Preliminary Nutrient Determination and Regeneration of Pancreatic Islet Cells by Extracts of Spondias mombin Leaves in Streptozotocin-Induced Diabetic Rats

Diabetes mellitus (DM) is a metabolic disorder marked by chronic high blood glucose, which results in an array of secondary complications of the heart, kidney, and eyes (1). DM is the predominant cause of death around the world affecting both developing and developed countries. DM is, therefore, a serious health challenge placing a heavy burden on the economy. The global prevalence of DM has shown an alarming projection in recent times and according to the World Health Organization (WHO), diabetes will be seen as the seventh leading cause of mortality by the year 2030 (2). Taking into account the pathology of diabetes, the prevention of beta cell from degenerating and enhancement of the endogenous regeneration of islets will be a crucial strategy in the management of diabetes. Despite the availability of therapy for diabetes management, there is a growing interest in using anti-diabetic compounds from natural sources because of the unwanted adverse effects of the available drugs (3). A wide range of studies are available on the advantageous effects of using phytotherapy for the management of diabetes (4). The mechanism of action of phytochemicals and active isolate(s) from plants is by decreasing intestinal absorption of glucose, inhibiting hepatic gluconeogenesis, enhancing uptake of glucose by tissues, stimulation of insulin release by the pancreas, and/or regenerating pancreatic tissues (5-7) Spondias mombin is a fructiferous plant belonging to a large family referred to as Anacardiaceae. All parts of the plant have been used in the management of various disease conditions. These conditions include cardiovascular diseases, dysentery, hemorrhoids, and diarrhea (8). Hosseini et al (9) in their reports on the “pancreatic beta cell protection/ regeneration with phytotherapy” recorded an increase in the number, percentage, and volume density of beta cells in islets of the treated diabetic animals. We, therefore, aimed to determine the improvement of insulin secretion and effects of leaf extracts (aqueous and ethanol) of S. mombin on pancreatic islet cell regeneration in streptozotocin-induced diabetic rats. Avicenna Journal of Medical Biochemistry


Plant Material
Fresh leaves of S. mombin were obtained from fields around the campus of the University of Benin in Edo State, Nigeria. Identification and authentication were done in the Department of Plant Biology and Biotechnology, University of Benin, Benin City. Voucher specimens were deposited in the Herbarium with voucher number UBH S 345.

Preparation of Plant Extract
The leaves were allowed to dry for few days. Then, they were pulverized with a mechanical grinder.
Afterwards, 1000 g of the pulverized sample was macerated in distilled water (3 L) for 48 hours, and 1000 g of the leaves were also macerated in ethanol (4 L). The crude extracts obtained were lyophilized. The freeze-dried extracts were kept in the freezer until use.
The mineral content of the powdered leave sample was investigated using the atomic absorption spectrometer (Shimadzu).

Vitamin Determination
The composition of the vitamins of the powdered leave sample was determined using a high-performance liquid chromatographic technique. Samples were separated using Acclaim PA column (3 µm, 120 A, 3.0 ×150 mm) for fatsoluble vitamins and Acclaim C18 column (3 µm, 120 A, 3.0 ×150 mm) for water-soluble vitamins.

Induction of Diabetes
Streptozotocin dissolved in carbonate buffer (0.1 M) with a pH value of 4.5 was administered by intra-peritoneal injection at a dose of 65 mg/kg of body weight after a 12hour fasting period. Diabetes was confirmed on day 7 by determining fasting blood sugar (FBS) using Accu-Chek glucometer. Only rats with FBS ≥ 250 mg/dL were used for this study

Feeding Pattern
A total of 25 male albino Wistar rats obtained from the animal house of the Department of Anatomy, University of Benin, Benin City, were used for this study. They were kept in galvanized cages and allowed to acclimatize for 2 weeks before extract administration under 12 hours light/ dark cycle. The room temperature was 22 o C to 25 o C and the animals had free access to food and water.
The rats were randomly divided into 5 groups (A-E) of 5 rats each.
• Group A: normal control • Group B: diabetic control (untreated diabetic rats) • Group C: positive control (diabetic rats treated with 5 mg/kg of body weight of glibenclamide as the standard drug) • Group D: diabetic rats treated with 200 mg/kg of body weight of aqueous extract • Group E: diabetic rats treated with 200 mg/kg of body weight of ethanol extract. Extracts were given orally using an orogastric tube daily for twelve weeks. At the end of the 12th week, the animals were anesthetized with chloroform and the blood samples were taken by cardiac puncture into plain sample tubes. Serum samples used for the biochemical assays were obtained after centrifuging the blood sample at 3000 g for 10 minutes. The sera obtained were used for insulin and C-peptide assays using insulin and C-peptide ELISA kits, respectively.

