CAJ | 학술논문

目的：探讨金银花活性成分异绿原酸对早期、成熟期烟曲霉生物被膜的体外作用。方法采用微量液基稀释法测定异绿原酸对受试烟曲霉菌株最低抑菌浓度(MIC)和最低杀菌浓度(MFC)；构建体外早期、成熟期静止生物被膜模型并用不同浓度(64、128、256、512、1024μg/mL)的异绿原酸组分别作用48 h，激光共聚焦显微镜(CLSM)检测生物被膜内存活菌；扫描电镜(SEM)观察生物被膜形态学改变；结晶紫染色法定量生物被膜。比较各组MIC和MFC。结果异绿原酸对受试烟曲霉菌株的MIC和MFC均大于1024μg/mL；不论早期或成熟期，空白对照组和异绿原酸各浓度组生物被膜内菌体在CLSM下均以绿色的活菌为主；电镜观察证实异绿原酸可破坏早期、成熟期的生物被膜结构，使胞外基质减少，菌体轮廓清晰；当异绿原酸浓度达到256μg/mL时，可以使载体上不同时期生物被膜的量明显降低，256、512、1024μg/mL异绿原酸组生物被膜定量与空白对照组及组间两两比较，差异均有统计学意义(P<0.05)。结论异绿原酸在体外不能杀死烟曲霉生物被膜内菌体，但可对烟曲霉早期和成熟期生物被膜产生破坏作用，其作用呈浓度依赖性。
목적：탐토금은화활성성분이록원산대조기、성숙기연곡매생물피막적체외작용。방법채용미량액기희석법측정이록원산대수시연곡매균주최저억균농도(MIC)화최저살균농도(MFC)；구건체외조기、성숙기정지생물피막모형병용불동농도(64、128、256、512、1024μg/mL)적이록원산조분별작용48 h，격광공취초현미경(CLSM)검측생물피막내존활균；소묘전경(SEM)관찰생물피막형태학개변；결정자염색법정량생물피막。비교각조MIC화MFC。결과이록원산대수시연곡매균주적MIC화MFC균대우1024μg/mL；불론조기혹성숙기，공백대조조화이록원산각농도조생물피막내균체재CLSM하균이록색적활균위주；전경관찰증실이록원산가파배조기、성숙기적생물피막결구，사포외기질감소，균체륜곽청석；당이록원산농도체도256μg/mL시，가이사재체상불동시기생물피막적량명현강저，256、512、1024μg/mL이록원산조생물피막정량여공백대조조급조간량량비교，차이균유통계학의의(P<0.05)。결론이록원산재체외불능살사연곡매생물피막내균체，단가대연곡매조기화성숙기생물피막산생파배작용，기작용정농도의뢰성。
Objective To investigate the effect of isochlorogenic acid on the biofilm of Aspergillus Fumigatus in early and mature stages in vitro. Methods The minimum inhibitory concentration (MIC) and the minimum fungicidal concen-tration (MFC) for isochlorogenic acid were measured by broth microdilution method according to CLSI M38-A2 guide-line. The biofilm models in early and mature stages established under static condition were affected by different con-centration gradients (64, 128, 256, 512, 1024μg/mL) of isochlorogenic acid for 48 h in vitro, then biofilm quantitation was performed by crystal violet staining assay. Scanning electron microscope (SEM) was used to observe the morpholog-ical change of biofilm structure. The viability of Aspergillus Fumigatus hyphae in biofilm was detected by confocal laser scanning microscope (CLSM). The MIC and MFC of different groups were compared. Results Both the MIC and the MFC of isochlorogenic acid on Aspergillus Fumigatus isolate were above 1024μg/mL. The results of CLSM demonstrat-ed that isochlorogenic acid showed hardly any fungicidal effect to the mycelia in early or mature stage of biofilm. Under SEM, the structure of Aspergillus Famigatustus biofilm treated with isochlorogenic acid was destructed and the outline of mycelia appeared to be more clear than that without isochlorogenic acid due to the reduction of extracellular matrix. The in vitro action was performed while the concentration of isochlorogenic acid achieved the level of 256μg/mL, with-in the concentrations of 256, 512, 1024 μg/mL, results of biofilm quantities indicated significant differences between serially increasing isochlorogenic acid concentration groups and untreated control group, with statistical significance (P<0.05). Conclusion Isochlorogenic acid exhibits destructive effect on the early and mature stages of Aspergillus Fami-gatustus biofilm in vitro, which shows a concentration-dependent manner.