| The Anemarrhena asphodeloides rhizomes is a kind of Liliaceae plant,which is rich in components,good for pharmacological effects and great potential to develop.With strengthen global demand for health care and prevention,the A.asphodeloides rhizomes was increasingly added to food as adjuvant at present.As an important component of the polysaccharide from A.asphodeloides rhizomes,its anti-inflammatory,anti-oxidation and hypoglycemic activity have been proved in the existing literature,but there were limited reports among its immune activities,antitumor activities and anticarcinogenic mechanism.At the same time,the structure-bioactivity relationship of polysaccharides,the interrelationship of immunity and antitumor had not been effectively studied.Therefore,the main research results on A.asphodeloides rhizomes were listed as follows:1.Optimization of hot water extraction process of A.asphodeloides rhizomes polysaccharide.The polysaccharides from A.asphodeloides rhizomes by hot water extraction method was named RAP1.The optimal extraction parameters of RAP1 were determined through single factor and orthogonal experiment as follows: solid-liquid ratio of 1:20 g/mL,extraction temperature 90°C,extraction time 2.0 h,with the yield of 12.81%.2.Purification and structure analysis of RAP1.The RAP1 was purified by DEAE Sepharose Fast Flow column purification to produce F1 and F2,with the yield of 50.01% and19.62%,respectively.The chemical constitutions of RAP1,F1 and F2 samples were measured to neutral sugars(60.54%,89.72% and 51.43%),protein(14.50%,1.94% and7.60%),sulfate esters(8.13%,3.42% and 5.92%),and uronic acids(15.61%,1.03% and30.20%).The Mw distribution of the three samples was in the range of130.00×103-576.51×103 g/mol,the Rg distribution 87.59-382.62 nm,and SVg distribution0.32-54.31 cm3/g.The proportion of monosaccharide in the three samples was rhamnose(0-6.12%),arabinose(7.10%-21.20%),xylose(0.20%-4.83%),mannose(39.90%-79.01%),glucose(6.01%-11.12%)and galactose(2.64%-22.01%),respectively.The main chain of F2was(1?4)-linked mannopyranosyl and(1?3)-linked galactopyranosyl.3.Immune activity evaluation of polysaccharides from A.asphodeloides rhizomes.Immune activities were analyzed by Griess assay and RT-PCR.The results indicated that the contents of NO,iNOS,COX-2,IL-1? and TNF-? in all treatment groups were increased significantly(P<0.05).It was confirmed that RAP1,F1 and F2 can promoted NO secretion by regulating the expression level of iNOS gene in RAW 264.7 cells,and regulate the gene expression of cellular immune factors to exert immune activities together.4.Inhibition activity and mechanism of A.asphodeloides rhizomes polysaccharides on gastric cancer cells.Anti-cancer activity was analyzed by MTT assay,Hoechst/PI staining,flow cytometry and western blotting.The experiment results showed that the RAP1,F1 and F2 were lethal to three gastric cancer cells.F2 can induce AGS cell apoptosis of human gastric cancer cells by MAPK/STAT3 signaling pathway,enhance the relative expression intensity of pro-apoptotic proteins,dramatic up-regulating the expression levels of p-p38 and p-JNK proteins(P<0.05),and significantly reduce the phosphorylation levels of ERK and STAT3(P<0.05),and then induce the apoptosis of human gastric AGS cancer cells.The above results determined the optimal process parameters by hot water extraction method,then identified the structure composition of polysaccharides from A.asphodeloides rhizomes,finally clarified that the RAP1,F1 and F2 polysaccharides exhibited good immune and anti-cancer activities,and illuminated the mechanism of F2 in AGS cells.In summary,the present research would offer theoretical support for the future development and application of foods and immune or anti-gastric cancer health care products associated with polysaccharide from A.asphodeloides rhizomes. |
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