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

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.

β-type glycosidic linkage. Conversely, Rusalan showed pronounced antioxidant activity in six in vitro assay systems with EC 50 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.

Introduction
Mushrooms have been appreciated since 3,000 BC due to their flavour, aroma and most importantly for medicinal metabolites. Consequently, they are long being used as dietary supplements for treatment and prevention of a multitude of diseases across the globe. This ethnically cultured health promoting concept has gradually matured over past decades through extensive scientific research for new and effective biomedicine [1]. Currently, investigators have isolated numerous active molecules from macrofungi and polysaccharides, especially β-glucan is considered as the most potent metabolite with diverse biological activities. These naturally occurring glucose polymers have been recognized as the best powerful immunostimulant and as a result fungal β-glucans are clinically used to activate the immune system function; particularly macrophages [2,3]. Macrophages act as the first line of defence against microbial infection, cancers and immunological diseases by initiating innate immune system followed by induction of adaptive immunity. They not only engulf foreign particulate matter, but also secrete several biological factors such as interleukin (IL) 6, IL 10, IL 1β, IL 12, transforming growth factor, (tumor necrosis factor) TNF-α and (nitric oxide) NO to invade foreign matters [4]. But when this macrophage-microbe interaction is excessively or inappropriately exploited application of agents able to improve host in born defence mechanism is highly recommended. Therefore, exhaustive research is now being directed in search of mushroom polysaccharides as effective immunomodulator [5,6]. Besides, there are previous report suggesting the high potential ability of β-glucans for inhibiting the growth of parasites, viruses and bacteria [7].
In recent years, significant increase of traditional antibiotics against antimicrobial infections has created an urgent health problem across the world. So, development of novel antimicrobial agents especially from natural sources can control infectious diseases along with decreasing emergence of new resistant strains [8]. In this regard, discovery of novel component that can inhibit growth of pathogenic microorganisms could be of considerable interest in search of products for therapeutic treatments.
Besides, mushroom polysaccharides have also emerged as an important source of antioxidants serving a critical role in protection of living organisms against free radical induced oxidative damage [9]. These radicals are known to be highly unstable and can readily react with surrounding molecules like protein, DNA, RNA, sugar and lipids. The reactions can lead to generate many severe disorders like cardiovascular, neurological, renal, liver, degenerative diseases and so forth [10]. Recently, there are several synthetic antioxidants available in market, though they are reported to execute several side effects including risk of carcinogenesis and liver damage. Thus, there is an urgent need for less toxic and more effective natural antioxidants [9]. In this regard, mushroom polysaccharides especially crude extracts appear to have the desired antioxidant activity [11].
Despite these therapeutic effects, research on macrofungi is not satisfactory as this diverse group still remains largely unexplored. To date, the number of mushrooms in world has been estimated approximately 150,000 of which only 10-15% have been scientifically described. It has also been postulated that to date, almost 60% of the novel species belong from tropics of which a large number are till to be described [12]. The Indian subcontinent is blessed with diverse agro-climatic zones harboring a treasure house of several mushrooms that in turn shaped indigenous knowledge. So far, approximately 850 mushroom species have been recorded only from India and more to discover yet [13]. In this context, our research group has been engaged to unveil the Indian myco-diversity mainly confining to the state, West Bengal and previously described three new species viz. Russula kanadii A.K. Dutta & K. Acharya [14], Russula hookeri S. Paloi, A.K. Dutta & K. Acharya [15] and Russula buyckii K. Acharya, S.
Paloi & A.K. Dutta [16], all of those belong to one of the most abundant and widely distributed ectomycorrhizal agaric genera [17].
During repeated field surveys, one member of Russula was collected with the help of inhabited tribal communities during collection of food for their daily square meal. After detailed morphological and molecular characterizations, the species was confirmed as a novel taxon. Therefore, the present study was aimed for authentication of this new macrofungus with proper description and comparison with other related taxa. The work was further elaborated in order to contribute the mushroom towards its effective utilization. Accordingly, a crude polysaccharide rich extract was isolated from the novel species for analysing chemical composition and medicinal effects in order to estimate its immunomodulatory, antibacterial and antioxidant efficacy.

