T.O. Obafemi A.C. Akinmoladun M.T. Olaleye Stephen O. Agboade Amos A. Onasanya
Abstract
Background:
Synsepalum dulcificum is a plant indigenous to West Africa. The fruit is used to modify taste of foods to sweetness.
Objectives:
This study aims to investigate the antidiabetic potentials of both methanolic and flavonoidrich leaf extracts of S. dulcificum (MSD and FSD respectively) in type 2 diabetic Wistar albino rats.
Materials and methods:
Sixty three rats were randomly distributed into nine groups of seven animals each with group 1 serving as the normal control. Groups 2 to 7 were given 10% fructose in their drinking water for 14 days, after which 40 mg/kg of streptozotocin was administered. Group 2 animals served as the diabetic control, while groups 3, 4, 5, 6 and 7 were treated with 30 mg/kg MSD, 60 mg/kg MSD, 30 mg/kg FSD, 60 mg/kg FSD and 5 mg/kg glibenclamide respectively. Groups 8 and 9, contained healthy animals, and were treated with only 60 MSD, and 60 mg/kg FSD respectively. Biochemical parameters such as liver and kidney function tests, lipid profile, as well as lipid peroxidation and antioxidant enzymes were assessed in addition to histopathology.
Results:
It was observed that daily oral administration of MSD and FSD for 21 days signifificantly (p < 0.05) improved the observed pathological changes as a result of type 2 diabetes.
Conclusion:
It could be deduced from results obtained in this study that methanolic and flflavonoid-rich leaf extracts of S.dulcifificum have antidiabetic potential in type 2 diabetic rats. © 2017 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Publishing Services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Introduction
Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia and alteration of carbohydrate, proteins and lipids metabolism as a result of abnormal secretion and/or activity ofinsulin. Over 346 million people have diabetes, of which, type 2 diabetes mellitus (T2DM) makes up 90% of these cases. Type 1 diabetes is caused by lack of insulin due to the destruction of b-cells in the pancreas usually as a result of autoimmune destruction. Some of the causes of type 1 diabetes include genetic susceptibility, environmental factors, viruses andinfections. Type 2 diabetes is caused by a combination of genetic factors related to impaired insulin secretion and insulin resistance; and environmental factors such as obesity, overeating, lack of exercise, stress as well as aging. T2DM patients live in a chronic state of hyperglycemia due to progression of pancreatic beta cell dysfunction and insulin resistance. About 40% of prescription drugs are derived from herbs and about half of the world's best-selling drugs are derived from plants. Moreover, several studies have shown that flavonoids are known to exhibit strong antidiabetic and antioxidant activities. Consumption of flavonoids or flavonoid-rich compounds protects the body against free radicals and other pro-oxidative compounds, thereby reducing the risk of diabetes. Therefore, search for new antidiabetic drugs from natural plants is still attractive because they contain substances which have alternative and safe effect on diabetes mellitus. Concurrently, phytochemicals identified from traditional medicinal plants are presenting exciting opportunities for development of new drug therapies for diabetes. Synsepalum dulcifificum is also known as miracle fruit, magic fruit, miraculous or flavor fruit. Compounds such as b-sitosterol, stigmasterol, pheophytin-a, pheophytin-b, lupeol, lupenone, lupeolacetate, and a-tocopheryl quinone were isolated from the leaves of S. dulcificum. It was reported that miracle fruit may be used as an adjuvant for treating diabetic patients with insulin resistance because this fruit has been shown to have the ability to improve insulin sensitivity. We have identified active principles in the methanolic extract of S. dulcifificum leaves using high performance liquid chromatography (HPLC). Rutin, quercetin, isoquercitrin, quercitrin, kaempferol, ellagic acid, caffeic acid, chlorogenic acid, catechin, gallic acid, epicatechin, tocopherol, b-carotene and lycopene have all been identified in the extract. This study reports the antidiabetic potentials of methanolic and flavonoid-rich extracts of S. dulcificum leaves in type 2 diabetic Wistar albino rats.
Materials and Methods
2.1. Chemicals and reagents
Glibenclamide and streptozotocin, Reduced glutathione and epinephrine and all other reagents used were of analytical grade.
2.2. Plant material and preparation
Fresh S. dulcifificum leaves , Avoucher number UIH-22457 was obtained for the leaf. The leaves were air-dried for three weeks and pulverized. A portion of the pulverized sample (700 g) was extracted in 80% methanol by maceration for 72 h. The methanolic extract was concentrated in a rotary evaporator, lyophilized and preserved for further use.
