ABSTRACT
The antioxidant effect on the liver of alloxan induced diabetic rats treated with aqueous stem bark extract (400 and 500mg/kg) of Treculiaafricanawas investigated using standard analytical methods. Forty adult albino rats were divided into 7 groups of five each;, group 1 served as the normal control group (received only food and water), group 2 received single dose of alloxan(120mg/kg) without treatment, group 3 received single dose of alloxan (120mg/kg) and treated with a standard drug (glucophage, 50mg/kg), group 4 received 400mg/kg dose of aqueous bark extract of Treculiaafricana without induction of diabetes, group 5 received 500mg/kg dose of aqueous bark extract of Treculiaafricanawithout induction of diabetes, group 6 received 400mg/kg dose of aqueous bark extract of Treculiaafricanaafter induction of diabetes and group 7 received 800mg/kg dose of aqueous bark extract of Treculiaafricanaafter induction. The experiments lasted for twenty-one days. The results revealed that there was no significant difference in the serum levels of catalase, ascorbic acid and malondialdehyde between the normal control and low and high treatments of plant extracts before and after induction. The findings of the present study showed that the aqueous extract of Treculiaafricanaeffectively restored the depleted antioxidant system due to induction of the experimental animals with alloxan.
TABLE OF CONTENTS
CHAPTER ONE
1.0 INTRODUCTION – – – – – – – 1
1.1 AIMS – – – – – – – – – 3
1.2 OBJECTIVES – – – – – – – 4
1.3 LITERATURE REVIEW – – – – – – 4
1.3.1 DESCRIPTION AND ORIGIN – – – – 4
1.3.2 BOTANICAL CHARACTERISTICS – – – 5
1.3.3VARIETES – – – – – – – – 5
1.4 DIABETES – – – – – – – – 7
1.5 TYPES OF DIABETES – – – – – – 8
1.5.1 DIABETES INSIPIDUS – – – – – 8
1.5.2 DIABETES MELLITUS – – – – – 8
1.6 CLASSIFICATION OF DIABETES – – – 9
1.6.1 TYPE 1 DIABETES MELLITUS – – – – 10
1.6.2 TYPE 2 DIABETES MELLITUS – – – – 10
1.6.3 GESTATIONAL DIABETES – – – – 11
1.6.4 OTHER TYPES – – – – – – 12
1.7 LIVER – – – – – – – – – 12
1.8 ALLOXAN – – – – – – – – 15
1.9 MECHANISM OF ALLOXAN – – – – – 16
1.10 BIOLOGICAL EFFECTSOF ALLOXAN – – – 18
CHAPTER TWO
2.0 MATERIALS AND METHODS – – – – 21
2.1 PLANT MATERIALS – – – – – – 21
2.2 PREPARATION OF EXTRACT – – – – 21
2.3 HANDLING OF EXPERIMENTAL ANIMALS AND GROUPING
………………………………………………………………………………..22
2.4 EXPERIMENTAL DESIGN – – – – – 23
2.5 REAGENTS AND CHEMICALS – – – – 24
2.6 EQUIPMENTS AND APPARATUS – – – – 25
2.7 INDUCTION OF DIABETES – – – – – 26
2.8 COLLECTION OF BLOOD FROM ANIMALS – – 26
2.9 DETERMINATION OF CATALASE ACTIVITY
(CAT) – – – – – – – – – 27
2.10 DETERMINATION OF SUPEROXIDE
DISMUTASE ASSAY (SOD) – – – – 28
2.11 DETERMINATION OF VITAMIN C (ASCOBIC)- 30
2.12 DETERMINATION OF LIPID PEROXIDATION (MDA) LEVEL
– – – – – – – 31
2.13 – – – – – – – – – – 33
CHAPTER THREE
3.0 RESULTS – – – – – – – – 34
3.1 EFFECTS OF AQUEOUS STEM BARK – –
EXTRACT OF TRECULIA AFRICANA
ON CATALASE CONCENTRATION – – –
3.2 ILLUSTRATES THE GRAPHICAL REPRESENTATION OF EFFECT OF TRECULIA AFRICANA ON THE SUPEROXIDE DISMUTASE CONCENTRATION OF ALLOXAN INDUCED DIABETIC RATS – – – – – – –
3.3 ILLUSTRATES THE GRAPHICAL REPRESENTATION OF THE EFFECT OF TRECULIA AFRICANA ON THE MALONDIALDEHYDE LEVEL OF ALLOXAN INDUCED DIABETIC RATS – – – – – – – – –
CHAPTER FOUR
4.0 DISCUSSIONS RECOMMENDATION AND CONCLUSIONS
4.1 DISCUSSIONS – – – – – – – –
4.2 RECOMMENDATION – – – – – – –
4.3 CONCLUSION – – – – – – – –
REFERENCES
APPENDIX
CHAPTER ONE
1.0 INTRODUCTION
Diabetes mellitus has been considered as one of the major health concerns all around the world today. Experimental animal model are one of the best strategies for the understanding of pathophysiology of any disease in order to design and develop the drug for its treatment. Numerous animal models have been developed for the past few decades for studying diabetes mellitus and testing antidiabetic agents that induce chemical, surgical and genetic manipulations (Noor et al., 2008).
