Abstract
The effect of a methanol extract of the seeds of Swietenia macrophylla King. (Meliaceae) was evaluated with streptozotocin-induced diabetic rats. Forty-eight hours after streptozotocin injection, the extract, at doses of 200 and 300 mg/kg, was administered orally for 12 consecutive days. Oral glucose tolerance test (OGTT) and normoglycemic activity were also measured during this course of experiment. The extract was found to be potent antidiabetic as evidenced by significant (p < 0.01) reduction of blood glucose level of diabetic rats from day 5 onward by both the doses (maximal effect of 59.69% reduction of blood glucose level, at 300 mg/kg, on day 12, p < 0.01). Results demonstrated a significant reduction of serum lipids (maximal effect of 45.41 and 37.78% reduction of cholesterol and triglyceride, respectively, at 300 mg/kg, p <0.01) and elevation of liver glycogen level (maximal effect of 58.14%, at 300 mg/kg, p < 0.01) in diabetic rats, comparable to that of standard antidiabetic glibenclamide at 10 mg/kg. In OGTT, the extract at different doses showed significant reduction in plasma glucose level (p < 0.01) at the second hour. The extract also revealed low reduction of fasting blood glucose level in normal rats (maximal effect of 19.64% reduction, at 300 mg/kg, on day 12, p < 0.01) while glibenclamide at 10 mg/kg exhibited maximum effect of 23.27% (p <0.01) on day 12. Improvement of body weight profile was also observed in extract-treated diabetic rats.
Introduction
According to the World Health Organization, diabetes mellitus is possibly the world’s largest metabolic disorder of our time, particularly with people in southeast Asia and the western Pacific region. It is a state of improper regulation of homeostasis of carbohydrate, protein, and lipid metabolisms characterized by insulin hyposecretion or insensitivity, resulting in the elevation of fasting and postprandial blood glucose level (CitationBalkau et al., 2000). Hyperglycemia can lead to disturbance in the structure and function of different organs (CitationKuyvenhoven & Meinders, 1999). Clinically, diabetes is associated with a number of complications, like polyuria, polyphagia, polydypsia, ketosis, retinopathy, as well as cardiovascular disorders. Phytochemicals identified from traditional medicinal plants are presenting an exiting opportunity for the development of new types of therapeutics against diabetes and its related complications. So, there is an urgent need to identify indigenous natural resources in order to design new therapeutics for diabetes.
The plant Swietenia macrophylla King. (Meliaceae) is a beautiful, lofty, evergreen tree native to tropical America, Mexico, South America, and India, usually 30-40 m in height and 3-4 m in girth. The seeds of Swietenia macrophylla has been reported to have anti-inflammatory, antimutagenecity, and antitumor activity (CitationGuevera et al., 1996). Local people of East Midnapore, West Bengal, India use a decoction of seeds of Swietenia macrophylla for the treatment of diabetic symptoms. Thus, the aim of present study lies in the investigation of antidiabetic potential of methanol extract of seeds of Swietenia macrophylla in normal and streptozotocin-induced diabetic rats.
Materials and methods
Plant material
Seeds of Swietenia macrophylla were collected in the months of December and January, 2006 from Midnapore (East), West Bengal, India. The plant was authenticated by H. J. Chowdhury, Joint Director, Botanical Survey of India, Shibpur, Howrah, India. A voucher specimen CNH/1-1(64) was deposited at our laboratory for future reference.
Preparation of methanol extract
Methanol extract of the seeds was prepared in accordance to the method of the National Institute of Health and Family Welfare (NIHFW), New Delhi, India. Fresh seeds of Swietenia macrophylla were dried in an incubator for two days at 40°C, crushed in an electrical grinder to fine powder of mesh 40. The 500 g of seed powder was then extracted with 90% methanol in a Soxhlet apparatus at 50°C until the powder became totally exhausted. The resulting extract was filtered with a course sieve filter paper. The filtrate was dried under reduced pressure with the help of rotary vacuum evaporator and finally lyophilized. The extractive value was 15% (w/w). The extract was stored in a refrigerator at 0-4°C for further use.
Animals
Healthy adult Wistar strain albino rats of both sexes between 2-3 months of age and weighing 180-240 g were used for the study. Animals were allowed to be acclimatized for a period of 2 weeks in our laboratory environment prior to the study. Rats were housed in polypropylene cages (4 animals per cage), maintained under standard laboratory conditions (i.e., 12:12 h light and dark sequence; at an ambient temperature of 25 ± 2°C; 35-60% humidity); the animals were fed standard rat pellet diet (Hindustan Liver Ltd. Mumbai, India) and water ad libitum. The principles of laboratory animals care (PHS, 1985) were followed and instructions given by our institutional animal ethical committee were followed throughout the experiment.
