自然界存在的複合性碳水化合物，為不同結構特性的大分子，同時也具備著不同類型的生物活性。幾十年來，它們的免疫增強活性引發了越來越多的科學關注。然而，由於其結構的複雜性，以及缺乏結構與活性關係的相關研究，以及個別多醣的生物活性作用機制不同，使得它們在臨床發展貢獻上受到限制。在本研究中，選用了二種真菌: 具有高藥物作用的牛樟芝，以及食用的黑木耳，探討它們的免疫刺激多醣體的化學結構及詳細活性作用機制。
從上述二種真菌的冷水萃取物中分離出多醣體，通過分子篩及離子交換層析管柱進行一連串純化。純化多醣的化學結構是通過氣相層析質譜儀（GC-MS）和核磁共振光譜（NMR）進行分析決定。免疫刺激活性則針對細胞中促進發炎細胞因子（TNF-α，IL-6）和一氧化氮（NO）產生能力進行探討。
牛樟芝的免疫活性多醣體 (ACP; 分子量大於7萬) 的化學結構鑑定為galactomannan，為八醣重複序列組成的多醣體，結構解析為{→6)-D-Manp-(α1→2)-D-Manp-(α1→2)[D-Manp-(α1→3)-D-Manp-(α1→2)-D-Manp-(α1→6)-D-Galp (α1→6)]-D-Manp-(α1→6)-D-Galp-(α1→}。牛樟芝多醣體ACP可活化小鼠巨噬細胞J774A.1、骨髓來源的小鼠巨噬細胞、以及人類單核細胞衍生的樹突狀細胞，具濃度依賴性的方式產生TNF-α和IL-6。類鐸受體4 (Toll-like receptor 4) 則被鑑定為ACP誘導巨噬細胞活化的主要細胞受體。除了能刺激免疫細胞活化外，ACP也能增強巨噬細胞的吞噬作用以及殺菌能力，同時也會增強對細菌內毒素LPS的耐受力 (LPS tolerance) 。
黑木耳的免疫刺激多醣體（AAPS，分子量大於7萬）的化學結構，鑑定為具有七醣重複單元結構的glucuronoxylomannan: {→3)-D-Manp-(α1→3)-[D-Xylp-(β1→6)]-D-Manp-(α1→3)-D-Manp-(α1→3)-[D-GlcAp-(β1→2)]-D-Manp-(α1→3)-D-Manp-(α1→}。在AAPS中，偵測到甘露糖及木糖上都具有O-乙醯基化修飾，而葡萄醣醛酸上則沒有乙醯基化修飾。AAPS在刺激TNF-α，IL-6和NO上，也具有劑量依賴性的特性，鑑定出類鐸受體4 (TLR4) 是主要參與在AAPS誘導巨噬細胞活化的細胞受體。而在AAPS誘導小鼠巨噬細胞的一氧化氮產生中，三個可能刺激一氧化氮產生的刺激訊息途徑: 包括TLR4→ROS→NF-κB→NO; TLR4→ROS→PI3K→Akt→NO 以及 TLR4→MAPK→NF-κB→NO。類似情況下，AAPS在誘導細胞因子產生，則藉由兩種可能的信號通路：TLR4→ROS→PI3K→Akt→TNF-α/IL-6 以及 TLR4→MAPK→ TNF-α/IL-6。
黑木耳多醣體AAPS，以濃度依賴性的方式增強巨噬細胞的吞噬和殺菌能力。此外，AAPS也能增強對內毒素LPS的耐受性。進行去乙醯基化修飾和羧基還原的AAPS中，則觀察到免疫刺激活性的消失，表明免疫刺激過程中乙醯基化修飾和羧基官能團的重要性。分子模擬數據則進一步證明了乙醯基化修飾和羧基部分在受體結合中的作用方式。我們的研究結果為兩種實用真菌的免疫增強性質，提供了堅實的科學依據，並且這兩種免疫多醣的開發潛力，在不久的將來，做為開發出新的治療免疫相關疾病的碳水化合物營養補充劑及佐劑的有力候選物。

Naturally occurring complex carbohydrates represent a structurally diverse group of macromolecules which shows a broad range of biological activities. Over decades they, have attracted growing scientific interest for their immunoenhancing properties. However, their clinical contribution is still limited because of the structural complexity, lack of studies on structure activity relationship and underlying mechanisms of biological activity of individual polysaccharide components. In the present study two mushroom species with high medicinal, dietary and economical reputation, Antrodia cinnamomea and Auricularia auricula-juadae, were selected for the investigation of their chemical structures of immunostimulatory polysaccharides and action mechanisms in detail.
