ABSTRACT
Cola nut is a caffeine-containing nut of evergreen trees of the genus Cola, primarily of the species Cola acuminata and Cola nitida. It is one of the most common chewable nut in Nigeria. It is a central nervous system stimulant used in folk medicine as an aphrodisiac, an appetite suppressant, to treat migraine headache and indigestion. The aim of the present study was to investigate the possible effects of Cola nitida extracts on the cerebral cortex, hippocampus and some biochemical parameters. Forty (40) adult Long Evans rats of both sexes, with average weight of 210-230grams were randomly divided into four groups: A (control) = four rats (administered normal feed and distilled water);B= caffeine fraction group containing three sub groups of four rats each, B1, B2 and B3 were administered with di-chloromethane caffeine fractionof 19.2, 38.4 and 57.6mg/kg body weight, respectively; C= aqueous extract group containing three sub groups of four rats each, C1, C2 and C3 and were administered cola nut aqueous extract orally at the concentration of 300, 600 and 900 mg/kg body weight respectively; D= cola nut supplement group containing three sub groups of four rats each, D1, D2 and D3 were administered 10%, 20% and 30% w/w cola nut supplemented feed, respectively. The administration lasted for a period of 21 days (3 weeks). Behavioural test for spatial learning and memory was carried out using Morris water maze and anxiety-like behaviours using Elevated Plus Maze. Blood was collected via cardiac puncture for determination of haematological indices, Brain tissues used for determination of oxidative stress parameters were homogenised in cold saline thoseused for histological studies were fixed in Bouin‟s fluid processed and stained using Haematoxylin and Eosin (H and E) and toluidine blue stains. The Morris water maze test showed a non-significant decrease (P˃0.05) in time to find the platform in all the treatment groups. A non- significant increase (P˃0.05) in the meantime spent in the open arm, number of entriesinto the open arm, rearing and number of head dips in the elevated plus maze test
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when compared with the control was observed. The result of the oxidative stress markers showedstatistical significant decrease insuperoxide dismutase (SOD) of the aqueous and caffeine fraction groups when compared with the control.No significant difference was observed with the other markers. The results of the haematological indices revealed a statistical significant increase(p˃0.05) anddecrease (P˃0.05) in lymphocytes and neutrophils counts respectively in the caffeine and aqueous extract groups. The histology results revealed neurodegenerative changes in the cerebral cortex and hippocampus of the treated groups, which range from neuronal degeneration, pyknosis and clumping of pyramidal cells in the hippocampus. It was concluded that ingestion of cola nut and its constituent by Long Evans rats caused no significant effect on learning and memory, decreased anxiety-like behaviours, anti-inflammatory effect by increasing lymphocytes and decreasing neutrophils level, non-antioxidant effect by significantly decreasing the level of superoxide dismutase (SOD) and neurodegenerative changes.
TABLE OF CONTENTS
TITLE PAGE ……………………………………………………………………………i
DECLARATION ……………………………………………………………………………………………… ii
CERTIFICATION …………………………………………………………………………………………… iii
DEDICATION ………………………………………………………………………………………………… iv
ACKNOWLEDGEMENT …………………………………………………………………………………. v
ABSTRACT …………………………………………………………………………………………………….. vi
TABLE OF CONTENTS ……………………………………………………………………………….. viii
List of Figures ………………………………………………………………………………………………….. xi
List of Tables ………………………………………………………………………………………………….. xii
List of Plates ………………………………………………………………………………………………….. xiii
List of Appendices …………………………………………………………………………………………. xvii
List of Abbreviations ……………………………………………………………………………………. xviii
CHAPTER ONE ………………………………………………………………………………………………. 1
1.0 Introduction ………………………………………………………………………………………………… 1
1.1 Statement of Research Problem …………………………………………………………………… 2
1.2 Justification of the Study ……………………………………………………………………………… 2
1.3 Significance of the Study ……………………………………………………………………………… 3
1.4.1 Aim of the Study ………………………………………………………………………………………. 3
1.4.2 Objectives of the Study ……………………………………………………………………………… 3
1.5 Research Hypothesis ……………………………………………………………………………………. 4
1.6 Scope of the Study ……………………………………………………………………………………….. 4
1.7 Limitations of the Study ………………………………………………………………………………. 4
CHAPTER TWO ……………………………………………………………………………………………… 5
2.0 Literature Review ……………………………………………………………………………………….. 5
2.1 Cola nitida. ………………………………………………………………………………………………….. 5
2.1.1 Plant Taxonomy …………………………………………………………………………………………. 6
2.1.2 Plant Description ………………………………………………………………………………………… 6
