Anti-Inflammatory Properties of the Medicinal Mushroom <i>Cordyceps militaris</i> Might Be Related to Its Linear (1→3)-β-D-Glucan

<div><p>The Ascomycete <i>Cordyceps militaris</i>, an entomopathogenic fungus, is one of the most important traditional Chinese medicines. Studies related to its pharmacological properties suggest that this mushroom can exert interesting biological activities. Aqueous (CW and HW) and alkaline (K5) extracts containing polysaccharides were prepared from this mushroom, and a β-D-glucan was purified. This polymer was analysed by GC-MS and NMR spectrometry, showing a linear chain composed of β-D-Glc<i>p</i> (1→3)-linked. The six main signals in the ¹³C-NMR spectrum were assigned by comparison to reported data. The aqueous (CW, HW) extracts stimulated the expression of IL-1β, TNF-α, and COX-2 by THP-1 macrophages, while the alkaline (K5) extract did not show any effect. However, when the extracts were added to the cells in the presence of LPS, K5 showed the highest inhibition of the pro-inflammatory genes expression. This inhibitory effect was also observed for the purified β-(1→3)-D-glucan, that seems to be the most potent anti-inflammatory compound present in the polysaccharide extracts of <i>C. militaris. In vivo</i>, β-(1→3)-D-glucan also inhibited significantly the inflammatory phase of formalin-induced nociceptive response, and, in addition, it reduced the migration of total leukocytes but not the neutrophils induced by LPS. In conclusion, this study clearly demonstrates the anti-inflammatory effect of β-(1→3)-D-glucan.</p></div

¹³C-NMR spectra of fractions PK5 (A), and SD-PK5 (B), in Me₂SO-<i>d</i>₆ at 70°C (chemical shifts are expressed in δ ppm).

<p>¹³C-NMR spectra of fractions PK5 (A), and SD-PK5 (B), in Me₂SO-<i>d</i>₆ at 70°C (chemical shifts are expressed in δ ppm).</p

Effect of β-(1→3)-D-glucan on neurogenic (A) and inflammatory phase (B) of nociception induced by formalin in mice. Footnote:

<p>Mice received vehicle (saline plus 5% Me₂SO, 10 mL/kg, i.p.) or β-(1→3)-D-glucan (3, 10 and 30 mg/kg, i.p.) 30 min before formalin administration. Statistical analyses were performed by means of one-way analysis of variance (ANOVA) followed by Newman–Keuls’ test. The results represent the mean ± SEM of 10–12 animals. *p<0.05 versus control group.</p

mRNA expression level of pro-inflammatory genes, after treatment with LPS+β-(1→3)-D-glucan (Glucan) for 3 h and 6 h. Footnote:

<p>Negative control (PBS) and positive control (LPS; 1 µg/mL). β-(1→3)-D-glucan was added at concentrations of 25, 50, and 100 µg/mL. Statistical analyses were performed by means of one-way analysis of variance (ANOVA) followed by Bonferronis' test, selected pairs. The results represent the mean ± SD of duplicate cultures of three representative experiments. *p<0.05; **p<0.01; ***p<0.001 versus positive control. ^#p<0.05; ^##p<0.01 versus different concentrations of glucan.</p

mRNA expression level of pro-inflammatory genes, after treatment with polysaccharide extracts for 3 h and 6 h. Footnote:

<p>Negative control (PBS) and positive control (LPS; 1 µg/mL). CW (cold water extract; 50 µg/mL), HW (hot water extract; 50 µg/mL), K5 (alkaline extract; 50 µg/mL). Statistical analyses were performed by means of one-way analysis of variance (ANOVA) followed by Bonferronis' test, selected pairs. The results represent the mean ± SD of duplicate cultures of three representative experiments. *p<0.05; **p<0.01; ***p<0.001 versus negative control.</p

Effect of β-(1→3)-D-glucan on number of total leukocytes (A) and myeloperoxidase levels (B) induced by LPS in mice. Footnote:

<p>Mice received vehicle (saline plus 5% Me₂SO, 10 mL/kg, i.p.), dexamethasone (DEXA, 0.5 mg/kg, i.p.), or β-(1→3)-D-glucan (30 mg/kg, i.p.) 30 min before LPS administration. Statistical analyses were performed by means of one-way analysis of variance (ANOVA) followed by Newman–Keuls’ test. The results represent the mean ± SEM of 6–8 animals. ^#p<0.05 versus saline group; *p<0.05 versus control group.</p

Monosaccharide composition of the fractions obtained from <i>C. militaris</i>.

<p>Footnote:</p>a<p>Calculated based on the initial dry mushroom weight.</p>b<p>Alditol acetates obtained on successive hydrolysis, NaBH₄ reduction, and acetylation, followed by GC-MS analysis.</p><p>Monosaccharide composition of the fractions obtained from <i>C. militaris</i>.</p

Extraction and purification steps of the β-D-glucan (SD-PK5) of <i>C. militaris</i>.

<p>Extraction and purification steps of the β-D-glucan (SD-PK5) of <i>C. militaris</i>.</p

Inhibitory effect of the extracts/glucan on the mRNA expression of pro-inflammatory genes by the THP-1 macrophages, after 3 h of incubation.

<p>Footnote:</p>a<p>Calculated based on the positive control (LPS) mRNA expression.</p><p>Inhibitory effect of the extracts/glucan on the mRNA expression of pro-inflammatory genes by the THP-1 macrophages, after 3 h of incubation.</p

Absortion spectra of Congo Red (control), Congo Red with dextran (random coil control), and Congo Red with β-D-Glucan from <i>C. militaris</i>.

<p>Absortion spectra of Congo Red (control), Congo Red with dextran (random coil control), and Congo Red with β-D-Glucan from <i>C. militaris</i>.</p