| Influenza is an upper respiratory tract disease caused by influenza virus.At present,the main means to prevent and treat influenza are the use of influenza vaccines and anti-influenza drugs.The extensive use of anti influenza drugs has led to the emergence of drug-resistant strains.Traditional Chinese medicine(TCM)has a long history in the prevention and treatment of diseases and contains rich ingredients.It can treat influenza from multiple targets and multiple mechanisms.Studies showed that hemagglutinin(HA)and neuraminidase(NA)on the surface of influenza virus played an important role in the process of viral adsorption and release.Sialylglycopeptide(SGP)is a natural glycopeptide with two sialic acids at the end of the molecule,which can be used as an inhibitor of hemagglutinin.Pentacyclic triterpene is an effective ingredient of traditional Chinese medicine with anti-influenza virus potential.It can be connected with other biological molecules to achieve the purpose of increasing efficiency and reducing toxicity.Therefore,in this experiment,sialylglycopeptide was selected as the backbone,which was connected with the pentacyclic triterpene molecules to form conjugates,and the anti-influenza virus activity was tested.Objectives:To investigate the method of extracting and separating sialylglycopeptide from egg yolk powder,prepare sialylglycopeptide,synthesize SCT-Asn-pentacyclic triterpenes,and test the anti-influenza virus activity of the conjugates.Methods:After defatting the egg yolk powder twice with 95%ethanol,the SGP was extracted with 40%ethanol and enriched by microcrystalline cellulose,and then purified by Sephadex LH-20 gel column and ODS reversed phase column.The purity of the obtained SGP was determined by high performance liquid chromatography(HPLC).SGP was hydrolyzed with pronase to obtain the glycan moiety(SCT-Asn),and then the SCT-Asn and pentacyclic triterpene molecules were connected to alkynyl and azide groups respectively.The conjugates were formed by copper-catalyzed azide alkyne cycloaddition reaction,and the intermediates and products were identified by nuclear magnetic resonance(NMR)technology and mass spectrometry(MS).The surface plasmon resonance technology(SPR)was used to assay the affinity of the conjugates with H1N1(A/WSN/1933)and H5N1(A/Hong Kong/483/97)hemagglutinin proteins,and cytopathic assay(CPE)was used to test the activity of conjugates against H1N1A/PR/8/34 influenza virus.Results:A method for preparing SGP from egg yolk powder was established,and 2.7 g of SGP was prepared.The purity of SGP was above 95%by HPLC detection.10 kinds of pentacyclic triterpenes were selected to form conjugates with SCT-Asn to synthesize SCT-Asn-ursolic acid,SCT-Asn-oleanolic acid,SCT-Asn-betulinic acid,SCT-Asn-glycyrrhetinic acid,SCT-Asn-celastrol,SCT-Asn-mesembryanthemoidigenic acid,SCT-Asn-3-O-acetyloleanolic acid,SCT-Asn-ganoderic acid A,SCT-Asn-maslinic acid and SCT-Asn-asiatic acid,respectively.The structure of intermediates and products were confirmed by NMR and MS.SPR test results showed that SCT-Asn-betulinic acid had the best affinity with H1N1(A/WSN/1933)hemagglutinin protein,and SCT-Asn-ganoderic acid A had the best affinity with H5N1(A/Hong Kong/483/97)hemagglutinin protein,with K_D values of 6.89μM and 7.13μM,respectively.The CPE test of the anti-influenza activity showed that pentacyclic triterpenoid monomer showed certain cytotoxicity,and the conjugate showed no cytotoxicity in the test concentration range.The CPE inhibition rate of SCT-Asn-oleanolic acid and SCT-Asn-betulinic acid against H1N1 PR8/34 influenza virus was 25%at the concentration of 25μM.Conclusions:Microcrystalline cellulose can be used as an enrichment material to prepare sialylglycopeptide.The combination of pentacyclic triterpenes and sialylglycopeptide glycan is beneficial to the improvement of anti influenza virus activity.In addition,the formation of conjugates can reduce the cytotoxicity of pentacyclic triterpenoids. |
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