ABSTRACT
The stem bark of Detarium microcarpum (Guill and Perr.) is used in traditional medicine for the treatment of liver disease in middle belt region of Nigeria. To substantiate this folkloric claim, ethyl-acetate and n-butanol fractions of Detarium microcarpum stem bark was investigated for its hepatocurative and antioxidant effect in CCl4 induced liver damage in rats. Aqueous extraction was carried out on Detarium microcarpum stem bark and the crude extract was further fractionated sequentially using ethyl-acetate and n-butanol solvents. In the in-vitro studies, phytochemical screening of the crude extract showed the presence of phenolic, flavonoids, tannins, saponins, alkaloids and glycosides while total phenolic content assay, total flavonoid content assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Reducing power and H2O2 free radical scavenging activities were carried out on ethyl-acetate and n-butanol fractions. The total phenol content for n-butanol and ethyl acetate fractions were 2.97±0.31 and 11.54±0.20 mg/g Gallic acid equivalents while total flavonoid content were 234.42±0.71 and 45.76±2.59 mg/g quercetin equivalents. Ethyl acetate fraction showed the highest DPPH free radical scavenging activity with 65.31% inhibition while n-butanol showed the highest reducing power and H2O2 free radical scavenging activities with 65.31% and 52.55% which informed the choice of n-butanol fraction for further studies. In the in-vivo studies, the LD50 of n-butanol fraction of Detarium microcarpum stem bark was >5000 mg/kg body weight of rats. CCl4 (1ml/kg body weight) as a 1:1(v/v) solution in olive oil was used to induce liver damage followed by subsequent treatment with n-butanol fraction of Detarium microcarpum stem bark at three different doses (100, 150 and 200 mg/kg bw/day) while silymarin (100 mg/kg bw/day) was used as standard drug for 28 days. The liver weight was significantly (p<0.05) increased in the negative control group when compared with the CCl4 treated groups. There was significant (p<0.05) reduction in the serum activities of alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase (ALP), direct and indirect bilirubin for CCl4 treated groups compared to the negative control group. Total protein (TP) and albumin (ALB) in the negative control group were reduced but not significantly (p>0.05) compared to the CCl4 treated groups. In endogenous antioxidant activities, there was significant (p<0.05) reduction of malondialdehyde (MDA) in CCl4 treated groups compared to the negative control group. A significant (p<0.05) increase was also observed in superoxide dismutase (SOD) and catalase (CAT) activities of CCl4 treated groups compared to the negative control group. These results may suggest hepatocurative and antioxidant effects of Detarium microcarpum stem bark in CCl4 induced liver damaged animals.
TABLE OF CONTENTS
Title Page
Abstract
Table of Contents
CHAPTER ONE
1.0 INTRODUCTION
1.1 Preamble
1.2 Statement of Research Problem
1.3 Justification
1.4 Aim and Objectives
1.4.1 Aim
1.4.2 Specific objectives
1.5 Null Hypothesis
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Detarium microcarpum. Guill and Perr
2.1.1 Classification of the plant
2.1.2 Description, distribution and habitat of Detarium microcarpum
2.1.3 General uses of Detarium microcarpum plant
2.1.4 Ethno-medicinal uses
2.2 Phytochemical profile of Detarium microcarpum plant
2.3 Pharmacological activities
2.3.1 Antidiabetic activity
2.3.2 Antibacterial and antifungal activities
2.3.3 Antiviral activity
2.3.4 Enzyme inhibition
2.3.5 Antisnake venom activity
2.4. The Liver
2.4.1 Structure and functions
2.4.2 Liver cells
2.4.3 Xenobiotics and liver metabolism
2.4.4 Mechanisms of hepatic injury
2.5 Mode of action of liver toxicants
2.5.1 Carbon tetrachloride (CCl4) induced hepatotoxicity
2.6 Liver injuries
2.6.1 Cholestatic liver injury
2.6.2 Fatty liver (Steatosis)
2.6.3 Cell death
2.7 Biochemical alterations in hepatic damage
2.7.1 Serum aminotransferase enzymes
2.7.2 Serum alkaline phosphatase
2.7.3 Serum total protein and albumin
2.7.4 Serum bilirubin
2.8 Silymarin
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Materials
3.1.1 Chemicals/reagents
3.1.2 Plant sample collection and identification
3.1.3 Experimental animals
3.2 Methodology
3.2.1 Preparation of plant sample
3.2.2 Aqueous extract preparation
3.2.3 Fractionation
3.2.4 Qualititative phytochemical analysis
3.2.5 Quantitative phytochemical analysis
3.2.6 In-vitro antioxidant activity
