Hypoglycemic effect of crude exopolysaccharides produced by Cerrena unicolor, Coprinus comatus, and Lenzites betulina isolates in streptozotocin- induced diabetic rats

Abstract

The objective of this study was to investigate the hypoglycemic activity of crude exopolysaccharides (EPS) produced by three mushroom isolates in streptozotocin-induced diabetic rats. The three experimental groups were fed EPS of Cerrena unicolor (Bull.) Murrill (Polyporaceae), Coprinus comatus (O.F. Müll.) Pers. (Agaricaceae), and Lenzites betulina (L.) Fr. (Polyporaceae) for 7 days. The serum glucose levels significantly decreased after oral administration of EPS by 61.23% with Cerrena unicolor, 42.78% with Coprinus comatus, and 42.08% with Lenzites betulina. According to histological observations based on staining in pancreatic tissues, Langerhans islet areas and cell numbers of diabetic animals increased in response to EPS treatment. In conclusion, our findings clearly suggest that exopolysaccharides produced by three mushroom isolates decreased blood glucose levels in STZ-induced diabetic rats. Therefore, the studied mushroom exopolysaccharides might be developed as potential oral hypoglycemic agents in the control of diabetes mellitus. This is the first attempted in vivo study using exopolysaccharides of local mushroom isolates for medicinal purpose in Turkey.

Keywords::

• Diabetes mellitus
• mushroom
• Cerrena unicolor
• Coprinus comatus
• Lenzites betulina

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Erratum

Introduction

Diabetes mellitus (DM) is one of the most important chronic diseases affecting ~250 million people worldwide. It is a group of metabolic disorders with different underlying etiologies, each characterized by hyperglycemia due to under utilization and/or overproduction of glucose. Hyperglycemia is a key clinical manifestation of diabetes mellitus (Li, 2007).

Different types of oral hypoglycemic agents are available for the treatment of DM, such as insulin. Insulin cannot be used orally and continuous use of the synthetic hypoglycemic drug causes side effects and toxicity. Therefore, because of their effectiveness, limited side effects, and relatively low cost, natural drugs are widely prescribed, even when their biologically active compounds are unknown (Hwang et al., 2005). Recently, dietary supplements, nutraceuticals, and functional foods have become a popular approach to prevent the occurrence of DM and to attenuate the complications of hyperglycemia in diabetic patients. Moreover, these natural agents reduce the costs of treating the disease. Growing evidence suggests that mushrooms have various biological activities (Lindequist et al., 2005).

Because of their high fiber and protein, and low fat content, mushrooms are known as ideal dietetic foods for antidiabetic therapy (Kim et al., 2001b). The hypoglycemic activities of ganoderan A and B (Hikino et al., 1985), coriolan (Ikuzawa et al., 1985), and an acidic glucuronoxylomannan (Kiho et al., 2000) have been reported. Although there are some reports on the hypoglycemic effects of mushroom exopolysaccharides (EPS) (Kim et al., 2001b, 2001c; Yang et al., 2002; Hwang et al., 2005), most of these studies were carried out with the fruiting bodies (Kubo et al., 1994; Gray & Flatt, 1998; Lo et al., 2006), submerged culture biomass (Kim et al., 2001a; Han et al., 2006; Lo et al., 2006; Yang et al., 2006) or intracellular polysaccharides (Kiho et al., 1993, 1994, 1995, 1996; Yuan et al., 1998).

In our study, the hypoglycemic effects of exopolysaccharides produced by Cerrena unicolor (Bull.) Murrill (Polyporaceae), Coprinus comatus (O.F. Müll.) Pers. (Agaricaceae), and Lenzites betulina (L.) Fr. (Polyporaceae) in submerged culture were investigated. For this aim, we measured serum glucose levels in the control, streptozotocin-induced diabetic, and EPS treatment groups. Additionally, pancreatic tissue samples were examined histologically.

Material and methods

Microorganisms

Native specimens of C. unicolor, C. comatus, and L. betulina were collected from their natural habitats within forests in Sakarya and Eskişehir, Turkey in 2006. M. Yamaç and M. H. Solak authenticated the fungus materials. The voucher specimens were prepared as herbarium materials and deposited in the fungiculture laboratory at the Department of Biology, Eskişehir Osmangazi University, Eskişehir, Turkey. All cultures were isolated from fruiting bodies of the mushroom samples. The stock cultures were maintained on potato dextrose agar slants at 4°C and subcultured periodically.

