Estimation of Serum Glutathione Peroxidase in Streptozotocin Induced Diabetic Rat Treated with Bitter Leaf Extract

Department of Medical Laboratory Science, Madonna University, Elele, Rivers State, Nigeria. 2 Department of Medical Laboratory Science, Imo State University, Owerri, Nigeria. Department of Medical Laboratory Science, University of Calabar, Cross River State, Nigeria 4 Department of Nursing Science, Imo State University, Owerri, Imo State, Nigeria. 5 Department of Pathology and Biochemistry, State University of Zanzibar, Tanzania. Department of Haematology and Immunology, Faculty of Clinical Medicine, Ebonyi State University, Abakaliki, Nigeria. Department of Medical Laboratory Science, Nnamdi Azikiwe University, Nnewi Campus, Anambra State, Nigeria. 8 Department of Medical Laboratory Science, Igbinedion University Okada, Edo State, Nigeria Department of Chemical Pathology and Immunology, Olabisi Onabanjo University, Ogun State, Nigeria.


INTRODUCTION
Antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction that transfers electron or hydrogen from substances to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions, when the chain reactions occur in a cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidative reactions. They do so by being oxidizing themselves. Antioxidants are often reducing agents such as, thiols, ascorbic acid or polyphenols.
Under physiological conditions, hydrogen peroxide, superoxide and hydroxyl radicals, collectively called reactive oxygen species, are continuously produced and kept under strict control by many enzymes and antioxidants within the cells [1]. Clinical and experimental studies have shown that disturbing of oxidant-antioxidant balance system is involved in the pathogenesis of chronic diseases such as cancer, coronary heart disease, diabetes and many diabetic complications [2].
Chemical drugs have many side effects; therefore, looking for new antidiabetic drugs from natural antioxidants sources is still attractive because they are safe and good alternative for treatment of diabetes mellitus. A growing body of research indicates that nutritional deficiencies of antioxidants contribute to the development of diabetes [3].
Among antioxidant micronutrients, selenium (Se) is an essential dietary trace element, which plays an important role in a number of biological processes in humans and other species. Deficiency of this element induces some pathological conditions, such as cancer, coronary heart disease, and liver necrosis. Researchers have shown selenium and zinc efficacy on immune system and increase response to influenza and HBV vaccine [4]. Also researchers have shown sodium selenite decrease levels of lipid peroxidation (LPO) and NOPs (nitric oxide products) and increase activities of superoxide dismutase, GR (glutathione reductase), and GPX (glutathione peroxidase) in heart diabetesinduced rats. Selenium is an essential component of several enzymes such as GPX, TR (thioredoxin reductase) and SeP (selenoprotein P), which contains Seas selenocysteine.
Diabetes-related dysfunctions are the major causes of mortality and morbidity for diabetic patients. Although the precise mechanism by which hyperglycemia induces organ dysfunction is not fully understood, one of the hypothesis to explain this phenomenon is mainly focused on the role of free radicals in these disease states [5]. Various type of diabetes mellitus include the following.
Type 1 DM results from the body's failure to produce enough insulin. This form was previously referred to as "insulin-dependent diabetes mellitus" (IDDM) or "juvenile diabetes". The cause is unknownType 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 form was previously referred to as "non insulin-dependent diabetes mellitus" (NIDDM) or "adult-onset diabetes". The primary cause is excessive body weight and not enough exercise. Gestational diabetes mellitus: refers to glucose intolerance with onset or first recognition during pregnancy.
The study was done to estimate enzymatic antioxidant activity; serum glutathione peroxidase in streptozotocin induced diabetic rat treated with bitter leaf extract.

Study Area
This experiment was carried out in Madonna University Teaching Hospital Animal Farm, Elele, Nigeria.

Preparation of Plant Material
The fresh leaves of Vernonia amygdalina (bitter leaf) was collected from the school surroundings daily, well sorted and only fresh leaves were used in preparation of extract juice.

Animal Handling
Thirty two (32) adult rats of Wister strain weighing between 120 g -200 g of both sexes equally were used. They were allowed to acclimatize for two weeks a room of suitable temperature in an animal house in the animal farm in Madonna University Elele. After which they were fed with high fat diet for seven weeks to induce obesity. They were further grouped into four groups with each group having 8 rats. The animals were given 37mg/kg of streptozotocin to induce diabetes.

