ABSTRACT
Buccholzias coriacea is a medicinal plant commonly known as wonderful kola. This study investigated neuroprotective potential of methanol extract of B. coriacea seeds in mercury- challenged Wistar albino rats. A total of 25 Wistar albino rats were used for the study and they were divided into 5 groups of five rats each. The rats in the first group received distilled water and served as the control. Group 2 rats received extract (400 mg/kg body weight). The rats in third group received mercury (4 mg/kg body weight). The rats in fourth group received the extract (200 mg/kg body weight) and mercury (4 mg/kg body weight) and the rats in the fifth group received the extract (400 mg/kg body weight) and mercury (4mg/kg body weight). The study lasted for a month and mercury was administered in the last two weeks. The extract and mercury were administered morning and evening. The rats were sacrificed and the cerebrum and cerebellum tissues harvested for biochemical and histological examinations. The results showed that there were significant (P < 0.05) increase in glutathione (GSH) concentration of the rats in the extract-treated groups and significant (P < 0.05) decrease of malondialdehyde (MDA) concentration of the rats in the extract-treated groups compared to the values obtained for the rats in group 3. Glutathione peroxidase (GPx) activities were found to be significantly (P < 0.05) higher in the cerebellum and cerebrum of rats in the treated groups compared to the rats in group 3. There was increase in catalase (CAT) activities in the cerebrum and cerebellum of the treated rats groups compared to the untreated rats (group 1). There was significant (P < 0.05) decrease in nitric oxide (NO) concentration in the cerebellum and cerebrum of rats in the treated group compared to those of untreated groups. The cholesterol concentration in the cerebellum of the treated rats (group 4 and 5) were significantly (P<0.05) higher compared to the values obtained in the rats of other groups. The adenosine deaminase (ADE) activities in the cerebellum and cerebrum of group 4 rats significantly (P < 0.05) reduced compared to the rats of other groups. Acetylcholinesterase (ACE) activities were higher in the cerebellum and the cerebrum of the rats in the treated group compared to the value obtained in the untreated rats (group 1). Histopathology showed that the extract reduced degeneration and necrosis in the cerebellum of rats in groups 4 and 5. The increase in the antioxidants and decreased lipid peroxidation, necrosis and degeneration observed in the rats of the extract-treated groups show that the extract has neuroprotective potential in mercury-challenged Wistar albino rats.
TABLE
OF CONTENTS
Title page i
Declaration ii
Certification iii
Dedication iv
Acknowledgments v
Table of Contents vi
List
of Tables x
List of Figures xi
List of Plates xiii
List of Abbreviations xiv
Abstract xv
CHAPTER
1: INTRODUCTION 1
1.1 Background of the Study 1
1.2 Statement of the Research Problem 4
1.3 Justification of the Study 4
1.4 Significance of the Study 4
1.5 Aim and
Specific Objectives of the Study 4
CHAPTER
2: LITERATURE REVIEW 6
2.1 Buccholzia. coriacea 6
2.1.1 Bioactive
components of Buccholzia. Coriacea 6
2.1.1.1 Alkaloid 6
2.1.1.2 Phenol 7
2.1.1.3 Flavonoid 7
2.1.1.4 Tannin 9
2.1.1.5 Saponin 9
2.1.1.6 Glycosides 10
2.2 Human Brain 11
2.2.1
Cellular mechanisms of protection
to the brain due to antioxidant
activity 13
2.3 Mercury 14
2.3.1 Chemical forms and properties of mercury 14
2.3.2 Sources of mercury 15
2.3.2.1 Natural
source of mercury 15
2.3.2.2
Anthropogenic source of mercury 16
2.3.3 Metabolism of mercury 16
2.3.4 Mercury poisoning 18
2.3.5 Mechanism of mercury toxicity 19
2.3.5.1 Neurotoxicity of mercury through generation of
free radicals 19
2.3.5.2 Glutamate and calcium dyshomeostasis mechanism
of neurotoxicity 21
2.3.6 Susceptibility to mercury toxicity 22
2.3.7 Treatment of mercury poisoning 23
2.3.8 Prevention of mercury poisoning 24
2.4 Biochemical Parameters 24
2.4.1 Glutathione 24
2.4.2 Catalase 26
2.4.3 Malondialdehyde 26
2.4.4 Nitric oxide 30
2.4.5 Glutathione peroxidase 31
2.4.6 Cholesterol 32
2.4.7 Acetylcholinesterase 35
2.4.8 Adenosine deaminase 37
CHAPTER 3: MATERIALS AND METHODS 38
3.1 Materials 38
3.1.1 Plant Material 38
3.1.2 Experimental animals 38
3.1.3 Equipment 38
3.1.4 Chemicals and reagents 38
3.2 Methods 39
3.2.1 Collection of the plant seeds 39
3.2.2 Preparation of plant materials 39
