ABSTRACT
The aim of the study was to evaluate the hepatoprotective effect of aqueous extract of bitter kola (garcinia kola) seeds on carbon tetrachloride (CCl4) induced liver damage in adult wistar rats. The experimental animals (36 male and female) weighing between 150-250g were randomly divided into six groups. Each group comprised of 6 rats and was labeled as groups I, II, III, IV, V and VI. Group I (control) animals were administered distilled water orally daily for 2 weeks (volume per body weight) while group II (CCl4 control) animals were administered distilled water orally daily for 2 weeks (volume per body weight) and carbon tetrachloride (CCl4) 0.4 m1/kg intraperitonially as a single application. Group III rats were administered 100 mg of silymarin / kg body weight once daily for 2 weeks followed by a single dose of CCl4 (0.4 m1) on day 14 of the experiment. Group IV rats were administered 800 mg aqueous extract bitter kola (Garcinia kola) / kg body weight orally once daily for 2 weeks. Group V and VI rats were administered 800 mg and 400 mg aqueous extract bitter kola (Garcinia kola) seed) orally once daily for 2 weeks followed by a single dose of CCl4 (0.4 m1) on day 14 respectively. At the end of the experiment, blood samples were a collected for serum analysis of levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), superoxide dismutase (SOD), reduced glutathione (GSH) and catalase (CAT). Hepatic tissues were also collected for histopathological assessment of liver damage. Results obtained showed that the CCl4 treated group caused significant increase in the levels of liver enzymes (AST, ALP and ALT). Bitter kola (Garcinia kola) seed aqueous extract caused significant decrease in the aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) levels in the serum of the extract treated groups. Histopathological and histochemical examinations of liver sections revealed distortion of histoarchitecture of the liver tissue, such as, sinusoidal congestion, necrosis, steatosis and fibrosis was observed in group II. The administration of aqueous extract of bitter kola (Garcinia kola) seed remarkably inhibited histoarchitectural distortion induced by CCl4 administration. Hepatoprotective activity of the extract at dose of 400mg/kg was comparable to the reference drug. Garcinia kola aqueous seed extract showed a remarkable hepatoprotective and antioxidant activity against CCl4-induced hepatotoxicity as observed from the serum marker enzymes and antioxidant levels in liver tissues. CCl4-induced a significant rise in AST, ALT, ALP with a reduction of superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH). Treatment of the rats with the extract significantly (p<0.05) altered serum marker enzymes and antioxidant levels to near normal compared with CCl4-treated rats (group II). The activity of the extract at dose of 400mg/kg (group VI) was comparable to the standard drug confirmed by histopathological examinations of liver sections. Results indicated that Garcinia kola aqueous seed extract has hepatoprotective and antioxidant properties against CCl4-induced hepatotoxicity in Wistar rats.
TABLE OF CONTENTS
Title page ————————————————————————————————ii
Declaration ———————————————————————————————iii
Certification ——————————————————————————————–iv
Dedication ———————————————————————————————–v
Acknowledgments ————————————————————————————vi
Abstract ————————————————————————————————vii
Table of Content ————————————————————————————–ix
List of Figures —————————————————————————————-xv
List of Tables —————————————————————————————–xvi
List of Plates —————————————————————————————–xvii
List of Abbreviations —————————————————————————–xviii
CHAPTER ONE
1.0 Introduction —————————————————————————————-1
1.1 Background —————————————————————————————-2
1.2 Statement of the problem ————————————————————————2
1.3 Justification of the study ————————————————————————-2
1.4 Significance of the Study ————————————————————————3
1.5 Hypothesis —————————————————————————————–3
1.6 Aims of the Study ———————————————————————————3
CHAPTER TWO
2.0 Literature Review ———————————————————————————5
2.1 The Liver —————————————————————————————-5
2.1.1 Functional Parts of the Liver——————————————————————-5
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2.1.2 Porta Hepatis————————————————————————————-5
2.1.3 Vessels and Nerves of the Liver—————————————————————6
2.1.4 Nerve Supply of the Liver———————————————————————-6
2.1.5 Lymphatic Drainage—————————————————————————–6
2.1.6 Histology of the Liver. ————————————————————————-7
