金银花对细菌生物膜的抑制作用及其化学成分研究
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摘要
目的:从中药金银花中筛选出对细菌生物膜有抑制作用的活性部位及单体化合物,并对单体化合物进行结构鉴定。
方法:以BBF为体外筛选模型,用染色法对BBF染色,经酶标仪测量特定波长下吸光度值的大小来考察样品对BBF的抑制能力;采用溶剂提取法对金银花进行提取,D101大孔树脂、硅胶、Sephadex LH-20等色谱方法进行分离纯化,采用BBF模型对各个提取部位进行活性跟踪来筛选金银花中抑制BBF的活性部位,并从中分离纯化单体化合物;通过多种波谱分析手段(IR、UV、1~H-NMR、(13)~C-NMR等)对分离出来的单体化合物进行结构鉴定,分别考察各单体化合物对大肠杆菌及金黄色葡萄球菌BBF的影响。
结果:①金银花醇提部分的正丁醇萃取物其95%的大孔树脂洗脱物具BBF抑制作用;②从物金银花醇提物中分离并确定7个化合物的结构,分别为白果醇(ginnol)、肉桂酸(cinnamic acid)、咖啡酸(Caffeic acid)、原儿茶酸、獐牙菜苷(sweroside)、7-表断马钱子甙半缩醛内酯(7-epi-vogeloside)、熊果酸(ursolic acid);③其中,sweroside、7-epi-vogeloside和ursolic acid对大肠杆菌及金黄色葡萄球菌形成的BBF具有较强的抑制作用。
结论:通过实验,初步确定了金银花中对大肠杆菌及金黄色葡萄球菌BBF有抑制作用的活性部位,并从中分离出7个单体化合物,为进一步研究金银花中具有对BBF有抑制作用的成分打下基础。
Purpose: To select the active ingredient(s) and compound(s) which can inhibit the formation of bacteria biofilm from Traditional Chinese Medicine Flos lonicerae, and to identify the structure of pure compound(s).
Method: Using the BBF as vitro model, color it by staining method, measure the OD value with Molecular Devices at a certain wave length to investigate the samples’capacity of inhibition on BBF. Extract the active ingredient(s) from Flos lonicerae with the method of solvent extraction. Separat and purify the compenent and compounds with varies of approaches (D101-macroporous resin, Silica gel, Sephadex LH-20.etc). Select the active compenents form the extracted parts through the BBF model. Separate and purify the compounds. Identify the structure with multi-anylysis instruments (IR、UV、1~H-NMR、(13)~C-NMR.etc.), and investigate the influence of pured compounds on E.coli and SA BBF.
Result:①T he alcohol extracted fract from Flos lonicerae through the n-butanol abstraction,the fract eluated by 95% macroporous resin display the function of inhibition on BBF.②7 compounds have been separated and the structures have been identified as following: ginnol, cinnamic acid, Caffeic acid, protocatechuic acid dihydroxy benzoic acid, sweroside, 7-epi-vogeloside and ursolic acid.③The result indicated that 7-epi-vogeloside, sweroside and ursolic acid can produce the stronger function of inhibition on BBF.
Conclusion: Through the experiment, the active compenents which can inhibit the formation on BBF of E.coli and SA has been found, and 7 compounds are separated and identified. The research can establish the substantial fundament in next step of selecting the active compounds from TCM Flos lonicerae.
