Effect Of Alstonia Boonei Stem Bark Extract On Liver Function In CCL4-Induced Liver Damage In Male Rabbits

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Abstract

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The aim of this study was to investigate the effect(s) of cold water stem bark extract of A. boonei on CCl4-induced liver damage in male rabbits; as well as to ascertain the immunological status of the animals. Twenty (20) animals were divided into 4 groups. Group 1 served as control and was given 1mL/kg body weight of normal saline for 7 days. Group 2 received a single dose of 1mL/kg of CCl4 , while group 3 received 0.5g/kg body weight of extract for 7 days before CCl4 administration. Group 4 received 0.5g/kg body weight of extract for 7 days. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, bilirubin, total protein and albumin were determined using standard colorimetric methods. The animals in group 3 did not significantly lose weight (p>0.05), while there was weight loss in groups 2 and 4 (p<0.05).

There was significant reduction (p>0.05) in the concentration of plasma and tissue homogenate of conjugated bilirubin, total bilirubin, and alkaline phosphatase activity. Furthermore, there was significant increase (p<0.05) in plasma and tissue homogenate of aspartate aminotransferase, alanine aminotransferase, total protein and albumin. All the haematological parameters increased (p>0.05) except WBC that decrease in group 3 (p>0.05). In group 4, there were decreases in RBC, Hb and HCT (p<0.05), and WBC and PLT (p>0.05).The plant extract aggravated toxicity effects in liver function with suppressed immunity. These results portend possible dangers for individuals who may be placed on long term therapy of this extract.

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Table Of Contents

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Title Page

• Title Page i
• Certification ii
• Dedication iii
• Acknowledgement iv
• Table of content v
• Abstract viii

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Chapter One

• 1.0 Introduction 41
• 1.1 Carbon Tetrachloride (CCl4) 42
• 1.1.1 Physical and Chemical Properties of CCl 43
• 1.1.2 Natural Occurrence of Ccl43
• 1.1.3 Environmental Occurrence of Ccl 44
• 1.1.4 Uses of Ccl 44
• 1.2 Mechanism of CCl4-Induced Nephrotoxicity 5
• 1.2.1 Phytochemical composition of A. boonei 8
• 1.2.2 Cultivation of A. boonei 8
• 1.2.3 Ethnobotanical Uses of A. boonei 9
• 1.3 Aims / Objectives 9

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Chapter Two

• 2.0 Literature Review 10
• 2.1 Anthelmintic Effect of A. Bonnel 10
• 2.2 Effects of A. Boonei on Renal Functions 10
• 2.3 Anti-Malaria Properties of A. Boonei 12
• 2.4 Effects of A. Boonei on Reproductive Function 13
• 2.5 Hypolipidaemic and Anti-Atherogenic Properties of A. Boonei 14

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Chapter Three

• 3.0 Materials and Method 16
• 3.1 Reagents16
• 3.2 Apparatus16
• 3.3 Plant Collection and Authentication18
• 3.4 Animals18
• 3.5 Animal Arrangement 18
• 3.6 Stabilization Treatment 18
• 3.7 Animal Feeding 19
• 3.8 Preparation of Plant Sample 19
• 3.9 Preparation of Administered Boses of the Plant Extract 19
• 3.10 Preparation of Ccl4 Solution for Administration 19
• 3.11 Determination of Extract Concentration 19
• 2.12 Plant Extract Administration 20
• 3.13 Experimental Design 20
• 3.14 Homogenization of Tissues 21
• 3.15 Histological Analysis of Liver Tisue 22
• 3.6 Full blood count 22
• 3.6.1 Assay for Total Protein 23
• 3.6.2 Assay for Albumin 24
• 3.6.3 Assay for Alkaline phosphatase 25
• 3.6.4 Assay for Alanine aminotransferase 26
• 3.6.5 Assay for Alanine Aminotransferase 27
• 3.6.6 Assay for Conjugated Bilirubin 28
• 3.21 Assay for Total Bilirubin 29
• 3.7 Statistical Analysis 31

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Chapter Four

• 4.0 Results 32
• 4.1 Plasma Biochemical Results 29
• 4.2 Full Blood Count Results 46
• 4.3 Tissue Homogenate Results 56
• 4.4 Histological Results 58

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Chapter Five

• 5.0 Discussion, Conclusion and Recommendation 74
• 5.1 Discussion 74
• 5.2 Conclusion 79
• 5.3 Recommendations 80
• References 81
• Appendix I – Results 88
• Appendix II – Weights, Volume of Extract and CCl4 Administration 94
• Appendix III – Statistics 97

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Chapter One

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Introduction

The liver is the largest internal organ in the body.It weighs about 1.5 kg in an adult human and 0.9kg inrabbits.It is also the most active and most complex organ. Suffice it to say, that, the liver plays a major role in metabolism by independently handling virtually all biochemical transformations. Consequently, the replenishing power of the liver is not in doubt because it carries out inter alia metabolic, secretory, excretory, storage, and detoxification functions (Edet et al., 2011). Needless to say that, the liver is a sine qua non for life.