Morphological identification
Fresh basidiocarps of the specimen were collected during subsequent field trips (2011-2013) from West Bengal, India. The morphological and ecological features were noted in the field. The places from which the samples were collected were neither a conserved area nor any privately owned land. Thus, no specific permissions were required. Also, none of the field studies involved any endangered or protected species.
The collected basidiocarps were then taken to the laboratory and dried in a desiccator (40-50˚C) for further preservation. The holotype specimen has been deposited in the Botanical Survey of India (CAL) and additional examined specimens in the Calcutta University Herbarium (CUH). Colour codes and terms (mostly) follow Methuen Handbook of Colour [18]. Microscopic features were performed following Dutta et al. [14] and Buyck [19]. Specimen was then examined with a Carl Zeiss AX10 Imager A1 phase contrast microscope. Q value denotes length/width ratio of the spores excluding ornamentation. Statistics for measurements of spores are based on 25 measurements from each of the four basidiocarps and given as a mean value (underlined); values in parentheses indicate minimum or maximum measured values. Scanning Electron Microscope (SEM) illustrations were obtained following Dutta et al. [14].

Extraction of crude polysaccharide (Rusalan)
The extraction procedure was followed according to Palacios et al. [25] with modifications. Dried and powdered fruit bodies were steeped with ethanol (approximately 10 volume) for 2 days to eliminate the alcohol soluble components and filtered residue was re-extracted with ethanol. The air-dried filtrate was suspended and refluxed with distilled water at boiling condition for 7 h. Subsequently the extract was filtered through nylon cloth and lyophilised to concentrate. Four volumes of absolute ethanol were added for harvesting polysaccharide and incubated overnight at 4˚C. After centrifugation, pellets were dissolved in water repeatedly to obtain crude polysaccharide fraction that is highly soluble in water. Alcohol precipitated pellets were recovered by centrifugation followed by washing with ethanol and acetone. The crude polysaccharide fraction was designated as Rusalan and kept in amber containers under dry condition until analysed.

Structural characterization of Rusalan
Determination of total carbohydrate, glucan and protein. Total sugar content was measured by phenol sulphuric acid method using glucose as standard [26] and results were expressed as gm of glucose equivalents/100 gm of the dry polysaccharide. Total glucan, α-Glucan and β-glucan of the crude polysaccharide were calculated using mushroom and yeast βglucan assay kit as per the protocol. All values of glucan contained were expressed as gm of glucose equivalents/100 gm of crude dry polysaccharide. Protein concentration was determined using the method described by Bradford [27]. Total protein content of the fraction was expressed as gm of BSA equivalents/100 gm of dry polysaccharide.
Depiction of helical conformation by congo red reaction. The helical structure of Rusalan was analysed by characterizing Congo red-polysaccharide reaction according to the method described by He et al. [28]. Different sets of solutions were prepared containing polysaccharide (0.5 mg/ml) in 0-0.5 M NaOH (increasing stepwise by 0.05 M increments) and 91 μM of Congo red. These were analysed in the range of 400-700 nm and the maximum absorption wavelengths were recorded as a function of NaOH concentration.
Fourier transform infrared spectroscopy (FT-IR). The FT-IR spectra were recorded on PerkinElmer Precisely Spectrum 100 Model (USA). The crude polysaccharides were powdered with potassium bromide and pressed forming pellets for FT-IR detection in frequency range 400-4000 cm -1 .
Determination of monosaccharide composition. 10 mg Rusalan was hydrolysed with 2M TFA (5 ml) at 100˚C for 2 h in a screw cap vial. TFA was removed by evaporation at 55˚C under reduced pressure (Rotavapor R3, Butchi, Switzerland). The hydrolysate was dissolved by adding 1 ml of 50% ethanol followed by centrifugation (12,000 rpm, 5 min) to remove nonhydrolysed polysaccharide and subjected for monosaccharide composition analysis by HPTLC and GC-MS as described in our previous publication [29].