Chemicals
Streptozotocin used for the induction of diabetes was procured from Sisco Research Laboratory Pvt. Ltd., India and other reagents used in the experiment were of analytical grade. Glibenclamide (Daonil™, Hoechst, India) tablets were purchased from a local medical store, Jadavpur, India.
Normoglycemic study
Twenty-four rats divided into 4 groups of 6 animals each were used for the normoglycemic study. Group I was kept as control and only received distilled water. Groups II and III were given methanol extract at 200 and 300 mg/kg body weight, respectively, by the oral route, once a day for 12 days, while group IV received glibenclamide as a standard drug at an oral dose of 10 mg/kg body weight (CitationMandal et al., 1997). After an overnight fast, on days 1, 5, 9, and 12, blood samples were withdrawn from the tail vein of each animal for fasting blood glucose estimation. Blood glucose level was estimated by one-touch glucometer.
Oral Glucose Tolerance Test (OGTT)
Animals were divided into 3 groups of 6 rats each. The OGTT was performed with overnight (18 h) fasted normal Wistar strain albino rats. Group I was administered distilled water. Groups II and III were treated with methanol extract at doses of 200 and 300 mg/kg body weight, respectively, by the oral route. Glucose (2 g/kg body weight) was fed orally half an hour prior to the administration of extracts (CitationBonner-Weir, 1988). Blood glucose levels were measured at 0, 1, and 2 h with the help of single touch glucometer.
Antihyperglycemic studies
Induction of diabetes
Hyperglycemia was induced in overnight fasted adult Wistar strain albino rats weighing 180-240 g by a single intraperitoneal injection of 65 mg/kg streptozotocin in a volume of 1 ml/kg body weight (CitationSiddque et al., 1987). Due to the instability of streptozotocin in aqueous media, the solution was made in citrate buffer (pH 4.5) immediately before injection (CitationKarunanayake et al., 1974). Hyperglycemia was confirmed by the elevated glucose level in plasma, determined at 48 h after injection. The rats found hyperglycemic were screened for the antihyperglycemic study.
Experimental design
One group of 6 normal rats was marked as group I and treated with distilled water. Diabetic animals were divided into 4 groups of 6 rats in each group and designed as groups II, III, IV and V. Group II was treated with distilled water. Groups III and IV were administered methanol extract at doses of 200 and 300 mg/kg body weight, respectively, by the oral route. Group V animals were treated with standard drug glibenclamide at a dose of 10 mg/kg body weight orally. All doses were started 48 h after streptozotocin injection. Fasting blood glucose levels were estimated on overnight fasted rats on days 1, 5, 9, and 12. Serum lipid profiles and liver glycogen profile (CitationCaroll et al., 1956) were measured after the animals were sacrificed measured. Initial and final changes in body weight were also measured (CitationShirwaikar et al., 2004).
Statistical analysis
Data were statistically calculated by utilizing one way ANOVA and expressed as mean ± SEM followed by followed by Dunnett’s t-test using computerized Graph Pad InStat version 3.05, Graph pad software, USA.
Results
Effect of methanol extract of seeds of Swietenia macrophylla on normal rats
The effect of different doses of methanol extract of the seeds of Swietenia macrophylla was assessed in normal rats at different day intervals. It was found that the extract is able to reduce significantly fasting blood glucose level in normal rats (). The percentage reduction of blood glucose levels were found maximum of 10.98% and 19.64%, p < 0.01, at the doses of 200 and 300 mg/kg, respectively, on day 12, while standard antidiabetic glibenclamide (10 mg/kg) showed maximum reduction of 23.27% on day 12.
Table 1. Effect of the methanol extract of seeds of Swietenia macrophylla on fasting plasma glucose level in normal rats.
In OGTT, the extract, at 2 h, showed a significant (p < 0.01) reduction in plasma glucose level ().
Table 2. Effect of methanol extract of seeds of Swietenia macrophylla on oral glucose tolerance test (OGTT).
Effect of methanol extract Swietenia macrophylla seeds on diabetic rats
The administration of streptozotocin induced diabetes in experimental rats as evidenced by elevation of fasting blood glucose level. The methanol extract of Swietenia macrophylla seeds was found to reduce elevated blood glucose level of diabetic rats in a dose-dependent manner (). The difference between experimental and diabetic control rats in lowering fasting blood glucose level was found to be statistically significant (p < 0.01) from day 5 onward, and comparable to that of standard drug glibenclamide. A maximal effect of 48.48% and 59.69% reduction of blood glucose level was found at 200 and 300 mg/kg, respectively, on day 12, (p < 0.01), while maximal effect of 61.95% reduction of blood glucose level was found on day 12, (p < 0.01) in glibenclamide treated diabetic rats.
