ABSTRACT
The present study was conducted to evaluate the effect of aqueous extract of Citrullus lanatus (water melon) seed on the liver of ethanol treated adult Wistar rats. Twenty Five number of Wistar albino rats were grouped, 1, 2, 3, 4 and 5 respectively. Group 1 and 2 (Negative Control group and Positive control group respectively) received 2ml/kg body weight with distilled water for eight days and 20ml/kg body weight of 40% ethanol on day eight, respectively. Group 3 and 4 received the extract 200mg/kg and 400mg/kg body weight for seven days, followed by 20ml/kg body weight of 40% ethanol on day eight. Group 5 received silymarin 100mg/kg body weight for seven days, followed by 20ml/kg body weight of 40% ethanol on day eight. All administration was once daily by oral gavage. At the end of the experiment, blood samples were collected for serum analysis for levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and Alkaline phoaphatate (ALP). Hepatic tissues were collected for analysis of protein concentration of the liver, while the histopathology of the liver was assessed using Haematoxylin and Eosin stain, histochemically using periodic Acid Schiff (PAS) stain detects glycogen, Feulgen stain detects DNA and Masson‘s Trichrome stain detect collagen fibres. Results obtained showed that C.lanatus seed extract did not affect weight gain or hepatic index, although the ethanol treated group showed decrease in weight gain and an increase in hepatic index. Treatment with C.lanatus seed extract decreased AST, ALT, ALP serum levels, and protein concentration in the liver, which was increased due to ethanol ingestion. From histopathological studies, ethanol-induced hepatotoxicity was characterized by hepatocytes degeneration, depletion in the pattern of glycogen storage in the cytoplasm of the liver hepatocytes, distortion in the presentation pattern of DNA materials in the liver hepatocytes and decrease in the distribution of collagen fibres in the
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liver parenchyma. Thus the study demonstrates that Aqueous extract of C.lanatus seed possess hepatoprotective effect against ethanol-induced hepatotoxi
TABLE OF CONTENTS
Title Page————————————————————————————– i
Declaration————————————————————————————- ii
Certification———————————————————————————– iii
Dedication ————————————————————————————- iv
Acknowledgement ————————————————————————— v
Table of Contents—————————————————————————– vii
List of Tables———————————————————————————- x
List of Figures——————————————————————————— xi
Abstract————————————————————————————- xiv
1.0 INTRODUCTION —————————————————————– 1
1.1 Background of the Study——————————————————— 1
1.2 Statement of the Problem——————————————————— 4
1.3 Justification ————————————————————————- 5
1.4 Aim and objectives ————————————————————— 5
1.4.1 Aims of the Study——————————————————————- 5
1.4.2 Objectives of the Study ———————————————————– 5
1.5 Significance of the Study———————————————————- 6
1.6 Study Hypothesis——————————————————————- 6
2.0 LITERATURE REVIEW —————————————————— 7
2.1 LIVER ——————————————————————————- 7
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2.1.1 Embryology of the liver ———————————————————— 8
2.1.2 Gross Anatomy of the Liver —————————————————– 9
2.1.3 Histology of the Liver ———————————————————— 11
2.1.4 Functions of the Liver ———————————————————- 13
2.2 Hepatotoxicity ——————————————————————– 14
2.2.1 Hepatotoxicity due to ethanol —————————————————- 15
2.2.2 Incidence of Hapatotoxicity —————————————————— 16
2.2.3 Causes of Hepatotoxicity ———————————————————- 17
2.2.4 Drugs used in the treatment of Hepatotoxicity ———————————– 24
2.3 ALCOHOL ———————————————————- ————— 26
2.3.1 Alcoholism ————————————————————————— 25
2.3.2 Effects of Alcohol on Mitochondrial Biomolecules —————————- 27
2.3.3 Pharmacokinetics ——————————————————————- 31
2.3.4 Alcohol Effects on the Liver —————————————————— 32
2.3.5 Liver Diseases ———————————————————————– 34
2.4 Citrullus lanatus ——————————————————————– 36
2.4.1 Taxonomy of Citrullus lanatus—————————————————— -36
2.4.2 Origin and distribution of Citrullus lanatus————————————— 37
2.4.3 Morphology of Citrullus lanatus ————————————————– —37
2.4.4 Phytochemistry of Citrullus lanatus ———————————————- —38
2.4.5 Pharmacological studies of Citrullus lanatus ———————————– —39
2.4.6 Toxicological studies of Citrullus lanatus ————————————— —43
3.0 MATERIALS AND METHODOLOGY
3.1 MATERIALS ———————————————————————– —45
3.1.1 Experimental Animals ————————————————————– —45
3.1.2 Experimental Plant —————————————————————— —45
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3.1.3 Drugs and reagents —————————————————————— —45
3.1.4 Equipment —————————————————————————- —46
3.2 Methods —————————————————————————— —44
3.2.1 Extraction of Plant —————————————————————— —44
3.2.2 Experimental design —————————————————————- —47
3.2.3 Morphological studies ————————————————————– —48
3.2.4 Biochemical studies —————————————————————– —48
3.2.5 Determination of protein concentration —————————————— —50
3.2.6 Histological Studies —————————————————————– —51
3.2.7 Histopathological Studies ——————————————————— —51
3.2.8 Tissue Preparation for Microscopy ———————————————– —51
3.3 Statistical analysis —————————————————————– —52
