Protective and curative effects of Cocos nucifera inflorescence on alloxan-induced pancreatic cytotoxicity in rats
Abstract
Objectives:
This study was planned to investigate the effects of pre and post-treatment of young inflorescence of Cocos nucifera (CnI) on alloxan-induced diabetic rats.
Materials and Methods:
Male albino Sprague Dawely rats were divided into five groups of six animals each. Group I was normal control, Group II was diabetic control, Cocos nucifera Inflorescence (CnI) was fed along with diet [20% (w/w)] orally (Group III) for a period of 11 days prior to alloxan injection (150 mg/kg i.p.). The curative effect of CnI was evaluated at the same feeding levels in alloxan-induced diabetic rats (Group IV) for a period of 30 days. The effects of both pretreatment and post-treatment (Group V) were also evaluated. Biochemical parameters such serum glucose, hepatic glycogen, and enzymes involving carbohydrate metabolism (hexokinase, phosphoglucomutase, pyruvate kinase, glucose-6-phosphatase, fructose 1, 6-diphosphatase, glucose-6 phosphate dehydrogenase, and glycogen phosphorylase) were assayed along with pancreatic histopathology. Data were analyzed using one-way analysis of variance followed by Duncan's post hoc multiple variance test. P < 0.05 was considered statistical significant.
Results:
Diabetic control rats showed significant increase in serum glucose (P < 0.05) and decrease in hepatic glycogen levels (P < 0.05) compared to normal rats, which was reversed to near normal in both CnI pretreated and post-treated rats. Treatment with CnI resulted in significant decrease (P < 0.05) in activities of gluconeogenic enzymes in Group III and IV on compared to the diabetic control group, while glycolytic enzyme activities were improved in these groups. The cytotoxicity of pancreatic islets also ameliorated by treatment with CnI on histopathological examination.
Conclusion:
The results obtained in the study indicate the protective and curative effects of CnI on alloxan-induced pancreatic cytotoxicity, which is mediated through the regulation of carbohydrate metabolic enzyme activities and islets cell repair.
Introduction
Diabetes mellitus (DM) is probably the single most important metabolic disease and is widely recognized as one of the leading causes of death and disability. Diabetes causes profound disturbances of carbohydrate, protein, and lipid metabolism that leads to pathological changes in the organs and subsequent vascular complications. DM arises due to a deficiency of insulin secretion from the beta cells of the pancreas or a deficiency of the insulin action.[1] Alloxan, a cyclic urea derivative, acts as a diabetogenic agent by its ability to cause selective cytotoxicity and necrosis of the beta cells of endocrine pancreas.[2] Enzyme activities of gluconeogenesis have been shown to increase during the course of diabetes, with a simultaneous increase in the glycogenolytic and lipolytic pathways. Diabetes also is accompanied by a decrease in the enzyme activities of glycolytic and pentose phosphate pathways.[3]
The treatment of DM in clinical practice has been confined to the use of oral hypoglycemic agents and insulin. But, oral hypoglycemic drugs have been reported to be endowed with serious side effects due to their continuous intake. Therefore, many studies have been undertaken to investigate the potential of natural medicines for the effective treatment of diabetes.[4] Though hundreds of plants are used in the world to prevent or to cure diseases, scientific evidence in terms of modern medicine is lacking in most cases. However, today it is necessary to provide scientific proof in order to justify the use of the plant or its active principles.[5] Coconut palm (Cocos nucifera L.) is a versatile plant that is cultivated in most tropical countries. It has various applications in medicinal, culinary, and commercial fields. People in Kerala largely depend on coconut and its products for dietary and economic purposes, because of its abundance. The peculiar type of floral arrangement in coconut palm is called the inflorescence. The young inflorescence of coconut is widely used for the production of neera, a natural drink and also for coconut sap sugar. The creamy preparation obtained from young inflorescence is used to cure back ache in Indian traditional medicine. Previous reports from our laboratory using the coconut kernel protein and fiber demonstrated their antidiabetic properties.[67] This study was conducted to evaluate the protective role of young inflorescence of coconut palm against alloxan-induced pancreatic β-cell damage in rats and also to modulate the abnormal biochemical parameters associated with alloxan-induced experimental diabetes.
