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ABSTRACT

Spondias mombin belongs to the family Anacardiacae. All parts of the tree are reported to be medicinally useful. The fruit juice is drunk as a diuretic and febrifuge. The main aim of the study is to determine the diuretic activity and toxicity of methanol stem bark extract of Spondias mombin.
The plant was collected, authenticated, and studied in Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria. Adult albino rats of Wistar strain of either sex were used for the experiments. Phytochemical screening of the methanolic extract of stem bark of Spondias mombin was carried out according to the methods described by Trease and Evans. The oral median lethal dose (LD50) of the extract was determined in rats according to Lorke’s method. The sub-chronic study was carried out in accordance with WHO and OECD 407 guidelines. Twenty four adult rats (Wistar strain) were randomly divided into four groups of six rats each. The rats in group I were administered with normal saline orally and served as the control. Groups II – IV were administered 250mg/kg, 500mg/kg, and 750mg/kg of the extract daily for twenty eight days.At the end of the study, the animals were euthanized and relative organ weight ratio (ROW) was determined.Histological examination of heart, liver and kidneys were also performed. Complete blood count, liver and kidney function tests were also determined. For diuretic screening, test animals were placed into metabolic cages with total withdrawal of food and water for 12 hours. They were then randomly divided into five groups of five animals each. Each animal was rehydrated with 25 ml/kg of normal saline just before the experiment. Group-I animals were provided only with 25ml/kg of Normal saline to serve as control. Group-II animals were provided with frusemide at a dose of 5 mg/kg body weight. Group-
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III, IV and V animals were given 250mg/kg, 500mg/kg and 750mg/kg of the extract respectively. These preparations were all given by the oral route. After administration of test samples, the urinary excretion was recorded at 3rd, 6th and 24th hour, from the graduated urine chamber of metabolic cage. Urine was also analyzed for electrolytes. The phytochemical screening of methanolic stem bark extract of S. mombin revealed the presence of flavonoids, alkaloids, tannins, saponins, cardiac glycosides, phenols, and carbohydrates,but anthraquinones were absent. The median oral lethal dose was found to be greater than 5000mg/kg. Sub-chronic toxicity studies revealed there was no significant statistical difference in the animal body weights at all weeks compared to week zero and in relation to different doses of extract compared to the control.A significant statistical reduction of bicarbonate was noted at the dose of 500mg/kg compared with control (p< 0.05).There was no statistically significant difference observed in the liver function test, haematological indices, and relative organ weight (ROW) ratio of the groups administered with the extract compared with control.Vascular congestion was noted in the heart at 500mg/kg and the kidneys at 250mg/kg. Vacuolar changes seen at 750mg/kg dose are most likely due to fatty liver.No significant statistical differences were observed in urine output between the test groups given the extract and the control groups during the first 6 hours and after 24 hours. There was significant excretion of potassium at 500mg/kg dose compared with the negative control. No other significant statistical differences were observed between the other test groups given the extract and the control groups. In conclusion methanolic extract of the stem bark of S.mombin has shown structural toxicity on the some organs, but has not been shown to have diuretic property.

 

 

TABLE OF CONTENTS

TITLE PAGE ……………………………………………………………………… Error! Bookmark not defined.