4.0 RESULTS ————————————————————————— —53
4.1 Morphological Studies ———————————————————— —55
4.2 Biochemical analysis of liver enzymes (AST, ALT, and ALP) ———– —-56
4.3 Protein concentration in the liver ———————————————- —-57
4.4 Histopathological Studies ——————————————————– —-59
5.0 DISCUSSION ———————————————————————- —-81
6.0 SUMMARY, CONCLUSION AND RECOMMENDATION ———— —-85
6.1 SUMMARY ————————————————————————- —-85
6.2 CONCLUSION ——————————————————————– —-85
6.3 RECOMMENDATION ———————————————————- —-85
REFERENCES ——————————————————————– —–87
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CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of Study
Hepatotoxicity implies chemical-driven liver damage (Singh et al., 2012). Drug-induced liver injury is a major health problem that challenges not only health care providers but also the pharmaceutical industry and drug regulatory agencies. According to the United States Acute Liver Failure Study Group, drug-induced liver injury accounts for more than 50% of acute liver failure, including hepatotoxicity caused by overdose of acetaminophen (39%) and idiosyncratic liver injury triggered by other drugs (13%) (Michael and Ju, 2006). Approximately 75% of the idiosyncratic drug reaction results in liver transplantation (or) death (Zimmerman, 1978). Drug-induced liver toxicity is a common cause of liver injury. It accounts for approximately one-half of the cases of acute liver failure and mimics all forms of acute and chronic liver disease (Kaplowitz, 2001). Different types of drugs such as acetaminophen, chloroquine and isoniazid are inducing hepatotoxicity in world. Isoniazid and rifampicin, the first line drugs used for tuberculosis therapy are associated with hepatotoxicity (Tasduq et al., 2005).
Medicinal plants play a key role in the human health care system. About 80% of the world population rely on traditional medicine which is predominantly based on plant medicine (WHO, 1993). Plant medicine (phyto-medicine) has been used in healthcare delivery in many parts of Africa and the rest of the world (Elujoba et al., 2005). Effective health cannot be achieved in Africa, unless orthodox medicine is complemented with traditional medicine (Elujoba et al., 2005). Fruits and vegetables have been recognized as natural sources of various bioactive compounds (Pennington and Fisher, 2010) which could be attributed to their phytoconstituents such as flavonoids, anthocyanins, vitamins
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C and E, phenolic compounds, dietary fiber, and carotenoids present in fruits and vegetables (Gonz´alez-Aguilar, et al 2008).
Health and well-being has been a subject of man‘s primary concern since time immemorial. There has been resurgence of scientific interest in medicinal plants during the past few decades due to their importance in traditional system of medicine. Herbal medicines are in great demand in the developed world for primary health care because of their efficacy, safety and lesser side effects. A detailed investigation and documentation of plants used in the traditions and pharmacological evaluation of these plant and their taxonomical relatives can lead to the development of invaluable plant based drugs for many dreaded diseases. A large number of plants and purified natural substances have been screened for liver disorders (Subramoniam and Pushpangadan, 1999).
Liver diseases are mainly caused by toxic chemicals, excess consumption of alcohol, infectious and autoimmune disorder. Most of the hepatotoxic chemical damage the liver cells mainly by lipid peroxidation and other oxidative damages (Wendel et al.,; 1987 Dianzani et al., 1991). Alcohol is the major culprit of liver disease in Western countries and this arises from excessive ingestion of alcohol (Lieber, 2000). Alcohol affects many organs of the body, including the nervous system and the liver (Adebisi, 2002, 2003, 2004, 2006). Liver is the most important organ where drugs are structurally altered; resulting biologically inactive or active metabolite and some of these are toxic. Liver is exposes to drugs in higher concentration as whole of the drug pass through the liver to reach systemic circulation. Thus, the liver is a vulnerable target of injury from various chemicals and drugs and disordered hepatic functions is an important cause of abnormal drug handling (Guyton and Hall, 2006).
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Further more; liver has capacity to recover from acute injury by hepatocellular regeneration with the production of new cells, which restore liver functions and normal tissue structure. Chronic liver injury, however after leads to fibrosis, scar formation and distortion of normal tissue architecture (Bennett and Brown, 2003). Ethanol consumption induces apoptosis in a variety of tissues including liver, (Nanji, 1998), buccal mucosa,(Slomiany et al., 1998), salivary gland,(Slomiany et al., 1997), gastric mucosa,( Piotrowski et al., 1997) brain,( Zhang et al., 1998), thymus,( Wang et al., 1997) and spleen( Coiler et al., 1998). In particular, ethanol consumption-induced hepatic apoptosis has been widely recognized in rats, (Benedetti et al., 1988; Baroni et al., 1994; Yacoub et al., 1995; Kurose et al., 1997; Sloamiany et al., 1999; Mi et al., 2000).
