Abstract
Mushrooms have been valued throughout the world as both food and medicine for thousands of years. In the current study, we report the hypoglycemic effects of aqueous extract of one variety of mushroom, Pleurotus pulmonarius. (Fr.) Quel.-Champ (Lentinaceae), in alloxan-induced diabetic mice. Pleurotus pulmonarius. extract was administrated orally at doses of 250, 500, and 1000 mg/kg to separate groups of mice (normal and alloxan-treated mice), and serum glucose and body weight were measured. In the separate group of mice, an oral glucose tolerance test (OGTT) was carried out. Acute oral toxicity data showed no mortality in the normal mice up to 5000 mg/kg. Oral administration of extracts reduced the serum glucose level in alloxan-treated diabetic mice in all the doses tested after acute and chronic (28 days) administration. The extract also showed increased glucose tolerance in both normal and diabetic mice. These results suggest that the extract possesses hypoglycemic activity.
Introduction
Mushrooms are a group of fleshy macroscopic fungi that until recently, as other fungi, were included in the plant kingdom because of cell wall and spores. Mushrooms have been valued throughout the world as both food and medicine for thousands of years (Tribe & Tosco, Citation1973; Wright, Citation2004). There are many varieties of mushrooms of which Pleurotus. are characterized by a white spore print, attached to gills, often with an eccentric stip, or no stip at all. They are commonly known as “oyster mushrooms” (Miles & Chang, Citation1997). Earlier studies have reported insulin release and insulin-like activity of other species of mushroom like Agaricus campestris. (Gray & Flatt, Citation1998). There is paucity of reports on the hypoglycemic activity of Pleurotus.. The objective of the current study was to evaluate the hypoglycemic effect of an aqueous extract of Pleurotus pulmonarius. (Fr.) Quel.-Champ (Lentinaceae) on serum glucose in alloxan-induced diabetic mice.
Materials and Methods
Drugs and chemicals
The mushroom Pleurotus pulmonarius. (Fr.) Quel.-Champ (Lentinaceae) was provided to us as a gift sample from Bajaj Orchard, Pvt. Ltd. (Mumbai, India). A.M. Mujumdar (Department of Botany, Agharkar Research Institute, Pune) authenticated it, and a voucher specimen was deposited at that institute. Glyburide (Ranbaxy Pharma. Ltd., New Delhi, India), alloxan monohydrate (Spectrochem, India), glucose estimation kit (Accurex Biomedical Pvt. Ltd., Mumbai, India), and D-glucose (S.D. Fine-Chem. Ltd., Mumbai, India) were purchased from respective companies.
Experimental animals
Swiss albino mice (25–30 g) were purchased from the National Toxicology Centre (Pune, India) and used for the study. They were maintained at a temperature of 25 ± 1°C and relative humidity of 45% to 55% under 12-h light:12-h dark cycle. The animals had free access to food pellets (Chakan Oil Mills, Pune, India), and water was given ad libitum.. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Poona College of Pharmacy, Pune, India.
Acute oral toxicity studies
Healthy adult albino mice of either sex, starved overnight, were subjected to acute toxicity studies as per guidelines (AOT no. 425) suggested by the Organization for Economic Cooperation and Development (OECD) (2001). The rats were observed continuously for 2 h for behavioral, neurological, and autonomic profiles for any lethality or death for the next 48 h.
Preparation of aqueous extract of Pleurotus pulmonarius.
Weighed quantity powder of air-dried Pleurotus pulmonarius. was added to distilled water (1:15), boiled for 20 min on water bath, cooled to room temperature, and filtered. The filtrate was dried on a tray dryer at 70°C (yield, 24% w/w).The dry extract powder was dissolved in distilled water to prepare the drug solution of concentration of 100 mg/ml and used for pharmacological studies.
Induction of experimental diabetes
Diabetes was induced in mice by a single intravenous injection of aqueous alloxan monohydrate (70 mg/kg, i.v.) by the method described by Rao et al. (Citation1999). After 48 h, the animals showing serum glucose levels above 200 mg/dl (diabetic) were selected for the study. All the animals were allowed free access to tap water and pellet diet.
Collection of blood and determination of serum glucose
Blood samples from the control and experimental mice were collected by orbital sinus puncture using heparinized capillary glass tubes. The blood samples so collected were analyzed for glucose levels by the glucose oxidase peroxidase (GOD/POD) method as described earlier (Abdel-Barry et al., 1997), and serum glucose levels were expressed in mg/dl.
