The ameliorative activity of Chrysobalanus orbicularis in streptozotocin-induced type II diabetes mellitus rat model.

Chrysobalanus orbicularis is a medicinal plant present in West Africa in the Itsekiri speaking part of Nigeria. It is used conventionally in diabetes mellitus management. This research investigates the ameliorative activity of the aqueous leaf extract of C. orbicularis in a streptozotocin-induced diabetic rat model. Freshly prepared streptozotocin (40 mg/kg body weight [BW]) was administered intraperitoneally to induce diabetes. Three diabetic groups were placed on aqueous leaf extract of C. orbicularis at 11.076, 22.134, and 44.268 mg/kg BW respectively; a group was placed on metformin (44.28 mg/kg BW), and the other two groups were the diabetic control and normal control. The experiment lasted for 28 days, thereafter, fasting blood glucose levels and body weight variations were recorded. Also, glycogen level, antioxidant enzyme, hexokinase and glucose-6-phosphatase activities, malonaldehyde (MDA) as well as glucose transporters 2 and 4 levels were analyzed using standard procedures. Diabetic rats administered aqueous extract of C. orbicularis leaf significantly (p < 0.05) decreased the fasting blood glucose and MDA levels, and glucose-6-phosphatase activity. In addition, administration of aqueous extract of C. orbicularis leaf to diabetic rats demonstrated a momentous increase in liver glycogen level, superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione, glutathione transferase, and hexokinase activities as well as GLUT-2 and GLUT-4 levels. The data from this study suggest that the aqueous extract of C. orbicularis leaf may be beneficial in the management of diabetic mellitus and its secondary effects.

Introduction

Diabetes mellitus is a significant issue on the earth's planet today and characterized by clinical abnormalities (Piero et al., 2015). This condition happens because of glucose homeostasis impairment, characterized by supreme or relative insufficiency of the hormone insulin or inefficacy of its fringe activity (Amira et al., 2016). This is showed with constant hyperglycemia a noteworthy side effect of diabetes mellitus, influencing carbohydrate, fat, and protein digestion (Sorour et al., 2018).

Among the various kinds of diabetes mellitus, type II is the most common, both at international and in sub-Sahara Africa, with Nigeria positioned at fifth (Ajiboye et al., 2020). Hindrance of insulin signalling in skeletal muscle, liver and fat tissues because of the advanced insulin resistance is the primary driver of type II diabetes mellitus. This disorder advances and prompts organ dysfunction and unexpected death (Joudaki and Setorki, 2019). Exogenous variables like ecological factors just as hereditary qualities, diet and weight contribute extraordinarily to the improvement of this ailment (Piero et al., 2015). Lately, characteristic spices have been utilized broadly for the administration of type II diabetes mellitus and its difficulties and it has been accounted for that it is more secure with next to zero side effects (Ibitoye et al., 2017) consequently the inclination of elective medications over manufactured drugs (Kooti et al., 2016).

In Africa there are a few restorative spices used to oversee diabetes mellitus and its intricacy, this organic assorted variety converts into an auxiliary decent variety (Pires et al., 2016) empowering the disclosure of new types of bioactive substances with antidiabetic potential documented by Paracampo (2011). In Nigeria, there are various plants under this classification and a model is Chrysobalanus orbicularis, which belongs to the family Chrysobalancea, known for its crucial therapeutic qualities locally in the Southern part of Niger Delta in Nigeria just as they are utilized for the mainstream Niger Delta pepper soup. The plant is generally called "Omillo", logical data on the impacts of this generally expended leaf has not been settled. Showing the scientific efficacy of the plant reinforcement as of late an investigation was done on the antioxidant and inhibitory activities of enzymes linked to type II diabetes the role of the novel Chrysobalanus orbicularis by Ekakitie et al. (2020) indicating the scientific efficacy of the plant. In light of an ethnobotanical study nonetheless, this existing study assesses the ameliorative action of aqueous leave extract of Chrysobalanus orbicularis in streptozotocin-prompted type II diabetes mellitus rats model.

