TMP对中药抗菌增效作用及其复方对免疫功能影响的研究
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摘要
本研究根据中药药理学和兽医药理学理论,对TMP与中药联用抗菌增效作用的量效关系及其联用对机体免疫机能的影响进行研究,从而为TMP与中药抗菌复方制剂的研制提供科学依据,为TMP与中药联用预防和治疗畜禽疾病奠定理论基础。
本试验采用试管二倍稀释法测定TMP与单味中药对金黄色葡萄球菌、大肠杆菌和沙门氏菌的最小抑菌浓度(MIC)。选择黄芩、秦皮、白头翁和苦参4味中药,依据其MIC值设定高剂量、中剂量、低剂量3种水平,按L9(34)正交设计9个组方,然后对各组方的体外抗菌活性进行测定,将最优组合确定为中药复方;采用倾注平板计数法测定TMP与中药联用作用于三种细菌1h、2h、4h和8h后的细菌数,并计算杀菌率,利用最小二乘法对杀菌率进行拟合,计算TMP的最优添加剂量;在此基础上组成中药复方合剂,再进行中药复方合剂的体内抗菌作用的研究,最后采用脏器系数测定法、腹腔巨噬细胞吞噬鸡红细胞法、比浊法以及MTT比色法分别对小鼠免疫器官指数、腹腔巨噬细胞吞噬功能、血清溶菌酶活性、血清溶血素水平、淋巴细胞增殖能力和血液学指标进行测定,检测中药复方合剂对小鼠免疫功能的影响。
试验结果表明:黄连、连翘、黄芩等14味中药均有抗菌活性,其中黄连的抗菌活性最强,MIC值依次为0.016、0.016和0.031g·mL-1;连翘次之,MIC值均为0.031g·mL-1。确定了组方6为中药复方(黄芩、秦皮、苦参和白头翁的配伍比例是1∶4∶2∶1 ),其对三种病原菌的MIC值均为0.125g·mL-1。计算出TMP的最优添加剂量按金黄色葡萄球菌、大肠杆菌、沙门氏菌的不同依次是:黄连为58.3、55.8、28.5mg·g~(-1);金银花为3.86、1.98、3.95mg·g~(-1);苦参为3.62、2.12、2.20mg·g~(-1);连翘为7.1、7.3、7.1mg·g~(-1);蒲公英为4.0、1.8、2.0mg·g~(-1);秦皮为7.62、4.62、3.98mg·g~(-1);白头翁为4.01、1.85、2.10mg·g~(-1);黄芩为27.25、13.81、7.14mg·g~(-1);中药复方为1.94、1.94、1.98mg·g~(-1)。在体内抗菌预防和治疗试验中,中药复方合剂高剂量对90%以上的感染小鼠有预防作用,能使小鼠死亡率降至20%以下。中药复方合剂的中剂量和高剂量可明显增加小鼠免疫器官指数、腹腔巨噬细胞吞噬功能和血清溶菌酶含量(P<0.05)、显著提高正常小鼠红细胞总数、白细胞总数和血红蛋白含量(P<0.05),对小鼠非特异性免疫功能有促进作用;同时还可明显提高血清溶血素水平和淋巴细胞增殖能力(P<0.05),增强小鼠的体液免疫和细胞免疫等特异性免疫功能。
The dose-effect relationship and the effects of immune function of TMP combined with Chinese Herbs were studied on the basis of theories of Pharmacology of Chinese Herbs and Veterinary Pharmacology. A scientific basis was provided for the development of the antimicrobial compound of TMP combined with Chinese Herbs, and a theoretical foundation was established for the prevention and treatment of animal and poultry diseases applied the compound of Chinese Herbs combined with TMP.
In this test, the MICs of TMP combined with Chinese Herbs against Staphylococcus aureus, Escherichia and Salmonella were detected by tube broth dilution. On the basis of MICs of Radix Scutellariae, Cortex Fraxini, Radix Sophorae Flavescentis and Radix Pulsatillae, the composing prescriptions were designed by L9(34) orthogonal test. the antibacterial effects of three kinds of test organisms were evaluated by uble dilution method in vitro. The optimal Chinese Herbal Compound were identified. The number of test organisms which had been inhabited by the combination of Chinese Herbs with TMP for 1h, 2h, 4h and 8h were enumerated by Plate Counts Methods. The bactericidal rates of Chinese Herbs combined with TMP were calculated. On this basis, Chinese Herbal Compound Mixture which was made of Chinese Herbs and TMP were formed. then to study its antimicrobial activity in vivo. Finally, the index of immune organ, the function of macrophage swallow CRBC, the activity of blood serum lysis enzyme, the level of blood serum hemolysin, the lymphopoiesis ability and the target of hematology were determined by the organ coefficient measuring method, the macrophage swallow CRBC method, the turbidimetric method and the MTT color method. The influence of Chinese Herbal Compound Mixture to the mice’s immunologic function was examined.
