|Year : 2016 | Volume
| Issue : 1 | Page : 25-31
Immunohistochemical evaluation of the antidiabetic potentials of S-allyl-cysteine (Garlic) and mangiferin (Mango) in type 2 diabetic rat models
IA Iliya1, B Mohammed2, SA Akuyam3, JD Yaro4, ZM Bauchi1, M Tanko1, J Idoko4, IL Aghemunu4, B Yusuf5
1 Department of Human Anatomy, Faculty of Human Medicine, Ahmadu Bello University, Zaria-Kaduna State, Nigeria
2 Department of Veterinary Pathology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria-Kaduna State, Nigeria
3 Department of Chemical Pathology, Ahmadu Bello University, Zaria-Kaduna State, Nigeria
4 Department of Histopathology, Ahmadu Bello University, Zaria-Kaduna State, Nigeria
5 Department of Integrated Science (Biology), College of Education, Minna, Niger State, Nigeria
|Date of Submission||01-May-2015|
|Date of Acceptance||22-Sep-2015|
|Date of Web Publication||12-Feb-2016|
I A Iliya
Department of Human Anatomy, Ahmadu Bello University, Zaria
Background: Diabetes mellitus is now one of the largest emerging pandemics of our time. Of the different types of diabetes mellitus, type 2 accounts for 90% of diabetic cases in humans worldwide. Insulin resistance followed by abnormal secretion of insulin from the pancreatic β-cells underlies the symptomatology of type 2 diabetes mellitus. Most investigations have assessed the hypoglycaemic potentials of s-allyl-cysteine and mangiferin by focusing on the biochemical and or pathophysiological changes. The histological and immunohistochemical changes have on the other hand received less attention. Aim: To evaluate the anti-diabetic potentials of s-allyl-cysteine (SAC), mangiferin (MAN) and a composite mixture of both (COM) in equal volume ratio (1:1) in type 2 diabetic Wistar rat models. Objective: This was achieved by evaluating the secretion of insulin in the pancreatic islets of diabetic and non-diabetic rats using immunohistochemistry techniques. Methodology: Eighteen (18) apparently healthy male albino Wistar rats (Rattus norvegicus) were grouped into 6 groups designated as non-diabetic control (NDC), diabetic control (DC), SAC, MAN, COM and glibenclamide (GLC). Insulin resistance was induced by first feeding the rats with a high-fat diet for a period of 10 weeks followed by a low dose of streptozotocin injection to induce type 2 diabetes mellitus. Therapeutic interventions was by the administration of 50 mg/kg body weight of SAC solution, 40 mg/kg body weight of MAN solution, equal volume ratio of both (SAC:MAN) and 5 mg/kg body weight of GLC. Results: Therapeutic interventions with the bioactive compounds significantly improved the glucose tolerance ability by ameliorating the hyperglycaemic condition in the diabetic rats which was significant at P < 0.05. Similarly, immunohistochemistry evaluation of the islet β-cells showed an increase in insulin secretion suggesting an improvement in glycaemic control and an eventual commitment of glucose to glycolysis. Conclusion: The amelioration of the type 2 diabetic mellitus by the bioactive compound therapies was due to the bioactive-mediated anti-hyperglycaemic and insulin release potentials. These potentials were observed to be more pronounced in the SAC group, followed by MAN group, then GLC group and lastly by COM group.
Keywords: Immunohistochemistry, insulin, mangiferin, mellitus, s-allyl-cysteine, type 2 diabete
|How to cite this article:|
Iliya I A, Mohammed B, Akuyam S A, Yaro J D, Bauchi Z M, Tanko M, Idoko J, Aghemunu I L, Yusuf B. Immunohistochemical evaluation of the antidiabetic potentials of S-allyl-cysteine (Garlic) and mangiferin (Mango) in type 2 diabetic rat models. Sub-Saharan Afr J Med 2016;3:25-31
|How to cite this URL:|
Iliya I A, Mohammed B, Akuyam S A, Yaro J D, Bauchi Z M, Tanko M, Idoko J, Aghemunu I L, Yusuf B. Immunohistochemical evaluation of the antidiabetic potentials of S-allyl-cysteine (Garlic) and mangiferin (Mango) in type 2 diabetic rat models. Sub-Saharan Afr J Med [serial online] 2016 [cited 2019 Oct 16];3:25-31. Available from: http://www.ssajm.org/text.asp?2016/3/1/25/176305
| Introduction|| |
Diabetes mellitus is the most common endocrine disorder in man. Prevalence and projection reports for the years 2013 and 2035, respectively, have shown it to currently affect over 382 million people worldwide, and this figure will likely increase potentially over 592 million by the year 2035.  A report of the World Health Organization in 1999  described in detail the physiologic impact of diabetes mellitus, thereby making it the sixth leading cause of death by disease globally.