方法:以BBF为体外筛选模型,用染色法对BBF染色,经酶标仪测量特定波长下吸光度值的大小来考察样品对BBF的抑制能力;采用溶剂提取法对金银花进行提取,D101大孔树脂、硅胶、Sephadex LH-20等色谱方法进行分离纯化,采用BBF模型对各个提取部位进行活性跟踪来筛选金银花中抑制BBF的活性部位,并从中分离纯化单体化合物;通过多种波谱分析手段(IR、UV、1~H-NMR、(13)~C-NMR等)对分离出来的单体化合物进行结构鉴定,分别考察各单体化合物对大肠杆菌及金黄色葡萄球菌BBF的影响。
结果:①金银花醇提部分的正丁醇萃取物其95%的大孔树脂洗脱物具BBF抑制作用;②从物金银花醇提物中分离并确定7个化合物的结构,分别为白果醇(ginnol)、肉桂酸(cinnamic acid)、咖啡酸(Caffeic acid)、原儿茶酸、獐牙菜苷(sweroside)、7-表断马钱子甙半缩醛内酯(7-epi-vogeloside)、熊果酸(ursolic acid);③其中,sweroside、7-epi-vogeloside和ursolic acid对大肠杆菌及金黄色葡萄球菌形成的BBF具有较强的抑制作用。
结论:通过实验,初步确定了金银花中对大肠杆菌及金黄色葡萄球菌BBF有抑制作用的活性部位,并从中分离出7个单体化合物,为进一步研究金银花中具有对BBF有抑制作用的成分打下基础。
Purpose: To select the active ingredient(s) and compound(s) which can inhibit the formation of bacteria biofilm from Traditional Chinese Medicine Flos lonicerae, and to identify the structure of pure compound(s).
Method: Using the BBF as vitro model, color it by staining method, measure the OD value with Molecular Devices at a certain wave length to investigate the samples’capacity of inhibition on BBF. Extract the active ingredient(s) from Flos lonicerae with the method of solvent extraction. Separat and purify the compenent and compounds with varies of approaches (D101-macroporous resin, Silica gel, Sephadex LH-20.etc). Select the active compenents form the extracted parts through the BBF model. Separate and purify the compounds. Identify the structure with multi-anylysis instruments (IR、UV、1~H-NMR、(13)~C-NMR.etc.), and investigate the influence of pured compounds on E.coli and SA BBF.
Result:①T he alcohol extracted fract from Flos lonicerae through the n-butanol abstraction,the fract eluated by 95% macroporous resin display the function of inhibition on BBF.②7 compounds have been separated and the structures have been identified as following: ginnol, cinnamic acid, Caffeic acid, protocatechuic acid dihydroxy benzoic acid, sweroside, 7-epi-vogeloside and ursolic acid.③The result indicated that 7-epi-vogeloside, sweroside and ursolic acid can produce the stronger function of inhibition on BBF.
Conclusion: Through the experiment, the active compenents which can inhibit the formation on BBF of E.coli and SA has been found, and 7 compounds are separated and identified. The research can establish the substantial fundament in next step of selecting the active compounds from TCM Flos lonicerae.
引文
[1] Hoiby Netal. Pseudomonasa and in vitro and invivo biofilm model of growth[J]. Microbes Infect. 2001; 3:23.
[2] Yanagihara K,etc. Diffuse panbronchiolitis-pathophsiology and treatmen mechanisms[J]. Antimicrob agent. 2001; 18:83.
[3] Kobayashi H.Airway biofirm disease[J]. Antimicrob Agents. 2001; 17:351.
[4]盛清.细菌生物膜.国外医学.流行病学传染病学分册[J]. 2003;1:04.
[5] Costerton J W,et al. Bacterial biofirms. A common cause of persis in persistent infections[J]. Science. 1999; 284:1318.
[6] Rodney M. Donlan Clinics. Microbiology Revies[M]. 2002; (4):167–193.
[7]李彤,庄辉.细菌生物膜的研究进展[J].中华微生物学河面医学杂志. 2002; 22(03):343.
[8]武建国.细菌生物膜及其临床问题[J].临床检验杂志. 2003; 21(4):244
[9] Arne Heydorn, Quantification of biofilm structures by the novel computer program Comstat[M]. Microbiology. 2000; 146:2395–2407.
[10]张良,黄云超.表皮葡萄球菌生物膜形成与生物材料感染[J].生物医学工程与临床. 2006;(10)3:194.
[11] Fang X, Zhao G, Cui L, etc. Interaction between biofilm formed by Pseudomonas aeruginosa and antibacterial agen[J]. Med. Microbio. 2006; 41:359-367
[12] Costerton, J. W., Z. Lewandowski, D. E. Caldwell etc. Microbial biofilms. Microbiol[M]. 1995; 49:711-745.
