甘草单体成分抗幽门螺杆菌研究
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摘要
目的:1.探讨幽门螺杆菌(helicobacter pylori,Hp)临床分离株对常用抗生素的耐药特点,进一步明确Hp对常用抗生素的耐药发生情况。2.从我国最常用中草药之一甘草入手,研究作为其主要药理成分的甘草酸和甘草黄酮对Hp的抗菌作用,并分别和克拉霉素及甘草等抗Hp的作用作对比研究。为临床应用甘草单体及甘草活性物质抗Hp提供理论依据。
方法:1.将来自不同患者、经快速尿素酶试验初筛阳性胃镜活检标本分别涂布接种幽门螺杆菌培养基后37℃微需氧环境下培养96h,培养分离实验需Hp临床株。2.参照美国临床实验室标准化委员会(National Committee for ClinicalLaboratory Standard,NCCLS)标准,采用Kirby-Bauer纸片扩散法药敏实验方行Hp临床分离株常用抗生素耐药情况的分析和甘草单体成分的抗Hp实验研究;其中甘草单体成分抗Hp药敏实验中甘草酸和甘草黄酮各分为10mg、1mg、100μg、50μg四个梯度组、并且各株菌药敏板均同步设置克拉霉素的抗生素药敏纸片对照组和甘草10mg对照组。
结果:1.获得实验需Hp临床分离株。2.Hp临床分离株对甲硝唑、克拉霉素、阿莫西林、呋喃唑酮等常用抗生素的耐药率分别为54.1%,37.7%,41.0%,45.9%,Hp对4种抗生素耐药率相比较差异无统计学意义(P＞0.05);Hp对抗生素耐药与年龄、疾病类型等比较均无统计学相关(P＞0.05),与近期用药史有关(P＜0.05)。3.甘草酸10mg组分别与甘草酸1mg、100μg、50ug组抗Hp效果相比较,无论抗Hp平均抑菌圈直径还是抗Hp有效率比较差别均有统计学意义(P＜0.05);甘草酸10mg组和克拉霉素对照组无论在抗Hp平均抑菌圈直径还是抗Hp有效率相比较差别均无统计学意义(P＞0.05);其余甘草酸组无论抗Hp平均抑菌圈直径还是抗Hp有效率和克拉霉素对照组比较差别均具统计学差异(P＜0.05)。4.甘草黄酮10mg组与甘草黄酮1mg组无论抗Hp平均抑菌圈直径还是抗Hp有效率相比较差别均无统计学意义(P＞0.05),甘草黄酮10mg组与其余甘草黄酮组相比较无论抗Hp平均抑菌圈直径还是抗Hp有效率差别均有统计学意义;甘草黄酮10mg组与克拉霉素对照组的抗Hp平均抑菌圈直径还是抗Hp有效率相比较差别均无统计学意义,其余各甘草黄酮组无论抗Hp平均抑菌圈直径还是抗Hp有效率和克拉霉素对照组比较差别均具统计学意义(P＜0.05)。5.甘草10mg组抗Hp的平均抑菌圈直径和抗Hp有效率均大于克拉霉素对照组,但差别均无统计学意义(P＞0.05)。6.甘草酸、甘草黄酮10mg含量组对Hp抑杀作用,无论从平均抑菌圈直径、还是有效率相互间比较,两者差别均无统计学意义(P＞0.05);甘草10mg组的平均抑菌圈直径大于甘草酸、甘草黄酮10mg组的平均抑菌圈直径,且甘草10mg组平均抑菌圈直径分别与甘草酸10mg组、甘草黄酮10mg组的平均抑菌圈直径比较,差别均有统计学意义(P＜0.05);甘草10mg组的抗Hp有效率最高、为69.7%,甘草黄酮10mg组的抗Hp有效率次之、为63.9%;甘草酸10mg组的抗Hp有效率为60.6%,但三者抗Hp有效率相比无统计学差异(P=0.734,P＞0.05)。甘草酸、甘草黄酮的100μg含量组尚分别有21.2%、24.2%的抗Hp的有效率。
结论:1.Hp临床分离株对甲硝唑、克拉霉素、阿莫西林、呋喃唑酮等常用抗生素均出现较高的耐药率,Hp抗生素耐药与近期用药史有关,与年龄、疾病类型等无关。2.甘草酸、甘草黄酮的各4个不同药物含量纸片组对Hp均表现出一定的抑杀效果,以各自10mg组的抗Hp效果最优,且甘草酸、甘草黄酮10mg含量组和克拉霉素对照组三者对Hp的抑杀作用相当;甘草酸和甘草黄酮的ug含量尚具有一定的Hp抑杀作用。3.甘草10mg组对Hp抑杀作用和克拉霉素抗Hp效果相当,但优于甘草酸10mg组、甘草黄酮10mg组对Hp抑杀作用。
Objective: To ingvestigate the drug resistance of Helicobacter pylori to antibiotic. To study the antimicrobial activity of Glycyrrhizic Acid and licoflavone Against Helicobacter Pylori and contrast the antimicrobial activity of Glycyrrhizic Acid and licoflavone Against Helicobacter Pylori to Clarithromycin and Liquorice respectively. Glycyrrhizic Acid and licoflavone is the mostly monomer or components extracted from Liquorice, one of the widespread used traditional Chinese medicine.The study may also be conduced to converting the drug into the cliclical application and give basic foundation for helping to cure Hp.
