奈米�次微米靈芝懸浮液多醣區分物對巨噬細胞活化之影響

Abstract

靈芝多醣的生理功能包含調節免疫、抗腫瘤等，最主要的活性多醣為 (1→3, 1→6)-β-D-葡聚醣。已有研究證實，利用濕式介質研磨（Media milling）製備奈米�次微米靈芝懸浮液所得多醣、蛋白質與 (1,3)-β-D-葡聚醣含量大於傳統熱水萃取組，但兩者(1,3)-β-D-葡聚醣結構鑑定結果差異大。因此，本研究分離純化出奈米�次微米靈芝懸浮液多醣區分物（Polysaccharide fractions of nano/submicron Ganoderma suspensions），並與巨噬細胞株（RAW 264.7）進行共培養，探討不同區分物對免疫反應之影響，進一步對有效活化巨噬細胞之多醣區分物做結構鑑定與組成分析。 本研究中，新鮮松杉靈芝子實體經介質研磨3小時後，樣品粒數粒徑約99.9%小於1 μm，且約有63.9%的粒子粒徑小於0.1 μm。以20%、40%、60%、80%酒精沉降獲得粗多醣F20、F40、F60、F80，總多醣、總 (1,3)-β-D-葡聚醣含量皆以F80最高，而總蛋白質以F60最高。每一組粗多醣經陰離子交換層析分離可得Fx-1（含醣）與Fx-2（含醣、蛋白質）兩組區分，總共八組多醣區分物F20-1、F20-2、F40-1、F40-2、F60-1、F60-2、F80-1、F80-2。於細胞存活率試驗結果除了F80-1存活率降至71%至79%外，其餘七組區分物皆無細胞毒性，且區分物F20-1、F40-1、F60-1有利於巨噬細胞生長；一氧化氮與細胞激素TNF-α試驗結果，於樣品濃度25 μg/mL時，F40-2為最有效的一組。多醣區分物以膠體過濾層析法所得分子量分布範圍介於2.9 kDa與126.2 kDa之間，而細胞試驗結果最有效的F40-2分子量範圍介於9.3 kDa與113.7 kDa之間。後續利用高效陰離子層析系統鑑定F40-2單醣組成以葡萄糖（68.2%）、葡萄糖醛酸（15.1%）為主，以及其 (1,3; 1,6)-β-D-葡聚醣分支度為0.27。

The biological functions of Ganoderma polysaccharide has been reported to be multi-functions, such as immune modulation, anti-tumor. (1→3, 1→6)-β-D-glucan is believed to be the main bioactive compound. Nano/submicron Ganoderma suspensions prepared by using wet media milling increased the contents of polysaccharides, proteins, and (1,3)-β-D-glucans in comparison with traditional hot-water extracts. However, the structures of (1,3)-β-D-glucans obtained from both preparation methods were significantly different. Thus, the present study was to fractionate polysaccharides from nano/submicron Ganoderma tsugae suspension, to investigate their effects on immunomodulatory activities and to identify the structure and composition of the fractions. Media milling resulted in 99.9% (number) of particles smaller than 1 μm. Crude polysaccharide F20, F40, F60, and F80 were obtained by using gradient precipitation method of 20%, 40%, 60%, and 80% ethanol. The content of total polysaccharides and (1,3)-β-D-glucans in F80 were the highest, while the content of protein in F60 was the highest among four samples. Two fractions Fx-1 and Fx-2 were isolated from each crude polysaccharide through DEAE (Diethylaminoethyl) anion-exchange chromatography and end up with eight polysaccharide fractions F20-1, F20-2, F40-1, F40-2, F60-1, F60-2, F80-1, and F80-2. These fractions were cultured with macrophage cell line (RAW 264.7) for 24 h. MTT assay showed that F80-1 resulted in lower the cell viability (71-79%), while other fractions showed no cell toxicity. Nevertheless, F20-1, F40-1, and F60-1 resulted in higher growth of macrophages. The results of nitric oxide assay and TNF-α assay indicated that at a concentration of 25 μg/mL, F40-2 was the most effective fraction among all with higher nitric oxide level in proportion to concentration. The molecular weight of polysaccharide fractions range from about 2.9 kDa to 126.2 kDa by gel filtration chromatography. The most effective fraction F40-2 has a molecular weight range from 9.3 kDa to 113.7 kDa, sugar composition mainly of glucose (68.2%) and glucuronic acid (15.1%), and its degrees of branching of (1,3; 1,6)-β-D-glucans was 0.27 by high-performance anion exchange chromatography.