2.3. Extraction of flavonoids A known gram of the methanol extract was dissolved in 20 ml of 10% H2SO4 and hydrolysed by heating in the water bath for 30 min at 100 C. The mixture was placed on ice for 15 min for precipitation of the flavonoid aglycones. The flavonoid aglycones were then dissolved in 50 ml of warm 95% ethanol, filtered and concentrated by rotary evaporation.
2.4. Induction of diabetes and experimental design
Type 2 diabetes was induced according to the method of Rachel and Shahidul. After giving water containing 10% fructose to rats for 14 days, streptozotocin (40 mg/kg body weight) in ice-cold 0.1 M citrate buffer (pH 4.5) was administered to the animals after an overnight fast. After 72 h of streptozotocin administration, blood glucose level was checked using Accu-check glucometer and animals with glucose levels 250 mg/dl were considered diabetic. The animals were then divided into nine groups with seven animals apiece. Group 1 served as the normal control, group
2 was the diabetic control while groups 3, 4, 5, 6 and 7 were diabetic animals treated with 30 mg/kg methanol extract, 60 mg/kg methanol extract, 30 mg/kg flavonoid-rich extract, 60 mg/kg flavonoid-rich extract and 5 mg/kg glibenclamide respectively. Groups 8 and 9 were administered 60 mg/kg methanol extract and
60 mg/kg flavonoid-rich extract respectively. All animals were administered the extract for 21 days, and sacrifificed 24 h after the last dose of the extract.
2.5. Oral glucose tolerance test (OGGT)
Oral glucose tolerance test was conducted to assess glucose utilization in experimental animals. On day 21, animals in groups 1e7 were fasted overnight prior to the administration of extracts and glibenclamide at 0 h. Glucose (2 mg/kg) was administered to all the groups and blood glucose was checked at 0 h (before any
treatment), 60 min and 120 min.
2.6. Biochemical assays
Glucose, urea, ALT, AST, ALP, HDL, total cholesterol, triglyceride and total protein levels were estimated. Catalase activity was estimated using the method of Sinha and assessment of lipid peroxidation was done. Glutathione S-transferase activity, superoxide dismutase activity, while glutathione peroxidase activity were all measured.
2.7. Histopathological study
Liver, pancreas and kidney tissues of animals were used for histopathological study. Tissues were fixed in 10% buffered formalin, routinely processed and embedded in parafin wax. Sections were cut on glass slides at a thickness of 4 mm and stained
with hematoxylin and eosin (H&E) (Culling, 1974). The slides were examined under a light microscope and the magnified images of the tissue structures were captured.
2.8. Statistical analysis
Results were expressed as mean value ± standard error of mean (SEM). Data analysis was done using GraphPad Prism 5 software by one-way analysis of variance (ANOVA) followed by Tukey-test. In all instances p values <0.05 were considered statistically significant.
Results
3.1. Effect of treatment on body weight Treatment of diabetic animals with MSD, FSD and glibenclamide led to increase in body weight in most of the animals. The diabetic control group showed a significant (p < 0.05) reduction in body weight when compared with the normal control, extracts and glibenclamide treated groups.
3.2. Serum glucose level
The extract and glibenclamide treated groups showed a significantly lower serum glucose levels when compared with the diabetic control group and their glucose levels were closer to that of the normal control. At the end of the study, the glucoselevels of animals treated with extracts only were not significantly (p < 0.05) different from that of control.
3.3. Oral glucose tolerance test
The extract and glibenclamide treated diabetic animals showed better utilization of glucose.
3.4. Serum level of ALP, AST, ALT, total protein, urea and creatinine There were significantly (p < 0.05) lower serum levels of ALP, AST, ALT, urea and creatinine in the MSD, FSD and glibenclamide treated diabetic animals when compared with the diabetic control. AST and ALT levels of the extract and glibenclamide
treated diabetic animals were significantly (p < 0.05) different from the normal control. Serum total protein level of the diabetic control group was however significantly (p < 0.05) lower than the normal control, extract and glibenclamide treated groups.
3.5. Lipid profile
Total-cholesterol, triglyceride and LDL-cholesterol levels of the diabetic control group were significantly (p < 0.05) higher when compared with the normal control, extract and glibenclamide treated groups. The HDL-cholesterol was however significantly (p < 0.05) lower in the diabetic control group than in the normal control group, extract and glibenclamide treated diabetic groups; and normal animals treated with extract only.
3.6. Liver total protein and MDA levels, and antioxidant enzyme activities
A significantly (p < 0.05) lower total protein level, SOD, GST and GPx and catalase activities were observed in the liver of diabetic control group when compared with the normal control and MSD treated groups. Catalse activity in FSD and glibenclamide treated groups were not significantly different from the diabetic
control group. However, a significantly (p < 0.05) higher hepatic MDA level was observed in the diabetic control group when compared with the other groups in the study.