Methods: H pylori strains were obtained by inoculating the specimens of diferrent patients in the selective media for 96 hours at 37℃under microaerophilic conditions. The drug susceptibility of clinical isolated Hp to antibiotic and the isolation of monomers from Liquorice were performed with Kirby-Bauer disk diffusion test respectively by the rules of NCCLS. Characters of susceptibility were analyzed. Among the drug susceptibility test of clinical isolated Hp to isolation of monomers from Liquorice , experimental group of Glycyrrhizic Acid and licoflavone were all divided into four different contained groups , which is the group of 10mg contained , 1mg contained, 100 ug contained and 50ug contained respectively; meanwhile, control group of Clarithromycin and Liquorice 10mg contained were set up. Results: The antibiotic resistant rates of Hpylori strains to metronidazole, clarithromycin, amoxicillin and furazolidone were 54.1%, 37.7%, 41.0% and 45.9%, respectively, and there were no significant difference among them (P > 0.05). Hpylori has high resistance to metronidazole, clarithromycin, amoxicillin, and furazolidone, which is associated with the history of antibiotic application, but not with the disease category and age of patients. There were not only the average diameter of inhibition zone no significant difference but also effective power between the group of Glycyrrhizic Acid 10mg contained and group of licoflavone10mg contained(P>0.05). No significant difference were found between the both contrast to clarithromycin either average diameter of inhibition zone or effective power respectively (P>0.05). The effective power of the groupe of Glycyrrhizic Acid 100ug contained and group of licoflavone100ug contained to isolated Hp were 21.2%, 24.2% respectively. The average diameter of inhibition zone between the group of Liquorice 10mg contained and the group of Glycyrrhizic Acid 10mg contained or group of licoflavone10mg contained were compared and significant difference were found(P<0.05); There was no significant difference among the effective power(P>0.05). No significant relationship existed between Not only average diameter of inhibition zone but also the effective power among Clarithromycin control group and The group of Liquorice 10mg contained(P >0.05).
Conclusion: The rate of antibiotic resistance of isolated Hp to metronidazole, clarithromycin, amoxicillin, furazolidone, were high. Both the 4 different group of Glycyrrhizic Acid contained and the 4 different group of licoflavone contained all take on a few effective power to isolated Hp , which are even found in group at level of ug contained. The effect of groupe of Glycyrrhizic Acid 10mg contained, groupe of licoflavone 10mg contained, and clarithromycin control group to isolated Hp are fairly. The effective power among of the group of Liquorice 10mg contained , the group of Glycyrrhizic Acid 10mg contained , and group of licoflavone 10mg contained to isolated Hp were similar and no significant difference were found(P>0.05). But the average diameter of inhibition zone of the group of Liquorice 10mg contained outweigh that of group of Glycyrrhizic Acid 10mg contained or group of licoflavone10mg contained(P<0.05). The group of Liquorice 10mg contained take on the better effect to isolated Hp than that of group of Glycyrrhizic Acid 10mg contained or group of licoflavone10mg contained. The group of Liquorice 10mg contained have the fairly antibacterial effect as that of the Clarithromycin control group.
