抗耐甲氧西林金黄色葡萄球菌的中药筛选
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摘要
目的从50种常用中药中筛选抗耐甲氧西林金黄色葡萄球菌(MRSA)的有效中药,为临床研制治疗MRSA感染的中药制剂提供依据。
方法常用的50种中药干燥粉碎成粗粉经水煎煮、浓缩、离心、无菌过滤后备用,每1mL浓缩液相当于生药1g。将本院临床检验中心提供的MRSA菌株经复苏、鉴定、传代后采用K-B纸片扩散法对50种中药抗MRSA菌株作用进行考察,并选择5种抗MRSA菌株作用最强的中药继续进行研究。按美国临床实验室标准化委员会(NCCLS)推荐的液体稀释法测定所筛选的5种中药抗MRSA菌株的最低抑菌浓度(MIC)。观察所筛选5种中药的杀菌作用,并绘制抗MRSA菌株的杀菌曲线。采用微量液体平板棋盘法研究5种中药两两联合应用的抗菌作用。
结果抗菌筛选实验中50种中药有35种产生抑菌圈,其中以黄连、黄柏、大黄、银柴胡和石榴皮的抗MRSA菌株的抑菌圈直径最大,抑菌直径依次为18.2±1.2 mm、17.5±1.5 mm、16.4±1.5 mm、15.3±1.4 m和14.6±1.2 mm。大黄、黄连、黄柏、银柴胡和石榴皮对5种MRSA菌种的平均MIC值分别为0.0125 g/mL、0.00365 g/mL、0.00625 g/mL、0.05g/mL和0.045 g/mL。大黄、黄连、黄柏、银柴胡和石榴皮均能杀灭一定数量的MRSA菌株,并且在12小时内MRSA数量不再增长,具有较强的抗菌效果。黄连和黄柏联合应用时抗菌指数(FIC)<0.5,表现为协同作用。
结论①中药黄连、黄柏、大黄、银柴胡和石榴皮水煎液具有较强的抗MRSA作用；②黄连、黄柏、大黄、银柴胡和石榴皮水煎液抗MRSA的MIC值分别为0.0125 g/mL、0.00365 g/mL、0.00625 g/mL、0.05g/mL和0.045 g/mL；③黄连、黄柏、大黄、银柴胡和石榴皮水煎液具有一定的杀菌作用；④两两联用的协同抗菌试验显示黄连和黄柏联合应用时协同作用为主,为中药复方抗MRSA的较好选择。
Objective:To screen traditional Chinese medicine with powerful antibacterial activity against methicillin-resistant Staphylococcus aureu (MRSA) from 50 Chinese medicine in common use, and provide basis of Chinese herbal preparation against MRSA.
Methods:50 Chinese medicine in common use were smashed in to coarse powder after drying, and then decocted by water, concentrated, centrifuged, and made into sterile solution after sterile filtration.1 ml of solution was equivalent to 1 g dried medicinal herbs. The bacterial strain of MARS provided by clinical examination centre of our hospital were recovered, identified and reproduced. The Chinese medicines with powerful antibacterial activity against MRSA were selected from 50 traditional Chinese medicines by Kirby-Bauer, and 5 Chinese medicines with most powerful antibacterial activity against MRSA were selected to carry on further research. The MICs of selected Chinese medicines with most powerful antibacterial activity against MRSA were performed according to liquid dilution method recommended by national committee for clinical laboratory standards. Bactericidal actions of the selected Chinese medicines were observed and killing curves of MRSA were drawn. Synergetic antibacterial test of each two of the selected 5 Chinese medicines against MRSA were carried out by trace multi-well culture plates.
