Study On Effect Of Against APEC Of Extract From Compound Chinese Medicine Sihuangzhili

ObjectiveThis paper directly perform a chain of experiments,optimization of methanol extract process,establish a method of multi-component detection,study the external antibacterial activity and effects on virulence genes of against avian pathogenic Escherichia coli?APEC?and internal treatment effect,to provide a theoretical basis to further clarify study of extract of Sihuangzhili compound.MethodsExtract of compound Chinese medicine Sihuangzhili?Scutellariae Radix,Phellodendri Chinensis Cortex,Coptidis Rhizoma,Rhei Radix Et Rhizoma,Glycyrrhizae Radix Et Rhizoma,Isatidis Radix?with methanol by dipping,taking extraction time,temperature,solid-liquid ratio and methanol concentration as argument,comprehensive score of concentration of baicalin and berberine,dry extract yield as dependent variable,concentration were determinded by HPLC,mathematical model was established by response surface methodology,prepare ECCS.Minimum inhibitory concentration?MIC?and the effect of the ECCS against the APEC-O₂ growth curve was explored by micropore-plate and absorptiometry.Effect of the extract against nucleic acid leakage and energy metabolism of APEC-O₂ was determined by spectrophotometry.The influence of the ECCS against virulence gene expression of tsh,iss,ibeA and fimC in APEC,were evaluated by fluorescence quantitative PCR.Bacterial fluid pretreated with subinhibitory concentration ECCS,to observe the effect of ECCS on the pathogenicity of APEC-O₂.Build pathological model of laying hens with APEC-O₂,ECCS treatment group?high,medium and low doses?,chemical and traditional Chinese medicine control group,to observe treatment effect and weight changes of chicks.Results1.HPLC chromatographic conditions:an C18 column?250 mm×4.6 mm,5?m?with methanol?A?-0.1%Phosphoric acid water?B?as mobile phase for gradient elution,detection wavelength was 278 nm,flow rate was 1.0 mL/min,and the column temperature was 25?with inaection volume of 10?L.Optimization of extraction conditions of Sihuangzhili Compound:extraction time146 min,soild-liquid ratio 1:333,methanol concentration 80%.In this conditions,dry extract yield was 17.44%,concentration of baicalin and berberine was209.28 mg/g and 168.17 mg/g.2.MIC of ECCS against APEC-O₂ was 125 mg/mL and 31.25¹25 mg/mL concentration could inhibit the growth of APEC-O₂ in the early stages,could lead to bacterial nucleic acid material overflow?p<0.01?,which showed dose-dependent.3.After treatment with APEC-O₂ by 15.62⁶2.5 mg/mL concentration of ECCS,the expression of virulence genes was regulated in a dose-dependent manner.Especially when the concentration of ECCS was 31.25 mg/mL,it could significantly inhibit the expression of tsh,iss and fimC genes?P<0.01?,and they all decreased by more than 40%compared with the control group.The expression of ibeA gene increased significantly when ECCS concentration was62.5 mg/mL?P<0.01?.31.25 mg/mL concentration of ECCS could reduce the morbidity and mortality of APEC-O₂,it is further confirmed that iss,tsh and fimC genes play an important role in the pathogenicity of APEC-O₂ infect host.4.2 g/L concentration of ECCS mixed drinks has a effective therapeutic effect on artificially infected avian colibacillosis and could significantly increase the weight gain of diseased chickens.ConclusionsThe multi-component detection method of ECCS by HPLC was established,the extraction process were optimized.The antibacterial mechanism of ECCS against APEC-O₂ could be by lead to bacterial nucleic acid material overflow,causing energy metabolism disorder and inhibiting virulence gene expression.ECCS has a effective therapeutic effect on artificially infected avian colibacillosis and could significantly increase the weight gain of diseased chickens.