方法:1.将来自不同患者、经快速尿素酶试验初筛阳性胃镜活检标本分别涂布接种幽门螺杆菌培养基后37℃微需氧环境下培养96h,培养分离实验需Hp临床株。2.参照美国临床实验室标准化委员会(National Committee for ClinicalLaboratory Standard,NCCLS)标准,采用Kirby-Bauer纸片扩散法药敏实验方行Hp临床分离株常用抗生素耐药情况的分析和甘草单体成分的抗Hp实验研究;其中甘草单体成分抗Hp药敏实验中甘草酸和甘草黄酮各分为10mg、1mg、100μg、50μg四个梯度组、并且各株菌药敏板均同步设置克拉霉素的抗生素药敏纸片对照组和甘草10mg对照组。
结果:1.获得实验需Hp临床分离株。2.Hp临床分离株对甲硝唑、克拉霉素、阿莫西林、呋喃唑酮等常用抗生素的耐药率分别为54.1%,37.7%,41.0%,45.9%,Hp对4种抗生素耐药率相比较差异无统计学意义(P＞0.05);Hp对抗生素耐药与年龄、疾病类型等比较均无统计学相关(P＞0.05),与近期用药史有关(P＜0.05)。3.甘草酸10mg组分别与甘草酸1mg、100μg、50ug组抗Hp效果相比较,无论抗Hp平均抑菌圈直径还是抗Hp有效率比较差别均有统计学意义(P＜0.05);甘草酸10mg组和克拉霉素对照组无论在抗Hp平均抑菌圈直径还是抗Hp有效率相比较差别均无统计学意义(P＞0.05);其余甘草酸组无论抗Hp平均抑菌圈直径还是抗Hp有效率和克拉霉素对照组比较差别均具统计学差异(P＜0.05)。4.甘草黄酮10mg组与甘草黄酮1mg组无论抗Hp平均抑菌圈直径还是抗Hp有效率相比较差别均无统计学意义(P＞0.05),甘草黄酮10mg组与其余甘草黄酮组相比较无论抗Hp平均抑菌圈直径还是抗Hp有效率差别均有统计学意义;甘草黄酮10mg组与克拉霉素对照组的抗Hp平均抑菌圈直径还是抗Hp有效率相比较差别均无统计学意义,其余各甘草黄酮组无论抗Hp平均抑菌圈直径还是抗Hp有效率和克拉霉素对照组比较差别均具统计学意义(P＜0.05)。5.甘草10mg组抗Hp的平均抑菌圈直径和抗Hp有效率均大于克拉霉素对照组,但差别均无统计学意义(P＞0.05)。6.甘草酸、甘草黄酮10mg含量组对Hp抑杀作用,无论从平均抑菌圈直径、还是有效率相互间比较,两者差别均无统计学意义(P＞0.05);甘草10mg组的平均抑菌圈直径大于甘草酸、甘草黄酮10mg组的平均抑菌圈直径,且甘草10mg组平均抑菌圈直径分别与甘草酸10mg组、甘草黄酮10mg组的平均抑菌圈直径比较,差别均有统计学意义(P＜0.05);甘草10mg组的抗Hp有效率最高、为69.7%,甘草黄酮10mg组的抗Hp有效率次之、为63.9%;甘草酸10mg组的抗Hp有效率为60.6%,但三者抗Hp有效率相比无统计学差异(P=0.734,P＞0.05)。甘草酸、甘草黄酮的100μg含量组尚分别有21.2%、24.2%的抗Hp的有效率。
结论:1.Hp临床分离株对甲硝唑、克拉霉素、阿莫西林、呋喃唑酮等常用抗生素均出现较高的耐药率,Hp抗生素耐药与近期用药史有关,与年龄、疾病类型等无关。2.甘草酸、甘草黄酮的各4个不同药物含量纸片组对Hp均表现出一定的抑杀效果,以各自10mg组的抗Hp效果最优,且甘草酸、甘草黄酮10mg含量组和克拉霉素对照组三者对Hp的抑杀作用相当;甘草酸和甘草黄酮的ug含量尚具有一定的Hp抑杀作用。3.甘草10mg组对Hp抑杀作用和克拉霉素抗Hp效果相当,但优于甘草酸10mg组、甘草黄酮10mg组对Hp抑杀作用。
Objective: To ingvestigate the drug resistance of Helicobacter pylori to antibiotic. To study the antimicrobial activity of Glycyrrhizic Acid and licoflavone Against Helicobacter Pylori and contrast the antimicrobial activity of Glycyrrhizic Acid and licoflavone Against Helicobacter Pylori to Clarithromycin and Liquorice respectively. Glycyrrhizic Acid and licoflavone is the mostly monomer or components extracted from Liquorice, one of the widespread used traditional Chinese medicine.The study may also be conduced to converting the drug into the cliclical application and give basic foundation for helping to cure Hp.