Results:35 Chinese medicines had inhibitory effects on the strain of MRSA with varied extent in 50 Chinese medicines, among which Coptis chinensis, Cortex Phellodendri, Rhubarb, Radix Stellariae and granatum had the most powerful effects respectively with the bacteriostatic diameter of 18.2±1.2 mm,17.5±1.5 mm,16.4±1.5 mm,15.3±1.4 mm and 14.6±1.2 mm. The mean MICs of Coptis chinensis, Cortex Phellodendri, Rhubarb, Radix Stellariae and granatum were resectively 0.0125 g/mL,0.00365 g/mL,0.00625 g/mL, 0.05g/mL and 0.045 g/mL. Coptis chinensis, Cortex Phellodendri, Rhubarb, Radix Stellariae and granatum could kill bacterial strain of MARS with varied extent, and made the amount of bacterial strain of MARS not increased in 12 hours and had powerful antibiosis effects. Furthermore, FIC index of Coptis chinensis and Cortex Phellodendri was below 0.5,so their interactions were synergetic.
Conclusion:(1) Coptis chinensis, Cortex Phellodendri, Rhubarb, Radix Stellariae and granatum have powerful antibacterial activity against MRSA. (2) The mean MICs of Coptis chinensis, Cortex Phellodendri, Rhubarb, Radix Stellariae and granatum were resectively 0.0125 g/mL,0.00365 g/mL,0.00625 g/mL,0.05g/mL and 0.045 g/mL (3) Coptis chinensis, Cortex Phellodendri, Rhubarb, Radix Stellariae and granatum could kill bacterial strain of MARS with varied extent. (4) Combined use of Coptis chinensis and Cortex Phellodendri has the most inhibitory effect on MRSA because of synergistic effect, and are the good choice in anti-MRSA Chinese medicines.
方法常用的50种中药干燥粉碎成粗粉经水煎煮、浓缩、离心、无菌过滤后备用,每1mL浓缩液相当于生药1g。将本院临床检验中心提供的MRSA菌株经复苏、鉴定、传代后采用K-B纸片扩散法对50种中药抗MRSA菌株作用进行考察,并选择5种抗MRSA菌株作用最强的中药继续进行研究。按美国临床实验室标准化委员会(NCCLS)推荐的液体稀释法测定所筛选的5种中药抗MRSA菌株的最低抑菌浓度(MIC)。观察所筛选5种中药的杀菌作用,并绘制抗MRSA菌株的杀菌曲线。采用微量液体平板棋盘法研究5种中药两两联合应用的抗菌作用。
结果抗菌筛选实验中50种中药有35种产生抑菌圈,其中以黄连、黄柏、大黄、银柴胡和石榴皮的抗MRSA菌株的抑菌圈直径最大,抑菌直径依次为18.2±1.2 mm、17.5±1.5 mm、16.4±1.5 mm、15.3±1.4 m和14.6±1.2 mm。大黄、黄连、黄柏、银柴胡和石榴皮对5种MRSA菌种的平均MIC值分别为0.0125 g/mL、0.00365 g/mL、0.00625 g/mL、0.05g/mL和0.045 g/mL。大黄、黄连、黄柏、银柴胡和石榴皮均能杀灭一定数量的MRSA菌株,并且在12小时内MRSA数量不再增长,具有较强的抗菌效果。黄连和黄柏联合应用时抗菌指数(FIC)<0.5,表现为协同作用。
结论①中药黄连、黄柏、大黄、银柴胡和石榴皮水煎液具有较强的抗MRSA作用；②黄连、黄柏、大黄、银柴胡和石榴皮水煎液抗MRSA的MIC值分别为0.0125 g/mL、0.00365 g/mL、0.00625 g/mL、0.05g/mL和0.045 g/mL；③黄连、黄柏、大黄、银柴胡和石榴皮水煎液具有一定的杀菌作用；④两两联用的协同抗菌试验显示黄连和黄柏联合应用时协同作用为主,为中药复方抗MRSA的较好选择。
Objective:To screen traditional Chinese medicine with powerful antibacterial activity against methicillin-resistant Staphylococcus aureu (MRSA) from 50 Chinese medicine in common use, and provide basis of Chinese herbal preparation against MRSA.
