Study Of Antifungal Activity Of Composition From Phellnodendro Chinense Schneid. Var. Yunnanense Huang In Vitro And Vivo

[Objective]In order to screen the efficacy,cheap and less side effect of antifungal agents,and combination therapy methods against fluconazole-resistant Candida albicans,we study the antifungal activity of Phellnodendro chinense Schneid.var. yunnanense Huang.[Methods]Study the antifungal activity of methanol and chloroform extracts from Phellnodendro chinense Schneid.var.yunnanense Huang against Candida albican, Candida glabratas,Candida parapsilosis,Candida krusei and Cryptococcus neoformans by hole agar diffusion test.The antifungal activity was evaluated by diameter of antibacterial circle.Determine the MICs in accordance with the National Committee for Clinical Laboratory Standards(NCCLS) guidelines J(M27-A).We investigated the combination effect between fluconazole(FLC) and extracts against FLC-resistant clinical isolates of Candida albicans.In vitro we tested by checkerboard microdilution assy,and the effect was evaluated by calculating the fractional inhibited concentration index(FICI).In vivo we tested by median survival time,colony count and histopathology examination of kidney of a mouse model of disseminated FLC-resistant clinical isolates of Candida albicans.Moreover,MIC of the compounds was determined in accordance with the NCCLS guidelines J(M27-A).We investigated the combination effect between fluconazole(FLC) and compounds against FLC-resistant clinical isolates of Candida albicans by checkerboard microdilution assy,and the effect was evaluated by calculating the FICI.Agar disk diffusion test,time-kill curve studies confirmed the synergism of them.[Results]The antifungal antivity of methanol and chloroform extracts are similar to each other,diameter of antibacterial circle was 14.5～24.5 mm.Both of them have antifungal activity,average of MICs was 80～320μg/mL.When extracts were tested by combination with fluconazole,both extracts and fluconazole displayed significant activity with lower MICs against FLC-resistant Candida albicans,synergic antifungal effect was observed when they were used in combination(FICI＜0.1).In vivo,the median survival time of methanol and chloroform extracts 9 mg/kg(i.p.) respectively combination with fluconazole 0.5 mg/kg(i.p.) therapy groups was 20,17 days,while fluconazole-alone 0.5 mg/kg(i.p.) therapy group was 9 days which was obviously longer than that of fluconazole only therapy group.The log₁₀CFU/g of colony count of methanol and chloroform extracts 9 mg/kg(i.p.) respectively combination with fluconazole 0.5 mg/kg(i.p.) therapy groups was 4.783±0.203, 5.034±0.279,while fluconazole-alone 0.5 mg/kg(i.p.) therapy group was 5.372±0.254 which was obviously lower than that of fluconazole only therapy group. The result of histopathology examination of kidney of methanol and chloroform extracts 9 mg/kg(i.p.) respectively combination with fluconazole 0.5 mg/kg(i.p.) therapy groups was better than that of control group.Palmatine(yh-12),Jatrorrhizine (yh-13) and Berberine(yh-14) form Phellnodendro chinense S chneid,var.yunnanense Huang have stroger antifungal activity,average of MIC was 80～160μg/mL.When they were tested by combination with fluconazole,both compounds and fluconazole displayed significant activity with lower MIC against FLC-resistant Candida albicans,synergic antifungal effect was observed when they were used in combination(FICI＜0.1).Agar disk diffusion test displayed that antifungal effect was poor when drug was used alonely and was stronger in combination.Time-kill curve studies also confirmed the synergic effect,and the lower concentration of fungal was the stronger synergic effect.[Conclusion]Our findings suggested that the combination of fluconazole and extracts produced potently synergistic action against fluconazole-resistant C.albicans in vitro and vivo.Palmatine(yh-12),Jatrorrhizine(yh-13) and Berberine(yh-14) form Phellnodendro chinense Schneid.var.yunnanense Huang have antifungal activity. Our findings suggested that the combination of fluconazole and those compounds produced potently synergistic action against fluconazole-resistant C.albicans in vitro.