Study On Anti-Shigella Dysenteriae Of Involucre Of Castanea Mollissima Blume And Its Antibacterial Mechanism

Castanea mollissima Blume is a chestnut plant in the Fagaceae family.Castanea mollissima Blume is the most coated burs of chestnut.Its extract has antioxidant,hypoglycemic,anti-tumor,anti-inflammatory,antibacterial and other physiological activities,and has high application value in the pharmaceutical industry.At present,the researches on the biological activities of chestnut burs mainly focus on the antioxidant and anti-inflammatory activities,but the researches on their antibacterial activities are relatively few,and the researches on their antibacterial mechanism are rarely reported.In this thesis,with Shigella dysenteriae as the test bacteria and antibacterial rate as the response value,the extraction process of antibacterial components of chestnut burs was optimized,and the antibacterial activity and mechanism of chestnut burs were systematically expounded,in order to provide theoretical basis for the treatment of bacterial diarrhea and industrial development and utilization of chestnut burs.The main research results are as follows:1.Through Single-factor experiment combined with response surface method to optimize the extraction process of antibacterial components from chestnut burs.Results:Under the optimal conditions of ethanol concentration 45%,solid to liquid ratio 1:20（g:m L）and extraction temperature 50℃for 120 min,the antibacterial rate of chestnut burs extract was 61.27±2.6%,which was consistent with the predicted value.2.The antibacterial activity was evaluated by antibacterial zone test,minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）test.The results showed that the ethyl acetate layer（CEY）had significant inhibitory effect on Shigella dysenteriae with MIC of 0.4 mg/m L and MBC of 1.6 mg/m L.The contents of total polyphenols and flavonoids in chestnut burs were determined.The results showed that the contents of polyphenols and flavonoids were positively correlated with the antibacterial activity.HPLC-MS was used to identify the components of CEY,and the components and contents of six main polyphenols were identified.They are Ellagic tannins acid（15.51%）,Procyanidin（0.13%）,myricetin（0.05%）,rutin（0.01%）,quercetin（0.03%）,kaempferol（0.22%）.Growth curve method was used to determine the inhibitory effect of CEY on Shigella dysenteriae.The results showed that the inhibitory rate of 0.4 mg/m L CEY on Shigella dysenteriae was more than 90%.Under the Scanning Electron Microscopy（SEM）,the forms of bacteria treated by CEY were shrunking and damaged.Flow cytometry also confirmed that CEY could inhibit the growth of Shigella dysenteriae.3.This part studies the bacteriostatic mechanism of CEY against Shigella dysenteriae at physiological level.The results showed that CEY damaged both the outer membrane and the inner membrane to a certain extent by targeting the cell wall and membrane of Shigella dysenteriae.The DIBAC₄（3）fluorescence staining experiment demonstrated depolarization of the cell membrane of Shigella dysenteriae.Leakage of alkaline phosphatase（AKP）,leakage of K⁺and internal macromolecular substances,protein release,ATP leak and 4,6-benzamidine 2-phenylindoles（DAPI）staining further confirmed that CEY causes the breakdown of cell wall and membrane barrier and the logistics of cytoplasmic contents,leading to apoptosis.4.The inhibitory mechanism of CEY on Shigella dysenteriae was studied at the molecular level,and the metabolomics was studied by liquid chromatoc-mass spectrometry（LC-MS）technology.The positive ion model found 362 different metabolites（DMs）in the cells of Shigella dysentery treated by CEY,148 of which were up-regulated and 214 of which were down-regulated.CEY affects membrane permeability through glycerophospholipid pathway,reduces purine nucleotide anabolism and hinders nucleic acid and protein synthesis by altering amino acid metabolism pathway.