Introduction

Mushroom is a macro fungus that has a characteristic fruiting body. Total 15,000 mushroom species were recorded worldwide, but only 2000 species were reported safe and edible for humans. However, limited (650) mushroom species were considered for potent medicinal uses (Petrovi et al., 2015). The Medicinal properties of macro fungi (belonging to Basidiomycota) have been known from the ancient era and commonly used as food and form of folk medicine. Many Mushroom varieties have striking for a portion of efficient food and useful for development of drugs and nutraceuticals materials. According to Chandrawanshi et al. (2017) mostly wild-growing mushrooms contain large amounts of secondary metabolites, minerals, carbohydrates, proteins, vitamins, and fibers that serving as the role of anti-diabetic, antitumor, and antioxidant properties. Mushrooms are rich in various biochemical compounds such as ascorbic acid, beta-glucans, tocopherols, carboxylic acids, lectins, and terpenoids (Babu et al., 2013). However, many wild Mushrooms are not consuming by the human population due to non-characterization of edible nature and/ or previously defined as poisonous nature, due to presence of a variety of the toxic group, which mostly harmful for human and animal. Mushroom toxins have classified into seven categories, such as orellanus (Cortinarius species), amatoxins (cyclopeptides), muscarine, ibotenic acid, gyromitrin (monomethylhydrazine), coprime and psilocybin, etc. Previous studies demonstrated that Japan, China and other countries from the Far East utilized the immuno-stimulatory, anti-inflammatory and anticancer activity of many fungal species. Nowadays, in many developed countries (United States of America, Israel), scientific achievements from these regions are used to implement complementary therapeutic methods (Wasser, 2002). The present study took the wild mushroom, S. commune, commonly known as split gill mushroom, belongs to family basidiomycete, demonstrating life cycle of only 10 days. It is found on dead wood having saprobic nature (Kuo, 2003). Patel and Goyal (2012) isolated Schizophyllan from                   S. commune ATCC 38548, which is a non-ionic, water-soluble homopolysaccharide, β-d-(1-6)-glucopyranosyl, lentynian, and polysaccharide-protein complexes. Furthermore, these biochemical molecules have the ability to prevent of anti-diabetic, promote normal phenomena of the human body, as well as they protect or inhibiting from carcinogenesis and tumors metastasis, etc. (Hilszczanska, 2012).

Two types of diabetes (type I and type II) were recognised in India. Type I diabetes occurred due to insulin deficiency. It may affect the pancreatic β–cells and generally occur in young ages, with acute phase. It may be severe to any age condition, with slight progression (Kuzuya et al., 2002). However, the majority of patients formally stated non-insulin-dependent diabetes mellitus (NIDDM), classified under Type II diabetes category. In this, the group of pancreatic β-cells became non functional and to maintain the normal physiological mechanism insulation have to do through injection. The previous studies depicted that free radicals might play a vital role in diabetes mellitus (Zimmet et al., 2001). Thus, it can be hypothesized that extracts of S. commune, may become an important and alternative natural bio resources for antioxidant and anti-diabetic medication product preparation. The aim of this study was the selection of efficient solvent for extraction of bioactive compounds of S. commune, and evaluation of anti-diabetic property of extracts.

Materials and method

Sample collection

S. commune dried mushroom was collected from the campus of Pt. Ravishankar Shukla University Raipur, India, on October 2015. The collected sample was preserved in an airtight polythene bag for future analysis. The sample was denoted as S. commune (SC) as a short name. The morphological identification was carried out according to the critical observations of sample and assessment with relevant literature (Kuo, 2003).

Standard and Reagent

The current study evaluated the antidiabetic property of various solvent extracts samples. For the analysis, various chemical and reagents such as Methanol, Ethanol, α-amylase and DNS (3, 5-dinitrosalicylic acid) were used. The chemical and reagents used in the present study were procured from Sigma-Aldrich, Bengaluru, India and HiMedia Pvt. Ltd. Mumbai, India.

Extraction Method

Water extraction

Water extraction method was carried out according to the modified method of Abdullah et al. (2011) and Chandrawanshi et al. (2017). Weight amount (25 g) of mushroom sample was cut into small pieces and indulged into 250 ml water. Then, this sample was incubated in water bath at 100°C for 2 hrs. After incubation, sample was filtered by Whatman filter paper no. 1 to obtain the water extract of bioactive compounds.

Methanol extraction

Methanol extraction of S. commune was done by the method of Elmastas et al. (2007) and          Chandrawanshi     et al. (2018). Dried sample of                S. commune was crushed by motor pestle to obtain the fine powder. Approximately 25 g fine powder of sample was mixed with 250 ml of methanol and incubated in shaker incubator for 24 hrs at 25°C in 150 rpm. After incubation, sample was filtered through Whatman filter paper no. 1 and collected methanolic extract sample was further incubated at 40°C to obtain the bioactive components.

Ethanol extraction

The ethanolic extraction was done by the modified protocol of Hu et al. (2009) and Chandrawanshi et al. (2017). In this procedure, 25 g of mushrooms fine powder was mix together with 250 ml of ethanol (99.9% purity) in the flask and incubated in shaking incubator at 150 rpm for 24 hrs. Then incubated sample centrifuged at 3000 rpm for 15 min. The supernatant was collected as an ethanolic extract of S. commune, which was further incubated at 40°C in the oven to get the dried bioactive compounds.

