Study On Isolation And Purification Of The Effective Anti Respiratory Syncytial Virus From Euonymus Alatus (Thunb.) Sieb And Its Anti-viral Mechanism

Objective The anti-RSV effective parts of Euonymus alatus(Thunb.)were screened by different isolation and purification methods using in vitro antiviral tracer technology.Further,the anti-RSV effects of the extracts were evaluated by in vitro and in vivo experiments,and the mechanism of anti-RSV action was priliminarily explored.Methods1.Water extraction and alcohol precipitation method were used to obtain the crude extract of Euonymus alatus(Thunb.),NKA-9、HPD-100、D-101、DM-301、AB-8 macroporous adsorption resin columns with different polarity were used for further separation and purification with distilled water,ethanol with volume fraction of 25%,50% and 75%respectively as flow.Antiviral experiments in vitro were carried out to investigate the antiviral effect of different isolated and purified parts,and to screen out the parts with good anti-RSV effect.2.Ultrafiltration method was used to ultrafiltration the solution of the better effective parts,and the sample solutions with different molecular weights were obtained.In vitro anti-RSV experiments were carried out respectively,and the effective parts with the best anti-RSV effect were obtained.3.Physical and chemical identification was carried out on the obtained effective parts to determine the types of its effective chemical constituents.4.The effective parts of Euonymus alatus(Thunb.)obtained were added to the cells via different time nodes to explore their anti-RSV mechanism initially.5.Establish models of mice of RSV pneumonia,and divide the effective parts of Euonymus alatus(Thunb.)into high,medium and low dose groups for pharmacodynamic experiments.Observe the changes of body weight and activity,lung index changes of each group and index inhibition rate,pathological section of lung tissue of the medication groups to explore their antiviral effect.6.The concentrations of the inflammatory factors tumor necrosis factor-α(TNF-α),tumor necrosis factor-β(TNF-β),interferon-α(IFN-α),and interleukin-4(IL-4)in the blood of mice were detected by ELISA to explore the anti-RSV mechanism in vivo of the effective parts of Euonymus alatus(Thunb.).7.Establish models of mice of RSV pneumonia,RT-PCR was used to detect the expression levels of viral load,TLR3 and TLR4 in lung tissue,and to investigate preliminarily the effect of the active site of Euonymus alatus(Thunb.)on the expression of RSV,TLR3 and TLR4 m RNA in lung tissue of mice.Results1.According to the treatment index(TI),the water eluent of HPD-100 macroporous adsorption resin had the best anti-RSV effect(TI=52.000),and the effect was better than ribavirin.2.Further separation and purification of effective parts by ultrafiltration experiment showed that the ultrafiltration extract with molecular weight less than 3000 had the best anti-RSV effect,TI=83.500,higher than ribavirin(TI=49.500).3.The physicochemical identification experiments showed that the chemical components of the effective ultrafiltration site were polyphenols.4.Through dosing experiments at different times,it was found that when the effective parts and the virus were added at the same time,the TI value was the maximum.It indicated that it had the strongest direct inactivation effect on the virus and played the preventive and therapeutic effects on the virus.5.In vivo experiments showed that the effective site could significantly reduce the lesion of lung tissue,reduce the lung index and reduce the virus titer in lung tissue of mice.6.Compared with the model group,the effective sites can increase the serum concentrations of IL-4 and IFN-α,and reduce the concentrations of TNF-α and TNF-β.7.Compared with the model group,the active site can reduce the viral load in the lung tissues of mice and reduce the expression levels of TLR3 and TLR4.ConclusionAfter separation and purification with water extraction and alcohol precipitation,HPD-100 macroporous adsorption resin and ultrafiltration,the effective parts obtained from Euonymus alatus(Thunb.)have a certain anti-RSV effect.Its chemical composition is mainly polyphenols.The results of in vitro experiments showed that the effective parts had the strongest direct inactivation effect on the virus.In addition,the results of in vivo experiments showed that the effective site may play an antiviral effect by reducing the levels of inflammatory factors in the serum of mice,increasing the levels of anti-inflammatory factors,and down-regulating the levels of TLR3 and TLR4.