Isolation,Purification And Preliminary Study Of The Effective Components Of Anti-EV71

Objective:To study the anti-EV71 effect of B.eucalyptus extract,select suitable sensitive virus strains,use cell tracing experiment as the screening method,isolate and purify the extract,preliminary determine the effective parts,and then use the virus infection in the body as an auxiliary method,combined The enzyme-linked immunosorbent assay test was carried out to investigate the inhibitory effect and preliminary mechanism of action of the effective parts of B.euonymus on EV71.Method:1.Extraction using distilled water reflux extraction method,ethanol reflux extraction method,ethyl acetate reflux extraction method and methanol cold immersion method,compare the TI value of each extract,select the best extraction method for virus screening of drugs: screening existing viruses Strains [Human Enterovirus 71(EV71),Respiratory Syncytial Virus(RSV),Herpes Simplex Virus(HSV-1),Influenza A Virus(H1N1)],using the antiviral index(TI value)as the investigation index,The cytopathic method,thiazole blue staining method(MTT method),etc.were used to determine the virus strain with the best antiviral effect.2.According to the principle of similar miscibility,choose distilled water,25%,50%,and 75% ethanol as the eluent to wash the DM130,X-5,AB-8,and D101 models.Resin column,through in vitro cell experiments,determine which eluent is used,and choose the macroporous adsorption resin with the best antiviral effect.3.Comparatively choose macroporous resin and silica gel column test.The silica gel column is separated and purified by 200-300 mesh,and different separationand purification parts are obtained.Using TI value as an index,the effective parts against EV71 are selected.4.The ultrafiltration of the effective parts is obtained by ultrafiltration with four molecular weights of different sizes,and the TI value is used as the index to select the best molecular weight segment for anti-EV71 effect.5.Through physical and chemical identification and high-performance liquid chromatography,initially determine the substance range of effective parts.6.Intraperitoneal injection of EV71 to establish a mouse virus model,conduct pharmacodynamic experiments in three dose groups with high,medium and low effective components of B.euglena,observe the changes in the body weight,food intake and other indicators of the mice.The tissues were sliced ? ? and observed to explore the antiviral effects of the effective parts of Bira moss in vivo.7.Use enzyme-linked immunosorbent assay to detect the concentration of TNF-α,TNF-β,IFN-α and IL-4 in mice,and explore the effective regulation of Bira true moss on mice.Result:1.Virus screening has the best effect of inhibiting EV71 with a TI value of20.37.2.The elution site of DM130 macroporous adsorption resin water 1 has the best inhibition effect on EV71,with a TI value of 56.06.3.The purified solution Q-1 after separation and purification by silica gel column has the best anti-EV71 virus effect,and the measured TI value is 124.4.After ultrafiltration,the ultrafiltrate with molecular weight greater than500 KDa and less than 1000 KDa C-2 has the best antiviral effect,with a TI index of131,which is higher than ribavirin(TI = 41.11).5.Using full-wavelength high-efficiency liquid phase scanning,absorption peaks appear at 335 nm and 268 nm,which are in accordance with the characteristics of the flavonoid spectrum;physical and chemical identification Molish reaction has a purple ring,indicating that the glycosides are contained,and the magnesium chloride powder reaction is positive,indicating Containing flavonoids,the effective substance is initially determined to be flavonoids or flavonoid glycosides.6.In vivo antiviral experiments showed that mice injected with virus intraperitoneally showed weight loss and decreased food intake,indicating successful modeling;after dissection,compared with the blank group,the lung color of the virus model group was darkened and lesions of varying degrees appeared;pathology The slice experiment showed that the effective part of Betula can effectively reduce the lesions of lung tissue and reduce the inflammatory exudation caused by infection.7.The experimental results of enzyme-linked immunosorbent assay showed that ultrafiltrate in the effective site can reduce the pro-inflammatory factor TNF,increase the anti-inflammatory factor IFN,and increase the benign factor IL-4 to enhance the immune function of mice.Conclusion:The best effective part of anti-EV71 effect is Q-1,the TI value is 124,and the molecular weight after ultrafiltration(less than 5KDa,greater than 10KDa)TI value is 131.Physical and chemical identification method combined with high performance liquid chromatography proves that the effective substance is flavonoids Or flavonoid glycosides,through in vivo experiments found that the large dose group of Piraeus can effectively control EV71,the high dose group can reduce the content of IFN-α and IL-2 by increasing TNF-α and TNF-β in the serum,and play a synergistic role Anti-EV71 effect.