| Objective:To understand the immunological, general pharmacological effects and security of the anti-tuberculosis Chinese medicine Niubeixiaohe--the aqueous extracts (NO.3) and the ethanol extracts (NO.4) of Niubeixiaohe, in order to provide the reliable experimental basis for it to be Chinese patent medicine.Methods:(1) The research on the immunological effects of the anti-tuberculosis Chinese medicine Niubeixiaohe:①The aqueous extracts and ethanol extracts of the the Niubeixiaohe in the normal mice:The64BALB/c mice were randomly divided into8groups as follow:blank control group, M.vaccae group, low dose group of Niubeixiaohe NO.3, middle dose group of Niubeixiaohe NO.3, high dose group of Niubeixiaohe NO.3, low dose group of Niubeixiaohe NO.4, middle dose group of Niubeixiaohe NO.4, high dose group of Niubeixiaohe NO.4. The mice were given distilled water or different doses of Niubeixiaohe extract by alimentary canal for6weeks, but the mice in M.vaccae group need be injected intramuscularly with M.vaccae vaccine and the blank group were also given saline by intramuscular injection one time at2weeks interval, totally injected3times. The mice were killed and taken blood by removing the eyeball, then anatomized and taken the thymuses and spleens. The ratios of CD3+、CD4+、CD8+T lymphocytes and the levels of cytokines IL-4, IL-6, IFN-y, TNF-a, IL-17A and IL-10in blood were detected by the flow cytometry. The spleen index and thymus index of the mice were calculated and assessed. The proliferative responses of spleen lymphocytes were determined by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay. The levels of IFN-γ and IL-4in the spleen culture supernatants were detected by ELISA assay.②The effects of the aqueous extracts and ethanol extract of Niubeixiaohe on the mouse peritoneal macrophages:The56BALB/c were randomly divided into7groups as follow:blank control group, low dose group of Niubeixiaohe NO.3, middle dose group of Niubeixiaohe NO.3, high dose group of Niubeixiaohe NO.3, low dose group of Niubeixiaohe NO.4, middle dose group of Niubeixiaohe NO.4, high dose group of Niubeixiaohe NO.4. The mice were given distilled water or Niubeixiaohe by alimentary canal for4weeks. On the second day of the last administration, the mice were killed by cervical dislocation and then collected the peritoneal macrophages. The BCG gene was amplified in the mouse peritioneal macrophages by PCR. The IL-1β, TNF-a and IL-6levels in macrophage culture supernatants were detected by ELISA assay.(2) The research on the general pharmacological effects of the aqueous extracts and ethanol extract of Niubeixiaohe:①The effects of the aqueous extracts and ethanol extract of Niubeixiaohe on the neurological system:The KM mice were randomly divided into7groups as follow:blank control group, low dose group of Niubeixiaohe NO.3, middle dose group of Niubeixiaohe NO.3, high dose group of Niubeixiaohe NO.3, low dose group of Niubeixiaohe NO.4, middle dose group of Niubeixiaohe NO.4, high dose group of Niubeixiaohe NO.4. There were10mice each group. The mice were given distilled water or Niubeixiaohe by alimentary canal for7days. After30min of the last administration, the general behavior, righting reflex, sleeping time, analgesia response of neurological system were observed.②The effects of the aqueous extracts and ethanol extract of Niubeixiaohe on liver functions:The KM mice were randomly divided into7groups as follow:blank control group, low dose group of Niubeixiaohe NO.3, middle dose group of Niubeixiaohe NO.3, high dose group of Niubeixiaohe NO.3, low dose group of Niubeixiaohe NO.4, middle dose group of Niubeixiaohe NO.4, high dose group of Niubeixiaohe NO.4. There were10mice each group. The mice were given distilled water or Niubeixiaohe by alimentary canal for7days. The mice were free to drink water without food for8hours before the last administration. After30min of the last administration, the mice were killed and taken blood by removing the eyeball. And then the sera were separated by centrifugating at3000rpm for5min. The liver function indexes including the levels of ALT, AST, TBIL, DBIL, TP, ALB, ALP, γ-GGT in sera were detected.