|Year : 2016 | Volume
| Issue : 1 | Page : 32-40
Histological and biochemical evaluation of the antidiabetic potentials of s-allyl-cysteine and mangiferin in type 2 diabetic rat models
IA Iliya1, B Mohammed2, SA Akuyam3, JD Yaro4, JA Timbuak1, M Tanko1, AJ Nok5
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 Biochemistry, Ahmadu Bello University, Zaria-Kaduna State, Nigeria
|Date of Submission||01-May-2015|
|Date of Acceptance||28-Aug-2015|
|Date of Web Publication||12-Feb-2016|
I A Iliya
Department of Human Anatomy, Ahmadu Bello University, Zaria
Biochemical and histological assessments was carried out on the anti-diabetic potentials of s-allyl-cysteine (SAC) and mangiferin (MAN) in Wistar rats models induced with type 2 diabetes mellitus by feeding the rats with a high fat diet for 10 weeks followed by a low dose injection of streptozotocin (40 mg/kg). Therapeutic interventions with 50 mg/kg body weight of (SAC) and 40 mg/kg body weight of (MAN) and a combined therapy (COM) of both SAC and MAN in equal volume ratios (1:1) for 14 days showed that there was a significant improvement in the glucose tolerance ability of the diabetic treated rats (P < 0.05). Consequently, the activities of hepatic pathophysiological enzymes (Alanine aminotransferase, ALT; Aspartate aminotransferase, AST; Alkaline phosphatase, ALP), as well as glycosylated haemoglobin levels were significantly ameliorated in the diabetic treated rat models (P < 0.05). Histomorphometrical examinations of stained pancreatic tissue sections showed a reduction in the total surface area of islets in the diabetic control rats which was however significantly improved in the diabetic treated rats (P < 0.05) except the COM treated group. Subsequent histomorphological evaluation also showed necrosis and vacuolization of islet β-cells to be reasonably reduced in the diabetic treated rats but to a lesser extent in the COM treated group.
Keywords: Glycosylated haemoglobin, histomorphological, histomorphometrical, insulin, liver enzymes, mangiferin, s-allyl-cysteine, type 2 diabetes mellitus
|How to cite this article:|
Iliya I A, Mohammed B, Akuyam S A, Yaro J D, Timbuak J A, Tanko M, Nok A J. Histological and biochemical evaluation of the antidiabetic potentials of s-allyl-cysteine and mangiferin in type 2 diabetic rat models. Sub-Saharan Afr J Med 2016;3:32-40
|How to cite this URL:|
Iliya I A, Mohammed B, Akuyam S A, Yaro J D, Timbuak J A, Tanko M, Nok A J. Histological and biochemical evaluation of the antidiabetic potentials of s-allyl-cysteine and mangiferin in type 2 diabetic rat models. Sub-Saharan Afr J Med [serial online] 2016 [cited 2019 Oct 16];3:32-40. Available from: http://www.ssajm.org/text.asp?2016/3/1/32/176306
| 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 to 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, 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 more common accounting for over 90% of diabetes cases worldwide, 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-like glibenclamide (GLC), metformin, 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 possess 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 have 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 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 C-glucoside xanthone compound in it called mangiferin (MAN) has been reported in various parts of the plant: Roots, leaves, stem bark, and fruits. ,, The effects of MAN on hyperglycemia, atherogenicity, and oxidative damage to cardiac and renal tissues in streptozotocin (STZ)-induced diabetic rats have been investigated. , The reported pharmacological activities of MAN include antioxidant, ,, antitumor,  anti-inflammatory,  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 histological aspects of the pancreatic islets in the disease have received less attention.
Objective of the Study
This was to evaluate the antidiabetic potentials of SAC, MAN and a composite mixture of both (COM) in equal volume ratio (1:1) in type 2 diabetic (T2D) rat models using histological and biochemical techniques.
| Materials and Methods|| |
Source of Experimental Animals and Husbandry
Thirty apparently healthy male albino Wistar rats (Rattus norvegicus) of approximately 5-week-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 growers mash 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 and illumination, sanitation, bedding materials as published by the Institute for Laboratory Animal Research. 
