| Neuraminidase(NA) is one of two glycoproteins expressed on the virus surface, andit is important for replication and spreading of the influenza virus. They are easy tomutate, while the active site of NA is highly conserved across all influenza A and Bvirus strains, so NA is an attractive target for an anti-viral strategy due to its key role.So far, zanamivir and oseltamivir as inhibitors of NA have been approved for humanuse by FDA. However, these drugs can cause nausea, vomiting and other side reaction,which have been proved to be the case. So, searching for new antivival compoundsfrom natural products is important for the development of new therapeutic agents.The main contents of this study were as follows:1. The collagen from cod skins was hydrolysed by nine proteases and the appropriateone was selected by neuraminidase inhibitory activity. Among the9kinds of proteases,the NA inhibition ratio of pepsin hydrolysates was the highest and showed adose-dependent manner with its IC508.86mg/mL (P<0.05).The addition level ofenzymes, substrate concentration and hydrolyzing time were studied. Then the threemain factors were optimized by response surface methodology(RSM). The optimalenzymolysis conditions were: enzyme dosage1600U/g, substrate concentration22mg/mL, hydrolyzing time6.8h, and IC50of the hydrolysates was7.95mg/mL. Theanti-virus rate was66.6%on MDCK cells infected by H1N1under the concentrationof10mg/mL.2. The enzymatic hydrolysate of cod skins collagen was utilized to purify potentinhibitory peptide of Neuraminidase, and neuraminidase activity was tested at eachpurification step. After identifing of the structure, its properties were studied. One NAinhibitory activity peptide was isolated from the mixture by ultrafiltration membrane(5000Da), gel filtration (Sephadex G-15), and high performance liquidchromatography (HPLC), and the sequence is PGEKGPSGEAGTAGPPGTPGPQGL.The stability of the peptide to simulated gastrointestinal digestion was tested in vitro. The results showed that the peptide was not resistant to proteinase digestion. Afterhydrolyzed by pepsin, chymotrypsin, trypsin consecutively, the NA inhibitory activitywas dropped makedly and even lost. So it is not suitable for oral use. Detailed kineticanalysis indicated that they were all competitive, the Ki value was0.29mmol/L.3. In the MDCK cell culture experiment in vitro, antiviral activity of the peptideagainst influenza virus was estimated in terms of cytopathic effect. The maximumnon-toxic concentration was250μg/mL by MTT method. In a certain concentrationrange, there was a positive correlation between the antiviral effect and the peptideconcentration. Its IC50value was471μg/mL.The virus was treated with different concentration of peptide at different timepoints (Pretreatment, Adsorption, After-adsorption), the peptide had an obviouslyinhibitory effect on influenza A virus with every treatment, which indicated thepeptide had a strong effect of direct inactivation, preventive and therapeutic effects onthe anti-influenza virus in vitro. The antiviral effects of the peptide in vitro might bemultitarget. However, the effects of the three treatments were different (P<0.05),pretreatment (138.7μg/mL)> adsorption (319.3μg/mL)> after-adsorption (417μg/mL). In the hemagglutination (HA) assay, they also displayed an inhibitory effecton HA, the agglutination titer reduced significantly along with the concentrationincrease, which further evidenced that there are more acting targets for NA inhibitorypeptide in cell. |
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