RJPS Vol No: 14 Issue No: 1 eISSN: pISSN:2249-2208
Mohammed Semol, Rema Razdan, Nathani Minaz*, Dhanashree Upadhye
Department of Pharmacology, Al-Ameen College of Pharmacy, Hosur Road, Near Lalbagh Main Gate, Bangalore-560027, Karnataka.
Author for Correspondence
Dr. Nathani Minaz
Department of Pharmacology
Al-Ameen College of Pharmacy
Hosur Road, Near Lalbagh Main Gate
Bangalore-560027, Karnataka, India.
E-mail: nathaniminaz@yahoo.co.in
Abstract
Cognitive impairment is one of the major and least identified complication of diabetes mellitus. The objective of our study was to evaluate the protective effect of allantoin, beta cyclodextrin and combination of both in diabetic cognitive impairment. Diabetes was induced by single intraperitoneal administration of streptozotocin (STZ) (53 mg/kg body weight). After 8 weeks of treatment, spatial memory, percentage of glycosylated haemoglobin and the levels of neurotransmitters in the cerebellum part of brain were measured. In addition to this hippocampal acetylcholinesterase activity, antioxidant enzymes and MDA levels were also assessed. Our results exhibit that treatment with allantoin, beta cyclodextrin and combination of both improved spatial memory, decreased glycosylated haemoglobin and increased neurotransmitters levels. Further, the treatment decreased acetylcholinesterase activity and lipid peroxidation and increased the antioxidant enzymes. Thus we conclude that allantoin, beta cyclodextrin and combination of both ameliorates the diabetes induced cognitive impairment in rats.
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INTRODUCTION
Diabetes mellitus (DM) is a complex metabolic disorder which has devastating effects on multiple organs.1 One of the organs which is affected by chronic hyperglycemia is brain. DM poses a wide range of changes in the neural structure of brain, neurotransmitter levels, electrophysiology and blood circulation.
Diabetic cognitive impairment is a clinical complication of DM that manifests as a deficit in reasoning ability, learning and memory, lack of attention and is a serious problem with rising prevalence worldwide.2 Studies suggests individuals with type 2 diabetes mellitus are more prone to the incidence of cognitive impairment than those without diabetes.3
The pathology of diabetes induced cognitive impairment includes, hyperglycemia, hypoglycemia, oxidative injury, neuronal apoptosis,2 abnormal expression of voltage gated potassium channels4-7 and increased formation of advanced glycation end products (AGEs).8
Allantoin is a heterocyclic organic compound9 and a product of uric acid. Increasing experimental and clinical data shows that uric acid plays a significant role in vivo as an antioxidant molecule10. The detailed radioligand studies confirmed the presence of imidazoline receptors such as I-1, I-2, and I-3 in the brain11 which regulates blood pressure12 glucose uptake into muscle cells13-14 and participation in insulin release15 respectively. Moreover, it has been documented that compounds with guanidinelike structures may bind to imidazoline receptors. Since, allantoin belongs to a guanidium derivative, it is of special interest to underst and the effect of allantoin in diabetic cognitive impairment.
Beta cyclodextrin is a cyclic heptamer composed of seven glucose units joined head to tail by alpha 1-4 links. It is produced by the action of enzyme cyclodextrin glycosyltransferases on hydrolysed starch syrups. β-cyclodextrins are one of those cyclodextrin derivatives that pose better water solubility16 In the pharmaceutical industry, cyclodextrins have mainly been used as complexing agents to increase the aqueous solubility of active substances poorly soluble in water, in order to increase their bioavailability and to improve stability. In addition, cyclodextrins can be used to reduce or prevent gastrointestinal and ocular irritation, reduce or eliminate unpleasant smells or tastes, prevent drug-drug or drug-additive interactions within a formulation (all these properties are based on reduction of the free drug in solution), or to convert oils and liquid drugs into microcrystalline or amorphous powders.17 Thus, aim of the study is to evaluate protective effect of allantoin, beta cyclodextrin and combination of both in diabetes induced cognitive impairment.
MATERIALS AND METHODS
Animals
Healthy male Wistar rats weighing about 200- 260 g were selected and housed in a group of three in polypropylene cages with clean paddy husk bedding. Animals were maintained under controlled temperature at 20±2 ºC and relative humidity of 45-55% with an alternative 12 h light/ 12h dark cycle with free access to water and standard pellet diet. The animals were used for the experiment after the approval by Institutional Animal Ethics Committee (IAEC, reference number: AACP/IAEC/Dec2016/03) of Al-Ameen College of Pharmacy and CPCSEA (The Committee for the Purpose of Control and Supervision of Experiments on Animals) guidelines were followed.
Chemicals
Streptozotocin was purchased from MP Biomedical India Pvt. Ltd., Allantoin from Himedia Pvt. Ltd. and Beta cyclodextrin from Spectrochem Pvt. Ltd. All the other chemicals and reagents used were of analytical grade.
