Study On The Antimicrobial Active Substance And Its Mechanism Of Action Of Carex Meyeriana Kunth

Objective:Candida is the most common human fungal pathogen,which can not only cause superficial skin and mucous membrane infection,but also induce systemic infection,seriously affecting the quality of life of patients,and even endanger their lives.The existing antifungal agents have a single mechanism of action,and their long-term use leads to a surge in drug resistance.Therefore,the clinical demand for multi-channel treatment of candidiasis is increasing day by day.Modern studies have shown that Carex meyeriana Kunth（CMK）has good antimicrobial activity,and its antimicrobial products are widely used,but the researches on antimicrobial active substances and mechanism of action are scarce.In this study,by conducting the antimicrobial activity oriented isolation of CMK against Candida albicans to screened the antibacterial active components.To carry out research on the mechanism,in vivo and in vitro activity and target of active components in inhibiting Candida albicans,and dig out the main active substances and mechanism of action of CMK against Candida albicans.To provide support for the development of CMK medicine resources and the related research on the formation of antimicrobial agents.Methods:1.To investigate the antibacterial activity of 70%ethanol extract（CM-70）of CMK against Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus and Candida albicans.The extract of CM-70 was separated by macroporous resin column chromatography,and screened the active component of anti-Candida albicans was,then preliminary analysis of the composition of the components.Silica gel column chromatography was used to separate the CM-D90 components,and the main active components for inhibiting Candida albicans were screened.2.The effect of 90%ethanol eluting component（CM-D90）on the growth and proliferation of Candida albicans was investigated by measuring the minimum inhibitory concentration and growth curve of the active component.By measuring the extracellular protein,ergosterol content,comparative adhesion effect,germ tube formation,secretion of extracellular phospholipase,determination of the minimum inhibition concentration of biofilm,observation of biofilm cell morphology,respectively to investigate the CM-D90components,virulence factors of Candida albicans membrane and the influence of the biological cell membrane,and then clear the components are activated to restrain activity of Candida albicans in vitro and the mechanism of action.In order to screen the main active substances that change the cell permeability,inhibit the growth of mycelia,and destroy the mechanism of biofilm formation of Candida albicans,the effects of silica gel isolated active components on extracellular protein content,germ tube growth,and biofilm formation were investigated.3.Oral candidiasis and systemic candidiasis models of mice were established respectively to investigate the survival and infection of mice,target organ damage and mycelium colonization status,serum cytokine level after intervention of CM-D90 and G-20,and to determine the inhibitory activity of CM-D90 and G-20 against Candida albicans in vivo and the mechanism of action.4.Analysis and preliminary identification of G-20 components,and the protein differences of Candida albicans before and after treatment were quantitated by Label-free proteomics.According to the function of the differential protein,the pathways and targets of G-20 components in inhibiting the survival,proliferation and hyphae formation of Candida albicans were identified.By molecular docking,the binding ability of the main components in G-20 components to the key protein of mycelium formation was compared,the binding mode of the best binding component to the target protein was analyzed,and the content of this component in G-20 components was measured,so as to preliminarily determine the active components of CMK affecting the formation of mycelium.Result:1.The MIC values of CM-70 extract against Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa and Candida albicans,were 125,125,500 and62.5μg/m L,respectively.The results showed that CM-D90 had significant inhibitory activity against Candida albicans,and 94 compounds of fatty acids,flavonoids and other compounds were identified.A total of 57 components were isolated from CM-D90 by gel column chromatography,of which 45 had antibacterial activity against Candida albicans.Group G-20,G-32,G-46,and G-51 were screened and identified as the main antibacterial active components.2.The mechanism of action has been studied that CM-D90 could inhibit the growth of Candida albicans,and inhibit the proliferation of Candida albicans within 24h.It can inhibit ergosterol biosynthetic（≥31.25μg/m L）,damage cell membrane structure,change cell permeability and cause intracellular protein leakage（≥62.5μg/m L）.It can inhibit virulency factors such as adhesion and germ tube growth（≥62.5μg/m L）of Candida albicans,and make the biofilm structure of the dense network of Candida albicans loose,thus inhibiting biofilm cells(SMIC₅₀is 125μg/m L).Further analysis of the mechanism of the main active components found that,G-51 had the strongest effect on cell permeability,G-20 had the most significant effect on the inhibition of germ tube formation,and the strongest ability to inhibit biofilm(SMIC₅₀was 15.625μg/m L).The G-20 group was divided into CM-D90 component,the main active component of inhibiting mycelium formation.3.In vivo activity studies have shown that after the intervention of CM-D90 with concentrations of 65,130 and 260 mg/Kg in oral candidiasis mice,the area of pseudo membrane of dorsal tongue and mycelium colonization were reduced,the bacteria in oral cavity were reduced,the lingual papilla injury was repaired,and the atrophy of spleen was controlled.However,after the intervention of mice with systemic candidiasis,the body weight,renal accumulation and mycelium colonization were reduced,the structure was restored,and inflammatory cell invasion was reduced.G-20 components of 32.5,65 and 130mg/Kg can inhibit oral and systemic Candida albicans infection in mice,clear bacterial colonies of target organs,alleviate mycelium invasion,repair target organ damage,reduce inflammatory cell infiltration.After treatment,the levels of serum cytokines such as IFN-γ,TNF-α,IL-4,IL-10,IL-17 and IL22 in the two models were significantly lower than those in the model group,which reduced the residual inflammatory mediators in vivo and alleviated the inflammatory injury.4.Label-free proteomic quantitative analysis technique was used to compare the protein differences of Candida albicans after G-20 treatment,and to analyze the protein function.Differentiated protein function showed that G-20 inhibited the growth and proliferation of Candida albicans by affecting ribosomal synthesis,one carbon pool by folate,and alanine,aspartate and glutamate metabolism,and affected the hypha formation of Candida albicans by c AMP-PKA pathway.The mycelial formation of Candida albicans was significantly inhibited by G-20 treatment under scanning electron microscope.This group was the main active component of CMK to inhibit the virulence factor of Candida albicans.QRT-PCR verified that G-20 could down-regulate ASR2 and EFG1 to block the c AMP-PKA pathway,thus preventing EFG1 from regulating the initiation of mycelium formation of SUN41,and blocking the formation and elongation of candida albics mycelia by down-regulating the expression of SUN41 and SEC2.The main components of G-20 can bind to As R2,EFG1,SUN41 and SEC2 proteins with good medicinal properties to varying degrees,and the hot residues are similar,showing synergistic and complementary co-regulation characteristics.Among them,indirubin,geniligin,α-linolenic acid,ethyl linolenic acid and linoleic acid have potential blocking activities.Conclusion:This study showed that the extract of CMK had good anti-Candida albicans activity in vitro and in vivo,and the G-20 group was divided into its main active components.This component mainly contains fatty acids,flavonoids,alkaloids and other components,which can affect the translation process by inhibiting ribosome synthesis,affect DNA replication by inhibiting nucleotide synthesis through folic acid circulation pathway,and cause abnormal amino acid metabolism,inhibiting the survival and proliferation of Candida albicans.This component can directly bind to mycelium specific proteins SUN41 and SEC2 and block c AMP-PKA pathway to affect the initiation of mycelium specific proteins,inhibit mycelium formation and elongation,and reduce pathogenicity.In this study,we screened out the drug target that could be used in the treatment of oral and systemic candidiasis.In this study,the antimicrobial active substance and its mechanism of action of CMK had been excavated,and the drug target that could be used in the treatment of oral and systemic candidiasis had been preliminary explored.This study provides a research basis for the development of antifungal agents and the comprehensive utilization of CMK.