Histological Evaluation
The excised pancreases were rinsed with normal saline and fixed for two days in 10 % buffered neutral formalin. The sections were 5 µm thick. They were paraffin-embedded and stained with hematoxylin and eosin. The sections of the pancreases were prepared and examined under Leica DM750 research microscope. Digital photomicrographs of the tissue sections were taken at ×40 and ×100 magnifications.

Statistical Analysis
Results were represented as mean ± SEM. The results were computed statistically using GraphPad Prism 5. The level of significance was determined at P<0.05 using analysis of variance. Table 1 shows the mineral composition of the pulverized leaves of S. mombin. Calcium was the highest mineral content, followed closely by phosphorus, sodium, and potassium with the lowest mineral content being copper while cadmium was not detected. Table 2 shows the vitamin composition of the pulverized leaves of S. mombin. Of the vitamins identified, vitamin C gave the highest content Figure 1 shows the chromatogram of vitamin composition Values represent the mineral composition of the powdered leave samples of S. mombin which were obtained from a single determination.

Regenerative Potential of Extracts of Spondias Mombin in Diabetic Rats
of pulverized leaves of Spondias mombin. Figure 2 shows the result of serum insulin and C-peptide assay. A significant decrease in serum insulin and C-peptide levels was seen in the diabetic control rats when compared to the normal control group.
Histological studies revealed that the normal pancreases were characterized by well-defined acinar cells, islet of Langerhans, interlobular ducts ad interlobular arteries in comparison to the diabetic control pancreases which had hypoplastic islets, distorted vascular cells and ducts filled with proteinaceous plugs. The pancreases of the positive control, ethanol extract treated and aqueous extract treated diabetic rats showed luxuriant islets and normal interlobular arteries and ducts. In addition, the number and sizes of the islet cells markedly increased in the treated rats when compared with the untreated animals (Supplementry file 1).

