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="" ccl="" compared="" control="" group="" in="" increased="" negative="" span="" the="" when="" with="">4 treated groups. There was significant (p<0 .05="" activities="" alanine="" alkaline="" aminotransaminase="" and="" aspartate="" bilirubin="" ccl="" direct="" for="" in="" indirect="" of="" phosphatase="" reduction="" serum="" span="" the="">4 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="" ccl="" in="" malondialdehyde="" of="" reduction="" span="">4 treated groups compared to the negative control group. A significant (p<0 .05="" activities="" also="" and="" catalase="" ccl="" dismutase="" in="" increase="" observed="" of="" span="" superoxide="" was="">4 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
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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