i 

 

UNIVERSITY OF GHANA 
 

COLLEGE OF HEALTH SCIENCES 
 

 

 

 

 

ANTICONVULSANT EFFECT OF ETHANOLIC LEAF EXTRACT OF 

EHRETIA CYMOSA THONN (BORAGINACEAE) 

IN MURINE MODELS 

 

 

 
 

BY 

 

 

LARTEY RICHARD NELSON 
 

(10552293) 

 

 

A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES                  

IN PARTIAL FULFILLMENT OF THE AWARD OF MASTER OF 

PHILOSOPHY DEGREE IN PHARMACOLOGY DEPARTMENT                        

OF PHARMACOLOGY AND TOXICOLOGY 

 

JULY, 2018  

 

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UNIVERSITY OF GHANA 

 

COLLEGE OF HEALTH SCIENCES 

SCHOOL OF PHARMACY 

 
 

ANTICONVULSANT EFFECT OF ETHANOLIC LEAF EXTRACT 

OF EHRETIA CYMOSA THONN (FAM: BORAGINACEAE) 

IN MURINE MODELS 

 

 

 

BY 

 

 

 

LARTEY RICHARD NELSON 
 

(10552293) 
 

 

 

A THESIS SUBMITTEDTOTHE SCHOOL OF GRADUATE STUDIES                     

IN PARTIAL FULFILLMENT OF THE AWARD OF MASTER OF 

PHILOSOPHY DEGREE IN PHARMACOLOGY DEPARTMENT                         

OF PHARMACOLOGY AND TOXICOLOGY 
 

 

JULY, 2018  

 

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ABSTRACT 

Purpose: Ehretia cymosa is used locally in Ghana to treat epilepsy. To validate this anecdotal 

information with scientific data, the anticonvulsant effect of the ethanolic extract of the leaves of 

Ehretia cymosa was studied in murine models.  

Materials and Methods: The potential anticonvulsant activity of an ethanol extract of Ehretia 

cymosa (ECE) (30, 100, and 300 mg kg
-1

) was tested employing the acute pentylenetetrazole 

(PTZ)-, PTZ-induced kindling, picrotoxin-induced seizures and maximal electroshock (MES) in 

mice. The extract‘s effect on motor co-ordination and nociception was also tested using the rota-

rod and the hot plate tests respectively. Acute and sub-chronic toxicity tests were also done to 

ascertain how safe the extract is in rodents. 

Results: This study showed that, the ethanolic extract of ECE possesses anticonvulsant effects in 

the various seizure threshold models used, except in the maximal electroshock seizure model. 

The latencies to the first myoclonic jerks were increased by ECE while both the duration and 

frequencies of seizures reduced significantly. There was however no effect on motor 

coordination even when highest dose of 300 mg kg
-1

 was used. No mortalities were recorded in 

the animals used during the period of this study. The ECE also did not have any significant effect 

on any of the serum biochemical parameters after the study. 

Conclusions: The ethanolic extract of the leaves of the Ehretia cymosa was found to possess 

anticonvulsant properties, and possibly acts through the GABAergic transmission pathway but 

has no muscle relaxant properties. The findings from this study, therefore, give scientific 

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credence to the traditional use of Ehretia cymosa to manage epilepsy. The 10-week oral 

administration of the extract of Ehretia cymosa under the prevailing laboratory conditions was 

found to be relatively safe in male Sprague–Dawley rats. 

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DEDICATION 

I dedicate this work to my wonderful parents, Mr. John Attuah Lartey (of blessed 

memory), and Mrs. Attuah Gloria Lartey. You have been a blessing in my life.  

 

  

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ACKNOWLEDGMENT 

I wish to express my utmost gratitude to God for His continuous blessings and mercies 

throughout this study.  

My profound appreciation goes to my mentor and supervisor Dr. Patrick Amoateng. Thank 

you for your support, expert direction, guidance, and great life lessons that you have taught 

me. For the mentorship I have received, I must say you have inspired me to aim far and 

beyond. I also say a hearty appreciation to Dr Kennedy Edem Kukuia, my co-supervisor 

for his mentorship and guidance as well. I say a very big thank you to all senior members, 

all lecturers, and technical support staff of the Department of Pharmacology and 

Toxicology, for the immense encouragement and emotional support. 

To the Head of Department of Animal Experimentation, Noguchi Memorial Institute for 

Medical Research (NMIMR), Dr. Samuel Adjei, I am glad to have been allowed to work in 

such an august department. I have learned some technical skills that would be with me 

forever. I thank all the staff and technical workers, especially Mr. Ahedor Believe, for his 

selfless assistance and help during my work there.  I am also grateful to the technical staff 

of the Pharmacognosy department of the School of Pharmacy of KNUST especially Mr. 

Osafo Asare for his help in the collection of my plant sample. 

I am also very thankful to my work colleague Ms. Authentia Sokpe who pushed me to 

complete this work when I felt like giving up. May the Almighty God bless you 

abundantly. 

To my parents, Mr. and Mrs. Lartey, may God continue to bless you. Thank you for your 

financial support and advice. A big thanks to my family, especially my sister Lily who has 

been a mother and friend to me throughout my work. To all my colleagues, thank you all for 

your encouragement and support. God bless you. To friends, family, strangers, and anyone 

who has contributed to the successful completion of this work, I say may the Lord richly 

bless you.

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TABLE OF CONTENTS 

DECLARATION ................................................................................................................... ii 

ABSTRACT ........................................................................................................................... ii 

DEDICATION .......................................................................................................................iv 

ACKNOWLEDGMENT......................................................................................................... v 

LIST OF FIGURES ...............................................................................................................ix 

LIST OF TABLES .................................................................................................................xi 

ABBREVIATIONS ............................................................................................................. xii 

CHAPTER 1 INTRODUCTION ......................................................................................... 1 

1.0 GENERAL INTRODUCTION ..................................................................................... 1 

1.1 PROBLEM STATEMENT ........................................................................................... 4 

CHAPTER 2 LITERATURE REVIEW .............................................................................. 7 
1.2 EPILEPTIC SEIZURES ................................................................................................ 7 

