Bioactive polysaccharides and small molecules from the native North American fungus Echinodontium tinctorium

Mushrooms, the fruiting bodies of fungi, are known to be powerful sources of nutraceuticals and pharmaceuticals but there are limited studies focusing on exploring the medicinal value of mushrooms native to North America. Here, I describe the isolation of two novel bioactive polysaccharides from the aqueous extracts of the fungus Echinodontium tinctorium: an immunostimulatory complex polysaccharide (EtISPFa) of 1354 kDa, and a growth-inhibitory β-glucan of 275 kDa. In addition, six small molecules including a phenol derivative, a new diphenylmethane derivative and three lanostane-type triterpenes were isolated from the organic extracts of E. tinctorium. The molar mass of these isolated small molecules (labelled 1-6) was determined to be 124, 260, 506, 498, 496, and 440 g/mol respectively. Phase separation, Sephadex LH-20 size exclusion, Sephadex DEAE ion exchange chromatography, Sephacryl S-500 HR size exclusion, silica column chromatography, and HPLC were used for bioactivity-guided purification. Chemical structures and linkages of EtISPFa and EtGIPL1a polysaccharides were determined by gas chromatography mass spectrometry (GCMS) and nuclear magnetic resonance (NMR). Final structures of small molecules were determined by Fourier transform infrared spectroscopy (FTIR), electrospray ionization mass spectrometry (ESI-MS), NMR, and X-ray crystallography. Immuno-stimulatory activity of EtISPFa was assessed by immunoassay in Raw 264.7 murine macrophage cells and growth-inhibitory activity of EtGIPL1a and small molecules were assessed by MTT growth-inhibitory assay in cancer cell lines. The mechanism of growth inhibition was assessed via apoptosis and cell cycle assays. EtISPFa stimulated the immune response by inducing TNF-α and other inflammatory cytokines in murine macrophage cells. In contrast, EtGIPL1a showed promising anti-proliferative activity against U251 glioblastoma cells and on ten other cancer cell lines. EtGIPL1a induced apoptosis in U251 cells with an increased cleaved caspase-3 apoptotic marker and significant DNA fragmentation in cell cycle analysis. Amongst the small molecules, compounds (2), (4) and (5) caused growth-inhibition in U251 cells; compound (4) also showed promising effects on multiple other cancer cell lines; all its bioactivities are reported here for the first time. The crystal structures of compounds (2), (4) and (5) have also been reported for the first time. Molecular targets of (1), (2), (4) and (5) by MolTarPred were predicted and warrants further experimental investigation.