Drug and Bioactive Compounds
SAC (1g), 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 (1g), 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 a period of 10 weeks. All measurements were done with the aid of a digital top-loader weighing machine (AccuLab E). At the end of the 10 th week, diabetes was induced by injecting the rats with a freshly prepared STZ injection (Zayo-Sigma, Nigeria) at a low-dose (40 mg/kg) in a citrate buffer with a pH of 4.5. ,,,, A pretreatment intraperitoneal glucose tolerance test (IPGTT) was performed on each rat 1 week after the induction of diabetes following an overnight fast. A sweet-tasting liquid containing 2g/kg glucose (Analar, BDH Chemicals, Poole-England) was administered intraperitoneally 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 5 groups namely, diabetic control (DC) group (treated with 1 ml/kg body weight normal saline via intragastric intubation for 14 days), SAC group (treated with SAC solution at a dose of 50 mg/kg body weight via intragastric intubation for 14 days), MAN group (treated with MAN solution at a dose of 40 mg/kg body weight via intragastric intubation for 14 days), composite 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 body weight for 14 days). A sixth group of rats was designated as non-DC (NDC) group and fed with normal pelletized rat chow and 1 ml/kg body weight distilled water via intragastric 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 Post-treatment IPGTT, the rats were Subjected to light anaesthesia via inhalation with chloroform soaked in cotton wool and placed in an anaesthetic box covered with lid. 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). Blood was collected from apex of the heart from rats in all the groups via cardiac puncture technique with the aid of a 2 ml syringe and needle. Some of the blood were collected into heparinized ethylenediaminetetraacetic acid bottles and stored in the refrigerator at 4°C, while the remainder of the blood was collected into plain bottles, allowed to clot, and centrifuged at 300 rpm for 10 min (Eppendorf 5417R) to obtain the serum.
Gross Morphometric Procedure
The final body weight (BW) of the rats was measured before sacrifice; likewise, liver tissue obtained from each rat was weighed immediately after dissection. Weights measurement was performed with the aid of a digital top-loading weighing balance (AccuLab E). The percentage relative organ weight was calculated by the mathematical formula: (Relative liver weight [%] = liver weight [L]/BW × 100). 
- Estimation of pathological liver enzymes: Sera from all the groups were collected with the aid of a disposable rubber pipette into clean plain bottles and analyzed for enzyme markers of liver toxicity (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and alkaline phosphatase [ALP] in the Department of Chemical Pathology, Ahmadu Bello University Teaching Hospital in Zaria, Nigeria). Diabetic and non-DC sera plus calibrators were loaded onto the sample tray inside the Bayer ® Express Plus Auto-analyzer. The machine was set; the progress monitored, and results were obtained
- Glycated hemoglobin assay (HbA1 C ) estimation via ion exchange resin method: Analytical procedure for HbA1 C was carried out with the aid of a kit (AccuCare) at the Center for Biotechnology, Ahmadu Bello University, Zaria, Nigeria, and consisted of three steps according to the manufacturer's instructions:
- Hemosylate preparation: A lysing reagent (250 μl) and whole blood (50 μl) were pipetted with the aid of a micropipette and into a test-tube and mixed
- HbA1 C separation and assay: HbA1 C resin tubes were brought to assay temperature by incubating in a water bath at 30°C (Grant OLS 200). About 100 μl of the hemosylate was added. Resin separators were positioned in the resin tubes 3cm above resin level and vortexed (MiniShaker MSI IKA) continuously for 5 minutes and allowed to settle. The supernatant was poured into a cuvette, and the absorbance was measured against deionized water on a digital colorimeter (Optima, AC-114) set at a wavelength of 415 nm
- Total hemoglobin (THb) assay: Deionized water (5 ml) and 20 μl of the hemosylate were pipetted into a test tube and mixed. Absorbance was measured against deionized water on a digital colorimeter set at 415 nm wavelength.
The results were calculated as follows:
(Percentage [%] HbA1 C = absorbance of HbA1c/absorbance of THb × 7.2 × temperature factor).
Excised pancreas tissues from all the groups were fixed in 10% neutral buffered formalin (JALLICA Scientific, Nigeria) and processed with an automatic tissue processor (Leica ATP 1020). Embedding was done in paraffin wax with the aid of an embedding machine bearing an embedding center (Leica EG1160) and sectioned at 5 μ on a rotatory microtome (Leica RM2125RT). The sections were stained with hematoxylin and eosin and mounted on distyrene plasticizer xylene. Photomicrographs at magnifications of ×250 were taken from all the groups with the aid of a digital microscope camera (ScopePhoto ® DCM 510 megapixels) and a Leitz Wetzlar light microscope. Histomorphometrical assessments of the pancreatic islets were done by measuring the total surface area of the islets in all the groups (10 islets/group) by placing transparent gridlines of equal dimensions (1 cm 2 × 1 cm 2 ) over the pancreatic islet area. The number of completed squares (S 1 ) within the boundary of the islet area was counted; furthermore, the innumerable number of uncompleted squares (S 2 ) was also counted. The final value was calculated by the mathematical formula: Total surface area of islet (S t ) = ∑ (S 1 + S 2 ).