Materials and Methods
Plant Material Collection And Preparation
Young inflorescence of coconut palm of West Coast Tall variety harvested from Kerala University campus was used for the study. The material was identified and confirmed at Department of Botany in the University and a voucher specimen (No. KUBH 5795) was deposited in the department herbarium. The inflorescence was then pulverized very well using an electric laboratory blender. It was then dried at 55°C in a hot air oven; the dry weight was taken and used for phytochemical analysis and in vivo experiments.
Preliminary Phytochemical Screening
The Cocos nucifera inflorescence (CnI) was subjected to various qualitative tests for the presence or absence of different phytochemical constituents.[8]
Animals
One month old male Sprague Dawley rats (150–200 g body weight) bred in our department animal house were used. The rats were housed individually in polypropylene cages in a room maintained at 25 ± 10°C with a 12 h light and 12 h dark cycle. All the animal cares and procedures were according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. This experimental protocol was approved by Institutional Animal Ethics Committee (IAEC).
Chemicals
Alloxan was purchased from Sigma Chemicals, St. Louis, USA. Kit for glucose estimation was purchased from Agappe Diagnostics, Thane, India. All other chemicals used were of analytical grade.
Experimental Design
The rats were randomly divided into five groups of six each. Group I served as the normal control while Group II served as the diabetic control. Rats in Group III (CnI pretreated) and V (CnI pretreated and post-treated) were fed CnI along with laboratory animal diet [20% (w/w)] for 11 days prior to alloxan injection. On day 12, diabetes was induced in rats of group II, III, IV, and V by injecting them with alloxan (150 mg/kg body weight, dissolved in normal saline) intraperitoneally after overnight fasting. The rats received 5% glucose solution for the next 24 h to prevent drug induced hypoglycemic phase. All rats were found to be diabetic after 72 h. Rats with a blood glucose level ≥ 250 mg/ dL were considered to be diabetic. The curative effect of CnI was then evaluated using the same feeding levels in rats of group IV and V for a period of 30 days after alloxan administration. The details of groups are as follows:
Group I – normal control
Group II – diabetic control
Group III – diabetes + CnI pretreated for 11 days
Group IV – diabetes + CnI post-treated for 30 days
Group V – diabetes + CnI pretreated+ post-treated
After the experimental period, animals were fasted overnight and sacrificed by intraperitoneal injection of thiopentone sodium (40 mg/kg body weight). Blood and liver were collected immediately for various biochemical estimations.
Serum Glucose Levels
Estimation of serum glucose was done using a commercial kit based on glucose oxidase/peroxidase enzymatic method.[9]
Hepatic Glycogen Content
Glycogen was isolated from fresh liver tissues by adding 30% KOH, 0.5 mL of saturated Na2SO4, 1 mL of 95% ethanol and centrifuged at 2000 rpm for 10 min. The resulting pellet was redissolved in saline. From this 0.1 mL aliquot was taken and 2.5 mL anthrone was added, boiled at 90°C for 10 min, cooled in ice and absorbance was measured at 660 nm.[10]
Activity of Glycolytic Enzymes in the Liver
The following procedures were used to estimate the activities of enzymes involved in glycolysis in the liver of control and treated animals. Hexokinase (EC 2.7.1.1; ATP-D-hexose-6-phosphotransferase) activity was measured by the method described by Crane and Sols.[11] Phosphoglucomutase (EC 2.7.5.1) by the procedure described by Najjar.[12] Pyruvate kinase (EC 2.7.1.40) by the method of Theodore Bucher and Pfleiderer.[13]
Activity of Gluconeogenic Enzymes
Gluconeogenic enzyme activities in the liver were assayed using the following procedures. The activity of glucose-6-phosphatase (EC 3.1.3.9; D-glucose-O-phosphate phosphohydrolase) was measured using the method described by Koide and Oda.[14] Fructose 1, 6-diphosphatase (EC 3.1.3.11) by the procedure described by Sandro Pontremoli.[15]
Activity of Glycogen Phosphorylase and Glucose-6-phosphate Dehydrogenase
Activity of glycogen phosphorylase (EC 2.4.1.1; α- l, 4-glucan orthophosphate glucosyl transferase) was assayed by the procedure described by Singh et al.[16] and glucose-6 phosphate dehydrogenase (EC 1.1.1. 49; D-glucose-O-phosphate NADP oxidoreductase) by the method of Kornberg and Horecker.[17]
Histopathological Studies
Pancreatic tissues from all groups were subjected to histopathological studies. The whole pancreas from each animal was removed after sacrificing the animal under anesthesia, collected in 10% formalin solution and immediately processed by the paraffin technique. Sections of 5 μm thickness were cut and stained with hematoxylin and eosin (H and E) for histological examinations. Stained sections were qualitatively evaluated using a photo microscope (Zeiss Axioscope 2 plus, USA) equipped with Canon Zoom Browser EX digital camera. (Japan)
Statistical Analysis
All the grouped data were statistically evaluated with Statistical Package for Social Sciences (SPSS) version 17. (SPSS Inc., Chicago, USA). Hypothesis testing methods included one way analysis of variance (ANOVA) followed by Duncan's post hoc multiple variance test. P < 0.05 was considered statistical significant. Data were expressed as mean ± S.D in each group.
