| Atherosclerosis(AS)is a chronic inflammatory disease characterized by lipid deposition and plaque formation in the arterial wall,which can further cause major diseases such as ischemic heart disease and peripheral vascular disease.At present,the efficacy of drugs used to prevent or treat AS is limited,and recurrence occurs occasionally.Researchers are increasingly committed to developing effective anti-atherosclerotic drugs from natural resources.Polysaccharide,as one of the main macromolecules of plants,animals and microorganisms,is not only the energy resources and components in organisms,but also have a variety of pharmacological activities,especially in the treatment of AS,which has attracted widespread attention.Cornus officinalis Sieb.et Zucc.,has been used as a traditional Chinese medicine to protect the liver and kidneys.Studies have shown that the active ingredients of Cornus officinalis can improve lipid metabolism,but the structure and anti-atherosclerotic activity of the polysaccharides from Cornus officinalis are scarcely reported.In this study,three homogeneous polysaccharides were isolated and purified from Cornus officinalis,and their physicochemical properties and primary structure were elucidated.The anti-atherosclerotic activities of polysaccharides from Cornus officinalis were preliminarily evaluated using in vitro and in vivo models,which provided a theoretical basis for further development and utilization of the polysaccharides from Cornus officinalis.Methods: 1.In this study,the total polysaccharides from Cornus officinalis(PFC)were prepared by alkali extraction and alcohol precipitation,and then removed protein and pigment by Sevag method and hydrogen peroxide method,respectively.Afterwards,three polysaccharides PFC-1,PFC-2,and PFC-3 were isolated and purified by ion exchange and size exclusion chromatography.High efficiency size exclusion chromatography was used to determine the homogeneity and molecular weights of three polysaccharides from Cornus officinalis.The total sugar,uronic acid,and protein contents in the three homogeneous polysaccharides were determined by the phenol sulfuric acid method,the carbazole sulfate method and the Coomassie brilliant blue method,respectively.Infrared spectroscopy(FT-IR)was used to identify the main characteristic functional groups of the polysaccharides.2.The monosaccharide compositions of these homogenous polysaccharides were determined by high performance liquid chromatography.Methylation and gas mass spectrometry were used to analyze glycosidic linkage types.One-dimensional and two-dimensional nuclear magnetic resonance(NMR)were used to analyze the connection types of glycosidic bonds.3.In vivo AS model of Apo E-/-mice was established to preliminarily explore the therapeutic effect of polysaccharides from Cornus officinalis on AS.The plaque formation level of AS was detected by haematoxylin-eosin staining and oil red O staining.The serum lipid level and antioxidant enzyme activity were determined by enzymatic method.The contents of macrophages,smooth muscle cells and collagen fibers in the plaques were detected by immunohistochemistry and Sirius scarlet staining to analyze the stability of the plaques.Oxidated low density lipoprotein(ox-LDL)induced RAW264.7 macrophage foaming in vitro model was established.The inhibitory level of RAW264.7 macrophage foaming was detected by oil red O staining method.Results: 1.Three homogeneous polysaccharides were obtained and named as PFC-1,PFC-2,and PFC-3,with molecular weights of 1.2×103,6.3×104,and 4.7×104 Da,respectively.Their total sugar contents were 93.1%,60.6%,and 51.3%,the contents of uronic acids were 3.0%,33.3%,and 46.7%,and the protein contents were 0.1%,0.1%,and0.1%,respectively.FT-IR results showed that the three polysaccharides had characteristic absorption peaks of polysaccharides.PFC-2 and PFC-3 had characteristic absorption peaks of