Screening Of Traditional Chinese Medicines Against Streptococcus Agalactiae And Study On Anti-Streptococcus Agalactiae Preliminary Mechanism Of Berberine

Streptococcus agalactiae (S. agalactiae) is the pathogens dairy cow mastitis, which takes the serious effect on the quality of milk. At present, the treatment of dairy cow mastitis mainly rely on antibiotics. However, the repeated use of antibiotics causes antibiotic residues in milk and the emergence of resistant strains makes the treatment fall. It is difficulty for Chinese herbal medicine to develop the drug resistance. Aslo, it has the advantages of low toxicity and low residue. Therefore, we screened the traditional Chinese medicine which have good antibacterial effects on S. agalactiae from 20 traditional Chinese medicines by this study. Then the traditional Chinese medicine extraction process was optimized. Under the conditions of the optimal extraction process, antibacterial activity of Coptis chinensis to the main pathogenic bacteria which can cause dairy cow mastitis was studied in vitro. Next, we regarded the monomer that exist in the traditional Chinese medicines with good anti-bacterial effect on S. agalactiae as the research object and studied on preliminary mechanism of the monomer against S. agalactiae. The main research content were as follows:1 Screening for the traditional Chinese medicines which have good antibacterial effects on Streptococcus agalactiae in vitro. Using ethanol reflux and ultrasonic method, the extracts of 20 different traditional Chinese medicines were obtained. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration(MBC) of Coptis chinensis and other 19 extracts of traditional Chinese medicines on S. agalactiae were detected by macrodilution broth method. The result of antimicrobial activity of active ingredients extracts of twenty kinds of tradition Chinese medicines to S. agalactiae in vitro showed that:The MICs and MBCs of the extracts obtained from Coptis chinensis, Scutellaria baicalensis, Polygonum cuspidatum and Sanguisorba officinalis against S. agalactiae ranged from 7.8mg/mL～15.625mg/mL; The MICs of the extracts obtained from Taraxacum mongolicum, Prunus mume, Artemisia scoparia, Fraxinus rhynchophylla, Artemisia argyi and Violae yedoensis against S. agalactiae ranged from 31.25mg/mL-62.5mg/mL and the MBCs ranged from 62.5mg/mL-125mg/mL; The MICs and MBCs of the extracts obtained from Phellodendron chinense, Houttuynia cordata and Pulsatilla chinensis against S. agalactiae were 125mg/mL; The MICs and MBCs of the extracts obtained from Sophora flavescens, Lonicera japonica, Andrographis paniculata, Eucommia ulmoides, Forsythia suspensa, Prunella vulgaris and Po. Multiflorum against S. agalactiae ranged from 250mg/mL-500mg/mL.Conclusion:Coptis chinensis had good anti-bacterial effect on S. agalactiae.2 Study on extraction process of Coptis chinensis against S. Agalactiae and antibacterial activity in vitro. L9(34) orthogonal was applied to select the best extraction process conditions of Coptis chinensis. In order to establish the best methods for the extraction of Coptis chinensis, we used extractive yield, minimum inhibition concentration (MIC) and minimal bactericidal concentration (MBC) against S. agalactiae as comprehensive evaluation index. Under the conditions of the optimal extraction process of Coptis chinensis, MIC and MBC of Coptis chinensis on the main pathogenic bacteria (S. agalactiae, Escherichia coli, Staphylococcus aureus) of dairy cow mastitis were detected by macrodilution broth method. The result of the best extraction conditions of Coptis chinensis showed that Coptidis Rhizoma was added to 8 times the amount of 80% ethanol, which were refluxed and extracted for 2 times and the each extraction time was 1.5h. The result of anti-bacterial activity in vitro showed that:the MIC and MBC of the extracts obtained from Coptis chinensis against S. agalactiae were 3.9mg/mL; the MIC and MBC against E. coli were 62.5mg/mL and 125mg/mL; the MIC and MBC against S. aureus were 31.25mg/mL.Conclusion:Bacterial effect of Coptis chinensis extraction under the best extraction conditions was better. Besides, it had the anti-bacterial activity against E. coli and S. aureus.3 The effect of Berberine on morphological structure and Lactate Dehydrogenase(LDH) activity of S. agalactiae. Using the method of plate dilution, the time-kill curves of Berberine on S. Agalactiae were obtained. The effect of Berberine on the ultratructure of S. agalactiae was observed by transmission electron microscope. In order to study the effect of Berberine on enzyme activity of S. agalactiae, the Lactate Dehydrogenase activity was determined. The result of time-kill curves showed that:At the concentration of MIC, Berberine can inhibit the growth of bacteria; At the concentration of 2MIC, Berberine can kill all the bacteria. The result of TEM showed that:normal S. agalactiae cells were shaped cells with intact cell walls, smooth membranes, a uniformly distributed cytoplasm and clear nuclear area in the middle of cells. Besides, cells stained evenly; The shape of S. agalactiae cells treated with berberine became irregular; Some cell walls and membranes were dissolved; There was loss of cell integrity and the cytoplasmic contents were leaking out of the cells; Besides, some cells stained unevenly and nuclear areas were straggling in the cells. The result of enzyme activity showed that:Lactate Dehydrogenase activity of S. agalactiae treated with berberine declined.Conclusion:The time-kill curves showed the correlation of concentration-time of Berberine against S. agalactiae. Berberine can damage the structure of bacterial cell membrane and the damage increased with time, as well as decreasing the Lactate Dehydrogenase activity.4 The effect of Berberine on Proteins and DNA of S. agalactiae. The effect of Berberine on Proteins and DNA of S. Agalactiae was studied by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) study and Nucleic acid fluorescent staining of 4’, 6-diamidino-2-phenylindole (DAPI). The result of SDS-PAGE showed that:Protein bands observed for untreated S. agalactiae were more than the treated cells; Some protein bands of treated bacteria became low and even disappeared. The result of fluorescence microscope showed that:The fluorescence intensity of treated S. agalactiae DNA were weaker than untreated S. agalactiae DNA.Conclusion:Berberine can inhibit synthesis of protein and DNA.