ABSTRACT
Tapinanthus globiferus is a semi-parasite mistletoe which grows on branches of large number of trees including Vitellaria paradoxa. It used locally in the management of insomnia, epilepsy, anxiety, headaches and hypertension. This study evaluated the toxicity and neuropharmacological properties of ethanol extract and fractions of T. globiferus. These were carried out using standard methods. Phytochemical screening of T. globiferus ethanol extract (TgE) revealed presence of alkaloids, anthraquinones, flavonoids, cardiac glycosides, saponins, steroids terpenoids and tannins variously distributed among the fractions. The intraperitoneal LD50 of TgE was 1,300 and 3,800 mg/kg in mice and rats respectively and ˃5,000 mg/kg orally. However, fractions of ethylacetate and ethylacetate water insoluble intraperitoneal LD50 were 1,400 and 1,100 mg/kg respectively, and 3,800 mg/kg each for butanol and residual aqueous in mice. Oral administration of TgE for 28 days in rats produced no effects on body weight and serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and total protein at 87.5, 175 and 350 mg/kg, but at 350 mg/kg, serum levels of total bilirubin and conjugate bilirubin were significantly (p ≤ 0.05) elevated while packed cell volume, haemoglobin and red blood cell were decreased. TgE produced significant (p ≤ 0.05) decreases in serum urea, creatinine and sodium concentrations, and increases in potassium and chloride concentrations at 175 and 350 mg/kg, whereas bicarbonate concentrations were unaffected at all doses compared to 1.0 ml/kg normal saline control. Liver sections at 175 and 350 mg/kg showed distortion and degeneration of hepatocytes but no toxic effects at 87.5 mg/kg. Kidney and spleen architecture were normal at 87.5 and 175 mg/kg, but 350 mg/kg resulted in degeneration of Bowman’s capsule, tubular degeneration and blood vessel distractions in kidney, as
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well as degeneration in red pulp and necrosis of epithelial cells in spleen sections (×100 H and E). TgE and fractions significantly (p ≤ 0.05) decreased onset and increased duration of diazepam-induced sleep but ethylacetate insoluble fraction had no effect at tested doses compared to control. Ethylacetate fraction (EF) was the most active among the fractions but produced no effect in ketamine-induced sleep at all tested doses. TgE and EF were significantly (p ≤ 0.05) reduced number of head dips in hole-board at tested doses. However, bicuculline at 5 mg/kg increased number of head dips which were antagonisted by EF and diazepam at 300 and 2 mg/kg respectively. TgE produced significant (p ≤ 0.05) increased in number of foot slips in beam-walk at 350 mg/kg and EF at 300 mg/kg, while BF showed no activity at 250, 500 and 1,000 mg/kg compared to control. TgE produced no effect in elevated plus-maze and number of entries and time spent in open arms at 175 and 350 mg/kg. TgE did not exhibit effect in number of rearing at tested doses but at 350 mg/kg reduced number of steps climbed in staircase. However, TgE significantly (p ≤ 0.05) reduced number of lines crossed and rearing at 175 and 350 mg/kg respectively in open field and offered significant effect in duration of haloperidol-induced catalepsy at 350 mg/kg, apomorphine-induced climbing behaviour at 87.5 and 175 mg/kg and no effect in duration of immobility in tail suspension at all doses compared to control. Antiseizure activities tested showed that TgE had no effect on onset of seizure compared to control in PTZ, STN, and PRT-induced seizures but offered 16.67, 33.33 and 50 % protection against PTZ and PRT at 87.5, 175 and 350 mg/kg. However, TgE did not protect mice and chicks against STN- and MES-induced seizures respectively. The study showed that extract and ethylacetate fraction of T. globiferus are less toxic and contained active constituents which have sedative effects and minimum anticonvulsant properties.