Data obtained were compared using means and standard error of the means. Student t-test was used to test the level of significance and a P < 0.05 was considered as significant. One-way analysis of variance was used to compare the mean values between the different groups. A post hoc test (Bonferonni) was also applied to assess where the difference lies between the groups. All statistical analysis was done with the aid of a Microsoft excel add-in statistical software tool, EZAnalyze Version 3.0. (Poyton, 2007). 
| Results|| |
Mean Body Weights Assessment
[Figure 1] shows a line graph depicting a significant shift from the baseline mean values of the BWs of the rats over a 10 weeks 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 intraperitoneal glucose tolerance of the nondiabetic and diabetic rats before and after treatment is shown in [Figure 2] and [Figure 3]. In [Figure 2], hyperglycemia was successfully induced in all experimental groups except the non-DC group. In the non-DC rats, the mean blood glucose level peaked to about 100 mg/dl after a glucose tolerance test (IPGTT) for a period of 120 minutes, a value still within the normal blood glucose range (70-110mg/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 blood glucose levels by 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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Effect on Relative Organ (liver) Weight
[Figure 4] shows the results of data of the relative organ (liver) weights from all the groups. None of the groups differ significantly when compared to the non-DC group (P > 0.05).
|Figure 4: Mean relative liver weights from all groups. None of the groups differed significantly (p > 0.05)|
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Effect on Percentage Glycated Hemoglobin
[Figure 5] summarizes the level of HbA1c in the non-DC and experimental rats. A significant rise in the level of HbA1c was observed in the DC rats. Oral therapeutic intervention with SAC, MAN and a combination of both in equal volume ratio (1:1) reduced the HbA1c levels in the diabetic rats.
|Figure 5: Mean % HbA1c for all experimental groups. indicates statistical significant difference between nondiabetic group with the 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 Liver Enzymes
[Figure 6], [Figure 7] and [Figure 8] show the serum levels of ALT, AST and ALP enzymes in the nondiabetic and DC rats. It was observed that the increase in the serum levels of these enzymes was significantly reduced in the diabetic rats after oral therapeutic intervention with SAC, MAN, and COM to levels.
|Figure 6: Mean alanine aminotransaminase (ALT) in the livers of animals in all the experimental groups. * indicates statistical significant difference between the 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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|Figure 7: Mean aspartate aminotransaminase (AST) in the livers of animals in all the experimental groups. *Indicates statistically significant difference between nondiabetic 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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|Figure 8: Mean alkaline phosphatase in the livers of animals from all the experimental groups. *Indicates statistically significant difference between the nondiabetic 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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[Table 1] shows the microanatomical changes in terms of the total surface area of the islet of Langerhans in the pancreas of the nondiabetic and DC rat models. [Table 1] showed the microanatomical changes in terms of the total surface area of the islet of Langerhans of the experimental rats. The histomorphometric evaluation revealed significant difference between the COM and DC islet areas in comparism to the NDC islet area.
|Table 1: Mean total surface area of the islets of Langerhans in the pancreas of all experimental groups|
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[Plates 1-6] indicate changes in the quality of the islet cells structure in all experimental groups. The pancreatic tissue sections showed that there was distortion of the normal histo-architecture of the cells in the islet area of the DC rats and such distortion was reduced and islet cells quality restored to near normal in the treated groups. The restoration was better in the SAC therapy group followed by the MAN therapy group, GLC therapy group, and lastly COM therapy group.