2.1.7 Functions of the Liver—————————————————————————8
2.1.8 Developmental Stages of Liver—————————————————————10
2.1.9 Liver Diseases———————————————————————————–10
2.2. Medicinal Plants———————————————————————————-12
2.2.1 Hepatoprotective Plants————————————————————————13
2.3. The Plant, Garcinia kola————————————————————————15
2.3.1 Medicinal properties and Uses of Garcinia kola——————————————-16
2.3.2 Physiological Properties of Garcinia kola————————————————–17
2.3.3 Phytochemicals———————————————————————————18
2.3.4 Functions of phytochemicals—————————————————————–19
2.3.5 Toxicology————————————————————————————–19
2.4. Silymarin—————————————————————————————- 19
2.4.1Antioxidant Properties of silymarin———————————————————-21
2.4.2 Mechanism of Action————————————————————————–21
2.4.3 Pharmacokinetics——————————————————————————-23
2.4.4 Toxicity——————————————————————————————23
2.4.5 Dosage and Administration——————————————————————-24
2.5. Carbon Tetrachloride—————————————————————————25
2.5.1Uses of Carbon Tetrachloride—————————————————————-25
2.5.2 Sources and Potential Exposure to CCl4—————————————————25
2.5.4 Health Hazard Information on CCl4——————————————————–26
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2.5.4.1 Acute Effects——————————————————————————–26
2.5.4.2. Chronic Effects (Non-cancerous) ——————————————————-27
2.5.4.3 Reproductive/Developmental Effects—————————————————-27
2.5.4.4 Cancer Risk———————————————————————————-27
CHAPTER THREE
3.0 Materials and Methods————————————————————————-28
3.1. Plant Material———————————————————————————–28
3.1.1 Extraction of plant material —————————————————————-28
3.2 Experimental Animals————————————————————————-28
3.3 Experimental Design—————————————————————————29
3.4 Sacrifice of Animals—————————————————————————-31
3.5 Morphological Studies————————————————————————-31
3.6 Biochemical Studies—————————————————————————-31
3.6.1 Liver Function Test—————————————————————————31
3.6.2 Estimation of Oxidative stress enzymes—————————————————-31
3.6.2.1 Superoxide Dismutase (SOD)————————————————————-31
3.6.2.3 Catalase (CAT)——————————————————————————-33
3.6.2.4 Principle————————————————————————————–33
3.6.2.5 Reagents—————————————————————————————33 3.6.2.6 Procedure————————————————————————————–33 3.6.2.7 Calculation————————————————————————————33
3.6.3 Reduced glutathione (GSH) ——————————————————————34
3.6.3.1 Reagents—————————————————————————————34 3.6.3.2 Procedure————————————————————————————-34
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3.7 Histopathological Analysis———————————————————————-35
3.8 Data Analysis————————————————————————————–35
CHAPTER FOUR
4.0 Results ———————————————————————————————37
4. l Physical Studies———————————————————————————–37
4.2 Biochemical Analysis—————————————————————————-39
4.2.14.2.1 Liver Enzymes: AST, ALT AND ALP————————————————39
4.2.2 Oxidative stress indicator———————————————————————-41
4.3 Histological Studies of Liver ——————————————————————-43
4.3.2 Histological Features of the control (untreated) Experimental Animals—————-43
4.3.2 Histological Features of the Treated Experimental Animals—————————–44
4.4 Histological Findings of the Liver (H and E) ————————————————45
4.5 Histochemical Findings of the Liver (Reticular fibres) ————————————-51
CHAPTER FIVE
5.0 Discussion —————————————————————————————–57
5.1 Physical Observation—————————————————————————–57
5.2 Biochemical Analysis—————————————————————————-58
5.2.1 Liver Enzymes (ALT, AST and ALP) ——————————————————58
5.2.2 Oxidative Stress Indicators——————————————————————–60
5.3 Histopathological Studies————————————————————————61
5.3.1 Histology of Treated Experimental Animals ———————————————–61
5.3.2 Reticular fibres———————————————————————————-62
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CHAPTER SIX
6.0 Summary, Conclusion and Recommendation ————————————————64
6.1 Summary and Conclusion ———————————————————————–64
6.2 Recommendations——————————————————————————–65
References—————————————————————————————–66
CHAPTER ONE
1.0 Introduction
Plants have been an important source of medicine for thousands of years. Today, the World Health Organisation (WHO, 2000) estimated that 80% of people still rely on traditional remedies such as herb for their medicines (Tripathi and Tripathi, 2003). Plants are also sources of many modern medicines. It is estimated that approximately a quarter of processed drugs contain plant extracts or active ingredient obtained from or modelled on plant substances (Tripathi and Tripathi, 2003).
