| Summary: | 博士 === 國立清華大學 === 化學系所 === 106 === 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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