uronic acids.2.The monosaccharide compositions of PFC-1 were Man,Glc,Gal,and Ara with a molar ratio of 1.00∶2.96∶6.25∶2.08.The methylation results showed that the main types of sugar residues and molar ratios of PFC-1 were 1,2-Araf,1,6-Manp,T-Galp,1,3-Galp and 1,6-Glcp(1.19 ∶ 1.00 ∶ 2.39 ∶ 4.41 ∶ 2.85).The monosaccharide compositions of PFC-2 were Man,Rha,Gal A,Glc,Gal,and Ara with the molar ratio of1.04∶8.41∶5.31∶1.00∶7.76∶25.84.The main types of sugar residues and molar ratios were T-Araf,1,3-Araf,T-Galp,2,3-Araf,1,2-Rhap,1,4-Galp,2,3,4-Araf,1,2,6-Glcp,1,3-Galp and 2,4-Manp(2.71∶1.41∶1.15∶1.00∶1.96∶3.75∶1.23∶0.16∶0.77∶1.73).PFC-3 was composed of Man,Rha,Gal A,Glc,Gal and Ara with a molar ratio of1.45∶5.44∶32.70∶1.00∶2.92∶10.76.The main types and molar ratios of glycogen residues were T-Araf,1,3-Araf,T-Galp,2,3-Araf,1,2-Rhap,1,4-Galp,1,6-Glcp,1,3-Galp,1,6-Galp,and 2,3,4-Manp(4.74∶6.04∶2.60∶1.00∶2.87∶15.93∶3.04∶0.51∶0.85∶0.39).PFC-2 and PFC-3 were analyzed by partial acid hydrolysis.The partial acid hydrolysates of PFC-2 were composed of Rha,Gal A,and Gal with a molar ratio of 1.53∶2.34∶1.00.The main sugar residue types and molar ratios were T-Galp,1,2-Rhap,1,4-Galp,and 1,3-Galp(1.00∶1.63∶1.58∶1.06).The partial acid hydrolysate of PFC-3was partially composed of Rha,Gal A,and Gal with a molar ratio of 1.23∶14.51∶1.00.The main sugar residue types and molar ratios were 1,2-Rhap,1,4-Galp,1,3-Galp,and1,6-Galp(1.16∶13.38∶1.90∶1.00).3.In vivo experiments showed that PFC significantly inhibited the formation of AS plaques in Apo E-/-mice fed with high-fat diet.The levels of serum low-density lipoprotein cholesterol(LDL-C)and total cholesterol(TC)were significantly decreased by 26.47% and37.39% in PFC-treated group compared with model mice,respectively.In addition,the activity level of superoxide dismutase(SOD)increased by 6.46%,while the activity of malondialdehyde(MDA)decreased by 63.17%.PFC significantly decreased the quantity of macrophages in plaques,and the quantity of smooth muscle cells were significantly increased.4.The three homogeneous polysaccharides had no obvious inhibitory effect on the proliferation of RAW264.7 cells when the dosage was less than 3 mg/L after 24-h treatment.However,when the concentration reached to 100 mg/L,the inhibition rates of PFC-1 and PFC-2 on RAW264.7 cells reached to 60.84% and 50.12%,respectively,while PFC-3showed no obvious inhibitory effect.In the evaluation of foaming inhibition activity of RAW264.7 cells induced by ox-LDL,it was found that both PFC-1 and PFC-3 significantly reduced the foaming of RAW264.7 cells in the concentration range of 0.3-3 mg/L,and the inhibitory rate of PFC-3 was higher than that of PFC-1.Conclusion: 1.In this experiment,three homogeneous polysaccharides from Cornus officinalis PFC-1,PFC-2,and PFC-3 were isolated.PFC-1 was a linear neutral oligosaccharide.The possible primary structure of PFC-1 was T-β-Galp-(1→3)-β-Galp-(1→6)-β-Glcp-(1→2)-α-Araf-(1→3)-β-Galp-(1→6)-α-Manp-(1→3)-β-Galp-(1→.PFC-2 was an acidic polysaccharide with highly branched chain.The primary structure unit of main chain was T-α-Galp-(1→3)-α-Galp A-(1→2)-α-Rhap-(1→4)-α-Galp A-(1→,and the main sugar residues in the branched chain were T-Araf and 1,3-Araf.PFC-3 was an acidic polysaccharide with branched chain,and the structural unit of the main chain was→6)-α-Galp-(1→2)-α-Rhap-(1→3)-β-Galp A-(1→[4)-α-Galp A-(1]13→,and the main sugar residues in the branched chain were T-Araf,1,3-Araf,T-Galp and 1,6-Glcp.2.PFC could inhibit the occurrence and development of atherosclerotic lesions by the regulating lipid metabolism in Apo E-/-mice fed with high fat diet.PFC-1 and PFC-3could concentration-dependently inhibit ox LDL-induced inflammatory response in RAW264.7 macrophages.At the same time,PFC-3 has the low toxicity effect,which could be further developed as an anti-AS agent. |
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