Table 3. Effect of methanol extract of seeds of Swietenia macrophylla on fasting plasma glucose level in streptozotocin induced diabetic rats.
The changes in initial and final body weight are listed in . Although the results indicated improvement of body weight for extract and glibenclamide treated diabetic rats with respect to diabetic control, statistical significance (p < 0.05) was only found in glibenclamide treated diabetic rats.
Table 4. Effect of methanol extract of seeds of Swietenia macrophylla on changes in body weight in streptozotocin induced diabetic rats.
A significant (p < 0.01) reduction of serum lipid profiles was observed in diabetic rats, with maximal effects of 45.41% and 37.78% reduction of cholesterol and triglyceride levels, respectively, at 300 mg/kg, while the standard antidiabetic glibenclamide at 10 mg/kg, exhibited maximum reductions of 55.18% and 45.65% (p < 0.01) for cholesterol and triglyceride, respectively, in diabetic rats. The maximum elevation of liver glycogen level was found to be 58.14% and 64.87% (p < 0.01), at 300 mg/kg of extract and glibenclamide (10 mg/kg), respectively, in diabetic rats ().
Table 5. Effect of methanol extract of seeds of Swietenia macrophylla on serum lipid profiles and liver glycogen level in streptozotocin-induced diabetic rats.
Discussion
Streptozotocin, a N-nitroso derivative of glucosamine (CitationLeslie & Elliott, 1994), is a potent beta cell toxin of islet of Langerhans of the pancreas and causes hyperglycemia in rats (CitationPalmer et al., 1998). These experiments focused on exploring the potential of a methanol extract of the seeds of Swietenia macrophylla for the treatment of diabetes to substantiate a folklore claim. The differences observed between initial and final fasting blood glucose levels of different groups revealed a significant diminution in blood glucose level in diabetic control as compared with normal animals at the end of day 12. In an oral glucose tolerance test, a hypoglycemic effect was observed at 2 h after administration of extract. This reflects the efficiency of the extract to control elevated blood glucose levels. Maintenance of blood glucose levels in both normal and diabetic rats with extract treatment indicates the effectiveness of the extract may involve increasing the peripheral utilization of glucose. The results also demonstrated significant control of serum lipid profiles in the extract treated diabetic rats and with responses comparable with the standard drug. Diabetes is associated with hyperlipidemia (CitationChase & Glasgow, 1976). It is well known that insulin activates enzyme lipoprotein lipase, which hydrolyzes triglyceride under normal conditions. Destruction of beta cells leads to depletion of plasma insulin, which results in hyperlipidemia. The significant control of plasma lipid levels suggests the extract may produce its action by improving insulin secretion.
Excessive hepatic glycogenolysis and gluconeogenesis associated with decreased utilization of glucose by tissue is the fundamental mechanism underlying hyperglycemia in the diabetic state (CitationLatner, 1958). Aberration of liver glycogen synthesis or glycogenolysis in diabetes may be due to a lack of or resistance to insulin, which is essential to activate the glycogen synthase system. The significant increase of liver glycogen level in extract treated diabetic groups may be due to reactivation of the glycogen synthase system by improving insulin secretion. Diabetes is associated with weight loss (CitationSwanston Flatt et al., 1990). The reversal of weight loss in the extract treated diabetic group indicates the restorative effect of the extract may result from reversal of gluconeogenesis and glycogenolysis. The experimental results indicate the seeds of Swietenia macrophylla possess significant antidiabetic activity and are capable of maintaining serum lipid profile, liver glycogen level and body weight.
The seeds of this plant contain swietenine, swietenolide (CitationGuha et al., 1951), swietemahonin, khayasin, andirobin, augustineolide, 7-deacetoxy-7-oxogedunin, 6-deoxy swietenine, proceranolide, 6-O-acetyl swietonolide, 2-hydroxy swietenine (CitationMootoo et al., 1999), but none of these substances has yet been reported to possess antidiabetic activity. It is planned to isolate potent antidiabetic phytomolecules from the seeds of the plant.
Conclusion
In summary, for the first time, we have scientifically explored the antidiabetic efficacy of the seeds of Swietenia macrophylla on animal models based on traditional literature. Experimental findings clearly indicate there is an exciting opportunity to develop a potent antidiabetic molecule from this plant. Our future research will be directed toward isolating a novel lead from the seeds of Swietenia macrophylla.
Acknowledgements
The authors are thankful to All India Council for Technical Education, New Delhi, India for financial assistance. We are also thankful to Jadavpur University, Kolkata, India for providing facilities for our research work.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Related Research Data
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