Development And Clinical Curative Effect Of Breast Injection Agent Cefquinme Sulfate And Extract Of Ligustrum Lucidum

Bovine mastitis is an inflammation of the udder caused by pathogenic microorganism infection,which can lead to the decrease of milk production and milk quality,and seriously affect the development of dairy farming.Antibiotics have always been the drug of choice for the treatment of cow mastitis caused by pathogenic microorganisms such as bacteria.Cefquinome sulfate is the fourth generation of cephalosporin antibiotics approved for the treatment of dairy cow mastitis in China.It has a great killing effect on the pathogenic bacteria causing dairy cow mastitis,such as Staphylococcus aureus,Escherichia coli and Streptococcus agalactiae.However,with the extensive use ofβ-lactam antibiotics,mastitis pathogens have developed resistance to most of theβ-lactam antibiotics,including cefquinome.The production ofβ-lactamase is the main mechanism by which bacteria develop resistance toβ-lactam antibiotics.Therefore,screening effective inhibitors of resistant enzymes to reverse the resistance of pathogens toβ-lactam antibiotics is a feasible strategy to solve the serious problem of resistance of pathogens of mastitis.Previous studies of our group found that oleanolic acid can enhance the antibacterial activity ofβ-lactam antibiotics againstβ-lactam producing pathogens.Therefore,screening effective drug-resistant enzyme inhibitors to reverse the resistance of pathogenic bacteria toβ-lactam antibiotics is a feasible strategy to solve the current serious drug resistance problem of mastitis pathogens.Previous studies by our group found that oleanolic acid could enhance the antibacterial activity ofβ-lactam antibiotics againstβ-lactamase-producing pathogens.Therefore,this thesis intends to carry out drug development on the Chinese herbal medicine Ligustrum lucidum containing oleanolic acid on the basis of existing research,aiming to develop a compound breast injection that combines Chinese and Western medicine（Cefquinome Sulfate and Ligustrum Lucidum）to target the clinical treatment of dairy cow mastitis caused by drug-resistant bacterial infection.Further,the preservation conditions and the rationality of prescription of the preparation were determined by investigating the stability of the preparation,and the safety evaluation and clinical treatment of the preparation were carried out.Firstly,the formulation and process research of breast injection agent cefquinome sulfate and extract of Ligustrum lucidum was carried out.In this study,the formula of the compound preparation was finally determined through formula screening and process research:8 g breast injection agent of cefquinome sulfate and extract of Ligustrum lucidum contains 60 mg cefquinome sulfate,300 mg Ligustrum lucidum extract（80%oleanolic acid）,80 mg glycerin monostearate,80 mg hydrogenated castor oil,8 mg vitamin E and soybean oil for injection.After preliminarily dispersing the above substances by a colloid mill,at a pressure of 1000 bar,under the condition of continuous cooling cycle,it is sheared by a high-pressure homogenizer,and finally a breast injection with uniform texture,high dispersion and stable properties is prepared.In order to effectively control the quality of the breast injection agent of cefquinome sulfate and extract of Ligustrum lucidum,this study established a content determination method by HPLC.Through the test of influencing factors,it was determined that the breast injection of cefquinome sulfate and extract of Ligustrum lucidum should be stored in a sealed,dark and cool place.Through accelerated test investigation,the results show that there is no significant change in each index within6 months,and it is a uniform milky white suspension with strong fluidity and stable preparation content.Through the long-term stability test,the results show that the breast injection agent of cefquinome sulfate and extract of Ligustrum lucidum should be stored at room temperature and away from light,and the validity period is tentatively set to 9months.To evaluate the safety of this preparation,the potential toxicity of cefquinome sulfate and extract of Ligustrum lucidum breast injection on primary bovine mammary epithelial cells was detected by the release of lactate dehydrogenase（LDH）,and the results showed that below 1 mg/m L had no toxic side effects on bovine mammary epithelial cells;The mucosal stimulation test of mice showed that the compound breast injection had no stimulation effect on the mucosal of mice after continuous administration for 3 days.Further,the potential acute toxicity of the compound preparation on mice was detected by oral gavage.The results showed that the compound preparation had an LD₅₀>5000 mg/kg in experimental mice,indicating that this preparation was safe and non-toxic,and it was a follow-up clinical trial.The successful development has laid a preliminary foundation for toxicology research.In order to further determine the clinical therapeutic effect of the breast injection of cefquinome sulfate and extract of Ligustrum Lucidum,this study selected dairy cows suffering from mastitis with similar clinical symptoms to investigate the therapeutic effect.Through observation and detection of continuous administration(12 h/time,Clinical symptoms,milk traits,somatic cell count（SCC）and clearance of pathogenic bacteria were evaluated.The results showed that the cure rates of the high dose group,medium dose group and low dose group were 80%,70%and 30%respectively on the14th day after treatment.To sum up,this study took dairy cow mastitis as the research object,established a breast injection preparation of the breast injection of cefquinome sulfate and extract of Ligustrum Lucidum,which can treat dairy cow mastitis,and determined the preparation technology and content determination method of the preparation.The storage conditions and validity period were determined;the breast injection of cefquinome sulfate and extract of Ligustrum Lucidum was prepared in this study is non-toxic and non-irritating;in the clinical efficacy study,the breast injection of cefquinome sulfate and extract of Ligustrum Lucidum can effectively treat clinical dairy cow mastitis.