| Objective:To study the antifungal effect of plasma-activated Ezhangfeng Cuji on Candida albicans and to explore fungicidal mechanism initially.Methods:After plasma treatment of Ezhangfeng Cuji（EC）and physiological saline（PS）and divided into four groups:physiological saline,plasma-activated physiological saline（PAPS）,Ezhangfeng Cuji and plasma-activated Ezhangfeng Cuji（PAEC）.The full paper is divided into four parts.The first part examines the liquid-phase reactive species（RS）（H2O2,NO3-and O3）in PS and PAPS and the p H values of the four groups,and detects the differences in the concentrations of the herbal representative ingredients oxymatrine and rhein in EC and PAEC by high performance liquid chromatography（HPLC）.The second part was to immerse PS/PAPS/EC/PAEC in Candida albicans for 1,5 and 10 min,respectively,and the time-inhibit curve was derived by calculating the colony formation units by plate counting method.In the third part,the changes of permeability to the cell wall and cell membrane of Candida albicans were measured indirectly by special staining of the cell wall and detecting the concentration of extracellular alkaline phosphatase（AKP）,UV absorption and the concentration of extracellular total protein（TP）,conductivity in extracellular,the changes to the nucleus were measured by DAPI staining,detection of cell morphological changes,apoptosis and ROS content in Candida albicans by PI staining,Annexin V-FITC/PI method and ROS kit by flow cytometry.The surface structure and ultrastructure of Candida albicans were observed by scanning electron microscopy（SEM）and transmission electron microscopy（TEM）.Determination of the effect on protein expression in mycobacterial cells by SDS-PAGE.The effect of PS/PAPS/EC/PAEC on the structure of Candida was thus measured.In the fourth part,detection of early adhesion and cell surface hydrophobicity（CSH）of Candida biofilm formation by CCK-8 and water-hydrocarbon two-phase assays.Inhibition of mycelium and shoot tubes by PS/PAPS/EC/PAEC observed by light microscopy.The secretory enzyme activities（secretory aspartic protease（Sap）,phospholipase（PL）,lipase（Lip））were measured by the bovine serum albumin agar plate method,the egg yolk agar plate method and the triglyceride agar plate method,respectively.From this,changes in the virulence factor of Candida were measured.Results:After plasma treatment,the concentrations of H2O2,NO3-and O3 were increased in PAPS,and the concentrations of oxymatrine and rhein were increased in PAEC.Compared to PS and EC,the p H values of PAPS and PAEC were decreased.Candida albicans was immersed in PS/PAPS/EC/PAEC for 1,5 and 10 min.Within 10 min,almost no change in the number of viable bacteria was seen in PS,and only a slight decrease in the number of viable fungi was observed by PAPS immersion,while immersion in EC/PAEC showed a significant and time-dependent decrease in fungi,and the effect of PAEC is more pronounced.Immersion of Candida albicans in PAEC resulted in cell wall,cell membrane,and nuclei were damaged leading to death,and the ROS content was increased;the early adhesion to tissue and cell surface hydrophobicity was all reduced,the formation of mycelial budding tubes was inhibited,and the secretase activity was inhibited.There was no obvious antifungal effect of PAPS on Candida albicans at 10 min,but a certain antifungal effect of PAPS could be seen at 48h.The antifungal effect of PAEC was more obvious at 10 min,and the antifungal effect of PAEC at 48 h effect was not as good as EC.Conclusion:The type and concentration of the main liquid phase active groups of EC changed after plasma treatment.PAEC has a more potent antifungal effect on Candida albicans than PAPS and EC,and its main antifungal mechanism may be related to the destruction cell structure and reduction the virulence factor of Candida albicans. |
