Structural Characterization And Bioactivity Of Polyphenol-polysaccharide Conjugates From Echinacea Purpurea

Echinacea purpurea（L.）Moench belongs to the genus Echinacea in the family Asteraceae and is an herb native to North America with a long history of medicinal use.However,several current studies on Echinacea are mainly limited to single extracts,such as phenolic acids,flavonoids,polysaccharides,etc.This paper takes the flowers of Echinacea purpurea as the research object,and the separation,extraction,purification,and structural characterization of Echinacea polyphenol-polysaccharide conjugates were carried out by combining chemical and instrumental analysis methods.Chemical antioxidant assay,macrophage RAW 264.7 model,and in vitro fermentation assay were used to study its antioxidant activity,immune activity,and fermentation properties,respectively.The main conclusions of this paper are as follows:1)Echinacea polyphenol-polysaccharide conjugates（EP）were extracted from Echinacea by alkaline extraction method with a yield of 2.10%.The crude extracts were separated into three fractions（EP1,EP2,and EP3）by Sephadex G-15 column chromatography.The molecular weights of EP1,EP2,and EP3 were 22.96 k Da,16.23 k Da,and 10.41 k Da,respectively.2)The results of chemical composition analysis,high-performance liquid chromatography analysis,and infrared spectroscopy analysis indicated that the conjugates were mainly composed of polysaccharides covalently bound to phenolics.The monosaccharide composition analysis showed that EP-1,EP-2,and EP-3 all consisted of five monosaccharides,including rhamnose,arabinose,mannose,glucose,and galactose,but their molar ratios were different,1:2.7:1.2:3.9:3.6,1:3.2:1:3.5:3.8,and 1.6:3.3:1:6.3:4.7,respectively.The result of methylation analysis showed that the major linkages of the glycosidic bonds of EP-1 are 6-Glcp,2,5-Araf,T-Araf,and 6-Galp,the major linkages of the glycosidic bonds of EP-2 are 3,4-Galp,6-Glcp,and 2,5-Araf,and the major linkages of the glycosidic bonds of EP-3 are 6-Glcp,T-Glcp,and 6-Galp.3)The total antioxidant capacity,DPPH·radical scavenging capacity,reducing power,·OH radical scavenging capacity,and O^2-·radical scavenging capacity of EP were investigated using in vitro chemical antioxidant assay.The results showed that the antioxidant capacity of EP was extremely strong.After comparison,there was no significant difference in the total antioxidant capacity of EP-1,EP-2,and EP-3.4)The immune activity of EP was studied using the RAW 264.7 macrophage model,and the effects of three fractions on the proliferation,phagocytosis of RAW 264.7 cells,and immune factor（IL-6、IL-10 and TNF-α）release were compared.The results showed that EP-2 could activate macrophages and increase the secretion of cellular immune factors at a concentration of 100μg/m L.5)The fermentation characteristics of EP were investigated by in vitro fermentation experiments of the porcine colonic digest.The experiment result indicated that EP could be utilized by intestinal microorganisms with a slow degradation rate.During the fermentation process,EP can increase the concentration of short-chain fatty acids,enhance the diversity of gut microbes and improve the structure of gut microbiota.The results of this study showed that the polyphenol-polysaccharide conjugates isolated from Echinacea had good antioxidant properties and immunological activity,but they fermented slowly in the intestinal tract.Echinacea macromolecule polysaccharide conjugates may be useful for the creation of new natural drugs or dietary supplements.