[13] Song Z.J. ,Wu H. Michael Givskov. Bacterial Biofilm And Antibiotics Resist[J].Natural Science Improvment. 2003;10(13):1017.
[14]邬强,马郁芳.细菌生物膜耐药机制与相关感染的治疗[J].现代预防医学. 2007 ;34(24):4667.
[15] Fumhata M., Wamura M., Baba S. Combined efect of clarithromycin and imipenem/cilastatin against urinary bioiflm infection after pyeloplasty[J]. J Urol. 2003; l0(4): 228-230.
[16] Knodoh K,Hashiba M. Inhibitory efcetfo nlaemlide anitbiotics on bioiflm formation by Pseudomonasn[J]. NipponJibiinkoka Gakkai Kaiho.1998;101(1):25-36.
[17]范春,牛其昌,王颖.铜绿假单胞生物膜耐药性机制[J].国外医药.抗生素分册. 2004; 5(05):363.
[18]朱莲娜,郑薛斌.细菌生物膜及其相关感染的研究进展[J].广西医学.2001; 23(05):1102.
[19]侯冬岩,回瑞华,杨梅等.抗病毒草药金银花的化学成分分析[J].鞍山师范学院学报. 2003;5(4):46-48.
[20]陈昌祥,王薇薇,倪伟.金银花花蕾中的新三萜皂苷[J].云南植物研究.2000; 22(2):201-208.
[21]黄喜茹,刘伟娜,曹冬.金银花的化学成分药理作用研究评析[J].中医药学刊. 2005; (03).
[22]朱斌.生物膜,细菌感染控制面临的新挑战[J].中国感染控制杂志. 2004;2(02):1005.
[23] Abraham SK. Protective effects of chlorogenic acid curcumi in and aα-carotene againstγ-radiation inducedin vitrochromosomal damage[J]. Mutation Res. 1993;303:109.
[24]娄红祥,郎伟君,吕木坚.金银花中水溶性化合物的分离与结构确定[J].中草药. 1996;27(4):195.
[25]王玉莉,杨立,罗国安等.金银忍冬果实非环烯醚萜苷类化学成分研究[J].天然产物研究与开发. 2007; 19(1):51.
[26]马春辉,李伯刚,许庆等.柳叶忍冬的化学成分研究[J].应用与环境生物学报.2006; 12(4):487.
[27]李会军,李萍.忍冬花蕾的化学成分研究[J].林产化学与工业. 2005;25(3):29.
[28]毕跃峰,田野,裴姗姗.金银花化学成分分析[J].郑州大学学报. 2007;39(2):184-185.
[29]李红娟,罗应刚,何志恒等.袋花忍冬的化学成分研究[J].应用与环境生物学报. 2007;13(2):188.
[30] NorikoM, MasaoK. Studies on the constituents ofLoniceraspeciesⅦ. Three new polyhydric alcoholglycosides from the leavesofLonicera gracilipesvar. glandulosamaxmi. Chem Pharm Bul[J]. 1995;43(6):1049.
[31]许小芳.灰毡毛忍冬花蕾中的化学成分.中国天然药物[J]. 2006;4(1):45.
[32]李道中,程村贵.金银花提取液的药理及临床应用.药学进展[J]. 2001;23(6):344.
[33]柴兴云,王林,宋越等.山银花中黄酮类成分的研究.中国药科大学学报[J]. 2007;8(4):24.
[34]高玉敏.金银花化学成分的研究[J].中草药. 1995; 26(11):568-569.
[35]茅青,曹东,贾宪生.灰毡毛忍冬化学成分的研究[J].药学学报.1993;28(4):273.
[36] Kakuda R., ImaiM., YaoitaY., etc. Secoiridoid Glyeosides from the Flower Buds of Lonicera japonica[J]. Phytochemistry. 2000;55(8):879-881.
[37] Li H. -J., Li P., WangM.-C. A New Secoiridoid Glucoside from Lonicera Japonica[J]. Chin.J.Nat.Med. 2003;l(3):132-133.
