Study On Isolation And Purification Of The Effective Anti Respiratory Syncytial Virus From Hibiscus Mutabilis Leaf And Its Anti-viral Mechanism

Objective: The anti-RSV effective parts of Hibiscus mutabilis Leaf were screened by different isolation and purification methods using in vitro antiviral tracer technology.Furtherly,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.Methods:1.The crude extracts of Hibiscus mutabilis Leaf were obtained by water extraction-alcohol precipitation method and ultrafiltration method.The cytopathic effect(CPE)was observed by MTT staining in vitro,and the therapeutic index(TI)was used as the index to determine the extract with the best anti-RSV effect.2.Separate and purify the crude extracts by five kinds of macroporous resin columns NKA-9,DM-301,D-101,X-5,AB-8 with the mobile phase as distilled water,volume fraction of 25%,50%,75% ethanol respectively.Simultaneously antiviral experiments were carried out at the cell level to screen the resin column and eluent with the best separation and obtain the effective part with the best anti-RSV effect.3.The effective parts of Hibiscus mutabilis Leaf obtained were added to the cells via different time nodes to explore their anti-RSV mechanism initially.4.Establish a mouse model of RSV pneumonia,and divide the effective parts of Hibiscus mutabilis Leaf 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 oflung tissue of the medication groups to explore their antiviral effect.5.The concentration of antiviral factors interleukin-2(IL-2),interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)in the blood of mice was detected by ELISA kit method to explore the in vivo anti-RSV mechanism.Results:1.The ultrafiltration extract with a molecular weight of 5000-10000 has the best anti-RSV effect with the TI was 51.8,higher than ribavirin(TI=41.11).2.D101 macroporous resin had the best separation and purification effect on Hibiscus mutabilis Leaf.The first elution site of 25% ethanol eluate had the best anti-RSV effect with the TI was 73.66.3.The TI was highest when the effective parts of Hibiscus mutabilis Leaf and virus were added into cells simultaneously.4.In vivo experiments have shown that the effective parts of Hibiscus mutabilis Leaf can improve the weight loss caused by viral infection,elevate the lung index and reduce lung tissue lesions.5.The effective parts of Hibiscus mutabilis Leaf can increase the concentration of inflammatory inhibitory factors IL-2 and IFN-γ in mouse serum and reduce the content of TNF-α.Conclusion: After the separation and purification of Hibiscus mutabilis Leaf by water extraction,ultrafiltration and D101 macroporous resin,the effective part obtained has certain anti-RSV effect.The results of in vitro experiments showed that the effective part of the leaves of Hibiscus mutabilis Leaf had the strongest direct inactivation effect on the virus.In addition,in vivo experiments showed that it can also increase the concentration of inflammatory inhibitory factors IL-2 and IFN-γ in mouse serum,and reduce the TNF-α level of inflammation-promoting factor.It is speculated that its antiviral mechanism may be related to immune regulation.