Induction of diabetes
STZ was administered intraperitonealy at a dose of 53 mg/kg body weight by dissolving in freshly prepared ice cold 0.1 M sodium citrate buffer (pH 4.5). 24 hours after the injection of STZ rats were given 10% w/v glucose solution to avoid hypoglycemic shock. After a week, fasting blood glucose (FBG) levels were estimated; rats with FBG >250 mg/dl were considered diabetic and divided into following groups.
Group 1: Normal rats (distilled water, p.o; for 8 weeks)
Group 2: Diabetic rats (53 mg/kg body weight, i.p; of single injection of STZ once)
Group 3: Diabetic rats + rats administered allantoin (200 mg/kg;p.o) for 8 weeks
Group 4: Diabetic rats + rats administered βetacyclodextrin (31.5 mg/kg;p.o) for 8 weeks
Group 5: Diabetic rats+ rats administered βetacyclodextrin (31. 5mg/kg;p.o)+allantoin (200 mg/ kg; p.o) for 8 weeks.
Measurement of body weight
Body weight of the animals were measured at the beginning of the experiment and every week using electronic balance throughout the study. Doses of the drugs were adjusted according to the body weight of the animals.
Evaluation of spatial memory by Barnes maze
The basic function of Barnes maze is to measure the ability of a rodent to learn and remember the location of a target zone using a configuration of distal visual cues located around the testing area18. The method was performed according to the method of Rosenfeld et al. 19
Estimation of Glycosylated haemoglobin (GHb) level
Glycosylated hemoglobin was determined by assay kit (Excel Diagnostics Pvt. Ltd. India) based on ion exchange method (Nathan et al.)20
Estimation of neurotransmitter levels in cerebellum
On the day of experiment rats were sacrificed, cerebellum part of brain was dissected out and was homogenized in 25 ml of HCl–butanol for about 1 min. The sample was then centrifuged for 10 m at 2000 rpm. The aqueous phase was used for dopamine (DA) and norepinephrine (NE) quantification by spectrofluorimetric method.21
Quantification of acetylcholinesterase (AChE) activity
The hippocampus were homogenized in 0.1M phosphate buffer separately and used for the quantification of activity of AChE according to the method of Jangra et al. 22
Estimation of lipid peroxidation and antioxidant enzyme
The hippocampus was washed with saline, chopped over ice and homogenates (10% w/v) were prepared with 0.1M phosphate buffer. The homogenates were centrifuged for 10 m at 3000 rpm at 4 ºC using SorvallTM. The supernatants were used for the estimation of MDA23 and GSH.24
Statistical analysis
All data were expressed as mean ± SEM. Statistical analysis was carried out by one-way anova followed by Dunnet’s test by using Graph Pad prism(version 5.03). $$$ p<0.001 Vs normal control and *** p<0.001 Vs diabetic control.
RESULTS
Effect of allantoin, beta cyclodextrin and combination of both on %change in body weight of diabetic rats
There was a significant reduction in body weight of diabetic rats when compared to normal rats. Diabetic rats treated with allantoin, beta cyclodextrin and combination of both exhibited a significant increase in the body weight than those of diabetic control rats. (Fig.1)
Effect of allantoin, beta cyclodextrin and combination of both on spatial memory by using Barnes maze
The time taken by the diabetic control rats to enter the escape cage was significantly increased when compared to normal rats. The time taken by the diabetic rats treated with allantoin, beta cyclodextrin and combination of both to enter the escape cage was significantly decreased than that of the diabetic control rats. (Fig. 2)
Effect of allantoin, beta cyclodextrin and combination of both on glycosylated haemoglobin in diabetic rats.
The percentage glycosylated haemoglobin of diabetic control rats were significantly increased when compared to normal rats, whereas all the treatment groups showed the significant decrease in the percentage glycosylated haemoglobin when compared with the diabetic control group. (Fig. 3).
Effect of allantoin, beta cyclodextrin and combination of both on neurotransmitter levels in the brains of diabetic rats.
The brain DA and NE levels in the diabetic control were significantly decreased when compared to normal rats. However, an elevation in the DA and NE levels was observed in the diabetic rats treated with allantoin, beta cyclodextrin and combination of both when compared to diabetic control rats. (Fig. 4)
Effect of allantoin, beta cyclodextrin and combination of both on brain acetylcholinesterase activity in diabetic rats.
The brain acetylcholinesterase activity of diabetic rats was significantly decreased when compared to normal rats. In contrast, the administration of allantoin, beta cyclodextrin and combination of both to the diabetic rats significantly increased the acetylcholinesterase activity compared to diabetic contol rats. (Fig. 5)
Effect of allantoin, beta cyclodextrin and combination of both on MDA levels and antioxidant enzymes in diabetic rats.
