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Antihyperglycemic, Insulin-sensitivity and Antihyperlipidemic Potential of Ganoderma Lucidum, a Dietary Mushroom, on Alloxan- and Glucocorticoid-induced Diabetic Long-evans Rats

Md. Moklesur Rahman Sarker
Department of Pharmacy, School of Science, Primeasia University, HBR Tower, 9 Banani C/A, Dhaka-1213, Bangladesh;

M.A.T.R. Zihad
Department of Pharmacy, School of Science, Primeasia University, HBR Tower, 9 Banani C/A, Dhaka-1213, Bangladesh;

Mohasiul Islam
Department of Pharmacy, School of Science, Primeasia University, HBR Tower, 9 Banani C/A, Dhaka-1213, Bangladesh;

Mazmun Nahar
Department of Pharmacy, School of Science, Primeasia University, HBR Tower, 9 Banani C/A, Dhaka-1213, Bangladesh;

Md. Mahfuzul Islam
Department of Pharmacy, School of Science, Primeasia University, HBR Tower, 9 Banani C/A, Dhaka-1213, Bangladesh;

Hasan Imam
Department of Pharmacy, School of Science, Primeasia University, HBR Tower, 9 Banani C/A, Dhaka-1213, Bangladesh;

Ahandra Ghosh
Department of Pharmacy, School of Science, Primeasia University, HBR Tower, 9 Banani C/A, Dhaka-1213, Bangladesh;

Mohd Shahimi Mustapha
Faculty of Pharmacy, Lincoln University

Nahlah Elkudssiah Ismail
Faculty of Pharmacy, Lincoln University

Abstract/ Executive Summary:

Reishi (Ganoderma lucidum) is a well-known and popular edible mushroom eaten all over the world. It has been used as an alternative medicine for many years in China, Korea, Japan, Malaysia, and in eastern Russia. This mushroom is reported to exhibit a number of medicinal properties including antitumor, antioxidant, immunomodulating, anti-inflammatory, hepatoprotective, and hypoglycemic activities, due to the presence of bioactive polysaccharide. Glucocorticoids, which are usually prescribed for the treatment of arthritis to protect inflammation and reduce pain, can induce hyperglycemia or aggravate the hyperglycemic condition. For example, causing very high glucose levels in diabetic patients. However, no report has been published for its effect on glucocorticoid-induced diabetes. The objective of the present study was to investigate the effect of Ganoderma lucidum on alloxan- and glucocorticoid- induced diabetes in Long-Evans rats. Alloxan monohydrate (150 mg/kg) was intraperitoneally administered to Long-Evans rats as a single dose. The same volume of normal saline was injected to control rats. Three days after alloxan injection, rats with plasma glucose levels higher than 12 mmoL /L were identified as diabetic and included in the study. Reishi mushroom was collected from the Mushroom Development Institute, Ministry of Agriculture, Savar, Dhaka, Bangladesh, where it was identified by a Taxonomist. Petroleum ether extract (PEE), known as petroleum spirit in the USA, and Methanol extract (ME) were prepared by maceration and distillation techniques. The extracts were orally administered once a day at doses of 200, 400, 600 and 800 mg/kg, respectively for 7 days. Metformin (150 mg/kg) was orally administered as a standard antidiabetic drug. Glucose levels were measured at 0 and 7th days of treatment. The rats were allowed to rest for 1 week without treatment. The animals were again injected with dexamethasone (2 mg/kg) through intra-muscular route for 3 days and glucose levels were monitored regularly. Rats were then further treated with PEE and ME and metformin for another 7 days and glucose levels were determined at 0 and 7th days of treatment. The PEE and ME of Reishi mushroom dose-dependently reduced the plasma glucose levels in alloxan-and steroid-induced fasting diabetic rats. The maximum reduction of fasting plasma glucose levels observed by PEE (800 mg/kg) and ME (800 mg/kg) were 55.57% and 36.01% in alloxan-induced and 51.41% and 32.02% in steroid-induced diabetic rats, respectively. On the other hand, metformin (150 mg/kg) resulted in the diminution of fasting blood glucose levels by 60.02 and 51.12% in the alloxan- induced and steroid-induced diabetic rats respectively. Both the PEE (800 mg/kg) and ME (800 mg/kg) significantly augmented plasma insulin levels (***P < 0.001 and **P < 0.01) and reduced HbA1c (**P < 0.01 and *P < 0.05) in alloxan-and steroid-induced diabetic rats. Additionally, treatment of diabetic rats with PEE (800 mg/kg) and ME (800 mg/kg) controlled the 2-h post prandial elevated glucose levels in blood plasma. The same dose of the extracts also significantly reduced the levels of total cholesterol (TC) (***P < 0.001 and ***P < 0.01), triglyceride (TG) (***P < 0.001 and **P < 0.01) and low-density lipoprotein-cholesterol (LDL-c) (***P < 0.001 and ***P < 0.001), as well as increased the level of high density lipoprotein cholesterol (HDL-c) (**P < 0.01 and **P < 0.01, respectively). Our study demonstrated that edible mushrooms-Reishi has antihyperglycemic, insulin-sensitivity, and hyperlipidemic effects against both alloxan and corticosteroid-induced diabetes rats. The bioactive chemicals responsible for those activities are most probably the polysaccharides available in the mushroom. Therefore, usage of Reishi mushrooms as vegetables or as extract will be beneficial for the management of diabetes.

