An Immunomodulatory Polysaccharide-Protein Complex Isolated from the Polypore Fungus Royoporus badius

Abstract

Many wild edible polypore mushrooms have medicinal value. In this study, we investigate the potential medicinal properties of the wild polypore mushroom Royoporus badius collected from north-central British Columbia, Canada. Water extract from R. badius was found to exhibit potent immunomodulatory activity. The extract was purified using DEAE-Sephadex anion-exchange chromatography as well as Sephacryl S-500 and HPLC BioSEC5 size-exclusion chromatography, to yield a novel polysaccharide-protein complex (IMPP-Rb).IMPP-Rb has a peak maxima molecular weight (M_p) of 950 kDa. GC-MS analyses showed that IMPP-Rb is composed predominantly of glucose (49.2%), galactose (11.3%), mannose (10.8%), rhamnose (9.6%), and galacturonic acid (8.2%), with smaller amounts of xylose (5.2%), fucose (2.8%), N-acetyl glucosamine (1.8%), and arabinose (1.2%). IMPP-Rb has multiple linkages, with 4-Glcp, 4-Manp, 6-Manp, 3,4-Manp, 4-Xylp, and 2-Rhap being the most prominent. IMPP-Rb is capable of inducing many cytokines in vitro and the protein component is indispensable for its immunomodulatory activity. IMPP-Rb has potential application as an immuno-stimulatory agent with pharmaceutical value.

Keywords: Picipes badius; Polyporus badius; Royoporus badius; immunomodulation; polysaccharide–protein.

Figure 1

Immunomodulatory and antiproliferative activities of crude extracts obtained from R. badius. (a) Dose- and (b) time-dependent MTT cell viability assays on HeLa cells. The concentration used for the time-dependent experiment was (b) 0.5 mg/mL. At 1 mg/mL, crude extracts from specimens (c) CL86 and (d) CL152 were assessed for their ability to induce TNF-α production in RAW264.7 macrophage cells, as an indicator of immunomodulation. Lipopolysaccharide (LPS) was used as a positive control, while media, water, and methanol were used as negative controls. Results presented are representations of two separate experiments (n = 2). Error bars show SD. One-way ANOVA (Tukey test) was used for statistical analysis. * p < 0.05 compared with the water control.

Figure 2

Purification of IMPP-Rb from R. badius. (a) Summary of the purification scheme used. (b) Purification using DEAE-Sephadex (column 2). Flow-through and eluent (post- 1 M NaCl) from a 750 mL DEAE column were pooled, dialyzed, and lyophilized. Various concentrations of samples were assessed for immunomodulatory activity, as indicated. Results shown are representative of two separate experiments.

Figure 3

Purification of IMPP-Rb from R. badius using Sephacryl S-500 (column 3). Collected fractions were assessed for their ability to induce TNF-α production in RAW264.7 cells. TNF-α levels plotted against (a) carbohydrate contents, and (b) protein contents. Results are representative of three separate experiments.

Figure 4

Purification of IMPP-Rb from R. badius using HPLC BioSEC5 (column 4). (a) Pooled, dialyzed, and lyophilized bioactive fractions from Sephacryl S-500 (column 3) were injected into BioSEC5. (b) Peak 1 (6–7.5 min) purified from (a) was reassessed for purity using BioSEC5. (c) Peak 2 (11–11.6 min) purified from (a) was reassessed for purity using BioSEC5. The top panel shows the refractive index spectrum and bottom panel indicates the UV spectrum at 280 nm.

Figure 5

FTIR spectrum of IMPP-Rb.

Figure 6

Effect of proteinase-K on immunomodulatory activity of polysaccharides. (a) PS-K (0.2 mg/mL) and (b) IMPP-Rb (1.5 mg/mL) were treated with 1 mg/mL proteinase-K. Samples were then heated at 80 °C for 20 min before being added to RAW264.7 cells. Results shown are mean ± SD from three independent experiments. One-way ANOVA (Tukey test) was used for statistical analysis. ns = not significant. * p < 0.05.