The brain MDA levels of normal rats was significantly lower when compared to diabetic rats. The brain MDA levels of diabetic rats treated with allantoin, beta cyclodextrin and combination of both was significantly lower when compared to diabetic rats. (Fig. 6.1)
The brain GSH levels of normal rats was significantly greater when compared to diabetic rats. The brain GSH levels of diabetic rats treated with allantoin, beta cyclodextrin and combination of both was significantly increased when compared to diabetic rats. (Fig. 6.2)
DISCUSSION
Cognitive impairment is one of the common complication of chronic hyperglycemia. It needs to be treated in the early stages in order to prevent further deterioration like dementia and alzheimer’s disease. In this study, we examined the effect of administration of allantoin, beta cyclodextrin and combination of both on diabetic cognitive impairment in rats. STZ induced diabetic rats were used as the animal models to study the diabetes induced cognitive impairment.
Body weight is the sign of good health. So, in the present study body weight was recorded throughout the study. A significant decrease in the body weight was observed in diabetic rats as compared to normal rats, which could be due to muscle wasting which causes severe damage to the energy reserves in the rats. Treatment with allantoin, beta cyclodextrin and combination of both significantly increased the body weight when compared with diabetic rats.
In this study, behavioral tests showed that persistent hyperglycemia declined the cognitive functions of spatial memory. Barnes maze test was carried out to measure the spatial memory of rats. The time taken by diabetic rats to locate the escape cage was significantly increased than those compared to normal rats. Our results are in accordance with the results of Minaz et al.25 However, the treatment with allantoin, beta cyclodextrin and combination of both improved spatial memory in diabetic rats.
In type 1 diabetes mellitus, hyperglycaemia is directly proportional to the rate of glycosylation of haemoglobin causing the formation of GHb (glycosylated haemoglobin). GHb is implicated in various diabetic microvascular complications like cognitive impairment, neuropathy, nephropathy, retinopathy etc. Glycosylation may lead to the formation of oxygen-derived free radicals in diabetes mellitus and its levels can be considered as one of the important marker of oxidative stress. Our results showed that the percentage GHb in diabetic control rats was significantly increased when compared to that of normal rats. However, the rats treated with drugs under study showed a significant reduction in percentage GHb when compared to that with the diabetic control rats, which indicates antihyperglycemic potential of allantoin, beta cyclodextrin and combination of both. So, improvement in cognitive function of diabetic rats by treatment may be due to their antihyperglycemic activity.
Neurotransmitters play an important role in cognitive functions. Any alterations in neurotransmitter levels leads to cognitive dysfunction. Dopamine (DA) levels in cerebellum of diabetic control rats were significantly increased as compared with the normal rats. Diabetes significantly increased DA levels of cerebellum. Treatment with allantoin, beta cyclodextrin and combination of both significantly decreased the DA levels in the cerebellum of the treated rats as compared with rats of diabetic control. The results of our work regarding decrease in DA levels were in agreement with previous works of Shimomura et al.26 Norepinephrine increases arousal and alertness, retrieval of memory and focuses attention. NE level was significantly decreased in the brains of diabetic control rats when compared with the normal rats. The decrease in levels of NE in the cerebellum was in harmony with the results of Ramakrishna et al.27 During our study we found that treatment with allantoin, beta cyclodextrin and combination of both significantly increased the levels of NE when compared with the diabetic control rats.
Acetylcholine is the principal neurotransmitter of cholinergic system and modulates attention, learning and memory. Acetylcholinesterase is the enzyme which is involved in the hydrolysis of acetylcholine, terminating its physiological action. In our study, we observed a decrease in AChE activity in diabetic rats in comparison to normal rats, whereas an increase in AChE activity was observed in the diabetic rats treated with allantoin, beta cyclodextrin and combination of both when compared to diabetic rats.
The key mechanism involved in diabetes induced cognitive impairment is oxidative stress. Lipid peroxidation of polyunsaturated fatty acids results in the formation of malondialdehyde (MDA).28 The degree of oxidative stress depends on the amount of MDA produced.29 In our study, diabetic rats showed increase in the MDA levels whereas treatment with allantoin, beta cyclodextrin and combination of both decreased the levels of MDA. Glutathione (GSH) is an antioxidant enzyme capable of destroying reactive oxygen species (ROS). Our study exhibited increased level of GSH in the diabetic rats treated with allantoin, beta cyclodextrin and combination of both which indicates antioxidant potential of drugs. So, by exerting antioxidant activity the treatment could have prevented the cognitive impairment in diabetic rats.
CONCLUSION
Our study provides evidence that treatment with allantoin, beta cyclodextrin and combination of both ameliorates the diabetes induced cognitive impairment in rats.
ACKNOWLEDGMENTS
We are thankful to Principal and Management of Al-Ameen College of Pharmacy for providing infrastructural facility to carry out research work.
Supporting Files
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