Effects Of Ethylacetate Extract From Huanglianjiedu Decoction On Virulence Factors Of Candida Albicans

Object:To research the effect of ethyl acetate extract from Huanglianjiedu decoction on virulence factors of Candida albicans.Methods:We use different polarity reagent to extract Huanglianjiedu decoction and to detect activity against C. albicans. XTT assay and agar plate assay were used to evaluate the effect of ethyl acetate extract of Huanglianjiedu decoction(EAHD)against adhesion of C.albicans to 96-well plates and polyester catheters. Inverted microscope was applied to observe the morphological changes of C. albicans in adhesion at different times;Inverted microscope,fluorescence microscope and scanning electron microscope(SEM)were applied to inspect the morphological change of C.albicans treated by EAHD.Solid plate was utilized to evaluate the colonies forms.Western blot was used to observe expression of mycelium-specific protein such as Nrg1p、Cdc28p;Egg-yolk medium, milk-plate medium and olive oil emulsification were used respectively to test the activities of phospholipase(PL),aspartic protease(Sap)and lipase(Lip)of C. albicans,The water-hydrocarbon two phase assay was applied to measure the cell surface hydrophobicity(CSH)of C. albicans;Catheter counting and scanning electron microscopy were applied to detect activities of effects of extracts on C.albicans biofilms dispersion.Microdilution method and time-kill experiment were adopted to observe the activity of EAHD in combination with Fluconazole(FLZ) on FLZ-resistant C. albicans,To further explore the mechanism,HPLC was utilized to evaluate the exchanges of ergosterol content. q RT-PCR was applied to evaluate expressions of virulence factors related genes.In vivo experiments to determine the treatment effect of EAHD to systemic and pulmonary infections.Results:(1) MIC of EAHD was 312μg/m L,which was better than other extracts.(2) 312 μg/m L EAHD could inhibit the adherence of C. albicans to 96-well plates and polyester catheters and the effect was time-indepent manner at 1,2,4h.(3) 1250μg/m L EAHD could inhibit the formation of C.albicans mycelium formating in different mediums,Inverted microscope,fluorescence microscope and SEMfurther confilmed intervation effect on morphological change.Western blot verified Nrg1 p was up-regulated and Cdc28 was down-regulated.(4) 1250μg/m L EAHD could significantly inhibit the activity of Sap and Lip but had no effect to PL. EAHD could reduce CSH of C.albicans in a dose-indepent manner.(5) SMIC80 of EAHD was 625 μg/m L;EAHD had no significant effect on the dispersion of biofilm.(6) MIC of EAHD were ranging from 156 to 1250 μg/m L, and the MIC of FLZ were ranging from 256 to 2048 μg/m L, SMIC80 of EAHD and FLZ were ≥1250μg/m L,≥512 μg/m L, respectively. Synergism between EAHD and FLZ, the minority FICI was0.066. Combination group also showed synergism effect except one group showing adding effect. The results of TK experiment also confirmed obviously synergistic effect when treated for 12 h. When compared with control groups, the ergosterol was reduced85% and 50% in the treatment of combination and EAHD groups by HPLC,respectively. FLZ group showed no significant difference compared to control group.(7) Most of genes about virulence factors were up-regulated and down-regulated at different folds especial in 1250 μg/m L group.(8) EAHD could effectively reduce the mortality rate and kidney colonies in mice of systemic infection of C. albicans, besides,EAHD could also improve Lung disease infected with acute lung infection.Conclusion: EAHD could inhibit adhesion,hypha formation,secretion of enzyme,hydrophobicity and biofilm formation of C.albicans at some extent..In addtion,the combination of EAHD and FLZ exhibited synergy against FLZ-resistant C.albicans.When infected with systemic and lung infection,EAHD showed therapeutic effect.