Inoculum preparation and culture conditions

Fresh potato dextrose agar (PDA) cultures were considered as pre-inocula and agar discs (5 mm diameter) of these cultures were transferred into the seed medium. The seed cultures were prepared in a 250 ml flask containing 50 ml of potato malt peptone medium (PMP: 2.4% potato dextrose broth, 1% malt extract, 0.1% peptone) at 25°C and 150 rpm, for 4 days. The submerged cultures of the isolates were grown in Erlenmeyer flasks containing 100 ml of the PMP medium after inoculating with 4% (v/v) of the seed culture. The cultures were incubated at 25°C and 150 rpm for 15 days.

Preparation of crude exopolysaccharide

The flask cultures were harvested at various intervals, centrifuged (8,000 g for 10 min), and filtered through Whatman no. 2 filter paper. To precipitate crude EPS, the resulting culture filtrates were mixed with ethanol (4:1, v/v), stirred vigorously, and left overnight at 4°C. The precipitated crude EPS were separated by centrifugation (8,000 g for 10 min). After the supernatant was discarded, the crude EPS was lyophilized and the weight estimated. The recovery of crude EPS of mushroom isolates is summarized in Figure 1.

Figure 1.  The recovery process of exopolysaccharides from a submerged culture of mushroom isolates.

Animals and breeding conditions

We obtained 35 adult female Sprague-Dawley rats weighting 160–180 g from TICAM (Medical and Surgical Experimental Research Center, Eskisehir, Turkey) and housed them in polycarbonate cages in a climate-controlled room (temperature: 22° ± 2°C, humidity: 50% ± 5%), with a 12-h cycle of light and dark (07.00-19.00 light). The rats were fed laboratory pellet chow and water was given ad libitum. All rats were clinically healthy. They adapted to the laboratory conditions for 7-10 days and then fasted for 12 h before the intramuscular injection of STZ (Sigma, 50 mg/kg body weight, dissolved in 0.01 M citrate buffer, pH adjusted to 4.5). Five days after the administration of STZ, diabetic rats were identified by the positive response of glucose in urine using test strips (Arkray, Japan) and they were used as the insulin-dependent diabetes mellitus (IDDM) model. The animals were divided into 5 groups as follows (n = 7):

Group 1: Non-diabetic animals given saline

Group 2: Diabetic animals given saline

Group 3: Diabetic animals given Cerrena unicolor

Group 4: Diabetic animals given Coprinus comatus

Group 5: Diabetic animals given Lenzites betulina

EPS (100 mg/kg body weight) was orally administered to the EPS treatment groups for 7 days. Control and diabetic groups were given saline. At the end of the oral administration period, the animals were fasted for 9 h and sacrificed by ether anesthesia.

The experimental design and procedures were approved by the Eskisehir Osmangazi University Institutional Ethical Committee for Animal Care and Use (protocol number: 2007/247).

Biochemical analysis

Serum samples were separated by centrifugation at 2,500 rpm for 15 min and stored at −70°C until assayed. Serum glucose levels were measured with a commercial kit using an auto-analyzer (Hitachi Boehringer, Mannheim). Food intake and body weight gain were also periodically measured.

Histological studies

A 5 mm piece of pancreatic tissue was fixed in neutral (10%) formalin for at least 24 h and processed for light microscopy. Each piece of the rat pancreas paraffin block was sectioned (7 μ) before staining with hematoxylin and eosin (H&E). Then micrographs of the slides were taken using an Olympus BX50 microscope fitted with an Olympus DP70 digital camera. Each digital micrograph was studied for surface area and number of cells of Langerhans islets using an image-analyzing program (Soft Imaging System LS in Olympus Com. v. 5.0).

Statistical analysis

Data are expressed as mean ± standard error (SE) and independent sample groups one-way analysis of variance (ANOVA) and Duncan’s multiple-range tests, and correlations were performed with the SPSS statistical package (v. 15.0) to compare the histological and biochemical values of the 5 study groups. P values <0.05 were considered to indicate statistical significance.