Results
Preliminary Phytochemical Screening
Preliminary phytochemical analysis of dried powdered CnI revealed the presence of proteins, carbohydrates, amino acids, terpenoids, fibers, phenolic compounds, flavanoids, tannins, and minerals.
Biochemical Parameters
Administration of alloxan on day 12 caused significant increase in serum glucose in Group II compared to Group I. Pretreatment with CnI had a moderate effect on reducing the serum glucose levels (Group III), while diabetic rats post-treated with CnI showed a significant reduction (P < 0.05) in the glucose levels compared to diabetic control (Group IV and V) [Table 1]. Hepatic glycogen in diabetic rats was found to be significantly reduced (P < 0.05) compared to the normal control. Treatment with CnI enhanced the glycogen storage efficiency of liver of diabetic rats (Group V) compared to diabetic control animals [Table 1].
Comparison of serum glucose and hepatic glycolytic enzymes in diabetic rats treated with Cocos nucifera Inflorescence
The activities of hexokinase, pyruvate kinase, and phosphoglucomutase were significantly decreased (P < 0.05) in Group II as compared to normal control animals. However, CnI pretreatment and post-treatment significantly increased (P < 0.05) these enzymes in diabetic rats as compared to Group II [Table 1]. On the other hand, there was an increase in the activities of gluconeogenic enzymes like glucose-6-phosphatase and fructose-1, 6 diphosphatase in Group II diabetic rats as compared to the normal rats. However, post-treatment with CnI showed restoration of these enzymes to the basal levels as compared to control rats [Table 2]. Administration of alloxan significantly (P < 0.05) elevated the activity of glycogen phosphorylase and reduced the activity glucose-6-phosphate dehydrogenase in diabetic control rats as compared to the normal animals. Post-treatment of CnI to diabetic rats restored the enzyme levels significantly (P < 0.05) to near the basal level [Table 2].
Comparison of hepatic gluconeogenic enzymes and glycogen phosphorylase in diabetic rats treated with Cocos nucifera Inflorescence
Histopathological Studies
Alloxan caused severe necrotic changes of pancreatic islets, especially in the centre of islets. Nuclear changes, karyolysis, and severe reduction of beta cells were observed in diabetic control rats. However, diabetic rats treated with CnI showed restoration of the altered architecture and number of islets, ameliorated karyolysis, and necrosis towards the normal morphology of pancreas [Figure 1].
Histopathology of pancreas of diabetic rats treated with Cocos nucifera Inflorescence (a) Group I – normal control, (b) Group II – diabetic control, (c) Group III – diabetic + CnI pretreated (d) Group IV – diabetic + CnI post-treated, (e) Group V – diabetic + CnI pretreated + CnI post-treated. Arrow shows islets β- cells
Discussion
This investigation aims to evaluate the preventive and modulatory effects of the young inflorescence of coconut palm against the pancreatic cytotoxicity produced by alloxan hydrate. Alloxan causes selective cytotoxicity and necrosis of the beta cells of endocrine pancreas.[2] The alloxanized rats showed severe hyperglycemia as well as metabolic stress due to progressive oxidative damage interrelated with a decrease in endogenous insulin secretion and release. Studies show that treatment with phytonutrients might be an effective strategy for reducing diabetes complications by influencing glucose metabolism and homeostasis by mechanisms such as modulation of glucose output from liver, inhibition of carbohydrate digestion and regulating the glucose metabolizing enzymes.
