Study On The Inhibitory Effect Of Centella Asiatica Extract On Aeromonas Veronii Biofilm And Preliminary Study Of Its Mechanism

Organisms on the earth have been under constant threat from bacteria for ages.Today,bacterial diseases have been largely controlled through the vaccines and antibiotics.The beginning of the antibiotic era had brought revolutionary changes to the treatment of infectious diseases.However,the prevalence of microbial resistance in the modern medical setting had also developed into a challenge for global health.As novel effective chemotherapeutic approaches continued to diminish,there was a pressing need to discover new antibacterial compounds and mechanisms for disease prevention and treatment.A competitive antibacterial advantage proposed for quorum sensing（QS）was that it weaken the virulence mechanism of bacteria without exerting selective pressure,while enabled bacterial infections more susceptible to eradication by the host immune system.From the perspective of alleviating the antibiotic resistance situation,this paper attempted to find inhibitors that could inhibit pathogen quorum sensing as well as biofilm formation.This study covers most of the methods that can be used to screen quorum sensing inhibitors through experiments,Chromobacterium violaceum（CV）ATCC 12472 and CV026 were used as indicators of QS.Lactic acid bacteria（LAB）with the ability to degrade N-acyl-l-homoserine lactone（AHLs）was screened from various sources by Oxford cup punching method,including freshwater fish intestine,mouse uterus and intestine,phasianus colchicus feces and pickled vegetables,The degradation activity was verified by separating LAB supernatant and cell disruption products,and it was found that its active site was intracellular.Subsequently,it was found that Centella asiatica extract demonstrated QS inhibitory activity from the extracts of 28different Chinese herbal medicines.The extract can significantly inhibit the purple pigment of Chromobacterium violaceum at the minimum inhibitory concentration（MIC）of 1400μg/m L,and does not affect the growth of Aeromonas veronii at sub-inhibitory concentration.The results showed that the ethyl acetate extract of Centella asiatica at the concentration of 1050μg/m L,700μg/m L and 350μg/m L could inhibit biofilm formation of Aeromonas veronii by 69.72±0.41%,53.71±1.15%and 42.35±0.73%,The state of the biofilm after staining was observed by electron microscopy,and the results also supported the inhibitory effect of the extract on biofilm.The content of extracellular polysaccharides was detected by sulfuric acid phenol method.The results showed that the inhibition rates of extracellular polysaccharides were 65.23±0.66%,41.94±1.23%and 19.28±0.19%,respectively.The addition of 1050μg/m L of the extract could also reduce the protease activity by 31.11±0.58%compared with the control group,and the inhibition rate of hemolytic activity was relatively reduced by 34.16±0.53%.Additionally,the extract also enabled to inhibit bacterial swimming and flagella formation.The active components of Centella asiatica extraction were analyzed by high performance gas chromatography-mass spectrometry,and the active Chinese medicine monomer was verified by indicator using Chromobacterium violaceum.It was found that dihydroactinidiolide and 2-ethyl-3-methylmaleimide had the QS inhibiting activity.The quorum sensing receptor protein model of Aeromonas veronii was predicted by Swiss-model,Alphafold2 and Modeller,respectively,and the protein model was evaluated.The crude extract of Aeromonas veronii extracted by ethyl acetate signal type was detected to be C₄-HSL by high performance liquid Chromatography mass spectrometry.The original ligand of the protein model was extracted and re-docking,and the parameters set by molecular docking were detected by RMSD value.The ligand C₄-HSL and the QS inhibitor furanone C-30 were used as positive control to dock with the predicted protein structure.It was speculated that 2-ethyl-3-methylmaleimide inhibited quorum sensing by competitively binding to the autoinducer binding site,however,its effect and influence need to be further explored in practical application.In this study,quorum-sensing inhibitors were screened through different methods,which has certain guiding significance for finding QS and biofilm inhibitors,and hoping to provide some ideas for future research on alleviating bacterial infection and the design of QS inhibitors.