Experimental Design
Rats were grouped into four (4) groups: Group 1 (Negative control): The animal in this group were fed with only animal feed and water throughout the experiment. Group 2 (Positive control): Were given 37mg/kg of Streptozotocin in addition to food and water. Group 3: In addition to food and water, received 37mg/kg of streptozotocin and 2ml of bitter leaf extract. Group4: In addition to food and water, were given 5mg/kg of glibenclamide (anti-diabetic).

Sample Collection
At the end the acute feeding with normal feed, high fat feed and then treatment with glibenclamide and bitter leaf extracts, cardiac puncture was used to collect blood samples from the diabetic rat.

Quantitative Determination of Rat
Glutathione Peroxidase

Methods
The samples were analyzed using Enzyme linked immunosorbent assay (ELISA) as modified by Bioassay technology laboratory, (2017) Cat number-E1759Ra.

Procedure
50u/l of standard was added to standard well. 40u/l of sample was added to sample well and then add 10u/l of anti GSH-PX antibody to sample well. Then 50u/l of streptavidin-HRP was added to sample wells and standard well (not to blank control well) and mixed well and covered the plate with a sealer and incubated for 60 minutes at 37c.The sealer was removed and washed plate 5 times with wash buffer. The wells were soaked with at least 0.35ml of washed buffer to 30seconds for each wash. 50u/l of substrate solution A was added to each well and then add 50u/l of substrate solution B to each well and incubated plate covered with sealer for 10minutes at 37c in the dark. 50u/l of solution was added to each well, the blue colour changed into yellow immediately.

DISCUSSSION
Type 2 diabetes mellitus is the most common form of diabetes mellitus characterized by hyperglycemia, insulin resistance and relative insulin deficiency.
The effects of bitter leaf extract on induced streptozotocin diabetic rats of both sexes were evaluated. The values of fasting blood sugar level, body mass index and glutathione peroxidase activities were checked and recorded before and after administration of treatment.  Table 2 shows no significant difference (p>0.05) in BMI of rats across the weeks. There is no significant difference (P>0.05) in group 1(0.95±0.75) compared to group 2(0.94±0.71), group 3(0.82±0.10) compared to group 4(0.89± 0.08) of week 1.There is no significant difference (p>0.05) in group 1(0.80±0.09) compared to group 3(0.82±0.05),group 3(0.82±0.05) compared to group 4(0.79± 0.05) of week 3.There is also a no significant difference (p>0.05) in group 1(0.91±0.06) compared to group 3(0.86±0.1),group 2(0.84±0.07) compared to group 3(0.86±0.13),group 3(0.86±0.13) compared to group 4(0.83± 0.08) of week 5. This result is in line with finding by [6] who states that Through life style and diet modification, that there was significant reduction in the incidence of type 2 DM with a combination of maintenance of body mass index of 25 kg/m2, eating high fiber and unsaturated fat and diet low in saturated and trans-fats and glycemic index. Table 3 shows a significant difference (P<0.05) in FBS level of rats across weeks. There is a significant decrease (P<0.05) at group 3(7.72±0.99) compared to group 4(9.93±1.22) in week 2.There is also a significant decrease (p<0.05) at group 3(7.72±0.99) compared to group 4(9.90±1.24) in week 3.There is also a significant decrease (p<0.05) at group 3(6.22±1.20) compared to group 46.50±0.70) in week 5.There is a significant increase (p<0.05) at group 1(7.63±0.71) compared to group 4(5.78±1.40), group 2(7.45±0.87) compared to group 4(5.78±1.40) in week 4. Crude chloroform extract of bitter leaf has an antidiabetic effect on rats with type 2 diabetes mellitus under laboratory conditions. Similar result was stipulated [7] on observation of bitter leaf extract reducing glycemic level significantly having peripheral action similar to insulin or glucose metabolism which is attributed to the bioactive molecule contained in the vegetables. The result above is also in line with [8] who stated that the nutrient composition also revealed that bitter leaf contains moisture and fibers which contribute less sugar to blood sugar pool, this is because study revealed that food rich in fiber content induces less glucose response. A similar finding also concur with the above [9] showing great evidence that a high intake of dietary fiber associates with enhanced insulin sensitivity and therefore may have a role in prevention and control of type 2 diabetes [10][11][12].

CONCLUSION
The result of this study revealed that the levels of glutathione peroxidase and level of fasting blood sugar were decreased in streptozotocin induced diabetic rats treated with bitter leaf extract. Due to its decrease in Glutathione peroxidase activity level, there no damage effect on liver.

CONSENT
It is not applicable.

ETHICAL APPROVAL
The experimental protocols were according to our Institutional Animal Ethics Committee guidelines as well as internationally accepted practices for use and care of laboratory animals as stated in US guidelines.