Research ArticleAbstract
The use of streptozotocin in experiments has given more rooms for researchers to test the effectiveness of new drugs from plants that might have some kidney protective and/or antidiabetic effects in diabetic victims, so as to help in the treatment of the disease and its associated complications. Thirty male Wistar rats were selected into six groups (n = 5). All the groups, excluding the negative control group were administered a single dose of streptozotocin intraperitoneally. The six groups include: non-induced and untreated group: (negative control)
A. Streptozotocin-induced and untreated group: (positive control)
B. Induced with streptozotocin and treated with Terminalia catappa leave extract
C. Induced with streptozotocin and treated with Persea americana seed extract
D. Induced with streptozotocin and treated with both extracts
E. Induced with streptozotocin and treated with glibenclamide: (standard drug)
F. A reversed assessment of kidney damage associated with streptozotocin-induced diabetes mellitus in male Wistar rats on treatment with leaves of Terminalia catappa and Persea americana seed extracts were investigated after 21 days of the experiment.
Administration of extracts of the T. catappa leaves and P. americana seed contribute significantly (p < 0.05) in exerting protective effects and bringing the levels of creatinine and urea; activities of alkaline phosphatase (ALP), alanine amino transferase (ALT) and aspartate amino transferase (AST) to normalcy and can be useful in the treatment of diabetes. Each plant extracts equally produced a significant (p < 0.05) protective effects, as well as regeneration of kidney cells and so are efficient in diabetes treatment.
Keywords: Terminalia catappa leave; Persia americana seed; Diabetes mellitus; Streptozotocinl Indian
Abbreviations: ALP: Alkaline Phosphatase; ALT: Alanine Amino Transferase; AST: Aspartate Amino Transferase; DM: Diabetes Mellitus
Introduction
Diabetes mellitus (DM) is a group of metabolic disorders characterized by high blood sugar levels over a prolonged period, frequent urination (polyuria), thirst (polydipsia), and increased hunger (polyphagia), inadvertent weight loss if left untreated, blurred vision, headache, fatigue, slow healing of cuts, and itchy skin [1,2]. DM could cause complications such as ketoacidosis, stroke, chronic kidney disease, foot ulcers, or even death [3]. Diabetes is due to either the pancreas not producing enough insulin, or the cells of the body not responding properly to the insulin produced [4]. Type 2 DM begins with insulin resistance, a condition in which cells fail to respond to insulin properly. As the disease progresses, a lack of insulin may also develop. This is referred to as “non insulin- dependent diabetes mellitus” (NIDDM) or “adult-onset diabetes”. The most common cause is a combination of sedative lifestyle, obesity and insufficient exercise. Type 2 DM is making up about 90- 95 % of the cases of diabetes worldwide [2]. Streptozotocin is an organic chemical that occurs naturally. It has been reported to be toxic to the insulin-producing β-cells of the pancreas in mammals [5]. However, it is a glucosamine-nitrosourea compound toxic to cells by causing damage to the nucleic acids. In structure, it is similar to glucose molecule, and as well transported into the cells by the glucose transport protein GLUT2, though not recognized by the other glucose transporters.
Figure 1: Streptozotocin(Source: Sithole, 2009).
The use of streptozotocin in experiments has given more rooms for researchers to test the effectiveness of new drugs that might have some hypoglycemic and/or protective effects in diabetic victims, so as to help in the treatment of the disease and its associated complications [6] (Figure 1). Over the past decades, herbal medicine has become a thing of global significance with medicinal and economic implications, and for treating different diseases [7,8]. Large utilization of herbs worldwide has raised thoughtful concerns over its quality, safety, and efficacy. Thus, exact scientific assessment has become a precondition for acceptance of herbal health claims. The use of Terminalia catappa (Indian almond) leave and Persea americana (avocado pear) seeds as a substitute for modern day treatment of increased blood sugar is not common. Termnalia catappa is widely grown in tropical regions of the world as an ornamental tree, grown for the deep shade its large leaves provide. The fruit is edible, tasting slightly acidic, and its leave extract has shown activity against Plasmodium falciparum chloroquine (CQ)-resistant (FcB1) and CQ-sensitive (HB3) strains [9]. Current research has shown that Persea americana seed may improve hypercholesterolemia, and be useful in the treatment of diabetes and other associated conditions [10]. Hence, this study aimed at finding the effects of the extracts of Terminalia catappa leaves and Persea americana seed on serum creatinine and urea levels; activities of alkaline phosphatase (ALP), alanine amino transferase (ALT), aspartate amino transferase (AST), and histology of kidney: a reversed assessment of kidney damage associated with streptozotocin-induced diabetes mellitus in male Wistar rats.
