An insight into antidiabetic properties of six medicinal and edible mushrooms: Inhibition of α-amylase and α-glucosidase linked to type-2 diabetes

a Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia b Plant Research International, Wageningen University and Research, P.O. Box 16, 6700 AA Wageningen, Netherlands c Mountain Research Centre (CIMO), School of Agriculture, Polytechnic Institute of Bragança, Campus de Santa Apolónia, apartado 1172, 5301-855 Bragança, Portugal


Introduction
Mushrooms are important dietary components in some cultures, consumed in daily diet as supplementary food since olden times . Mushroom extracts are increasingly consumed because of their health beneficial effects, including the enhancement of immune function and antitumor activity (Popović et al., 2013). It is confirmed that mushroom extracts contain diverse compounds such as alkaloids, fibers, lectins, protein, polyphenols and polysaccharides, which are contributing to their biological activities (Popović et al., 2013). Mushrooms are thought to exert many pharmacological functions such as antitumor, immunomodulatory, antigenotoxic, antioxidant, anti-inflammatory, hypocholesterolemic, antihypertensive, antiplatelet-aggregating, antihyperglycemic, antimicrobial and other activities (Lindequist, 2013;Paterson and Lima, 2014). Mushrooms as functional food and dietary supplements can help in the intervention of sub-health states and may prevent the full-blown consequences of life-threatening diseases. An equilibrated diet linked with the mushroom consumption has an advantage of the nutritional/medicinal features of mushrooms  and their pharmacological applications. The term "mushroom" is used here for a medicinal/edible fruiting body of higher fungi.
Diabetes mellitus is a chronic disorder of metabolism followed by abnormal rise in plasma glucose levels, as a consequence of unequilibrated insulin production and/or insensitivity to the effect of this hormone in signal transduction of cellular receptors. These metabolic changes are accompanied by modifications in carbohydrate, lipid and protein metabolism. Most of the diabetes type 2 complications in patients are due to hyperglycemia as their main cause (Oritz et al., 2007;Shobana et al., 2009). One of the effective strategies for diabetes type-2 management is the inhibition of complex polysaccharide hydrolysis by pancreatic α-amylase and absorption limitation of glucose by inhibiting intestinal α-glucosidase enzyme. Acarbose, miglitol, voglibose are commercial drugs used for α-glucosidase inhibition in the control of the diabetic disease (Saito et al., 1998). Chronic α-amylase and α-glucosidase inhibitions may be useful in the management of type-2 diabetes and obesity (Melo et al., 1999;Koike, 2005). The drugs currently used as reversible inhibitors of α-amylase and α-glucosidase for the treatment of diabetic patients exhibit side effects such as abdominal distension, meteorism, bloating, flatulence and possibly diarrhea (Fujisawa et al., 2005).
To continue our investigations Reis et al., 2014;Stojković et al., 2014;Bizarro et al., 2015;Glamočlija et al.,  In vitro assays on α-amylase and α-glucosidase enzyme inhibition were performed with methanolic extracts of the selected mushrooms.

Mushroom samples
Six edible and medicinal mushroom species were used in this study: (collected -wild growing in Serbia) and P. linteus Berk. & M.A. Curtis (commercial sample). The origin of samples, identification and preparation of dried powder were described previously Reis et al., 2014;Stojković et al., 2014;Bizarro et al., 2015;Glamočlija et al., 2015;Vieira et al., 2016).

Extraction procedures
Mushroom samples (~5 g) were extracted by stirring with 150 mL of methanol (− 20°C at 150 rpm) for 24 h and subsequently filtered through Whatman No. 4 paper. The residue was then extracted with an additional portion of methanol. The combined methanolic extracts were evaporated under reduced pressure (rotary evaporator Büchi R-210; Flawil, Switzerland) to dryness.