TABLE OF CONTENTS
TITLE PAGE………………………………………………………………….………..iii
DECLARATION ………………………………………………………………………………………….. iii
CERTIFICATION …………………………………………………………………………………………. iv
DEDICATION ………………………………………………………………………………………………..v
ACKNOWLEDGEMENTS …………………………………………………………………………….. vi
ABSTRACT ………………………………………………………………………………………………… vii
TABLE OF CONTENTS ……………………………………………………………………………….. ix
LIST OF TABLES ………………………………………………………………………………………..xvi
LIST OF FIGURES ……………………………………………………………………………………….xix
LIST OF PLATES …………………………………………………………………………………………. xx
LIST OF APPENDICES ………………………………………………………………………………..xxi
ABBREVIATIONS …………………………………………………………………………………….. xxiv
CHAPTER ONE …………………………………………………………………………………………….1
1.0 INTRODUCTION …………………………………………………………………………………..1
1.1 Statement of the Research Problem …………………………………………………………….1
1.2 Justification of the Study…………………………………………………………………………..3
1.3 Theoretical Framework …………………………………………………………………………….3
1.3.1 Toxicity study ………………………………………………………………………………………..4
1.3.2 Diazepam-induced sleep test ……………………………………………………………………4
1.3.3 Ketamine-induced sleep test …………………………………………………………………….5
1.3.4 Hole-board test ……………………………………………………………………………………..5
1.3.5 Mouse beam walking assay …………………………………………………………………….6
1.3.6 Elevated plus-maze test ………………………………………………………………………….6
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1.3.7 Elevated staircase test ……………………………………………………………………………6
1.3.8 Open field test………………………………………………………………………………………7
1.3.9 Haloperidol-induced catalepsy test …………………………………………………………..7
1.3.10 Apomorphine-induced climb test …………………………………………………………….8
1.3.11 Tail suspension test ……………………………………………………………………………….8
1.3.12 Pentylenetetrazole-induced convulsion test ……………………………………………….9
1.3.13 Strychnine-induced convulsion test ………………………………………………………….9
1.3.14 Electroshock-induced convulsion test…………………………………………………….. 10
1.3.15 Picrotoxin-induced seizure …………………………………………………………………… 10
1.3.16 Phytochemical screening ……………………………………………………………………… 11
1.3.17 Statistical analysis ………………………………………………………………………………. 11
1.4 Aim and Objectives ……………………………………………………………………………. 11
1.4.1 Aim…… ……………………………………………………………………………………………. 11
1.4.2 Objectives ………………………………………………………………………………………….. 11
1.5 Statement of Research Hypothesis …………………………………………………………. 12
CHAPTER TWO…………………………………………………………………………………………. 13
2.0 LITERATUREREVIEW ………………………………………………………………………. 13
2.1 Toxicity Study …………………………………………………………………………………….. 13
2.1.1 Haematology ………………………………………………………………………………………. 13
2.1.2 Biochemistry ………………………………………………………………………………………. 14
2.1.3 Histopathology ……………………………………………………………………………………. 14
2.2 Drugs that Acts on the Central Nervous System ……………………………………….. 15
2.2.1 Benzodiazepines ………………………………………………………………………………… 17
2.2.2 Barbiturates ………………………………………………………………………………………. 18
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2.2.3 Butyrophenones …………………………………………………………………………………. 18
2.2.4 Phenothiazines ……………………………………………………………………………………. 19
2.2.5 Hydantoin ………………………………………………………………………………………….. 19
2.2.6 Other drugs ………………………………………………………………………………………… 20
2.3 Medicinal Plants with Central Nervous System Activity ……………………………. 22
2.4 The Plant Tapinanthus globiferus…………………………………………………………… 23
2.4.1 Ethnomedicinal uses …………………………………………………………………………… 24
2.4.2 Mistletoe plant ………………………………………………………………………………….. 24
2.4.3 Vitellaria paradoxa (Tapinanthus globiferus host plant) …………………………… 25
CHAPTER THREE …………………………………………………………………………………….. 28
3.0 MATERIALS AND METHODS ……………………………………………………………. 28
3.1 Preparation of the Plant Materials …………………………………………………………… 28
3.2 Fractionation of Ethanol Extract of T. globiferus ……………………………………….. 28
3.3 Animals ……………………………………………………………………………………………… 30
3.4 Drugs, Chemicals and Equipment ………………………………………………………….. 30
3.4.1 Preparation of drug solutions ……………………………………………………………….. 30
3.4.2 Routes of drug administration ………………………………………………………………. 31
3.5 Qualitative Phytochemical Screening ……………………………………………………… 31
3.5.1 Test for alkaloids ………………………………………………………………………………… 31
3.5.2 Test for anthraquinones (Borntrager’s Test) ……………………………………………. 32