Antidiabetic assay (α-amylase inhibitory activity)

This analysis process was performed according to Rubilar et al. (2011). Initially, 50 µl mushroom extracts were mixed with 50 µl of α-amylase and allowed for the reaction at 37°C for 5 min. when finished the incubation period add 100 µl of starch solution and yet again incubated at room temperature for 3 min. and mix 100 µl of DNS reagent and incubated further in the water bath at 100⁰C for at 8 min., afterward it was cooled in room temperature and absorbance was recorded at 450 nm.

Antidiabetic potential assay (%) = (absorbance of Control - absorbance of extracts)/ (absorbance of Control) ×100

The antidiabetic property of mushroom extracts represented as the inhibition concentration at 50% (IC₅₀). Consequently, it is essential to obtain 50% radical scavenging ability at that particular concentration.

Statistical analysis

Present experiment was performed in three replicate (n=3). Collected data were analyzed through one way-ANOVA using SPSS software version 16.0. Graph was constructed by Origin Pro Lab and Microsoft Excel 2013.

Results and Discussion

Yield of Mushroom extracts

In the present study, the yield of bioactive components varies with varying amounts and type of extraction medium (Table 1). The yield was recorded from 14.00 to 19.60% using various extraction medium. The quantity determination method was employed to calculate the yield of mushroom extracts. The results revealed that the water extraction medium showed the highest yield (19.06g) and followed the other solvents like methanol and ethanol gave (14.00 g) in 100 g dried weight (dw) of mushroom.

The IC₅₀ values were determined for various extracts, analyzed for anti-diabetic potency. The significant IC₅₀ values gave by methanol (50.98); followed by ethanol (54.61) and least value recorded for water extract (54.92). The IC₅₀ depict in radar evaluated in the 2-dimensional graph (figure 2).

 Table 1: Yield of Mushroom Extracts

In vitro Anti-diabetic Assay

Diabetes mellitus embrace a heterogeneous group of hyperglycemic disorders, which is characterized by disturbances of lipid and carbohydrate metabolism, with loss of glucose homeostasis. Patients with diabetic condition faced a high of atherosclerosis and an array of other clinical disorder like coronary, renal, cerebrovascular and peripheral vascular diseases, etc.    In the present study, in vitro anti-diabetic property of various extracted of S. commune was evaluated, which depict of phytochemical and pharma quality validation in very short periods of times.

Hydrolysis of α-1,4-glycosidic internal links in starch by α-amylase is major pathway to produce simple sugars such as glucose, maltose, and dextrins in humans. The inhibitors of this enzyme can delay carbohydrate metabolism and reduce the rate of glucose absorption. Consequently, these α-amylase inhibitors can decrease postprandial plasma glucose levels and improve glucose tolerance in diabetes patients. Several side effects related to the intake of synthetic α- amylase inhibitors have been reported including abdominal distension, flatulence, bloating, and possible diarrhea.  

Figure 1: Antidiabetic effect of various extracts of           S. commune

Results revealed that increasing the concentration of extracts also increases the anti-diabetic activities (Fig.1). Water extract exhibited (21.49±0.0) anti-diabetic rate. However, methanolic extract showed 22.98±0.06, while ethanolic extracts exhibited 23.74±0.09 anti-diabetic rate, which was highest among others. Figure 1 detailed about the linear graph that showed about the relationship between concentrations with scavenging (%) rate. 

The IC₅₀ values were determined for various extracts, analyzed for anti-diabetic potency. The significant IC₅₀ values gave by methanol (50.98); followed by ethanol (54.61) and least value recorded for water extract (54.92). The IC₅₀ depict in radar evaluated 2-dimensional graph (figure 2).The IC₅₀ facts described from axis values distance range in contiguous surface and the developed triangle defined proximity of potency of an extract with the fitness of selection with suitable solvents. Rushia et al. (2013) worked on anti-diabetic assay in Rats using methanolic extract of Pleurotus citrinopileatus mycelium. They found decrease in blood glucose levels at 500 mg/kg and 1000 mg/kg body weight in STZ induced diabetic Rats after 45 days of treatment. Similarly, Johnny and Okon (2013) observed that the maximum reduction result at 5 hrs phase with the upper limit dose of the oyster mushroom extract (1341.63mg/kg),  giving at 83.21+5.2mg/dl. Babu et al. (2013) reported that some secondary metabolites (ascorbic acid, beta-glucans, tocopherols, and lectins) played a very important role in pharmaceutical validating.

Figure 2: Anti-diabetic potency of various extracts of      S. commune at IC₅₀

Conclusion

The S. commune naturally occurs in a dead tree trunk. It is rich sources for numerous biological metabolites, which is essential for sources for human nutrition’s. In the present study, extraction efficiency of various solvents was compared and their anti-diabetic capacity was analyzed. The data interpretation was done by an inhibitory graph with radar axis value presentation. The study reveals that S. commune, wild mushroom having highly nutritional and important medicinal properties. Therefore, it may be used as a therapeutic agent, especially for medication preparation targeted for diabetic other related human illness. As well as, it will be useful for natural bioproduct, in the place of the synthetic medical product.

Acknowledgment

The authors are grateful to the Chhattisgarh Council of Science & Technology, Raipur for granted a Mini-Research Project (Project Sanction No. 722/CCOST/MRP/2015, dated on 23/07/2015). Also have grateful to the Head of the department, School of Studies in Biotechnology for providing the essential facilities for conduct the experiment and analysis.

Conflict of interest

Authors had no conflict of interest.

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