Anti-Influenza Mechanism Study Of Chlorogenic Acid From Honeysuckle Extractand Its Protein Conjugate

Influenza,the short form of influenza,is a highly contagious disease of the upper respiratory tract that is initiated by influenza viruses as pathogens.Influenza is characterized by high transmissibility,low lethality,high morbidity and distinct morbidity features.Its typing is generally of four types:type A（commonly known as a-stream）,type B,type C,and type D.There are two filamins on the surface of the viral envelope:one is hemagglutinin（HA）fibrillary protein which acts on the early stage of virus infection;The other is neuraminidase（NA）fibrillary protein,which acts on the late stage of virus infection.Most of the anti influenza drugs now available on the market target the HA and NA proteins.Honeysuckle is rich in chlorogenic acid（CHA）in traditional anti influenza traditional Chinese medicine.As the main active component of honeysuckle extract,it has neuraminidase inhibitory activity,but the inhibitory effect of chlorogenic acid on virus in vitro is not high.It is found that the terminal of the six membered ring in the structure of chlorogenic acid has carboxyl group,which can condense with amino group.Through the previous research of the research group,bovine serum albumin（BSA）has the characteristics of availability,biodegradability,non toxicity and non immunogenicity,and there are multiple amino groups on BSA that can be condensed with it.In order to enhance the inhibitory activity of CHA,the natural protein BSA in plasma was used as the skeleton to construct multivalent protein conjugate without changing the original activity of chlorogenic acid.Firstly,the chlorogenic acid component in honeysuckle was purified,and the extract was multivalent coupled with natural protein BSA to obtain chlorogenic acid bovine serum albumin（CHA-BSA）macromolecule.Its antiviral activity was evaluated and its mechanism was studied.The results of NA inhibition experiment（NAI）at the individual level showed that its IC₅₀value reached the micromolar level.At the cellular level,its inhibitory effect on virus induced cytopathy（CPE）was studied.It was found that the EC₅₀value was at the micromolar level.The two levels corroborate each other.CHA-BSA shows good neuraminidase NA inhibitory activity.In order to further study the action mechanism of conjugates,time of addition（TOA）,hemagglutination inhibition（HAI）,binding inhibition and capture experiments were carried out.The results showed that the protein conjugate could not only specifically bind to the viral NA target and inhibit its NA enzyme activity,but also synergistically inhibit the early stage of influenza virus infection.This inhibitory effect was achieved by capturing influenza virus and aggregating it.In this study,the anti influenza activity of chlorogenic acid（CHA）and its protein conjugate（CHA-BSA）was systematically studied,and their anti influenza mechanism was further determined at the individual level and cell level.The results showed that in the inhibition of influenza virus life cycle,compared with chlorogenic acid,protein conjugate CHA-BSA showed higher inhibitory activity against NA targets in the later release stage,and could also inhibit pre infection.Therefore,the study on the anti influenza mechanism of chlorogenic acid protein conjugate is helpful for the prevention and treatment of influenza,and lays a foundation for the further development of multivalent anti influenza drugs.