The results showed that: all of fourteen kinds of Chinese Herbs had antibacterial activity against three kinds of test organisms in vitro, the antibacterial activities of Rhizoma Coptidis were strongest, the MICs were 0.016, 0.016 and 0.031g·mL-1; the composing prescriptions 6 was identified as the optimal Chinese Herbal Compound(the proportion of Radix Scutellariae, Cortex Fraxini, Radix Sophorae Flavescentis and Radix Pulsatillae was 1∶4∶2∶1), its MICs against three kinds of test organisms were 0.125g·mL-1.The optimal doses of TMP about the combination of Chinese Herbs with TMP on Staphylococcus aureus, Escherichia Coli and Salmonella in vitro were: Rhizoma Coptidis: 58.3, 55.8, 28.5mg·g~(-1); Flos Lonicerae: 3.86, 1.98, 3.95mg·g~(-1); Radix Sophorae Flavescentis: 3.62, 2.12, 2.20mg·g~(-1); Fructus Forsythiae: 7.1, 7.3, 7.1mg·g~(-1); Herba Taraxaci: 4.0, 1.8, 2.0mg·g~(-1); Cortex Fraxini: 7.62 4.62, 3.98mg·g~(-1); Radix Pulsatillae: 4.01, 1.85, 2.10mg·g~(-1); Radix Scutellariae: 27.25, 13.81, 7.14mg·g~(-1); Chinese Herbal Compound: 1.94, 1.94, 1.98mg·g~(-1). In trials of prevention and treatment in vivo, the high-dose of the Chinese Herbal Compound Mixture could protect more than 90% of the infected mice, and could also cut the mortality rate to below 20%. The index of immune organ, the function of macrophage swallow CRBC and the activity of blood serum lysis enzyme were increased obviously, and the number of RBC and WBC was also increased obviously used the moderate dose and high-dose of Chinese Herbal Compound Mixture (P<0.05). There were promoter actions for mice's Non-specificity immunologic function. Simultaneously, The mice's level of blood serum hemolysin and ability of lymphopoiesis were also increased obviously (P<0.05). There were promoter actions for mice's specificity immunologic function which included humoral immunity function and cellular immunity function.
本试验采用试管二倍稀释法测定TMP与单味中药对金黄色葡萄球菌、大肠杆菌和沙门氏菌的最小抑菌浓度(MIC)。选择黄芩、秦皮、白头翁和苦参4味中药,依据其MIC值设定高剂量、中剂量、低剂量3种水平,按L9(34)正交设计9个组方,然后对各组方的体外抗菌活性进行测定,将最优组合确定为中药复方;采用倾注平板计数法测定TMP与中药联用作用于三种细菌1h、2h、4h和8h后的细菌数,并计算杀菌率,利用最小二乘法对杀菌率进行拟合,计算TMP的最优添加剂量;在此基础上组成中药复方合剂,再进行中药复方合剂的体内抗菌作用的研究,最后采用脏器系数测定法、腹腔巨噬细胞吞噬鸡红细胞法、比浊法以及MTT比色法分别对小鼠免疫器官指数、腹腔巨噬细胞吞噬功能、血清溶菌酶活性、血清溶血素水平、淋巴细胞增殖能力和血液学指标进行测定,检测中药复方合剂对小鼠免疫功能的影响。
试验结果表明:黄连、连翘、黄芩等14味中药均有抗菌活性,其中黄连的抗菌活性最强,MIC值依次为0.016、0.016和0.031g·mL-1;连翘次之,MIC值均为0.031g·mL-1。确定了组方6为中药复方(黄芩、秦皮、苦参和白头翁的配伍比例是1∶4∶2∶1 ),其对三种病原菌的MIC值均为0.125g·mL-1。计算出TMP的最优添加剂量按金黄色葡萄球菌、大肠杆菌、沙门氏菌的不同依次是:黄连为58.3、55.8、28.5mg·g~(-1);金银花为3.86、1.98、3.95mg·g~(-1);苦参为3.62、2.12、2.20mg·g~(-1);连翘为7.1、7.3、7.1mg·g~(-1);蒲公英为4.0、1.8、2.0mg·g~(-1);秦皮为7.62、4.62、3.98mg·g~(-1);白头翁为4.01、1.85、2.10mg·g~(-1);黄芩为27.25、13.81、7.14mg·g~(-1);中药复方为1.94、1.94、1.98mg·g~(-1)。在体内抗菌预防和治疗试验中,中药复方合剂高剂量对90%以上的感染小鼠有预防作用,能使小鼠死亡率降至20%以下。中药复方合剂的中剂量和高剂量可明显增加小鼠免疫器官指数、腹腔巨噬细胞吞噬功能和血清溶菌酶含量(P<0.05)、显著提高正常小鼠红细胞总数、白细胞总数和血红蛋白含量(P<0.05),对小鼠非特异性免疫功能有促进作用;同时还可明显提高血清溶血素水平和淋巴细胞增殖能力(P<0.05),增强小鼠的体液免疫和细胞免疫等特异性免疫功能。
The dose-effect relationship and the effects of immune function of TMP combined with Chinese Herbs were studied on the basis of theories of Pharmacology of Chinese Herbs and Veterinary Pharmacology. A scientific basis was provided for the development of the antimicrobial compound of TMP combined with Chinese Herbs, and a theoretical foundation was established for the prevention and treatment of animal and poultry diseases applied the compound of Chinese Herbs combined with TMP.