[38] Machida K., Kikuchi M. Aniridoid Glucoside from Lonicera caeruea[J]. Phytochemistry. 1995; 40(2):603-604.
[39] Machida K., Asano J., Kikuehi M.Phytochemistry[M]. 1995;39:111.
[40]吴二喜,章乃荣,葛维宝等.忍冬与灰毡毛忍冬微量元素的分析[J].中草药.1988; 19(6): 45.
[41]黄丽瑛,吕植桢,李继彪等.中药金银花化学成份的研究.中草药. 1996; 27(11):645.
[42]董克满.金银花的化学成分及生物活性[J].齐齐哈尔医学院学报.2003;24(6):288.
[43]林丽美,王智民等.金银花、连翘及银翘药对水煎剂的抗炎解热作用研究[J].中国中药杂志. 2008;33(4):203.
[44]谢新华,蒋绍祖等.金银花对发热新西兰兔解热作用机制的研究[J].时珍国医国药2009;20(3):690.
[45]樊宏伟,肖大伟等.金银花及其有机酸类化合物的体外抗血小板聚集作用及作用强度[J].中国医院药学杂志. 2006;26 (2):145 .
[46]潘竞锵,刘惠纯,刘广南.金银花能降低小鼠血糖血脂水平[J].广州医药.1998;29(3):59.
[47]朱振宝,田宾,易建华.金银花不同提取物的油脂抗氧化效果研究[J].食品与发酵工业. 2008;34(2):69.
[48]李斐,黎海芪.金银花水提物对卵清蛋白致敏小鼠的抗过敏作用研究[J].重庆医科大学学报. 2004,29(3):288.
[49]罗中华等.几种中药对烫伤小鼠中性粒细胞的作用[J].解放军医学杂志. 1994; 19(4):271-273.
[50]刘恩荔,李青山.金银花的研究进展[J].山西医科大学学报. 2006; 37(3):331.
[51]周洁,韦莉萍.细菌生物膜的形成及其耐药性研究进展[J].第一军医大学分校学报. 2005;28(02):194-195.
[52]葛剑平,岳松龄,胡涛.生物膜细菌耐药机制的研究近况[J].国外医药.口腔医学分册,2004;25(5):294.
[53]唐于平,楼风昌,王景华等.银杏叶中黄酮类成分的研究[J].中国药学杂志.2001;36(4): 231-233.
[54]杨学东,徐丽珍,杨世林.蝉翼藤茎中有机酸成分的研究[J].中国中药杂志. 2001;26(4):258.
[55]徐丽萍,刘建生,敏德等.金龙胆草的化学成分研究[J].中国中药杂志. 1998;23(4):293.
[56]高晓忠,周长新,张水利等.毛蓑科植物石龙芮的化学成分研究[J].中国中药杂志. 2005;30(2):124-126.
[57]戴好富,周俊,彭再刚等.北五味子的水溶性化学成[J].天然产物研究与开发.2001;13(l):24-26.
[58]贺清辉,田艳艳,李会军等.红腺忍冬藤茎中环烯醚萜苷类化合物的研究[J].中国药学杂志.2006;41(9):656.
[59] Recio-Iglesias M,Marston A,Hostettmann K.Xanthones and Secoiridoid Glucosides of Halonia Campanulata[J].Phytochemistry.1992;31(4):1387.
[2] Yanagihara K,etc. Diffuse panbronchiolitis-pathophsiology and treatmen mechanisms[J]. Antimicrob agent. 2001; 18:83.
[3] Kobayashi H.Airway biofirm disease[J]. Antimicrob Agents. 2001; 17:351.
[4]盛清.细菌生物膜.国外医学.流行病学传染病学分册[J]. 2003;1:04.
[5] Costerton J W,et al. Bacterial biofirms. A common cause of persis in persistent infections[J]. Science. 1999; 284:1318.
[6] Rodney M. Donlan Clinics. Microbiology Revies[M]. 2002; (4):167–193.
[7]李彤,庄辉.细菌生物膜的研究进展[J].中华微生物学河面医学杂志. 2002; 22(03):343.