Materials and Methods
Collection of Plant Materials
The tender leaves of Terminalia catappa and the Persea americana seeds were collected from Bells University of Technology, Sango-ota, Ogun State, Nigeria. They were shade dried at room temperature. The dried leaves and seeds were subjected to coarse powder using a clean and dry grinder; powder was later sieved respectively. Approximately about 200 g of the powdered leaves and seeds were soaked into 2000 ml distilled water and 140 ml of ethanol for 48 hours respectively. The extracts were filtered through muslin cloth, marc was discarded, and filtrates were dried in a hot air oven at 45 °C respectively, till a semi solid mass was produced.
Experimental Animals
Thirty (30) male Wistar rats, weighing approximately 150 g were purchased from a rearer. They were housed in plastic cages with net covererd for ventilation and subjected to a standard environmental condition (12:12 hour light: dark cycle). They were fed with rat pellet feed ad libitum. Wood shavings were used as beddings for the animals; beddings were changed daily to avoid build up of toxic ammonia levels. The rats were acclimatized for 2 weeks prior to exposure.
Animal Grouping and Exposure
The 30 rats were divided into 6 groups, including the negative and positive control groups. Each group contained five animals (n = 5) to avoid overcrowding and to allow easy accessibility and identification of the animals in the course of the research. All the groups, excluding the negative control group were administered a single dose of streptozotocin intraperitoneally. The blood glucose and body weight of the experimental rats were monitored for 1 week. Thereafter, the animals were treated with the extracts of Terminalia catappa leaves and Persea Americana seed, and a standard drug for 21 days (Table 1).
Table 1: Experimental Design (n = 5).
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Sacrifice of Animals and Collection of Blood and Organs
on the 22nd day of treatment, the animal was subjected to a 12-hours food fast at the end of which the blood samples were collected into floride oxalate bottle. The liver and kidney were isolated and preserved in 10% formalin for histopathology study.
Biochemical Analysis
On the 22nd day of the experiment the rats were fasted over night, they were sacrificed by cervical dislocation and blood samples were collected by retro-ocular method. The blood samples were centrifuged at 3000rpm for 15 minutes. The serum obtained was then analyzed to determine the creatinine and urea levels; activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) using Randox kits following manufacturer’s instructions.
Histopathology Procedure
Histopathology is the microscopic study of diseased tissue. It is performed by examining a thin tissue section under light microscopes. The tissue were fixed with neutral formalin (10 %), embedded in paraffin, and then manually sectioned with a microtome to obtain 4-5 µm thick paraffin sections. Dewaxed sections were then stained with hematoxylin and Eosin blue (H and E). After staining, a very thin glass was placed over the tissue section to protect it and to enhance the optical evaluation of the tissue.
Statistical Analysis
All values were expressed as the mean ± standard error of mean (SEM). The data were analyzed using one-way analysis of variance (ANOVA) followed by post hoc Duncan’s multiple range tests by statistical software package (SPSS, version 17.00). Differences were considered significant at p < 0.05. Alphabetic superscripts indicate statistical differences.
Results (Tables 2 & 3)
Table 2 showed effects of Terminalia catappa leaves and Persea americana seed extracts on creatinine and urea levels (mg/dL) in the serum of streptozotocin-induced diabetes rats (Table 3). The creatinine level in serum of the positive control (2) showed a significant increase (p < 0.05) when compared to the negative control (1). There was no significant (p > 0.05) difference in the creatinine levels in serum of groups 4, 5 and 6, when compared to the negative control (1). Group 3 showed a significant decrease (p < 0.05) towards the negative control when compared to positive control (2). There was a significant (p < 0.05) increase in the urea level of positive control (2) when compared with the negative control (1). Group 5 showed a significant (p < 0.05) decrease towards the negative control when compared to the positive control (2). There was no significant (p > 0.05) difference in the urea levels in serum of groups 3, 4 and 6, when compared to the negative control (1). The positive control (2) showed a significant (p < 0.05) increase in ALP activity, when compared to the negative control (1). Group 3 showed no significant (p > 0.05) difference in ALP activity when compared with the negative control (1). Groups 4, 5 and 6 showed a significant (p < 0.05) decrease towards the negative control when compared to the positive control (2). There was a significant (p < 0.05) increase in the in ALT activity of the positive control (2) when compared to the negative control (1). Groups 3, 4, 5 and 6 showed significant (p < 0.05) decrease towards the negative control when compared to the positive control (2). The positive control (2) and group 6 showed significant (p < 0.05) increase in AST activities, when compared to the negative control (1). Group 3, 4 and 5 showed no significant (p > 0.05) difference in AST activities when compared with the negative control (1).
Table 2: Effects of erminalia catappa leaves and Persea americana seed extracts on creatinine and urea levels (mg/dL) in the serum of streptozotocin-induced diabetes.