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Figure 1; Showing the anterior and posterior view of the liver (www.liverpic.com)

The figure is shown in the complete material which you will download before the end of this page

Despite its many interesting functions, the liver is prone to suffer a lot of damage.

These damages give rise to various forms of liver diseases. The end result is that the liver malfunctions and its efficacy in carrying out its functions is hampered. There exists a myriad of liver diseases such as alcohol liver disease, hepatic cirrhosis, hepatocellular carcinoma, haemochromatosis, autoimmune hepatitis, fulminant hepatitis, hepatic encephalopathy, etc.

These defects in liver function could have implications on other organs. For instance, acute liver failure correlates with acute renal failure with an approximately 55% frequency of occurrence, although the relationship between cirrhosis and renal dysfunction has not been fully established (Jaramillo-Juarez et al., 2008). The defects in liver function could be as a result of alcohol, drugs (acetaminophen), chemical compounds (e.g. thioacetamide, carbon tetrachloride) etc.

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1.1 Carbon Tetrachloride

Carbon tetrachloride (also known as carbon tet, with IUPAC name: tetra chloromethane) is an organic compound with the chemical formula CCl4. It has adverse health effects because of its toxicity. Researchers have shown that CCl4 is one of the most potent toxins; especially, those of hepatic forms. This explains its wide usage in experimental models for the evaluation of the abilities of hepatoprotective agents (Seifert et al., 1994). CCl4-induced hepatic damage is principally caused by lipid peroxidation and decreased activities of antioxidant enzymes. This gives rise to the generation of free radicals (Shahjahan et al., 2004). These effects could however be mitigated by hepatoprotective agents. These hepatoprotective agents are usually common medicinal plants.(Etim et al., 2008).

1.1.1 Physical And Chemical Properties

• Description; Colourless liquid
• Molecular formula; CC14
• Molecular Weight; 153.8g/mol
• Density:1.59g/cm3 @ 20oCBoiling point; 76.7oC
• Melting point:-23oC
• Vapour pressure; 91.3 torr @ 20oC
• Solubility; Soluble in acetone, ethanol, benzene, carbon disulfide, slightly soluble in water.(HSDB, 1995; CRC, 1994).

1.1.2 Natural Occurrence Of Ccl4

There are suggestions that carbon tetrachloride can be formed in the troposphere by the photochemical reactions of chlorinated alkenes that are solar-induced (Singh et al., 1975). However, this reaction has so far remained solely laboratory-based since it can only be demonstrated in the laboratory. Even if it could happen in nature, there are indications that it would not be a major source of environmental carbon tetrachloride. Also, Carbon tetrachloride has been detected in volcanic emission gases (Isidorov et al., 1990). Several other studies have postulated that global atmospheric levels of carbon tetrachloride can be clearly explained by anthropogenic sources alone (Singh et al., 1976). This implies that humans are major determining factors in CCl4 concentration and pollution in the world.

1.1.3 Environmental Occurrence Of Ccl4

The major source of carbon tetrachloride in air is industrial emissions. It has also been detected in surface water, groundwater and drinking-water. This is due to industrial and agricultural activities. The occurrence of carbon tetrachloride has also been reported in wastewater arising from iron and steel manufacturing, foundries, metal finishing, paint and ink formulations, petroleum refining and nonferrous metal manufacturing industries (United States National Library of Medicine, 1997).

1.1.4 Uses Of Ccl4

A large percentage of the carbon tetrachloride produced is principally used in the production of Chlorofluorocarbons (CFCs). These CFCs are primarily used as refrigerants, propellants, foam-blowing agents and solvents and in the production of other chlorinated hydrocarbons. Carbon tetrachloride has also found wide usage as a grain fumigant, pesticide, solvent for oils and fats, metal degreaser, fire extinguisher and flame retardant, and in the production of paint, ink, plastics, semi-conductors and petrol additives. Carbon tetrachloride also found wide usage previously as a cleaning agent.

However, these many uses have apparently phased-out since production has dropped (ATSDR, 1994). The drop in production in recent years is due to the Copenhagen Amendment to the Montreal Protocol (1992) (UNEP, 1996). Furthermore, carbon tetrachloride is a known and potent hepatotoxin, hence its wide usage in experimental models for the evaluation of the properties of hepatoprotective agents (Seifert et al., 1994). This justifies the central position it occupies in this study.

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1.2 Mechanism Of Carbon Tetrachloride Induced Toxicity

It has been reported that the cytochrome P-450-dependent monoxygenase system is a principal site responsible for carbon tetrachloride reductive metabolism (Wolf et al., 1980). In the study of CCl4 metabolism, a great emphasis is placed on the involvement of covalent binding of CCl4 metabolites to cell components and/or peroxidative damage as the cause of injury. Binding has been reported to occur preferentially to triacylglycerols and phospholipids, with phosphatidylcholine containing the highest amount of binding. CCl4 is also known to decrease the rate of triacylglycerol secretion as very low density lipoproteins (Boll et al., 2001).