Methods: H pylori strains were obtained by inoculating the specimens of diferrent patients in the selective media for 96 hours at 37℃under microaerophilic conditions. The drug susceptibility of clinical isolated Hp to antibiotic and the isolation of monomers from Liquorice were performed with Kirby-Bauer disk diffusion test respectively by the rules of NCCLS. Characters of susceptibility were analyzed. Among the drug susceptibility test of clinical isolated Hp to isolation of monomers from Liquorice , experimental group of Glycyrrhizic Acid and licoflavone were all divided into four different contained groups , which is the group of 10mg contained , 1mg contained, 100 ug contained and 50ug contained respectively; meanwhile, control group of Clarithromycin and Liquorice 10mg contained were set up. Results: The antibiotic resistant rates of Hpylori strains to metronidazole, clarithromycin, amoxicillin and furazolidone were 54.1%, 37.7%, 41.0% and 45.9%, respectively, and there were no significant difference among them (P > 0.05). Hpylori has high resistance to metronidazole, clarithromycin, amoxicillin, and furazolidone, which is associated with the history of antibiotic application, but not with the disease category and age of patients. There were not only the average diameter of inhibition zone no significant difference but also effective power between the group of Glycyrrhizic Acid 10mg contained and group of licoflavone10mg contained(P>0.05). No significant difference were found between the both contrast to clarithromycin either average diameter of inhibition zone or effective power respectively (P>0.05). The effective power of the groupe of Glycyrrhizic Acid 100ug contained and group of licoflavone100ug contained to isolated Hp were 21.2%, 24.2% respectively. The average diameter of inhibition zone between the group of Liquorice 10mg contained and the group of Glycyrrhizic Acid 10mg contained or group of licoflavone10mg contained were compared and significant difference were found(P<0.05); There was no significant difference among the effective power(P>0.05). No significant relationship existed between Not only average diameter of inhibition zone but also the effective power among Clarithromycin control group and The group of Liquorice 10mg contained(P >0.05).
Conclusion: The rate of antibiotic resistance of isolated Hp to metronidazole, clarithromycin, amoxicillin, furazolidone, were high. Both the 4 different group of Glycyrrhizic Acid contained and the 4 different group of licoflavone contained all take on a few effective power to isolated Hp , which are even found in group at level of ug contained. The effect of groupe of Glycyrrhizic Acid 10mg contained, groupe of licoflavone 10mg contained, and clarithromycin control group to isolated Hp are fairly. The effective power among of the group of Liquorice 10mg contained , the group of Glycyrrhizic Acid 10mg contained , and group of licoflavone 10mg contained to isolated Hp were similar and no significant difference were found(P>0.05). But the average diameter of inhibition zone of the group of Liquorice 10mg contained outweigh that of group of Glycyrrhizic Acid 10mg contained or group of licoflavone10mg contained(P<0.05). The group of Liquorice 10mg contained take on the better effect to isolated Hp than that of group of Glycyrrhizic Acid 10mg contained or group of licoflavone10mg contained. The group of Liquorice 10mg contained have the fairly antibacterial effect as that of the Clarithromycin control group.
引文
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1.管远志、王艾琳、李坚主编.医学微生物学实验技术.第一版.北京:化学工业出版社,2006:105-146
2.胡文华,徐采朴.幽门螺杆菌菌株的7种药物体外药敏试验研究.第三军医大学学报.1996;18(5):457-458.