Materials and methods

Chemicals and reagents

Streptozotocin and other chemicals used in this study were purchased from Sigma Chemical Co., St Louis, MO, USA. Although, the enzyme assay kits were secured from Randox Laboratories Ltd., Antrim, UK. It is important to note that analytical grade was taken into consideration for all the chemicals and reagents.

Plant materials and authentication

Chrysobalanus orbicularis leaf was secured in March 2019 at exactly 9 a.m in Itsekiri layout, Sapele, Delta State, Nigeria. The leaf was subsequently authenticated at Forestry Research Institute of Nigeria (FRIN) Ibadan, Nigeria, and FHI:112232 was assigned as its herbarium number.

Preparation of plant materials

Firstly, the obtained leaves were washed under running water and dried at 25 °C for two weeks and then pulverized into fine particles (using a food processor). Then, two hundred grams of the powdered sample was extracted in two thousand millilitres of distilled water by maceration, according to the method of Nwozo and Oyinloye (2011) for 72 h, filtered with cheesecloth, and freeze-dried to obtain the dried extract. Based on the ethnobotanical survey, a cup of the filtrate was also dried and 0.2523 g was obtained as the yield. This is corresponding to the dose consumption of a 70 kg man. This was further extrapolated to get 22.134 mg/kg. The obtained yield was kept at 4 °C using a universal bottle for further studies.

Experimental animal

Sixty male albino rats, weighing between 130 to 140 g were secured from the Animal Holding unit of Afe Babalola University, Ado-Ekiti, Nigeria. The animals were acclimatized for seven days in a laboratory environment of 12 h light/dark phase and had free access to feed and water before the commencement of the experiment at a temperature of 25 °C. This research study was approved by the Animal Ethical Committee of Afe Babalola University, Ado-Ekiti, Ekiti State, Nigeria (ABUAD/PG/SCI/022).

Induction of diabetes mellitus

Streptozotocin (STZ) was liquefied in citrate buffer at a concentration of 15 mg/mL. The animals fasted overnight and the initial fasting blood glucose (FBG) level for each of the animals was checked before inducing diabetes by injecting 40 mg/kg of STZ intraperitoneally. Forty-eight hours after STZ administration, FBG levels of all the experimental animals were determined using the ACCU check glucometer. As documented previously by Ajiboye et al. (2018), animals with FBG levels greater than or equal to 250 mg/dL were employed in this study.

Animal groupings

This was divided randomly into six groups of ten animals in each group, which was housed in a pathogen-free environment.

Group 1: non-diabetic control (normal control)

Group 2: diabetic rats without treatment (diabetic control)

Group 3: diabetic rats managed on 11.076 mg/kg body weight of leaf extract of C. orbicularis

Group 4: diabetic rats managed on 22.134 mg/kg body weight of leaf extract of C.orbicularis

Group 5: diabetic rats managed on 44.268 mg/kg body weight of leaf extract of C. orbicularis

Group 6: diabetic rats managed on 44.280 mg/kg body weight metformin

Treatment of animal with plant extract and metformin

The extract of C.orbicularis leaf and metformin were orally administered to the corresponding animals using a needle and intubator. This administration route was preferred due to its safety, ease of ingestion as well as preventing pain. Notably, the administration was carried out between 9 and 10 am during the experimental period.

Tissues processing

The animals were fasted overnight (12 h) and euthanized with halothane anesthesia on the 28th day of the experiment. The whole blood of each rat was collected through cardiac puncturing, permitted to clot, centrifuged at 3000 rpm for 900 s to achieve a clear solution called serum, and finally conserved by a refrigerator. The liver of the experimental animals was expunged washed, wiped and homogenized using 0.1 M potassium phosphate buffer. Successively, centrifuged at 4000 rpm for 900 s to achieve supernatant, and employed for different analyses (Ekakitie et al., 2020).

Biochemical analysis

FGB level was carried out according to the procedure of Ahmad et al. (2007) via ACCU check glucometer. The obtained blood from the tail end of each rat was dropped into the testing area of the glucometer. Then the actual FBG level was revealed in the screen of the glucometer (mg/dL). The FGB was recorded before STZ induction, at 48 h induction of STZ, on the 14th day of the experiment, and finally on the 28th day of the experiment before the sacrifice of the animals.