Results

The body weight gain of the experimental groups was not significantly different from that of the control group (Table 1). Serum glucose levels in all groups are shown in Table 2. The effects of EPS derived from the three different fungal cultures on serum glucose level retention in diabetic rats were evaluated in comparison to the saline-administered diabetic group. EPS of different fungal cultures behaved differently in a serum glucose level retention pattern in diabetic animals given saline. In the present study, the serum glucose levels significantly decreased after oral administration of EPS by 61.23% with Cerrena unicolor, 42.78% with Coprinus comatus, and 42.08% with Lenzites betulina (Table 2).

Table 1.  Effects of mushroom exopolysaccharides (EPS) on the body weight gain in STZ-treated diabetic rats.

Experimental groups^a	Initial body weight (g)	Final body weight (g)
NC	164.40 ± 2.85	170.00 ± 1.89
STZ	173.42 ± 4.31	164.24 ± 2.41
Cerrena unicolor	164.32 ± 2.48	161.58 ± 3.54
Coprinus comatus	148.57 ± 3.94	146.00 ± 2.94
Lenzites betulina	151.45 ± 3.08	147.96 ± 2.84

Values are means ± SE (n = 7).

^aFor details, see Material and methods section.

Table 2.  Effects of crude exopolysaccharides produced from submerged mycelial culture of mushrooms on serum glucose levels and Langerhans islets in diabetic and non-diabetic animals.

Experimental groups^a	Average values of glucose in blood serum for 7 rats (mg/dl)	Average area of Langerhans islands (nm^2)	Average cell numbers of Langerhans islands
NC	140.14 ± 15.17	17271.50 ± 1966.70	123.00 ± 7.96
STZ	563.71 ± 14.39 *	5271.25 ± 113.89 *	37.25 ± 3.41 *
Cerrena unicolor	218.50 ± 31.26 * . **	42074.33 ± 1811.72 * . **	295.33 ± 41.28 * **
Coprinus comatus	322.50 ± 25.39 * . **	40424.50 ± 393.50 * . **	277.50 ± 19.44 * **
Lenzites betulina	326.50 ± 26.56 * . **	34569.50 ± 392.80 * . **	210.50 ± 3.89 * **

Values are means ± SE (n = 7).

^aFor details, see Materials and methods section.

*Significantly different from the NC group, p <0.05.

**Significantly different from the STZ group, p <0.05.

In the islets of Langerhans, all cells strongly stained with H&E and appeared to be mainly homogenously scattered. Identification of specific cell types without special stains is difficult. However, Figure 2 clearly shows that there were large histological differences between islet of Langerhans areas and differences in the average number of cells between Langerhans islets areas. Specifically, Langerhans islets of EPS experimental groups were bigger than the control group (Table 2, Figure 2C–E). Langerhans islets in the experimental groups were highly vascularized, with small blood vessel entering the core. Such an arrangement of blood flow allows a high concentration of cells (Figure 2C–E). Additionally, the capillaries within the islets were fenestrated, facilitating hormone entry into the blood stream.

Figure 2.  The histological effects of exopolysaccharide in pancreas of streptozotocin treated rats. Non-diabetic animals given saline as negative control (A), diabetic animals animals given saline as diabetic control (B), diabetic animals animals given Cerrena unicolor exopolysaccharide at 100 mg/kg dose (C), diabetic animals animals given Coprinus comatus exopolysaccharide at 100 mg/kg dose (D), diabetic animals animals given Lenzites betulina exopolysaccharide at 100 mg/kg dose (E). Scale bar: 125 μm

The present data clearly demonstrated that there was a significant increase in the average number of Langerhans islet cells in the Cerrena unicolor EPS treatment group (295.33) in comparison to the diabetic group (37.25). Also, the area of Langerhans islets increased to 42074.33 nm² in the Cerrena unicolor EPS treatment group. This value was 5271.25 nm² in diabetic control group.