Supporters of herbal medicine claim that herbs may treat and prevent diseases. This adds to a deep belief that these treatment are safe as they are natural and fit into the image of gentle and therefore harmless alternative to conventional medicine. Modern medicine offers limited success in providing effective cure and there is a severe need to develop new drugs capable of healing toxic liver damages. In traditional system of medicine, plants were claimed to be effective and used successfully to alleviate multiple liver disorder (Hikino and Kiso 1998). But, evidence for efficacy is sparse. In spite of limitations, a number of herbal show promising effects, either experimentally in cell culture (invitro models), animal studies (invivo models), or even in clinical trials. Some hepatoprotective plants include: Silybum marianum Lnn., Adhatoda vasica, Apium graveolens Linn., Ballota glandulosissima, Carica papaya Linn., Cleome viscose Linn., Phyllanthus niruri Linn., ( Chaudhary et al., 2010).
Citrullus lanatus of family Cucurbitaceae is commonly known as water melon and in local name Tarmuz (Hindi), Puchakaya (Telugu), Kankana (Hausa), Eso-ba‘ra‘(Yoruba). The ripe fruits are edible and largely used for making confectionary. Its nutritive values
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are also useful to the human health. It also reported having analgesic and anti-inflammatory activity of roots and leaves, antimicrobial activity, laxative activity of fruit (, anti-oxidant and antiulcerative activity (Chopra, 1958; Chiej, 1984; Rahman et al 2008; Deng et al, 2010; Loiy et al., 2011). Water melon seeds have both nutritional and cosmetic importance, the seed contain vitamins B2, minerals, riboflavin, fat carbohydrates and protein (Lazos, 1986). Watermelon is a rich natural source of lycopene, a carotenoid of great interest because of its antioxidant capacity and potential health benefits (Rhodes and Zhang, 1999). Cucurbitaceae plants are known to contain bioactive compounds such as cucurbitacin, triterpenes, sterols and alkaloids (Yuan et al., 2006). The seed is also a good vermifuge and has a hypotensive action. Preliminary research indicates that the consumption of watermelon may have antihypertensive effects (USA, 2012).
1.2 Statement of Research Problem
The liver plays a pivotal role in the metabolism of toxic substances that enter the body. Alcohol is one of these toxic substances, which is ultimately broken down into simple end-products for easy elimination (Fernandez-Checa et al., 1997; Fernandez-Checa, 2003). However, certain by products generated during alcohol metabolism may be more toxic than alcohol itself and may contribute to the development of alcohol liver disease (Ashak et al., 1991).Almost all ingested alcohol is metabolized in the liver and excessive alcohol consumption may lead to acute and chronic liver disease (Cederbaum, 2001; Cederbaum et al., 2009).
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1.3 Justification of the Study
Herbal medicinal products play an important role in the management of liver diseases for the lack of satisfactory liver protective drugs in allopathic medical practices. Searching for hepatoprotective drugs with high efficacy and safety is of great need. Over the years, Citrullus lanatus (water melon) seed have been use traditionally for the management of different ailments, thus there should be a pharmacological justification on the management and control of induced liver toxicity. This study will evaluate the effect of aqueous extract of Citrullus lanatus (water melon) seed on the liver of ethanol treated Aldult wistar rats.
1.4 Aim and Objectives
1.4.1 Aims of the Study
To evaluate the effect of aqueous extract of Citrullus lanatus (water melon) seed on the liver of ethanol treated aldult Wistar rats.
1.4.2 Objectives of the Study
The specific objectives were to determine the following:
1. The changes in morphology and morphometry of the liver due to ethanol treated adult Wistar rats.
2. Histopathological changes in the liver using routine histological techniques of haematoxylin and eosin (H and E) and the use of special stains such as Periodic Acid Schiff for glycogen deposition, Masson‘s Trichrome stain for collagen fibres, Feulgen stain for DNA.
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3. The changes in liver enzymes activities such as alanine amino-transferase (ALT), aspartate amino-transferase (AST) and alkaline phosphates (ALP) using biochemical analysis.
4. The changes in protein concentration of the liver in treated and untreated rats using Bradford method.
1.5 Significance of the Study
The result of the study will be of importance to complement the efforts of researchers in the identification and evaluation of available natural medicinal plants that may possibly be used as alternatives to currently used hepatoprotective drugs which are expensive and not free from adverse effects.
1.6 Study Hypothesis
Aqueous extract of Citrullus lanatus (water melon) seed has effect on the liver of ethanol treated aldult Wistar rats.
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