[8]武建国.细菌生物膜及其临床问题[J].临床检验杂志. 2003; 21(4):244
[9] Arne Heydorn, Quantification of biofilm structures by the novel computer program Comstat[M]. Microbiology. 2000; 146:2395–2407.
[10]张良,黄云超.表皮葡萄球菌生物膜形成与生物材料感染[J].生物医学工程与临床. 2006;(10)3:194.
[11] Fang X, Zhao G, Cui L, etc. Interaction between biofilm formed by Pseudomonas aeruginosa and antibacterial agen[J]. Med. Microbio. 2006; 41:359-367
[12] Costerton, J. W., Z. Lewandowski, D. E. Caldwell etc. Microbial biofilms. Microbiol[M]. 1995; 49:711-745.
[13] Song Z.J. ,Wu H. Michael Givskov. Bacterial Biofilm And Antibiotics Resist[J].Natural Science Improvment. 2003;10(13):1017.
[14]邬强,马郁芳.细菌生物膜耐药机制与相关感染的治疗[J].现代预防医学. 2007 ;34(24):4667.
[15] Fumhata M., Wamura M., Baba S. Combined efect of clarithromycin and imipenem/cilastatin against urinary bioiflm infection after pyeloplasty[J]. J Urol. 2003; l0(4): 228-230.
[16] Knodoh K,Hashiba M. Inhibitory efcetfo nlaemlide anitbiotics on bioiflm formation by Pseudomonasn[J]. NipponJibiinkoka Gakkai Kaiho.1998;101(1):25-36.
[17]范春,牛其昌,王颖.铜绿假单胞生物膜耐药性机制[J].国外医药.抗生素分册. 2004; 5(05):363.
[18]朱莲娜,郑薛斌.细菌生物膜及其相关感染的研究进展[J].广西医学.2001; 23(05):1102.
[19]侯冬岩,回瑞华,杨梅等.抗病毒草药金银花的化学成分分析[J].鞍山师范学院学报. 2003;5(4):46-48.
[20]陈昌祥,王薇薇,倪伟.金银花花蕾中的新三萜皂苷[J].云南植物研究.2000; 22(2):201-208.
[21]黄喜茹,刘伟娜,曹冬.金银花的化学成分药理作用研究评析[J].中医药学刊. 2005; (03).
[22]朱斌.生物膜,细菌感染控制面临的新挑战[J].中国感染控制杂志. 2004;2(02):1005.
[23] Abraham SK. Protective effects of chlorogenic acid curcumi in and aα-carotene againstγ-radiation inducedin vitrochromosomal damage[J]. Mutation Res. 1993;303:109.
[24]娄红祥,郎伟君,吕木坚.金银花中水溶性化合物的分离与结构确定[J].中草药. 1996;27(4):195.
[25]王玉莉,杨立,罗国安等.金银忍冬果实非环烯醚萜苷类化学成分研究[J].天然产物研究与开发. 2007; 19(1):51.
[26]马春辉,李伯刚,许庆等.柳叶忍冬的化学成分研究[J].应用与环境生物学报.2006; 12(4):487.
[27]李会军,李萍.忍冬花蕾的化学成分研究[J].林产化学与工业. 2005;25(3):29.
[28]毕跃峰,田野,裴姗姗.金银花化学成分分析[J].郑州大学学报. 2007;39(2):184-185.
[29]李红娟,罗应刚,何志恒等.袋花忍冬的化学成分研究[J].应用与环境生物学报. 2007;13(2):188.
[30] NorikoM, MasaoK. Studies on the constituents ofLoniceraspeciesⅦ. Three new polyhydric alcoholglycosides from the leavesofLonicera gracilipesvar. glandulosamaxmi. Chem Pharm Bul[J]. 1995;43(6):1049.
[31]许小芳.灰毡毛忍冬花蕾中的化学成分.中国天然药物[J]. 2006;4(1):45.
[32]李道中,程村贵.金银花提取液的药理及临床应用.药学进展[J]. 2001;23(6):344.
