The Study Of Structural Identification,selenized Modification And Biological Activity Of Polysaccharides From The Pulp Of Rosa Laevigata Michx Fruit

Rosa laevigata Michx,belonging to the Rosaceae,is widely distributed in the eastern,central,southern and southwestern regions of China.It is a berry with high food and medicine value,and is rich in polysaccharides and other functional factors.In this study,the isolation and purification,structural identification,physiological activities（such as antioxidant,immunomodulatory and polarized regulation of macrophages,and inhibition of its foaming）and their mechanisms of polysaccharides from the pulp of Rosa laevigata Michx fruit（PPRLMF-2）were systematically studied.At the same time,the selenized PPRLMF-2（Se-PPRLMF-2）was successfully prepared by selenized modification,and its immunomodulatory and antioxidant activities as well as related mechanisms were systematically explored.The main research results were as follows:（1）The crude polysaccharides were obtained by water extraction,deproteinization,decolorization,alcohol precipitation and freeze-drying,its yield was 10.36%.Two polysaccharide fractions,PPRLMF-1 and PPRLMF-2,were isolated by DEAE-Sepharose Fast Flow.PPRLMF-2 was further purified by Sephadex G-100 gel chromatography,and the purity of PPRLMF-2 was 96.8%.（2）The structure of PPRLMF-2 was characterized by high-performance gel permeation chromatography,monosaccharide composition,methylation analysis,1D and 2D nuclear magnetic resonance（NMR）spectra,infrared spectroscopy,congo red assay and scanning electron microscopy.The average molecular weight（Mw）of PPRLMF-2 was 137.1 k Da,and it was composed of rhamnose（7.6%）,arabinose（26.5%）,xylose（3.5%）,mannose（0.9%）,glucose（5.7%）,galactose（31.9%）and galacturonic acid（23.9%）.PPRLMF-2 had 16 kinds of glycosidic bonds,mainly for the T-Araf-(1→,T-Rhap-（1→,→5）-Araf-（1→,→4）-Galp-（1→,→4）-Glcp-（1→,→6）-Galp-（1→and→3,6）-Galp-（1→.The main type of uronic acid in PPRLMF-2 was→4）-α-D-Gal PA-(1→.PPRLMF-2 hadαandβpyranose ring configurations with three helical structures and helical folds on its surface.（3）PPRLMF-2 was a good electron donor with good ABTS^·+scavenging ability,oxygen radical absorbance capacity and Fe³⁺reducing power.In addition,PPRLMF-2 could significantly inhibit the oxidative hemolysis of erythrocytes induced by APPH,reduce the overexpression of ROS and the production of lipid peroxidation product MDA in erythrocytes caused by APPH,and maintain the antioxidant enzymes（CAT,SOD and GSH-Px）activities and content of GSH at normal levels,and keep physiological homeostasis in erythrocytes.（4）PPRLMF-2 had good immunomodulatory activity,and could significantly enhance the pinocytosis and phagocytosis ability of RAW264.7 cells.PPRLMF-2 can significantly enhance the immune response of RAW264.7 cells and promote the transcription,synthesis and secretion of cytokines（TNF-α,IL-6 and NO）by binding with the main recognition receptors of RAW264.7 cell membrane,including SR（CD36）,GR,TLR2 and TLR4（mainly TLR2）,and further promoting the phosphorylation of MAPKs（including p38,ERK and JNK）and related factors of NF-κB（p65）signaling pathway.（5）PPRLMF-2 can moderately induce the transformation of M0 type and M2 type macrophages to M1 type and can not induce the transformation of M1 type macrophages to M2 type,but can inhibit the LPS-induced inflammatory response.PPRLMF-2 synergically inhibited the proliferation of foam cells.In addition,PPRLMF-2 inhibited the ox LDL uptake of macrophages by the one or more of three ways（interacting with ox LDL,or transforming M0 type macrophages into M1 type macrophages,or competitively binding with ox LDL to CD36 site）to prevent foaming and improve its lipid accumulation and inflammatory status caused by ox LDL.（6）The selenized PPRLMF-2（Se-PPRLMF-2）was successfully prepared by HNO₃-Na₂SeO₃ method,and its yield was 32.67%.The morphology of Se-PPRLMF-2 was changed into irregular filaments,the average Mw was reduced to 82.2 k Da,and its selenium content was 862μg/g.Compared with PPRLMF-2,the characteristic absorption peaks of Se=O and C-O-Se appeared at 763.78 cm^-1 and 915.19 cm^-1 in the infrared spectra of Se-PPRLMF-2.NMR ¹³C spectrum showed a new absorption peak at 65.7 ppm,indicating a non-selective selenium-substituted reaction at the C-6 position of PPRLMF-2.These results indicated that polysaccharides were successfully selenized.Se-PPRLMF-2 significantly improved the ability of free radical scavenging,and effectively inhibited the oxidative damage of erythrocytes induced by AAPH,and reduced the demand for high-activity antioxidant enzymes（CAT,GSH-Px,SOD,etc.）and GSH.Se-PPRLMF-2 significantly enhanced the immune response of RAW264.7 macrophages,improved its pinocytosis and phagocytosis abilities,and promoted the secretion of cytokines（TNF-α,IL-6 and NO）.The enhanced antioxidant activity of Se-PPRLMF-2 was not related to the pure selenium element,but was closely related to the conformational change of polysaccharide and the enhanced ability of supplying hydrogen and hydroxyl caused by the bonding mode of organic selenium.Se-PPRLMF-2 enhanced the immune response of macrophages,but the effect of organic selenium was significantly stronger than that of inorganic selenium,and its toxicity of selenium was significantly reduced.In conclusion,PPRLMF-2 is a new polysaccharide from the pulp of Rosa laevigata Michx,which had many good activities,such as antioxidant,immunomodulatory and polarized regulation of macrophages,and inhibition of its foaming.It can be developed and utilized as a health care drug and functional food for the prevention of immunodeficiency,atherosclerosis and oxidative stress-related diseases.At the same time,Se-PPRLMF-2 can be used as a new dietary supplement of selenium source to enhance immunity and antioxidant.