1.3 SEIZURES AND EPILEPSIES .................................................................................... 8 

1.4 MECHANISMS OF EPILEPTOGENESIS ................................................................ 11 

1.4.1 VOLTAGE-GATED SODIUM CHANNEL SUBTYPES AND EPILEPSY ...... 13 

1.4.2 GABAA RECEPTOR SUBUNITS AND EPILEPSY .......................................... 14 

1.5 DISEASE MODIFICATION AND ANTIEPILEPTOGENESIS ............................... 16 

1.6 SIDE EFFECTS OF ANTIEPILEPTIC DRUGS ....................................................... 19 

1.7 ANIMAL MODELS OF EPILEPSY .......................................................................... 23 

1.7.1 CHEMOCONVULSANT MODELS ................................................................... 24 

1.7.2 ELECTRICAL STIMULATION ......................................................................... 25 

1.8 EHRETIA CYMOSA THONN BORAGINACEAE..................................................... 26 

1.8.1 PLANT DESCRIPTION ...................................................................................... 26 

2.6.2 TRADITIONAL USES ............................................................................................ 27 

CHAPTER 3 MATERIALS AND METHODS ................................................................... 30 

1.9 DRUGS AND CHEMICALS ..................................................................................... 30 

1.10 PLANT COLLECTION ............................................................................................ 30 

1.11 PREPARATION OF EXTRACT .............................................................................. 30 

1.12 QUALITATIVE PHYTOCHEMICAL ANALYSIS ON CRUDE EXTRACT ....... 30 

1.12.1 TEST FOR TANNINS ....................................................................................... 31 

1.12.2 TEST FOR REDUCING SUGARS ................................................................... 31 

1.12.3 TEST FOR SAPONINS ..................................................................................... 31 

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1.12.4 TEST FOR ALKALOIDS .................................................................................. 31 

1.12.5 TEST FOR FLAVONOIDS ............................................................................... 32 

1.12.6 TEST FOR STEROLS........................................................................................ 32 

1.12.7 TEST FOR TERPENOIDS ................................................................................ 32 

1.13 HANDLING OF ANIMALS .................................................................................... 32 

1.14 IRWIN OBSERVATION TEST ............................................................................... 33 

1.15 HOT PLATE TEST ................................................................................................... 34 

1.16 SKELETAL MUSCLE EFFECTS OF ECE ............................................................. 34 

1.16.1 ROTAROD TEST .............................................................................................. 34 

1.17 ANTICONVULSANT EFFECT OF EHRETIA CYMOSA ....................................... 35 

1.17.1 PENTYLENETETRAZOLE (PTZ)-INDUCED SEIZURES ............................ 35 

1.17.2 PICROTOXIN-INDUCED SEIZURES ............................................................. 36 

1.17.3 PENTYLENETETRAZOLE-INDUCED KINDLING ...................................... 36 

1.17.4 MAXIMAL ELECTROSHOCK TEST.............................................................. 37 

1.18 TOXICITY STUDIES .............................................................................................. 38 

1.18.1 ANIMAL GROUPINGS AND EXTRACT ADMINISTRATION.................... 38 

1.18.2 HISTOPATHOLOGY AND BIOCHEMICAL ANALYSIS ............................. 38 

CHAPTER 4 RESULTS.................................................................................................... 40 
1.19 PHYTOCHEMISTRY SCREENING ....................................................................... 40 

1.20 IRWIN TEST ............................................................................................................ 40 

1.21 EFFECT OF EXTRACT ON NEUROMUSCULAR ACTIVITY ........................... 41 

1.21.1 ROTAROD ......................................................................................................... 41 

1.21.2 HOT PLATE TEST ............................................................................................ 42 

1.22 ANTICONVULSANT THRESHOLD TESTS ......................................................... 43 

1.22.1 PTZ-INDUCED SEIZURE TEST ...................................................................... 43 

1.22.2 PICROTOXIN INDUCED SEIZURE TEST ..................................................... 46 

1.22.3 MAXIMAL ELECTROSHOCK TEST.............................................................. 48 

1.22.4 PTZ KINDLING TEST ...................................................................................... 50 

1.23 TOXICITY STUDIES .............................................................................................. 53 

1.23.1 BIOCHEMICAL PARAMETERS ..................................................................... 53 

1.23.2 HISTOLOGICAL EXAMINATION OF ISOLATED TISSUES ...................... 56 

CHAPTER 5 DISCUSSION ............................................................................................. 60 

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CHAPTER 6 CONCLUSIOS AND RECOMMENDATIONS ........................................ 68 
1.24 CONCLUSION ......................................................................................................... 68 

1.25 RECOMMENDATIONS .......................................................................................... 68 

REFERENCES ..................................................................................................................... 70 

 

 

  

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LIST OF FIGURES 

Figure 2.1: The ILAE 2017 classification of types of seizures (Robert S. Fisher, 2017) 10 

Figure 2.2: A figure showing the process of epileptogenesis (Alyu & Dikmen, 2017) 12 

Figure 2.3: A figure showing the mechanism of anti-epileptic drugs (Shih, Tatum, & A 

Rudzinski, 2013) 19 

Figure 2.4: A clinical side effect of phenytoin manifesting as gingival hypertrophy 

(Khorsand & Saaveh, 2007) 23 

Figure 2.5:  An overview of models for specific types of epilepsy or epileptic seizures 

(Löscher, 2011) 26 

Figure 2.6 An image of Ehretia cymosa Thonn (Boraginaceae) http://tropical.theferns.info29 

Figure 4.1: Effect of Ehretia cymosa extract ECE 30, 100, and 300 mg kg
-1

 on 

neuromuscular coordination in mice in the rotarod test.  Data are Mean ± SEM 

(n=5) 42 

Figure 4.2: Effect of ECE (30, 100, 300 mg kg
-1

) on latency [time for which mouse 

remained on the hot plate (55˚C ± 0.1˚C) without licking or flicking of the hind 

limb or jumping in seconds. 43 

Figure 4.3: Effect of ECE 30, 100, 300 mg kg
-1

 and phenobarbitone 3, 10, 30 mg kg
-1

 on 

the latency to first myoclonic jerks, the total frequency of the seizures, and total 

frequency of the seizures induced by PTZ. Each column represents the mean ± 

SEM (n = 5). *P < 0.05, **P < 0.01compared with vehicle-treated group (one-

way analysis of variance followed by Newman–Keuls post hoc test). 45 

Figure 4.4:Effect of ECE 30, 100, 300 mg kg-1 and phenobarbitone 3, 10, 30 mg kg-1 on 

the latencies to first myoclonic jerks, the total frequency of the seizures, and 

total frequency of the seizures induced by picrotoxin. Each column represents 

the mean ± SEM (n = 5). *P < 0.05, **P < 0.01compared with vehicle-treated 

group (one-way analysis of variance followed by Newman–Keuls post hoc 

test). 47 

Figure 4.5: Effect of ECE 30, 100 and 300 mg kg
-1

 and carbamazepine 3, 10 and 30 mg kg
-

1
 on the duration of First HLE induced by Maximal Electroshock Test (MEST).49 

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Figure 4.6: Effect of ECE 30, 100 and 300 mg kg
-1

 and carbamazepine 3, 10 and 30 mg kg
-

1
 on the total duration of Hind Limb Extensions (HLE) induced by Maximal 

Electroshock Test (MEST). 50 

Figure 4.7:The dose-response effects of ECE 30, 100, and 300 mg kg
-1

 (A and B) and 

phenobarbitone 3, 10, and 30 mg kg
-1

 (C and D) on the PTZ-kindled mice. 52 

Figure 4.8: The Photomicrographs of Brain isolated from rats after 72-day continuous 