Discussion

The pancreas has exocrine and endocrine functions and Langerhans islets are responsible for endocrine functions. Histological observations of Langerhans islets following mushroom polysaccharide treatment have not been previously reported. However, both insulin deficiency and its excess have been discussed (Genovese, 2005). Though α-cells, which secrete the glucagon hormone that raises blood glucose levels are present within the islets of Langerhans, they become functionally insensitive to hypoglycemia due to unknown mechanisms.

Most drugs for treating DM have focused on controlling and lowering blood glucose to a normal level (Hwang et al., 2005). It was reported that polysaccharides from hundreds of plants had blood-glucose lowering potential (Ding et al., 2004). Moreover, it was reported that various mushroom species have hypoglycemic effects (Yang et al., 2002). Edible mushrooms have been studied extensively and are proven to be ideal foods for the prevention of hyperglycemia due to their high fiber and protein content, and low fat content (Yang et al., 2006).

In our study, serum glucose levels in the STZ-induced diabetic group significantly increased as compared to the control group (P < 0.05). We observed decreased serum glucose levels in all three EPS-treated groups, as compared to the diabetic group (P < 0.05). Glucose levels in the EPS-treated groups were close to the control group levels; hence, our study clearly demonstrated that EPS produced by Cerrena unicolor, Coprinus comatus, and Lenzites betulina have hypoglycemic activity.

Among the studied mushroom isolates in the present study, EPS of C. unicolor caused a marked decrease in the serum glucose levels of STZ-induced diabetic rats, as compared to the control group. The ability of the EPS of this isolates to decrease blood glucose level by 61% is impressive. Thus, the EPS of C. unicolor may be useful for preventing chronic complications in DM. This isolates followed by C. comatus and L. betulina. Coprinus comatus is a mushroom claimed to benefit glycemic control in diabetes (Han et al., 2006), but there are no previous studies of the hypoglycemic activity of the EPS of these mushrooms we studied.

The hypoglycemic activity of various mushroom species is well documented in the literature (Table 3). The fruiting body, and intracellular and extracellular polysaccharides of mushrooms have been shown to decrease serum glucose level. Although Hwang et al. (2005) suggested that the hypoglycemic effects of EPS were generally lower than those of the mycelia of fruiting bodies, in the present study comparatively high hypoglycemic activity was observed with the EPS of the studied mushroom isolates. Submerged mycelia of C. comatus caused a 7.5% reduction in blood glucose level (Han et al., 2006), whereas, in the present study EPS of this species reduced blood glucose by 42.78%. As shown in Table 3, the hypoglycemic activity of the EPS of the three mushroom isolates, especially Cerrena unicolor, was higher than most reported in the literature, even if they were used as intravenously and intraperitoneally.

Table 3.  Hypoglycaemic activities of various mushrooms in diabetic animals.