3.2.3 Extraction of the plant
seeds 39
3.2.4 Collection of the experimental animal 39
3.2.5 Acclimatization of the experimental animals 40
3.2.6 Experimental design 40
3.2.7 Laboratory analysis 40
3.2.7.1 Mercury preparation 40
3.2.7.2 Determination of the acute toxicity and
lethality 41
3.2.7.3Assay of glutathione
concentration 41
3.2.7.4 Assay of catalase 42
3.2.7.5 Determination of malondialdehyde 43
3.2.7.6 Determination
of nitric oxide concentration 44
3.2.7.7 Assay of glutathione
peroxidase activity 44
3.2.7.8 Determination of cholesterol
concentration 45
3.2.7.9 Assay of acetylcholinesterase activity 46
3.2.7.10 Assay of adenosinse deaminase activity 46
3.2.7.11 Histological examination 47
3.2.7.11.1 Tissue preparation 47
3.2.7.11.2 Slide examination
and photomicrography 48
3.3 Statistical Analysis 48
CHAPTER 4: RESULTS AND DISCUSSION 49
4.1 Results 49
4.1.1 Effect
of methanol extract of B.coriacea
seed on glutathione (GSH)
concentrations of the cerebellum and
cerebrum of Wistar albino rats 49
4.1.2 Effect
of methanol extract of B.c oriacea
seed on catalase activity in the
cerebellum and cerebrum of Wistar albino
rats 51
.
4.1.3 Effect of methanol extract of B.coriacea seed on malondialdehyde
concentration in the cerebellum and cerebrum of Wistar albino rats 52
4.1.4 Effect
of methanol extract of B.coriacea
seed on nitric oxide
concentration in the cerebellum and
cerebrum of Wistar albino rats 54
4.1.5 Effect of methanol extract of B.coriacea seed on glutathione
peroxidase
GPx concentration in the cerebellum and
cerebrum of Wistar
albino rats.
56
4.1.6
Effect of methanol extract of B.coriacea seed on cholesterol
concentration
in the cerebellum
and cerebrum of rats Wistar albino
rats 57
4.1.7.
Effect of methanol extract of B.coriacea seed on acetylcholinesterase
(ACE) activity in the cerebellum and cerebrum of Wistar albino rats 59
4.1.8 Effect of methanol extract of B.coriacea seed on adenosine deaminase
(ADE) activity in the cerebellum and cerebrum of Wistar albino rats 60
4.1.9 Effect of methanol extract
of Buccholzia coriacea seed on the
histology of the cerebellum and cerebrum of
Wistar albino rats. 62
4.2 Discussion 69
CHAPTER 5: CONCLUSION AND RECOMMENDATION 77
5.1 Conclusion 77
5.2 Recommendation 77
References 78
Appendix 92
LIST OF TABLES
2.1: Biological activity of phyto-components
identified in the aqueous
extracts of Bulchholzia
coriacea seed using GC-MS method. 10
LIST
OF FIGURES
1.0 Structure of neuron 3
2.1: Basic
phenolics and flavoniods 8
2.2:
The basic structure of tannin. 9
2.3:
Basic structure of saponin 10
2.4: Buccholzia
coriacea fruit, seed and leaf 11
2.5: Human Brain 12
2.6: Fate
of mercury vapor 17
2.7: Free
radical scavenging pathway of the antioxidant system. 25
2.8: Structure of glutathione. 25
2.9:
Structure of Malondialdehyde 28
2.10: Malondialdehyde modification of nucleic acid 29
2.11: The
MDA reaction with amino acid 29
2.12: Biosynthetic pathway of
nitric acid 30
2.13: Reaction Pathway of
gluthatione peroxidise 32
2.14: Structure of cholesterol 33
2.15: Major pathway of cholesterol
oxidation 35
2.16. Reaction of the cleavage of
acetylcholine by acetylcholinesterase 36
2.17: Reaction pathway of adenosine deaminase 37
4.1 Effect
of methanol extract of B.coriacea
seed on Glutathione (GSH)
Concentrations of the cerebellum and cerebrum of Wistar
albino rats. 50
4.2: Effect
of methanol extract of B. coriacea
seed on catalase activity in the
cerebellum and cerebrum of Wistar albino rats 51
4.3 Effect
of methanol extract of B. coriacea
seed on malondialdehyde
Concentration in the cerebellum and cerebrum of
Wistar albino
rats. 53
4.4 Effect of methanol extract of B. coriacea seed on Malonaldehyde (MDA)
concentration in the cerebellum and cerebrum of Wistar albino rats 55
4.5
Effect of methanol extract of B. coriacea seed on glutathione
peroxidase
GPx
concentration in the cerebellum and cerebrum of Wistar albino rats. 56
4.6 Effect
of methanol extract of B.c oriacea
seed on cholesterol concentration
in
the Cerebellum and Cerebrum of Wistar albino rats. 58
4.7. Effect of methanol extract of B.c oriacea seed on acetylcholinesterase
(ACE) activity in the cerebellum and cerebrum of Wistar albino rats. 59
4.8 Effect of methanol extract of B.coriacea seed on Adenosine Deaminase
(ADE)
activity in the
cerebellum and cerebrum of Wistar albino rats 61
LIST OF PLATES
1A
Histology
of Cerebrum of Wistar albino
rats administered water and feed. Mag.x400.