Garcinia kola belongs to the family Guittiferae, and it is commonly referred to as ‘Bitter Kola’. The plant has the popular acronym “wonder plant” amongst the South-Western Nigerian people because every part of it have been found to be of medicinal importance (Olaleye et al., 2000). In Nigeria, it is referred to as Namijin-goro in Hausa, Orogbo in yoruba, Akiilu in Igbo and Oro in Ebira languages. Garcinia. kola is used in folklore remedies for the treatment of ailments such as liver disorders, hepatitis, diarrhoea, laryngitis, and bronchitis (Iwu, 1993; Adesina et al., 1995).
The seed is masticatory and is used to prevent chest colds, and cough and can be used as well to treat headache (Ayensu, 1978). Iwu (1993) reported the use of this plant for the treatment of jaundice, high fever, purgative and chewing stick. The plant has also been found useful in the treatment of stomach ache and gastritis (Ajebesone and Aina, 2004).
Liver diseases are some of the fatal diseases in the world today, posing a serious challenge to the international public health (Ahsan et al., 2009). Increase in the incidence of alcoholism, substance abuse (toxic chemicals) and other unhealthy life style such as eating fatty foods have contributed to the morbidity and mortality due to liver diseases (Franchesca et al., 2010). Some
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of the commonly known disorders of the liver include viral hepatitis, alcoholic liver diseases, non-alcoholic liver diseases, auto-immune liver diseases, metabolic liver disease, drug-induced liver injury, gall stones, etc. Most of the hepatotoxic chemicals damage liver cells mainly by inducing lipid peroxidation and other oxidative damages (Dianzani et al., 1991).
Carbon tetrachloride (CCL4) has been used extensively to study liver injury induced by free radicals in an animal model system. CCL4 -treated rats are widely used to study liver damage where it was reported that CCL4 induced not only necrosis but also apoptosis in rat liver (Shi et al., 1998; Sun et al., 2001). Although the mechanism by which CCL4 causes liver damage is unclear, several lines of evidence suggest that the liver damage could be caused by free radical metabolites (Williams et al., 1990). CCL4 is converted to the trichloromethyl radical by cytochrome P-450 through a 1-electron reduction. A fatty acid radical is generated by the reaction between trichloromethyl radical and unsaturated fatty acids, and lipid peroxidation follows, Shi et al., 1998; Sun et al., 2001). .
1.1 Statement of the Research Problem
In recent times, hepatotoxicity and liver related diseases are becoming prevalent especially in developing countries, such as, Nigeria. Some of these diseases result from the kind of diet and life style of the individuals, (Franchesca et al., 2010).
Over the years, the crude extracts of Garcinia kola seeds have been used traditionally in the management of ailments especially liver disorders, (Iwu, 1990). Clinical investigations using Garcinia kola are few and a clear understanding of how it exerts its effect is unknown.
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1.2 Aim of the study
To evaluate the hepatoprotective effect of aqueous extract of bitter (Garcinia kola) seeds on CCL4-induced hepatic toxicity.
1.3 Objectives of the study To evaluate the hepatoprotective effect of aqueous extract of bitter kola seeds on CCL4-induced hepatic toxicity using established hepatoprotective drug, silymarin as a standard. To investigate the histological changes of the liver resulting from oral consumption of Garcinia kola extract using H & E stain. To investigate the activities liver enzymes (ALT, AST, ALP, SOD, GSH and CAT) using biochemical techniques.
1.4 Justification of the study
Popular and indigenous medical systems, often, are the only source of immediate help for populations that lack access to formal health resources. Because plants are integral elements of these medical systems, it is important to understand indigenous therapeutic objectives in using plant medicine.
The improvement of health in developing nations requires an understanding of indigenous and popular concepts of liver related diseases, including its etiology and treatment.
1.5 Hypothesis
Oral administration of aqueous extract of bitter kola (Garcinia kola ) seeds has protective effect on CCl4 induced liver toxicity in adult wistar rats.
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1.6 Significance of the study
The study is significant as it may lead to the design of plant based remedy for hepatoprotection that are natural, easily accessible, cheap with minimal side effects when compared to the convectional hepatoprotective drugs e.g erythromycin, alphaamethyldopa (Shah, 2010).
Results from this study may be important as it may enlighten the public on the effect of ingesting G.kola particularly because it is an inexpensive and effective prophylactic and/or treatment against liver toxicity/ diseases
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