Mushrooms	Fungal materials (methods)	Dose	Diabetic animals	Period	eduction ratio in blood glucose level (%)	References
Agaricaceae
Agaricus campestris L.	Extract of fruiting body (DW) and fruiting body powder (FD)	62.5^§	Mice (STZ)	12 d	50.4	Gray & Flatt, 1998
Coprinus comatus (O.F. Müll.) Pers.	EPS (OA)	100^‡	SD rat (STZ)	7 d	42.78	This study
Auriculariaceae
Auricularia auricula- judae (Fr.) Quél.	Extract of fruiting body (FD)	30^§	KK-Ay-TA mice	13 d	50.5^*	Yuan et al., 1998
Bolbitiaceae
Agrocybe cylindracea (DC.) Gillet	IPS (IP)	50^†	ddy mice (STZ)	5 h	51.0	Kiho et al., 1994
Clavicipitaceae
Cordyceps militaris (L.) Link	EPS (OA)	50^‡	SD rat (STZ)	7 d	14.0	Kim et al., 2001b
	Extract of fruiting body (OA)	100^‡	Rat (STZ)	48 h	60.0 – 82. 0^**	Zhang et al., 2006
	EPS (OA)	10^‡	Rat (STZ)	24 h	70.0 – 85.0^**	Zhang et al., 2006
Cordyceps sinensis (Berk.) Sacc.	IPS (IP)	50^†	KK-Ay-TA mice	48 h	13.7	Kiho et al., 1996
	IPS (IV)	1 – 10^†	KK-Ay-TA mice	6 h	18.8 – 46.4	Kiho et al., 1996
	IPS (IP)	100^†	ddy mice (STZ)	3 h	48.6 - 62.3	Kiho et al., 1993
	IPS (OA)	500^‡	ddy mice (STZ)	24 h	6.9 – 24.5	Kiho et al., 1993
	EPS (OA)	10^‡	Rat (STZ)	24 h	55.0 – 65.0^**	Zhang et al., 2006
Hymenochaetaceae
Phellinus baumii Pilát	EPS (OA)	200^‡	SD rat (STZ)	14 d	52.3	Hwang et al., 2005
Phellinus linteus (Berk. & M.A. Curtis) Teng	EPS (OA)	50 – 200^‡	SD rat (STZ)	7 d	19.4 - 50.0	Kim et al., 2001c
	EPS (OA)	50^‡	SD rat (STZ)	7 d	18.0	Kim et al., 2001b
	Submerged biomass (FD)	2.5 – 10^#	SD rat (STZ)	7 d	19.0 - 28.0^**	Kim et al., 2001a
Marasmiaceae
Lentinus edodes (Berk.) Singer	EPS (OA)	100^‡	SD rat (STZ)	7 d	17.8	Yang et al., 2002a
	EPS (OA)	50 ^‡	SD rat (STZ)	7 d	18.0	Kim et al., 2001b
Meripilaceae
Grifola frondosa (Dicks.) Gray	Extract of fruiting body (FD)	25^#	KK-Ay-TA mice	14 d	54.0 ^*	Kubo et al., 1994
Pleurotaceae
Pleurotus pulmonarius (Fr.) Quél.	Extract of fruiting body (OA)	250 - 1000^‡	SA mice (ALL)	acute or 28 d	73.0	Badole et al., 2006
Polyporaceae
Cerrena unicolor (Bull.) Murrill	EPS (OA)	100^‡	SD rat (STZ)	7 d	61.23	This study
Lenzites betulina (L.) Fr.	EPS (OA)	100 ^‡	SD rat (STZ)	7 d	42.08	This study
Tremellaceae
Tremella aurantia Schw. ex Fr.	IPS (IP)	30-100^†	KK-Ay-TA mice	24 h	6.6 – 18.0	Kiho et al., 1995
Tricholomataceae
Collybia confluens (Pers.) P. Kumm.	Submerged biomass (OA)	100 – 400^‡	SD rat (STZ)	21 d	18.0 – 34.0^**	Yang et al., 2006
Omphalia lapidescens (Horan.) Cohn & J. Schröt	Extract of fruiting body (OA)	100^‡	Rat (STZ)	24 h	60.0 – 65.0^**	Zhang et al., 2006
	EPS (OA)	10^‡	Rat (STZ)	24 h	55.0 – 60.0^**
Tricholoma mongolicum S. Imai	EPS (OA)	10^‡	Rat (STZ)	24 h	7.0 – 45.0^**	Zhang et al., 2006

DW: drinking water; EPS: Extracellular polysaccharide; FD: feeding with diet; IP: intraperitoneal administration; IPS: Intracellular polysaccharide; IV: Intravenous; OA: oral administration; ALL: diabetic animals induced by the injection of alloxan monohydrate for the induction of type 1 diabetes; STZ: diabetic animals induced by the injection of streptozotocin for the induction of type 1 diabetes; KK-Ay-TA mice: one of the animal models for type 2 diabetes, SA mice: Swiss albino mice; SD rat: Sprague-Dawley rat.

^§g/kg diet; ^†mg/kg, ip; ^‡mg/kg/day; ^#fungal material ratio in diet.

^*The data indicate approximate values calculated by Hwang et al. (2005) from figures in the references.

^**The data indicate approximate values calculated by the authors from figures in the references.