One of the most potent methods to induce experimental diabetes mellitus is chemical induction by alloxan. It’s a urea derivative which causes selective necrosis of the beta cells of the pancreatic islets. In addition, it has been widely used to produce experimental diabetes in animals such as rabbit, rat, mice and dogs with different grades of diseases severally by varying the dose of alloxan used. As it has been widely accepted, alloxan selectively destroys the insulin-producing beta – cells found in the pancreas, hence it is used to induce diabetes in laboratory animals (Walter, 2007).
In recent times, there has been growing interest in exploiting the biological activities of different ayurvedic medicinal herbs due to their natural origin, cost effectives and lesser side effects (H. Mohan, et al.,2005). Plants produce vast array of secondary metabolites as defense against environmental stress or other factors like pest attacks, wounds and injuries. The complex secondary metabolites produced by plants have found various therapeutic uses in medicine (Senthikumar and A. Padhiari, 2006).
The major goals in the treatment of Diabetes mellitus has been to keep both short and long term glucose levels within acceptable limits thereby reducing the risk of long term complications. This could be achieved by optimizing both fasting blood glucose and also postprandial glucose levels which has been found very important in achieving near normal glucose levels (Park et al., 2009).
Herbs have been used for medicinal purposes for the treatment of various diseases. Plants and herbs derived medicine popularly known as ‘Herbal Medicine’ is now commonly employed to manage such ailment as diabetes mellitus that currently could not be cured with allopathic medicines. Herbal Medicine is in use by about 60% of the world population both in the developing and in the developed countries where modern medicines are predominantly used (Ogbonnia et al., 2008). Whole plants are used locally by the traditional herbalist for the treatment of various diseases including diabetes and stroke. Also different parts of Treculia Africana are employed for the treatment ofvarious diseases including bacterial infections and diabetes mellitus locally (Yinegar et al., 2008).
1.1 AIMS
The aim of this research work is to investigate the effects of aqueous extract of the bark of Treculia on the liver function of alloxan diabetic induced rats.
1.2 OBJECTIVES
- To determine the changes in the blood glucose level in alloxan diabetic induced rats treated with the bark of aqueous extract of treculia Africana.
- To determine the weight changes of the rats.
- To access the effect if extract in serum liver enzymes in alloxan induced rats.
1.3 LITERATURE REVIEW
1.3.1 DESCRIPTION AND ORIGIN
Treculia Africana, the African breadfruit is tree specie in the genius Treculia. It is used as afford plant. The fruits are hard and fibrous, can be size of volleyball and weighs up to 8.5kilogram. Many names are given to this specie but the most common is ‘UKWA’. The geographical distribution of treculia Africana extends through West and Central Africa. The specie can grow below altitudes of 1,500metre (4,900ft) (Nuga et al., 2010).