Evaluation of antioxidant activities of Rusalan in vitro
The assay of total antioxidant capacity was carried out as described by Prieto, Pineda and Aguilar [30] and activity was expressed as number of equivalents of ascorbic acid. Besides, superoxide radical scavenging activity of Rusalan (100-1000 μg/ml) was evaluated using riboflavin-light-NBT system based on the method of Martinez et al. [31]. The technique explained by Halliwell, Gutteridge and Arumo [32] was followed for determination of hydroxyl radical scavenging activity. These free radicals were produced by Fenton's reaction in existence of variable concentrations (500-1500 μg/ml) of Rusalan and absorbance was measured at 535 nm. In addition, the antioxidant activity was also evaluated using DPPH radical based on assay described by Ekanayake et al. [33]. DMSO solution of the radical (0.1 mM) was evaluated against various concentrations of crude polysaccharide (1000-2000 μg/ml) and absorbance was detected at 517 nm. Moreover, the ability of investigated extract to chelate ferrous ion was determined [34] using different concentrations of Rusalan (100-300 μg/ml). A modified method of reducing power as described by Oyaizu [35] was also considered. Variable doses of Rusalan (500-1500 μg/ml) were mixed in 1.5 ml reaction mixture and the absorbance was measured at 700 nm. Ascorbic acid was used for comparison in reducing power and total antioxidant capacity techniques. BHA was considered as standard in superoxide, DPPH and hydroxyl radical scavenging assays; while EDTA was adopted as a positive control in chelating ability of ferrous ion method. The sample concentrations providing 0.5 of absorbance or 50% of antioxidant activity were calculated from graphs of antioxidant activity percentages and regarded as EC 50 value.  [36]. The six investigating bacteria were cultured freshly and 1×10 5 CFU/ml concentrated dilutions were prepared separately. Reactions were performed in 96 well plate consisting of 200 μl of NB, 20 μl of inoculum and different dilutions of Rusalan. Following incubation for 24 h at 37˚C, 40 μl of INT dye (0.2 mg/ml) was added to each well and incubated for another 30 min. Concentration that inhibited 50% growth of bacteria in comparison with positive control was calculated as MIC value. Streptomycin was used as a standard drug.

Determination of immuno-modulatory activity of Rusalan
Cell culture. RAW 264.7 mouse macrophage cells were purchased from NCCS, Pune, India. The cell line was maintained in DMEM supplemented with 10% (v/v) FBS, 0.5% (v/v) PenStrep (5,000 IU/ml penicillin and 5 mg/ml streptomycin) and 0.25% (v/v) amphotericin B (250 µg/ml). Cell line was maintained at 37˚C in a humidified atmosphere with 5% CO 2 . In all experimental sets, LPS at 5 µg/ml concentration was used as a positive control. The studies were carried out on commercially available murine cell line; thus, no ethical clearance applies in this case.
Effect on Raw 264.7 cell proliferation. Increase in cell proliferation was determined using WST assay. Briefly, about 3000 cells were seeded in a 96-well plate overnight, Rusalan (50, 100, 200 and 400 µg/ml) was added to the 200 µl reaction mixture and incubated for different time intervals at 37˚C. 20 µl WST reagent was added after 24h as well as 48 h treatment and absorbance was measured using a spectrophotometer at a wavelength of 450 nm.
Effect on phagocytic activity of macrophage cells. 3000 cells/well were plated in 96 well plate overnight and then exposed to serial concentrations of Rusalan (50, 100, 20, 400 μg/ml) as the experimental group in 200 μl reaction mixture for 24 and 48 h. After treatment, reaction mixture was discarded and incubated with 100 μl DMEM media containing 0.07% (w/v) neutral red for 1 h followed by washing the cells with PBS twice. Then 100 μl cell lysate solution (ethanol and 0.01% acetic acid at the ratio of 1:1) was added into each well to lyse cells at room temperature for 2 h. The optical density at 575 nm was measured by a microplate reader [37].
Effect on NO production in macrophages. 3000 cells/well were seeded in 96 well microplate overnight and then exposed to serial concentration of Rusalan (50, 100, 200, 400 μg/ml) as the experimental group in 200 μl reaction mixture for 24 h. After incubation, 100 μl of culture supernatant was mixed with an equal volume of the Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 5% H 3 PO 4 ). The mixture was incubated at room temperature for 10 min and absorbance was measured at 545 nm. The nitrite concentration was determined by extrapolation based on sodium nitrite (1-200 μM in culture medium) curve [38].
Detection of morphological changes of macrophages. Raw cells (1 × 10 4 cells/well) were treated with various concentrations of Rusalan (50, 100, 200 and 400 µg/ml) and allowed for incubation of 24 h. The adherent cells were fixed with methanol for 10 min, washed with PBS and incubated with DAPI solution (1 µg/ml in PBS) for 15 min in dark. Excess dye was removed by washing cells three times with PBS. Cellular morphology was viewed and photographed using fluorescent microscope (FLoid Cell Imaging Station, Life Technologies, India).
Effect on production of intracellular reactive oxygen species (ROS). RAW 264.7 cells were seeded (6 × 10 5 cells/well) in 6 well plates and treated with increasing concentrations of Rusalan (50, 100, 200, 400 μg/ml) for 24 h. After treatment, cells were harvested and incubated with ROS indicator, DCFDA, to a final concentration of 5 μM in dark at 37˚C for 30 min [39]. The intracellular ROS levels were measured with flow cytometry (BD Bioscience, USA) and analysed by BD CellQuest Pro software.