Keywords: Antihyperglycemic, Antidiabetic, Insulin-sensitizer, Anti-hyperlipidemic, Gano mushroom, Reishi mushroom, Steroid induced diabetes,

Location: Animal Ethical Committee of Primeasia University, Dhaka, Bangladesh.

Start Date:

End Date:

Program Area: Development of Host and Medicinal Plants

Commodity: Mushroom

Objectives:

To evaluate the antihyperglycemic and insulin sensitivity potential of petroleum ether extract (PEE) and methanol extract (ME) of GL on alloxan- as well as corticosteroid-induced diabetic Long-Evans rats.

Methodology:

Collection of mushroom: Fresh Reishi mushroom (Ganoderma lucidum) was collected from the Mushroom Development Institute, Ministry of Agriculture, Sobhanbag Savar Dhaka, Bangladesh, where it was identified by a Taxonomist and a voucher specimen was retained for further reference.

Preparation of extract: The mushrooms were cut into small pieces and shed dried with a good air circulating system for 2 weeks. The dried mushrooms were grinded to coarse powder with a mechanical grinder (multi-mill) and screened through mesh to remove large particles. The extracts were prepared by using maceration technique. Twenty gram of mushroom powder was soaked in 200 mL of 99.8% methanol (chemical ID: 106009; boiling point range: 64-65 ºC; Merck, Germany) and 99.7% of petroleum ether (chemical ID: 101769; boiling point range: 40-80 ºC; Merck, Germany) separately at room temperature for 2 days with occasional shaking. The extracted solution was then filtered with Whatman No. 1 and stored at refrigerator. The extraction process was repeated 5 times with the incorporation of the same solvent until the extract depicts no faint discoloration and the filtrates were accumulated in a beaker. PEE and ME of GL were then concentrated for complete removal of extra solvents under reduced pressure by using a rotary evaporator at 40°C. The yields of PEE and ME were 17.05 (3.41 g) and 12.35 % (2.47 g) respectively. The extracts were stored at 4°C in a refrigerator until use.