H&E stain. 62
1B Histology
of Cerebellum of Wistar albino
rats administered water and
feed. Mag.X400.H&E
Stain 63
2A
Histology of the Cerebrum of Wistar albino rats treated with
methanol
Extract
of B.coriacea seed. Mag.x400, H&E
stain 64
2B
Histology of Section of the
Cerebellum Wistar albino
rats treated with
methanol extract of B.coriacea seed. Mag.x400, H&E stain 64
3A Histopathology of Section of the Cerebrum
of mercury induced Wistar
albino rats Mag. X400,
H&E Stain 65
3B Histology of Section of the Cerebellum of
mercury induced Wistar
albino rats. Magx400, H&E
stain 65
4A Histology of Section of the Cerebrum of
mercury induced Wistar
albino rats treated with
methanol extract of B.coriacea seed.
Mag.x400,
H&E
Stain 67
4B Histology
of Section of the Cerebellum of mercury induced Wistar
albino rats treated with
methanol extract of B.coriacea seed.
Mag.x400,
H&E
Stain 67
5A
Histology
of Cerebrum of mercury induced Wistar
albino rats treated with methanol extract of B.coriacea
seed. Mag.x400, H&E Stain 68
5B Histology of the Cerebellum of mercury
induced Wistar albino
rats treated
With
methanol extract of B.coriacea seed.
Mag.x400, H&E Stain 69
LIST OF ABBREVIATION
CAT Catalase
MDA Malondialdehyde
NO Nitric oxide
GPx Glutathione peroxidase
GSH Reduced glutathione
ACE Acetylcholinesterase
ADA Adenosine deaminases
H and E Hemoxylene and eosin
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Buccholzia
coriacea is a medicinal plant. The use
of medicinal plants is accepted all over the world and can be used in detection,
prevention and treatment of diseases (Rickert et al., 1999). Useful information on the importance of herbal
medicine had been traced back to previous studies since the beginning of man
(Sofowora, 1984). Plants with useful ethno-medicinal potentials which have been
scientifically validated are important for alternative drug production (Nazeruddin
et al., 2011).
Buccholzia
coriacea grows to about 5 cm in
height. It has bipinnate leaves, white flowers and yellowish fruit containing
few seeds (Ajaiyeoba et al., 2001; Ezekiel and Onyeoziri, 2009) and these seeds are usually
eaten raw or cooked (Lemmens, 2015). Buccholzia coriacea seed extract is
composed of phytochemicals such as alkaloids, glycoside, saponin, steroids, tannin,
flavonoids, terpenes and phenols, macronutrients such as carbohydrate and
micromolecule such as, iron, phosphorus, calcium, magnesium, potassium,
zinc, sodium (Ibrahim
and Fagbohun, 2012). It is considered as brain food due to its
capability to enhance the memory, treat and prevent premature aging, hence
neuroprotective (Ibrahim and Fagbohun, 2012).
The previous studies on methanol seed
extract of B. coriacea had shown that it has hypoglycaemic effect due to
the synergistic potentials exhibited by the extract with metformin an agent of
hypoglycaemia (Erhirhie et al., 2015). Ezekiel and
Onyeoziri, (2009) reported that this plant extract has antifungal and antibacterial
effect, due to the inhibitory zones showed by extract of Buchholzia coriacea
with test bacteria and the retarded growth showed by test fungi on administration
of B. coriacia extract. Extract of Buchholzia
coriacea has also been found to have antihypercholesterolemic activity due to its ability to reduce
total cholesterol concentration and peroxidation of lipid in the liver and
serum of hypercholesterolemic rats (Erhirhie
et al., 2015).
Erhirhie et al (2015) further reported that this
plant extract has anti-ulcer and anti-secretion of gastric
mucosa activities due to its
ability to reduce gastric acid secretion mediated by histamine and blockage of contractile
responses induced by histamine.
Mercury (hydrargyrium, Hg) is
classified as a toxic heavy metal (Clarkson et
al., 2003; World Health Organisation, 2007). There are different types of
mercury but for this research study inorganic mercury will be emphasized. Inorganic
mercury is used in the preparation of medicine such as anti-bacterial and anti-fungi
drugs (Jain et al., 2013), body
cream, soaps (Guzzi and La Porta, 2008) and also infant teething powder, hence
human are easily exposed to mercury.