| Discussion|| |
The present study investigated the antidiabetic effects of SAC a bio-active compound found in garlic (Allium sativum), MAN another bioactive compound found in mango leaves (Mangifera indica), and a COM of these two compounds (COM) in equal volume ratio on T2D rat models. The diabetic model used in this study was developed by feeding rats a high-fat diet to induce obesity and consequently insulin resistance in combination with a low dose of intraperitoneal STZ injection, resulting in partial β-cell dysfunction and lowered insulin secretion. This model is widely used for investigating the T2D Mellitus state due to the fact that it is a noninsulin-dependent model, with insulin resistance, hyperglycemia, and abnormal lipid profiles, ,, though like all the other models, it is not without minor drawbacks.  In the IPGTT of the present study, oral administration of SAC, MAN, and COM significantly reduced the blood glucose level in the hyperglycemic rats after 120 min of a glucose challenge. The mechanism of the hypoglycemic action of SAC, MAN, and COM probably involves direct or indirect stimulation of insulin secretion from the remnant of β-cells or regenerated β-cells, ,,,, Furthermore, it is also suggested that SAC, MAN, or COM treatment interventions might enhance glucose utilization by restoring delayed insulin response at the level of the insulin receptors or along the insulin pathway up to the glucose transporter in glucose recipient cells.
Hyperglycemia is the clinical hallmark of poorly controlled diabetes, which is known to cause protein glycation, also known as nonenzymatic glycosylation.  It has been reported that various proteins, including hemoglobin, albumin, collagen, low-density lipoprotein, a crystalline fibronectin, undergo nonenzymatic glycation in diabetes.  Glycosylated hemoglobin was found to be significantly increased in the diabetic rats, and the amount of this increase is directly proportional to the fasting blood glucose level.  The highly significant decreases in glycosylated hemoglobin indicate the efficiency of SAC, MAN, and COM in hyperglycemic control in the diabetic rats.
Increases in the activities of plasma AST, ALT, and ALP indicate that diabetes may be induced due to liver dysfunction/damage and therefore, an increase in the activities of AST, ALT, and ALP in serum may be mainly due to the leakage of these enzymes from the liver cytosol into the blood as reported by other authors, , who found that the liver was necrotized in STZ-induced diabetic rats. The present study indicated significant increases in the activity of AST, ALP, and ALT in the high fat diet fed and STZ-induced diabetic rats and these levels were significantly reduced after treatment intervention with SAC, MAN, and COM which are concomitant with other findings. ,
The histological observation of pancreatic tissues provided additional support to the antidiabetic potencies of SAC, MAN, and COM. The induced type 2 diabetes mellitus produced noticeable pancreatic injury culminating in a decrease in the pancreatic islets total surface area that was perhaps due to decrease in the number of β-cells. STZ is highly specific to β-cell toxicity and consequently causes diabetes mellitus; hence, it is widely used to study β-cell damage in vivo in animal experiments. ,,, In the present study, necrosis and vacuolization of pancreatic islet β-cells was observed in the pancreas of the DC rats. However, oral administration of SAC and MAN to the diabetic rats ameliorated the pancreatic islet injuries and preserve the remaining β-cells and perhaps cause a regeneration of some β-cells in the islets and subsequently an increase in the total surface area of the islets as indicated by the histomorphometrical analysis in the present study even though, the COM therapy group (COM) showed to a lesser extent an increase in the total surface area of the islets. This could be due to herb-herb interaction thereby hampering the regenerative powers of the bioactive compounds as compared to when administered singly. Histomorphologically, SAC and MAN reduced significantly the vacuolization and necrosis of pancreatic islets β-cells as compared to the COM and GLC treated rats. SAC and MAN possibly could have ameliorated the pancreatic islets from free radicals and hyperglycemic-mediated oxidative stress and thus preserve the integrity of the remaining pancreatic β-cells and stimulate the cells to synthesize and secrete insulin to maintain glucose homeostasis. Such a sequence of events has been proposed by other investigators to strengthen the antioxidant defense system.  These histomorphological observations in the diabetic rats treated with SAC and MAN in the present study appear to correlate with the histomorphometrical observations, which would have brought about increased levels of insulin secretion and subsequently lowered levels of blood glucose in the rats except in the COM treated group where such improvements in the histomorphological and histomorphometrical features were observed to be less pronounced compared to the SAC and MAN treated groups. These findings suggest the antidiabetic nature of SAC and MAN in restoring and preserving the structural and functional integrity of pancreatic β-cells especially when administered singly.
| Conclusion|| |
The amelioration of type 2 diabetes mellitus and its complications by SAC, MAN, and COM therapies may be due to SAC-mediated, MAN-mediated, and COM-mediated insulin release, hypoglycemia, and β-cells regeneration potencies. Histopathological observations showed that STZ partially destroyed pancreatic β-cells, but these were reasonably revived with SAC and MAN treatment and to lesser extent by COM therapy treatment. However, while these antidiabetic mediated effects 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], [Figure 5], [Figure 6], [Figure 7], [Figure 8]