| In recent years, drug resistance of E. coli, especially to the quinolones resistance (Qs) is becoming more and more serious, which lowered the Qs clinical treatment effect seriously, leaded to higher costs of treatment, increased breeding risk and drug dosage, caused food safety issues such as drug residues. Therefore, how to improve the therapeutic effect of Qs, not only relates to the healthy development of animal husbandry, but also social problems about food safety and human health. From characteristics and whether it can reverse of Qs drug resistance, resistance controling and reverse research focuses on new drug development and screening ideal efflux inhibitors. New drug research and development requires a lot of manpower, material resources, financial resources and time, and a new drug could quickly develop resistance, so it is difficult to solve the increasingly serious problem of drug resistance. Efflux inhibitors are materials which can effectively inhibit activity of drug transporters to bacteria, cancer cells (efflux pump), stop drug efflux, thereby eliminating the multiple drug resistance. Traditional Chinese medicine effective component which has an obvious advantage in product development and clinical application, because of safety, low residue, no resistance, etc. relatively.So the natural medicine which has the outside inhibitory activity can not only increase the sensitivity to drugs of pathogenic bacteria by efflux pump mediated drug-resistant, but also be conducive to reducing the generation of mutant strains of drug-resistant by outside efflux, has great clinical application value, has far-reaching significance for the treatment of drug-resistant bacteria infection and the healthy development of animal husbandry. This experiment made traditional Chinese medicine Mahonia Bealei as the research object which was screened preliminarily, extracted its main effective component berberine, studied whether it has multiple drug-resistant E. coli efflux quinolones, laid the ground for confirmtion of efflux pump inhibitor later, also provided theoretical basis for development of new efflux inhibitors. 1The preparation of berberine extractionUsing ethanol reflux extraction method to extract the effective components berberine in Mahonia Bealei, to identify with reference to the pharmacopoeia methods, bying high performance liquid chromatography (HPLC) for content determination of berberine extraction.Results:The berberine extraction rate was1.2%in Mahonia Bealei by ethanol refluxing extraction; By identifying all accord with berberine hydrochloride identification in Chinese pharmacopoeia2010version under experiment; The regression equation of the reference substance solution is:A=170931+167702C, the linear range of15to135μg/mL (r=0.9996), the content of berberine extraction was97.3%.Conclusion:Berberine extracted with the use of ethanol refluxing extraction, its purity was97.3%after refining, the method for extract is feasible; The results of established HPLC test are accurate and reliable in this test, can be used as the method for content determination of berberine extract.2Berberine extraction in vitro bacteriostasis experimentUsing broth dilution method recommended by U.S. clinical laboratory standards committee (NCCLS), determinating minimum bacteriostatic concentration (MIC)of ciprofloxacin lactate (Cip) and berberine extraction on quality control bacteria ATCC25922and drug-resistant E. coli73-2respectively, and making berberine extraction and ciprofloxacin lactate togather to detect the bacteriostatic effect of ciprofloxacin lactate, making the energy inhibitors carbonyl cyanide chlorobenzene hydrazone (CCCP) as a drug control.Results:The MIC of ciprofloxacin lactate on ATCC25922and drug-resistant E.coli73-2were0.0078μg/mL and256μg/mL, berberine extraction on ATCC25922and drug-resistant E. coli73-2were>500mg/mL and>500mg/mL. The MIC of Berberine extraction with ciprofloxacin lactate joint application on ATCC25922did not change, on resistant E. coli73-2to4μg/mL, reduced to1/64of the original; The MIC of CCCP with ciprofloxacin lactate joint application on ATCC25922did not change,on resistant E. coli73-2to8μg/mL, reduced to1/32of the original.Conclusion:Berberine extraction in combination with ciprofloxacin lactate can increase sensitivity of drug-resistant e. coli to ciprofloxacin lactate, showed good inhibitory effection of resistance, and the effect is better than that of the CCCP.3Berberine extraction effected on the tested strains of ciprofloxacin lactate accumulation volumeUsing high performance liquid chromatography (HPLC) method to detect volume of ciprofloxacin (Cip) in ATCC25922and drug-resistant E. coli73-2with and without berberine extraction, and making the CCCP as the drug control.Results:After joining the berberine extraction, in vivo ciprofloxacin lactate volume almost no change of quality control bacteria, increased significantly of drug-resistant E. coli73-2, it was about2.62times than that of without berberine extraction group; After joining CCCP, in vivo ciprofloxacin lactate volume almost no change of quality control bacteria, increased significantly of drug-resistant E. coli73-2, it was about2.25times than that of without CCCP group. Conclusion:Berberine extraction and CCCP both can increase the volume of ciprofloxacin lactate in drug-resistant E. coli73-2, showed good outside inhibition, and the former is better than the latter.4Berberine extraction effected on strain73-2gene expression quantity of acrABy berberine extraction processing drug-resistant E. coli73-2, using real-time fluorescent quantitative PCR to detect the quantity of acrA efflux pump gene expression.Results:Berberine extraction processing of drug-resistant E. coli model73-2acrA expression decreased, the quantity of efflux pump gene expression change to the original0.19times.Conclusion:Berberine extraction had obvious inhibitory effection on gene expression quantity of efflux pump in drug-resistant E. coli. |
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