1.3.2 Botanical characteristics
Treculia Africana is a large tree and is part of the family Moracea. It grows in wet areas and forests. The species can grow up to a height of 30 meters(98ft).The girth of the stem can attain 6 meters (20 ft). The bark is grey and discharges cream latex. The leaves are large and dark green above and lighter below. Trees dioceous (sexes on separate leaves) or sometimes monocieous. Leaves in tworanksstipulates amplexiciual (enclosing bud). Its flowering period is from October to February. The fruit is big round and greenish yellow. The texture of the fruit is spongy when it is ripe and it contains abundant seeds which are edible part of this fruit. Under good environmental conditions, the yield from one tree attains 200kg dried seeds (Ofodile E.A.U, 2010).
1.3.3 VARIETES
Based on detailed field observations, 3 varieties are distinguished within the subspecies. Their taxonomic differences are based mainly on the size of the fruit head and the hairness of branchlets and leaves. There is a striking variation in the number of fruit heads produced by trees belonging to treculia Africana (with large fruit heads and with sadstmall fruit heads (Uchendu C.B, 2011)
Freshly extracted treculia fruit
Treculia Africana tree
Fluted nature and fruits of treculiaAfricana
1.4 DIABETES
The term diabetes, without qualification, usually refers to as diabetes mellitus, which roughly translates to excessive sweet urine (known as glycosuria). Several rare conditions are also named diabetes. The most common of thus known as diabetes insipidus, in which large amount of urine is formed (polyuria), which is not sweet. (Insipidus meaning “without taste” in Latin).
1.5 TYPES OF DIABETES
Two types of diabetes are diabetes mellitus and diabetes insipidus.
1.5.1 DIABETES INSIPIDUS
These are diseases caused by deficiency of vasopressin, one of the hormones of the posterior pituitary gland, which controls the amount of urine secreted by the kidney. The symptoms of diabetes insipidus are marked, taste and. Excretion of large quantities of urine as much as 4-10 liters a day. The urine has a low specific gravity and contains no excess sugar. In many cases, injection or nasal inhalation of vasopressin controls the symptoms of the disease (Microsoft Encarta, 2009).
1.5.2 DIABETES MELLITUS
Diabetes mellitus is a complex, chronic disease. It is a condition characterized by an elevation of the level of glucose in the blood. Insulin, a hormone produced by the pancreas, controls the blood glucose level by regulating the production and storage of glucose. In diabetes there may be a decrease in the body’s ability to respond to insulin or a decrease in the insulin produced by the body which leads to abnormalities in the metabolism of carbohydrates, proteins and fats. The resulting hyperglycemias may lead to acute metabolic complications (Smelters & Bare 2012).
The prevalence of diabetes is higher in minority groups among those who are socio-economically disadvantaged, but the reason for that has not been given yet. Unfortunately, there is no cure for diabetes yet but by controlling blood sugar levels through a healthy diet, exercise and medication of the risk of long-term diabetes complication can be decreased. Long term complications that can be experienced are:
* Eyes – cataracts and retinopathy (gradual damaging of the eye) that may lead to blindness
* Kidney – kidney disease and kidney failure
* Nerves – Neuropathy (gradual damaging of nerves)
* Feet – ulcers, infections, etc
* Cardiovascular system – hardening of arteries, heart disease and stroke (Heart foundation, 2003).
1.6 CLASSIFICATION OF DIABETES
Diabetes mellitus is classified into four broad categories: Type 1 diabetes, Type 2 diabetes, gestational diabetes and other types.
1.6.1 TYPE 1 DIABETES MELLITUS
It is generally classified by the abrupt onset of severe symptoms, dependent on exogenous insulin to distant life and proneness to me to is even in the basal state, all of which is caused by absolute insulin deficiency. It is a catabolic disorder in which circulating insulin is virtually absent, plasma glucagon is elevated; hence fail to respond to all insulinogenic stimuli (Nolte and Karam 2001). Most affected people are otherwise of healthy weight when onset occurs. Sensitivity and responsiveness to insulin are usually normal in the early stages. Type 1 diabetes can affect children or adults but was traditionally termed juvenile diabetes because majority of this diabetes were in children (Harris and Zimmet, 2007).
1.6.2 TYPE 2 DIABETES MELLITUS
Type 2 diabetes greatly out numbers all other forms of diabetic patient. However they may require insulin for the correction of fasting hyperglycemia if this cannot be achieved with the use of diet or oral agents and they may develop ketosis under special circumstances such as severe stress precipitated by infections or trauma (Harris and Zimmet, 2007).