3.5.3 Test for cardiac glycosides (Keller- Kiliani’s Test) ………………………………….. 32
3.5.4 Test for coumarins (Feigl’s reaction) ……………………………………………………… 32
3.5.5 Test for flavonoids (Shinoda’s Test) …………………………………………………….. 32
3.5.6 Test for saponins (Frothing Test) …………………………………………………………. 33
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3.5.7 Test for tannins …………………………………………………………………………………… 33
3.5.8 Test for terpenoids and steroids (Liebermann-Burchard Test) …………………….. 33
3.6 Acute Toxicity Studies …………………………………………………………………………. 34
3.7 Sub-acute Toxicity Studies……………………………………………………………………. 34
3.7.1 Haematological study…………………………………………………………………………… 35
3.7.2 Biochemistry study ……………………………………………………………………………… 35
3.7.3 Histopathological study………………………………………………………………………… 36
3.8 Neuro-behavioural Studies ……………………………………………………………………. 37
3.8.1 Diazepam-induced sleep in mice ……………………………………………………………. 37
3.8.2 Ketamine-induced sleep in mice…………………………………………………………….. 37
3.8.3 Hole board test for exploratory behaviour in mice …………………………………….. 38
3.8.4 Beam walk assay in mice for motor coordination ……………………………………… 39
3.8.5 Elevated plus-maze test in mice for anxiolytic behaviour ………………………….. 40
3.8.6 Staircase test in mice for anxiolytic behaviour ………………………………………… 41
3.8.7 Open field test in mice for locomotor activity …………………………………………. 41
3.8.8 Haloperidol-induced catalepsy in mice ………………………………………………….. 42
3.8.9 Apomorphine-induced climbing behaviour in mice …………………………………. 43
3.8.10 Tail suspension test in mice for antidepressant screening …………………………. 43
3.8.11 Pentylenetetrazole-induced convulsion test in mice ………………………………… 44
3.8.12 Strychnine-induced convulsion test in mice ……………………………………………. 44
3.8.13 Maximal electroshock-induced convulsion test in chicks ………………………….. 45
3.8.14 Picrotoxin-induced convulsion test in mice ……………………………………………. 45
3.9 Statistical Analysis ……………………………………………………………………………… 46
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CHAPTER FOUR ……………………………………………………………………………………….. 47
4.0 RESULTS…………………………………………………………………………………………. 47
4.1 Percentage Yield of Plant Material ………………………………………………………….. 47
4.2 Phytochemical Constituents of Ethanol Extract and Fractions of Tapinanthus
globiferus……………………………………………………………………………………………. 47
4.3 Toxicity Studies on Ethanol Extract and Fractions of T. globiferus ………………. 50
4.3.1 Acute toxicity studies …………………………………………………………………………… 50
4.3.2 Sub-acute toxicity studies …………………………………………………………………….. 50
4.3.2.1 Effect of ethanol extract of T. globiferus on body weight of rats following
twenty eight days daily oral treatment…………………………………………50
4.3.2.2 Effect of ethanol extract of T. globiferus on haematological indices in rats
Following twenty eight days daily oral administration……………………..….51
4.3.2.3 Effect of ethanol extract of T. globiferus on serum biochemical parameters in
rats following twenty eight days daily oral administration…..…….………..….51
4.3.2.4 Effect of ethanol extract of T. globiferus on functional and structural integrity of
rat liver following twenty eight days oral daily administration………….…….57
4.3.2.5 Effect of ethanol extract of T. globiferus on functional and structural integrity of
rat kidney following twenty eight days oral daily treatment…………………..57
4.3.2.6 Effect of ethanol extract of T. globiferus on functional and structural integrity of
rat spleen following twenty eight days oral daily administration………………57
4.4 Neuro-behavioural Studies …………………………………………………………………….. 61
4.4.1 Effect of ethanol extract and fractions of T. globiferus on diazepam-induced
sleep in mice…. ………………………………………………………………………………….. 61
4.4.2 Effect of ethanol extract and fractions of T. globiferus on exploratory behaviour
in mice using hole-board test………………………………………………………………… 69
4.4.3 Effect of ethanol extract and fractions of T. globiferus on motor coordination
using beam walking assay in mice…………………………………………………………. 74
4.4.4 Effect of ethanol extract of T. globiferus on elevated plus-maze test in mice …. 79
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4.4.5 Effect of ethanol extract of T. globiferus on staircase test in mice ……………….. 79