administration of (A) Vehicle, (B) ECE (30, 100 and 300 mg kg
-1

), (H&E 

staining, 40×). 56 

Figure 4.9:The Photomicrographs of livers isolated from rats after 72-day continuous 

administration of (A) Vehicle, (B) 30 (C) 100 and (D) 300 mg kg
-1

) (H&E 

staining, 40×). 57 

Figure 4.10:Photomicrographs of kidneys isolated from rats after 72-day continuous 

administration of (A) Vehicle, (B) ECE 30, (C) 100 and (D) 300 mg kg
-1

 

showing normal renal tubule and glomeruli (H&E staining, 40×). 58 

Figure 4.11:Photomicrographs of hearts isolated from rats after 72-day continuous 

administration of (A) Vehicle, (B) 30, (C) 100, (D) ECE 300 mg kg
-1

) showing 

normal myocardial fibers with characteristic central nuclei and branching 

arrangement as indicated by the arrows. 59 

 

 

  

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LIST OF TABLES 

Table 4.1: Primary phytochemical screening of ECE 40 

Table 4.2: IRWIN TEST RESULTS 41 

Table 4.3: A table showing the biochemical analysis of a single administration of ECE (30, 

100 and 300 mg kg
-1

) after a 72-day study period in Sprague-Dawley male rats54 

 

 

 

 

 

 

 

 

 

 

 

 

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ABBREVIATIONS 

AED Antiepileptic drugs  

CBZ Carbamazepine 

CNS Central Nervous System 

DZP Diazepam 

ECE Ehretia cymosa Extract 

ED50 Effective dose for 50% 

EEG Electroencephalography 

GABA γ- aminobutyric acid 

GABA-T GABA Transaminase 

GAT1, GABA transporter 1 

HLTE Hind limb Tonic extensions                                    

ILAE International League Against Epilepsy 

LD50 Lethal dose which causes the death of 50%  

MEST Maximal Electroshock Threshold test 

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MRI Magnetic resonance imaging 

NMDA N-Methyl-D-aspartate 

PIC Picrotoxin 

PTZ Pentylenetetrazole 

SE Status epilepticus 

SV2A Synaptic vesicle protein 2A 

TLE Temporal lobe epilepsy 

VPA Valproic acid 

 

 

 

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CHAPTER 1 INTRODUCTION                                                                              

1.0 GENERAL INTRODUCTION 

Epilepsy is a disease condition that dates back to the very existence of mankind with 

references in ancient writings including the Bible (WHO, 2019; King James Version, 

Matthew 17:14-15). In the pre-Hippocratic era, the condition was however thought of as a 

retribution for offences committed. Thus, its treatment was sought in the temples (York, 

2005). This can be seen where one man pleaded with the Messiah to cure his son who usually 

exhibited the symptoms of epilepsy (King James Version, Matthew 17:14-15). By the man‘s 

description, it can be inferred that he believed the disease had a spiritual cause and as such 

needed spiritual healing. The Messiah went ahead and healed the said boy who was described 

by the father as a lunatic; sorely vexed and often times fell into the fire (King James Version, 

Matthew 17:17 -18). However, Hippocrates, the father of medicine, disputed the mystical 

cause of epilepsy. To this, he said that the only reason men thought epilepsy was divine was 

that there was no understanding of the disease (York, 2005) Modern physicians also agree 

with this view. It is therefore incumbent on the scientist to explore the scientific dimension to 

the disease epilepsy.  

The International League Against Epilepsy (ILAE) defines epilepsy as a disease of the central 

nervous system (CNS) which is also characterized by seizures. The seizures are transitory, 

unprovoked, sudden, and recurrent episodes of abnormal hypersynchronous neuronal 

discharge. In other words, it is a brain condition which is associated with the occurrence of 

more than one epileptic seizure. There is also an increased susceptibility to generate more 

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seizures in addition to psychological, cognitive, neurobiological and social disturbance (R S.  

Fisher et al., 2005). Epilepsies are classified into four groups namely: partial (focal) seizures, 

generalized seizures, combined generalized and focal, and the unclassified (unknown) 

seizures. Categorization of the condition is determined by the type of seizure. The partial type 

of seizures stem from networks linked to one hemisphere of the brain and this is usually 

accompanied by an underlying structural disease. On the other hand, generalized seizures 

originate from bilaterally distributed networks which lead to the concurrent start of extensive 

electrical discharge with no localizing characteristics referable to a single hemisphere 

(Wilkinson et al., 2017). The combined generalized and focal type are seizures which have 

characteristics of both focal and generalized types of seizures. The unknown epilepsy refers to 

one with little information on the clinical condition. The unknown type of epilepsy may be re-

classified as focal, generalised or combined generalized and focal types of epilepsy if later 

enough clinical information becomes available (Chang, Leung, Ho, & Yung, 2017). 

Though epilepsy is one of the oldest documented conditions dating as far back as the year 

4000 BC (WHO, 2019), successful cure has not been achieved. Thus, an estimated sixty-five 

(65) million still live with the condition and out of this number, the active epilepsy cases with 

recurrent seizures requiring regular treatment at any given time is estimated to be around 4 to 

10 per 1000 people (in developed countries) and 7 to 14  in developing countries. In addition 

to this, the number of people that are estimated to be diagnosed with epilepsy each year from 

these countries is said to be around 2.4 million relative to 30 to 50 per every 100 000 people 

(Vogel, Franz, Jochen, & Dieter, 2015).  

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To compound this worrying statistics, there is fear, discrimination, and social stigma attached 

to the disease and this can even be used as grounds to annul or prohibit marriages in some 

countries (Vogel et al., 2015). Aside from these, the treatment and management of epilepsy 

with various anticonvulsant drugs have a lot of side effects. Some of these unwanted effects 

such as aberrant gum growth (gum hyperplasia), hardening of the face, and excessive hair 

growth or hirsutism affect the physical appearance or the beauty of the patients. There are also 

recorded cases of cognitive impairment, hypersensitivity reactions, behavioural changes, and 

peripheral neuropathy. There are also issues like Dupuytren's contractures, gastrointestinal 

problems, hyponatremia, leukopenia, weight gain, and endocrine changes reported as side 

effects of taking some of these antiepileptic drugs. There is also the issue of tolerance and 

dependence associated with the use of antiepileptic drugs (Löscher & Schmidt, 2006). These 

effects are experienced depending on the type of medication being used to manage the 

condition (Vogel et al., 2015). The numerous adverse effects associated with anti-seizure 

drugs have negatively impacted the clinical effectiveness of the medications. 