Discussion
Beta cell degeneration caused by streptozotocin resulted in a reduction in serum insulin and C-peptide levels ( Figure 2). The measurement of C-peptide and insulin concentration has been reported to be an important marker of insulin secretion (10). As the estimation of serum insulin without assaying C-peptide may not be ideal because insulin goes through first-pass clearance by the liver once secreted by the pancreas. The increase of serum insulin and C-peptide concentration observed in the treated diabetic rats may have been due to the up-regulation of insulin synthesis by the pancreatic β-cells or regeneration of the islet cells as confirmed by the histological studies. The high concentration of serum insulin seen in the diabetic treated rats suggests that S. mombin can be used as an anti-diabetic agent that enhances the secretion of insulin. We recorded 68.1%, 47.58%, and 50.99% increase in serum insulin concentration of the glibenclamide, aqueous, and ethanol-treated diabetic rats, respectively, when compared with the diabetic control. On the other hand, 71.13%, 54%, and 45.24% increase in serum C-peptide concentration were reported for glibenclamide, aqueous, and ethanol-treated diabetic rats, respectively. This, therefore, shows that the ethanol extract better increased serum insulin concentration, while aqueous extract better increased serum C-peptide level.
It is proposed that the increased insulin levels noted in the present study could be due to the ability of the phytochemicals, amino acid contents, minerals, and vitamins in S. mombin to stimulate insulin release, inhibit insulin breakdown and/or rejuvenate βcells that were destroyed by streptozotocin in the diabetic state. Studies have shown that the anti-diabetic effects of many phytochemicals from plant materials have been attributed to their antioxidant properties (11). In our previous study, we have established that the rich polyphenols present in the extracts of S. mombin have antioxidant effects (12).
C-peptide is an important marker for assessing pancreatic beta cell function. It has a long half-life and is not subject to hepatic clearance. Some researchers prefer c-peptide concentration to insulin in recognizing any variation in beta cell secretion of insulin. Our results indicated that there was a remarkable reduction in C-peptide level in STZ induced diabetic rats. Cong and Chen (13) and Amin et al (14) reported the same findings.
Regeneration of β-cells has also been reported by several authors following treatment of STZ-induced diabetic  animals with medicinal plants (15)(16)(17). Other reports have established that diabetes induction in animals can lead to 50% reduction in pancreatic β-cells (18,19). The increase in the number and size of the pancreatic β-cells observed in the aqueous and ethanol extract treated animals in this study can be regarded as the β-cells regenerative effects of S. mombin.
Our result also showed evidence of beta cell recovery in the glibenclamide treated group when compared with the untreated animals. Onakpa and Asuzu (20), and Alese et al (21) reported similar findings. Glibenclamide has been used as an antidiabetic drug to compare the efficacy of various hypoglycemic medicinal compounds (22) Certain minerals and vitamins play a vital function in controlling diabetes. They may have directly or indirectly led to the improvement of insulin secretion and regeneration of the pancreatic beta cells observed in this study. Of the minerals identified in Spondias mombin (Table 1), magnesium and zinc play important roles in diabetic individuals. Magnesium plays a role in the metabolism of glucose. Certain conditions such as insulin resistance and carbohydrate intolerance occur often as a result of low magnesium in the body (23). Owing to the role of magnesium in controlling diabetes, decreased magnesium levels in the blood can be compelling.
Zinc is an important mineral useful to diabetics. In humans and animals, diabetes causes alterations in vital micronutrients including zinc (24). Zinc is required for the production and storage of insulin. It plays a role in preserving the structure of insulin (25).
Vitamins C and E are important antioxidants which help protect the cell from oxidative damage (26). A crucial link exists between the level of vitamin C in the body and the development of diabetes (27).
Niacin (vitamin B3) is a co-enzyme needed in reduction and oxidation reactions. Niacin plays a vital role in protecting the cardiovascular system in diabetics. Niacin has been shown to protect the pancreas, by maintaining intracellular levels of NAD + , leading to the prevention of autoimmune destruction of beta cells. It also mops up nitric oxide radicals in the pancreas. This in turn protects the pancreas from oxidative damage.
It was shown that the presence of vitamin E in S. mombin may be helpful in eliminating byproducts of lipid peroxidation. A decreased incidence of diabetes has been reported in people with high vitamin E (28) and researches have shown that diabetic patients have decreased antioxidants in the body (29). Therefore, the presence of vitamin E in S. mombin will be useful to individuals with diabetes.
Morphometric analysis of the sections of the pancreas showed a remarkable increase in the surface area of islets of the treated diabetic rats (4904.00±4.00 pixel, 3307.00±2.00 pixel, and 3293.00 ±318.00 pixel for the glibenclamide, aqueous, and ethanol treated diabetic groups, respectively) compared to the untreated group (1324.50±2.50 pixel).
The diameter of islet also increased in the treated diabetic animals (21.86±0.10, 20.47±0.23 µm 3 , and 20.45±0.33 µm 3 for the glibenclamide, aqueous, and ethanol treated diabetic groups, respectively) when compared to the untreated rats (17.53±0.59 µm 3 ) (Tables 3 and 4) (Figures 3 and 4).   Values represent serum insulin and C-peptide level of rats treated with aqueous and ethanol extracts for 12 weeks which are expressed as mean ± SEM and were obtained from 3-5 determinations

Conclusion
The improvement in insulin concentration and pancreatic β-cells regeneration observed in this study may not be unconnected to the presence of important minerals and vitamins detected in the leaves of Spondias mombin.

Authors' Contributions
IOO designed the experiment and supervised the work. NE performed the experimental work and wrote the manuscript.

Conflict of Interest Disclosures
None.

Ethical Issues
Treatment of the animals conformed to the guidelines of the Principles of Laboratory Animal Care (NIH Publication 85-23, revised 1985).