[33]柴兴云,王林,宋越等.山银花中黄酮类成分的研究.中国药科大学学报[J]. 2007;8(4):24.
[34]高玉敏.金银花化学成分的研究[J].中草药. 1995; 26(11):568-569.
[35]茅青,曹东,贾宪生.灰毡毛忍冬化学成分的研究[J].药学学报.1993;28(4):273.
[36] Kakuda R., ImaiM., YaoitaY., etc. Secoiridoid Glyeosides from the Flower Buds of Lonicera japonica[J]. Phytochemistry. 2000;55(8):879-881.
[37] Li H. -J., Li P., WangM.-C. A New Secoiridoid Glucoside from Lonicera Japonica[J]. Chin.J.Nat.Med. 2003;l(3):132-133.
[38] Machida K., Kikuchi M. Aniridoid Glucoside from Lonicera caeruea[J]. Phytochemistry. 1995; 40(2):603-604.
[39] Machida K., Asano J., Kikuehi M.Phytochemistry[M]. 1995;39:111.
[40]吴二喜,章乃荣,葛维宝等.忍冬与灰毡毛忍冬微量元素的分析[J].中草药.1988; 19(6): 45.
[41]黄丽瑛,吕植桢,李继彪等.中药金银花化学成份的研究.中草药. 1996; 27(11):645.
[42]董克满.金银花的化学成分及生物活性[J].齐齐哈尔医学院学报.2003;24(6):288.
[43]林丽美,王智民等.金银花、连翘及银翘药对水煎剂的抗炎解热作用研究[J].中国中药杂志. 2008;33(4):203.
[44]谢新华,蒋绍祖等.金银花对发热新西兰兔解热作用机制的研究[J].时珍国医国药2009;20(3):690.
[45]樊宏伟,肖大伟等.金银花及其有机酸类化合物的体外抗血小板聚集作用及作用强度[J].中国医院药学杂志. 2006;26 (2):145 .
[46]潘竞锵,刘惠纯,刘广南.金银花能降低小鼠血糖血脂水平[J].广州医药.1998;29(3):59.
[47]朱振宝,田宾,易建华.金银花不同提取物的油脂抗氧化效果研究[J].食品与发酵工业. 2008;34(2):69.
[48]李斐,黎海芪.金银花水提物对卵清蛋白致敏小鼠的抗过敏作用研究[J].重庆医科大学学报. 2004,29(3):288.
[49]罗中华等.几种中药对烫伤小鼠中性粒细胞的作用[J].解放军医学杂志. 1994; 19(4):271-273.
[50]刘恩荔,李青山.金银花的研究进展[J].山西医科大学学报. 2006; 37(3):331.
[51]周洁,韦莉萍.细菌生物膜的形成及其耐药性研究进展[J].第一军医大学分校学报. 2005;28(02):194-195.
[52]葛剑平,岳松龄,胡涛.生物膜细菌耐药机制的研究近况[J].国外医药.口腔医学分册,2004;25(5):294.
[53]唐于平,楼风昌,王景华等.银杏叶中黄酮类成分的研究[J].中国药学杂志.2001;36(4): 231-233.
[54]杨学东,徐丽珍,杨世林.蝉翼藤茎中有机酸成分的研究[J].中国中药杂志. 2001;26(4):258.
[55]徐丽萍,刘建生,敏德等.金龙胆草的化学成分研究[J].中国中药杂志. 1998;23(4):293.
[56]高晓忠,周长新,张水利等.毛蓑科植物石龙芮的化学成分研究[J].中国中药杂志. 2005;30(2):124-126.
[57]戴好富,周俊,彭再刚等.北五味子的水溶性化学成[J].天然产物研究与开发.2001;13(l):24-26.
[58]贺清辉,田艳艳,李会军等.红腺忍冬藤茎中环烯醚萜苷类化合物的研究[J].中国药学杂志.2006;41(9):656.
[59] Recio-Iglesias M,Marston A,Hostettmann K.Xanthones and Secoiridoid Glucosides of Halonia Campanulata[J].Phytochemistry.1992;31(4):1387.