Also, the socio-economical differences between the developed and developing world impact 

clinical outcomes largely. Epilepsy places a heavy economic burden on both patients and their 

countries. Consequently, it is pertinent to assess the treatment and management of epilepsy so 

as not to only to increase quality of life, but also consider the financial or economic burden of 

the prescribed treatments for this disease condition (Liu, Liu, & Meng, 2013). For these 

reasons, there should be a pragmatic research into novel plant-based medicines that are readily 

available and safer to use for the control of convulsions. 

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Ehretia cymosa, known as Okusua in the Twi, is a shrub or small tree with drooping branches 

and can be found in the savannah, forest thickets, and cultivated lands (Mshana, 2000). The 

infusion of the leaves is used as a bath to manage fevers and convulsions. The roots are used 

to cure tetanus and dysentery, whereas the leaves are employed in the treatment of headaches, 

constipation in children, and fractures (Mshana, 2000). The hydroethanolic extract of the 

leaves has been reported to have antidiabetic, antimicrobial, and antioxidant properties (H. M.  

Burkill, 1985). Despite its folkloric use for convulsions, there is no scientific data confirming 

its anti-convulsant or antiepileptic properties. There is therefore the need to investigate its 

antiepileptic and related neuropharmacological properties as well as its safety. 

 

1.1 PROBLEM STATEMENT 

An estimated 65 million people suffer from epilepsy worldwide and it is the most 

predominant cause of chronic neurological diseases. Even more frightening is that, about 30 

percent of the people who live with epilepsy are unresponsive to clinical management 

(Łukawski et al., 2018). Unfortunately, a third of these people in this category live in Africa, 

and this accounts for nearly 16% of people afflicted globally. Furthermore, the problem is 

compounded by a wide treatment gap that can even be as high as 100 percent in some suburbs 

in the African region  (Mugumbate & Zimba, 2018). People living with epilepsy have, 

throughout history, been associated with the divine, demonic, and supernatural attributes 

(Devinsky & Lai, 2008). The mystical associations with the disease coupled with poor 

scientific understanding of the disease have resulted in serious social implications for patients, 

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especially for women due to the stigma attached to the condition (Kpobi, Swartz, & 

Keikelame, 2018). In addition to these, antiepileptic drugs (AEDs) have side effects and poor 

tolerability which greatly affects patients‘ compliance. Patients on antiepileptic drugs go 

through a strained social and emotional wellbeing due to physical changes like acne, alopecia, 

hirsutism and weight gain which in turn lead to a low quality of life (Chen et al., 2015). These 

side effects lead to increased drug non-adherence which in turn lead to reduced effectiveness 

of AED regimen leading to higher rates of road traffic accidents, injuries and fractures 

(Faught, Duh, Weiner, Guerin, & Cunnington, 2008). The consequence of this AED non-

adherence is increased mortality rates and adverse clinical outcomes. This can lead to patients 

being incorrectly classified as having a refractory epilepsy (O‘Rourke & O‘Brien, 2017). In 

addition to these, non-adherent adults with epilepsy (AWE) are known to run the risk of 

developing convulsive status epilepticus (Skinner et al., 2010). The most serious outcome of 

AED non-adherence is the increased risk of sudden unexplained death in AWE (Lathers, 

Koehler, Wecht, & Schraeder, 2011). It is therefore imperative to identify the barriers to AED 

and remove those hurdles to enable a better management of this disease condition. 

Aside from the cosmetic side effects, some of the currently available anti-seizure drugs like 

felbamate, phenytoin and valproic acid have been implicated in liver toxicity (Vidaurre, 

Gedela, & Yarosz, 2017). With regards to primary health care, an estimated 80 percent of the 

worldwide population depend on herbal medicines (Sam, 2019) hence herbal medicines can 

be a good source of antiepileptic drugs that are easily accessible, able to prevent 

epileptogenesis and with few side effects. 

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AIM 

This research seeks to investigate the anti-epileptic effect of the ethanolic extract of Ehretia 

cymosa. 

OBJECTIVES 

1. To determine the effects of Ehretia cymosa extract on motor co-ordination and 

nociception. 

2. Investigate the effect of the extract of Ehretia cymosa extract on convulsive threshold 

tests using the acute and chronic models of convulsion.  

3. Perform acute, sub-acute, and sub-chronic toxicity tests. 

 

  

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CHAPTER 2 LITERATURE REVIEW 

1.2 EPILEPTIC SEIZURES 

Epileptic seizures are defined as transient occurrences of signs and/or symptoms as a result of 

excessive, aberrant or hypersynchronous neuronal brain activity (Falco-Walter, Scheffer, & 

Fisher, 2018). Approximately 65 million people in the world are affected by this condition 

(Ngugi, Bottomley, Kleinschmidt, Sander, & Newton, 2010) and the prevalence of this 

condition is around 6.4 cases per 1000 people according to data available (Fiest et al., 2017). 

Even though it is one of the most predominant and disabling neurologic diseases, there is still 

not a complete comprehension of the pathophysiology and proven treatment regimen to 

combat the condition (Stafstrom & Carmant, 2015). Case management of the condition is 

usually by the use of antiepileptic drugs. However, there are about one-third of such patients 

who do not attain seizure control with the available drugs (Devinsky et al., 2018).  

One of the most effective ways to help eliminate these seizures is through surgeries but only a 

small percentage of these patients are qualified for such surgeries (Wiebe, Blume, Girvin, & 

Eliasziw, 2001). A lot of the patients whose conditions have become drug-resistant are not 

eligible and the options available for such patients are the use of neurostimulation devices, 

adherence to the good dietary regimen, or partaking in clinical trials for new anti-seizure 

drugs (ASDs) (Wiebe et al., 2001) patients may relapse. An estimated 10 percent of the 

people in the world will experience a seizure at least once in their life (Hauser & Beghi, 

2008). Epilepsy can lead to death directly through events like head injuries from falls, burns, 

drowning, vehicular accidents, sudden death that occurs in epilepsy and status epilepticus. 

There can be indirect causes like suicide, pneumonia, or through the adverse drug reactions of 

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anti-seizure drugs like obesity and cardiovascular reactions (Devinsky, Spruill, Thurman, & 

Friedman, 2016). 

 

1.3 SEIZURES AND EPILEPSIES 

Seizures are defined as paroxysmal change of neurologic function due to excessive, 

hypersynchronous neuronal discharge in the brain. It is usually unprovoked and recurrent and 

the cause can be attributed to an underlying brain disorder (Shorvon, Andermann, & Guerrini, 

2011). When the cause of seizures is through reversible condition like fever or hypoglycemia, 

it is not considered epilepsy (Shorvon et al., 2011). Epilepsy syndrome on the other hand 

refers to a pattern of clinical manifestations that consistently occur together with seizures as a 

primary manifestation. Some of the features of an epilepsy syndrome include similar age of 

onset electroencephalogram (EEG) findings, etiology, inheritance pattern and response to 

particular antiepileptic drugs  (Stafstrom & Carmant, 2015). 

