Screening Of Antibacterial Synerists From Chinese Herbal Medicine For Tuberculosis Bacteria In Deer & Effect Of Coptis Chinensis Alkaloid On Transcription Level Of PhoPR Two-component System Genes

Tuberculosis(TB)is a type of chronic and wasting zoonosis disease wich is caused by Mycobacterium tuberculosis wich is gram-positive bacteriam. Tuberculosis is not only a threat to the life and safety of animals, but also affects the survival of human beings. At present, the way to treat tuberculosis is to take first and second-line drugs. However, the unresonable use of drugs results in the difficulty of the treatment of drug-resistant tuberculosis. Therefore it is imperative to find a new treatment plan. Chinese herbal medicines as the abundant natural resource in China hardly produce drug resistance and side effects for the treatment of diseases.The aim of this study was to screen out ideal and novel multi-drug resistance reversal agents and Antibacterial Synerists from traditional Chinese medicine to inhibit mycobacterium tuberculosis. The mechanism of antibacterial and synergistic effect was also explored preliminarily.44 strains of TB in deer were used to detect multi-drug resistance by ratio method. The result of the study showed that MT-7 and MB-1 were defined to be multi-drug resistant Mycobacterium tuberculosis and used as model bacteria. 24 kinds of Chinese herbal medicines extracted by water and ethanol were applied to detect sensitivity MT-7, MB-1 and H37 Rv by Microdilution alamar blueassay(MABA) method. The data clearly demonstrated that Coptidis rhizoma, Angelicae pubescentis radix and Platycladi cacumen showed greater antimicrobial activity against drug-resistance Mycobacterium tuberculosis and H37 Rv. The antibacterial effects of extracts from Coptidis rhizoma were identical to that of berberine. The extracts combined with INH and RFP were applied to detect sensitivity to MT-7, MB-1 and H37 Rv by method of checkerboard. The result demonstrated synergistic effect when ethanol extracts from Coptidis rhizome were combined with RFP, indicating that ethanol extracts from Coptidis rhizome was Antibacterial Synerists of RFP.Extraction and purification of total alkaloids from the aqueous and ethanol extracts of Coptidis rhizoma were testified to detect its active ingredient concentration by nucleic acid dye method and compare with standard variety by MABA method. The results showed that the total alkaloid content of purified was the highest and was the most effective against Mycobacterium tuberculosis in all extractions of Coptidis rhizome. Compared with the berberine, the Coptis chinensis total alkaloid had better antibacterial effect.The Coptis chinensis total alkaloid which was combined with 6 kinds of anti tuberculosis drugs were applied to detect sensitivity toMT-7、MB-1 and H37 Rv by method of checkerboard. Meanwhile, the Coptis chinensis total alkaloid and berberine were used to detect cytotoxicity of cell. The FIC of alkaloids from Coptidis rhizoma combined with RFP was ≤0.5, which demonstrated that both had a synergistic role. This result indicated that Coptis chinensis total alkaloid was Antibacterial Synerists of RFP.Coptis chinensis total alkaloid and berberine showed toxicity when the concentration of total alkaloids from Coptidis rhizoma and berberine were≥100μg/mL, which was far higher than MIC.The different concentrations of Coptis chinensis alkaloid or in combination with RFP were applied to detect sensitivity to MT-7, MB-1 and H37 Rv. Then the total RNA was extract. We analyzed and compared with expression level of PhoPR and PhoP gene belonging to the PhoPR two-component system which was related to drug resistance with realtime fluores-cence quantitative PCR. The results showed that the expression of level oPhoP and phoR of MT-7,MB-1 was decreased significantly with the high concentration of total alkaloids from Coptidis rhizoma, which may reduce drug resistance of MB-1and MT-7. The expression of level oPhoP and phoR of MT-7and MB-1 was decreased significantly by Coptis chinensis total alkaloid combined with RFP. The results showed that Coptis chinensis alkaloid was Antibacterial Synerists of RFP. The antibacterial efficiency mechanismof Coptis chinensis total alkaloid or in combination with RFP needs to be further studied.Coptidis rhizoma as the Antibacterial Synerist was screened successfully from 24 kinds of Chinese herbal medicines which was foundation on solving drug resistance of Mycobacterium tuberculosis from animals.