Measurement of gene expression by reverse transcriptase-PCR analysis
Raw cells (6 × 10 5 cells/well) in 6 well plates were treated with various concentrations of Rusalan (50, 100 and 200 µg/ml) and allowed for incubation of 24 h. RNA was extracted from cultured cells using TRIzol reagent and reverse transcription was carried out with 1 µg of total RNA using RT-and GO Mastermix. The reaction was carried out at 42 ºC for 60 min followed by 70 ºC for 10 min. COX-2, IL 6, iNOS, TNF-α genes were co-amplified with β-actin gene as control. The sequences of PCR primers are listed in Table 1. The PCR cycle conditions were as follows: 95 ºC for 4 min, then 35 cycles of 94 ºC for 20 s, annealing temperature (Tm) for specific primers for 30 s and 72 ºC for 45 s with a final extension step of 7 min at 72 ºC. The PCR products were separated in agarose gel, visualized under UV transilluminator and then photographed. For each gel, ImageJ software (http://rsbweb.nih.gov/ij/index.html) was applied for quantitative estimation of band intensity.
Within the same subsect. Aurantinae, the present species appears close to Russula aurea Pers. but, easily distinguish from the later by its smaller size of the basidiospores (6.5-7.5 × 5.4-7.2 μm), very distinct (reticulate-winged up to 2.1 μm high) and unique (in this group) spore ornamentation. In addition, the occurrence of the new species concerned in tropical region and in association with Shorea robusta is very striking. In the Indian context, under subgen. Russula, among species with more or less same colouration of pileus and smaller size of the basidiospores: Russula rosea Pers. differs by the presence of light yellow to pale orange yellow coloured lamellae, pale yellow spore print, and fertile lamellae edge [43,44]; Russula minutula var. robusta Saini, Atri & Singer has forked lamellae with red coloured margin, and a spore ornamentation that is more typical of subsect. Roseinae (partial reticulum or warts connected by veins); Russula xerampelina differs by the presence of forked lamellae, white tinged with vinaceous red coloured stipe which turns brown at maturity, ochraceous yellow coloured spore print and basidiospores ornamentation composed of conic to spinoid isolated warts [44]. Among other species belonging to the subgen. Russula with white coloured spore print, R. emetica (Schaeff.) Pers., previously reported to be common in temperate mixed forest of India, differs by its acrid taste, having lamellae forked near the stipe, presence of lamellulae, slightly different size (7.4-9.8 × 6-7 μm; Q = 1.14-1.46) basidiospores, and presence of cylindrical subclavate to clavate pileocystidia [44]; Russula vaurasiana K. Das & J.R. Sharma, originally described from India [44], differs by having a pileus coloured deep yellowish pink to reddish orange with light orange yellow tinge towards centre, forked and interveined lamellae, Discovery of an edible, folk and medicinal mushroom presence of cylindrical to subclavate pileocystidia, and distinctly broader basidiospores (7.3-9.8 × 6.2-8 μm).

Phylogenetic analysis
Sequencing products of the newly described species ranged from 662 to 673 nucleotides. Based on the nrITS sequences obtained in this study and from GenBank, the phylogenetic relationships of R. alatoreticula was inferred from MCMC analyses. ITS sequences were aligned and the ends trimmed to create a dataset of 662 base pairs. Bayesian analyses reached a standard deviation of split frequencies of 0.004 after 10 6 generations. The initial 25% trees recovered were excluded as the burn-in and the remaining trees obtained were then used to estimate the posterior probabilities of the group (Fig 3). Accession numbers of newly generated nrITS sequences, as well as other GenBank sequences used for conducting the phylogenetic analysis, are presented in

