ISOLATION, CHARACTERIZATION, AND IMMUNOMODULATORY EVALUATION OF A BRANCHED GALACTOMANNAN OF THE MEDICINAL MUSHROOM CORDYCEPS MILITARIS
The mushrooms of genus Cordyceps are considered exotic and medicinal fungi, being C. militaris the second most commercialized species in China, Korea and Japan. Many of the beneficial effects provided by mushrooms were attributed to its polysaccharides, including immunomodulatory activities. An heteropolysaccharide from C. militaris was isolated and chemically characterized. The dried fungus was extracted using a 5% KOH solution. The purification steps included freeze-thawing, dimethylsulfoxide treatment and dialysis (100 kDa cut-off), giving rise to a homogeneous polysaccharide. Its monosaccharide composition was mannose (56.7%), galactose (34.5%), and glucose (8.8%). The Mw was estimated at 23,000 g/mol by HPSEC. A complex polysaccharide was identified by NMR and methylation analysis. The anomeric region of HSQC spectrum showed signals at d 107.7/5.06; 106.2/5.14; 105.9/5.13 relative to β-D-Galf non-reducing ends, O-2 and O-3 substituted β-D-Galf units, respectively. Other signals corresponded to C-1 of a-D-Manp O-6 and O-2 substituted (d 100.2/4.94; 100.5/5.27; 100.6/5.23; 100.7/5.16), and a-D-Manp 2,6-di-O-substituted (d 99.5/5.20; 99.7/5.37). This galactomannan is mainly constituted by (1®6)- and (1®2)-linked a-D-Manp fragments. Their presence was confirmed by methylation analysis, that showed the derivatives: 2,3,4-Me3-Manp (12.3%) and 3,4,6-Me3-Manp (29.6%). The main chain of mannose, which is still in analysis, is highly substituted. These data were confirmed by the presence of highly amounts of 3,4-Me2-Manp. The side chains are composed of small fragments containing β-D-Galf, β-D-Galp, and β-D-Glcp. Partial hydrolysis, COSY and NOESY experiments are in progress to elucidate the main chain and the side chains of this polymer. The anomeric configurations were determined by their chemical shifts and coupling constants (JH-1,C-1) via coupled HSQC spectrum. The galactomannan was tested on THP-1 macrophages to verify its effects on the cytokine gene expression. RT-PCR analysis showed an increase of the expression of pro-inflammatory genes, as IL-1β, TNF-a, COX-2, and NF-kB by these cells. Supported by CNPq, and Fundação Araucária.
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