Structural Identification and the Immunological Activity of Polysaccharides from Maca Root(Lepidium Meyenii Walp)

Author:Zhang Meng Meng

Supervisor:wu zuo


Degree Year:2019





China has become the main producer of Maca in the world since the introduction of Maca,although maca originated in Peru.However,in recent years,Maca market has fallen into a trough.One of the reasons is that people don’t have a correct and comprehensive understanding to the composition and physiological activity of Maca.As one of the active components of maca,Maca polysaccharides have received increasing attention in recent years.However,most of the current researches just focused on Maca in China,and the structural information and physiological activity of polysaccharides in the Peruvian maca still lacked.In this paper,two purified polysaccharides were isolated from the Peruvian maca root.The structural information and physiological activity,especially immunoregulatory activity,of the two polysaccharides were researched.The basic chemical composition of the Peruvian maca root,including cellulose,starch,protein and lipids,was investigated.The water extraction method was used to isolate the polysaccharide.The best conditions for extraction after optimization was that the raw materials(solid-liquid ratio,1:30)was once extracted in boiling water for 2 h.The yield of the maca polysaccharide was 6.13% after the optimization.Three maca polysaccharides,MC-1,MC-2 and MC-3 were receiverd by the purification of DEAE Fate Flow column chromatography and Sephadex G50 column chromatography.In this study,oour research mainly foucs on MC-1 and MC-2.The purity of MC-1 and MC-2 was 97.5% and 98.3% respectively.The ultraviolet spectral scanning and endotoxin detection confirmed that MC-1 and MC-2 didn’t contain the nucleic acids,proteins and the endotoxin.The results of TG-DSC indicated that of MC-1 and MC-2 had good thermal stability.And the thermal stability of MC-2 is better than that of MC-1.Structural characterization revealed that MC-1 had an average molecular weight of 11.3 kDa and consisted of arabinose(26.21%),mannose(11.81%),glucose(53.66%),and galactose(8.32%).The main linkage types of MC-1 were proven to be(1→5)-α-L-Ara,(1→3)-α-L-Man,(1→2,6)-α-L-Man,(1→)-α-D-Glc,(1→4)-α-D-Glc,(1→6)-α-D-Glc and(1→6)-β-D-Gal by HPGPC,FTIR,GC-MS,methylation analysis and NMR analysis.The immunostimulating assay indicated that MC-1 could significantly enhance the pinocytic and phagocytic capacity and promote the NO,TNF-α and IL-6 secretion of RAW 264.7 cells,involving toll-like receptor 2,complement receptor 3 and mannose receptor mainly.The same structural characterization revealed that MC-2 has an average molecular weight of 9.83 kDa and is composed of arabinose(20.9%),mannose(4.5%),glucose(71.9%),and galactose(2.7%).The main linkage types of MC-2 were proven to be(1→5)-α-L-Ara,(1→3)-α-L-Man,(1→)-α-D-Glc,(1→4)-α-D-Glc,(1→6)-α-D-Glc,and(1→6)-β-D-Gal by methylation and NMR analyses.Congo red assay showed that MC-2 possesses a triple helix conformation.Immunostimulating assays indicated that MC-2 could induce M1 polarization of original macrophages and convert M2 macrophages into M1 phenotype.Although MC-2 could not shift M1 macrophages into M2,it could still inhibit inflammatory reactions induced by LPS.Furthermore,toll-like receptor 2,toll-like receptor 4,complement receptor 3,and mannose receptor were confirmed as the membrane receptors for MC-2 on macrophages.It was found that MC-1 and MC-2 were difficult to be absorbed in Caco2 cell monolayer.However,MC-1 and MC-2 could promote the secretion of IL-8,IL-10,IL-12 and INF-γ in Caco2 cells,and promote the secretion of TNF-α,IL-6 and NO in RAW264.7 cells via Caco2 cell monolayer.In addition,MC-1 and MC-2 could inhibit the damage induced by LPS to the densification of Caco2 monolayer membrane by the expression of TLR4,ZO-1 and occludin.MC-1 and MC-2 could also inhibit the secretion of inflammatory factors induced by LPS such as TNF-α,IL-8 and INF-γ in Caco2 cells,and increase the secretion of anti-inflammatory factors,IL-10.The antitumor,antioxidant,antiviral and anti-fatty liver activities of MC-1 and MC-2 were also explored.MC-1 and MC-2 were found to have weak antitumor directly.However,they exhibited strong anti-tumor activity by activating macrophages.MC-1 and MC-2 had weak free radical scavenging ability.However,they could protect red blood cells and liver cells from the oxidative damage indcued by AAPH.MC-1 and MC-2 didn’t have significant anti-HBV activity.However,MC-2 at a high concentration had an inhibitory effect on the secrection of HBeAg.MC-2 showed the inhibitory effect on oleic acid and palmitic acid-induced hepatic steatosis,and MC-1 didn’t have the activity.The above results indicate that MC-1 and MC-2 are new maca polysaccharides which have significant immunomodulatory activity to macrophages and the intestinal immune system.They also show the potential in anti-tumor,anti-oxidant and anti-fatty liver.These results will lay the foundation for the further research and application of maca polysaccharides.