2.1.3 Cultivation ………………………………………………………………………………………………… 7
2.1.4 Distribution of Cola Nut ……………………………………………………………………………… 8
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2.1.5 Reports on the effects of Cola nitida. ……………………………………………………………. 9
2.1.6 Phytochemical composition ……………………………………………………………………….. 11
2.1.7 Side effects of Cola nitida …………………………………………………………………………. 11
2.2 Caffeine; the active component of Cola nitida ……………………………………………… 12
2.2.1 Reports on the effects of caffeine ……………………………………………………………….. 13
2.2.1 Mechanism of Action of Caffeine ………………………………………………………………. 14
2.3 Cerebrum ………………………………………………………………………………………………….. 15
2.3.1 The Gross anatomy of cerebrum …………………………………………………………………. 15
2.3.2 Histology of the cerebrum …………………………………………………………………………. 18
2.3.3 Embryology of the Cerebrum …………………………………………………………………….. 23
2.3.4 Functions of the Cerebrum ………………………………………………………………………… 23
2.4 The Hippocampus ……………………………………………………………………………………… 24
2.4.1 Functions of the Hippocampus …………………………………………………………………… 27
2.4.2 Learning and Memory ………………………………………………………………………………. 27
CHAPTER THREE ………………………………………………………………………………………… 29
3.0 Materials and Methods ………………………………………………………………………………. 29
3.1 Materials …………………………………………………………………………………………………… 29
3.2 Plant Material …………………………………………………………………………………………… 29
3.3 Extraction of Plant Material ………………………………………………………………………. 29
3.4 Experimental Animals ……………………………………………………………………………….. 30
3.5 Acute toxicity study …………………………………………………………………………………… 30
3.6 Experimental Design………………………………………………………………………………….. 30
3.7 Neurobehavioral Studies ……………………………………………………………………………. 32
3.7.1 Spatial memory and learning (Morris water maze). ………………………………………. 32
3.7.2 Elevated Plus Maze …………………………………………………………………………………… 34
3.8 Animal Sacrifice ………………………………………………………………………………………… 36
3.9 Determination of Haematological Indices …………………………………………………… 36
3.10 Tissue Preparation …………………………………………………………………………………… 37
3.10.1 Fixation …………………………………………………………………………………………………. 37
3.10.2 Tissue processing ……………………………………………………………………………………. 37
3.10.3 Sectioning ……………………………………………………………………………………………… 38
3.10.4 Staining …………………………………………………………………………………………………. 38
3.10.5 Photomicrograph …………………………………………………………………………………….. 38
3.11 Estimation of Oxidative Stress Parameters ……………………………………………….. 38
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3.11.1 Determination of Catalase activity (CAT) ………………………………………………….. 38
3.11.2 Determination of Superoxide Dismutase (SOD) …………………………………………. 39
3.11.3 Assessment of Lipid Peroxidation …………………………………………………………….. 40
3.11.4 Assay of Reduced glutathione (GSH) ………………………………………………………… 40
3.12 Statistical Analysis …………………………………………………………………………………… 41
CHAPTER FOUR …………………………………………………………………………………………… 42
4.0 Result ………………………………………………………………………………………………………… 42
4.1 Acute Toxicity: ………………………………………………………………………………………….. 42
4.2 Physical Observation …………………………………………………………………………………. 42
4.3 Body Weight of the Animals ………………………………………………………………………. 42
4.4 Morris water maze test ………………………………………………………………………………. 44
4.5 Elevated plus maze …………………………………………………………………………………….. 46
4.6 Haematological indices ………………………………………………………………………………. 48
4.7 Oxidative stress …………………………………………………………………………………………. 51
4.8 Histological Studies ……………………………………………………………………………………. 54
CHAPTER FIVE ……………………………………………………………………………………………. 98
5.0 Discussion …………………………………………………………………………………………………. 98
CHAPTER SIX …………………………………………………………………………………………….. 104
6.0 Conclusion and Recommendations …………………………………………………………… 104
6.1 Conclusion ………………………………………………………………………………………………. 104
6.2 Recommendations for Further Research ………………………………………………….. 105
References …………………………………………………………………………………………………….. 106
Appendices ……………………………………………………………………………………………………. 115
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CHAPTER ONE
1.0 Introduction
Plants have been an important source of medicine for thousands of years. The world health organization (WHO) estimated that 80% of people still rely on traditional remedies such as herbs for their medicines (WHO, 2000; Tripathi and Tripathi, 2003). Plants are also a source of modern medicines. It is estimated that approximately a quarter of processed drugs contain plant extracts or active ingredients obtained from or modelled on plant substances (Tripathi and Tripathi, 2003). Plant-derived substances have recently become of great interest due to their versatile applications. Medicinal plants are the richest resource of drugs of traditional system of medicine, modern medicines, food supplements, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs (Ncube et al.,2008). Medicinal plants were used by people from different cultures without the knowledge of their active ingredients. This is common practice which is laden with hazards as the extract may contain some toxic constituents (Lown, 1993).