3.7. Pancreatic total protein and MDA levels, and antioxidant enzyme activities
A signifificantly (p < 0.05) lower pancreatic total protein, SOD, GST, GPx and catalase levels were observed in the diabetic control group when compared with the normal control, FSD and glibenclamide treated groups. Catalase activity in diabetic
animals treated with MSD was not significantly different from diabetic control group. However, a significantly (p < 0.05) higher MDA level was observed in the diabetic control group when compared with the other groups in the study.
3.8. Kidney total protein level and antioxidant enzyme activities
Total protein, SOD, GST, GPx and catalase activities in the kidney of diabetic control group was significantly (p < 0.05) lower when compared with the normal control and both extract and glibenclamide treated groups.
Discussion
Reduction in body weight of diabetic animals has been linked to degradation of structural proteins and muscle wasting. There was a significant reduction in the body weight of diabetic control animals. Treatment with MSD and FSD however improved body weight to a reasonable extent as observed from the percentage change in body weights of experimental animals in the course of the study. Research has shown that sustained reduction in hyperglycemia associated with diabetes will decrease the risk of developing microvascular diseases and reduce diabetes complications. Furthermore, the antihyperglycemic property of several medicinal plants. The diabetic control group showed a persistently higher serum glucose levels throughout the experimental period while administration of MSD, FSD and glibenclamide significantly (p < 0.05) reduced the serum glucose levels in the treated diabetic animals. It has been severally stated that treatment of diabetic animals with medicinal plants could activate b-cells and normalize their granulation. This could result in potentiation of insulin secretion from the remaining pancreatic b-cells, or increased utilization of glucose by tissues. We propose that our extracts may exert their antihyperglycemic effect through the aforementioned mechanisms.
The results of the OGTT that the glucose levels of the animals reached a peak 60 min after glucose load. These observations indicate that the diabetic control group had a slower glucose utilization rate than other groups in this study, a hallmark of diabetes. We therefore suggest that both FSD and MSD could be useful in improving glucose intolerance and insulin resistance associated with type 2 diabetes.
Experimentally induced diabetes indicates several alterations of amino acid metabolism, which may be attributed to increased muscle proteolysis, reduced protein synthesis which is an energy dependent process in the liver, and stimulated hepatic gluconeogenesis utilizing glucogenic amino acids. Renal damage consequent upon persistent hyperglycemia associated with diabetes is linked with increased levels of urea and creatinine.
The World Health Organization states that diabetes is a leading cause of kidney failure which is responsible for 10%e20% of deaths in diabetic people. The fact that increase in the serum levels of biomarker enzymes, such as AST, ALT, and ALP (as observed in diabetic rats) indicates organ damage. This is usually as a result of leakage of the enzymes from organs where they are located into the blood stream. Our results corroborates this fact as a significantly higher level of the enzymes were observed in the serum of animals in the diabetic control group when compared with the other groups. Diabetes is a metabolism-associated disease, particularly closely
related to lipid metabolism, affecting the serum lipid and lipoprotein profile.
Type 2 diabetes-associated cardiovascular complications are due to lowered HDL and elevated triglyceride, lowdensity lipoprotein (LDL) and cholesterol levels. Antidiabetic agents have been reported to reduce the cardiovascular risk by controlling the lipid profile levels in diabetic patients. In the present study, shows that lipid metabolism in diabetic control rats was markedly deranged.
Insulin resistance further exacerbates this atherogenic dyslipidemia by increasing the hepatic secretion of VLDL and other apolipoprotein (apo) B-containing lipoprotein particles such as LDL.
Administration of glibenclamide, MSD and FSD signifificantly (p < 0.05) increased the antioxidant enzymes activities in the liver, pancreas and kidney respectively to those comparable with that of the normal control group. Catalase activity in the liver of diabetic animals treated with FSD and glibenclamide, and pancreas of diabetic animals treated with MSD were not significantly (p < 0.05) different from diabetic controls. Treatment for 21 days with both extract and glibenclamide was observed to protect/prevent the organ damage that was still evident in the diabetic control group at the end of the study. This further corroborates the protective effect of the extracts on the organs of diabetic animals as observed from biochemical analyses.
Conclusion
The methanolic and flavonoid-rich extracts of S. dulcificum leaves have definite antidiabetic activities, and compared favorably with glibenclamide. We suggest that the antidiabetic potential of both MSD and FSD is due to the presence of the identified polyphenols in the extract. However, further studies should focus on the characterization of these active principles. This will enhance studies on the mechanism of action of the principles which may be acting singly or in synergy to bring about the observed activity.
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