Synergistic Effect of Combined Leaf Extract of Vernonia amygdalina, Ocimum gratissimum, and Zingiber officinale Tuber on Phytochemical Profile, Antioxidant Activity, Serum Insulin, and Biochemical Parameters in Streptozotocin-Induced Diabetic Rats

Abbrevations

WBC: White Blood Cell; RBC: Red Blood Cell; HB: Hemoglobin; HCT: Hematocrit; MCV: Mean Corpuscular Volume; MCH: Mean Corpuscular Hemoglobin; MCHC: Mean Corpuscular Hemoglobin Concentration; PLT: Platelets.

Introduction

The metabolic illness known as diabetes causes elevated blood sugar levels, irregular metabolism of lipids and proteins, and many long-term consequences that might impact the kidneys, neurological system, and retina [1]. An abnormal increase in blood glucose after food, beta-cell malfunction, and relative insulin insufficiency all contribute to hyperglycemia. Pancreatic α-amylase and α-glucosidases both play a role in the breakdown of starch and other big polysaccharides into smaller, more easily absorbed sugars [2, 3]. Given the multi-lesion nature of diabetes’ pathophysiology, it would need more than one pharmacological therapy to undo the disease’s effects in their entirety. Because of the abundance of active components in just one plant, traditional medicinal herbs have gained favor as part of a multimodal approach to treatment [4, 5, 6]. Phytochemicals are a class of bioactive, non- nutritive plant compounds with a wide range of protective and disease-preventive effects, including hormone action, antioxidant, enzyme stimulant, antibacterial, and anti-cancer effects. Medicinal plants are often plants that contain a high concentration of these compounds. Some organic chemicals found in medicinal plants are recognized to have specific physiological effects on humans [5].

The use of poly-herbal therapy, which involves combining agents from different plants for therapeutic purposes, has garnered significant attention for several decades. This approach has the potential to be very effective in treating a wide range of diseases, and it is also safer than conventional medicine, with few or no side effects [7, 8]. Given the multi-factorial pathogenicity of diabetes mellitus, polyherbal therapy is favored as a therapeutic approach to its control [7]. Because they provide promising prospects for the creation of novel treatments for the control of diabetes mellitus, traditional medicinal herbs are believed to be the source of this increased effectiveness. For example, saponins slow the transfer of glucose from the stomach to the small intestine [9] and polyphenols in tea reduce postprandial hyperglycemia and glucose transport throughout the small intestine [10].

According to Bounouham M, et al. [8], pancreatic β-cells are protected from alloxan damage by epicatechin, and plant flavonoids have anti-diabetic effects due to their antioxidant capabilities. Ayo VI, et al. [11] and Umaru IJ, et al. [12] found that medicinal plants with high concentrations of phytochemicals that could help fight diabetes and its problems have sped up attempts to collect and process them.

Vernonia amygdalina is an Asteraceae family shrub or small tree that can grow to be 2-5 meters tall. Its petiolate, elliptically-shaped leaves are approximately 6 mm in diameter. The leaves have a bitter flavor and a distinct smell; they are green in color [13]. Ezeonu CS, et al. [14] noted that the plant has been domesticated in numerous regions of Nigeria. In Yoruba, it is called “Ewuro,” in Ibibio, “Onugbu,” and among the Hausa people of Nigeria, it is called “Chusa- diki” [15].

Villagers often use Vernonia amygdalina as a fence post or pot herb since it thrives in a variety of African biological zones, produces a lot of forage, and can withstand drought [16]. Vernonia amygdalina is known to have a diverse range of phytochemicals. Some studies have found tannins, oxalates, and phytates in it [13, 17]. The ethnomedical and pharmacological effects of extracts from Vernonia amygdalina are extensive, and include antibacterial, anti-diabetic, anti- malarial, and anti-helminthic capabilities [18]. Another plant with therapeutic importance is Ocimum gratissimum, also known as smell leaf.

There is a whole system of roots, stems, and leaves in this fully grown flowering plant [19]. The Lamiaceae family includes the aromatic medicinal herb Ocimum gratissimum. Scent leaf is the common name for it. Because of its fragrant minty flavor, it finds application in cookery. Effraim K, et al. [20] listed many Nigerian names for the plant: “Effinrin-na” for the Yoruba, “Alumokho” for the Esan, “Nchanwu” for the Igbo, “Aramogbo” for Edo, and “Daidoya” for the Hausas of northern Nigeria. The medicinal ginger plant, scientifically known as Zingiber officinale, features slender, verdant, grass- like leaves and fragrant, yellowish-green blossoms spotted with purple. The rhizome of ginger, which is harvested in tropical regions when it is around 10 months old, is edible and has several uses, both in the kitchen and in medicine [21]. Egedigwe C [15] states that ginger’s fragrant, carminative, and absorbent qualities are responsible for its effectiveness. Both as a spice and a functional food, ginger has several uses.