Cola nitida commonly called cola nut in English, “Goro” in Hausa, “Obi” in Yoruba, “Oji” in Igbo, “Irevu” in Ebira and “Ebi” in Nupe is a genus of 125 species of trees native to tropical rainforest of Africa. Cola is one of the major tree crop grown in Nigeria today, and the tree on which it grows is believed among some Nigerian tribes to be the first tree on the earth(Lowor, 2010). It is a smooth fruit consisting two cotyledons of the family Sterculoideae. It is cultivated in the tropical West Africa. Some species are also cultivated for their nuts in Madagascar, Brazil, Jamaica and other humid tropics. The seed contains caffeine and is bitter. The first taste of Cola nitida is bitter, but it
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sweetens upon chewing (Lovejoy et al., 1980). It isan important part of the traditional spiritual practice of culture and religion in West Africa, particularly in Nigeria.Cola nut has been reportedly used in folk medicine as an aphrodisiac, an appetite suppressant, to treat migraine headache and indigestion (Esimone et al., 2007).
1.1 Statement of Research Problem
Cola nitida has neurodegenerative effects on the cerebellum of Wistar rats(Buraimoh et al., 2014). Memory and cognition enhancement procedure is not well understood in the field of neuroscience and due to high consumption of Cola nitida by Nigerians, especially students to increase alertness and probably memory, there is need to investigate and compile empirical evidence to either proof or disprove this belief. Furthermore, considering the use of the fruit by people in West Africa, especially Nigeria, who believe that it has a divine power of curing some diseases, there is need to investigate the effects of Cola nitida on neurobehavioral activities and vital tissues of the body such as cerebrum and hippocampus at microscopic level.
1.2 Justification of the Study
Despite reported evidence of neurodegeneration that Cola nitida has on the cerebellum (Buraimoh et al., 2014), traditional, cultural and social use in our environment is still on the rise. Therefore, there is need to investigate further; the effect of Cola nitida on other parts of the nervous system, such as the cerebral cortex and hippocampus.
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1.3 Significance of the Study
The study could be of great importance inidentifying and evaluating the regions of the brain that may be affected by Cola nitida, give insight to people, especially students on the effect of the plant on memory, cognition, anxiety-like behaviours and its relationship with biochemical parameters. 1.4 Aim and Objectives of the study
1.4.1 Aim of the Study
The aim of the study was to investigate the effects of supplement, aqueousextract and di-chloromethane caffeine fraction extract administration of Cola nitida on the cerebrum and hippocampus of adultLong Evans rats.
1.4.2 Objectives of the Study
The objectives of this study were to:
I. Investigate the effect of Cola nitida on spatiallearningand memory, andanxiety-like behaviour using Morris water maze and elevated plus maze, respectively.
II. Investigate the effect of Cola nitida on haematological indices (PCV, WBC, Hb, and differential cell counts) of Long Evans rats.
III. Evaluate the comparative effect ofCola nitida on the oxidative stress markers(GSH, MDA, CAT and SOD) of the cerebrum of Long Evans rats.
IV. Examine the effect of Cola nitida on the histology of cerebral cortex and hippocampus of Long Evans rats.
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1.5 Research Hypothesis Cola nitida has no effect on the histology of the cerebrum and hippocampus of adult Long Evans rats. Cola nitida has effect on neurobehavioursof adult Long Evans rats.
1.6 Scope of the Study
This work would be limited to the effects of Cola nitida and its active ingredient caffeine on the histology of cerebral cortex and hippocampus, neurobehaviours, haematological indices and some biochemical parameters.
1.7 Limitations of the Study Electron microscopy was not done to show the detailed structure of the cerebral cortex and hippocampus due to lack of electron microscope. Neurotransmitters were not assayed due to lack of fund.
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