The pathogenesis in type 2 diabetes is that the pancreas produces insulin but the body does not utilize the insulin correctly. This is primarily due to peripheral tissue insulin resistance where insulin receptors or other intermediates within the body cells are insensitive to insulin and consequently, glucose does not readily enter the tissue leading to hyperglycemia or elevated blood glucose concentrations (Albright, 2007). Obesity which generally results in impaired insulin action, is a common risk factor for this type of diabetes and most patients with type 2 diabetes are obessed and will ultimately require adequate glycogenic control (Gerich, 2011).
1.6.3 GESTATIONAL DIABETES
The onset of gestational diabetes mellitus is during pregnancy, usually in the second or third trimester, as a result of hormones secreted by the placenta, which inhibit the action of insulin. It occurs in about 2-5% of all pregnancies. About 30-40% of patients with gestational diabetes mellitus will develop type 2 diabetes within 5-10 years (especially if obese). Impaired glucose tolerance and statistical risks groups are one example of gestational diabetes mellitus. Statistical risks groups are individuals at greater risk than the general population of developing diabetes and the risk factors include immediate family members with the disease and presence of islet cell antibodies (Royle& Walsh 2002).
1.6.4 OTHER TYPES
This is where diabetes is associated with other conditions for example, pancreatic disease, hormonal disorders, drugs and estrogen – containing preparations. Depending on the ability of the pancreas to produce insulin (Smeltzer& Bare, 2002).
1.7 LIVER
The liver which is anatomically structured as
is the second largest gland in the body that performs astonishing large number of tasks that impact all body systems. It is a meaty organ that sits on the right side of the belly. Weighing about three pounds thus reddish – brown in colour and feels rubbery to touch.
It has two large sections called the right and left lobes. The gall bladder sits under the liver, along with parts of the pancreas and intestines hence these organs with the liver work together to digest, absorb and process food (Crook, 2006).
Some biological assays are done known as the liver function tests; whichare groups of clinical biochemistry laboratory blood assay designed to give information about the state of an individual’s liver. Different test give different information on hepatic dysfunction. Some tests are associated with rationality; some with cellular integrity and some are conditions linked to the biliary track. Several biochemical tests are useful in the elevation and management of patients with hepatic dysfunction. These tests can be used to detect the presence of liver disease, distinguish among different types of liver disorders, determine the extent of liver damage, follow the response to treatment, and to detect hepatotoxicity caused by drugs (Laker, 2000).
The liver plays a major role in the filtering of blood coming from the digestive tract, before passing it to the rest of the body. It also detoxifies chemicals and metabolises drugs. As it does so, the liver secretes the bile that ends up back in the intestines. The liver also makes protein important for blood clotting and other functions hence, regulates carbohydrate metabolism as it uses glucose as fuel; it has the capability to store glucose from non-carbohydrate sources. This key function of liver makes it vulnerable to diseases in subjects with metabolism disorders, particularly diabetes (Levinthal and Tavil, 2009).
1.8 ALLOXAN
Alloxan which has its structure as is sometimes referred to as alloxan hydrate, an organic compound with the formularoc (N(H)Co)2C(OH)2. It is classified as a derivative of pyrimidine. The anhydrous derivate is also known as well as a dimeric derivative. These are some of the earliest known organic compounds. They also exhibit a variety of biological activities.
