Studies On The Combination Between Natural Products And Fluconazole Against Drug-Resistant Candida Albicans And Its Mechanisms

We adopted the M27-A2 project recommended by the National Committee For Clinical Laboratory Standard(NCCLS).Twelve natural products were tested and selected with several clinical candida albicans with different susceptibilities to fluconazole by micro dilution test method. The susceptibility results reveal that,among the 12 natural products, just artemisinin and sodium houttuyfonate have anti-candia albicans activity,the range of their eighty percent of minimal inhibitory concentrations(MIC80) are 8μg/ml to 16μg/ml and 8μg/ml to exceed 64μg/ml respectively.But by checkerboard microdilution assay,6 natural products,which are forsythiaside, quercetin, luteolin, rutin, artemisinin, sodium houttuyfonate,have synergetic effects with fluconazole against clinical fluconazole-resistant candida albicans,Especially forsythiaside.The range of fractionary inhibitory concentration index (FICI) of forsythiaside and fluconazole is 0.01 to 0.024. After in combination with forsythiaside, MIC80 of fluconazole reduced to=0.125-2μg/ml from >64μg/ml against FLC-resistant candida albicans. Forsythiaside and fluconazole also have synergistic effects against candida tropicalis and cryptococcus neoformans. Besides,when in combination with the other azoles such as itraconazole, ketoconazole, miconazole and so on, forsythiaside appeared synergistic effects against FLC-resistant candida albicans. At the same time, forsythiaside have synergism with amphotericin B against FLC-resistant candida albicans,candida tropicalis,candida parapsilosis,candida krusei and candida glabratas. Increasing the number of clinical FLC-resistant candida albicans strains,we further confirmed the vitro synergism between forsythiaside and fluconazole by checkerboard microdilution assay,time-growth curve and WST-8 test.But in the treatment of systemic infection with FLC-resistant candida albicans in normal mice,the combination therapeutic effect between forsythiaside and fluconazole is not more potential than the group treated by fluconazole alone, which might be closely related to its instable structure. No significant effect from forsythiaside occurred at the active absoption and efflux of rhodamine 6G,but it dramatically promoted the production of intra-cellular reactive oxygen species (ROS) with dose-dependent mode. The results of GC/MS assay indicate that the combination between forsythiaside and fluconazole can obviously decrease the amount of ergosterol and lead to obvious accumulation of lanosterol and its analog eburico compared to fluconazole alone. After treated with the combination, cell membrane was destroyed more heavily through electron microscope.Mechanisms of the synergism between forsythiaside and fluconazole may be mainly related to its augmentation of intra-cellular ROS and impact on the synthesis of ergosterol.This research offer a new way for the treatment and finding a new leading product for resistant candida albicans.