4.4.6 Effect of ethanol extract of T. globiferus on open field test in mice ……………… 79
4.4.7 Effect of ethanol extract of T. globiferus on haloperidol-induced catalepsy in
mice ………………………………………………………………………………………………… 83
4.4.8 Effect of ethanol extract of T. globiferus on apomorphine-induced climbing
behaviour in mice……………… …………………………………………………………… 83
4.4.9 Effect of ethanol extract of T. globiferus on tail suspension test in mice ……….. 83
4.4.10 Effect of ethanol extract of T. globiferus on pentylenetetrazole-induced
convulsion test in mice ………………………………………………………………………… 87
4.4.11 Effect of ethanol extract of T. globiferus on strychnine-induced convulsion test
in mice …………………………………………………………………………………………….. 87
4.4.12 Effect of ethanol extract of T. globiferus on maximal electroshock-induced
convulsion in chicks …………………………………………………………………………… 87
4.4.13 Effect of ethanol extract of T. globiferus on picrotoxin-induced convulsion test
in mice ……………………………………………………………………………………………… 88
4.4.14 Effect of bicuculline on activities of diazepam and ethylacetate fraction of T.
globiferus on hole-board test in mice……………………………………………………. 88
4.4.15 Effect of ethylacetate fraction of T. globiferus on ketamine-induced sleep in
mice ……………………………………………………………………………………………….. 88
CHAPTER FIVE …………………………………………………………………………………………. 95
5.0 DISCUSSION ……………………………………………………………………………………… 95
CHAPTER SIX………………………………………………………………………………………….. 109
6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS …………………… 109
6.1 Summary …………………………………………………………………………………………… 109
6.2 Conclusion…………………………………………………………………………………………. 111
6.3 Recommendations ………………………………………………………………………………. 111
6.4 Contributions to Knowledge …………………………………………………………………. 112
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REFERENCES……………………………………………………………………………………………. 113
APPENDICES ……………………………………………………………………………………………. 129
CHAPTER ONE
1.0 INTRODUCTION Neuro-pharmacological agents induce changes in the function of cells of the nervous system. However, nervous system is the most important physiological part that differentiate human from other species. Disorders of nervous system function may lead to malfunction of other systems, which are the major concern to human society, and a field in which pharmacological intervention plays a key role. Drugs acting on the central nervous system (CNS) were among the first to be discovered by primitive humans and are still the most widely used group of pharmacological agents (Nicoll, 2007). Plant products or extract may produce similar pharmacological activity with orthodox medicine on CNS. However, substances that can be able to act on CNS may selectively relieve pain, depression, anxiety, insomnia and other neuropsychiatric and neurological disorders (Pandy et al., 2012). Herbalists use leaves, flowers, stem and root barks of plants to prevent, relieve and treat illnesses and also to improve one’s sense of wellbeing, but the mechanisms by which herbal medicine acts in the CNS have not been elucidated. However, herbal medicines are believed to be more potent, safe and affordable.
1.1 Statement of the Research Problem
Most people in the world have experienced at least one neuropsychiatric or neurological problem during their lifespan (WHO, 2017a). However, several new drugs for the treatment of neurological and neuropsychiatric disorders have been put into clinical practice, but up to one third of psychiatric patients remain resistant to optimum drug treatment (Kwan and Brodie, 2000) and only few individuals can afford to buy the
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drugs (Nisar et al., 2011). Insomnia is a significant public health problem which affects large segment of the population at one point or another in life (Morin and Jarrin, 2013). Some of the drugs used in insomnia such as diazepam, midazolam and flurazepam are drugs of tolerance, dependence and abuse potential (Charles and Robert, 1997). Depression is a serious mood disorder which affects most population worldwide (Nisar et al., 2011). Nigeria was ranked the most depressed in Africa in which 3.9% of the population suffered from depression and 2.7% of the population suffered from anxiety disorder (WHO, 2017b). However, depression and anxiety disorders cost the global economy one trillion US dollar each year (WHO, 2016). Despite the improvements and other developments in the treatment of schizophrenia and other neuropsychiatric disorders, schizophrenia still the most devastated neuropsychiatric disorder that affect men and women in the world (Ramesh and Reddy, 2014). Nigeria with a population of about 170 million has about 1.7 million people suffering from schizophrenia (Adegbaju, 2014) and nearly 80% of them live in low and middle income countries (WHO, 2017a).
