The present study evaluates anti-hyperglycemic activity of fractionated
Diabetes mellitus, a metabolic disorder, is a major global health concern with a projected rise in prevalence from 171 million in 2000 to 366 million in 2030 [
While a number of reports have been put forth demonstrating the anti-hyperglycemic activity of Mc, systematic studies to evaluate the effect of prolonged treatment with the fractionated seed extracts on the blood glucose levels together with acute toxicity studies have not been carried out.
Therefore, the present study focuses on the bioassay-guided fractionation of the acid-ethanolic extract of Mc seeds to identify the fraction that contains the active principle(s) responsible for the anti-diabetic activity. Further, the effect of long-term treatment with the active fraction on glycemic control and on the liver and kidney functions in diabetic rats was also investigated, as these studies would aid in evaluating the hepatotoxic and nephrotoxic effects, if any, of the prolonged treatment and proving to be nontoxic will facilitate the use of the active fraction(s) for diabetes management.
Random bred male Wistar rats (12–14 weeks) were housed in the Small Animal Facility of the Jawaharlal Nehru University. The animals were provided with rat feed (Hindustan Lever Ltd, India) and water ad libitum. The use of animals was duly approved by the Institutional Animal Ethics Committee of the JNU, and the guidelines prescribed by the Institutional Animal Ethics Committee, JNU, New Delhi, were followed while handling animals.
Extraction of seeds was done essentially as described earlier [
The male Wistar rats were made diabetic by using alloxan. Briefly, alloxan was administered i.p. after starving the animals for 36 hrs at a dose of 150 mg/kg body weight (b.wt.). Animals were stabilized for three days by insulin administration, 1-2 units per day for 2 days. Only those animals having blood glucose level more than 300 mg per 100 mL blood were selected for further analysis.
The diabetic animals were grouped into experimental groups each containing minimum 5 rats. The doses of different fractions are expressed in terms of their protein content. Different groups were treated with different Mc seed fractions (15 mg/kg b.wt.). Diabetic animals treated with saline (vehicle) were included in the study as negative control. A group of diabetic animals treated with protamine zinc insulin (10 IU/kg b.wt., s.c., Boots Pharmaceuticals Ltd., India) served as standard reference control. A group of normal untreated non-diabetic animals was also included in the study. Fasting serum glucose (6 h fasting) was measured in blood drawn from the tail vein during the study period using glucose oxidase-peroxidase method [
In order to determine the time by which the active fraction is able to bring about anti-hyperglycemic effect, a short-term (0–4.5 h) study was conducted by measuring the blood glucose levels within the indicated periods after administration.
Short-term time kinetics of the active fraction in diabetic rats was determined with a dose of 15 mg/kg b.wt. administered intraperitoneally (i.p.) or insulin (10 IU/kg b.wt). The blood glucose levels were measured at different time intervals. Optimum dose of the active fraction was determined by administering the animals with different concentrations (5–25 mg/kg b.wt.) of the active fraction. Blood glucose levels were measured at 3 h after administration.
The rats were divided into different groups (five rats in each group): Group I—PBS-treated normal non-diabetic controls, Group II—PBS-treated diabetic rats, Group III—diabetic rats treated with 15 mg/kg b.wt. of the active fraction, and Group IV—the diabetic rats treated with protamine zinc insulin (10 IU/kg b.wt.). The first two groups of rats were given saline (vehicle) daily. The extract and insulin were administered at the selected dosage to Groups III and IV, respectively, every day for 18 days. Body weights of the untreated and treated animals were monitored throughout the study period. The rats were bled prior to sacrifice on the last day of the treatment by cervical dislocation. Serum was collected and subjected to biochemical analysis for hepatic function markers using assay kits from AutoZyme, India, by the method of Penttila et al. [
The enriched fraction showing maximum biological activity was further fractionated by gel filtration chromatography using Sephacryl S-100 HR in 0.2 M NH4HCO3 (pH 7.2–7.4). The collected fractions were analyzed by SDS-PAGE and silver staining. The peak fractions designated as Mc-3.1, Mc-3.2, and Mc-3.3 were analyzed for anti-hyperglycemic activity using experimental diabetic rats as described before.