Extraction parameters and physico-chemical characterization of Rusalan
The extraction procedure is considered to have a significant impact on yield and structural characteristics of polysaccharide as well as their biological activities. However, conventional heated reflux extraction is the most common method for isolation of polysaccharide [48]. Therefore, in the present study crude polysaccharide from R. alatoreticula was prepared using the common method in a modified way. The first step of methodology included an incubation of mushroom powder in ethanol for eliminating unwanted molecules such as lipids, pigments, monosaccharides, oligosaccharides and other small molecular weight components. Afterwards, filtered powder was subjected to hot water extraction, where boiling temperature was adopted with simultaneous shaking for about 6 h. Finally, the water soluble crude polysaccharide, Rusalan, was obtained as a whitish powder following ethanol precipitation with a yield of 3.07% of crude material. The observation was in agreement with previous reports where quantity of harvested polysaccharide from mushrooms have been reported to be in the range of 1.51-5.24% [29,49,50].
To depict the structural characteristics, Rusalan was evaluated using several physico-chemical parameters. As shown in Table 2, the crude polysaccharide was consisted mainly of carbohydrate with small amounts of protein. Besides, glucan was detected as the main constituents where glucose linked by β-glycosidic linkages were detected as the predominant form. According to previous literature, the chemical composition of Rusalan was found to be better than Macrocybe gigantea [29], Pleurotus florida [51], Ganoderma applanatum [52] and closely related to Termitomyces eurhizus [49]. Consequently, R. alatoreticula could be regarded as a good source of crude polysaccharide enriched with β-glucan.
Depiction of helical conformation by congo red reaction. Glucans with high degrees of β (1!6) or (1!3) glycosidic bonds exist generally in triple helical conformation and can form complexes with Congo red in dilute alkaline solution. The complex formation is usually characterized by means of shift in visible absorption maxima (λmax) of Congo red spectrum. However, a strong alkaline environment can break the helical structure into a disordered form, as a result λmax changes [53]. As shown in Fig 4A, λmax of the mixture of Congo red and Rusalan presented a shift of 9 nm (from 493 to 502 nm) in 0.1 M NaOH solution and further it decreased with increment of alkali concentration. Therefore, the result suggested that Rusalan might had triple helical conformation which lost at higher concentrations of 0.3 M NaOH. This ordered helical structure has been recently reported as the most stable form of polysaccharide. At present, all the major therapeutic mushroom polysaccharides available in market such as schizophyllan, lentinan and scleroglucan are known to possess that specific construction [54].
FT-IR of Rusalan. FT-IR spectroscopy was performed herein to identify structure of polymers present in Rusalan and the infrared spectra confirmed existence of functional groups corresponding to carbohydrate (Fig 4B). Presence of broad and strong absorption band at 3433 cm −1 was due to stretching vibrations of hydroxyl groups. Peak at 2921.28 cm −1 corresponded to symmetric stretching vibrations of CH group. The signal of amide band at 1627.45 cm −1 indicated presence of some residual protein in crude polysaccharide. Band at 1394.76 cm −1 resembled to OH group of phenolic compounds. Absorption peaks between 1250 and 950 cm -1 (i.e. 1239.63 cm -1 , 1029.32 cm -1 ) indicated that sugar rings were of pyranose forms. The characteristic peak at 871.79 cm −1 denoted existence of β-glycosidic bonds. Band at 720.8 cm −1 indicated presence of mannose in Rusalan. The band visible in between 500 and 749 cm -1 (i.e. 519.15 cm -1 ) signified presence of R-CH group [55][56][57][58]. Overall, the spectra showed high absorbance at wavenumbers characteristic of polysaccharide: 3400 cm −1 OH and 1200-800 cm −1 carbohydrate. Based on aforementioned results, it can be concluded that Rusalan was composed of sugar units in β-configuration and pyranose rings. Small amount of lipid and protein were also extracted together with polysaccharides.
Determination of monosaccharide composition. It is well known that the biological properties of carbohydrate are greatly influenced by monosaccharide ratio that forms the backbone of polysaccharides. For preliminary characterization of the monomer types present in Rusalan, HPTLC was performed followed by orcinol-H 2 SO 4 spray. The reagent reacts differently to each monosaccharide showing a specific colour and colour intensities vary depending on sugar concentration [59]. In the present study, the chromatogram of Rusalan demonstrated to be consisted of three monosaccharides i.e. galactose, glucose and mannose while, other six monomers were not detected (Fig 4C). The result was further confirmed by using GC coupled with MS and the approach required derivatization of Rusalan by hydrolysis, reduction followed by acetylation. The fingerprint profile was detected as similar to previous result as identified by HPTLC (Fig 4D). Discovery of an edible, folk and medicinal mushroom

