Extraction,Structural Characterization And Anti-Tumor Activity In Vivo Of Polysaccharide From Eucommia Folium

As a traditional Chinese medicine,Eucommia ulmoides has many good physiological functions,but due to its low comprehensive utilization efficiency,researchers began to explore the bioactive components and functions of Eucommia Folium.It has been confirmed that the main active compounds in Eucommia Folium are almost the same as those in Eucommia ulmoides bark,which have antioxidant,anti-inflammatory,and immune-regulating effects.As its main active ingredient,polysaccharide has attracted more and more researchers to explore its structure and activity.Therefore,in this paper,a homogeneous polysaccharide EFP was isolated and purified from Eucommia Folium by water extraction and alcohol precipitation(100℃),and its basic structure and anti-tumor activity in vivo were preliminarily studied.The optimized extraction process of crude polysaccharide from Eucommia Folium was obtained through single factor and response surface experiments: extraction temperature was 100°C,liquid-solid ratio was 20:1,extraction time was 3 h,extraction was performed 3times,and the concentration of alcohol precipitation was 60%.Under these conditions,the yield was 4.79 ± 0.26%.The pure polysaccharide EFP was obtained after separation and purification by Sephadex G-200 gel column.The total sugar content was 89.12 ± 0.92%,the protein content was 2.03 ± 0.16%,the uronic acid content was measured by related experiments is 9.45 ± 0.35% and the relative molecular weight was 9.98×105Da.EFP was composed of Rha,Ara,Gal,Glc,Xyl,Man,Gal A,Glc A in molar ratio: 0.226: 1.739: 2.183:1: 0.155: 0.321: 0.358: 0.047.Infrared and NMR showed that EFP was an acidic polysaccharide containing both α-glycosidic bonds and β-glycosidic bonds,and its main chain contained α-L-Araf-(1→,→3,5)-α-Araf-(1→,→3)-β-Galp-(1→,→3,6)-β-Glcp-(1→,→2)-α-D-Manp-(1→,→4)-α-Galp A-(1→ and →2,4)-α-Rhap-(1→.Experiments such as scanning electron microscope and TGA showed that EFP presented a dense,smooth,curly network structure and had good thermal stability below 200°C.At the same time,EFP did not have a triple helix structure and was weak in crystallinity.By establishing the H22 tumor-bearing mice model,the in vivo anti-tumor activity of Eucommia Folium polysaccharide was explored.The results showed that Eucommia Folium polysaccharide can effectively inhibit the growth of solid tumors in mice and protect the immune organs of mice in a dose-dependent manner.H&E staining also proved that Eucommia Folium polysaccharide can destroy the complete shape of tumor cells,but have a certain protective effect on liver tissue,and have no obvious toxic side effects on the body.Annexin V-FITC/PI double-staining experiments proved that Eucommia Folium polysaccharide could induce tumor cells apoptosis;PI single-staining experiments proved that Eucommia Folium polysaccharide could block tumor cells in S phase and prevent their further proliferation;JC-1 experiment results showed that Eucommia Folium polysaccharide could destroy the mitochondrial membrane potential of cells,indicating that polysaccharides from Eucommia Folium may induce tumor cells apoptosis through the mitochondrial pathway.In conclusion,Eucommia Folium polysaccharide can be used as a potential drug for the prevention and preliminary treatment of liver cancer,providing a new possibility for future anticancer drugs.