Seizures are classified into 3 groups: generalized type seizures, focal type seizures which was 

formerly called partial, and epileptic spasms. Focal seizures stem from neuronal networks 

specific to one area of the cerebral hemisphere. Generalized seizures originate in bilateral 

distributed neuronal networks. A focal seizure can later become a generalized seizure. For an 

appropriate diagnosis to be made, a physician must combine a comprehensive history in 

addition to results from an electroencephalogram (EEG) and response to anti-seizure 

medications (Stafstrom & Carmant, 2015). 

Generalized seizures are classified as absence, generalized tonic-clonic, myoclonic and atonic 

seizures. Absence seizures which were formerly named petit mal is a type of seizure in which 

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the patient stares into space for a few seconds and also becomes unresponsive to external 

verbal stimuli but blinks the eye or nods the head occasionally. Generalized tonic-clonic 

(GTC) seizures which were previously named grand mal comprise of bilateral symmetric 

convulsive movements in addition to the body becoming stiff. After this, jerking of all the 

limbs take place. There is no loss of consciousness during such an episode. In myoclonic 

seizures, there is an abrupt, short very fast movement that is not associated with any loss of 

consciousness. The muscle contractions associated with this kind of seizure are usually brief 

and involuntary and may impair one or several other muscles. For this reason, myoclonic 

seizures can either be classified focal type seizures or generalized type seizures. On the other 

hand, atonic seizures are characterized by body tone loss, which lead to a head drop or fall 

(Stafstrom & Carmant, 2015). 

When the brain has an imbalance between excitation (E) and inhibition (I), then, seizures can 

be said to be occurring (Rho, Sankar, & Stafstrom, 2010). The excitation/inhibition variation 

can be as a result of a change at various stages of the function of the brain from subcellular to 

genetic signaling cascades to general neuronal circuits. This imbalance can either have a 

genetic cause or can be acquired. The genetic pathological causes of this disease can be 

ubiquitous in nature. Again, cerebral injuries that are acquired are able to alter the circuit 

function, for example, change in the structure of the hippocampal circuitry due to persistent 

head injury or febrile seizures (Berkovic, 2015). Synaptic excitatory role matures before 

inhibitory function in a young brain leading to increased excitation and seizure generation. 

More so, due to the propensity of the neurotransmitter GABA to cause excitation instead of 

inhibition early in life (Ben-Ari, 2002; Pitkänen, Lukasiuk, Dudek, & Staley, 2015) it can be 

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partly deduced the brains of the very young people are easily prone to having seizures (G. L. 

Holmes & Ben-Ari, 1998).  

Focal seizures can be classified based on a level of awareness. The term awareness is used as 

a substitute for consciousness because consciousness comprises many aspects that can be 

difficult to fully evaluate. The level of consciousness and awareness are therefore two of 

many possible features of a seizure, but they play an important role with regards to seizures 

(Robert S. Fisher, 2017). Awareness is basically, the knowledge of the environment and of 

self. The ability to determine awareness is a practical tool utilized to assess if consciousness 

level is compromised or not. It is therefore the awareness during a seizure, and not awareness 

of whether or not a seizure has occurred (Robert S. Fisher, 2017).  

 

Figure 2.1: The ILAE 2017 classification of types of seizures (Robert S. Fisher, 2017) 

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1.4 MECHANISMS OF EPILEPTOGENESIS 

Epileptogenesis is the process through which a formerly functional normal brain changes such 

that it becomes biased towards the production of the aberrant electrical events which in turn 

result in chronic seizures. In other words, epileptogenesis is the process whereby there is 

growth and extension of tissue which is able to generate seizures that are spontaneous in 

nature, which ultimately result in an epileptic condition (Łukawski et al., 2018). Various 

mechanisms have been proposed to be the mode of initiation of the disease. The ability to 

prevent chronic epileptic disorder by using the necessary procedures would be touted as the 

most ideal goal in the clinical management of an epileptic condition but this, has not been 

successful to this day. Due to this, various clinical trials aimed at preventing chronic epilepsy 

have often yielded bleak, discouraging results (Di Maio, 2014). There are various 

antiepileptogenic drugs like eslicarbazepine and diazepam but the connection between 

inhibition of epileptogenesis and neuroprotection is not apparent (Łukawski et al., 2018). The 

ability to prevent epileptogenesis after brain injury is currently a challenge that is still unmet 

(Pitkänen & Lukasiuk, 2011). Research in animal models is currently the best source of 

information in the research into epileptogenesis. As a result, a lot of animal models are being 

researched into, to better understand the pharmacological and pathophysiological causes of 

the disease (Russo & Citraro, 2018). The process of epileptogenesis happens in three phases. 

It first of all starts with, causative injury or event; after which there is a latent period during 

which the alterations that were caused by the said injury also change the formerly normal 

brain into an epileptic brain. Lastly, the third phase follows and this is the point where the 

epilepsy becomes fully established and chronic. The best point in the epileptogenesis whereby 

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various medical management can be utilized to stop the ultimate occurrence of a chronic 

epilepsy is at the latent period during which an acquired epileptogenesis is believed to 

coalesce (Goldberg & Coulter, 2013).  

 

Figure 2.2: A figure showing the process of epileptogenesis (Alyu & Dikmen, 2017) 

 

2.3.1 THE EMERGING TARGETS AND NOVEL STRATEGIES FOR FUTURE 

TREATMENT 

The discovery of novel antiepileptic drugs which would help to better control patients‘ 

seizures may require a fundamental change in the strategies of drug development. Instead of 

randomly screening compound libraries, rational drug design could rather be based on 

scientific understanding of the pathophysiological mechanisms of ictogenesis of epilepsies. 

After this is achieved, new compounds can be made to target specific established epileptic 

brain defects, which can lead to the direct control of causative pathways without causing harm 

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to the normal neuronal function. Aside this, there could also be the use of more innovative 

ways that mimic one or more characteristics of pharmacoresistance in humans and integrate 

them into epileptogenesis models thereby aiding in characterizing possible disease-modifying 

therapies (Brodie et al., 2011; Walker, White, & Sander, 2002).  

