Study On The Against Candida Albicans Activity Of Traditional Herbal Monomers By Cell Wall Remodeling And The Antifungal Mechanism Of Sodium Houttuyfonate Combined With Fluconazole

Objective:To study the interaction between Sodium Houttuyfonate（SH）、Cinnamaldehyde（CIN）、Berberine（BER）、Jatrorrhizine（JAT）、Palmatine（PAL）five traditional herbal monomers（THMs）and Candida albicans SC5314、Candida auris 12372 cell wallβ-glucan;At the same time,in-depth study of SH and fluconazole（Fluconazole,FLU）with C.albicans（C.albicans SC5314/fks1Δ/FKS1 knockout strain）biofilmβ-glucan interaction,analysis of the two anti-C.albicans mechanism.Methods:The minimal inhibitory concentration（MIC）of Sodium Houttuyfonate（SH）,cinnamaldehyde（CIN）,berberine（BER）,jatrorrhizine（Jat）and palmatine（PAL）against C.albicans SC5314 and C.auris 12372 was determined by microdilution method,At the same time,the MIC of the above five kinds of THMs was determined by chessboard method;C.albicans SC5314,C.auris 12372（1×10³CFU/m L）were incubated with 5 kinds of THMs（alone/synergistic MIC）and the control group,then were observed and photographed by Gram staining;Take 5μL C.albicans SC5314,C.auris 12372（1×10³、1×10⁴、1×10⁵CFU/m L）were inoculated on YPDA agar plate containing 5 kinds of THMs（alone/synergistic MIC）and control group,spot test to observe changes in colonies;C.albicans SC5314 and C.auris 12372（1×10³CFU/m L）were with 5 kinds of THMs（alone/synergistic MIC）were incubated in 96 well flat bottomed microplate and the control group,the optical density（OD）was measured at600 nm of microplate reader to analyze the survival rate of fungi;Five kinds of THMs（synergetic MIC）were used to treat C.albicans SC5314,C.auris 12372 and Alexa fluor 488 coupled with WGA,then the chitin exposed to the cell wall and drug treat C.albicans SC5314,C.auris 12372 with the first antibody（anti-β-glucan）incubated for 4hours,the second antibody（Cy3 labeled Goat anti mouse IgG）was incubated for 1hour,then theβ-glucan exposed to the cell wall.Microdilution method to detect SH and/or FLU against C.albicans（C.albicans SC5314 and fks1Δ/FKS1 knockout strain）MIC and minimum inhibitory concentration of biofilm（Sessile MIC）SMIC₅₀and SMIC₈₀in the presence or absence of exogenousβ-glucan.By preparing C.albicans static,flow dynamic and subcutaneous catheter biofilms,separating biofilm extracellular matrix（EM）and cell wall（CW）,co-incubating with SH and/or FLU,UPLC（Ultra Performance Liquid Chromatography,Ultra Performance Liquid）Chromatography,UPLC))to detect the remaining drug content in the supernatant;Glucose Oxidase Kit method were used to detect EM and CWβ-glucan content;UPLC detects the drug content after the interaction of exogenousβ-glucan（Zymosan A）with SH and/or FLU.Results:The results of drug sensitivity showed that the MICs of SH、CIN、PAL、BER and JAT on C.albicans SC5314 were 128、100、128、128、256μg/m L,and the MICs of C.auris 12372 are 64、50、256、256、256μg/m L.In the THMs combination,the FICI range of C.albicans SC5314 is 0.313 to 1.5,and The FICI range for C.auris 12372 is0.5 to 1.125;the results of Gram staining showed that compared with the control group and the THMs（MIC）alone group,the mycelial microaggregates of the THMs（synergy MIC）combined treatment group disappeared,and only scattered bacterial cells can be seen;the results of spot experiment found that compared with the control group and alone THMs（MIC）,the colonies formed by Candida in the combined action of THMs（synergy MIC）shrank and dispersed more significantly;in the strain survival rate analysis experiment,THMs（single/synergy MIC）combined effect on the strain inhibition rate reached 52%～83%;at the same time,five THMs（synergy MIC）can induce different degrees of C.albicans SC5314 and C.auris 12372 cell wall componentsβ-glucan and chitin Exposed.The anti-C.albicans（C.albicans SC5314 and fks1Δ/FKS1 knockout strain）test results of drug sensitivity against SH and/or FLU showed that SH and/or FLU have a certain inhibitory potential on C.albicans biofilm,SH and/or FLU on C.albicans、fks1Δ/FKS1 knockout strain has lower SMIC than C.albicans SC5314;In the presence of exogenousβ-glucan,β-glucan can greatly enhance the MIC of C.albicans on SH and/or FLU,while exogenousβ-glucan（Zymosan A）and SH and/or FLU co-incubation,the drug content decreased significantly;in the C.albicans biofilm experiment of the three models,it was found that compared with the control group,the drug content decreased after the SH and/or FLU co-incubation with CW and EM.Conclusion:These studies show that the five THMs of SH、CIN、PAL、BER and JAT have certain inhibitory effects on Candida alone or in combination.Among them,the five THMs of SH、CIN、PAL、BER and JAT interact with the cell wall of Candida at a coordinated concentration.It can induce different degrees of exposure of Candida cell wallβ-glucan and chitin,which helps the immune system to recognize and eliminate Candida,thereby exerting the antifungal effect of THMs.At the same time,this study also shows that the effective drug concentration of SH and/or FLU in the combined action of the two drugs is significantly higher than the effective drug concentration of the single drug action,suggesting that the antifungal activity of SH and/or FLU is comparable to that of C.albicans biofilm EM、CWβ-glucan interaction（p Hysical adsorption）is related.