Evaluation of antioxidant activities of Rusalan in vitro
The antioxidant activity of a component is dependent on its variable characteristics such as scavenging or quenching free radicals, chelation of transition metals, inhibition of lipid peroxidation and reduction power [59]. Thus, it would be difficult to predict the potentiality of antioxidants by performing a single assay. In this circumference, six different in vitro techniques were executed in the present study to evaluate antioxidant ability of Rusalan and the effects have been presented in Table 3. For estimation of radical scavenging activity three methods were performed such as superoxide, hydroxyl and DPPH radical scavenging assays. For the first process, riboflavin-light-NBT method was followed based on generation of blue coloured formazan by action of superoxide radicals. Presence of antioxidants prevented formazan formation and as a result colour intensity decreased. Results demonstrated that Rusalan exhibited strong quenching effects of the radicals which increased dose-dependently. As presented in Fig 5A, Rusalan was able to inhibit 19.05% and 35.23% radicals at concentrations of 100 and 500 μg/ml respectively which reached up to 63.98% at the dose of 1000 μg/ml. Based on the outcome, it can be postulated that Rusalan may possess stronger potentiality than crude and pure polysaccharides of Russula virescens [60]. Besides, another approach was adopted to determine radical scavenging properties of the extract. In this case, hydroxyl radicals were generated using Fenton's reaction that gradually produced MDA-TBA pink chromogen and presence of antioxidants scavenged radicals causing inhibition of the whole process. Results of the assay showed that Rusalan was able to scavenge the radicals at relatively low concentrations in a dose dependent manner (Fig 5B). The quenching activities of Rusalan were found to be 25.29%, 39.88%, and 52.5% at the concentrations of 500, 1000 and 1500 μg/ml respectively. On comparison to previous publications, the extract exhibited better radical scavenging potentiality than that of crude polysaccharides extracted from Auricularia auricula, Agaricus bisporus, Lentinus edodes and Flammulina velutipes [61]. DPPH . , a commercially available free radical, was evaluated against the formulation for better visualization of radical scavenging activity.
Results indicated that at 1000 μg/ml concentration, Rusalan exhibited strong quenching potentiality (31.09%) that gradually increased to 48.37% and 60.67% at the doses of 1500 and 2000 μg/ml respectively (Fig 5C). However, the potentiality was found to be less effective than crude polysaccharide extracted from M. gigantea [29]. Further, metal ion binding capacity of the fraction was estimated following method of chelating ability of ferrous ion. The assay was based on formation of ferrous ion-ferrozine complex which became interrupted in presence of antioxidative component. In this technique, the extract demonstrated marked Fe 2+ binding ability at relatively low concentrations that incremented with increase of concentrations ( Fig  5D). At the level of 100, 200 and 300 μg/ml, the formulation was able to chelate 35.1%, 51.09% and 61.55% of ferrous ions respectively. Recently in a previous study, antioxidant activity of partially purified crude polysaccharide extract from the mushroom, Schizophyllum commune, have been reported which appeared to be much poorer than Rusalan [62]. Further, ferricyanide/prussian blue and total antioxidant assay by phosphomolybdenum methods were carried out to evaluate the reducing power of Rusalan. The previous assay was based on electrondonating ability of antioxidants to reduce yellow coloured ferric to blue coloured ferrous form. According to results, the crude polysaccharide exhibited strong reduction power which incremented dose dependently (Fig 5E). At the concentration of 500 and 1000 μg/ml the reducing power have been detected as 0.26 and 0.47 that gradually incremented to 0.62 at the level of 1500 μg/ml. However, the sample exhibited antioxidant activities less effective than a purified β-glucan of Russula albonigra [63]. Finally, total antioxidant capacity assay was performed based on reduction of Mo(VI) to Mo(V) in presence of antioxidant. Results indicated that 1000 μg of Rusalan acted as the equivalent to 4.9 μg of ascorbic acid. Thus, based on the results of all aforementioned assays, it could be stated that Rusalan may possess comparatively stronger antioxidant ability.  50 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 (p < 0.05). Discovery of an edible, folk and medicinal mushroom