α-Amylase and α-glucosidase inhibition assays
Mushroom extracts at different concentrations dissolved to 500 μL and 500 μL of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) containing Porcine pancreatic α-amylase (Sigma Aldrich, Germany) (0.5 mg/mL) were incubated at 25°C for 10 min. Then, 500 μL of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) was added to each tube. The reaction mixtures were incubated at 25°C for 10 min and stopped with 1.0 mL of dinitrosalicylic acid color reagent. Thereafter, the mixture was incubated in a boiling water bath for 5 min and cooled to room temperature. The reaction mixture was then diluted by adding 10 mL of distilled water, and absorbance measured at 540 nm using the spectrophotometer (Agilent 8453, Agilent Technologies, Waldbronn, Germany).
Mushroom extracts at different concentrations dissolved to 50 μL and 100 μL of α-glucosidase (Sigma Aldrich, Germany) solution (1.0 U/mL) in 0.1 M phosphate buffer (pH 6.9) was incubated at 25°C for 10 min. Then, 50 μL of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in 0.1 M phosphate buffer (pH 6.9) was added. The mixtures were incubated at 25°C for 5 min. Then, 2 mL of Na 2 CO 3 was added to terminate the reaction before reading the absorbance at 405 nm in the spectrophotometer (Agilent 8453, Agilent Technologies, Waldbronn, Germany).

Results and discussion
The results of α-amylase and α-glucosidase inhibition linked to diabetes type-2 therapy are summarized in Table 1. C. comatus methanolic extract had the most prominent inhibitory activity on α-amylase with IC 50 714.45 μg/mL, while C. militaris and M. conica did not have inhibitory potential on this enzyme. All of the investigated mushroom species inhibited α-glucosidase with I. obliquus as the most potent inhibitor among the studied species (IC 50 220.31 μg/mL). Table 1 also summarizes calculations of the daily intake of mushroom methanolic extracts and dry mushroom powders necessary to achieve the same effect as commercial therapeutic drug acarbose on the daily basis. Acarbose is given to diabetic patients in an initial dose of 3 × 25 mg per day. Calculations were done on the basis of αglucosidase inhibition by acarbose. The lowest daily intake of mushroom powder was predicted for I. obliquus with the dose of 3 × 1.148 g/day, while the highest was predicted for P. linteus 3 × 2.215 g/day (Table 1). Kim et al. (2005) demonstrated that β-glucans and their enzymatically hydrolyzed oligosaccharides from Agaricus brasiliensis (syn. A. blazei) show the activities of antihypercholesterolemic, antihypertriglyceridemic, antiarteriosclerotic and antihyperglycemic, indicating antidiabetic activity in diabetic rats. The data suggested that both β-glucans and oligosaccharides might enhance insulin secretion from pancreatic islets as well as proliferation of islets in diabetic or normal rats (Kim et al., 2005). The results of our study showed that A. blazei could also have an inhibitory influence on key enzymes involved in the degradation of complex sugars (Table 1), thus delaying the process of glucose uptake. Our previous study (Stojković et al., 2014) on chemical composition of A. blazei revealed that this species possessed oxalic, malic and fumaric organic acids. The total content of organic acids was 1.87 g per 100 g dw. The phenolic acids found in the studied mushroom were p-coumaric acid (0.28 mg per 100 g dw) and cinnamic acid (0.12 mg per 100 g dw), (Stojković et al., 2014). Different compounds present in methanolic extracts and their different combinations are probably linked to A. blazei enzyme inhibitory potential.
Recent studies described in vivo antidiabetic activity of different extracts and fractions of C. comatus (Yamaç et al., 2009;Ding et al., 2010;Zhou et al., 2015). According to our knowledge, no previous records were published on α-amylase and α-glucosidase enzyme inhibition with C. comatus methanolic extract. Methanolic extract of C. comatus studied here had inhibitory potential on both enzymes (Table 1). Regarding C. comatus, our previous study  revealed organic acid profile, with five different compounds namely oxalic, Table 1 Inhibitory potential of mushroom species on α-amylase and α-glucosidase linked to type-2 diabetes treatment.

