抗马链球菌马亚种组方加味优选及其体内抑菌作用研究

【目的】筛选可抑制马腺疫主要致病菌——马链球菌马亚种的最优中药组方,为临床马腺疫的防治提供参考。【方法】选用赤芍、大黄、山豆根等27种中药,首先通过体外抑菌试验(牛津杯法、最小抑菌浓度(MIC)、最小杀菌浓度(MBC)、联合药敏试验)结合L₈(2⁶)正交试验初步筛选抗马链球菌马亚种中药组方的最佳优选因素,再通过L₁₈(3⁶)正交试验进一步筛选基础组方最佳配比;最后在基础组方的基础上,根据中兽医方剂配伍理论,添加黄芪和当归2味补虚药,通过马腺疫小鼠模型体内抑菌试验筛选加味组方配比,结合组方保护率结果及组织脏器病理变化验证加味组方的体内抑菌作用。【结果】试验筛选出了马链球菌马亚种超敏和高敏的7味中药:浙贝母、赤芍、甘草、大黄、雪白睡莲、山豆根和苦参。大黄、雪白睡莲、山豆根、甘草、苦参和浙贝母的MIC分别为0.078、0.078、0.125、0.156、0.156和0.250 mg/mL。雪白睡莲、赤芍、山豆根、甘草、大黄、苦参和浙贝母的MBC分别为0.078、0.125、0.125、0.156、0.156、0.156和0.250 g/mL。根据各单味药物的MIC和两两联合后的MIC计算得出浙贝母与雪白睡莲联合药敏指数＞2,二者存在颉颃作用,故剔删浙贝母。通过2次正交试验得出基础组方最佳配比为:赤芍∶甘草∶大黄∶雪白睡莲∶山豆根∶苦参＝4∶2∶2∶4∶4∶1;结合加味组方体内抑菌试验可知,黄芪∶当归=1∶1、配伍浓度为0.5 g/mL时,保护率可达87.5%,且中药组方对马链球菌马亚种所致的小鼠肺脏炎性损伤修复良好。【结论】本试验成功优选出1种对马腺疫主要致病菌——马链球菌马亚种具有良好抑菌、组织保护作用的中药组方,配比为:赤芍∶甘草∶大黄∶雪白睡莲∶山豆根∶苦参∶黄芪当归(1∶1)=4∶2∶2∶4∶4∶1∶1。

Study on the Optimization of Modified Formula for Inhibiting Streptococcus equi subspecies equi and Its Bacteriostatic Effect in Vivo

【Objective】 The aim of this study was to screen the optimal Chinese herbal formula that could inhibit the main pathogen of strangles Streptococcus equi subspecies equi,and provide a new reference for the prevention and treatment of strangles.【Method】 27 kinds of herbs including Radix Paeoniae Rubra,Rhubarb and Radix Sophorae Sinensis etc.were used as materials in this experiment,antibacterial Chinese herbal medicines was preliminary screened by in vitro antibacterial test (Oxford cup method,minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC),combined drug susceptibility test) and L₈(2⁶) orthogonal test,and then the formula for inhibiting Streptococcus equi subspecies equi was composed.Secondly,the optimal ratio of basic formula was further screened by L₁₈ (3⁶) orthogonal test.Finally,under the premise of basic formula,according to the theory of compatibility theory of Chinese herbal formula,two tonifying drugs were added,the ratio of modified formula was screened by in vivo bacteriostasis test of strangles mice model,and the antibacterial effect of modified formula was verified in combination with formula protection rate and pathological changes of tissues and organs.【Result】 In the experiment,seven herbs (Fritillaria thunbergii,Radix Paeoniae Licorice root,Rhubarb,Nymphaea candida,Sophora root,Sophora flavescens) were selected,which were super sensitive and highly sensitive to Streptococcus equi subspecies equi,The MIC of Rhubarb,Nymphaea candida,Sophora root,Radix Paeoniae Rubra,Sophora flavescenswere 0.078,0.078,0.125,0.156,0.156 and 0.25 mg/mL,respectively.The MBC of Nymphaea candida,Radix Paeoniae,Sophora root,Licorice root,Rhubarb,Sophora flavescens,Fritillaria thunbergii were 0.078,0.125,0.125,0.156,0.156,0.156 and 0.25 g/mL,respectively.According to the MIC values of each single herb and the MIC values after pairwise combination,it was calculated that the combined herb sensitivity index of Fritillaria thunbergii and Nymphaea candida was＞2,which was antagonistic,so Fritillaria thunbergii was deleted.Then the optimal ratio of the basic formula was obtained by two orthogonal tests as follows:Rhubarb∶Sophora flavescens∶Sophora root∶Radix Paeoniae Rubra∶Licorice root∶Nymphaea candida= 4∶2∶2∶4∶1.Combined with the in vivo bacteriostasis test of modified formula,the protection rate was 87.5% when the compatibility concentration of Astragalus membranaceus∶Angelica sinensis = 1∶1 was 0.5 mg/mL,and Chinese herbal formula could repair inflammatory injury of lung induced by Streptococcus equi subspecies equi in mice.【Conclusion】 In conclusion,the study successfully screened out a new Chinese medicine formula with good antibacterial and tissue protection effects on Streptococcus equi subspecies equi.The ratio was Rhubarb∶Sophora flavescens∶Sophora root∶Radix Paeoniae Rubra:Licorice root∶Nymphaea candida∶Astragalus-Angelica(1∶1)= 4∶2∶2∶4∶4∶1∶1.