Estimation of antibacterial potentiality of Rusalan
Antibacterial activity of Rusalan was assessed against six different bacteria, some of which are involved in diseases related to respiratory and gastrointestinal tracts. Results indicated that crude polysaccharide was the most effective in inhibiting growth of S. aureus; while B. subtilis was also detected as sensitive to the fraction. In contrast, Rusalan did not show any activity against rest of the organisms especially Gram negative bacteria even when higher concentration was applied (Table 4). According to previous literature, Gram positive bacteria such as S. aureus has revealed high susceptibility to mushroom extracts. While, Gram negative bacteria are difficult to be inhibited due to their cell wall structure [64]. However, our findings indicated that Rusalan was more active in inhibition of bacterial growth than methanol fraction of Russula delica [65].

Determination of immuno-modulatory activity of Rusalan
Effect on Raw 264.7 cell proliferation. Activation of macrophages is an important step in defence mechanism system to protect the host from pathogenic infection. Thus, at first influence of Rusalan on macrophage cellular viability was examined using WST reagent which produces orange coloured formazan dye in direct proportional to number of living cells. As shown in Fig 6A, proliferation of cells was enhanced significantly at all concentrations of the crude polysaccharide applied. It has been estimated that after treatment of polysaccharides (50,100,200 and 400 μg/ml) for 24 h, proliferation of macrophage cells increased to 187.65%, 172.85%, 109.36% and 39.08% respectively in comparison to negative control. While after 48 h, the viability elevated to 478.6%, 562.68%, 363.5% and 183.32% at those above mentioned concentrations. Data also suggested that the extract might activate macrophages more efficiently than the positive control because LPS incremented proliferation up to 127.57% and 147.74% after 24 and 48 h of treatment respectively. However, the results allowed to conclude that Rusalan presented a non-toxic safety profile to murine macrophages over the entire concentration range evaluated.
Effect on phagocytic activity of macrophage cells. To eliminate foreign matters, macrophages have ability to phagocytize the pathogens that plays an important role in host defence. Thus, phagocytosis is considered as an indication of macrophage activity and it represents the final essential step of immunological defence system [4]. In the present assay, influence of Rusalan on phagocytic activity of macrophages was determined by measuring the amount of neutral red internalized in cells. Results clearly indicated that crude polysaccharide promoted phagocytosis of macrophages in a dose and time dependent manner (Fig 6B). After 24 h of treatment, the activity was increased by 107.41%, 134.17%, 128.72% and 115.25% due to treatment of 50, 100, 200 and 400 μg/ml of Rusalan while after 48 h, the activity was 126.93%, 156.72%, 132.86% and 102.02% in comparison with negative control. On the other hand, LPS induced 109.76% and 116.67% after treatment of 24 and 48 h respectively. Thus, it could be predicted that the extract could induce innate immune response by regulating phagocytosis of macrophage. Discovery of an edible, folk and medicinal mushroom Effect on NO production in macrophages. Production of NO is another killing mechanism of an activated macrophage that works in combination with hydrogen peroxide or superoxides. The reaction leads to formation of peroxynitrite radicals that kill phagocytosed microbes inside macrophages. Thus, generation of NO can be used as an indication for activation of macrophages [66]. In the present assay, nitrite concentrations in the supernatant of stimulated macrophages were determined by Griess reagent as a reflection of NO production. As shown in Fig 6C, Rusalan had a strong potential on stimulating NO production in macrophages in a concentration dependent trend. In case of control and LPS stimulated sets 13.33 and 24.47 μM NO was quantified; while the crude polysaccharide induced 26.92, 31.08, 28.2 and 19.77 μM NO production in RAW 264.7 cells at the level of 50, 100, 200 and 400 μg/ml respectively.
Detection of morphological changes of macrophages. When macrophages encounter a stimulus, they are known to undergo certain morphodynamics such as increase in cell size, production of thin sheets of cell edges called filopodia or lamellopodia [67,68]. Recently, it has been evidenced that NF-κB plays an important role in regulation of the formation of lamellipodia and induction of morphological changes [67]. Consequently, to investigate effects of Rusalan on macrophage cell morphology, Raw 264.7 cells were analysed in presence of crude polysaccharide at different concentrations for 24 h. LPS was used a standard as it induces structural changes that are definite sign of macrophage activation. Microscopic analysis showed that after LPS stimulation, a large number of thin sheet extensions were produced Discovery of an edible, folk and medicinal mushroom from surface of macrophage along with increased spreading of cells. Rusalan treatment also induced similar kind of morphological changes in all examined concentrations, in contrast to unstimulated cells that did not show any morphological alterations (Fig 7).
Effect on production of intracellular ROS. In addition to phagocytosis, generation of ROS inside phagocytes also plays a key role in the process of bacterial killing as it signals for cytokine production via activating NF-κB pathway. In fact, it has been proved that persons with lacking ability of ROS production suffer from chronic granulomatous disease as they are inefficient to clear pathogens properly. Thus, enhanced ROS formation may be essential for host survival against microbial and fungal pathogens [69]. Result of the present study showed that Rusalan was capable of inducing ROS production inside macrophages as measured by fluorescent oxidative product of DCFDH. In case of 50, 100, 200 and 400 μg/ml treated cells, the fluorescence intensity increased to 154.82%, 192.9%, 77.52% and 75.26% respectively in comparison with control. While the standard at the concentration of 5 μg/ml could enhance the oxidative bursts only by 135.722% in RAW 264.7 cells (Fig 8).
Measurement of gene expression by Reverse Transcriptase-PCR analysis. Cytokines and chemokines are considered as potent signalling molecules produced by macrophages which connect both innate and adaptive immunity. However, secretion of them requires activation of intracellular signalling cascades such as MAPK and NF-κB pathways that regulate expression of iNOS, TNF-α and IL-6 genes [70]. Among them, TNF-α has been noticed as the first cytokine to be secreted in response to pathogens that in turn activates the macrophages itself. Gradually, stimulated macrophages produce other effectors such as IL-6 which is Discovery of an edible, folk and medicinal mushroom important for differentiation of B cells into plasma cells and activation of cytotoxic T cells [4]. In addition, macrophages express iNOS that signals for production of NO encouraging a highly microbicidal environment [69]. The overproduction of NO further stimulates synthesis of COX-2 that functions in conversion of arachidonic acid to prostaglandins [71,72].
In order to investigate the mechanism of action of Rusalan towards stimulation of macrophages, the crude polysaccharide was incubated with Raw 264.7 cells for 24 h. In this experiment, concentration of 50, 100 and 200 μg/ml of crude polysaccharide were selected as the highest dose as (400 μg/ml) could not activate macrophages effectively according to previous observations. As shown in Fig 9, the levels of all four genes were visibly increased in treatment of Rusalan in contrast to negative control and LPS. Analysis by ImageJ software suggested that Rusalan at 100 μg/ml concentration increased the mRNA levels of iNOS, TNF-α, COX-2 and IL-6 by 478.3%, 278.72%, 524,12% and 146.89% respectively over control. Conversely, LPS upregulated these genes only by 140.48%, 201.15%, 491.38% and 137.85% indicating strong promoting effect of Rusalan. So, the results demonstrated that these genes are definitely involved in Rusalan mediated immune-stimulatory activities in murine macrophage cells.