Herbalists use plant remedies in the management of neuropsychiatric and neurological disorders (Andrews, 1982). WHO and UNO have acknowledged the fact that, about 80% of world rural population relies on herbal medicine for the management of psychiatric and other neurological problems but about 40% of the problems have remained unresolved and 60% of plants used lack scientific merit (WHO, 2013). However, the problems associated with herbal medicine include, lack of scientific
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proof, unpredictable dosage, intangible practices of traditional medicine and possibly improper diagnosis of ailment among others (Sofowora, 1993).
1.2 Justification of the Study
Most of the people in rural areas use plants as their source of medicine. Plants might form a virgin field for research work, but focus is yet to receive the attention of researchers (Sofowora, 1993). Among these plants are Tapinanthus globiferus that grows on Vitellaria paradoxa tree which has great medicinal importance and is used in traditional medicine for the management of neuropsychiatric and neurological disorders. Tapinanthus globiferus is used in traditional medicine of Northern Nigeria for the treatment of insomnia, epilepsy, anxiety and headache (personal communication) but its effectiveness needs to be investigated scientifically. Neuropharmacological study of ethanol extract and fractions of T. globiferus in rats, mice and chicks may provide a justification for the traditional uses and also may provide an important source for the development of better and safer drugs for the treatment of neuropsychiatric disorders (e.g. depression, schizophrenia, insomnia) or neurological (e.g. epilepsy, alzheimers, stroke, parkinsonism).
1.3 Theoretical Framework
The screening process of medicinal agents usually starts with index of acute toxicity studies (LD50). The result of the LD50 determination enabled the conduct of specific
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behaviour tests to evaluate the neuropharmacological properties of the extract and fractions of Tapinanthus globiferus.
1.3.1 Toxicity study
Toxicity study is conducted first to investigate the acute and sub-acute toxicities of ethanol extract of T. globiferus. The index of acute toxicity (LD50) of the plant is determined using Lorke (1983) method. This method can be used for every route of administration and it uses fewer animals to obtain adequate information on the acute toxicity. However, the result of acute toxicity study enabled the selection of range of doses used in conducting specific behavioural tests. Sub-acute toxicity is conducted to further observe the toxicity or safety of the extract. The method of Hodge and Sterner (1943) was adopted for sub-acute toxicity study. The observation and analysis of haematological, biochemical and histopathological components or toxicity index of the extract were carried out using acceptable techniques.
1.3.2 Diazepam-induced sleep test
Diazepam-induced sleep test is used to illustrate central nervous system (CNS) properties of drugs. The pharmacological tests of sedative, hypnotics, tranquilizers, neuroleptics as well as antidepressants are based on the potentiation of sleeping time induced by other sedative agents (Vogel, 2008). However, analeptics and stimulants shorten sleeping time. In this study, diazepam-induced sleep in mice is used as described by Rakotonirina et al. (2001). Diazepam acts at the level of the limbic, thalamic and hypothalamic regions of the CNS through potentiation of gamma amino butyric acid (GABA). GABA is known to be an important inhibitory neurotransmitter in
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the brain. GABA interacts with GABA-receptors (GABAA, GABAB and GABAC-receptor). GABAA-receptor controls the opening of chloride channel for the entering of chloride anions resulting in the neuronal hyperpolarisation (Mirshafa et al., 2013).
1.3.3 Ketamine-induced sleep test
Ketamine-induced sleep is used to evaluate antidepressant-like effects (Razmjou et al., 2016). Ketamine is a widely used anaesthetic that exerts its depressant effect by reducing neuronal excitation via N-methyl-D-aspartate (NMDA) receptor as a non-competitive antagonist and mediating sympathetic responses. Besides the direct action of ketamine on NMDA receptor, it also affects dopaminergic, noradrenergic, cholinergic and serotonergic neurotransmission (Manocha et al., 2001).
1.3.4 Hole-board test
This method is employed to evaluate certain components of behavior in mice such as curiosity or exploration (Crawley, 1985). It has been accepted as an experimental animal model for the evaluation of psychosis, sedation and anxiety condition (Crawley, 1985; Goodman et al., 2006). Poking the nose into a hole is a typical behavior of mice and indicating a certain degree of curiosity. An agent that decreases this parameter reveals a more sedative property (Mandal et al., 2001). Hence, anxiolytics have been shown to increase the number of head dips (Takeda et al., 1998). Benzodiazepines tend to suppress nose-poking at relatively low doses (Vogel, 2008).