DEDICATION ……………………………………………………………………………………………………………… ii
DECLARATION …………………………………………………………………………………………………………. iii
CERTIFICATION ……………………………………………………………………………………………………….. iv
ACKNOWLEDGMENT ……………………………………………………………………………………………….. v
ABSTRACT………………………………………………………………………………………………………………… vi
TABLE OF CONTENTS ……………………………………………………………………………………………………. viii
LIST OF TABLES ……………………………………………………………………………………………………….. xi
LIST OF PLATES ………………………………………………………………………………………………………. xii
LIST OF APPENDICES ……………………………………………………………………………………………… xiv
LIST OF ABBREVIATIONS ……………………………………………………………………………………….. xv
CHAPTER ONE …………………………………………………………………………………………………………………..1
1.0 INTRODUCTION ………………………………………………………………………………………………….1
1.1 Statement of Research Problems ………………………………………………………………………………5
1.2 Justification ……………………………………………………………………………………………………………5
1.3 Research Hypothesis ……………………………………………………………………………………………….7
1.4 Aim: ……………………………………………………………………………………………………………………..7
1.5 Specific Objectives: ………………………………………………………………………………………………..7
CHAPTER TWO ………………………………………………………………………………………………………………….8
2.0 LITERATURE REVIEW ………………………………………………………………………………………..8
2.1 Diuretics ………………………………………………………………………………………………………………..8
2.1.1 Introduction …………………………………………………………………………………………………….8
2.1.3 Clinical uses and side effects …………………………………………………………………………. 13
2.1.4 Time course of diuresis …………………………………………………………………………………. 16
2.1.5 Preclinical studies on plants that have been shown to have diuretic properties ……………. 17
2.2 Phytochemical Screening ……………………………………………………………………………………… 18
2.3 Toxicology ………………………………………………………………………………………………………………. 18
2.3.1 Importance of dose-response relationship in toxicology ………………………………………….. 19
2.3.2 Routes of exposure to toxic substances………………………………………………………………….. 20
2.3.3 Some target organs in toxicology …………………………………………………………………… 21
2.3.4 Preclinical toxicology studies ………………………………………………………………………… 25
2.4 Traditional Medicine ……………………………………………………………………………………………. 27
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2.4.1 Safety of traditional medicine ………………………………………………………………………… 28
2.5 The plant; Spondias mombin …………………………………………………………………………………. 30
2.5.1 Ethnomedical uses ……………………………………………………………………………………….. 31
2.5.2 Non medical uses …………………………………………………………………………………………. 31
CHAPTER THREE …………………………………………………………………………………………………………… 33
3.0 MATERIALS AND METHODS …………………………………………………………………………… 33
3.1 Plant Collection and Authentication ………………………………………………………………………. 33
3.2 Preparation of the Extract …………………………………………………………………………………….. 33
3.3 Experimental Animals …………………………………………………………………………………………. 33
3.4 Materials ……………………………………………………………………………………………………………. 34
3.5 Phytochemical Screening of the methanol extract of Spondias mombin ……………………… 34
3.5.1 Test for cardiac glycosides …………………………………………………………………………….. 34
3.5.2 Test for tannins ……………………………………………………………………………………………. 35
3.5.3 Test for saponins ………………………………………………………………………………………….. 35
3.5.4 Test for flavonoids ……………………………………………………………………………………….. 35
3.5.6 Test for anthraquinones…………………………………………………………………………………. 36
3.6 Acute toxicity studies: …………………………………………………………………………………………. 37
3.7 Sub chronic toxicity study ……………………………………………………………………………………. 38
3.7.1 Calculation of relative organ weight ratio (ROW) …………………………………………….. 38
3.7.2 Biochemical studies ……………………………………………………………………………………… 38
3.7.3 Haematological studies …………………………………………………………………………………. 39
3.7.4 Histopathological Study ………………………………………………………………………………… 39
3.8 Screening of Diuretic Activity ………………………………………………………………………………. 39
3.9 Data analysis ………………………………………………………………………………………………………. 40
CHAPTER FOUR……………………………………………………………………………………………………………… 41
4.0 RESULTS ………………………………………………………………………………………………………….. 41
4.1 Phytochemical Screening ……………………………………………………………………………………… 41
4.2 Toxicity studies …………………………………………………………………………………………………… 43
4.2.1 Acute toxicity studies (LD50 determination) …………………………………………………….. 43
4.2.2 Sub chronic toxicity studies …………………………………………………………………………… 44
4.2.3: Diuretic Screening …………………………………………………………………………………………….. 68
CHAPTER FIVE ………………………………………………………………………………………………………………. 78
5.0 DISCUSSION …………………………………………………………………………………………………….. 78
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CHAPTER SIX …………………………………………………………………………………………………………………. 85
6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS …………………………………. 85
6.1 Summary ……………………………………………………………………………………………………………. 85
6.2 Conclusion …………………………………………………………………………………………………………. 85
6.3 Recommendations ……………………………………………………………………………………………….. 86
REFERENCES …………………………………………………………………………………………………………………. 87
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CHAPTER ONE

1.0 INTRODUCTION
Plants have fed the world and cured its ills since time immemorial. Ecology has shown us the intense dependence of man on plants for his basic needs of food, shelter, clothing and even medicine. The use of plants for curing and healing is as old as man himself. Therefore, a vast knowledge of medicinal plants have accumulated,but most of the knowledge only exists as verbal tradition and only a fraction has gotten scientific validation till date(Osai, 1998). A plant becomes a medicinal plant only when its biological activity has been ethnobotanically reported or scientifically established (Elujoba, 1997). Since 1978 the World Health Organisation (WHO) commenced the evolution of scientific confirmation of medicinal effect of herbs. World Health Organization has estimated that over 75% of the world’s population relies on plant-derived medicines, usually obtained from traditional healers, for basic health-care needs (Patel et al., 2009). It is estimated that about 25% of all modern medicines are directly or indirectly derived from plants (Craig et al., 1997).