The medicinal properties of ginger have been extensively studied and documented. Some of these properties include anti-arthritic, anti-thrombotic, anti-inflammatory, hypolipidaemic, hypocholesterolaemic, and anti-nausea effects. The anti-diabetic effects of Vernonia amygdalina, Ocimum gratissimum, and Zingiber officinale tuber extracts have been proven individually, but new research suggests that their combined effects are much more effective [7]. Thus, it became critical to study the biochemical and histological impacts of a mixture of plant extracts. This study aims to examine the effects of a combination of extracts from Verninia amygdalina, Ociumum gratissimum, and Zingiber officinale tuber on the histology (morphology) of the pancreas, as well as on blood glucose, insulin, lipid profile, and streptozotocin- induced diabetes in wistar rats.

Discussion

Since poly-herbal therapies are safer and have few or no side effects, they have received a lot of attention for a number of years. This is because they maximize therapeutic efficacy against a wide range of diseases [5, 7]. The purpose of this study was to examine the effects of a combination of extracts from Verninia amygdalina, Ociumum gratissimum, and Zingiber officinale tuber on hyperglycemia, insulin resistance, lipid profiles, and pancreatic histology in rats with streptozotocin diabetes.

The study’s findings showed that diabetic rats (Group 2), which were not treated, lost significantly more weight than non-diabetic rats (Group 1) and rats treated with a combination of plant extracts (Group 4). This could be because of the excessive breakdown of tissue protein and fatty acids, which leads to the loss of muscle and adipose tissue [7]. For every gram of glucose excreted, there is a considerable loss of calories, leading to severe weight loss despite increasing hunger. This is particularly true when combined with the breakdown of protein, which causes muscle and adipose tissue to lose weight as well. One symptom of diabetes mellitus is weight loss, particularly in cases of poor glycaemic control. Untreated diabetic rats also showed a considerable decrease in body weight, according to the studies [1]. Group 3, which received metformin, gained more weight than Group 4, which received extract, according to the weight results.

This is in line with the findings of Mäkimattila S, et al. [23], who found that metformin’s enhanced glycaemic control leads to weight gain through a decrease in metabolic rate and glycosuria. Probably as a result of interactions between several bioactives, the extract-treated group was able to avoid such drastic weight loss. Nonetheless, when contrasted with the diabetic control animals (Group 2), the mice treated with extracts (Group 4) exhibited a notable rise in weight. This gain in mass is evidence that the treatment enabled the tissues to use glucose for energy and even set aside some to construct growth-related tissue components.

Research conducted by Ebong PE, et al. [7] demonstrated that mice treated with Verninia amygdalina extracts experienced a weight gain after 14 days. When taken together, the leaf extracts of Vernonia amygdalina, Ocimum gratissimum, and Zingiber officinale tuber reduced blood sugar levels to a level comparable to those of metformin. According to Joseph I, et al. [24], plant extracts are believed to have a blood sugar reducing impact due to the phytochemicals they contain, such as alkaloids, polyphenols, tannins, saponins, and dietary fiber. One possible explanation for the hypoglycemic effect of plant extracts is the presence of micronutrients like vanadium, which have actions similar to insulin. The majority of plant extracts reportedly work by way of insulin-like elements that are already present in the extracts, according to Yeh GY, et al. [25]. This study’s findings of pancreatic islet cell proliferation and regeneration, in addition to the presence of phytochemicals and micronutrients in plant extracts that have a direct impact on blood sugar, may explain why the treated animals’ blood glucose levels dropped.

Liver function test results showed that treated diabetic rats had higher serum insulin levels compared to control diabetic rats, and that combined plant extracts considerably reduced serum AST and ALT. In Group 4, diabetic rats, the results demonstrated that the combination plant extract reduced serum urea and creatinine levels. Ife AV, et al. [26] found that renal impairment is associated with elevated serum creatinine and urea levels in diabetic hyperglycemia. When diagnosing liver diseases, blood enzymes like AST and ALT are employed. Elevated levels of these enzyme activity indicate that the liver is actively damaged. Levels of transaminase are dramatically increased in inflammatory hepatocellular diseases [21]. Based on the significant rise (P<0.05) in total cholesterol, triglyceride, and LDL-cholesterol levels compared to the control group, this research shows that Streptozotocin alters the lipid profile of diabetic rats (Group 1). The levels of total cholesterol, triglycerides, and LDL-cholesterol in the animals treated with the mixed plant extract in Group 4 were significantly lower (p<0.05). The mixed plant extract may have a hypocholesterolemic impact because of the high polyphenol content. Cholesterol production, cholesterol clearance from the bloodstream, dietary cholesterol absorption, and cholesterol excretion through bile and feces are all known to impact plasma cholesterol content [27].

