Study On The Effect Of Resistance To Bovine Viral Diarrhea Virus By Baicalin In Vitro And The Synthesis Of Its Derivatives

Bovine viral diarrhea disease（BVD）is an acute infectious disease that can spread and infect cattle,sheep,deer and other domestic animals and wildlife.The disease’s course is complex,the clinical and sub-clinical symptoms are diverse,and it lacks effective prevention and control measures.It has spread rapidly in the world and caused huge economic losses.Bovine viral diarrhea virus（BVDV）belongs to the Flaviviridae and Prion genus and is a direct cause of bovine viral diarrhea.Therefore,finding and screening out active substances against BVDV is extremely necessary for the prevention and treatment of BVD.More and more studies have shown that Chinese medicine can overcome drug resistance problems in terms of antibacterial and anti-virus,and the prevention and treatment of such diseases can be achieved through multiple pathways and multiple targets.Among them,the Chinese medicine Huanghua has attracted increasing attention.Baicalin is a characteristic component and the main functional ingredient of Astragalus membranaceus,and its remarkable antiviral effect has attracted increasing attention.Based on this,this paper focuses on the study on the anti BVDV effect of baicalin and takes into account the low bioavailability of baicalin,and studies its structural modification to provide new ideas and directions for the development and application of baicalin.First,we carried out baicalin activity against bovine viral diarrhea virus in vitro.The TCID₅₀ of the virus was determined by immunoperoxidase assay at 10^-4.375/0.1 mL.The CCK-8method was combined with the CPE method to determine that the maximum drug safe concentration of baicalin on MDBK cells was 12.5μg/mL,and that Within a safe concentration range,low-dose baicalin has a certain role in promoting the proliferation of cells;at the same time,the four different modes of administration of baicalin have a significant role in vitro about anti-BVDV activity.Among them,baicalin antiviral has the most significant effect of invasive blocking,and its anti-BVDV effective rate is as high as 85.81%.Followed by direct killing,comprehensive role and inhibition of replication.Furthermore,we carried out the mechanism of baicalin against bovine viral diarrhea virus in vitro.The TaqMan fluorescent probe method was used to detect the replication level of BVDV in MDBK cells.Real-time fluorescent quantitative PCR was used to detect the relative expression levels of autophagy genes Beclin-1,Atg-12,and LC3B using SYBR Green fluorescent dye.The results showed that baicalin inhibited the replication of viral RNA;the expression of autophagy genes Beclin-1,Atg-12,and LC3B increased significantly after viral infection.Baicalin reduced the expression of autophagy genes Beclin-1,Atg-12,and LC3B after viral infection of cells;at the same time,flow cytometry was used to further detect the apoptosis of infected cells and cells after infection.It was found that the cell apoptosis was significantly reduced when the cells were given baicalin and virus compared with cells given only with the virus.Finally,we aim at the structure of baicalin.Alcohol compounds were introduced into the6-position carboxyl group of the high active group-7-β-D-glucuronic acid of baicalin,Baicalin methyl ester,baicalin ethyl ester,baicalin propyl ester,baicalin butyl ester,baicalin amyl ester,baicalin hexyl ester and baicalin heptyl ester were synthesized.Among them,7 kinds of baicalin ester derivatives,Among them,baicalin hexyl ester and baicalin heptate have not been reported in the literature.