Diuretics are a class of drugs that increases the rate of urine formation. They are used in many clinical conditions including edematous disorders and hypertension. Historically, the classification of diuretics has been made using multiple of ideas like: place of action (loop diuretics), efficiency (high ceiling diuretics), chemical structure (thiazide diuretics), similarity of action to other diuretics (diuretics similar to thiazides), the effects upon the potassium excretion (potassium-sparing diuretic), and others (Florez, 2003; Rang et al., 2008).
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The diuretic effectiveness of medicinal plants needs to be experimentally proved, because diuresis could be influenced by the form of administration (infusion or decoction) which implies the consumption of a great amount of liquids that can provoke an increase in the amount of urine excreted without a true evidence of a diuretic action. This is illustrated by the following equations (Abeywickramaet al., 2010):
1. Urine excretion = mean urine volume of test x100
total fluid administered
2. Diuretic action:= urinary excretion in test x100
urinary excretion in standard
3. Diuretic activity= diuretic action of test x100
diuretic action of standard
4. Diuretic index= urine volume of test group x100
urine volume of control group =diuretic action of test x100 diuretic action of control
5. Saluretic index= urinary excretion of electrolyte of test group
urinary excretion of electrolyte of control group
6. Natriuretic index (Aldosterone index)= urinary excretion of Na+
urinary excretion of K+
7. Ion quotient (carbonic anhydrase inhibition index)= urinary excretion of Cl
sum of urinary excretion of Na+ and K+
8. Thiazide diuretic index= urinary excretion of Na+
urinary excretion of Cl-
9. Lipschitz value = mean urine volume of test
mean urine volume of standard
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= urine excretion of test urine excretion of standard Even though all diuretics are used to increase renal excretion of sodium and water, they differ considerably in chemical derivation, efficacy, sites and mechanism of action (Florez, 2003; Rang et al., 2008). The choice of a diuretic clinically depends on the objective of therapy and the pathophysiology of the patient’s disease. Patients with renal insufficiency require loop diuretics because they do not respond to other agents to a clinically relevant degree. Patients with cirrhosis are reported to have sodium retention secondary to hyperaldosteronism and diuretic treatment in such patients is initiated with an inhibitor of aldosterone, spironolactone. Effective use of diuretics requires knowledge of the pharmacology of each diuretic agent coupled with an understanding of the pathophysiology of the patient’s disease.
Several preclinical studies have been reported in Nigeria to assay the diuretic action of the following plants: Agave sisalana (Omodamiro et al., 2014), leaf extracts of Irvingia gabonensis (Nosiri et al., 2009), stem-bark extracts of Steganotaenia araliacea hochst [Apiaceae] (Agunu et al., 2005).
Toxicology is the science that deals with the study of the adverse effects caused by chemicals or physical agents in living organism under specific conditions of exposure (Doull et al., 2008). It is a science that attempts to qualitatively identify all the hazards, such as organ toxicities associated with a substance, as well as to qualitatively determine the exposure conditions under which those hazards are induced. It also experimentally
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determines the occurrence, nature, incidence, mechanism, and risk factors for the adverse effects of a toxic substance (James et al., 2000). Toxicity studies are conducted to provide greater understanding of the potential intrinsic hazard of the test item and to estimate the safety margins (Robinson et al., 2013). These safety margins are used to determine an initial safe starting dose for clinical trials, a safe dose for continued use in humans through longer clinical trials, and ultimately to achieve successful review of registration dossiers to support marketing approval.
