Study On The Anti-bovine Viral Diarrhea Virus And Antimicrobial Activity Of Baicalin And Its Ester Derivatives

Baicalin is an active representative component of Scutellaria bailensis Georgi,and its remarkable role in antiviral and antimicrobial activities has also attracted increasing attention.Bovine viral diarrhea virus（BVDV）is a direct cause of bovine viral diarrhea disease（BVD）.The positive detection rate in China is as high as 90.0%,and the mortality rate is as high as 50%,which has caused huge economic losses to cattle industry.In this paper,the anti-BVDV and bacteriostasis of baicalin and its ester derivatives were studied.Firstly,the TCID₅₀ of different biotypes of BVDV were investigated by immunoperoxidase assay.The TCID₅₀ of TC（NCP）BVDV and NADL（CP）BVDV were 10^7.25/0.1mL and 10^4.75/0.1mL,respectively.The maximum safe concentration of Baicalin and ester derivatives on MDBK cells was determined by CCK-8 method combined with CPE method.Secondly,we studied the anti-NCP BVDV and CP BVDV activity of baicalin and its ester derivatives in vitro.TaqMan fluorescence probe method was used to detect the replication level of NCP BVDV RNA in MDBK cells under four different drug treatments,including the replication inhibitory effect,the direct killing effect,the invasion blocking effect and the comprehensive inhibition effect.The results showed that baicalin and ester derivatives inhibited the RNA replication of NCP BVDV,and there were significant differences in the inhibitory effects of different drug delivery methods and different drug delivery methods.At the same time,the anti-CP BVDV activity of baicalin and ester derivatives in vitro was studied by CCK-8 method combined with CPE method.The results showed that baicalin and ester derivatives had significant inhibitory effects on CP BVDV in vitro under four different administration modes.Furthermore,with baicalin,baicalin methyl ester and baicalin ethyl ester with significant activity and stable chemical properties as the research objects,the apoptotic status of the cells infected with the virus and the cells treated were further detected by flow cytometry using the best drug delivery method selected.Real-time fluorescent quantitative PCR and SYBR Green fluorescent dye method were used to detect the relative expression of autophagy genes Beclin-1 and ATG-14.Detection of Beclin-1 and ATG-14 protein expression by Western blot technique and preliminary study on antiviral mechanism of Baicalin and ester derivatives.The results showed that the apoptotic rate of cells treated with baicalin,baicalin methyl ester and baicalin ethyl ester plus virus was significantly lower than that of cells only treated with virus.The expression of autophagy genes increased after NCP BVDV and CP BVDV infected cells.Baicalin,baicalin methyl ester and baicalin ethyl ester reduced the expression of autophagy genes Beclin-1 and ATG-14 after CP NCP BVDV infected cells;down-regulated the expression of Beclin-1 and ATG-14 protein.Finally,the in vitro bacteriostatic activities of baicalin and ester derivatives against Escherichia coli,Pseudomonas aeruginosa,Salmonella paratyphi A,Salmonella paratyphi B,Salmonella typhi and Staphylococcus aureus were evaluated.The results showed that baicalin and ester derivatives had bacteriostatic effects on 6strains of bacteria in vitro.The MIC values of baicalin methyl ester,baicalin ethyl ester,baicalin hexyl ester and baicalin hexyl ester in inhibiting Salmonella paratyphi A（0.625 mg/mL）were similar,and lower than that of Baicalin（1.25 mg/mL）.Baicalin ethyl ester inhibited the low MIC value（0.625 mg/mL）of Salmonella typhimurium,which was lower than baicalin MIC value（1.25 mg/mL）.