The most active fraction obtained after gel filtration chromatography was assessed for its ability to induce glucose tolerance in normal Wistar rats. The rats were administered with 2 g/kg of glucose (i.p.) after 30 min of the test fraction at a dose of 15 mg/kg b.wt. (i.p.). Serum glucose levels were measured in the blood samples collected from the tail vein prior to glucose administration (considered as 0 h) and at different time intervals after glucose administration.
All the results were analyzed statistically using one-way ANOVA or Student’s paired
The crude acid ethanolic extract upon centrifugation gave rise to fractions Mc-0 (insoluble fraction) and Mc-1. Since fraction Mc-0 contained insoluble material, it was not administered in the animals. The fraction Mc-1 and the fractions Mc-2 and Mc-3 derived from Mc-1 were tested for their anti-hyperglycemic potential in experimental diabetic rats. As evident, fraction Mc-1 at the tested dose (15 mg/kg b.wt.) was able to lower blood glucose levels (Table
Effect of different fractions of
Period (days) of treatment | 0 | 1 | 3 |
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Treatment | |||
Normal |
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PBS |
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Mc-1 |
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Mc-2 |
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ND |
Mc-3 |
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Insulin |
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The diabetic animals were treated with different fractions (Mc-1, Mc-2, and Mc-3) at a dose of 15 mg/kg b.wt. and protamine zinc insulin (10 IU/kg b.wt), once daily for 3 days. Control normal and control diabetic animals were treated with corresponding volume of PBS. The data represent mean ± S.D. Each group consisted of at least 5-6 animals.
Since fraction Mc-3 was found to possess enriched anti-hyperglycemic activity, it was desirable to study how quickly fraction Mc-3 could exert its anti-hyperglycemic effect after administration. Therefore, blood glucose levels of the Mc-3 treated animals were measured at different time after administration for a short period of 4.5 h. As shown in Figure
Short term anti-hyperglycemic effect of fraction Mc-3 of
In order to assess the optimum concentration of Mc-3 that was able to bring about significant reduction in serum glucose levels of diabetic animals, the animals were administered with different concentrations of the fraction Mc-3 (5–25 mg/kg b.wt.) and the blood glucose levels were determined at 3 hr after administration (the time point determined earlier for visualizing the effect). It was observed that Mc-3 showed an increased reduction in blood glucose levels till 15 mg/kg b.wt. No further reduction in the blood glucose levels was observed when the animals were treated with a higher concentration (20 mg/kg b.wt.) of fraction Mc-3 (Table
Determination of the optimum dose of fraction Mc-3 for anti-hyperglycemic activity in diabetic animals.
Period (hr) |
0 | 3 | Reduction % |
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Normal control PBS |
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5.5 |
Diabetic control PBS |
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4.26 |
Mc-3 (5 mg/kg b.wt.) |
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22.84 |
Mc-3 (10 mg/kg b.wt.) |
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33.55 |
Mc-3 (15 mg/kg b.wt.) |
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43.6 |
Mc-3 (20 mg/kg b.wt.) |
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41.3 |
Mc-3 (25 mg/kg b.wt.) |
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38.6 |
Insulin (10 IU/kg b.wt.) |
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44 |
Diabetic animals were treated with different doses of fraction Mc-3. Fasting serum glucose (mg/dL) was measured before and 3 h post-administration. PBS-treated normal and diabetic animals were included as controls. Protamine zinc insulin-treated animals were included as positive controls. The data represent mean ± S.D. Each group consisted of at least 5-6 animals.
