Isolation,Identification And Anti-Aeromonas Hydrophila NJ-35 Mechanism Of Antibacterial Component Extracted From The Potentilla Chinesis Ser. (Wei Ling Cai)

Potentilla chinensis Ser.（Wei Ling Cai）belonged to Rosaceae,mainly distributed in the north and southwest area.The gas of Wei Ling Cai was slight,and flavour was acerbity,slightly bitter.It had the effect of clearing away heat and detoxifying,cooling blood and stopping dysentery for dysentery,abdominal pain,dysentery,hemorrhoids bleeding,carbuncle swelling sore.At present,many scholars had studied the chemical constituents and pharmacological activities of Wei Ling Cai.However,there were few researches on the antimicrobial activity of Wei Ling Cai.Therefore,in this study,the antibacterial active components was isolated and identified,and its antibacterial mechanism was studied.The research content of this paper was divided into the following three parts:1 Preparation of ethyl acetate extract and isolation and identification of antimicrobial active components of Wei Ling CaiThe dry powder of Wei Ling Cai was extracted by soaking in ethyl acetate,then was concentrated by rotary evaporation.The filtrate was pumped by vacuum pump,and the filtrate was concentrated by rotary evaporation,dried at 50℃and stored at-20℃.the components were isolated and identified by high performance liquid chromatography（HPLC）and nuclear magnetic resonance（NMR）.The results showed that the effective antimicrobialcomponentwerecompound（1R,2S,5R,6S）-2,6-bis（3-Ethyl-4-hydroxy-5-methoxyphenyl）-1,7-dioxabicyclo[3.3.0]octane（P-3）,which molecular formula was C₂₄H₃₀O₆.2 The antibacterial mechanism of P-3 against Aeromonas hydrophila NJ-35 in vitroMicrodilution method was used to determine the minimum inhibitory concentration（MIC）and determination time-kill curve to evaluate the antibacterial activity.The permeability of cell wall and cell membrane were determined,and the protein expression differences were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）combined with Nano LC-ESI-MS/MS mass spectrometry,and the m RNAexpression of the different proteins was determined by Real-time PCR.Scanning electron microscopy（SEM）and transmission electron microscopy（TEM）were used to observe the ultrastructural changes of Aeromonas hydrophila NJ-35（NJ-35）to explore its antibacterial mechanism.The results showed that the MIC of the extract P-3 against NJ-35was 0.125 mg/m L;After P-3 treatment,the permeability of bacterial cell wall and membrane were significantly enhanced（P<0.01）;The protein and m RNA expressions of phosphoglucomutase（PGM）,pyruvate dehydrogenase E1 and alkyl hydroperoxide reductase subunit C（Ahp C）were up-regulated,while the protein and m RNA expressions of porin and 30S ribosomal S3 were down-regulated;The morphology changes of NJ-35were observed by SEM and TEM as wrinkled depression,and the contents leaked,and the cytoplasm contracted.The results showed that antimicrobial mechanism of P-3 was to destroy the cell wall and cell membrane of bacteria,inhibit the ability of NJ-35 to obtain nutrients,interfere with normal protein synthesis,and affect normal energy metabolism of bacteria through glycolysis and gluconeogenesis pathways.3 Effect of P-3 on resistance of carp to Aeromonas hydrophila NJ-35 infectionIn this study,carp was used as the research object to study the control effect of adding P-3 in the basal diet on Aeromonas hydrophila NJ-35（NJ-35）infection in experimental fish.The experimental fish were intraperitoneally injected with NJ-35,and fed with 0.5%,1%and 2%extract P-3 in the basal diet,respectively.The fish were fed for 2 weeks.The growth performance of the fish was measured,the changes of body surface were observed,and the intestinal sections were observed by hematoxylin-eosin staining（HE）.The results showed that the weight gain rate of low dose group（0.5%P-3）,medium dose group（1%P-3）and high dose group（2%P-3）increased by 8.09%（P<0.05）,13.09%（P<0.05）,16.26%（P<0.05）,the mortality rate was significantly decreased（P<0.05）,from 100%in the control group to 40%in the low-dose group and 10%in the medium does group,and the high does group was no mortality.The P-3 extracts in medium dose group and high dose group also had a protective effect on the intestinal tract of carp,showed good antibacterial and bactericidal effects against Aeromonas hydrophila in vivo.