A very early investigation has already suggested that carbon tetrachloride toxicity depends on cleavage of the carbon-to-chlorine bond (C-Cl bond). There is also a very strong and established link between the metabolism of carbon tetrachloride and the peroxidative decomposition of cytoplasmic membrane structural lipids (Recknagel, 1967).

In the final analysis, covalent binding of the CCl3* radical to cell components initiates the inhibition of lipoprotein secretion and thus steatosis, whereas reaction with oxygen, to form CCl3OO*, initiates lipid peroxidation. The two processes are independent of each other, and the extent to which they occur depends on partial oxygen pressure. The formation of adduct and ultimately, cancer initiation, is a possible consequence of the former process whereas the latter process results in loss of calcium homeostasis and ultimately, apoptosis and cell death (Boll et al., 2001).

Right from time immemorial, many herbal medicines have found great relevance in the treatment of liver diseases; this leads to the exploration of the plant kingdom and a search for common medicinal plants for the development of new phytotherapeutic agents for liver diseases (Thirupathi et al., 2007). Some of these common medicinal plants are Alstonia boonei, Gongronema latifolium, Vernonia amygdalina, etc.

Alstonia boonei De Wild is a herbal medicinal plant of West African origin, popularly known as God’s tree.In Nigeria it is called ukpukuhu in URHOBO, EDO call it úkhú, whileIGBO call it égbú-ọ̀rà. Within West Africa, it is considered as sacred in some forest communities; consequently the plant parts are not eaten. The plant parts have been traditionally used for its anti-malarial, aphrodisiac, anti-diabetic, antimicrobial, and antipyretic activities, which have also been proved scientifically.

Figure 6; showing the leaves of A.boonei (www.Astonia boonei_pic).

The figure is shown in the complete material which you will download before the end of this page

The plant parts are rich in various bioactive compounds such as echitamidine, Nα-formylechitamidine, boonein, loganin, lupeol, ursolic acid, and β-amyrin among which the alkaloids and triterpenoids form a major portion. The 2 alkaloids have diuretic, spasmolytic and hypotensive properties (Arhoghro et al., 2009).

An infusion in cold water of the stem bark is drunk as a cure for venereal diseases, worms, snakebiteandrheumatic pains and to relax muscles. It is also taken internally or used as a bath as a remedy for dizziness. An infusion of root and stem bark is drunk as a remedy for asthma; a liquid made from the stem bark and fruit is drunk once daily to treat impotence. A decoction of the bark is given after childbirth to help the delivery of the placenta (Arhoghro et al., 2009).

1.2.1 Phytochemical Composition Of A. Boonei

The ethanolic extract has been shown to contain more metabolites. This implies that ethanol being of high polarity than hexane but similar to that of water (solvent commonly used traditionally), Phytochemical profile of ethanol extract will be similar to that of water. Alkaloids, tannins, saponins, flavonoids and cardiac glycosides were among the phytochemicals detected together with the vitamin, ascorbic acid in A. boonei (Akinmoladun et al., 2007).

1.2.2 Cultivation Of A. Boonei

A. boonei grows into a giant tree in most of the evergreen rain forests of tropical West Africa. The plant thrives very well in damp riverbanks. It is well known to all the traditional healers practicing along the west coast of Africa. It occurs in deciduous and fringing forest of Ghana (Gosse et al., 1999). A. boonei De Wild is a deciduous tree up to 35 meters high. The flowers are white with lax terminal cymes. The fruits are paired with slender follicle up to 16cm long with brown floss at each end (Adotey et al., 2012).

1.2.3 Ethnobotanical Uses Of A. Boonei

All the parts of the plant are very useful. Various documented and undocumented claims have it that alcoholic or aqueous preparations from some parts of the plant especially the stem bark have medicinal uses for treating febrile illness, jaundice, painful micturition, rheumatic conditions (Ojewole, 1984; Asuzu and Onaga, 1991), as an anti-venom against snake bite, as antidote against arrow poisoning, etc. The extract of the stem bark is commonly used as a febrifuge in treating malaria and is listed in the African pharmacopoeia as an antimalarial drug (Olajide et al., 2000). It is highly effective when used in its fresh form; however, the dried one could equally be used. It has also been found to possess anti-rheumatic, anti-inflammatory (Abbiw, 1990), analgesic/pain-killing, anti-malaria/antipyretic, anti-diabetic (mild hypoglycemic), anti-helminthic, antimicrobial and antibiotic properties (Kamet al., 1997). A decoction could be sweetened with pure honey and be taken up to 4 times daily as an effective painkiller (Adotey et al., 2012).

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1.3 Aims / Objectives

This study is aimed at evaluating the in-vivo effects of cold water extract of A. boonei on liver function in CCl4 induced- liver damage by determining total protein, albumin, plasma bilirubin, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase.

To evaluate the immunological status by determining the full blood count.

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