| Objective: To isolate,purify and basically analyze p H-modified Citrus Pectin(MCP),analyze the main active components in MCP that inhibit galectin-8,and explore the preliminary structure-activity relationship between the active components in MCP and galectin-8.Methods: MCP was prepared by p H modification.After the initial classification of MCP by gradual alcohol precipitation of 30%,50% and70% ethanol in volume fraction,three components were obtained,including MCP-30,MCP-50 and MCP-70.The most active component MCP-30 was further separated and purified by DEAE Sepharose Fast Flow anion exchange column chromatography and multiple molecular sieves,and one neutral sugar component MCP-30-N and three acidic sugar components MCP-30-1,-3,-5 were obtained.The monosaccharide composition of the above MCP components was analyzed by HPLC pre-column derivatization,and the relative molecular weights of the above components were analyzed by HPGPC.Galectin-8 expression plasmid was constructed with vector p ET-28 a by modern molecular biology method.Galectin-8 protein was expressed in E.coli prokaryotic.Galectin-8 protein with higher purity was isolated by lactosyl-Sepharose CL-6B affinity chromatography column.Galectin-8-mediated hemagglutination(G8H)and fluorescence spectroscopy were used to analyze the interaction between MCP components and galectin-8,and the inhibitory activity of MCP components on galectin-8 was comprehensively evaluated.MCP-30-3 was hydrolyzed byβ-galactosidase and pectinase respectively,and the enzymatic products MCP-30-3-Gal and MCP-30-3-Gal A were obtained respectively.The structure-activity relationship between MCP-30-3 and galectin-8 was preliminarily discussed by studying the inhibition of galectin-8 activity by enzymatic hydrolysis products.Results: MCP was prepared after p H modification,with the yield of about 82.2%.After gradual alcohol precipitation,three components including MCP-30(with yield of about 60.0%),MCP-50(with yield of about 17.5%)and MCP-70(with yield of about 1.4%)were prepared from MCP.After further separation and purification of MCP-30,four components were obtained,namely neutral sugar component MCP-30-N(with yield of about 35.0%),acidic sugar component MCP-30-1(with yield of about 13.8%),MCP-30-3(with yield of about 29.2%)and MCP-30-5(with yield of about 14.8%).The monosaccharide composition results showed that MCP-30-N was mainly composed of Glc(95.4%)and a small amount of Gal(4.6%),MCP-30-1,-2,-3 was mainly composed of Gal,Gal A and Glc,among which the Gal residue content of MCP-30-3was the highest,about 46.1%,followed by that of MCP-30-5,and the Gal residue content of MCP-30-1 was the lowest.MCP-30-3 has good homogeneity measured by Sephadex G-75,Sephadex G-150 and Superdex G-200,and its molecular weight is about 168 k Da.Galectin-8protein was prepared,with higher purity verified by SDS-Page protein electrophoresis.The results by G8 H method showed that the MIC value of MCP was 0.313 mg/m L;The MIC of MCP-30 was 0.078 mg/m L;The MIC of MCP-50 was 0.625 mg/m L;The MIC of MCP-70 was greater than 5.000 mg/m L;The MIC of MCP-30-N was greater than 5.000mg/m L;The MIC of MCP-30-1 was 0.313 mg/m L;The MIC of MCP-30-3 was 0.039 mg/m L;The MIC of MCP-30-50 was 0.625 mg/m L.After the hydrolysis of MCP-30-3 by β-galactosidase,and the G8 H measurement showed that the MIC of MCP-30-3-Gal was greater than 5mg/m L,and the activity of galectin-8 was significantly reduced.After being hydrolyzed by pectinase,The MIC of MCP-30-3-Gala was 1.25mg/m L measured by G8 H,and the inhibitory activity of galectin-8 was also significantly reduced.Conclusion: In this article,MCP was systematically isolated and purified by various methods,and the main active component(MCP-30-3)inhibiting galectin-8 in MCP and its basic structure were basically clarified.The important effects of Gal and Gal A residues in MCP-30-3 on activity and structure-activity relationship were discussed. |
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