Conclusions
The present investigation provided information to confirm novelty of the studied mushroom, R. alatoreticula, based on morphological characteristics and molecular phylogenetic study Discovery of an edible, folk and medicinal mushroom using ITS region. In addition, the work also demonstrated that crude polysaccharide extracted from the macrofungus, Rusalan, as a potent therapeutic agent with several therapeutic effects. Evaluation of antioxidant activity indicated that the fraction exhibited significant potentiality in hydroxyl, superoxide and DPPH radicals scavenging activity, chelating ability of metal ions along with donating electron. Besides, the extract also revealed to be capable of inhibiting growth of certain pathogenic bacteria. Nevertheless, the crude polysaccharide showed high immune-modulatory activity without cytotoxicity in terms of stimulation of phagocytic activity, production of NO by activating iNOS gene expression, generation of intracellular ROS and upregulation of transcription level of cytokines in macrophage Raw 264.7 cells. Interestingly, the fraction was determined to be mainly consisted of carbohydrate in triple helical conformation where glucose (especially β-glucan), galactose and mannose were the main constituents. Based on the outcome of the present study, Rusalan could be regarded as a novel ingredient for pharmaceutical use against free radical, antibiotic resistant pathogens and hypo-immunity. Further studies on isolation of active component and its relation with bioactivity are presently in progress.

Acknowledgments
Authors would like to thank DST (FIST) for providing instrumental facilities.