Isolation,Purification,Structure Identification And Bioactivity Of Neutral Polysaccharides From Polygonatum Mill.

Polygonti Rhizome belongs to the genus Polygonatum of the Lilium family,and is an important medicinal and food homologous plant in China.Its main components include polysaccharides,saponins,total phenols,flavonoids and so on.Polysaccharides are one of the main active components of Polygonti Rhizome,which has significant effects in lowering blood glucose,anti-aging and regulating immunity.This research carried out systematic separation,purification,structural characterization and in vitro activity studies on Polygonatum polysaccharides,in order to provide a theoretical basis for the research of the pharmacological activity and structure-activity relationship of Polygonatum polysaccharides,and lay a foundation for the research and development of Polygonatum resources.The research results were as follows:1.The extraction process of Polygonatum polysaccharide was optimized by orthogonal test.The optimization of water extraction and alcohol precipitation method were as follows:solid-liquid ratio of 1:20（g/m L）,extraction time of 2 h,extraction temperature of 75℃,extraction times of 3 times.Under these conditions,the extraction rate of polysaccharide was 14.621%.The optimization of ultrasonic extraction method were as follows:ultrasonic time of 50 min,solid-liquid ratio of 1:35（g/m L）,ultrasonic power of 450 W,ultrasonic times of 2 times.Under these conditions,the extraction rate of polysaccharide was 15.813%.Comparing with water extraction and alcohol precipitation method,ultrasonic extraction method had higher yield,convenient operation and shorter extraction time,which was more suitable for the extraction of Polygonatum polysaccharide.2.The separation and purification technology of Polygonatum polysaccharide was studied.The results show that the protein removal effect of TCA method was better than that of Sevage method.When the concentration of TCA was 6%,the protein clearance rate and polysaccharide retention rate were 73.69%and 74.93%,respectively.The decolorization effect of the activated carbon method was better than that of the H₂O₂method,the macroporous resin D101 method and the macroporous resin AB-8 method.When the activated carbon content was 2.5%,decolorization rate and polysaccharide retention rate were 72.63%and 84.23%,respectively.Polygonatum polysaccharide was separated and purified by DEAE-52 and Sephadex G-100 to obtain a single neutral polysaccharide component.3.The structure of Polygonatum polysaccharides was characterized preliminarily.Four neutral polysaccharides isolated and purified from four different regions of Chun’an（Zhejiang province,China）,Xixia（Henan province,China）,Danfeng（Shanxi province,China）,and Pan’an（Zhejiang province,China）were named CAZJ,XXHN,DFSX,and PAZJ,respectively.Monosaccharide composition analysis showed that CAZJ was composed of Ara,Gal,Glc,Man and Fru,while XXHN,DFSX and PAZJ were composed of Rha,Ara,Gal,Glc,Man and Fru in different proportions.The results of FT-IR and NMR confirmed the existence ofα-glycosidic bond andβ-glycosidic bond in polysaccharides.Among them,CAZJ,XXHN and DFSX had pyranose ring structure,while PAZJ had furanose ring structure.The results of periodate oxidation indicated that there were different proportions of（1→）or（1→6）glycosidic bond,（1→2）or（1→4）glycosidic bond and（1→3）glycosidic bond in polysaccharides.Congo red test indicated that XXHN,DFSX and PAZJ had triple-helix structure.TGA analysis showed that polysaccharids had good thermal stability.X-ray diffraction illustrated that polysaccharides existed in both crystalline and amorphous forms,while scanning electron microscopy revealed the morphological characteristics of polysaccharides from different origins.CAZJ displayed microporous structure with smooth surface,and XXHN was honeycombed with rough surface.DFSX was fibrous and irregular in structure,while PAZJ exhibited irregular stripe with smooth surface.4.The antioxidant and hypoglycemic activities of Polygonatum polysaccharide were evaluated in vitro.The results showed that all the four polysaccharides exhibited different degrees of antioxidant and hypoglycemic activities with a dose-dependent manner in the concentration range of 1 to 10.0 mg/m L.The antioxidant and hypoglycemic activities of the four polysaccharides were significantly different（P<0.05）.XXHN and PAZJ possessed signficantly higher biological activities than other polysaccharides.Correlation analysis revealed that the bioactivity of polysaccharides was significantly related to its monosaccharide composition,uronic acid and protein content.