The structural features and viscosity of mushroom polysaccharides also affect hypoglycemic activity. The (1→4)-linked and/or (1→6)-linked residues in a β-(1→6)-branched (1→3)-β-d-glucan were needed for the hypoglycemic effect in diabetic mice (Kiho et al., 1994). Several studies (Johnson & Gee, 1981; Kiho et al., 2001) suggest that a water-soluble dietary fiber showing high viscosity increases gastric emptying time. In addition, it suppresses and/or delays intestinal digestion and absorption of carbohydrates to prevent rapid blood glucose increase. Our earlier studies showed that Cerrena unicolor and Coprinus comatus, which were both used in the present study, have higher viscosity than other mushroom isolates (unpublished data).

In the present study we used an STZ-induced diabetic rat model to investigate the hypoglycemic effect of Cerrena unicolor, Coprinus comatus, and Lenzites betulina EPS. Streptozotocin (STZ: N-nitroso derivate of glucosamine) is a broad spectrum antibiotic extracted from Streptomyces acromogenes. Previous reports suggested that STZ inhibits insulin secretion by the pancreas through selective destruction of β-cells in the pancreatic islets. Additionally, it is widely used to induce IDDM in experimental animal models (Hwang et al., 2005). However, Kim et al. (2006a, 2006b) reported that fungal EPS might neutralize the ability of STZ to cause β-cell damage or induce regeneration of islet β-cells. EPS administration after STZ treatment restored serum insulin and glucose levels. Our biochemical and histological results verify this assumption not only by decreasing the serum glucose level, but also by increasing both the size of the Langerhans islets area and the number of its cells. In the present study Cerrena unicolor, Lenzites betulina, and Coprinus comatus EPS might have been effective in repairing damaged pancreatic β-cells and promoting insulin synthesis, thereby lowering the level of serum glucose. On the other hand, Zhang and Lin (2004) reported that Ganoderma lucidum polysaccharides could not stimulate insulin synthesis, but they could stimulate the insulin release from the pancreatic islets directly due to facilitation of Ca²⁺ inflow to the pancreatic β-cells. However, our results of histological examinations clearly showed that Langerhans islets were induced by tested fungal EPS, thereby increasing cell number and total area of the Langerhans islets. The rates of increase in cell number and total area of Langerhans islets were higher than the rate of decrease of serum glucose level in the diabetic control and experimental groups; therefore, fungal EPS must have directly affected the Langerhans islets by repair or regeneration.

In order to investigate whether the hypoglycemic activity of EPS correlates with total area and/or cell number of Langerhans islets, statistical studies were performed, and it was demonstrated that all of the values correlated with each other (Table 4).

Table 4.  Statistically correlations on values of experimental groups.

Correlations		Glucose level	Area of Langerhans islets	The cell number of in Langerhans islets
Glucose level	Pearson correlation	1	0.607(*)	0.539(*)
	Sig. (2-tailed)	.	0.017	0.038
	N	33	15	15
Area of Langerhans islets	Pearson correlation	0.607(*)	1	0.967(**)
	Sig. (2-tailed)	0.017	.	0.000
	N	15	15	15
The cell number of in Langerhans islets	Pearson correlation	0.539(*)	0.967(**)	1
	Sig. (2-tailed)	0.038	0.000	.
	N	15	15	15

*Correlation is significant at the 0.05 level (2-tailed).

**Correlation is significant at the 0.01 level (2-tailed).

In conclusion, our findings showed that oral administration of Cerrena unicolor, Coprinus comatus, and Lenzites betulina EPS (100 mg/kg body weight) could play an important role in the prevention of STZ-induced diabetes. EPS of these mushroom isolates might be developed as oral hypoglycemic agents for diabetic patients and/or for persons at high risk for developing DM. In further experiments, comprehensive pharmaceutical and chemical studies should be performed to elucidate the mode of action and to identify the active fractions of these isolates’ EPS. Further investigation on the optimization of submerged culture conditions of Cerrena unicolor, Coprinus comatus, and Lenzites betulina are underway for EPS production.

Acknowledgments

This study was supported by the Eskişehir Osmangazi University Research Foundation (Project no: 200619016), Scientific Research and Application Center (FBAM) and Biology Department of Eskisehir Osmangazi University. The authors wish to thank Dr. Berna Yazıcı for statistical analysis and Burcu Altınay, Dr. Onur Koyuncu, Dr. M. Halil Solak, and Mr. Ö. Koray Yaylacı for their technical support.

Declaration of17957 interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.