Purification And Identification Of Quorum Sensing Inhibitors From Lentinus Edodes And Flammulina Velutipes

Some bacteria can produce,release,and detect a chemical signaling molecule called autologous inducer?autoinducers,AI?,whose concentration will increase as the bacterium population gets dense.Based on AI concentration variation,bacteria can sense the change of their community.When the concentration of AI reaches a certain threshold,bacteria reciprocally communicate,initiate,or regulate some related gene expression,which results in the change of their population behavior.And this AI-mediated bacterial population behavior is called quorum sensing?QS?.Pathogenic gene expressions of a considerable number of pathogenic and conditional pathogenic bacteria are regulated by QS mechanisms,and thus quorum sensing inhibitors?QSI?possess the potential to be explored as a new antimicrobial agent.However,the chemical synthesis QSI,such as bro minated furan,was found to be highly toxic,which limits its clinical application.Therefore,it is an essential research field to look for such QSIs that manifest lower toxic than compound derived QSIs,even no toxic to human beings.In this study,compounds that possess inhibit QS activities were isolated and purified by a biological activity-guided method from Lentinus edodes and Flammulina velutipes.Two monomeric compounds were successfully obtained,and their relative structures were elucidated.Furthermore,the actions that they inhibited the population behavior of the conditional pathogenic bacterial were preli minarily analyzed.The main conclusions obtained are as follows.1.Four kinds of edible fungi possessing the QSI potentialsMethanol extracts were prepared from fruit bodies of L.edodes,F.velutipes,Tremella aurantialba,and Pleurotus eryngii,respectively.Chromobacterium violaceum CV026 was used as the QSI activity indicator bacterium,which was applied to test the QSI activities of methanol extracts from four those edible fungi.And the corresponding results demonstrated that all four methanol extracts displayed the QSI activity to some extent,implying that compounds inhibiting the QS system,exist in the methanol extracts.Furthermore,these methanol extracts extracted from L.edodes and F.velutipes,displayed relatively high QSI activities.2.Construction of minimal QSI activity detection and analysis systemTo pursue the fractions containing QSI activities during the isolation and purification of extracts,minimal QSI activity detection and analysis system of the indicator bacterium CV026 was constructed.Besides,the effects of the kinds of organic solvent and the concentration of AI used in this study on QSI activity detection were analyzed.Our results showed that,first,the QS system of CV026could be efficiently initiated by the concentration of N-acyl-homoserine lactonase?C₆-HSL?over 10mM.Second,200?L to 500?L of CV026 culture in 2 m L Eppendorf tube,possesses the potential that discri minates which fraction includes the QSI activity compounds.Third,there is no effect of the minimal QSI activity detection and analysis system on the result analysis,when the methanol content and the ethyl acetate content were lower than 3%?V/V?and 2%?V/V?,respectively.3.Identification of the QSI activity compound from L.edodes fruit bodyThe QSI activities from different solvent extracts from L.edodes were compared,and the result showed that the ethyl acetate extract displayed the highest QSI activity.Large amounts of ethyl acetate extract were prepared.In the combination of silica-gel column chromatography with semi-prepared high-performance Liquid Chromatography technologies,one monomeric compound was successfully isolated and purified from L.edodes ethyl acetate extract,which inhibits the CV026 QS system.And then,high-performance Liquid Chromatography-Mass Spectr?LC-MS?and Nuclear Magnetic Resonance?NMR?were applied to analyze its molecular weight and relative structure,respectively.Its molecular weight is 280.23,which was provided by LC-MS analysis.Moreover,¹³C spectra and DEPT 135°spectra uncovered that it consists of 18 carbon atoms,including 4 unsaturation carbon atoms?128.05-130.38 ppm?,and 12 methylene groups?22.73-34.09 ppm?,and 1 methyl group?14.22 ppm?.In the combination of other spectra information?¹H spectra,COSY spectra,HMBC spectra,and HSQC spectra?with its molecular weight information,this compound was preli minarily identified as linoleic acid?all cis-9,12-Octadecarbondienoic acid?.4.Identification of the QSI activity compounds from F.velutipes fruit bodyIdentification of the QSI activity compounds from F.velutipes fruit body was performed,which was similar to the workflow used in looking for the QSI activity compound from L.edodes fruit body.And the corresponding result manifested that two monomeric compounds possessing QSI activities were identified from F.velutipes,and named compound 1 and compound 2,respectively.Two compounds were analyzed by MS,and the results showed that the molecular weight of compound1 and 2 are 280.23 and 278.23,respectively.And then,two compounds were further analyzed by NMR.The corresponding results showed that compound 1 is linoleic acid.¹³C spectra and DEPT 135°spectra of compound 2 revealed that it consists of 18carbon atoms,including 6 unsaturation carbon atoms?128.05-130.38 ppm?,and 10methylene groups?22.73-34.09 ppm?,and 1 methyl group?14.22 ppm?.In the combination of other spectra information with its molecular weight information,this compound was preli minarily identified asa-linolenic acid?all cis-9,12,15-Octadecarbontrienoic acid?.5.Effects of linoleic acid on pigment formation or swar ming behavior of conditional pathogenic bacterialAll L.edodes and F.velutipes derived and commercial linoleic acid samples were identified and compared by Gas Chromatograph Mass Spectrometer?GC-MS?.And the results showed that all their main ion peaks appeared at 14.2 min.Furthermore,their main ion fragments and characteristic fragments were highly consistent,indicating that they are the same compound.Biological activity assays were performed by using commercial linoleic acid.And the corresponding results exhibited that 61%of pigment production was inhibited by 0.2mM linoleic acid in Serratia marcescens?red?.Moreover,swarming behaviors of Pseudomonas aeruginosa and S.marcescens MG1 were efficiently inhibited by 5 m M linoleic acid.