In order to assess the long-term effect of fraction Mc-3, the diabetic animals were maintained on fraction Mc-3 for a period of 18 days in order to assess if the continued administration of the active fraction Mc-3 had some toxic or undesirable effect on the liver and kidney functions. As expected, the blood glucose levels of the animals treated daily with Mc-3 were lower when compared to initial levels prior to the treatment (Table
Effect of prolonged Mc-3 treatment on serum glucose levels.
Period (days) of treatment | 0 | 3 | 6 | 9 | 12 | 18 |
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PBS-treated normal |
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PBS-treated diabetic |
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Mc-3-treated diabetic |
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Insulin-treated diabetic |
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Diabetic animals (
After treatment, the serum activities of hepatic function markers: serum glutamic pyruvic transaminase (SGOT), serum glutamic oxaloacetic transaminase (SGPT), gamma-glutamyl transpeptidase (GGT), and the levels of renal function markers (urea and creatinine) were measured. Daily administration of insulin for 18 days resulted in significantly reduced levels of SGOT, SGPT, and GGT in comparison to PBS-treated diabetic controls. Similarly, the Mc-3-treatment of the diabetic animals for a period of 18 days also resulted in a significant reduction in the serum glutamic pyruvic transaminase (SGOT), serum glutamic oxaloacetic transaminase (SGPT), and gamma-glutamyl transpeptidase (GGT) when compared to PBS-treated control diabetic animals (Table
Effect of prolonged Mc-3 treatment on biochemical parameters in diabetic rats.
Parameters |
PBS-treated normal | PBS-treated diabetic | Mc-3-treated diabetic | Insulin-treated diabetic |
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Liver function | ||||
SGOT (IU/L) |
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SGPT (IU/L) |
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GGT (IU/L) |
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Kidney function | ||||
Urea (mg/dL) |
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Creatinine (mg/dL) |
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Total cholesterol (mg/dL) |
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Diabetic animals were treated with fraction Mc-3 (15 mg/kg b.wt.) or protamine zinc insulin (10 IU/kg b.wt.) once daily for 18 days. The serum was analyzed for biochemical parameters related with liver and liver and kidney function. The data represent mean ± S.D. The control animals received corresponding volume of PBS. Each group consisted of 5-6 animals each.
In order to determine if the reduction in serum glucose levels by Mc-3 treatment is due to increased glucose utilization, liver and muscle glycogen levels were measured. A significant reduction in both the liver and muscle glycogen levels was observed in the diabetic animals when compared to normal controls. Mc-3 treatment resulted in an increase of ~63% and ~80% in glycogen content of liver and muscle when compared to the PBS-treated diabetic animals (Figure
Effect of fraction Mc-3 of
The effect of Mc-3 treatment on key enzymes of glucose utilization in the livers of treated animals was also evaluated. To understand the possible mechanism of action, the diabetic animals were administered with either fraction Mc-3 or same volume of saline and maintained for 3 days on respective treatment. On day 4, the animals were injected with the test samples and were sacrificed after 3 hr of injection. Activities of the various regulatory enzymes were estimated in the livers of the control (saline treated) and Mc-3-treated diabetic animals (Figure
Control diabetic animals showed reduced levels of all the enzymes when compared to normal controls. It was observed that fraction Mc-3 resulted in a significant increase (~2 fold) both in pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G6PDH) activities, whereas only ~1.3 and ~1.6 fold increase in the specific activity of hexokinase (HK) and malic enzyme (ME) was noted. However, no significant change in lactate dehydrogenase (LDH) activity was observed. Insulin-treatment also resulted in an increase in the activities of all the enzymes and restored their levels to that observed in normal non-diabetic animals.
