The Study On The Antibacterial Effect Of Niu Bei Xiao He Of Traditional Chinese Medicine In Vivo And In Vitro Experiments

Objective:To study the effects of different processing products of Niubeixiaohe on Mycobacterium tuberculosis (M.tb) in vitro and in vivo, to provide a new Chinese medicine for the clinical treatment of tuberculosis especially multiple resistant tuberculosis. To study the effects of of different processing products of Niubeixiaohe on respiratory common bacteria in vitro, to find their antibacterial spectrum and provide a basis for the prevention and treatment of tuberculosis complicated with bacterial infection.Method:1. The research on the inhibitory effects of Niubeixiaohe on BCG, M.tb and multidrug-resistant M.tb in vitro:10-3mg BCG,10’3mg M.tb H37Rv, and10-3mg MDR-M.tb were inoculated respectively on improved L-J culture medium without the drug and with different concentrations of drugs, incubated at37℃for4weeks, and then observed the results.2. The research on the inhibitory effects of Niubeixiaohe on the respiratory common bacteria in vitro:104CFUs of clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, Enterobacter cloacae were inoculated respectively on MH culture medium without drug and with different concentrations of drugs, incubated at35℃overnight, and then observed the results of drug sensitivity.3. The treatment effect of Niubeixiaohe on the mouse tuberculosis model. One hundreds and three of female BALB/c mice were challenged via tail vein with5×105CFUs of M.tb H37Rv suspension, and then the mice were randomly divided into the following15groups to treat at the seventh day after infection:the blank control group (0.5ml water), M. vaccae treatment group (22.5μg/100μl/each mouse/time), INH treatment group (0.4mg/day), the low dose of Niubeixiaohe Ⅰ group (3.3mg/day), the middle dose of Niubeixiaohe Ⅰ group (6.7mg/day), the high dose of Niubeixiaohe Ⅰ group (13.3mg/day), the low dose of Niubeixiaohe Ⅱ group (2mg/day), the middle dose of Niubeixiaohe Ⅱ group (4mg/day), the high dose of Niubeixiaohe Ⅱ group (8mg/day), the low dose of Niubeixiaohe Ⅲ group (1.67mg/day), the middle dose of Niubeixiaohe Ⅲ group (3.35mg/day), the high dose of Niubeixiaohe Ⅲ group (6.67mg/day), the low dose of Niubeixiaohe Ⅳ group (0.75mg/day), the middle dose of Niubeixiaohe Ⅳ group (1.5mg/day) and the high dose of Niubeixiaohe Ⅳ group (3mg/day). The mouse’s dosages were determined according to clinical drug effective dose in adult and the weight conversion.0.5ml of Niubeixiaohe and INH were given by intragastric administration for5times each week (except Saturday and Sunday) and continuous3months. The mice in M. vaccae treatment group were injected intramuscularly for3times at2week intervals. The body weights of the mice were observed weekly. The pathological changes (for example, organ size, extent of the lesion, surface nodules, etc.) of lungs, livers, spleens each mouse were observed after the treatment for3months. The lung histopathological changes in mouse tuberculosis model group were observed by HE staining. The colony counts of spleens each group were performed on improved L-J culture medium for4weeks.Result:1. The minimum bactericidal concentrations (MBCs) of Niubeixiaohe Ⅱ, Ⅲ, Ⅳ on BCG were50mg/ml,25mg/ml and12.5mg/ml, respectively. Compared with the blank control group, the minimum inhibitory concentrations (MICs) of Niubeixiaohe Ⅱ, Ⅲ, Ⅳ on BCG were12.5-25mg/ml,12.5-20mg/ml and10mg/ml, respectively. The MBCs of Niubeixiaohe Ⅱ, Ⅲ, Ⅳ on M.tb H37Rv were100mg/ml,100mg/ml and25mg/ml, respectively. Compared with the blank control group, the MICs of Niubeixiaohe Ⅱ, Ⅲ, Ⅳ on M.tb H37Rv were50mg/ml,50mg/ml and12.5-25mg/ml, respectively.200mg/ml of Niubeixiaohe Ⅱ, Ⅲ, Ⅳ had some inhibitory effects on multidrug-resistant M.tb.2. Compared with the blank control group, the bactericidal concentrations of Gentamicin, Sulfamethoxazole, Amikacin, Niubeixiaohe Ⅱ, Ⅲ, Ⅳ on Staphylococcus were1-100μg/ml,0.1-10μg/ml,1-100μg/ml,160mg/ml,160mg/ml and≤40mg/ml, respectively; on Acinetobacter baumannii were10μg/ml,1-10μg/ml,10μg/ml,160mg/ml,80-160mg/ml and40mg/ml, respectively; on Staphylococcus epidermidis were1μg/ml,0.1-20μg/ml,0.1-100μg/ml,40mg/ml,80mg/ml,<20mg/ml, respectively; on E. coli were100μg/ml, resistant,10μg/ml,160mg/ml,160mg/ml and80mg/ml, respectively; on Enterococcus were≤0.01μg/ml,≤1μg/ml,100μg/ml,160mg/ml,160mg/ml,80mg/ml, respectively; on Klebsiella pneumoniae were1μg/ml,1-20μg/ml, not done, contaminated, contaminated,40mg/ml, respectively; on Pseudomonas aeruginosa were10μg/ml,20μg/ml,10μg/ml,160mg/ml,160mg/ml,40mg/ml, respectively; on Enterobacter cloacae were100μg/ml, resistant,100μg/ml, contaminated, contaminated, 80mg/ml, respectively.3. The body weights of the mice each group increased at different degree after treatment than before treatment, in which Niubeixiaohe nuclear groups were higher than the distilled water group. Compared with the distilled water group, the spleen colony counts in M. vaccae treatment group, INH treatment group, the low dose group of Niubeixiaohe Ⅰ, the middle dose group of Niubeixiaohe Ⅰ, the high dose group of Niubeixiaohe Ⅰ, the low dose group of Niubeixiaohe Ⅱ, the middle dose group of Niubeixiaohe Ⅱ, the high dose group of Niubeixiaohe Ⅱ, the low dose group of Niubeixiaohe Ⅲ, the middle dose group of Niubeixiaohe Ⅲ, the high dose group of Niubeixiaohe Ⅲ, the low dose group of Niubeixiaohe IV, the middle dose group of Niubeixiaohe Ⅳ and the high dose group of Niubeixiaohe Ⅳ reduced by0.37,3.04,0.43,0.22,0.38,0.24,0.29,0.46,0.73,0.17,0.35,0.21,0.36,0.58logs. Pulmonary general pathology displayed that the lung lesions in treatment group were ameliorated at certain degree and the normal lung structure could be observed.Conclusions:1. Different processing products of Niubeixiaohe had obviously inhibitory effects in vitro on BCG and M.tb H37Rv, in which the ethanol extracts of Niubeixiaohe had best inhibitory effect. Three extracts of Niubeixiaohe had some inhibitory effect on multidrμg-resistant M.tb.2. Different processing products of Niubeixiaohe all had some inhibitory effects in vitro on Staphylococcus aureus, Staphylococcus epidermidis, Bauman Acinetobacter, Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae,in which the ethanol extracts of Niubeixiaohe had best inhibitory effect.3. Different processing products of Niubeixiaohe had therapeutic effects on the mice infected with M.tb in vivo, could inhibit or kill M.tb, reduce the severity of tuberculosis, improve the general condition.