Experimental animals: Long-Evans male rats (4 weeks age, weight 83.5 g) were purchased from the Animal Resource Branch of International Centre for Diarrheal Disease Research in Bangladesh (ICDDRB). The animals were kept and maintained under standard environmental conditions (temperature at 25 ± 5°C, relative humidity 40-60%, 12 hour dark and light cycle), and fed ad libitum with normal animal pellet diet. The animals were allowed to acclimatize for 2 weeks before commencing the experiments. All the studies were conducted according to the guidelines of Animal Ethical Committee of Primeasia University, Dhaka, Bangladesh.
Induction of diabetes by Alloxan monohydrate: Diabetes was induced to Long-Evans rats by intraperitoneal administration of a single dose of Alloxan monohydrate (150 mg/kg). Blood glucose levels were monitored each day using a glucometer (Accu-Chek, Roche). Three days after alloxan injection, rats with plasma glucose levels higher than 12 mmoL/L were considered as diabetic rats and they were included in the study.
Induction of diabetes by Corticosteroid (Dexamethasone): At the end of first round treatment of alloxan-induced diabetic rats with GL extracts, the rats were allowed to rest for 1 week without treatment. Diabetes was again induced in the rats with intra-muscular injection of dexamethasone (3 mg/kg/day) for 3 consecutive days. Glucose levels were measured and we discovered that the plasma glucose levels of all rats were potentially elevated. The glucose levels of all the rats were found to be higher than 12 mmoL/L and they were used for further treatment with the extracts.
Experimental Design: A total of 55 male Long-Evans rats were used for this study. The animals were randomly divided into the following groups:
Group I: Nondiabetic rats
Group II: Diabetic rats (control)
Group III: Diabetic rats treated with GL-ME (200 mg/kg)
Group IV: Diabetic rats treated with GL-ME (400 mg/kg)
Group V: Diabetic rats treated with GL-ME (600 mg/kg)
Group VI: Diabetic rats treated with GL-ME (800 mg/kg)
Group VII: Diabetic rats treated with GL-PEE (200 mg/kg)
Group VIII: Diabetic rats treated with GL-PEE (400 mg/kg)
Group IX: Diabetic rats treated with GL-PEE (600 mg/kg)
Group X: Diabetic rats treated with GL-PEE (800 mg/kg)
Group XI: Diabetic rats treated with Metformin (150 mg/kg)
The extracts and metformin were administered to the respective groups by oral gavage using stainless steel feeding needle. Normal saline was used as a vehicle of extract for administration; control group was administered with the same volume of saline only. The animals were sacrificed at the end of the experiments and the collected serum was used for the determination of glucose, insulin, and lipid levels.
Oral Glucose Tolerance Test (OGTT): Oral glucose tolerance test was performed as described by Kumar et al. Alloxan-induced diabetic rats were treated with ME and PEE regularly. At day 7, overnight fasted (14 hours) diabetic Long-Evans rats were treated with ME or PEE or metformin or with normal saline. Glucose (2g/kg) was fed 30 minutes after the administration of extracts or metformin doses. Blood was collected from the tail vein to measure the glucose levels at 0 and 2 hours of glucose administration. Similarly, OGTT was performed to dexamethasone induced diabetic rats at day 7 of treatment, using the method described above.
Determination of fasting glucose, insulin and HbA1c in blood serum: Alloxan-induced diabetic rats were administered with different doses of ME and PEE of GL or metformin daily for 7 days, similar to standards of other researchers. The animals were kept at rest for 1 week. Dexamethasone (3 mg/kg/day) was injected for 3 consecutive days to induce diabetes or elevate the glucose levels. Dexamethasone-induced diabetic rats were again treated with GL-extracts and metformin for another 7 days. Blood samples were collected from tail vein of overnight fasting rats on days 0 and 7 (alloxan-induced diabetic rats), and 18 and 25 (dexamethasone-induced diabetic rats). Serum glucose level was measured by Glucose oxidase method and serum insulin level was assayed by using an Enzyme Linked Immunosorbant Assay (ELISA) kit. Glycated haemoglobin (HbA1c) was estimated by a colorimetric method with some modification.
Measurement of serum lipid levels: On day 25, overnight fasted rats were anesthetized by ether and blood samples were collected by cardiac puncture. The blood was then centrifuged at 1500 × g for 15 min at 4°C to collect the supernatants for the determination of lipid levels. Triglycerides (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-c), and high density lipoprotein cholesterol (HDL-c) were determined by using a bioanalyzer with standard kits.
Statistical analysis: The data was analyzed by SPSS software package Version 22.0. The values were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test. The data are means ± standard error of mean (SEM). P values less than 0.05 (P < 0.05) were considered as significant.

Source of Information: Functional Foods in Health and Disease 2015; 5(12): 450-466

Article Link (Online Journal): http://ffhdj.com/index.php/ffhd/article/view/220/447

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The results of our present investigation clearly demonstrate that petroleum ether and methanol extracts of Ganoderma lucidum (Gano mushroom) have the potential to reduce hyperglycemia with the enhanced glucose uptake, induce insulin production and secretion by pancreatic beta-cells, improves insulin sensitivity, and ameliorate lipid profile in alloxan and steroid-induced diabetic Long-Evans rats. Dietary intake or administration of extracts of Reishi mushroom will effective for the management of diabetes and hyperlipidemia.

Abbreviations Used: GL, Ganoderma lucidum; PEE, Petroleum ether extract; ME, Methanol extract; GL-PEE, Petroleum ether extract of Ganoderma lucidum; GL-PE, Methanol extract of Ganoderma lucidum; OGTT, Oral glucose tolerance test; LDL-c, low-density lipoprotein cholesterol; TC, Total cholesterol; TG, Triglycerides; HDL-c, High-density lipoprotein cholesterol; i.p., Intraperitoneal; HbA1c, Glycated hemoglobin; PPG, Post-prandial plasma glucose.
Competing Interests: The authors declare that they have no financial or intellectual conflicts of interest.

Knowledge Management: Journal