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1.3.5 Mouse beam walking assay
The method of beam walking assay is used to evaluate the activity of drugs that interfere with motor coordination and is more sensitive than the rota-rod in determining benzodiazepines-induced motor coordination deficits (Stanley et al., 2005). However, the method has increased ability in predicting doses of extract and diazepam that cause sedation (Vogel, 2008) and increase foot slips in mice reveals sedative activity of the extract or drugs.
1.3.6 Elevated plus-maze test
Elevated plus-maze is regarded as a reliable measure of anxiolytic activity. It has been proposed for selective identification of anxiolytic and anxiogenic drugs. Anxiolytic drugs increase the number of entries into open arms and the time spent in the open arms whereas anxiogenic agents do the opposite (Vogel, 2008). Benzodiazepines and valproate decrease motor activity and increase open arm exploration time. The method was validated by Lister (1987) and it was adopted for this research.
1.3.7 Elevated staircase test
This model was first described by Thiebot et al. (1973); it evaluates anxiolytic activity of chemical agents. The modified model of Simiand et al. (1984) was used in this study. The model can determine the effects of psychotropic agents on rearing and climbing separately and can detect behavioural effects of agents active at the GABAA receptor (Simiand et al., 1984). Mouse staircase test is an efficient paradigm for studying agents active at the GABAA receptor complex (Weizman et al., 2001). Step-climbing is
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claimed to reflect exploratory or locomotor activity, while rearing behavior is an index of anxiety state (Vogel, 2008).
1.3.8 Open field test
The Open Field Test provides simultaneous measures of locomotion, exploration and anxiety. The model is usually used as measures of locomotor activity, but also measures of exploration and anxiety. A high frequency of these behaviours indicates increased locomotion and exploration and lower level of anxiety. The number of central square entries and the time spent in the central square are measures of exploratory behaviour and anxiety. A high duration of these behaviours indicates high exploratory behaviour and low anxiety levels.
1.3.9 Haloperidol-induced catalepsy test
Haloperidol-induced catalepsy test is used to measure the reduced ability to initiate movement and the failure to achieve correct posture in mice, which is assessed by means of a standard bar test. Mice are considered to be cataleptic if they remain on posture position for 30 seconds or more (Salam, 2011). Catalepsy is a sign of extrapyramidal effect of drugs that inhibit dopaminergic transmission or increase histamine release in brain (Pathan et al., 2009). Haloperidol is a dopamine D2-receptor antagonist (Pandy et al., 2012). Haloperidol induces catalepsy by blocking the postsynaptic striatal D1- and D2-receptors (Klemm, 1993). The phenomenon of catalepsy can be used for measuring the efficacy and the potential side effects of neuroleptics (Vogel, 2008). Neuroleptics which have an inhibitory action on the nigrostriatal dopamine system induce catalepsy (Costall and Naylor, 1974) while
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neuroleptics with little or no nigrostriatal blockade produce relatively little or no cataleptic behaviour (Honma and Fukushima, 1976).
1.3.10 Apomorphine-induced climb test
Apomorphine administration produced a peculiar climbing behaviour in mice which is characterized initially by rearing and then spontaneous climbing activity (Costall et al. 1978). The ability of a drug to antagonize apomorphine-induced stereotyped climbing behaviour in the mouse has been correlated with neuroleptic potential (Protais et al., 1976, Costall et al., 1978). Reduction or suppression of climbing behaviour in mice after apomorphine administration can be used for neuroleptic drugs evaluation (Vogel, 2008).
1.3.11 Tail suspension test
This test is sensitive to antidepressant drugs such as tricyclic, monoamine oxidase inhibitors (MAOI), serotonin specific reuptake inhibitors and atypical antidepressant (Porsolt et al., 1977; Steru et al., 1985). Tail suspension was found to be an easy method to evaluate potential antidepressant compounds (Vogel, 2008). Stressed mouse would become immobile after an initial period of struggling to escape. This immobility signifies behaviour that resembles a state of mental depression which can be reduced by antidepressant drugs (Porsolt et al., 1977; Steru et al., 1985). However, several mouse strains are essentially resistant to tail-suspension induced immobility (Vogel, 2008).
