藥用菇類水溶性多醣體的物化性質及免疫受體結合特性研究

Abstract

藥用菇類在亞洲地區已廣泛應用於傳統醫藥及機能性食品。水溶性及非消化性多醣體，例如β-D-葡聚醣，為菇類中重要的活性成分之一；此類多醣體具有多種生物活性，包含免疫調節，抗癌及抗發炎等。多醣體所具有的生物活性會受多醣本身的物化性質及其與免疫受體作用的特性影響，而這些影響因子又會因菇類來源和多醣製備條件的不同而有所差異。本研究分析五種常見藥用菇類的水溶性多醣體物化性質，分別為舞菇（Grifola frondosa）、靈芝（Ganoderma lucidum）、雲芝（Coriolus versicolor）、桑黃（Phellinus linteus）和牛樟芝（Antrodia cinnamomea），其中β-D-葡聚醣含量及分子量較高，且抗發炎活性最佳的水溶性多醣體則進一步用於探討免疫受體結合特性。結果顯示，在相似的製備條件下，自不同菇類製備而得的水溶性多醣體之單醣組成（葡萄糖：53–93%）、分子量（10–723 kDa），以及(1→3, 1→6)-β-D-葡聚醣的含量（3–25%）和(1→6)支鏈分支度（0.21–0.38）等物化性質具明顯差異。由較低的萃取溫度製備之水溶性多醣體的(1→3, 1→6)-β-D-葡聚醣含量較少，但分子量則較高。在脂多醣活化之RAW264.7巨噬細胞試驗中，舞菇多醣體具有較佳的抗發炎活性，且其70°C低溫萃取之多醣體（GF70）對促發炎介質的生成具有最佳的抑制效果，但此抑制效果不受細胞上主要的β-葡聚醣受體Dectin-1的表現量影響，也與水溶性多醣體中的(1→3, 1→6)-β-D-葡聚醣含量無關。GF70的高分子量區分（GF70-F1）可能透過與toll-like receptor 2（TLR2）結合作用進而啟動抗發炎效果，而非透過Dectin-1或complement receptor 3（CR3）受體；推測GF70-F1中含量最豐富的多醣應為TLR2的配體，其結構特徵為具有以β-(1→4)鍵結之葡萄糖主鏈骨架並含(1→6)分支，而GF70-F1中的(1→3, 1→6)-β-D-葡聚醣則可能為Dectin-1的拮抗劑。由本研究結果得知，來自不同藥用菇類的水溶性多醣體具有不同的物化性質，其中菇類水溶性多醣體的分子量及鍵結型式（例如含有(1→6)分支的(1→4)-β-D-葡聚醣）皆可能為其免疫受體結合特性及抗發炎活性的關鍵影響因子，而TLR2則可能為菇類抗發炎活性多醣的作用目標受體。

Medicinal mushrooms have been popularly used as folk medicine and functional foods in Asia. Water soluble and non-digestible polysaccharides especially β-D-glucans are one of the major bioactive components in mushrooms; they possess various biological activities, such as immunomodulation, anti-cancer, anti-inflammation and others. These bioactivities are mainly affected by the physicochemical characteristics of polysaccharides and their interaction with immune receptors, which may be different due to the mushroom sources as well as the preparation conditions and downstream processing of polysaccharides. In this study, the physicochemical characteristics of water soluble polysaccharides (WSP) from five well-known medicinal mushrooms, including Grifola frondosa, Ganoderma lucidum, Coriolus versicolor, Phellinus linteus and Antrodia cinnamomea were analyzed; the WSP that possessed higher molecular weight, higher (1→3, 1→6)-β-D-glucan content, and the strongest anti-inflammatory activity was selected for the investigation on immune receptor-binding properties. Results showed that WSP prepared from different mushrooms under similar preparation conditions possessed different physicochemical properties, including monosaccharide composition (glucose molar %: 53–93%) and molecular weight (10–723 kDa), as well as the content (3–25%) and degree of (1→6) side chain branching (0.21–0.38) of (1→3, 1→6)-β-D-glucan. Lower extraction temperature may lead to a WSP with a lower (1→3, 1→6)-β-D-glucan content but a higher molecular weight. The WSP from G. frondosa was found to possess the strongest anti-inflammatory activity in lipopolysaccharide-stimulated RAW264.7 macrophages, and the one prepared under lower extraction temperature (70°C; GF70) was shown to possess the most potent inhibitory effect on pro-inflammatory mediator production. Surprisingly, the inhibitory potency of WSP was not related to the expression level of the major β-glucan receptor Dectin-1, and no relationship was found between the anti-inflammatory activity and the content of (1→3, 1→6)-β-D-glucan of WSP. Further studies showed that the high molecular weight fraction of GF70 (GF70-F1) may contribute to the anti-inflammatory activity through interacting with toll-like receptor 2 (TLR2) but not Dectin-1 or complement receptor 3 (CR3). The major glycan consisting of β-(1→4)-linked glucose backbone with (1→6) branches in GF70-F1 was considered to be the TLR2 ligand, whereas the (1→3, 1→6)-β-D-glucan in GF70-F1 may act as Dectin-1 antagonist. In conclusion, WSP from different medicinal mushrooms possessed diverse physicochemical characteristics, and their molecular weights as well as linkage pattern [such as (1→6)-branched (1→4)-β-D-glucan] could be the key factors affecting their immune receptor-binding properties and anti-inflammatory activities, and TLR2 may be the target receptor for mushroom anti-inflammatory polysaccharides.