Polysaccharides were isolated from the cold water extracts of above two fungal species, and sequentially purified by size exclusion chromatography and ion exchange chromatography. Chemical structures of purified polysaccharides were elucidated by Gas Chromatography-Mass spectroscopy (GC-MS), and Nuclear Magnetic Resonance (NMR) spectroscopy. Immunostimulatory properties of each polysaccharide were investigated targeting the proinflammatory cytokines (TNF-α, IL-6) and nitric oxide (NO) producing abilities in selected immune cell models.
Immunologically active polysaccharide of A. cinnamomea (ACP; MW>70 kDa) was chemically identified as galactomnnan, which was composed of branched octasaccharide repeating unit, {→6)-D-Manp-(α1→2)-D-Manp-(α1→2)[D-Manp-(α1→3)-D-Manp-(α1→2)-D-Manp-(α1→6)-D-Galp (α1→6)]-D-Manp-(α1→6)-D-Galp-(α1→}. ACP activated J774A.1 murine macrophage, bone marrow-derived murine macrophages and human monocyte derived dendritic cells to produce, TNF- and IL-6, in a concentration dependent manner. Toll like receptor 4 was identified as the main receptor involved in ACP mediated macrophage activation. Further to immune cell activation, ACP enhanced the phagocytic activity, bactericidal potential of macrophages and elicited the endotoxin tolerance like effect against lipopolysaccharides (LPS)
Immunostimulatory polysaccharide of A. auricula-judae (AAPS, MW>70 kDa) was identified as glucuronoxylomannan with the repeating unit structure {→3)-D-Manp-(α1→3)-[D-Xylp-(β1→6)]-D-Manp-(α1→3)-D-Manp-(α1→3)-[D-GlcAp-(β1→2)]-D-Manp-(α1→3)-D-Manp-(α1→}. O-acetyl modification at mannose and xylose residues was observed in AAPS while glucuronic acid residues remained unacetylated. Native AAPS showed dose dependent immunostimulatory activity to secrete TNF-α, IL-6 and NO in Raw 264.7 cells. TLR4 was identified as the sole receptor involved in AAPS induced macrophage activation. Three possible signaling pathways, TLR4→ROS→NF-κB→NO; TLR4→ROS→PI3K→Akt→NO and TLR4→MAPK→NF-κB→NO were identified in AAPS induced NO production in murine macrophages. In similar vein, two possible signaling pathways identified in AAPS induced cytokine production: TLR4→ROS→PI3K→Akt→TNF-α/IL-6 and TLR4→MAPK→ TNF-α/IL-6.
AAPS enhanced the phagocytosis and bactericidal potential of macrophages in concentration dependent manner. Further, AAPS elicited the endotoxin tolerance like effect against LPS. Complete abrogation of immunostimulatory properties was observed in deacetylated and carboxyl reduced AAPS indicating the essentiality of both acetyl and carboxylic functionalities in immunostimulatory process. Molecular modelling data further demonstrated the role of acetyl and caroboxyl moieties in receptor binding. Our findings have provided firm scientific evidences for the immunoenhancing properties of two mushroom species, and the potential of these two polysaccharides to be strong candidates for the development of new carbohydrate-based nutraceutical supplements and adjuvants in the treatment of immunity related disorders in near future.

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