3.2.7 Acute toxicity studies
3.2.8 Induction of liver damage
3.2.9 Experimental design
3.2.10 Biochemical analysis
3.2.11 Determination of oxidative stress parameters
3.3 Statistical Analysis
CHAPTER FOUR
4.0 RESULT
4.1 Qualitative Screening of Phytochemicals
4.2 Total flavonoid / total phenolic content
4.3 In-vitro Antioxidant Activity
4.3.1 DPPH radical scavenging activity
4.3.2 Reducing power assay
4.3.3 Hydrogen peroxide (H202) radical scavenging activity
4.4 Lethal Dose Determination
4.5 Effect of n-butanol fraction on body weight / organ weight
4.4.1 Effect of n-butanol fraction on body weight
4.4.2 Effect of n-butanol fraction on relative organ weight
4.5 Biochemical Parameters
4.5.1 Effect of n-butanol fraction on serum liver damage biomarkers/liver function parameters in CCl4 induced liver damage in rats
4.5.2 Effect of n-butanol fraction on kidney function parameters of CCl4 induced liver damage in rats
4.6 Oxidative Stress Parameters
4.6.1 Effect of n-butanol fraction on oxidative stress parameters in CCl4 induced liver damage in rats
CHAPTER FIVE
5.0 DISCUSSION
CHAPTER SIX
6.0 Summary, Conclusion and Recommendations
6.1 SUMMARY
6.2 CONCLUSION
6.3 RECOMMENDATIONS
REFERENCE
CHAPTER ONE
INTRODUCTION
1.1 Preamble
Herbal medicines are herbal preparations produced by subjecting plant materials to extraction, fractionation, purification, concentration or other physical or biological processes which may be produced for immediate consumption or as a basis for herbal products (WHO, 2001). Notwithstanding the extent of significant advancement in modern medicine in recent decades, plants still make an important contribution to health care. Traditionally they are used worldwide for the prevention and treatment of disease. Herbal plants were prescribed even when their active compounds were unknown because of their effectiveness and relatively low cost (Bhawna and Kumar, 2010). This observation is particularly more relevant to people in the developing countries of the world where the majority of the populations are living in the rural areas.
The liver plays an important role in regulating various physiological processes. It is essential in the body for maintenance, performance and regulating homeostatic functions. It is involved with almost all the biochemical pathways for growth, fight against diseases, nutrient supply, energy provision and reproduction. In addition, it aids metabolism of carbohydrate, protein and fat, detoxification, secretion of bile and storage of vitamins (Ahsan et al., 2009). Because of its central role in drug metabolism, it is the most vulnerable tissue for drug toxicity (Sunil et al., 2012). The role played by the liver in the removal of substances from the portal circulation makes it susceptible to persistent attack by offending foreign compounds, culminating in liver dysfunction (Bodakhe and Ram, 2007). The liver secretes bile, prothrombin, fibrinogen, blood-clotting factors and heparin, a mucopolysaccharide sulfuric acid ester that prevents
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blood from clotting within the circulatory system (Bhawna and Kumar, 2010). Toxic chemicals, xenobiotics, alcohol consumption, malnutrition, anaemia, medications, autoimmune disorders (Marina, 2006), viral infections (hepatitis A, B, C, D, etc.) and microbial infections (Sharma and Ahuja, 1997) are harmful and cause damage to the hepatocytes.
Reactive oxygen species (ROS) are continuously generated during metabolic processes to regulate a number of physiological functions essential to the body (Valko et al., 2007). These reactive oxygen species are prone to withdraw electrons from biological macromolecules such as proteins, lipids, nucleic acids in order to gain stability in the biological system. This disruption may be attributed to a number of factors such as the inability of the cells to produce sufficient amounts of antioxidants, nutritional deficiency of minerals or vitamins (Abd Ellah, 2010). When the production of ROS exceeds the capability of the body to detoxify these reactive intermediates, oxidative stress would develop (Mena et al., 2009). Oxidative stress can be induced by variety of factors such as radiation or exposure to heavy metals and xenobiotics (e.g carbon tetrachloride). This may lead to drastic harm to the body such as membrane damage, mutations due to attenuation of DNA molecules, and disruption to various enzymatic activities in metabolism of the body (McGrath et al., 2001; Valko et al., 2006; Chanda and Dave, 2009).