Liver glycogen was evaluated via a method of Murat and Serfaty (1975). Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GSH), glutathione transferase (GST), GLUT 2, GLUT 4 and lipid peroxidation [measured as malondialdehyde (MDA)] were determined via commercial kits. Also, hexokinase and glucose-6-phosphatase activities were respectively carried out via Akinyosoye et al. (1987) and Baginskyi et al. (1974).

Data analysis

Data were accepted as a mean ± SD of ten replicates. For the statistical analysis of all the results obtained, graph pad prism 5 (GPP) software was used. A one-way ANOVA was used to conduct Tukey's post hoc examination. The meaningful difference was set at p < 0.055.

Results

Body weight of diabetic rats administered aqueous extract of C. orbicularis leaf

The body weights of the rats were not substantially different from each other at the beginning of the experiment (Figure 1). However, there was a substantial decline in the body weight of diabetic control rats at days 14 and 28 compared with other classes. Whereas there was a substantial increase in body weight of diabetic rats, administered at different doses of C. orbicularis leaf.

Figure 1

Aqueous leaf extract of C. orbicularis on body weight of streptozotocin-induced diabetic rats. Values are expressed as mean ± standard deviation (SD) of ten replicates. Bar with the same letters are not significantly different at p > 0.05. Bar with different letters are significantly different at p < 0.05. NC: normal control; DC: diabetic control; Diab: diabetic; metf: metformin; AACO: C. orbicularis leaf extract; BW: body weight; IBW: Initial Body Weight.

Fasting blood glucose level of diabetic rats administered aqueous extract of C. orbicularis leaf

The result (Figure 2) indicates that before the injection of STZ, fasting blood glucose levels were not substantially different from each other. Important changes in fasting blood glucose levels occurred at 48 h of STZ induction in all except in the normal control group. Three doses of the aqueous extract of C. orbicularis leaf showed (at 14th and 28th days) a substantial decrease with non-significant (p > 0.05) difference in the fasting blood glucose levels when compared with the control group.

Figure 2

Aqueous leaf extract of C. orbicularis on fasting blood glucose levels in streptozotocin-induced diabetic rats. Values are expressed as mean ± standard deviation (SD) of ten replicates. Bar with the same letters are not significantly different at (p > 0.05). Bar with different letters are significantly different at (p < 0.05). NC: non-diabetic control; DC: diabetic control; Diab: diabetic; metf: metformin; AACO: C. orbicularis leaf extract; IFBGL: Initial Fasting Blood Glucose Level; FBGL: Fasting Blood Glucose Level.

Liver glycogen of diabetic rats administered aqueous extract of C. orbicularis leaf

In contrast to other groups, there was a substantial reduction in liver glycogen in diabetic control rats. However, a substantial increase in liver glycogen levels in diabetic rats administered three separate doses of aqueous extract of C. orbicularis leaf was observed at the end of the experiment (Figure 3).

Figure 3

Liver glycogen level in streptozotocin-induced diabetic rats after administration of aqueous extract of C. orbicularis leaf. Values are expressed as mean ± standard deviation (SD) of ten replicates. Bar with the same ∗ are not significantly different at p > 0.05. Bar with different ∗ are significantly different at p < 0.05. Normal control: non-diabetic control; met: metformin; diab: diabetic; AACO: Chrysobalanus orbicularis aqueous leaf extract.

Oxidative stress biomarkers of diabetic rats administered aqueous extract of C. orbicularis leaf

STZ administration resulted in substantial depletion in superoxide dismutase, catalase, glutathione peroxidase, glutathione (reduced) and glutathione transferase concentrations, as well as augmented levels of MDA relative to other groups. However, the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione (reduced) and glutathione transferase increased significantly (p < 0.05) as well as a decrease in MDA levels in diabetic rats (Figure 4).

