for4weeks in vitro environment at37℃incubator, the inhibitory effects of baicalin on Mycobacterium tuberculosis was observed and results was analyzed with absolute concentration gradient method. Observed inhibitory effect of varied baicalin concentration till no colony growth in culture media to determine the minimum inhibitory concentration (MIC) and calculated minimum concentration of baicalin with inhibition effect on50%,90%TB strains respectively to obtain MIC50and MIC90. By preparation of modified L-J drug-containing medium with various baicalin concentration, and inoculation of clinical TB strains for4weeks in vitro environment in37℃incubator, mycobacterium tuberculosis after the inhibitory effects of varied concentration of baicalin is further laboratory processed (centrifugal rinsed, fixed, embedded, sectioned and double stained) then its cell morphological change observed with electronic microscope.ResultsAfter bacteria inhibition test of herbal extract baicalin, of60clinical Mycobacterium tuberculosis strains’MIC,1strains’MIC is grater than48mg/ml,1.67%;7strains’is12mg/ml,11.67%;25strains’is6mg/ml,41.67%;22strains’is3mg/ml,36.67%;5strains’is1.5mg/ml,8.33%. The strains whose MIC is between3mg/ml and6mg/ml is more than78.34%, while strains’MIC ranging from3mg/ml to12mg/ml more than90.01%. The number of strains’MIC at3mg/ml is close to that of MIC6mg/ml but number of strains’MIC at6mg/ml has been the largest proportion, indicating that most of the clinical Mycobacterium tuberculosis can be inhibited in this level in vitro. Mycobacterium tuberculosis after the inhibitory effects of varied concentration of baicalin and normal Mycobacterium tuberculosis cultured in blank media were further laboratory processed then its cell morphological change observed with electronic microscope. Normal cell morphology and inner structure of Mycobacterium tuberculosis can be clearly observed with projection electronic microscope. M. tuberculosis cell atrophied even degraded with the increase of inhibitory baicalin concentration, cell wall and membrane gradually dissolved partially or in total, part of cytoplasm condensed or scattered, electronic density reduced and silk-shaped DNA disappeared. All cell morphology of M. Tuberculosis becomes abnormal as baicalin concentration at3/4MIC, with inner structure obscured in whole, cell wall disappeared, cell membrane dissolved partially or in total, ribosomes particles rare, silk-shaped DNA not visible, nucleoplasm concentrated and electronic density reduced.ConclusionsBacteria inhibition tests on varied concentration of baicalin in vitro showed that Baicalin can effectively inhibit the growth of Mycobacterium tuberculosis, with Min MIC1.5mg/ml, Max MIC grater than48mg/ml, MIC50at6mg/ml and MIC90at12mg/ml. Observations cell morphology change of mycobacterium tuberculosis before and after the baicalin inhibition effect with electronic microscope showed that as baicalin concentration increased, damage to the mycobacterium tuberculosis was aggravating and number of damaged tuberculosis increased, especially to cell membrane and cell wall, which indicates the baicalin inhibits mycobacterium tuberculosis probably by shattering capsule, cell membrane and wall structure to degrade bacteria germs, concentrating its nucleoplasm.