3.YakooB J,Fan XG,Liu L,Hu GL,Zhang Z.Antibiotic susceptibility of Helicobacter pylori in the Chinese population.Gastroenterology and Hepatology.2001;16(9):981-985.
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10.Wu H,Shi XD,Wang HT,Liu GX.Resistance of Helicobacter pylori to metronidazole,tetracycline and amoxicillin.J Antimicrob Chemother.2000;46:121-123.
11.郝庆,李岩,高红,张显忠.幽门螺杆菌对克拉霉素耐药的分子基础.世界华人消化杂志.2003;11(10):1485-1487ⅰ.
12.姜葵,何利华,赵飞,王邦茂,张建中.一种新的幽门螺杆菌克拉霉素耐药相关基因.世界华人消化杂志2006;14(15):1516-1519.
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1.管远志、王艾琳、李坚主编.医学微生物学实验技术.第一版.北京:化学工业出版社,2006:105-146
2.胡文华,徐采朴.幽门螺杆菌菌株的7种药物体外药敏试验研究.第三军医大学学报.1996;18(5):457-458.
3.YakooB J,Fan XG,Liu L,Hu GL,Zhang Z.Antibiotic susceptibility of Helicobacter pylori in the Chinese population.Gastroenterology and Hepatology.2001;16(9):981-985.
4.Toracchio S,Marzio L.Primary and secondary antibiotic resistance of Helicobacter pylori strains isolated in central Italy during the years 1998-2002.Dig Liver Dis.2003;35(8):541-545.[PMID:14567457]
5.Taneike I,Goshi S,Tamura Y,Wakisaka-Saito N,Matsumori N,Yanase A,Shimizu T,Yamashiro Y,Toyoda S,Yamamoto T.Emergence of clarithromycin-resistant Helicobacter pylori(CRHP)with a high prevalence in children compared with their parents.Helicobacter.2002;7(5):297-305.[PMID:12390209].
6.McMahon B J,Hennessy TW,Bensler JM,Bruden DL,Parkinson AJ,Morris JM,Reasonover AL,Hu.rlburt DA,Bruce MG,Sacco F,Butler JC.The relationship among previous antimicrobial use,antimicrobial resistance,and treatment outcomes for Helicobacter pylori infections.Ann Intern Med,2003;139(6):463-469.[PMID:13679322]
7.Perez,AL,Kato M,Nakagawa S,Kawarasaki M,Nagasako T,Mizushima T,Oda H,Kodaira J,Shimizu Y,Komatsu Y,Zheng R,Takeda H,Sugiyama T,Asaka M.The Relationship Between Consumption of Antimicrobial Agents and the Prevalence of Primary Helicobacter pylori Resistance.Helicobacter.2002;7(5):306-309.[PMID:12390210]
8.郝庆,李岩,张智杰,刘勇,王晓.沈阳地区幽门螺杆菌耐药情况的研究.世界华人消化杂志.2002;10(4):480-481.
9.Godoy APO,Ribeiro ML,Benvengo YHB,Vitiello L,Miranda CBM,Mendonca S,Jr JP.Analysis of Antimicrobial susceptibility and virulence factors in Helicobacter pylori clinical isolates.BMC Gastroenterol.2003;3:20.
10.Wu H,Shi XD,Wang HT,Liu GX.Resistance of Helicobacter pylori to metronidazole,tetracycline and amoxicillin.J Antimicrob Chemother.2000;46:121-123.
11.郝庆,李岩,高红,张显忠.幽门螺杆菌对克拉霉素耐药的分子基础.世界华人消化杂志.2003;11(10):1485-1487ⅰ.
12.姜葵,何利华,赵飞,王邦茂,张建中.一种新的幽门螺杆菌克拉霉素耐药相关基因.世界华人消化杂志2006;14(15):1516-1519.
13.Glupczynski Y,Megraud F,Lopez-Brea M,ANDERSEN L P.European multicenter survey of in vitro antimicrobial resistance in Helicobacter pylori.Eur J Clin Microbiol Infect Dis.2001;20(11):820-823.[PMID:11783701]
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