巴西洋菇菌絲發酵物中含岩藻糖之多醣特質

Abstract

岩藻糖是誘導細胞產生免疫反應的重要抗原結合位；在本研究中以活性導向分離純化了巴西洋菇中含岩藻糖(fucose)之酸性多醣F3_FA；F3_FA具刺激巨噬細胞Raw 264.7分泌TNF-α的生物活性，經活性測試結果顯示岩藻糖在F3_FA刺激小鼠巨噬細胞RAW 264.7分泌TNF-α有著重要的角色；試驗結果並得知F3_FA在RAW 264.7細胞的接受器為TRL-4且其刺激可能為受NF-κB的調控，進一步影響下游TNF-α細胞激素的表現。巴西洋菇多醣F3_FA的化學分析結果顯示其分子量為3.45 x 105 Da.；糖組成莫耳百分比為6.4% L-fucose, 15.6% L-arabinose, 28.7% D-galactose, 14.8% D-xylose 及 24.3% 的D-galacturonic acid 並含少量的D-glucose, D-mannose, L-rhamnose 及 D-glucuronic acid ；利用甲基化衍生試驗得知F3_FA的結構中含大量1-4GalAp (24.3%)、1-4 Galp (22.1%)的鍵結結構並含有末端Fuc (6.4%)、1-5Ara (5.4%)、1-2Rha (5.1%)，推論為其結構為類似果膠多醣之結構；以果膠多醣單株抗體辨識也得到F3_FA含有(1, 4)-β-D-galactans、 (1, 5)-α-L-arabinans 及 (1, 4)-α-D-galacturonans的結構特徵；結構特徵結果在NMR圖譜中也得到呼應。本研究以四級銨鹽的選擇性沉降為分離酸性多醣、提高岩藻多醣含量的策略；以去氧糖呈色試驗估算樣品中岩藻糖的含量，結果顯示樣品中去氧糖含量由粗多醣中的4.6%經沉降區分後提升至酸性區分中含15.6%；且經比對證實F3_FA為巴西洋菇菌絲多醣非由發酵基質而來。

Fucose is one of important residues of recognition pattern for many immune cells. In this study, we characterized bioactive fucose-containing acidic polysaccharides from submerged fermentation of Agaricus blazei Murill. We obtained the polysaccharides through a cell-based activity-guided strategy, and used carbohydrate recognition monoclonal antibodies based Enzyme-Linked Immuno Sorbent Assay (ELISA) along with methylation and NMR analyses to investigate the structural characteristics of the polysaccharides. The polysaccharides had Mw of 3.45 × 105 Da. The major sugars were L-fucose, L-arabinose, D-galactose, D-xylose, and D-galacturonic acid in the molar ratio of 6.4, 15.6, 28.7, 14.8, and 24.3% with a small amount of D-glucose, D-mannose, L-rhamnose, and D-glucuronic acid. Results indicated that the bioactive polysaccharides consisted of a (1,4)-Galp and (1,4)-GalAp back bone; (1,2)-Xyl and (1,2)-Rha might also comprise backbone or constitute side chain; linkage (1,5)-Ara and terminal fucosyl residues were also involved in the polysaccharides. Regarding bioactivity, removal of the terminal L-fucosyl residues reduced the TNF-α cytokine stimulating activity of the polysaccharides in a RAW264.7 macrophage cell-line test, whereas NF-kapaB and TLR4 affected the polysaccharide-induced TNF-α production. Quanternary selecting precipitation as a strategy to rapid enriches fucose-containing acidic polysaccharide, deoxyl sugar contain from 4.2% upgrade to 15.6% by one step separated.