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1.3.12 Pentylenetetrazole-induced convulsion test
The test is used primarily for screening antiepileptic drugs. However, it has been shown that most anxiolytic agents are able to prevent or antagonize metrazol-induced convulsions. Stimulant, antidepressant, neuroleptic and some antiepileptic drugs do not show metrazol-antagonism at tolerable doses (Lippa et al., 1979). Tonic extension of the hind limbs is an indication of epilepsy in mice but absence of clonic spasms in the 30 minutes observation period indicated the compounds have ability to abolish the effect of pentylenetetrazole (PTZ) on seizure threshold (Swinyard et al., 1989). PTZ-induced seizures are further characterized into different patterns that are one or more generalized myoclonic body twitches, generalized body seizure with loss of righting reflex, loss of righting reflex with tonic forelimb extension and loss of the righting reflex with tonic forelimb and hind limb extensions (Loscher et al., 1991). Pentylenetrazole exert its convulsive effect by inhibiting the activity of gamma amino butyric acid (GABA) at GABAA receptor. Sodium valproate is effective in the treatment of myoclonic and absence seizures (McNamara, 2006) and active against both pentylenetrazole and maximal electroshock seizures.
1.3.13 Strychnine-induced convulsion test
The test is used to screen agents that are active in the central nervous system (CNS) as well as chemical that induced seizure. The convulsing action of strychnine is due to interference with postsynaptic inhibition mediated by glycine (Vogel, 2008). Glycine is an important inhibitory transmitter to motor neurons and inter-neurons in the spinal cord, and strychnine acts as a selective, competitive antagonist to block the inhibitory effects of glycine at all glycine receptors (Rajendra et al., 1997). Strychnine-sensitive
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postsynaptic inhibition in higher centres of the CNS is also mediated by glycine. Compounds which reverse the action of strychnine have been shown to have anxiolytic properties (Costa et al., 1975) as well as effective anti-epileptics (Larson, 1969). Phenobarbitone, valproate and diazepam are effective against strychnine-induced convulsion.
1.3.14 Electroshock-induced convulsion test
The electrically induced seizures are useful models for detecting compounds with anticonvulsant activity. Administration of maximal electroshock in chicks through corneal electrodes or ear-clip electrodes in mice is used to deliver the stimuli. Tonic hind-limb extensions are evoked by electric stimuli which are suppressed by anti-epileptics such as phenytoin, carbamazepine and phenobarbitone (Vogel, 2008). The behavioural and electrographic seizures generated in this model are consistent with the human disorder (Swinyard et al., 1989).
1.3.15 Picrotoxin-induced seizure
Picrotoxin-induced convulsions are used to further evaluate CNS-active compounds (Vogel, 2008) and a model of recurring focal epilepsy screening (Freitas et al., 2006). Picrotoxin is regarded as a GABAA receptor antagonist that produces seizures by blocking the chloride ion channels linked to GABAA receptor complex (Nicoll, 2007). Drug effective against picrotoxin-induced convulsion includes phenobarbitone and diazepam.
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1.3.16 Phytochemical screening
The relationship between phytochemical constituents and pharmacological activity has been shown by many authors. Previous studies revealed that, chemical constituents such as alkaloids, flavonoids, saponins and tannins obtained from plants produce pharmacological activity (Kporou et al., 2010; Bassey, 2012; Ajao and Akindele, 2013).
1.3.17 Statistical analysis
Analysis of data generated and significant differences using One-way Analysis of Variance (ANOVA) followed by Dunnett’s post-hoc test gives portable presentation of results as tables and figures.
1.4 Aim and Objectives
1.4.1 Aim
The aim of this research is to study some neuropharmacological effects of the ethanol extract and fractions of Tapinanthus globiferus that grows on Vitellaria paradoxa (host) in rats, mice and chicks with a view to find out the most active fraction.
1.4.2 Objectives
The objectives of the study are:
i. To identify the phytochemical constituents present in ethanol extract and fractions of T. globiferus using standard methods.
ii. To determine safety (through acute and sub-acute toxicity studies) of the ethanol extract of T. globiferus in mice and rats using oral and intraperitoneal routes of administration.
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iii. To evaluate some neuropharmacological properties such as sedative, anxiolytic, antipsychotic, antidepressant and anticonvulsant of ethanol extract of T. globiferus in mice and chicks.
iv. To determine fractions of ethanol extract of T. globiferus and identify the most active among them.
v. To investigate the possible pathway and mechanisms of the most active fraction.
1.5 Statement of Research Hypothesis
The ethanol extract and fractions of T. globiferus contain active phytochemical constituents which have some neuropharmacological activities.
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