Note: Data were expressed as mean ± SEM (n = 5). Values having different alphabetic superscripts within the same column are significantly different (p < 0.05).
A. Group 1 Non-induced and untreated (negative control)
B. Group 2 Strptozotocin-induced and untreated
C. Group 3 Strptozotocin -induced and treated with Terminalia catappa
D. Group 4 Strptozotocin -induced and treated with Persea americana
E. Group 5 Strptozotocin-induced and treated with Terminalia catappa and Persea americana
F. Group 6 Strptozotocin -induced and treated with glibenclamide
Table 3: Effects of Terminalia catappa leaves and Persea americana seed extracts on alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities in the serum of streptozotocin-induced diabetes (g/L).
Note: Data were expressed as mean ± SEM (n = 5). Values having different alphabetic superscripts within the same column are
significantly different (p < 0.05).
A. Group 1 Non-induced and untreated (negative control)
B. Group 2 Streptozotocin-induced and untreated
C. Group 3 Streptozotocin -induced and treated with Terminalia catappa
D. Group 4 Streptozotocin -induced and treated with Persea americana
E. Group 5 Streptozotocin-induced and treated with Terminalia catappa and Persea americana
F. Group 6 Streptozotocin -induced and treated with glibenclamide
Discussion
Medicinal plants have been used as an accepted complementary medical option for centuries to improve the health condition of the folk through decreasing the adverse effects and costs of the synthetic medicines [11,12]. In developing countries, more than 80 % of the populace use herbal medicine to treat a variety of diseases [12]. Herbs are regarded as ‘natural’ and safe, there is limited evidence regarding the use of herbal medicines during pregnancy as well as in pediatric and geriatric populations [13]. The kidney-protective effects of the extracts of T. catappa leaves and P. americana seeds were investigated in streptozotocin-induced diabetic rats. Creatinine and urea levels, including alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important biomarkers which activities are important in the diagnosis of kidney and other tissues’ damage caused during toxicity or infection. An increase in ALP, ALT and AST activities in serum of untreated group compared with the negative control was as a result of streptozotocin toxicity. Treatment with Terminalia catappa leave extract and Persea americana seed extract may have contributed to the decrease in the their activities in the experimental animals. ALP, ALT and AST measures the activities of intracellular kidney enzymes that have leaked into the blood circulation and serve as biomarkers of kidney injury. This is in line with the the work of Ezejiofor, et al. [14,15] which concentrated on the mechanism of the antidiabetic activities of Terminalia catappa P. americana respectively.
The insulin-stimulative and antioxidative effects of Persea americana were evaluated in streptozotocin (STZ)-treated rats. This group found that the activities of pathophysiological enzymes such as serum aspartate transaminase (AST), serum alanine transaminase (ALT), and serum alkaline phosphatase (ALP) were altered in the serum of rats that were treated with glyclazide, which was used as the standard reference drug. These results revealed the tissue-protective nature of Persea americana. The histopathological study of the kidney of negative control showed normocellular glomerular tufts disposed on a background containing renal tubules. No abnormality was observed in the negative control (1). This study confirmed the significant kidney protective effects of Terminalia catappa leave and Persea americana seed extracts [16] (Plate 1), that both extracts equally had significant protective effects and reversed the histopathological damage that occurred in in streptozotocin-induce diabetic rats, comparable with the effects of glibenclamide [14,17-20].
Figure 2: Effects of Terminalia catappa leave and Persea americana seed extracts on the kidney of experimental animals: Negative control
1. Kidney showing normocellular glomerular tufts disposed on a background containing renal tubules, and compact tissues. No abnormality was observed in group 1; streptozotocin-induced and untreated positive 2. Kidney showing profuse infiltration by inflammatory cells, areas with dissolved cells and an absence of glomerulus; induced with streptozotocin and treated with Terminalia catappa leave extract 3. Kidney showing Bowman’s space, glomeruli and inflammatory cells; induced with streptozotocin and treated with Persea americana seed extract 4. Kidney showing Bowman’s space, glomeruli and inflammatory cells; induced with streptozotocin and treated with both extracts 5. Kidney showing glomeruli and a near compact kidney architecture; induced with streptozotocin and treated with glibenclamide (standard drug) 6. Kidney showing glomeruli and a near compact kidney architecture.
Conclusion
The results obtained revealed that the plant extracts exerted protective effects on the damaged kidney of streptozotocin-induced groups. Terminalia catappa leave and Persea americana seed extracts have enzyme regulatory effects in the streptozotocin-induced rats. These extracts showed improvement in parameters like creatinine and urea levels as well as regeneration of kidney cells and so might are efficient in diabetes treatment.
Authors’ Contribution
Authors contributed equally to this work.