Methods:50 Chinese medicine in common use were smashed in to coarse powder after drying, and then decocted by water, concentrated, centrifuged, and made into sterile solution after sterile filtration.1 ml of solution was equivalent to 1 g dried medicinal herbs. The bacterial strain of MARS provided by clinical examination centre of our hospital were recovered, identified and reproduced. The Chinese medicines with powerful antibacterial activity against MRSA were selected from 50 traditional Chinese medicines by Kirby-Bauer, and 5 Chinese medicines with most powerful antibacterial activity against MRSA were selected to carry on further research. The MICs of selected Chinese medicines with most powerful antibacterial activity against MRSA were performed according to liquid dilution method recommended by national committee for clinical laboratory standards. Bactericidal actions of the selected Chinese medicines were observed and killing curves of MRSA were drawn. Synergetic antibacterial test of each two of the selected 5 Chinese medicines against MRSA were carried out by trace multi-well culture plates.
Results:35 Chinese medicines had inhibitory effects on the strain of MRSA with varied extent in 50 Chinese medicines, among which Coptis chinensis, Cortex Phellodendri, Rhubarb, Radix Stellariae and granatum had the most powerful effects respectively with the bacteriostatic diameter of 18.2±1.2 mm,17.5±1.5 mm,16.4±1.5 mm,15.3±1.4 mm and 14.6±1.2 mm. The mean MICs of Coptis chinensis, Cortex Phellodendri, Rhubarb, Radix Stellariae and granatum were resectively 0.0125 g/mL,0.00365 g/mL,0.00625 g/mL, 0.05g/mL and 0.045 g/mL. Coptis chinensis, Cortex Phellodendri, Rhubarb, Radix Stellariae and granatum could kill bacterial strain of MARS with varied extent, and made the amount of bacterial strain of MARS not increased in 12 hours and had powerful antibiosis effects. Furthermore, FIC index of Coptis chinensis and Cortex Phellodendri was below 0.5,so their interactions were synergetic.
Conclusion:(1) Coptis chinensis, Cortex Phellodendri, Rhubarb, Radix Stellariae and granatum have powerful antibacterial activity against MRSA. (2) The mean MICs of Coptis chinensis, Cortex Phellodendri, Rhubarb, Radix Stellariae and granatum were resectively 0.0125 g/mL,0.00365 g/mL,0.00625 g/mL,0.05g/mL and 0.045 g/mL (3) Coptis chinensis, Cortex Phellodendri, Rhubarb, Radix Stellariae and granatum could kill bacterial strain of MARS with varied extent. (4) Combined use of Coptis chinensis and Cortex Phellodendri has the most inhibitory effect on MRSA because of synergistic effect, and are the good choice in anti-MRSA Chinese medicines.
引文
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[2]李娟,韩艳.连续5年金黄色葡萄球菌耐药性监测[J].中华医院感染学杂志,2010,20(24)：4008-4010.
[3]Jordens J Z, Duckworth G J, Williams R J. Production of virulence factors by epidemic methicillin- resistant Staphylococcus aureus in vitro[J]. J Med Mieorbial,1989,30 (4):245-252.
[4]张珏,乔昀,倪语星.医院感染的耐甲氧西林金黄色葡萄球菌耐药基因分析[J].检验医学,2007,22(4)：390-392.
[5]侯晓娜,丁雪松,付炜听,等.耐甲氧西林金黄色葡萄球菌的基因多态性研究[J].中华医院感染学杂志,2003,13(10)：919-921.
[6]TE Zaoutis, P Toltzis, J Chu, et al. Clinical and molecular epidemiology of community-acquired methicillin-resistant Staphylococcus aureus infections among children with risk factors for health care-associated infection:2001-2003 [J]. Pediatr Infect Dis J,2006,25:343-8.
[7]陈世敏.耐甲氧西林葡萄球菌耐药特点极其分子生物学基础[J].国外医学流行病学传染病学分册,2003,30(3)：188-189.
[8]卓超,苏丹虹,钟南山.2007年广州地区细菌耐药性监测分析[J].中华检验医学杂志,2009,32(4)：397-402.
[9]FX Lescure, M Biendo, Y Douadi, et al. Changing epidemiology of methicillin-resistant Staphylococcus aureus and effects on cross-transmission in a teaching hospital[J]. Eur J Clin Microbiol Infect Dis,2006,25:205-7.
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