This study showed that pretreatment with CnI moderately reduced serum glucose levels in diabetic rats, while, CnI post-treatment significantly reduced glucose levels indicating the antihyperglycemic action of CnI. These are in agreement with the previous studies, where phytonutrients exerts antihyperglycemic effects by regulating the glucose output.[18]
The liver glycogen level may be considered as one of the best markers for assessing the insulin action. The decrease in hepatic glycogen of diabetic rats suggests the increased glucose output during insulin deficiency. Depletion of hepatic glycogen is due to the loss of glycogen synthase activating system and/or the increased activity of glycogen phosphorylase in diabetic rats. Because alloxan causes selective destruction of β-cells in the islets of Langerhans, resulting in a marked decrease in insulin levels, it follows that glycogen levels in the liver decrease because they depend on insulin for the influx of glucose. Administration of CnI to diabetic rats significantly improved hepatic glycogen levels. This is possibly due to the inactivation of the glycogen phosphorylation system following CnI treatment of diabetic rats. These results are in correlation with the previous reports.[19]
Carbohydrates, particularly glucose, are important source of energy for living organisms. In DM, the glucose homeostasis is altered and the major metabolic defects that contribute to persistent hyperglycemia are due to increased hepatic glucose release, defective insulin secretion and sensitivity and inability of insulin to stimulate glucose uptake in the peripheral target tissues. Liver functions as a “glucostat” and plays a vital role in the maintenance of the blood glucose level and it is the main site for glycolysis, a process where glucose is degraded and gluconeogenesis, where glucose is synthesized from lactate, amino acids, and glycerol. Blood glucose levels are maintained through these two different routes.[20] Hence, it is of interest to examine the possible role of CnI on key enzymes of carbohydrate metabolism in the liver.
In DM, the activity of enzymes involved in these pathways; such as hexokinase, pyruvate kinase, glucose-6- phosphatase and fructose 1, 6-diphosphatase is markedly altered, resulting in hyperglycemia, which leads to the pathogenesis of diabetic complications.[21] Hexokinase and pyruvate kinase, two important enzymes in the catabolism of glucose, are found to be decreased in the liver of diabetic control rats in the present study. Administration of CnI to alloxan-induced diabetic rats increased these enzyme activities; this in turn may result in increased glycolysis and increased utilization of glucose for energy production. The activities of regulatory enzymes in gluconeogenesis; glucose-6- phosphatase and fructose 1, 6-diphosphatase are elevated in diabetes mellitus[22] and increased activities of these enzymes in alloxan induced diabetic rats may be due to insulin insufficiency and have been reported in experimental diabetes previously.[23] In CnI, pretreated and post-treated rats, these two key enzymes were seen significantly reduced in the liver.
The glucose-6-phosphate dehydrogenase activity was decreased in diabetic condition can result in the diminished functioning of the pentose phosphate pathway and thereby the production of reducing equivalent such as NADH and NADPH. The significant increase in the activity of glucose-6-phosphate dehydrogenase in CnI treated hyperglycemic rats suggests that the hydrogen shuttle systems and the redox state of the cell become more oxidized, which results in the increased formation of NADPH for increased utilization in lipogenesis and in turn activates the enzyme, as NADPH is a strong inhibitor of glucose-6-phosphate dehydrogenase.[24] The regulation of glycogen metabolism occurs in vivo by the multifunctional enzyme glycogen synthase and glycogen phosphorylase and the reduced glycogen store in diabetic rats has been attributed to the increased activity of glycogen phosphorylase.[25] In this study, diabetic rats treated with CnI restored the levels of glycogen, probably by means of decreasing the activity of glycogen phosphorylase. Histopathological analysis of pancreatic tissue shown that pretreatment with CnI to diabetic rats ameliorated the altered architecture and number of islets, restored the pancreatic tissue integrity, and was able to regenerate the alloxan damaged pancreatic β-cells.
From this study, it can be concluded that the young inflorescence of coconut palm exhibited protective and ameliorative effects against alloxan-induced pancreatic cytotoxicity and severe hyperglycemia by enhancing the peripheral utilization of glucose, correcting the impaired liver glycolysis and limiting gluconeogenic formation and also repairing and rejuvenating the residual beta cell population. These effects may be due to the presence of phenolic acids, flavonoids, and other phytochemical constituents, which could act synergistically or independently in modulating the activities of glycolytic and gluconeogenic enzymes. Thus, the findings of the present study provide scientific validation for the use of CnI as a promising candidate in folk medicine in the treatment of diabetes.
Footnotes
Source of Support: Kerala State Council for Science Technology and Environment for the financial assistance in the form of KSCSTE Fellowship.
Conflict of Interest: None declared.
Acknowledgment
The authors are grateful to Kerala State Council for Science Technology and Environment for the financial assistance in the form of KSCSTE Fellowship.
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