Extraction,purification,characterization Of Polysaccharides From Panax Ginseng C.A. Meyer And The Mechanisms Underlying Its Immunity And Anti-tumor

Ginseng(Panax ginseng C.A.Meyer)is a plant belonging to the family of Araliaceae.In China,it has been applied for clinic for a long time.It has long been listed as the top-grade in the"Shen Nong Materia Medica",and it was showed that"ginseng tastes bitter and slightly cold,mainly tonifying the five internal organs,calming the nerves,calming the soul,stopping the shock,eliminating the evil spirit,brighting eyes,happiness and wisdom".Ginseng has other names such as yellow ginseng,god grass,goblin and king of herbs.It is mainly produced in northeastern China and is listed as the"Three Treasures of Northeast China"alongside deer antler and mink.Therefore,it has been extensively studied the medicinal value and application value.The objective of the present study was to clarify the immunomodulatory and anti-tumor effects of ginseng polysaccharides as well as the cell signal pathway using four parts of experiments.Test 1:Extraction of ginseng polysaccharidesThe ginseng polysaccharides were extracted by hot water reflux extraction method(WRE)and microwave extraction method(MAE),respectively.Fitting the optimal extraction process conditions of microwave assistance extraction was as follow:extraction time,6 min;extraction temperature,77?;microwave power,557 w;liquid-solid ratio,37 ml/g.The extraction rate under this condition was 42.4%.Under the same conditions,the extraction rate of ginseng polysaccharide extracted with hot water reflux was 28.5%.This result indicated that the extraction rate of ginseng polysaccharide extracted by microwave is higher than that of hot water reflux method.The MAE method is simple,efficient,and could improve the utilization rate of raw materials.The antioxidant activity was evaluated using hydroxyl,ABTS and DPPH free radical scavenging assays.The experimental results show that ginseng polysaccharides have strong reducing ability.In the in vitro antioxidant experiments,ginseng polysaccharides extracted by MAE have higher antioxidant activity than that extracted by WRE.The antibacterial activity of ginseng polysaccharides was measured by calculating their MIC₅₇ value.The results showed that ginseng polysaccharide had strong inhibitory effect on Staphylococcus aureus 7.25 mg/mL,Bacillus pumilus 7.71 mg/mL,Escherichia coli 7.725mg/mL and Bacillus subtilis 7.5 mg/mL.It has highest antibacterial activity against Escherichia coli.The extraction rate of ginseng polysaccharide extracted by MAE method is higher than that of WRE method.The ginseng polysaccharide extracted by microwave has better in vitro antioxidant activity and antibacterial activity than that obtained by hot water reflux extraction.Test 2:Separation,purification and structural characterization of ginseng polysaccharideThe microwave-assisted extraction of ginseng polysaccharide was preliminarily purified by cotton-ike DEAE column chromatography and eluted with NaCl.The fraction was further dialyzed against distilled water and lyophilized.The re-dissolved polysaccharides were separated by DEAE-52 column chromatography and eluted with different concentrations of NaCl.Four fractions(GSP-7,GSP-1,GSP-2,and GSP-3)were obtained.The structure of ginseng polysaccharide was determined by UV-visible spectrophotometer,infrared spectrophotometer and high-performance liquid chromatography.UV analysis:There are no absorption peaks of protein and nucleic acid found in GSP-2 and GSP-3.Infrared analysis:GSP-1 is a polypyranose.The absorption peak at 1733 cm^-1indicates the presence of ester carbonyl group in the polysaccharide.GSP-7 and GSP-2 have mannose absorption peaks at 857 cm^-1 and 865 cm^-1.HPLC result analysis:The average molecular weights of GSP-7,GSP-1,GSP-2,GSP-3 were approximately 9.24×17⁵Da?5.26×17⁵Da?1.76×17⁵ Da?8.32×17⁵Da,and the molecular weights of the four ginseng polysaccharides in the order of GSP-7>GSP-3>GSP-1>GSP-2.The monosaccharide composition of GSP-7,GSP-1,GSP-2,GSP-3 were similar,mainly including rhamnose,glucose,galactose,arabinose,galacturonic acid,glucuronic acid,as well as a small amount of mannose and fucose,but there is a certain difference in molar ratio.XPS result analysis indicated that GSP contains high content of C and O elements.It is speculated that polysaccharides may contain glycoproteins.Compared with GSP-7,the peaks in the C-O form of GSP-2 are significantly reduced,indicating that part of-OH of GSP-2 is substituted.Congo red experimental results analysis:the four components of ginseng polysaccharides have triple helix conformation.Test 3:Study on the immunomodulatory activity and mechanism of ginseng polysaccharides on RAW264.7 cells in vitroThe studies of the effects of GSPs on macrophages RAW264.7 showed that ginseng polysaccharide can enhance the viability of RAW264.7 cells,the phagocytic function and promote NO secretion.Therefore,ginseng polysaccharide has an immunomodulatory effect.The immunomodulatory activities of GSPs were different due to the difference of its components.GSP-2 has the highest activity,while the GSP-3 has the weakest or no activity.Enzyme-linked immunosorbent assay(ELISA),real-time PCR(Real-time PCR)and western blotting(Western Blot)were used to detect the cytokines productions including TNF-?and IL-6,iNOS,TNF-?and IL-6 m RNA expression levels,and signal pathway key molecules p38,ERK1/2,JNK protein phosphorylation levels in GSPs stimulated RAW264.7 cells.The results showed that ginseng polysaccharide can significantly promote the secretion of NO,TNF-?and IL-6 in RAW264.7 cells(p<7.71),and can up-regulate the phosphorylation levels of p38,ERK,and JNK proteins in the MAPK signaling pathway.Therefore,GSP-2 can promote the secretion of cytokines in macrophages,and through the binding of cytokines to corresponding receptors,activating the corresponding signal pathways to exert its immune function.Test 4:Research on anti-tumor activity of ginseng polysaccharideThe CCK-8 method was used to screen the inhibitory effects of ginseng polysaccharide GSPs on the colon cancer cells.The colon cancer cells include HCT-116,HCT-8,Caco2,SW48,HT-29 human colon cancer cells.The results showed that GSP-2 could significantly inhibit the proliferation of HCT-116 colon cancer cells.It has the highest cell proliferation inhibition rate among GSPs.The WB experiment found that ginseng polysaccharide GSP-2 has an anti-colon cancer effect.It could induce apoptosis of tumor cell by affecting the lysis of PARP protein.The effects of GSP-2 on the expression of Caspase-3,Cyclin B1,p21,and CDC25C was determined.The results showed that GSP-2 has no significant effect on the expression of these proteins in the HCT-116 cells.