Effect of aqueous extract of Pleurotus pulmonarius. on serum glucose in alloxan-induced diabetic mice
The method described by Dunn and McLetchie (1943) was adopted. Diabetic Swiss albino mice of either sex were divided into five groups (n = 6), viz.: group I, vehicle (distilled water, 10 ml/kg); group II, glyburide (10 mg/kg); group III, extract (250 mg/kg); group IV, extract (500 mg/kg); group V, extract (1000 mg/kg). All drugs were given orally.
The acute study involved estimation of serum glucose at 0, 2, 4, 6, and 24 h after drug administration.
The subacute study involved repeated administration of drug for 28 days at prefixed times, and serum glucose levels were estimated on the 7th, 14th, 21st, and 28th days. The data were represented as mean serum glucose level and standard error of mean (SEM). The mice were weighed daily during the study period of 28 days, and their body weights were noted and presented as mean change in body weights. The death of mice was also noted during the study periods, and percentage mortality was calculated.
Effect of aqueous extract of Pleurotus pulmonarius. on oral glucose tolerance test (OGTT) in normal and diabetic mice
Normal (nondiabetic) and diabetic mice were divided into five groups (n = 6), viz.: group I, glucose (2.5 g/kg); group II, glyburide (10 mg/kg); group III, extract (250 mg/kg); group IV, extract (500 mg/kg); group V, extract (1000 mg/kg). The animals were fasted overnight before commencing the experiment. The animals were loaded with D-glucose (2.5 g/kg) solution after half an hour of drug administration. Serum glucose levels were estimated prior to drug administration and at 30, 60, and 120 min after glucose loading.
Statistical analysis
The results are expressed as mean ± SEM. Comparison between the groups was made by two-way analysis of variance (ANOVA) followed by post hoc. Dunnett's test.
Results
Acute toxicity studies revealed that the extract was safe up to a dose level of 5000 mg/kg of body weight. No lethality or any toxic reactions were found up to the end of the study period. Single administration of aqueous extract of Pleurotus pulmonarius. (250, 500, and 1000 mg/kg) as well as glyburide (10 mg/kg) reduced serum glucose levels at 2, 4, and 6 h after extract administration (). Maximum reduction in serum glucose level was seen at 6 h after extract administration. Subacute administration (once a day for 28 days) of the extract as well as glyburide causes significant (p < 0.001) reduction in the serum glucose as compared with vehicle-treated group. Maximum activity of extract (reduction from 444.29 to 174.32 mg/dl) was seen with a significant decrease (p < 0.001) in serum glucose levels at the dose of 500 mg/kg on the 28th day ().
Table 1. Effect of Pleurotus pulmonarius. on serum glucose level in alloxan-induced diabetic mice (acute study)
Table 2. Effect of Pleurotus pulmonarius. on serum glucose level in alloxan-induced diabetic mice (subacute study)
Administration of vehicle (distilled water, 10 ml/kg, p.o.) in alloxan-induced diabetic mice resulted in a decrease in the body weight during the period of 28 days (). Extract (250, 500, and 1000 mg/kg) prevented a decrease in body weight in alloxan-treated mice. On the other hand, mice gained body weight as compared with the vehicle-treated group, which indicated the beneficial effect of the extract in preventing further loss of body weight. Administration of vehicle in alloxan-induced diabetic mice resulted in death of 44.4% of the total animals during the 28-day study period. Administration of the extract (250, 500, and 1000 mg/kg) reduced mortality (14.29%, 14.29%, and 25%, respectively) in mice. It was thus apparent that when no drug was administered, progression of diabetes resulted in mortality of mice, whereas the extract treatment resulted in reduction of mortality. Extract (500 mg/kg) produced a significant (p < 0.05) increase in the glucose threshold, 30-min post–glucose loading in both normal () as well as diabetic mice ().