Much more worrisome is the report of the International Diabetic Federation  describing diabetes mellitus as the fourth leading cause of death in developing countries. Type 2 diabetes mellitus is now one of the largest emerging pandemics of this modern age and is associated with several complications such as cardiomyopathy, neuropathy, nephropathy, and retinopathy.  It is tightly linked to dietary habits, obesity, physical inactivity, and sedentary lifestyle and characterized by insulin resistance and reduced or abnormal insulin secretion after some time. ,,, Globally, of the two types of diabetes mellitus, type 2 is the most common accounting for over 90% of diabetes cases worldwide and the less common type 1 accounts for about 5% of diabetes cases worldwide. ,, The prevalence of type 2 diabetes mellitus is even increasing in young children (5-12 years) and adolescents (13-19 years) accounting for approximately 45% of new cases among these groups.  About 50% of people with type 2 diabetes mellitus are unaware of their condition and in some countries, especially developing countries, this figure is even higher.  At present, oral therapy for type 2 diabetes mellitus relies on treatments including the use of insulin secretagogues such as sulfonylurea compounds and insulin sensitizers such as thiazolidinedione, but these are not without undesirable side effects or contraindications. 
Allium sativum commonly called garlic has been reported to pose many medicinal properties including antidiabetic and antilipidemic properties.  S-allyl-cysteine (SAC), a sulfur-containing amino acid derived from garlic, has been reported to account for most of its medicinal properties. ,,, Literature has shown that this SAC, a sulfur-containing amino acid in garlic, had a potential to reduce diabetic condition in rats almost to the same extent as did glibenclamide (GLC) and insulin. ,, Mangifera indica commonly referred to as mango (family-Anacardiaceae) is also a medicinal plant widely distributed in tropical regions and is used to cure a range of diseases. ,,,, The natural xanthone c-glucoside compound in it is called mangiferin (MAN) has been reported in various parts of the plant such as roots, leaves, stem bark, and fruits. ,,,, The effects of MAN on hyperglycemia, atherogenicity, and oxidative damage to cardiac and renal tissues in streptozotocin-induced diabetic rats have been investigated. , The reported pharmacological activities of MAN include antioxidant, ,,,, antitumor,  and anti-inflammatory,  and antidiabetic activity of MAN could involve pancreatic β-cell insulin release/secretion. ,,
Most studies on the effect of SAC or MAN in diabetes mellitus (type 1 or 2) have mainly focused on the biochemical and pathophysiological changes that occur. However, in comparison, the immunohistochemical and/or histological aspects of the pancreatic islets in the disease have received less attention.
Objective of the Study
This was to evaluate the antidiabetic potencies of SAC and MAN on insulin secretion in the pancreatic islets of both diabetic and nondiabetic rat models using immunohistochemistry (IHC) techniques.
| Materials and Methods|| |
Source of Experimental Animals and Husbandry
A total of 18 apparently healthy male albino Wistar rats (Rattus norvegicus) of approximately 5 weeks old were purchased from the Laboratory Animal Resource Center in the Department of Pharmacology, Ahmadu Bello University, Zaria, and then transferred to the Laboratory Animal Facility in the Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University. The rats were allowed to adapt to the new environment for 2 weeks before the commencement of the study. All the rats were fed with pelletized form of rat chow and allowed free access to water in plastic bottles with stainless steel nozzles. Housing of the rats was done after adhering to recommended guidelines based on animal welfare laws pertaining micro and macro environment conditions in-terms of space recommendations, temperature, humidity, ventilation, illumination, sanitation, and bedding materials as published by the Institute for Laboratory Animal Research. 
Drug and Bioactive Compounds
SAC 1 g, a pure white powder from garlic (98.68% purity by high-performance liquid chromatography [HPLC]) with a product ID PCM-0-006 and CAS No. 4773-96-0 and MAN 1 g, a pure yellow powder from mango leaves (98.46% purity by HPLC) with a product ID PCM-AA-003 and CAS No. 4773-96-0 were purchased from Phytomarker Ltd., Tianjin, China. CLEZIDE (GLC 100 tablets 5 mg; batch no. 141221) was purchased from Jiangsu Ruinian Qianjin Pharmaceutical Co., Ltd., China.