In this test, the MICs of TMP combined with Chinese Herbs against Staphylococcus aureus, Escherichia and Salmonella were detected by tube broth dilution. On the basis of MICs of Radix Scutellariae, Cortex Fraxini, Radix Sophorae Flavescentis and Radix Pulsatillae, the composing prescriptions were designed by L9(34) orthogonal test. the antibacterial effects of three kinds of test organisms were evaluated by uble dilution method in vitro. The optimal Chinese Herbal Compound were identified. The number of test organisms which had been inhabited by the combination of Chinese Herbs with TMP for 1h, 2h, 4h and 8h were enumerated by Plate Counts Methods. The bactericidal rates of Chinese Herbs combined with TMP were calculated. On this basis, Chinese Herbal Compound Mixture which was made of Chinese Herbs and TMP were formed. then to study its antimicrobial activity in vivo. Finally, the index of immune organ, the function of macrophage swallow CRBC, the activity of blood serum lysis enzyme, the level of blood serum hemolysin, the lymphopoiesis ability and the target of hematology were determined by the organ coefficient measuring method, the macrophage swallow CRBC method, the turbidimetric method and the MTT color method. The influence of Chinese Herbal Compound Mixture to the mice’s immunologic function was examined.
The results showed that: all of fourteen kinds of Chinese Herbs had antibacterial activity against three kinds of test organisms in vitro, the antibacterial activities of Rhizoma Coptidis were strongest, the MICs were 0.016, 0.016 and 0.031g·mL-1; the composing prescriptions 6 was identified as the optimal Chinese Herbal Compound(the proportion of Radix Scutellariae, Cortex Fraxini, Radix Sophorae Flavescentis and Radix Pulsatillae was 1∶4∶2∶1), its MICs against three kinds of test organisms were 0.125g·mL-1.The optimal doses of TMP about the combination of Chinese Herbs with TMP on Staphylococcus aureus, Escherichia Coli and Salmonella in vitro were: Rhizoma Coptidis: 58.3, 55.8, 28.5mg·g~(-1); Flos Lonicerae: 3.86, 1.98, 3.95mg·g~(-1); Radix Sophorae Flavescentis: 3.62, 2.12, 2.20mg·g~(-1); Fructus Forsythiae: 7.1, 7.3, 7.1mg·g~(-1); Herba Taraxaci: 4.0, 1.8, 2.0mg·g~(-1); Cortex Fraxini: 7.62 4.62, 3.98mg·g~(-1); Radix Pulsatillae: 4.01, 1.85, 2.10mg·g~(-1); Radix Scutellariae: 27.25, 13.81, 7.14mg·g~(-1); Chinese Herbal Compound: 1.94, 1.94, 1.98mg·g~(-1). In trials of prevention and treatment in vivo, the high-dose of the Chinese Herbal Compound Mixture could protect more than 90% of the infected mice, and could also cut the mortality rate to below 20%. The index of immune organ, the function of macrophage swallow CRBC and the activity of blood serum lysis enzyme were increased obviously, and the number of RBC and WBC was also increased obviously used the moderate dose and high-dose of Chinese Herbal Compound Mixture (P<0.05). There were promoter actions for mice's Non-specificity immunologic function. Simultaneously, The mice's level of blood serum hemolysin and ability of lymphopoiesis were also increased obviously (P<0.05). There were promoter actions for mice's specificity immunologic function which included humoral immunity function and cellular immunity function.
引文
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