Hyperglycemia, insulin insufficiency, and hyperketonemia are the three main causes of electrolyte and water imbalances in people with diabetes mellitus [27]. The electrolyte result reveals a significant decrease in serum Na, Cl, and K levels as well as an increase in serum Ca in the diabetic subjects (Group 2), but no difference between the normal control animals (Group 1) and the animals treated with the combined plant extract (Group 4). This suggests that the electrolyte imbalance seen in diabetic animals can be alleviated by combining plant extracts. Consistent with other investigations, this outcome was reported by Atangwho IJ, et al. [28]. The proximal tubule of the kidney is responsible for reabsorption of the majority of Na under normal conditions [27]. Dehydration, diabetic nephropathy, or renal failure could cause this electrolyte loss in animals with diabetes mellitus (Group 2). Inhibition of the rennin-angiotensin- aldosterone pathway, which is essential for maintaining proper fluid and electrolyte balance, is another possible cause of this electrolytes imbalance. Numerous endocrine and cardiovascular disorders have been found to impact this enzyme system. Some disturbed hematological parameters of diabetic rats may be improved by treating diabetes with Verninia amygdalina, Ociumum gratissimum, and Zingiber officinale. Anemia is thought to occur in diabetes mellitus as a result of increased non-enzymatic glycosylation of red blood cell (RBC) membrane proteins, which is associated with hyperglycemia [29].

A rise in lipid peroxide generation, leading to RBC hemolysis, occurred as a result of oxidation of these glycosylated membrane proteins and hyperglycemia in diabetes mellitus. When it comes to the quality of the feed that animals consume and the nutrients that are accessible to them to fulfill their physiological needs, hemostatic indices are indications and reflections of the impacts of dietary therapies. Hemoglobin (Hb) values, which are defined as an iron-containing conjugated protein that carries oxygen and carbon dioxide throughout the body, showed no significant changes in the diabetic or diabetic treated groups compared to the normal control group. This suggests that the animals in both groups did not experience a decrease in respiratory capability, suggesting that the oxygen-carrying capacity of their blood was unaffected. The reduced lipid peroxide level in RBC membrane, which in turn decreased the susceptibility of RBC to hemolysis, may explain why animals treated with the mixed plant extract (Group 4) had an enhanced RBC count. One possible explanation for the action of the combined plant extracts in treating diabetic rats is a reduction in the increased glucose concentration, since hyperglycemia is associated with non-enzymatic glycosylation of membrane proteins [30]. Group 4 diabetic rats were found to have normal red blood cell (RBC) counts after receiving the mixed plant extract, which suggests that it stimulates erythropoiesis. They may have a function in erythropoiesis, since their PCV (Packed cell volume) decreased significantly in diabetic mice and then normalized after treatment with a combination of plant extracts. Based on the findings of RBC, Hb, and PCV tests, it appears that Verninia amygdalina, Ociumum gratissimum, and Zingiber officinale tuber have antioxidant properties.

These plants also aid in stabilizing the RBC membrane by binding to proteins and carbohydrates that make up the RBC membrane. As a result, they may prevent the RBC membrane from breaking down and counteract Streptozotocin’s anemic effects. According to Ganong WF [31], neutrophils are the initial line of defense against bacterial infections because they consume and destroy microorganisms. One theory is that the immune system’s inability to properly operate as a result of diabetes-related neutrophil dysfunction undermines the body’s ability to fight off infections [28]. This study showed that diabetic rats were able to restore their normal white blood cell (WBC) neutrophil percentage after being treated with a mixture of Verninia amygdalina, Ociumum gratissimum, and Zingiber officinale tuber extracts. This finding provided compelling evidence that diabetic rats treated with a mixture of plant extracts may have an enhanced immune response to infections. A normalization of RBC count in diabetes treated rats may explain the recovery of blood indices (MCV, MCH, MCHC), as previously noted, RBC count increased to control level in group 4.