Induction of Experimental Diabetes Mellitus (Type 2)
Obesity, insulin resistance, and hyperglycemia are the hallmarks of type 2 diabetes mellitus. Thus, induction of obesity in the rats was achieved by mixing 100 g of pelletized rat chow with 40 g of animal derived fats ,,, for 10 weeks. All measurements were done with the aid of a digital top-loader weighing machine (Accu Labs). At the end of the 10 th week, diabetes was induced by injecting the rats with a freshly prepared streptozotocin injection (Zayo-Sigma, Nigeria) at a low-dose (40 mg/kg) in a citrate buffer with a P H of 4.5. ,,, A pretreatment intra-peritoneal glucose tolerance test (IPGTT) was performed on each rat 1 week after the induction of diabetes following an overnight fast. A sweet-tasting liquid (2 g/kg glucose) (Analar, BDH Chemicals, Poole-England) was administered intra-peritoneally to the experimental rats.  Glucose concentrations were subsequently measured in the blood collected from the tail veins of the rats at 0 min (fasting blood glucose level), and then measured at regulated postprandial intervals of 30 min up to a maximum of 120 min with the aid of an Accu-Chek Active ® Glucometer (Roche). Rats that displayed a sustained rise in blood glucose level up to ≥200 mg/dl were confirmed to be diabetic. ,,,
Experimental Design and Treatment
Diabetic rats were divided into five groups namely diabetic control (DC) group (treated with normal saline via intra-gastric intubation for 14 days), SAC group (treated with SAC solution at a dose 50 mg/kg via intra-gastric intubation for 14 days), MAN group (treated with MAN solution at a dose 40 mg/kg via intra-gastric intubation for 14 days), combined therapy (COM) group (treated with a combined solution of SAC and MAN in equal ratio by volume 1:1 for 14 days), and GLC group (treated with GLC at a dose 5 mg/kg for 14 days). The 6 th group of rats was designated as non-DC (NDC) group and fed with normal pelletized rat chow and distilled water via intra-gastric intubation for 14 days.
At the end of the treatment period, the rats were fasted overnight and a posttreatment IPGTT was conducted to check for the level of hyperglycemia.
At the end of the posttreatment IPGTT, the rats were anesthetized via an intra-muscular injection with Ketamine Hydrochloride USP at a dose of 50 mg/kg. Pancreatic tissue samples were collected by dissection via a mid-line incision through the anterior abdominal wall of the rats. Excised pancreatic tissues were fixed in 10% neutral buffered formalin (JALLICA Scientific, Nigeria).
Fixed tissue samples were processed with an automatic tissue processor (Leica TP 1020) and embedded in paraffin wax with the aid of an embedding machine bearing an embedding center (Leica EG 1160). Sections were cut with a rotatory microtome (Leica RM2125RT) at a thickness of 5 μ and then deparaffinized in xylene and rehydrated in graded changes of alcohol. Antigen retrieval was performed with a retrieval solution (P H 8.0) and then neutralized with an endogenous peroxidase (Leica Novocastra™ Peroxidase) after which sections were washed in a tris-buffered saline solution. Initial incubation was performed with the primary antibody (Lyophilized mouse monoclonal insulin antibody (Leica Biosystems, NCL-INSULIN). A second incubation was performed with a secondary antibody (Leica Novocastra™ postprimary) to form a complex (TAG) with the primary antibody. A peroxidase activity was developed with horseradish peroxidase, a linkage polymer (Leica Novolink polymer). The labeled insulin target molecule was eventually visualized via reaction with diaminobenzyldehyde hydrochloride working solution. Sections were finally counterstained with hematoxylin, dehydrated in graded changes of alcohol, cleared in xylene, and mounted on DPX.
Photomicrographs were taken with the aid of digital microscope camera (DCM 510 mega pixels, ScopePhoto ® , China) and a Leitz Wetzlar light microscope.
Immunohistochemical assessment score of the insulin secreted from the islets was done by 3 different professionals independently (2 histoscientists and 1 histopathologist) using the Allred IHC scoring method for all groups (10 fields/group)  and the final immunohistochemical score in percentage was obtained from the mathematical formula:
Total % IHC score = Intensity of stain + staining proportion × 100.
Data obtained were compared using means and standard error of the means. Student's t-test was used to test the level of significance, and a P < 0.05 was considered statistically significant. Statistical analysis was done with the aid of the statistical software tool, EZAnalyze Version 3.0. 
| Results|| |
Mean Body Weights Assessment
[Figure 1] shows a line graph depicting a significant shift from the baseline mean values of the body weights of the rats over a 10-week period during which the experimental rats were fed on a high-fat diet to induce obesity while the nondiabetic rats received normal rat chow.
|Figure 1: Mean weights of the rats over a 10-week period. *Indicate a statistically significant difference between nondiabetic control group and other experimental groups at p<0.05. NDC = Nondiabetic control, DC = Diabetic control, GLC = Glibenclamide, SAC = S-allyl-cysteine, MAN = Mangiferin, COM = Combined|
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Effect on Mean Blood Glucose Level
The intra-peritoneal glucose tolerance of the nondiabetic and diabetic rats before and after treatment are shown in [Figure 2] and [Figure 3]. As shown in [Figure 2], hyperglycemia was successfully induced in all experimental groups except NDC group. In NDC rats, the mean blood glucose level peaked to about 100 mg/dl after 120 min a value still within the normal blood glucose range (70-110 mg/dl).  Whereas, after treatments with the bioactive compounds, glucose utilization became enhanced and hyperglycemic condition of the diabetic rats was significantly reduced as shown in [Figure 3].