MECHANISM OF ACTION OF ALLOXAN
Alloxan-induced diabetes has been commonly employed as an experimental model of insulin dependent diabetes mellitus. The mechanism of alloxan action has been thoroughly studied which currently can be characterized quite well. Several experimental studies have demonstrated that alloxan evokes a sudden rise in insulin secretion in the presence or absence of glucose which appeared just after alloxan treatment (Lachin and Reza,2012). This particular alloxan induced insulinrelease occurs for short duration followed by the complete suppression of the islet response to glucose even when high concentrations of glucose were used (Kilberet al., 2015). Further, the alloxan action in the pancreas is preceded by its rapid uptake by pancreatic beta cells, the reduction process occurs in the presence of different reducing agents like reduced glutathione(GSH), cysteine, ascorbate and protein-bound sulfhydryl(-SH) groups (Lenzen and Munday,2010). Ailoxan reacts with two -SH groups in the sugar binding site of glucokinase resulting in the formation of the disulfide bond and inactivation of the enzyme. As a result of alloxan reduction, dialuric acid is formed which is then re-oxidized back to alloxanestablishing a redox cyclefor the generation of reactive oxygen species(ROS) and superoxide radicals (Das et al., 2012). The superoxide radicals liberate ferric ionsfrom ferritin and reduce them to ferrous and ferric ions (Sakurai and Ogiso,2007). In addition, superoxide radicals, undergo dismutation to yield hydrogen peroxide (H202) in the presence of superoxide dismutase. As a result, highly reactive hydroxyl radicals are formed according to the fenton reaction in the presence of ferrous and H202. Another mechanism that has been reported in the effect of ROS on the DNA of pancreatic islets. The fragmenatation of DNA takes place in the beta cells exposed to alloxan that causes DNA damage, which stimulates poly ADP-ribosylation, a process participating in DNA repair. Antioxidants like superoxide dismutase,catalase and non-enzymatic scavangers of hydroxyl radicals have been found to protect against alloxan toxicity.In addition, the disturbance in intracellular calcium homeostasis has also been reported to constitute an important step in the diabetogenic action of alloxan. It has been noted that alloxan elevates cytosolic free ca2+ concentration in the beat cells of pancreatic islets. The calcium is resulted from the ability of alloxan to depolarize pancreatic beta cells that further opens voltage dependent calcium channels and enhances calcium entry into pancreatic cells. The increased concentration of ca2+ ion further contributes to supraphysiological insulin release that along with ROS has been noted to ultimately cause damage of beta cells of pancreatic islets(Lenzen, 2008).
BIOLOGICAL EFFECTS OF ALLOXAN
Alloxan is a hydrophilic and unstable chemical compound which has similar shape as that of glucose, which is responsible for its selective uptake and accumulation by the pancreatic beta cell. Similarity in shape allows it to transport into the cytosol by the glucose transporter (GLUT2) in the plasma membrane of the beta cell. (Eisner et al., 2002). Another biological effect of alloxan has been attributed to the thiol group reactivity that allows selective inhibition of glucose-induced insulin secretion through inhibition of glucokinase. This inhibition of glucose-induced insulin secretion has been regarded as the major pathophysiological effect of alloxan, which results from the thiol group reactivity of alloxan. The thiol group of glucokinase, the glucose phosphorylating enzyme,are particularly sensitive to oxidation by alloxan. Glucose inhibition reduces glucose oxidation and ATP generation that further suppresses glucose-induced insulin secretion (Tiedgeet al., 2000). Moreover, the insulin biosynthesis is also inhibited by alloxan through the same mechanisms. Alloxan inhibits many cellularfunctions at higher concentrations such as ability to oxidize thiol groups of many functionaly important enzymes hexokinase, phosphofructokinase, calmodulin-dependent protein kinase, acotinase and other proteins(Lenzen and Mirzaie, 2008). Hence, it is evident that the inhibition of glucose induced insulin secretion by alloxan is pancreatic beta cell toxicity and diabetogenicity that may be attributed to alloxan-induced redox cycling and ROS generation. The mechanism underlying the cytotoxic action of alloxan to insulin-producing cells may be ascribed as the reduction by interaction with intracellular thiols such as glutathione (Eisner et al., 2006). The resultant formation of cytosolic ROS is the result of a cyclic reaction between alloxan and its reduction product, dialuricacid, which by auto-oxidation generates superoxide radicals, hydroxyl radicals and H202. Induction of diabetes in the laboratory animals by alloxan injection is the result of selective uptake of alloxan via GLUT2 into a pancreatic beta cell (Winterbourn and Munday, 2006). The effective prevention of redox cycling and generation of ROS can prevent pancreatic beta cell death and counteract the development of alloxan diabetes in vivo (Zhang et al., 2009). Hence, it can be summarized that the alloxan-induced pancreatic beta cell toxicity and the resultantdiabetogenicity is due to the redox cycling and the toxic ROS generation in combination with the hydrophilicity and the glucose similarity of the molecular shape of alloxan.
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