Isolation,Purification,Structure Identification And Biological Activity Of Polysaccharide From The Plumula Nelumbinis

Plumula Nelumbinis was the green young leaves and radicles in the middle of the mature seeds of the Nelumbo nucifera Gaertn of the Nymphaeaceae family.Plumula Nelumbinis has multiple nutritional values.As one of the main active ingredients,Plumula Nelumbinis polysaccharide has good antioxidant,anti-inflammatory,hypoglycemic abilities and can regulate glucose and lipid metabolism.At present,the research on Plumula Nelumbinis polysaccharide mainly focuses on crude polysaccharide,and there is a lack of in-depth research on the structure characterization and biological activity of pure Plumula Nelumbinis polysaccharide.In this paper,polysaccharides were extracted from Plumula Nelumbinis and further purified.Then,the structure of the purified Plumula Nelumbinis polysaccharide（PNP）was analyzed,and the in vitro biological activities of Plumula Nelumbinis polysaccharide,such as antioxidant activity,reduction of oxidative damage and anti-inflammatory activity,were evaluated.The main research contents and results are as follows:（1）The effects of hot water extraction（HWE）,ultrasonic-assisted extraction（UAE）,enzyme-assisted extraction（EAE）and microwave-assisted extraction（MAE）on the physical and chemical properties,structural characteristics and functional properties of crude Plumula Nelumbinis polysaccharides were compared.The four polysaccharides prepared by different extraction methods were named PNP_HWE,PNP_UAE,PNP_EAE,and PNP_MAE.The results show that PNP_MAE has higher yield,purity and uronic acid content.The results of high performance gel permeation chromatography（HPGPC）showed that different extraction methods have an effect on the molecular weight and distribution of crude PNP,and the content of high molecular weight components of PNP_MAE was the highest.The surface of PNP_MAE was rough and hollow by SEM,which was quite different from other crude PNP obtained by the other three methods.PNP_MAE has better rheological properties.In short,the MAE method has shown greater advantages in the extraction of polysaccharides from lotus seed heart.（2）The response surface analysis method was used to optimize the microwave-assisted extraction process of crude PNP.The results showed that the polysaccharide yield was 4.84±0.11%under the conditions of microwave time 4.5 min,microwave power 680 W,and liquid-to-material ratio 28 m L/g.The Sevag method and AB-8 macroporous resin were used to remove proteins and pigment impurities in PNP_MAE.After DEAE-52 column chromatography and Sephadex G-200 column chromatography,purified PNP with a uniform molecular weight was obtained.The purity of PNP was 88.48±1.58%,and the uronic acid content was 22.74±0.87%.（3）After monosaccharide composition,methylation and nuclear magnetic analysis,the results show that PNP was composed of five monosaccharides,including rhamnose（18.41%）,galacturonic acid（18.24%）,xylose（16.92%）,galactose（14.26%）,and arabinose（32.16%）.The main glycosidic bond types are→5)-α-L-Araf-（1→、→3）-α-D-Galp-(1→、α-D-Xylp-（→1、→3,4）-α-D-Rhap-（1→、→4）-α-D-Galp A-(1→.The Congo Red test shows that PNP does not have a triple helix structure.Atomic force microscopy results show that PNP presents a block structure of different sizes and irregular shapes.（4）The results of antioxidant experiments show that PNP has good DPPH,ABTS,and superoxide free radical scavenging ability.In the concentration range of 100-400μg/m L,PNP can significantly reduce the production of intracellular reactive oxygen species（ROS）and malondialdehyde（MDA）,and reduce the secretion of lactate dehydrogenase（LDH）,indicating that PNP can protect oxidative damage.In addition,PNP can enhance the activity of superoxide dismutase（SOD）,catalase（CAT）and glutathione peroxidase（GSH-PX）in cells.At the same time,it can significantly increase glutathione（GSH）content and total antioxidant capacity（T-AOC）levels.It shows that PNP samples can protect cells from damage caused by oxidation through enhancing the activity of the enzymatic antioxidant defense system and the level of the non-enzymatic antioxidant system in the cell.（5）The inflammation model established by LPS-incuded RAW264.7 mouse macrophages was used to study the anti-inflammatory activity of PNP.In the concentration range of100-1200μg/m L,PNP will not have a toxic effect on LPS-incuded RAW264.7 mouse macrophages.And it can significantly inhibit the expression of NO,TNF-α,INF-γ,IL-1βand IL-6 in macrophages of RAW264.7 mice stimulated by LPS（p<0.05）,and the inhibitory effect increased with the increase of sample concentration.PNP can reduce the phosphorylation of ERK,JNK,and p38 in the MAPK cell pathway.And at the same time,PNP can reduce the phosphorylation of p65 and IκB-αand the content of c-Jun in the NF-κB cell pathway,and increase the level of IκB-αin the cell.It shows that PNP can exert anti-inflammatory activity by affecting the MAPK/NF-κB/AP-1 signaling pathway in cells.