| Owing to its high nutritional values and attractive taste, it is one of the most popular edible mushrooms worldwide. Its production and consumption ranked at fourth place in the edible mushrooms in the world. Many studies have demonstrated that Flammulina velutipes(Curtis) Singer has various biological activities, Including anti-tumor, immunomodulatory activities and antidiabetics activities. In this paper, modern isolation and analysis methods were used to study on the extraction, purification, characteristics and bioactivities of F. velutipes polysaccharides, The research offers some scientific basic for comprehensive utilization for F. velutipes. 1、Extraction、Isolation and purification of F. velutipes polysaccharidesThe fruiting bodies of F. velutipes were first exhaustively extracted with 95% ethanol under reflux for 12 h to remove lipids. This step was repeated three times. After filtration, the residue was air-dried at room temperature, and then extracted with boiling distilled water twice(for 2 h each time). The liquid extracts were combined, and concentrated to one-tenth of the original volume. 95% Et OH was added until the final alcohol concentration reached 30% for precipitation. The resulting precipitate was separated out, defined as FVP30. 95% ethanol was subsequently added until the final alcohol concentration reached 60%. The resulting precipitate was separated out, defined as FVP60.The crude polysaccharide was fractionated and purification by anion-exchange chromatography and gel filtration chromatography. Finally, four homogeneous polysaccharide(FVPA2, FVPB2, FVPA1, FVPA2). All of these were identified by HPLC. 2、Study on structures of homogeneous polysaccharides from F. velutipes(1) Analyzed by HPLC, the molecular mass of FVPA2, FVPB2, FVPA1 and FVPA2 were as follows: 3.4×104 Da, 1.502×104 Da, 1.8×104 Da and 2.044×104 Da. Analyzed by HPAEC, FVPA2 contained galactose, fucose, mannose in the ratio 5:1:1. FVPA1、FVPB2 and FVPB1 contained galactose, mannose, fucose, glucose in the ratio of 2.6:1.4:1.1:1, 1.9:1.2:1:2.5, 2.2:1.38:1.2:18.52;(2) Character analysis of homogeneous polysaccharides from F. velutipesMethylation analysis and NMR and GC-MS to analysis the homogeneous polysaccharides, then we know the structure characterization of FVPA2, FVPB2, FVPA1 and FVPB1.(1) Structural of FVPA2Sugar analysis, methylation analysis, and 1D and 2D NMR spectroscopy revealed FVPA2(2) Structural of FVPA1Sugar analysis, methylation analysis, and 1D and 2D NMR spectroscopy revealed FVPA1 to have the following repeat unit:(3)Structural of FVPB2Sugar analysis, methylation analysis, and 1D and 2D NMR spectroscopy revealed FVPB2 to have the following repeat unit: 3、Studies on immunocompetence in vivo of the polysaccharides from the fruting bodies of Flammulina velutipeIn the immunocompetence tests, FVPB2 and FVPB1 showed effect on immunomodulatory effects.(1) FVPB2 and FVPB1 can stimulated the proliferration of mouse spleen lymphocytes, at the same time activated cells in mouse spleen lymphocytes, after activation, immunoglobulin was released.(2) FVPB2 and FVPB1 stimulated the proliferation of B lymphocytes, at the same time activated cells in mouse spleen lymphocyte, Breg, a subpopulation in B cell, was activated to releaseIL-10, Intracellular IL-10 analysis was used to analysis the pathway play: FVPB2 induced IL-10 was at least partially dependent on ERK1/2 and NF-κB to release IL-10; FVPB1 induced IL-10 was at least partially dependent on JNK, P38 and ERK1/2 to release IL-10.Our results show that FVPB2 and FVPB1 can stimulate T cells and macrophage at the same time. To make the activated immune cells to perform their respective functions. |
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