Table 3. Effect of extract on body weight in alloxan-induced diabetic mice
Table 4. Effect of Pleurotus pulmonarius. on oral glucose tolerance test (OGTT) in normal mice
Table 5. Effect of Pleurotus pulmonarius. on oral glucose tolerance test (OGTT) in diabetic mice
Discussion
Mushrooms are highly nutritive as they contain good-quality proteins, vitamins, and minerals (Flegg & Maw, Citation1976; Khanna & Garcha, Citation1984). Mushrooms are a low-calorie food with very little fat and are highly suitable for obese persons. With no starch and very low sugars, they are the “delight of the diabetics” (Bano, Citation1976). In adequate quantities and being low in sugars, they can serve as medicinal foods for diabetes (Rai, Citation1986). Preliminary phytochemical analysis of the Pleurotus pulmonarius. showed the presence of proteins, minerals, vitamins, and carbohydrates (Food and Agriculture Organization of the United Nations, 1968).
In the past, many mushroom varieties have been reported to possess hypoglycemic activities in animals (Gray & Flatt, Citation1998; Swanston-Flatt et al., 1989), as well as in diabetic patients (Konno et al., Citation2001). In normal and streptozotocin diabetic mice treated with the mushroom variety Agaricus bisporus. (J. Lange) Imbach, the mushroom was shown to retard the development of hyperglycemia, hyperphagia, polydipsia, body weight loss, and glycated hemoglobin in the streptozotocin-treated mice (Swanston-Flatt et al., 1989) by counteracting reduction in plasma and pancreatic insulin concentration and by improving the hypoglycemic effect of exogenous insulin.
In the current study, the hypoglycemic activity of the aqueous extract of Pleurotus pulmonarius. was evaluated in alloxan-induced diabetic mice. Significant reduction in serum glucose level was seen at the second hour and maximum reduction occurred at the sixth hour by treatment with the extract in an acute study, and also on 28-day administration. The extract showed short onset and prolonged duration of hypoglycemic action. A dose of 500 mg/kg of extract showed optimum activity, as compared with 250 and 1000 mg/kg doses of extract.
Subacute treatment for 28 days with the extract in the tested doses brought about improvement in body weights of alloxan-treated diabetic mice, indicating its beneficial effect in preventing loss of body weight in diabetic mice. Administration of extract lowered mortality (14.29%) as compared with alloxan-induced diabetic mice (44%). The protective effects against diabetes-induced weight loss is supported by earlier studies (Swanston-Flatt et al., 1989).
In the oral glucose tolerance test, the doses increased the tolerance for glucose suggesting increased peripheral utilization of glucose in both diabetic as well as nondiabetic (normal) mice. The extract showed optimum activity at the dose of 500 mg/kg. Agaricus campestris. L. Fr., a mushroom variety, was reported to counter the hyperglycemia of streptozotocin-diabetic mice probably by an insulin-releasing mechanism (Gray & Flatt, Citation1998).
Water-soluble fraction obtained from maitake mushroom is reported to lower fasting blood glucose and, thus, is useful to treat insulin resistance in animals (Talpur et al., Citation2002aCitation2002b), as well as in diabetic patients (Konno et al., Citation2001). This effect of maitake mushroom was suggested to be through glucose/insulin metabolism and/or by enhancing peripheral insulin sensitivity. A large amount of glycogen was also observed after treating rats with maitake mushrooms, suggesting the possibility of increased glycogen formation by mushroom as a probable mechanism of their hypoglycemic effect.
The ability of lectins isolated from mushrooms (A. campestris., A. bisporus.) to enhance insulin release by isolated rat islets of Langerhans has been documented (Ewart et al., Citation1975). The nature of the active principle(s) and mechanism of action of insulin secreting cells and muscles remain to be established (Gray & Flatt, Citation1998).
Some variety of mushrooms were also shown to possess antihypertensive effects in spontaneous hypertensive rats (Talpur et al., Citation2002b), indicating the possibility of antidiabetic potential of mushrooms for hypertensive patients.
Conclusions
In conclusion, an oral dose of 250 and 500 mg/kg of aqueous extract of Pleurotus pulmonarius. possesses strong hypoglycemic activity against alloxan-induced diabetes and increased oral glucose tolerance in diabetic (OGTT model) mice. An upper dose (1000 mg/kg) caused an increase in mortality and loss of body weight without an increase in the hypoglycemic effect in diabetic mice.
Acknowledgments
The authors would like to acknowledge Dr. S.S. Kadam, Principal, and Dr. K.R. Mahadik, Vice-Principal, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, for providing necessary facilities to carry out the study. We are also thankful to Bajaj Orchard, a division of Trinity Bio-Tech, Mumbai, India, for a gift sample of Pleurotus pulmonarius..
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