Analysis of mechanism of action by crude polysaccharide, Rusalan, isolated from <i>Russula alatoreticula</i> in Raw 264.7 cells.

<p>(A) Total RNA was isolated from macrophage cells after 24 h incubation either with LPS (5 μg/ml concentration) or Rusalan (50, 100 and 200 μg/ml concentration) along with untreated cells. cDNA was prepared from respective RNA samples and semi-quantitative reverse transcriptase PCR was performed to analyse the expression of four different genes where β-Actin was considered as a house keeping gene. Further, the band intensities were quantified by ImageJ software to signify increase in transcription level of corresponding genes in relation (%) to control: (B) COX-2, (C) iNOS, (D) IL-6 (E) TNF-α. Values were represented as mean ± standard deviation of two independent experiments. (*<i>p</i>,0.05, **<i>p</i>,0.01, ***<i>p</i>,0.001, unpaired t-test).</p

Structural and molecular characterization of crude polysaccharide, Rusalan, isolated from <i>Russula alatoreticula</i>.

<p>(A) Changes in absorption maximum of Congo red-polysaccharide complex at various concentrations of sodium hydroxide solution (B) FT-IR spectra (C) Identification of monosaccharides in hydrolysed polysaccharides by HPTLC. Lanes: 1: L-arabinose, 2: D-fructose, 3: D-fucose, 4: D-galactose, 5: Rusalan, 6: D-glucose, 7: D-mannose, 8: D-rhamnose, 9: D-xylose (D) GC-MS chromatogram of derivatized Rusalan (Retention time of D-mannose: 16.6 min, D-glucose: 16.7 min, D-galactose: 16.8 min).</p

Antibacterial activity of crude polysaccharide, Rusalan, isolated from <i>Russula alatoreticula</i> as determined by minimum inhibitory concentration (MIC) value (mean ± standard deviation; n = 3).

<p>Antibacterial activity of crude polysaccharide, Rusalan, isolated from <i>Russula alatoreticula</i> as determined by minimum inhibitory concentration (MIC) value (mean ± standard deviation; n = 3).</p

Chemical characterization of crude polysaccharide, Rusalan from <i>Russula alatoreticula</i>, (mean ± standard deviation; n = 3).

<p>Chemical characterization of crude polysaccharide, Rusalan from <i>Russula alatoreticula</i>, (mean ± standard deviation; n = 3).</p

Antioxidant activity of crude polysaccharide, Rusalan, isolated from <i>Russula alatoreticula</i>.

<p>The results are presented in EC₅₀ values (mean ± standard deviation; n = 3) corresponding to 50% of antioxidant activity or 0.5 of absorbance for reducing power assay. BHA was used as standard in superoxide, hydroxyl and DPPH radical scavenging methods. Ascorbic acid was considered as standard in reducing power and total antioxidant capacity assays, while EDTA was adopted as a positive control in chelating ability of ferrous ion method. In each row, different letters mean significant differences between sample and standard (<i>p</i> < 0.05).</p

Effect of crude polysaccharide, Rusalan, isolated from <i>Russula alatoreticula</i> on morphology of macrophages.

<p>Cells were incubated for 24 h with different concentrations of Rusalan where LPS at 5 μg/ml concentration was used as a positive control. Afterwards, cells were fixed, stained with DAPI, subjected to fluorescence microscopy and images were captured. (A) Negative control (B) LPS (C) 50 μg/ml (D) 100 μg/ml (E) 200 μg/ml (F) 400 μg/ml.</p

Effect of crude polysaccharide, Rusalan, from <i>Russula alatoreticula</i> on activity of macrophages.

<p>(A) Proliferation was monitored in treatment of Rusalan for 24 and 48 h by WST method and expressed in relation (%) to negative control. (B) Phagocytosis in relation (%) to control was determined by neutral red method. (C) Release of nitric oxide in cell supernatant was quantified using Griess reagent. In all assays LPS at the concentration of 5 μg/ml was used as positive control. Values were presented as mean ± standard deviation of at least three independent experiments. (*<i>p</i>,0.05, **<i>p</i>,0.01, ***<i>p</i>,0.001, unpaired t-test).</p

<i>Russula alatoreticula</i> (CAL-1271).

<p>(A) Fresh Basidiomata in the field. (B-C) Habitat—<i>Shorea robusta</i> dominated areas of Gurguripal and Khairulachak, West Bengal from where basidiomata of <i>Russula alatoreticula</i> were collected (photographs by S. Paloi). Scale bars: 10 mm in A.</p

Microscopic features of <i>Russula alatoreticula</i> (CAL-1271, holotype).

<p>(A-B) Scanning Electron Micrograph of basidiospores. (C) Basidia. (D) Hymenial cystidia (gill sides) as observed in Congo red. (E) Pileipellis. (F) Sphaerocytes. Scale bars: 10 μm in C-F.</p

Antioxidant activity of crude polysaccharide, Rusalan, prepared from <i>Russula alatoreticula</i>.

<p>(A) Superoxide radical scavenging activity (B) Hydroxyl radical scavenging activity (C) DPPH radical scavenging activity (D) Chelating ability of ferrous ion (E) Reducing power. Results were represented as mean ± standard deviation of triplicate experiments.</p