 

1.4.1 VOLTAGE-GATED SODIUM CHANNEL SUBTYPES AND EPILEPSY 

Voltage-gated sodium channels (VGSCs) are necessary for the production and propagation of 

coordinated action potentials in the whole nervous system. For this reason, they are believed 

to have a vital part in alleviation and occurrence of epilepsy. There have been several genetic 

studies on people living with epilepsy and these have helped characterized several mutations 

of more than 700 among the genes that code for voltage-gated sodium channels validating the 

role they play in pathogenesis. Again, a lot of anti-seizure medications are known to act on 

VGSCs to reduce seizures (Czuczwar & Patsalos, 2001). VGSCs are imperatively necessary 

for moderating neuronal excitability and subsequently, network activity. They can be found 

all over the central neuron compartments and their population is increased at the nodes of 

Ranvier and axon initial segments (AIS). In myelinated neurons, the collection of voltage-

gated sodium channels at the nodes of Ranvier is important, the increase of the speed of action 

potential transmission by saltatory conduction (Conti, Hille, Neumcke, Nonner, & Stämpfli, 

1976; Kaplan et al., 2001). Research into the genetics of epileptic patients has found a vast 

number of mutations out of the genes known to encode for these ion channels. A significant 

amount of these mutations are in VGSC genes with most discovered in SCN1A, and fewer in 

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SCN2A, SCN8A, and SCN1B (Herlenius et al., 2007; Mulley et al., 2005; Striano et al., 2006; 

Wallace et al., 1998). VGSCs are very important mediators of intrinsic neuronal and muscle 

excitability. Any aberrant activity is key to the pathophysiology of seizures, and a lot of 

widely used antiepileptic medications, like carbamazepine and phenytoin are known inhibitors 

of VGSC activity. Many of the known antiepileptic medications block the VGSC but the 

drugs currently available are mostly non-selective and as such do not utilize the discrete 

attributes of the brain channels. VGSC are thought to be found at various regions\on the 

neuronal membranes and populations. For this reason, other compounds can be produced that 

can target only sodium channel conformations and its subtypes studied to be over-expressed in 

epileptic tissue, in so doing, sparing normal neuronal function. Lacosamide is one example of 

a blocker of sodium channel with distinct attributes with regards to selectivity for a specific 

biophysical state or subunit but unfortunately have a lot of side effects (Brodie et al., 2011; 

Errington, Stöhr, Heers, & Lees, 2008; Remy et al., 2003).  

 

1.4.2 GABAA RECEPTOR SUBUNITS AND EPILEPSY 

This is a ligand-gated ion channel receptor that has a centrally placed anion pore around 

which five separate protein subunits are arranged. The anion pore is permeable to bicarbonate 

and chloride ions. There are 19 GABAA receptor subunits discovered so far and it has been 

shown that the composition of the subunit can affect the pharmacology and physiology of the 

receptor (Garcia, Kolesky, & Jenkins, 2010). In the brain, the receptors of GABAA possess an 

age-adapted role. In its early development, they moderate excitatory activities which result in 

the trigger of signalling processes that are calcium-sensitive necessary for brain 

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differentiation. They however transmit inhibitory signals during later phases of their 

development (ILAE, 2018). These inhibitory effects have been significantly utilized in the 

management of diseases where there is the need of silencing neuronal activity. The function 

and physiology in the brain of epileptic patient or a status epilepticus subject change 

according to literature available (ILAE, 2018) and as such may have an influence on the 

propensity to seizures. For this reason, many available anticonvulsants have GABAA receptors 

as their primary or secondary targets (Chang et al., 2017). Some of these drugs act by 

increasing GABAA receptor activity through direct interaction with the receptor for example 

barbiturates, carbamazepine and benzodiazepines. Others indirectly increase the available 

GABA such as valproate and vigabatrin (Czuczwar & Patsalos, 2001; Granger et al., 1995; P. 

Kumar, A. Jhanjee, & M. Bhatia, 2010; Meldrum & Rogawski, 2007; Quilichini, Chiron, Ben‐

Ari, & Gozlan, 2006). Furthermore, anticonvulsants such as topiramate, zonisamide, 

acetazolamide can inhibit carbonic anhydrase through the reduction of the depolarizing 

outcome of GABAA receptors (Davis, Penschuck, Fritschy, & McCarthy, 2000; Dodgson, 

Shank, & Maryanoff, 2000; Nishimori et al., 2005; Reiss & Oles, 1996). Barbiturates which 

are part of the examples of available GABAA receptor medications are relatively non-selective 

(Walker et al., 2002). The ability to produce drugs that are of subunit specificity could help 

avoid unwanted side effects which are related to non-selective modulation of GABAA. There 

are records for subunit-specific AED: like stiripentol which has been shown to preferentially 

activate α3-β3-γ2-containing receptors. Hence, the ability to have drugs that are GABAA 

selective and can specifically act on subunits studied to be upregulated in epileptic brain 

tissue, may demonstrate being efficacious against seizures with less unwanted drug reactions 

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(Brodie et al., 2011; Brooks-Kayal, Shumate, Jin, Rikhter, & Coulter, 1998; Johnston, 2005; 

Stell, Brickley, Tang, Farrant, & Mody, 2003). 

 

1.5 DISEASE MODIFICATION AND ANTIEPILEPTOGENESIS 

The recent antiepileptic medications generally manage ictogenesis, or the commencement of 

paroxysmal activity (Reiss & Oles, 1996). Due to these, various classes of AEDs act by 

suppressing excitability of neuronal activity by blocking sodium channels or increasing 

inhibitory GABAergic activity (Czuczwar & Patsalos, 2001; White, 1999). Both cognition 

and ictogenesis are moderated by excitability of neuronal activity. For this reason, using the 

normal methods of AEDs screening may be difficult to find drugs that are non-impairing. 

However, employing chronic animal models of epilepsy may be used to improve drug 

screening. Thus, by using kindled or genetically modified animals (Matagne & Klitgaard, 

1998) it may be possible to find novel AEDs that prevent the neuronal hypersynchronization 

which leads to an ictal event, without obstructing normal neuronal excitability (Margineanu & 

Klitgaard, 2000). When a normal brain undergoes multiphase events which end up producing 

alterations that enhance the development of spontaneous seizures, we term it as 

epileptogenesis. This can be as a result of brain damage caused by things like head trauma 

(Golarai, Greenwood, Feeney, & Connor, 2001), growth (Goldberg et al., 2013), stroke 

(Margineanu & Klitgaard, 2000), infection (Golarai et al., 2001), or status epilepticus. 

Usually, brain damage does not immediately lead to seizures and this is a latency period that 

may last for weeks or even years. In these latency periods, the normal seizure thresholds are 

lowered due to continuous alterations to the brain which also subsequently lead to 

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spontaneous seizures (Golarai et al., 2001; Santhakumar, Ratzliff, Jeng, Toth, & Soltesz, 

2001). Immediately seizures start occurring, the epileptic disease state worsens. Each seizure 

then has the propensity to produce extra neuronal changes that may additionally reduce 

seizure threshold (Goddard, McIntyre, & Leech, 1969). Most anticonvulsants act by either 

reducing seizure frequency or duration of seizure by reducing neuronal excitation, but ideal 

antiepileptogenic agents would either block the first epileptogenic development or alter the 

epileptic disease state after the onset of seizure (McNamara, 1984). The ideal screening for 

antiepileptogenic drug action would be studies that have the ability to reduce changes in 

cellular network and molecular properties that happen during the process of epileptogenesis. 

