Study On Screening And Pharmacodynamic Evaluation Of Chinese Herbal Formula For Prevention And Treatment Of Strangles

Strangles is a bacterial epidemic disease caused by Streptococcus equi subspecies equi（S.equi）,which is highly contagious and acute contagious,showing typical clinical symptoms such as fever,mental depression,mandibular swallow lymph node swelling,and respiratory distress in severe cases.At present,the treatment of strangles is mainly controlled by antibiotics and vaccines.However,with the emergence of drug-resistant strains and mutant strains,the control effect of antibiotics and vaccines is still not ideal.Chinese veterinary drugs have the characteristics of small toxicity and broad spectrum,which provides a new idea for the prevention and treatment of strangles.Therefore,in this study,we intend to create a traditional Chinese medicine formula that can prevent and treat strangles.At the same time,the pharmacodynamics of formula was verified by experiments in vitro and in vivo.The antibacterial mechanism of the formula against S.equi was preliminarily investigated.According to relevant literature and book records,27 single herbs with high frequency and strong efficacy were selected as experimental materials,and S.equi isolate in Xinjiang preserved in the laboratory was used as the target strain to carry out relevant experiments.The optimal factors of anti-S.equi herbs were preliminarily screened by in vitro antibacterial test（Oxford cup method,minimum inhibitory concentration,minimum bactericidal concentration,combined drug sensitivity test）,combined with L₈（2⁶）orthogonal test.Then through L₁₈（3⁶）orthogonal test,the optimal ratio of the base formula was optimized.Finally,according to the theory of Chinese veterinarians,two tonifying herbs were added to the basic formula,and the optimal ratio of the modified formula was obtained through the in vivo protection rate test combined with the pathological changes of tissues and organs to verify the in vivo antibacterial effect of each group of the modified formula.The results showed that S.equi was sensitive to 7 herbs,and the combined drug sensitivity index of Fritillaria thunbergii and Nymphaea candida was>2,which was antagonistic,so Fritillaria thunbergii was deleted.The optimal ratio of the basic formula was Chishao:Glycyrrhiza uralensis:Rheum officinale:Nymphaea candida:Sophora flavescens root:Sophora flavescens=4:2:2:4:4:1;from the results of the modification test,when the compatibility concentration of Astragalus membranaceus:Angelica sinensis=1:1 was 500 mg/m L,the protective rate of the optimal formula could reach 87.5%,and the modified groups of formula repaired the inflammatory injury of the lungs of mice induced by S.equi well,and the final optimized modified formula was named formula-1.Based on the requirements for new drug development,this study verified the safety and effectiveness of formula clinical medication through acute toxicity test,antibacterial test in vitro（Oxford cup method,minimum inhibitory concentration,minimum bactericidal concentration）and treatment test in vivo of Strangles mice model.The acute toxicity test showed that formula-1 had very low toxicity,the maximum tolerated dose was 480 g/kg,which could ensure the safety of normal clinical medication.antibacterial test in vitro showed that S.equi was highly sensitive to formula-1,with an inhibition zone diameter of 15.56mm,a minimum inhibitory concentration of 0.3125 g/m L,and a minimum bactericidal concentration of0.1563 g/m L.treatment test in vivo showed that formula-1 had a protection rate of 75%against Strangles mouse model,It can effectively reduce the invasiveness of S.equi to mice.Finally,the effects of formula-1 on the growth（cell membrane and cell wall integrity,permeability,bacterial morphological changes）,and virulence factor expressions（hyaluronidase,hemolysin S activity,FNEB-anchored protein expression）of S.equi were evaluated,and the Antibacterial mechanism of formula-1 in the treatment of Strangles was preliminarily explored.The results showed that formula-1 could inhibit the growth of S.equi by destroying the integrity of cell wall and increasing the permeability of cell membrane,while formula-1 could effectively inhibit the activities of hyaluronidase and hemolysin S,and significantly reduce the expression level of FNEB-anchored protein.In summary,this study successfully developed a traditional Chinese medicine formula that can effectively inhibit S.equi,which can provide some technical support for clinical treatment of Strangles.