Introducing a novel mushroom from mycophagy community with emphasis on biomedical potency

Abstract

Mushrooms have been prized by humankind as medicine and culinary wonder since antiquity. Though several species are ethnically valued; many prospective species are still being discovered. One such wild macrofungus has recently been discovered during subsequent field surveys in West Bengal, India which in turn exposed as a traditionally consumed popular myco-food. The collected taxon was found to be unique with regard to its morphological as well as genetical features. After detailed characterizations, the fungus was identified as a novel taxon belonging to the genus Russula (Russulaceae, Basidiomycota). Besides, the investigation was further extended in search of new functional ingredients and in this context, a water soluble crude polysaccharide rich extract (Rusalan) was isolated from dried basidiocarps. Accumulating evidences from GC-MS, HPTLC, FT-IR along with several spectrophotometric methods postulated that the fraction consisted mainly of carbohydrate in triple helical conformation, where glucose was the major monosaccharide mostly with β-type glycosidic linkage. Conversely, Rusalan showed pronounced antioxidant activity in six in vitro assay systems with EC50 value ranging from 190-1328 μg/ml concentration. The crude polysaccharide was also evaluated against six bacterial strains using microdilution method and the growth of Staphylococcus aureus and Bacillus subtilis were found to be inhibited effectively. In addition, immune-stimulatory assays demonstrated that Rusalan could evidently promote proliferation, induce phagocytosis, release NO, produce intracellular ROS and upregulate mRNA expression of iNOS, TNF-α, COX-2, as well as IL-6 genes in in mouse macrophage cells. Therefore, aim of the present study was not only to describe a new taxon to the world mycoflora but also to introduce a potent therapeutic agent that could be explored for food and pharmaceutical purposes. However, isolation of active component and in vivo studies need to be designed further.

Fig 1. Russula alatoreticula (CAL-1271).

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

Fig 2. Microscopic features of Russula alatoreticula (CAL-1271, holotype).

(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.

Fig 3. Phylogenetic tree.

Consensus phylogram (50% majority rule) resulting from a Bayesian analysis of the nrITS sequence alignment of Russula species, showing mean branch lengths, obtained from 10⁶ generations of an MCMC analysis. Bayesian posterior probabilities (≥ 0.50) has been indicated above the branches. The scale bar represents number of expected changes per site. Categorizations of Russula species within the phylogenetic tree follows classification of Sarnari [42].

Fig 4. Structural and molecular characterization of crude polysaccharide, Rusalan, isolated from Russula alatoreticula.

(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).

Fig 5. Antioxidant activity of crude polysaccharide, Rusalan, prepared from Russula alatoreticula.

(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.

Fig 6. Effect of crude polysaccharide, Rusalan, from Russula alatoreticula on activity of macrophages.

(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. (*p,0.05, **p,0.01, ***p,0.001, unpaired t-test).

Fig 7. Effect of crude polysaccharide, Rusalan, isolated from Russula alatoreticula on morphology of macrophages.

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.

Fig 8. Effect of crude polysaccharide, Rusalan, isolated from Russula alatoreticula on intracellular production of ROS in macrophages.

Raw 264.7 cells were treated with Rusalan or LPS and after 24 h intracellular ROS generation was determined by flow cytometry using DCFDA dye. Red coloured graphs represent log fluorescence intensity of oxidative product of DCFDA treated with (A) LPS at 5 μg/ml concentration and Rusalan at variable doses such as (B) 50 μg/ml (C) 100 μg/ml (D) 200 μg/ml (E) 400 μg/ml concentration in comparison with negative control denoted by black coloured graph. (F) Relative fluorescence intensity was also analysed in detail. Data were represented as mean ± standard deviation of three independent experiments. (*p,0.05, **p,0.01, ***p,0.001, unpaired t-test).

Fig 9. Analysis of mechanism of action by crude polysaccharide, Rusalan, isolated from Russula alatoreticula in Raw 264.7 cells.

(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. (*p,0.05, **p,0.01, ***p,0.001, unpaired t-test).