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
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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