Study Of Butanol Extract Of Paris Polyphylla Var.Yunnanensis Against Candida Albicans Biofilm And The Related Mechanism

Object:To investigate the effect of Butanol extract from Paris polyphylla var.yunnanensis on the Candida albicans biofilm and explore the related mechanism.Methods:We used Butanol to extract Paris polyphylla var.yunnanensis and detect activity against Candidas. Microdilution method was applied to determine the minimum inhibitory concentration(MIC) and sessile minimal inhibitory concentration80(SMIC80) of Butano extract of Paris polyphylla var.yunnanensis(BEP)against C.albican; XTT assay and agar plate assay were used to evaluate the effects of BEP against adhesion of C.albicans to different materials. Scanning electron microscope(SEM) was applied to observe the morphological changes of C. albicans; Inverted microscope was applied to inspect the morphological change of BEP on C.albicans and the detected the absorbance;Gram staining method was used to observe the mycelial morphology; YPD Solid plate was utilized to evaluate the colony morphology; XTT method was used to detect the inhibition rate of C.albicans biofilm under different concentrations of BEP and at different points; the time-kill curve was made to evaluate the effect of BEP on C.albicans. SEM was used to observe the morphological changes of biofilm at different times. Confocal laser scanning microscopy(CLSM) was applied to determine the thickness of the C.albicans biofilm.Microplate Reader and plate counting method were used to detect the effect of BEP on C.albicans mature biofilm dispersion. QRT-PCR was applied to dertermine the expression changes of the related genes of biofilm formation and dispersion.Microdilution method was used to determine the MIC and SMIC80 of C.albicans SC5314; fluorescence microscopy was applied to detect the effect of BEP on the cell membrane of C.albicans; flow cytometry was used to observe the reactive oxygen species(ROS) of C.albicans; FITC-VAD-FMK staining assay was performed to evaluate the metacaspase activity;DAPI staining was performed to determine thenuclear fragmentation; qRT-PCR was applied to determine expression changes of genes related biofilm.Results:1?Anti Candida activity results showed that the MIC of BEP against C.albicans is 8?g/mL,which is better than others Candida strain.2?The MIC of BEP against C.albicans is determined as 8-16?g/mL, The SMIC80 of BEP against the biofim of C.albicans is determined as 32-128?g/mL; at 4h,32?g/mL BEP can inhibit the adherence of C.albicans to the polyester catheter and polystyrene and the morphological transformation of C.albicans; 32?g/mL BEP can repress the morphological changes of C.albicans in liquid and solid environment;Furthermore,the inhibition rate of BEP on biofilm formation of C.albicans was about 80%, and the inhibitory effect of BEP in the early stage is better than the late stages;Time sterilization curve results showed that BEP has a promise bactericidal effect; The morphology of Candida albicans biofilm is affected and the thickness of the biofilm is reduced by BEP according to SEM and CLSM results.BEP can induce C.albicans biofilm cell dispersion and affect the expression of genes related biofilm formation and dispersion.3?The MIC of BEP against C.albicans SC5314 is determined as 8?g/mL, The SMIC80 of BEP against C.albicans SC5314 is determined as 32?g/mL; BEP can damage the cell membrane of C.albicans and increase intracellular ROS; Under BEP treatment, the metacaspase enzyme activity of the cell membrane is activaed and the nucleus appears fragmentation.Conclusion:Our presented results suggested that,BEP may inhibit adhesion, transformation of hyphae, biofilm formation of C.albicans, and promote the dispersion of mature biofilm.And our results also showed that,C.albicans biofilm cell is damaged by BEP,including cell membrane, nuclear and so on.Therefore, our results suggested that BEP has a moderately antifungal effect on C.albicans.