EFFECT OF ALSTONIA BOONEI STEM BARK EXTRACT ON LIVER FUNCTION IN CCl4-INDUCED LIVER DAMAGE

IN MALE RABBITS

TABLE OF COTENT

TITLEPAGE

Title Pagei

Certificationii

Dedicationiii

AcknowledgementivTable of contentv

Abstractviii

CHAPTER ONE

1.0Introduction1

1.1Carbon Tetrachloride (CCl4)2

1.1.1Physical and Chemical Properties of CCl43

1.1.2Natural Occurrence of Ccl43

1.1.3Environmental Occurrence of Ccl44

1.1.4Uses of Ccl44

1.2Mechanism of CCl4-Induced Nephrotoxicity5

1.2.1Phytochemical composition of A. boonei8

1.2.2Cultivation of A. boonei8

1.2.3Ethnobotanical Uses of A. boonei9

1.3Aims / Objectives9

CHAPTER TWO

2.0Literature Review10

2.1Anthelmintic Effect of A. Bonnel10

2.2Effects of A. Boonei on Renal Functions10

2.3Anti-Malaria Properties of A. Boonei12

2.4Effects of A. Boonei on Reproductive Function13

2.5Hypolipidaemic and Anti-Atherogenic Properties of A. Boonei 14

CHAPTER THREE

3.0Materials and Method16

3.1Reagents16

3.2Apparatus16

3.3Plant Collection and Authentication18

3.4Animals18

3.5Animal Arrangement18

3.6Stabilization Treatment18

3.7Animal Feeding19

3.8Preparation of Plant Sample19

3.9Preparation of Administered Boses of the Plant Extract19

3.10Preparation of Ccl4 Solution for Administration19

3.11Determination of Extract Concentration 19

2.12Plant Extract Administration20

3.13Experimental Design20

3.14Homogenization of Tissues21

3.15Histological Analysis of Liver Tisue22

3.6Full blood count 22

3.6.1Assay for Total Protein 23

3.6.2Assay for Albumin24

3.6.3Assay for Alkaline phosphatase 25

3.6.4   Assay for Alanine aminotransferase 26

3.6.5Assay for Alanine Aminotransferase27

3.6.6Assay for Conjugated Bilirubin28

3.21Assay for Total Bilirubin29

3.7Statistical Analysis31

CHAPTER FOUR

4.0Results32

4.1Plasma Biochemical Results29

4.2Full Blood Count Results46

4.3Tissue Homogenate Results56

4.4Histological Results58

CHAPTER FIVE

5.0Discussion, Conclusion and Recommendation74

5.1Discussion74

5.2Conclusion79

5.3Recommendations80

REFERENCES 81

APPENDIX I – Results88

APPENDIX II – Weights, Volume of Extract and CCl4 Administration94

APPENDIX III – Statistics97

ABSTRACT

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.

CHAPTER ONE

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)

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.

1.1Carbon 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).

1.1.2Natural 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.

1.1.3Environmental 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).

1.1.4Uses 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.

1.2Mechanism 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 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).

1.2.1Phytochemical 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).

1.2.2Cultivation 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).

1.2.3Ethnobotanical 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).

1.3AIMS /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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