|Figure 2: Mean intra-peritoneal glucose tolerance test for all animals before treatment at p <0.05. NDC = Nondiabetic control, DC = Diabetic control, GLC = Glibenclamide, SAC = S-allyl-cysteine, MAN = Mangiferin, COM = Combined|
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|Figure 3: Mean blood glucose levels by intra-peritoneal glucose tolerance test of all experimental animals after treatment at p<0.05. NDC = Nondiabetic control, DC = Diabetic control, GLC = Glibenclamide, SAC = S-allyl-cysteine, MAN = Mangiferin, COM = Combined|
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[Plates 1-6] show the insulin immunostained islets of Langerhans from the pancreas of all experimental rats including the NDC. There was a homogenous distribution of insulin in the β-cells from the islets of the NDC. Whereas the insulin immunostained islets from the DC rats showed a substantial decline in the insulin content. This decrease in insulin content was however significantly ameliorated in the immunostained islet of diabetic rats treated with the bioactive compounds (SAC, MAN and a combination of both, COM) to a near normal homogenous distribution. [Figure 4] on the other hand shows the quantity of insulin produced at the islet of the experimental rats after treatment in percentages based on the subjective IHC assessment method.  The assessment revealed a substantial increase in the quantity of insulin produced at the islets to be higher in SAC treated rats followed by MAN treated rats, GLC and finally COM treated rats.
|Figure 4: A pie chart showing the mean percentage immunohistochemistry score in all experimental groups|
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| Discussion|| |
The present study dealt with the immunohistochemical evaluation of SAC, MAN and a combination therapy of both on type 2 diabetic mellitus Wistar rats. It was shown that the bioactive compounds exerted a real hyperglycemic lowering effect in vivo which was preceded by an increase in insulin content. Hyperglycemia induced by a high-fat diet and low dose of streptozotocin injection in rats causing a differential wreck action on the β-cells of the pancreas is considered to be a reasonable model for the evaluation of drugs or herbal plants active against type 2 diabetes mellitus ,, despite its drawbacks  and an ideal model that depicts the exact pathogenic characteristics as seen in humans is still not available.  The severity of the experimental diabetes and its persistence depends on the dose of streptozotocin used. ,,, The general strategy adopted by most scientists is to first of all feed the experimental rats with a high-fat diet for a period of time with the purpose of inducing mild insulin resistance then followed by an injection of low-dose streptozotocin to achieve partial dysfunction of the β-cells of the pancreas in-order to suppress the insulin secretion which works as a compensation to the insulin resistance with the result of persistent hyperglycemia. Therefore, in this regard, it is generally recognized that an antidiabetic agent be it conventional drugs or herbal plants should exert a beneficial effect on the diabetic condition by enhancing insulin secretion and/or insulin action or both. ,,,
The present study revealed that SAC, MAN, and COM, as well as GLC, caused significant lowering of blood glucose levels in the hyperglycemic rats at the end of a glucose tolerance test spanning a period of 120 min as shown in [Figure 2]. This hypoglycemic potency of the bioactive compounds is in accordance with other findings for SAC and MAN. ,,,, In the immunostained islets, Plates 1-6 revealed restoration in the quantity of the insulin content released at the islet areas of the diabetic treated rats. SAC and MAN could have ameliorated the pancreatic islets from free radicals and hyperglycemic-mediated oxidative stress, and thus preserve the integrity of the remaining and\or regenerated pancreatic β-cells thus stimulating the cells to synthesize and secrete insulin to maintain glucose homeostasis. This reaffirms the secretagogue potentials of SAC, MAN as reported earlier. , Even the combination therapy of the two compounds (COM) also caused an increase in the insulin content of the diabetic rats. However, the quantity of insulin content released by the combination therapy was found to be slightly less compared to SAC and MAN treated groups. This could be due to herb-herb interaction that may have interfered with the secretagogue action of the bioactive compounds when acting in combination as compared to when administered singly.
| Conclusion|| |
The oral administration of SAC, MAN, and COM from this study showed the antidiabetic potency of these bioactive compounds by stimulating insulin production from the β-cells of the islets of Langerhans in the pancreas of diabetic rat models and reversing hyperglycemia suggesting glucose utilization and commitment to glycolysis. However, while this insulin-release and antihyperglycemic mediated effect in the rat models may reflect similar effects in humans, it does not necessarily mean clinical improvement on the overall, as such, it is still necessary to confirm the present findings in humans.
Financial Support and Sponsorship
Conflicts of Interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]