Earlier reports have indicated that the Mc fruit/seeds contain polypeptides that are capable of reducing blood glucose levels [
SDS-PAGE analysis of different fractions of
(a) Gel filtration chromatography of fraction Mc-3 of
In order to determine which of the major peak fractions consisted of the anti-hyperglycemic activity, these fractions were dialyzed and tested for their anti-hyperglycemic potential in diabetic animals. Fractions corresponding to different peaks were designated as fractions Mc-3.1 (fractions 31-32), Mc-3.2 (fractions 35–40), Mc-3.3 (fractions 43–46), Mc-3.4 (fractions 47–49), and Mc-3.5 (fractions 50–53). Fraction Mc-3.1 could not be evaluated for its anti-hyperglycemic activity due to small amounts. Of the remaining fractions, only fraction Mc-3.2 resulted in significant decrease (~40%) in blood glucose levels (Figure
(a) Determination of the anti-hyperglycemic effect and putative nature of fraction Mc-3.2 of
To check the ability of fraction Mc-3.2 to maintain normoglycemia in normal animals, glucose tolerance test was performed. Administration of Mc-3.2 resulted in faster clearance of blood glucose levels in normal rats when compared to PBS-treated controls, without causing hypoglycemia (Figure
Glucose tolerance test (GTT) with fraction Mc-3.2 in normal rats. After glucose load of 2 g per kg b.wt., normal Wistar rats were treated with fraction Mc-3.2 (15 mg/kg body wt) and equal volume of PBS (control). Changes in serum glucose levels were determined at different time intervals. Values are plotted as mean ± SD from at least five rats in each group.
Hyperglycemia associated with diabetes mellitus can be controlled by diet management, exercise, oral hypoglycemic agents, and insulin therapy. Both insulin therapy and oral hypoglycemic agents have their own side effects and adaptation problem. The secondary complications of diabetes that appear with lapse of time are actually the major cause of morbidity and mortality. Therefore, development of new approaches for treatment of diabetes that can reduce blood sugar level with better adaptation is desirable.
The evaluation of plants and especially of their active principles is a logical way of searching for new drugs to treat the disease. However, the presence of undesirable hyperglycemic substances along with the hypoglycemic components in
Many small molecular weight proteins, polypeptides with hypoglycemic and/or anti-hyperglycemic activities have been isolated from
The anti-hyperglycemic activity of the fraction Mc-3 at a much lower dose (15 mg/kg b.wt.) is significantly higher than that observed with acid-ethanol extract (fraction Mc-1) or other fraction Mc-2 derived from Mc-1. It appears that fraction Mc-2 consists of some toxic compounds that caused skin lesion and necrosis at the site of injection. Over a period of 18 days treatment daily, the blood glucose levels of fraction Mc-3-treated diabetic rats were significantly reduced, a desirable criterion for any potential anti-diabetic agent. Also, no hypoglycemic condition was observed in the treated animals. The fraction Mc-3 was effective at much lower concentration (15 mg/kg b.wt.) when compared to that of the crude ethanolic extracts of other plants which were found to be effective in the range of 100–500 mg/kg b.wt. in diabetic rats [
Hyperglycemic condition due to partial or total lack of insulin arises because of disturbances in glucose metabolism caused by a decrease in several key enzymes of glycolysis, namely, glucokinase, phosphofructokinase, and pyruvate kinase, thus resulting in impaired peripheral glucose utilization and augmented hepatic glucose production [
It is of interest to note that the treatment of diabetic animals with Mc-3 resulted in reduction in the serum levels of marker enzymes of liver function, namely, SGOT, SGPT, and GGT. An increase in the serum levels of hepatic function marker enzymes generally arises due to necrotized liver conditions caused by chronic diabetes [
Earlier reports on the anti-diabetic potential of
Body weight
Glucose-6-phosphate dehydrogenase
Hexokinase
Pyruvate kinase
Lactate dehydrogenase
Glucose tolerance test.
The Department of Biotechnology, Ministry of Science and Technology, New Delhi, is acknowledged for providing financial support. The Indian Council of Medical Research and the University Grants Commission are acknowledged for providing research fellowships to G. Chhabra and A. Vashishta, respectively. The Council of Sceintific and Industrial Research is acknowledged for providing research fellowships to D. Sharma and S. Ohri. Mr. Samaresh Singh and Mr. Amaresh Kumar Singh are acknowledged for technical assistance.