Medicinal plants are important sources of antioxidants (Rice, 2004). Antioxidants stabilize or deactivate free radicals, often before they attack targets in biological cells (Nunes et al., 2012). Natural antioxidants either in the form of raw extracts or their chemical constituents are very effective in preventing the destructive processes caused by oxidative stress (Zengin et al., 2011). Recently interest in naturally occurring antioxidants has considerably increased for use in food, cosmetic and pharmaceutical
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products, because they are multifaceted in their multitude and magnitude of activity and provide enormous scope in correcting imbalance (Djeridane et al., 2006; Wannes et al.,
2010). The beneficial medicinal effects of plant materials typically results from the combinations of secondary products present in the plant (Wink, 1999). Phytochemical constituents of medicinal plants (e.g. polyphenols, carotenoids, flavonoids, phenolics, vitamins C and E), act as antioxidants by preventing damages to cell membrane due to cellular oxidative processes that may result in diseases (Omoregie and Osagie, 2011). They are found in all parts of plants such as leaves, fruits, seeds, roots and bark (Mathew and Abraham, 2006).
Antioxidants are broadly divided into enzymic antioxidants and non enzymic antioxidants. Enzymic antioxidants include the superoxide dismutases, glutathione peroxidase and catalase (Klaunig and Kamendulis, 2004). Non-enzymic antioxidants, which include vitamin E, vitamin C, β-carotene, reduced glutathione, and coenzyme Q function to quench reactive oxygen species (Clarkson and Thompson, 2000). Antioxidants have various mechanisms such as prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction and radical scavenging (Rao et al., 2004). Many chemicals damage mitochondria, an intracellular organelle that produces energy, its dysfunction release excessive amount of oxidants which in turn damage hepatic cells. Activation of some enzymes in the cytochrome P450 system, such as CYP2E1, also leads to oxidative stress (Jaeschke et al., 2002).
Carbon tetrachloride (CCl4) is a well known hepatotoxin used in diverse experimental models (Singh et al., 2008). In addition to hepatic problems, it causes dysfunction of the kidneys, lungs, testis, brain, and blood by generating free radicals (Ozturk et al., 2003; Khan et al., 2009). Carbon tetrachloride (CCl4) is rapidly transformed to trichloromethyl
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radical (CCl3*) and its derivative trichloromethyl peroxy radical (CCl3OO*), generated by cytochrome P450 of liver microsomes (Brent and Rumack, 1993). These free radicals react with membrane lipids leading to their peroxidation (Singh et al., 2008). Membrane disintegration of hepatocytes with subsequent release of membrane associated enzymes and necrosis are some of the consequences of CCl4 induced liver damage.
1.2 Statement of Research Problem
Hepatotoxicity is one of the very common ailments resulting into serious debilities ranging from severe metabolic disorders to even mortality (Anil et al., 2010). Liver injury due to chemicals or infectious agents may lead to progressive liver fibrosis and ultimately cirrhosis and liver failure (Anand, 1999). According to the report published by USFDA, more than 900 drugs, toxins, and herbs have been reported to cause liver injury, and drugs account for 20 – 40% of all instances of hepatic failure (Soni et al.,
2011).
Liver ailments represent a major global health problem. Chronic liver cirrhosis and drug induced liver injury is the ninth leading cause of deaths in western and developing countries (Baranisrinivasan et al., 2009; Saleem et al., 2010).
In Nigeria, as in other parts of sub-saharan Africa, the major causes of liver cirrhosis include infections particularly chronic hepatitis B virus (HBV) infection (Otu, 1987 and Cook, 1980) and hepatitis C virus infection (Bojuwoye, 1996). There is increasing evidence that free radicals and reactive oxygen species play a crucial role in various steps that initiate and regulate the progression of liver diseases independently of the original agent (Jemal et al., 2007).