Structural Characterization And Immunoregulatory Activity Of Glycoprotein From Lilium Davidii Var.unicolor Salisb.

Glycoprotein is a kind of biological macromolecule that carbohydrates and proteins bind together by covalent bonds.Glycoproteins are widely distributed in natural animals,plants and microorganisms.They exist in many forms in organisms and play a complex and important role in life activities.Studies have shown that glycoproteins have a variety of biological activities and play an important role in immune regulation,antioxidation and anti-cancer.Therefore,glycoproteins have broad prospects for development in functional food-grade drug research and other fields.In this paper,the glycoprotein components of Lilium davidii var.unicolor Salisb.were extracted by ultrasound-assisted buffer solution.The structure and physicochemical properties of glycoprotein components were studied.The immunoregulatory activity of glycoprotein components was studied using RAW 264.7,a mouse macrophage line,as a cell model.The paper mainly includes the following parts:(1)After a series of pretreatments,fresh Lilium davidii var.unicolor Salisb.scales were degreased by petroleum ether,extracted with 0.01 mol/L PBS buffer solution of pH 7.4,salted out and separated,and lyophilized by lyophilization to obtain LGP,the yield was 2.09%;After reconstitution of the crude product,it was further purified by Sephadex G-200 gel column and DEAE-Cellulose 52 anion exchange column,and the purity of HPLC and Sephadex G-100 was used to obtain two purified components of glycoprotein LGP-1 and LGP-2.(2)The structural characterization and physicochemical properties of purified LGP-1 and LGP-2 were studied.The sugar content and protein content of LGP-1 were55.80%and 43.04%,respectively.While that of LGP-2 were 62.52%and 35.28%,respectively.FT-IR results showed that LGP-1 and LGP-2 have characteristic absorption peaks of sugar and protein;the molecular weight measurement result is that the Mw of LGP-1 is 3.639×10~4,while that of LGP-2 is 9.311×10~4,and the dispersion coefficient LPG-1 is 1.950.LGP-2 was 2.927 smaller;?-elimination reaction showed that both LGP-1 and LGP-2 contained O-linked glycopeptide bonds;LGP-1 was composed of Man and Glc as determined by GC-MS.The molar ratio is1.07:1.01,and LGP-2 is composed of three monosaccharides:Man,Glc and Gal.The molar ratio is 1.04:4.64:5.43.The amino acid composition analysis shows that LGP-1 is composed of 16 amino acids,lacking methionine.While LGP-2 consists of15 amino acids,lacking methionine and tyrosine,LGP-1 and LGP-2 contain a large number of essential amino acids;The hierarchical structure indicated that the secondary structure of LGP-1 and LGP-2 were mainly?-sheets;scanning electron microscopy revealed LGP-1 exhibits a spherical or ellipsoidal shape with uniform size and smooth surface,while LGP-2 exhibits an apparent morphology of the cube.The particle size distribution of LGP-1 is in the range of 0.3-0.9?m.For 0.51?m,the particle size distribution of LGP-2 is in the range of 0.6 to 1.8?m with an average value of 0.97?m.(3)The immunomodulatory activities of LGP-1 and LPG-2 were studied using the mouse macrophage cell line RAW 264.7.The effect of CCK-8 experimental study on cell viability showed that both of them could promote the proliferation of RAW264.7 cells in a concentration-dependent manner.The Griess method was used to determine the release ability of NO,and the secretion of TNF-?was determined by ELISA.The immunomodulatory effect of LGP on RAW 264.7 cells was determined.The results showed that when the cell culture time was 48 h and the concentration of the sample reached 100?g/mL,the secretion of NO and TNF-?reached the maximum.It can be seen that both LGP-1 and LGP-2 can promote the proliferation of RAW264.7 cells and activate the immune function of the cells.It is speculated that the glycoprotein from Lilium davidii var.unicolor Salisb.component has good immunomodulatory and antitumor activities.The structural characterization and immunomodulatory activity of the above-mentioned glycoprotein from Lilium davidii var.unicolor Salisb have not been reported in the same literature so far.The structural characterization and immunomodulatory activity of the above-mentioned glycoprotein from Lilium davidii var.unicolor Salisb have not been reported in the same literature so far.