Exposure
to mercury leads to loss in the integrity of the membrane and consequently to cellular
necrosis which results from its effect on the anti-oxidant system which
are either enzymatic, such as superoxide dismutase (SOD), catalase, glutathione
peroxidase GPx, or non-enzymatic, such as the tripeptide glutathione. High
level of reactive oxygen species produced from the condition of antioxidant
system depletion can lead to peroxidation of lipid (Farina et al., 2011b). Delayed detoxification of
mercury prevents methylation of DNA, RNA, histones and prevents synthesis of
methylcobalamin, phosphatidylcholine and neurotransmitters (Deth, 2004) and
examples of neurotransmitters includes acetylcholine, serotonin, glutamate,
dopamine, gama amino butyric acid (GABA) (NIMH, 2017). Mercury poisoning has
been reported to have the capability of crossing the blood brain barrier and
this can lead to shrinking of the neurons that contains pyknotic nuclei of the
cerebral cortex (Ghusson et al.,
2012) and degeneration and necrosis in purkinje cells of cerebellum.
High
exposure to mercury and its compounds causes disorders associated
with neurodegeneration such as Alzheimer’s disease (Coppede and Migliore, 2010)
and amyotrophic lateral sclerosis (Johnson and Atchison 2009). Alzheimer
disease is a condition associated with memory loss due to brain death, dementia
and decline in cognitive and it occurs from deficiency in acetylcholine and
choline acetyltransferase (Bhushan et al.,
2018).
Neurodegenerative disorders are
disorders that results from neurodegeneration which involves the loss of the
structure and function of the neurons. A feature of neurodegeneration is the
progressive cell degeneration in specific neuronal cells of the central nervous
system (CNS), this is often accompanied with changes in cytoskeletal protein
that lead to intranuclear and intracytoplasmic inclusions in glia and neurons. The
neurological effects of neurodegeneration are devastating and can lead to
severe mental disorders (Alessia et al.,
2014).
Fig 1: structure of neurone.
1-Dendrite,
2-Axon, 3-Node of ranvier, 4-Axon terminal, 5-Schwann cells, 6-cell body. 7- Nucleus. (Gorazd
and Lawrence, 2016)
1.2 STATEMENT
OF THE RESEARCH PROBLEM
Humans are easily exposed to mercury and its compound and
B.coriacea seed extract component has been
reported to have nutritive and medicinal importance and it is therefore
considered probably a detoxifying agent of the effect of mercuric chloride in the
cerebellum and cerebrum. The histological examination is to provide more
reliable pictures of the effect of mercury and methanol extract of B. coriacea
seed in the cerebellum and
cerebrum.
1.3 JUSTIFICATION
OF THE STUDY
Several
research studies have been carried out on the health potentials of Buccholzia coriacea seed extract and
they have proven effective due to its health beneficial components.
This
study was carried out to evaluate the potentials of this seed extract in the
prevention of neurodegenerative conditions caused by mercury poisoning.
1.4 SIGNIFICANCE OF THE
STUDY
This study will provide useful informations on the
potentials of methanol Buccholzia
coriacea seed extract on the biochemical indices and histological
implications of mercury-challenged Albino Wistars.
1.5 AIM AND SPECIFIC OBJECTIVES OF
THE STUDY
This study
was aimed at investigating the neuroprotective potentials of the methanol extract
of Buccholzia coriacea seed extract in
mercury challenged albino Wistar rats.
Specific objectives of the study
The specific objectives of study were.
ü To determine
the acute toxicity profile of methanol extract of B. coriacea seeds.
ü To determine
the effect of methanol extract of B. coriacea seeds on antioxidant
enzymes such as catalase and glutathione peroxidase in the cerebellum and
cerebrum of mercury-challenged rats.
ü To determine
glutathione concentration in the cerebellum and cerebrum of mercury-challenged
rats treated with methanol extract of B.
coriacea seed.
ü To determine
concentration of malondialdehyde in the cerebellum and cerebrum of mercury-challenged
rat treated with methanol extract of B.
coriacea seed.
ü To determine
adenosine deaminase activity in the cerebellum and cerebrum of mercury-challenged
rats treated with methanol extract of B.
coriacea seed.
ü To determine
the level of total cholesterol in the cerebellum and cerebrum of mercury -challenged
rat treated with methanol extract of B.
coriacea seed.
ü To determine
the nitric oxide concentration in the cerebellum and cerebrum of mercury-challenged
rat treated with methanol extract of B.
coriacea seed.
ü To determine
acetylcholinesterase activity in the cerebellum and cerebrum of mercury-
challenged rat treated with methanol extract of B. coriacea seed.
ü To carry out
histological examination in the cerebellum and cerebrum of mercury- challenged rat
treated with methanol extract of B.
coriacea seed.
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