Figure 4

Oxidative stress biomarkers level in streptozotocin-induced diabetic rats after administration of aqueous extract of C. orbicularis leaf. Values are expressed as mean ± standard deviation (SD) of ten replicates. Bar with the same ∗ are not significantly different at p > 0.05. Bar with different ∗ are significantly different at p < 0.05. Normal control: non-diabetic control; met: metformin; diab: diabetic; AACO: Chrysobalanus orbicularis aqueous leaf extract; GPx: Glutathione peroxidase; GSH: Glutathione reductase; GST:Glutathione transferase SOD: superoxide dismutase; CAT: catalase; MDA: Malondialdehyde.

Hexokinase and glucose-6-phosphatase specific activities of diabetic rats administered aqueous extract of C. orbicularis leaf

The result revealed a substantial reduction in diabetic control of hexokinase-specific activity as well as a momentous upsurge in glucose-6-phosphatase-specific activity of diabetic control rats linked to other groups. On the other hand, a substantial increase in hexokinase activity was observed with a significant (p < 0.05) decrease in glucose-6-phosphatase activity in the liver of diabetic rats given three separate doses of aqueous extract of C.orbicularis leaf (Figure 5).

Figure 5

Hexokinase and Glucose-6-phosphatase specific activity in streptozotocin-induced diabetic rat after administration of aqueous extract of C. orbicularis leaf. Values are expressed as mean ± standard deviation (SD) of ten replicates. Bar with the same ∗ are not significantly different at p > 0.05. Bar with different ∗ are significantly different at p < 0.05. Normal control: non-diabetic control; DC: diabetic control; Diab: diabetic; met: metformin; AACO: Chrysobalanusorbicularis leaf.

Glucose transporters (GLUT 2 and GLUT 4) of diabetic rats administered aqueous extract of C. orbicularis leaf

Figure 6 displayed a substantial decline in diabetic control levels of GLUT 2 and GLUT 4 when compared with other groups. On the 28th day of this study, there was a substantial increase in the GLUT 2 and GLUT 4 levels of diabetic animals given three separate doses of aqueous extract of C. orbicularis leaf, with no distinct significance (p > 0.05) concerning the non-diabetic control group.

Figure 6

Glucose transporter (GLUT-2 and GLUT 4) levels after administration of aqueous extract of C. orbicularis leaf. Values are expressed as mean ± standard deviation (SD) of ten replicates. Bar with the same ∗ are not significantly different at p > 0.05. Bar with different ∗ are significantly different at p < 0.05. Normal control: non-diabetic control; met: metformin; Diab: diabetic; AACO: C. orbicularis leaf; GLUT: Glucose Transporter.

Discussion

Oxidative stress has been associated with the aetiology of numerous illnesses including diabetes mellitus. Oxidative stress causes membrane harm driving at long last to membrane rupture in various cell types (Kirubananthan et al., 2019). Cells have a proficient antioxidant defence agent protection framework against ruinous prompted by oxidative stress. A rise in blood glucose incites oxidative stress bringing about an expanded creation of oxygenated free radicals and diminished antioxidant enzyme activities (Liu et al., 2008). This may bring about intracellular structure adjustment and eventually influence the normal cellular functions, prompting pathogenesis and the development of diabetic complexities. The current study portrays the ameliorative effect of the ethnobotanical doses of aqueous leaf extract of C. orbicularis in the therapeutic management of diabetes mellitus, a significant piece of information in drug advancement.

Currently, an aqueous extract of C. orbicularis leaf had the potential to increase the weight of induced diabetic rats. Khaleel et al. (2015) reported among numerous scientists that streptozotocin-induced diabetic rats are known by body weight loss, probably due to protein breakdown and the incompetence to provide gluconeogenesis amino acid linked to insulin deficiency, resulting in muscle wasting and tissue breakdown in diabetic rats (Anand et al., 2008). Whereas, administration of various doses of aqueous extract of C. orbicularis leaf to the diabetic rat was able to restore weight loss probably due to insulin secretion enhancement.