The animal model that is mostly employed for assessing the anti-convulsive characteristics of 

AEDs is the focal kindling. In this model, initial exposure to repeated sub-convulsive stimulus 

ultimately evokes seizures (Albright & Burnham, 1980; Goddard et al., 1969; McNamara, 

1984). Initially, stimuli in the kindling process only elicit short-duration after-discharges as a 

result of a synchronous neuronal discharge near the stimulation. Further kindling stimulation 

produces prolonged after-discharges involving larger brain compartments with the quick 

involvement of the limbic system. With longer and repeated stimuli, behavioural seizures 

accompany the after-discharges and become more complex. It takes a number of days or 

weeks before there can be an increased sensitivity to a formerly sub-convulsant stimuli which 

subsequently reach a peak during which the kindling stimuli result in seizures and after-

discharges (McIntosh & Levy, 2021). Since kindled seizures are inducible and have durations 

and behavioural and electrographic characteristics, kindling can be employed as a screening 

method for anti-seizure effects that are characterized easily. Potential AEDs can be 

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administered to animals that have been fully kindled and the consequence on electrographic 

and behavioural seizures measured (Teskey, 2020). Kindling is progressive in nature and for 

this reason, repeated seizures over time can cause a reduction in seizure thresholds. This may 

be similar to the process of human epileptogenesis. The long interval between trauma and 

seizure manifestation in posttraumatic epilepsy may be a reflection of a slow kindling process 

(Skinner et al., 2010). This idea is buttressed  by the generation of generalized seizures in a 

patient undergoing electrical stimulation of the thalamus (Lathers et al., 2011). There is data 

showing that the current antiepileptic drugs have antiseizure properties but not 

antiepileptogenic ones (Suchomelova et al., 2006). In other words, the symptoms can be 

managed but the ultimate development of epilepsy is not affected. Contrary to this, the 

priority of management of epilepsy is the discovery of therapeutic agents that can prevent or 

correct the neuropsychological and neurological deterioration accompanied with chronic 

seizures or, even stop epilepsy in individuals who are at risk (Brodie et al., 2011; Walker et 

al., 2002). FIG 2.3 illustrates some of the mechanisms of some the antiepileptic medications. 

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Figure 2.3: A figure showing the mechanism of anti-epileptic drugs (Shih, Tatum, & A Rudzinski, 2013) 

 

1.6 SIDE EFFECTS OF ANTIEPILEPTIC DRUGS 

The core aim of the management of people with epilepsy is to completely abolish or 

significantly reduce frequency of seizures, reduce the unwanted side-effects of drug therapy 

and greatly enhance medical and neuropsychiatric comorbidities (Pitkänen, 2010) Anti-

seizure drugs (ASDs) aim to subdue the development, severity and the propagation of 

epileptic seizures but these medications are to be taken up to 4 times a day for years, and 

sometimes for throughout one‘s lifetime (Faught et al., 2008; Ryvlin, Cucherat, & Rheims, 

2011). Drug noncompliance to ASDs treatment is not uncommon and this usually results in 

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the seizure relapse which in turn leads to increased mortality, injuries, and frequent hospital 

visits and admissions with added costs (Faught et al., 2008). Unlike other disorders, such as 

hypertension in which an 80% compliance is accompanied with good disease management, a 

single missed dose of an ASD can result in a fatal seizure (Devinsky et al., 2018). 

Antiepileptic drugs (AEDs) can effectively manage patients with epilepsy but there is usually 

treatment failure and poor drug compliance due to the numerous side-effects experienced by 

taking these AEDs. These side-effects can lead to about 25% of the patients discontinuing 

treatment (Kwan & Brodie, 2000; Perucca, Carter, Vahle, & Gilliam, 2009; Uijl et al., 2009) 

and this lead to a significant reduction in their quality of life (Luoni et al., 2011; Uijl et al., 

2009). Some of the frequently reported adverse drug reactions of AEDs are fatigue, tremors, 

gastrointestinal symptoms and memory problems. While others too experience dizziness, 

osteoporosis, drowsiness, depression, changes to their weight, and nausea (Carpay, 

Aldenkamp, & Van Donselaar, 2005). The impact of these adverse effects may further 

necessitate the need for specialist care which brings additional financial burden to the patient 

(de Kinderen et al., 2014). A lot of adverse drug effects are reported with AEDs but the most 

prevalent is CNS effects. When the regimen of an antiepileptic drug fails, it can lead to 

untoward effects which may be due to drug intolerance or to inadequate seizure control also 

due to drug inefficacy or a combination of both (Kwan & Brodie, 2000). The AEDs are either 

a first-generation or a second generation. Examples of the first-generation are valproate, 

phenobarbital, phenytoin, carbamazepine and primidone. The second-generation AEDs offer 

better response over first-generation drugs nonetheless, either of them can cause significant 

adverse effects (Cramer, Fisher, Ben‐Menachem, French, & Mattson, 1999). Poorly managed 

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or controlled seizures may lead to a combination of drug therapy which further leads to 

potential pharmacokinetic or pharmacodynamic interactions leading to greater side-effects 

than when monotherapy is used (Hirsch et al., 2004). There are situations where seizures are 

not adequately controlled or there can be an imbalance between the control of seizures and 

adverse effects. In such instances, there may have to be a discontinuation of the drug which 

leads the patient  being put on a new drug with their unique side effects (Lawal, Ogunwande, 

Salvador, Sanni, & Opoku, 2014). Some of the cognitive effects experienced by patients 

include loss of memory, diminished intelligence, attention and language skills. Even though 

most AEDs can cause cognitive impairment, phenobarbital and topiramate have been reported 

to have the most effects on cognitive abilities. Furthermore, the same phenobarbital has been 

shown to cause mental slowing which studies have shown that this diminished cognitive 

ability improves once the medication is stopped. Exposure of children to phenobarbital leads 

to intelligence scores less than those taking valproate (Lawal, Opoku, & Ogunwande, 2015). 

Anxiety and depression are common among epileptic patients (Blumer, Montouris, & 

Hermann, 1995). When these conditions exist before the diagnosis of epilepsy, the said 

conditions may worsen subtly with treatment thereby making it difficult to recognize (Ohemu 

et al., 2014).  

Carbamazepine has known side effects such as impaired balance, leucopenia, diplopia, blurred 

vision and drowsiness (Wilkinson et al., 2017). There is also been vertigo and loss of 

coordination, aplastic anemia and agranulocytosis have also been reported (Pellock, 1987). 