③The effects of the aqueous extracts and ethanol extracts of Niubeixiaohe on the renal function:The KM mice were randomly divided into7groups as follow:blank control group, low dose group of Niubeixiaohe NO.3, middle dose group of Niubeixiaohe NO.3, high dose group of Niubeixiaohe NO.3, low dose group of Niubeixiaohe NO.4, middle dose group of Niubeixiaohe NO.4, high dose group of Niubeixiaohe NO.4. There were10mice each group. The mice were given distilled water or Niubeixiaohe by alimentary canal for7days. After last administration, the mice were put into metabolic cage to collect the urine for240min. Then the mice were killed and taken blood by removing the eyeball. The sera were separated by centrifugating at3000rpm for5min. The levels of creatinine and urea nitrogen in the sera from the mice and the levels of Na+, K+in urine were determined.(3) Research on the acute toxicity test of the aqueous extracts and ethanol extract of Niubeixiaohe:The KM mice were divided into control group and experimental groups. The experimental groups were given5times,10times and20times clinical dosage of Niubeixiaohe’s by alimentary canal, all drugs must be finished within24hours. The mice were observed for14days after administration. The body weight, general behavior and the morphological changes of heart, liver, spleen, lung, kidney, and organ index in mice were evaluated.Results:(1) The research on the immunological effects of the anti-tuberculosis Chinese medicine Niubeixiaohe:①The aqueous extracts and ethanol extracts of the Niubeixiaohe in the normal mice:There was no significant difference in spleen index and thymus index between the drug groups and the blank control group (P>0.05). After the mouse spleen lymphocytes each group were stimulated by PHA (Phytohemagglutinin) and BCG, respectively for48hours, the spleen lymphocyte proliferation indices in drug groups had no significant difference compared with blank control group (P>0.05). The mouse spleen lymphocytes each group were stimulated by PHA and incubated for72hours, the levels of IFN-y and IL-4in spleen lymphocyte culture supernatants showed no significant difference between drug groups and blank control group (P>0.05). The percentages of CD3+, CD4+, CD8+T lymphocytes and the levels of cytokines IL-4, IL-6, IFN-γ, TNF-α, IL-17A and IL-10in the blood showed no statistical difference between drug groups and blank control group (P>0.05).②The effects of the aqueous extracts and ethanol extracts of Niubeixiaohe on the mouse peritoneal macrophages:The BCG gene in Mφ+BCG wells each group could be amplified and showed positive bands by PCR, while there were no obvious band in Mφ wells and BCG wells. In macrophage culture supernatants, the levels of IL-6and IL-1βeach group had no significant difference comparing Mφwells with MφBCG wells (P>0.05), but the levels of TNF-α in Mφ+BCG wells each group were significantly higher than that in Mφ wells (P<0.05). The levels of IL-6, IL-1βand TNF-α in Mφ wells and Mcp+BCG wells showed no significant difference comparing drug groups with blank control group (P>0.05).(2) The research on the general pharmacology of the aqueous extracts and ethanol extracts of Niubeixiaohe:There were no obvious effects of Niubeixiaohe on the general behavior, righting reflex, sleeping time, analgesia response of neurological system, levels of creatinine and urea nitrogen, ALT, AST, TBIL, DBIL, TP, ALB, ALP, γ-GGT in mouse sera and the levels of Na+, K+in urines.(3) Research on the acute toxicity test of the aqueous extracts and ethanol extract of Niubeixiaohe:It is an increasing trend on the body weight, the hair of mice is shiny, the movement is freedom. There is no statistical difference of organs index among all groups.Conclusions:(1) The aqueous extracts and ethanol extracts of the Niubeixiaohe in the normal mice had no obvious effect on the immune function of normal mice.(2) There was no obvious effect on neurological system and the function of liver and kidney.(3) The aqueous extracts and ethanol extract of Niubeixiaohe had no acute toxicity reaction. It is safe. |
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