Spondias mombin Linn or Spondias purpurea var. lutea, is a tree species of the flowering plant in the family Anacardiaceae. It is native to the tropical Americans, including the West Indies. The tree has been naturalized in parts of Africa, India, Sri Lanka and Indonesia. It is rarely cultivated. Traditionally, it has been used for a lot of local clinical indications. The fruit-juice is used as a febrifuge and diuretic. The roots are well-known febrifuge. The bark is used as a purgative and in local applications for leprosy and for severe cough, causing relief through vomiting. The dry pulverized bark is applied as a dressing to the circumcision wound.. A decoction of the mashed leaves is used by the Ibos (Nigeria) for washing a swollen face. A leaf infusion is a common cough remedy and it is used as a laxative for fever with constipation and decoction is used for gonorrhea. All these leaf preparations are used for leprosy. Crushed, with lemon they are effective for worms in children. A decoction of pounded leaves is used as an eye lotion and the juice pressed from young, warm leaves is given to children for stomach troubles. The young leaves are used as an infusion taken internally or as a warm astringent lotion by women in confinement in Sierra Leone (Aiyelojaet al., 2006; Ayoka et al., 2008).
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1.1 Statement of Research Problems
Diuretics are indicated in different clinical conditions such as hypertension, oedematous states like congestive heart failure, nephrotic syndrome, renal failure, acute pulmonary oedema, cerebral oedema, and dyselectrolytaemia like acute hypercalcaemia, hypokalaemia. Other uses are glaucoma, urine alkalanization, etc. These conditions are some of the commonest reasons for physician consultation in various health care delivery systems in Nigeria, United States and other parts of world. The combined prevalence of hypertension in Nigeria by metanalysis, for instance, is said to be 22% (Obinna and Cletus, 2011). Unfortunately significant percentages of Nigerians do not have access to specialized health facility or cannot even afford to sustain the bill for abovementioned chronic illnesses. Like every part of the world, for so many reasons, most of the people depend on traditional herbs for their medications (Patel et al., 2009). Unfortunately, the use of commonly available orthodox drugs is sometimes associated with some side effects (Vincent and Furnham, 1996). Most of the medicinal plants that have been identified have not been subjected to studies to ascertain their side effects. Toxicological studies of medicinal plants will add more value to the therapeutics.
1.2 Justification
Nigerians require different alternative therapies because of their different beliefs, limited health facilities, and large number of people with low socio-economic status. World Health Organization statistics project that nearly 75% of population worldwide still depend upon herbals (Patel et al., 2009). This percentage may be higher in Nigeria. So far literature search shows few studies done on medicinal plants with diuretic properties in Nigeria.
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A number of diuretics like mannitol, thiazides, frusemide, and spironolactone are used in practice. However, most diuretic drugs have been associated with numerous adverse effects such as electrolyte imbalance, metabolic alterations, development of new-onset diabetes, activation of the renin-angiotensin and neuroendocrine systems, and impairment of sexual function. According to Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure, JNC VIII, the choice of antihypertensive agent(s) in blacks must include diuretic to achieve good goal (Paul et al., 2014). Hence there is a need for novel diuretics such as plant-based substances, which are considered to be relatively safe and possessing lower potential for adverse effects.’ The fruits of Spondias mombin have been demonstrated to have diuretic property (Ayoka et al., 2008), but no literature has shown such properties on the stem bark. Fruits of Spondas mombin are seasonal, unlike the stem bark which can always be sourced as long as the plant exists. Additionally, fruits are perishable unlike stem bark which can be more easily preserved. There is a continuous need to carry out research into these useful medicinal plants which are used as potent diuretics with fewer side effects, cost effectiveness and are accessibility. This research will also provide additional information for further studies in the area of pharmacology, phytochemistry, pathology, and toxicology. Toxicological studies on the renal, hepatic and hematological systems will provide information on the effects of this plant on these tissues and organs.
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1.3 Research Hypothesis
Methanol stem bark extract of Spondias mombin has diuretic activity associated with minimal renal, liver and haematological adverse effects.
1.4 Aim:
To determine the diuretic activity and toxicity of methanol stem bark extract of Spondias mombin.
1.5 Specific Objectives:
The specific objective is to determine the following parameters of methanol stem bark extract of Spondias mombin:
1. To determine the phytochemical components
2. To ascertain the median lethal dose
3. To conduct a sub-chronic toxicity study for 28 days
4. To determine the diuretic property of the extract
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