Mushroom species Yield of extracts (%) α-Amylase inhibition;
IC 50 (μg/mL) quinic, malic, citric and fumaric acids. Concerning phenolic acids, the studied sample revealed the presence of dominant p-hydroxybenzoic acid (0.09 mg/100 g) and presence of p-coumaric acid, as also the related compound cinnamic acid . Phenolic compounds and organic acids are probably related to the anti-diabetic activity investigated herein. Water and polysaccharidic extracts of C. militaris previously showed antidiabetic properties in diabetes induced rats (Liu et al., 2016). Our study showed that methanolic extract of C. militaris did not inhibit the activity of α-amylase (Table 1), while it had inhibitory potential on α-glucosidase. A recent study showed that water extract of C. militaris had inhibitory activities on α-glucosidase with values of 45.77% with 0.1 mg/mL, 54.76% with 0.25 mg/mL, 58.74% with 0.5 mg/mL, and 60.5% with 1.0 mg/mL (Kim et al., 2017). The organic acid we found previously in higher amounts in C. militaris was citric acid 7.97 g/100 g (Bizarro et al., 2015). Regarding phenolic acids, p-hydroxybenzoic acid was the only compound found in the studied species (0.02 mg/100 g dw). However, cinnamic acid was also found (0.11 mg/100 g dw), (Bizarro et al., 2015). The link between chemical composition of methanolic extract of C. militaris and the enzyme inhibitory activity certainly exists.
Water extracts and polysaccharidic fractions of I. obliquus are recorded to possess in vivo antidiabetic properties (Qin et al., 2015). Extract of I. obliquus tested in our study possessed inhibitory potential of both enzymes investigated (Table 1). A previous study by Ying et al. (2014) revealed terpenoids from this species as inhibitors of glucosidase. In our previous study (Glamočlija et al., 2015) oxalic acid was the only organic acid detected in the extract of I. obliquus (72 mg/g extract). The phenolic acids found were gallic, protocatechuic and p-hydroxybenzoic acids, as also the related compound cinnamic acid (Glamočlija et al., 2015). Relation between organic, phenolic acids, their combination in the extract and antidiabetic activity explored herein certainly exists.
According to our knowledge, no previous studies were published on M. conica antidiabetic activity. Our results indicate that methanolic extract of this species did not inhibit α-amylase, while α-glucosidase was inhibited (Table 1). Regarding M. conica organic acid profile, previously (Vieira et al., 2016) we have quantified three different organic acids: oxalic, quinic, and fumaric acids. The phenolic compounds gallic and p-hydroxybenzoic acids were only present in the wild growing sample, as well as cinnamic acid.
A polysaccharide isolated from P. linteus has previously shown hypoglycemic properties in alloxan-induced diabetic mice (Zhao et al., 2014). P. linteus methanolic extract exhibited enzyme inhibitory potential in this study (Table 1). The hydrophilic compounds recorded in P. linteus earlier  were only organic acidsoxalic acid was the only detected molecule (0.295 g/100 g dw).
The investigated mushroom species possessed antidiabetic properties regarding the inhibition of the enzymes investigated. C. militaris and M. conica failed to inhibit the activity of α-amylase, while inhibiting the activity of α-glucosidase. I. obliquus methanolic extract was the most potent inhibitor of α-glucosidase, while C. comatus has the highest potential in inhibition of α-amylase. The lowest antidiabetically effective daily intake of mushroom powder was predicted for I. obliquus with the dose of 3 × 1.148 g/day, while the highest was predicted for P. linteus 3 × 2.215 g/day. The activity of the extracts investigated herein could be linked to their chemical compositions previously published by our research groups. Organic acids, phenolic acids, fatty acids and their specific combination in each species contributed to unique in vitro antidiabetic activity of investigated mushrooms.
Although majority of previous studies showed in vivo antidiabetic potential of water and polysaccharidic mushroom extracts by different experimental approaches, our study is the first highlighting in vitro antidiabetic potential by inhibition of α-amylase and α-glucosidase with methanolic extracts; which makes the investigated species more promising for the diabetes type-2 treatment by another additional and different mechanism of action.