Pancreatic histopathology in non-diabetic animals reveals intact endocrine and exocrine pancreatic tissues (Figure 1). The islet cells in the endocrine section were normal. In contrast, the pancreatic cyto-architecture was significantly distorted with necrotic and degraded islet cells in the diabetic control mice (Figure 2). Streptozocin causes damage to the pancreatic tissues, particularly the islet cells, leading to a type 1 diabetes prototype, as reported by Bolkent S, et al. [32]. Full-blown diabetes was caused by STZ-induced pancreatic lesions [33]. Figure 4 shows that mice given a mixture of plant extracts had islet cell growth, a nearly normal pancreatic cyto-architecture, and other pancreatic cell proliferation. This is in line with the previous study that focused on the effects of giving people Vernonia amygdalin extracts [28]. The prior report did show some improvement, but it was only a partial recovery. The current study suggests a synergistic interaction of the numerous phytochemicals and antioxidants from Vernonia amygdalina, Ocimum gratissimum, and Zingiber Officinale, tuber, as the extract is co-administered with these three plants. The results are full and holistic. This potential synergistic activity utilizing the extracts of Azadirachta indica and Vernonia amygdalina was previously described by Ebong PE, et al. [7]. Because the regenerated islet cells may improve insulin synthesis and secretion, the antihyperglycemic/ hypoglycemic actions of the extracts may be explained by their effects on pancreatic lesions.

The capacity of extracts from Vernonia amygdalina and Ocimum gratissimum to reverse streptozotocin- induced pancreatic damage in mice may be due to active components such as vernoniosides, glucosides, flavonoids, and antioxidants [34]. Antioxidant characteristics and components of Vernonia amygdalina, Ocimum gratissimum, and Zingiber officinale tuber have been shown in multiple research. Ezeonu CS, et al. [14] found that Vernonia amygdalina has a high concentration of antioxidants, while Kesavulu M, et al. [35] found that Zingiber officinale has antioxidant characteristics. Reactive Oxygen Species (ROS) are produced in diabetes mellitus, which damages tissues because they interfere with the antioxidant reaction that enzymes used to scavenge ROS do their work. Kesavulu M, et al. [35] found that diabetes mellitus is associated with enzyme deficiencies that scavenge ROS. Through one way or another, a plant extract that may alleviate or repair hypoglycemia-causing pancreatic lesions would also address the process of ROS formation. So, these plants need to be antioxidants that can either mop up ROS in the blood or turn back the cytotoxic cycle of STZ in the pancreas. Vacuolations in the exocrine parts of the pancreatic islet cells gave the impression of distortion and degeneration in the metformin-treated mice (Figure 3). Therefore, it’s possible that metformin has no direct impact on the pancreas. Pancreatic islet cyto-architecture did alter slightly from the diabetic control group, but only little. This is in line with previous findings from Pirola L, et al. [36, 37, 38] that metforming lowers blood sugar levels by acting on peripheral tissues rather than the pancreas. Metforming helps with glucose uptake into cells and inhibits hepatic glucose output. The destruction of insulin-producing cells in the pancreatic islets may explain the hyperglycemia seen in the Diabetic Control animals (Figure 2). This is in line with the findings of Kesavulu M, et al. [35, 39], who found that streptozotocin causes diabetes by targeting and killing the pancreatic cells that produce insulin.

Conclusion

The presence of phytochemicals and micronutrients in plant extracts exert direct effect on blood sugar, the proliferation/regeneration of the pancreatic islet cells which was evident in this study could account for the reduction in blood glucose of animals treated with the extract which leads to the regeneration of the beta-cells of the pancreas. The results obtained from the electrolyte analysis it could be concluded that the diabetic patients have electrolyte imbalance characterized by depletion in the levels of sodium, potassium and chlorine and increased in the levels of the calcium ions. The result obtained from the Liver function, Kidney function biomarkers there was a significant improvement in the liver and kidney function of the diabetic rats treated with the combined extract of Verninia amygdalina, Ociumum gratissimum and Zingiber officinale tuber. It also exhibit antihyperglycemic and antihyperlipidemic properties. From the hematological results, it is apparent that oral administration of the combined extracts might decrease the diabetes-induced disturbances of hematological parameters in streptozotocin-induced diabetic rats. Therefore this combined plant extracts must be considered as excellent candidate for future studies on diabetes mellitus. In addition, further comprehensive pharmacological investigations, including experimental chronic studies, should be carried out, and further comprehensive studies should be carried out to confirm how they exact their mechanism of action.

Acknowledgments

We acknowledge all the authors of this work for their contributions.

Conflicts of Interest

All authors declare that they have no conflict of interest associated with this research work.

Funding

No special funding was received for this research work.