Streptozotocin has been employed as an inducing agent to cause diabetes mellitus by causing selective damage to insulin generating pancreatic β-cells (Zhang et al., 2015). Pancreatic beta-cell damage affects insulin secretion and eventually leads to a disorder known as hyperglycaemia (Sicree et al., 2006). The ability of the C. orbicularis leaf aqueous extract to reduce hyperglycaemia to normoglycaemia may be due to its high antioxidant content, which may regenerate the damaged beta cells (of the pancreas) that were impaired (Ekakitie et al., 2020). Administration of aqueous extract of C. orbicularis leaf to induced diabetic rats exhibited brilliant performance probably by inhibiting the absorption of glucose from the intestine, thereby helps to release glucose from the liver, or it could increase the number of insulin receptors, which in turn increases the sensitivity to insulin levels of the target tissues.

Liver glycogen is an essential biomarker for the assessment of the normoglycaemic effect of plant extracts or drugs (Huang et al., 2000). The rise in liver glycogen levels of the aqueous extracts of C. orbicularis leaf administered to diabetic rats can be due to its action in inhibiting glycogen phosphorylase. This is a central enzyme in glycogenolysis that, by inhibiting glycogen phosphorylase, causes glucagon and epinephrine to release glucose into the blood. This activity inhibits the entry of glucose into the blood, thereby reducing the amount of glucose in large quantities (Ajiboye et al., 2018). Besides, the aqueous extract of C. orbicularis leaf administered to diabetic rats trigger an enhancement in liver glycogen related to an increase in the secretion of insulin.

Prolonged hyperglycemia observed in uncontrolled diabetes mellitus leads to glucose oxidation, the main source of free radicals. It is known that diabetes mellitus reduces the activity of antioxidant enzymes (especially SOD, CAT, GPx, GSH and GST). Oxidative stress can inhibit insulin signalling and cause insulin resistance via hyperglycemia. Declined cellular antioxidants may also result from hyperglycemia. However, a momentous increase in SOD, CAT, GPx, GSH and GST activities in diabetic rats administered an aqueous extract of C. orbicularis leaf, indicates that it can reverse the action of free radicals. This is in accordance with the Mahmoud et al. (2002) study. Also, the antioxidant nature of aqueous extract of C. orbicularis leaf, as well as the antioxidant enzyme boosters observed in this study, may be linked to a decline in lipid peroxidation levels.

The increased hexokinase-specific activity observed in diabetic rats administered aqueous extract of C. orbicularis leaf. The ability of the extract to directly induce glycolysis may be due to an upsurge in insulin secretion. Hexokinase is the primary regulating enzyme in the metabolism of intracellular glucose, thereby increased glucose removal catalyzes the phosphorylation of glucose to glucose-6-phosphate in the tissues (Huang et al., 2000). The last step of the gluconeogenesis pathway that catalyzes the breakdown of glucose-6-phosphate into glucose is glucose-6-phosphatase. The glycolytic metabolite pools are therefore modulated by these key enzymes, hence they are essential for maintaining normal blood glucose levels by sustaining stability between the production of glucose and its use in the body (Anand et al., 2008; Murray et al., 2000). The ameliorative potential of these enzymes in diabetic rats administered aqueous extract of C. orbicularis leaf might be associated with an increase in insulin secretion triggers an increase in glycogen synthesis. The findings obtained are in line with Kirubananthan et al. (2019) previous studies.

The noticed rises in the amount of GLUT 2 and GLUT 4 in diabetic rats placed on aqueous extract of C. orbicularis leaf is probably linked to an enhancement in insulin secretion. Though the reduction in these glucose transporters could be associated with declining glucose uptake in diabetic mellitus states (Taderera et al., 2019). Thus this could be one of the mechanisms of antidiabetic potential of C. orbicularis.

Conclusions

The results of this research show that an aqueous extract of C. orbicularis leaf has antidiabetic activities, as it ameliorates the streptozotocin-induced type II rats model. Therefore, using ethnobotanical dosage, the plant extract may be useful for treating diabetic mellitus and its complications. To the best of our understanding, this is the first study reporting the ethnobotanical value of this plant in diabetes mellitus management.

Declarations

Author contribution statement

Basiru Ajiboye, Lisa Ilobekemen Ekakitie, Babatunji Emmanuel Oyinloye: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data will be made available on request.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.