Patients on lamotrigine can develop Stevens-Johnson syndrome or toxic epidermal necrolysis 

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especially those on valproic acid. Other unwanted effects include photosensitivity, diplopia, 

blurred vision, agitation, tremors, aplastic anemia and vomiting. There are reports indicating 

patients on levetiracetam to develop psychiatric side effects such as depression and agitation 

and other side effects like blood dyscrasias, dyspepsia, drowsiness, diplopia whiles the 

teratogenic effects of sodium valproate has also been reported (Alyu & Dikmen, 2017) and 

can also cause tremors, thrombocytopenia, nausea, pancreatitis, hair loss, edema, and ataxia 

(McIntosh & Levy, 2021). Phenytoin has been removed as a first-line drug because of its 

numerous toxicity issues which include gum hyperplasia (fig. 2.4), nystagmus, diplopia, 

tremor, dysarthria, and ataxia (Teskey, 2020). It can also lead to a decrease in intellect, 

depression, acne, and gum hypertrophy. Patients taking this drug can also develop coarse 

facial features polyneuropathy and blood dyscrasias (Wilkinson et al., 2017). Aside from 

these, lacosamide has been found to cause dizziness and ataxia and may even increase the risk 

of suicidal thoughts in patients taking these medications for any indication (P. Kumar, A. 

Jhanjee, & M. S. Bhatia, 2010). 

 

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Figure 2.4: A clinical side effect of phenytoin manifesting as gingival hypertrophy (Khorsand & 

Saaveh, 2007) 

 

1.7 ANIMAL MODELS OF EPILEPSY 

Temporal lobe epilepsy (TLE) is mostly accompanied by variable and recurrent seizures and 

this make this neurological disorder very difficult to understand. More often than not, seizures 

in TLE are often resistant to anti-epileptic medications. There is the option of resection of the 

epileptogenic tissue surgically but it is expensive and at times unfeasible. For these reasons, 

animal models that mimic the neuropathological, behavioural and electroencephalographic 

attributes of epilepsy have been developed and researched into in the past decades to better 

understand the pathophysiology of TLE and to also help develop better AEDs (York, 2005). 

Understanding epileptogenesis and seizure generation as well as their complex mechanisms in 

temporal lobe epilepsy and other forms of epilepsy is not completely possible in human 

clinical studies. Because of this, there has to be the use of appropriate animal models which 

will aim to mimic the symptoms of focal epilepsy with an epileptogenic injury or insult. The 

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research into seizure mechanisms can give a better knowledge about the general functions and 

consciousness of the brain. Thus animal models can help with the advancement of the 

research into both neurophysiology and epilepsy (Löscher & Schmidt, 1988).  FIG. 2.5 shows 

some of the various animal models employed in the discovery of antiepileptic medications. 

 

1.7.1 CHEMOCONVULSANT MODELS 

Research into the chemoconvulsant pentylenetetrazole (PTZ) started in the later part of 1940s 

and continued through the early 1950s. During this period, properties of PTZ seizure threshold 

model were described by various publications, and this helped to make a comparison between 

the anticonvulsant attributes of antiepileptic medications (AEDs) in the PTZ and other animal 

models. This lead to the PTZ model being used in addition to other various animal models to 

discover most of the drugs currently utilized in epileptic seizure management (Yadeta, 2016). 

PTZ is believed to employ an antagonistic mechanism at the picrotoxinin-sensitive site at the 

GABAA receptor complex (Stegaroiu, 2016). Furthermore, (Bourgeois, 2014) has shown that 

PTZ and picrotoxin interact with distinct overlapping regions of the GABAA receptor.  

Chemoconvulsants are compounds that activate seizures and they can be employed in 

assessing various AEDs that act on different types of seizures. For example, compounds like 

N-methyl-D, L-aspartate and strychnine have been shown to trigger generalized tonic-clonic 

seizures, whereas PTZ generates non-convulsive myoclonic or absence seizures (Porter et al., 

1984). Through these models, drugs like trimethadione, ethosuximide, valproate, and other 

efficacious medications have been discovered (Liu et al., 2013). Administrations of several 

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doses of flurothyl or PTZ have also been employed to imitate recurrent generalized tonic-

clonic seizures in young rodents (Pitkänen, Buckmaster, Galanopoulou, & Moshé, 2017).  A 

single administration of PTZ can induce seizures and when it is given in sufficient amounts, 

status epilepticus can even be induced (Gregory L Holmes, Sarkisian, Ben‐Ari, & Chevassus‐

Au‐Louis, 1999; Pitkänen et al., 2017).
 

 

1.7.2 ELECTRICAL STIMULATION 

To better understand the mechanisms underlying epileptogenesis and ictogenesis, various 

animal models of seizures and epilepsy have contributed immensely to this effect. 

Furthermore, these models have played a significant part in the preclinical development and 

discovery of new antiepileptic drugs (AEDs) (Löscher, 2011). A lot of animal models of 

chronic brain disorders that are known to elucidate the pathophysiology of epilepsy and some 

of these chronic models of epilepsy are the kindling model of temporal lobe epilepsy (TLE), 

post-status models of TLE and genetic models of different types of epilepsy. Furthermore, 

―the results from these models can be used to assess the effects of antiepileptic medications. A 

differentiation of the study of chronic models with models of acute seizures in previously 

healthy animals is the maximal electroshock seizure test. This can aid in testing of drugs in 

chronic models of epilepsy to provide data which is more predictive of clinical efficacy and 

unwanted drug reactions. Given this, chronic models can be employed early in the 

development and discovery of drugs to help reduce false positives (Loscher, 2002).  

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Figure 2.5:  An overview of models for specific types of epilepsy or epileptic seizures (Löscher, 2011) 

 

1.8 EHRETIA CYMOSA THONN BORAGINACEAE 

1.8.1 PLANT DESCRIPTION 

Ehretia cymose (see fig. 2.6) is a small tree that belongs to the Boraginaceae or borage family. 

It is found over a wide area of habitat throughout central, eastern, and western Africa, 

including Ghana, Côte d'Ivoire, Benin, Cameroon, Ethiopia and Kenya (Wikipedia 

Contributors, 2018, June 19). The plant is referred to as Okosua by the Akan-Twi speaking 

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https://en.wikipedia.org/wiki/Boraginaceae
https://en.wikipedia.org/wiki/Borage
https://en.wikipedia.org/wiki/Africa
https://en.wikipedia.org/wiki/C%C3%B4te_d%27Ivoire
https://en.wikipedia.org/wiki/Benin
https://en.wikipedia.org/wiki/Cameroon
https://en.wikipedia.org/wiki/Ethiopia
https://en.wikipedia.org/wiki/Kenya


 

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tribes from Ghana (Humphrey Morrison Burkill, 1985). The leaves of the plant are used 

among the people of south western Nigeria for the treatment of measles (Oladunmoye & 

Kehinde, 2011). The leaves have also been reported to be used for the management of 

epilepsy, spasms and convulsions, as a laxative, febrifuge, analgesic and for paralysis (H. M.  

Burkill, 1985). It is a small to medium deciduous tree or shrub that can grow up to 20 to 25 m 

tall. It has low and crooked branches that can grow up to up to 30 cm in diameter. The surface 

of the bark is grey to pale brown, with protruding lenticels. The leaves are spirally arranged 

and are simple and entire without any stipules present. The petiole is 1 to 3.5 cm long with an 

elliptical blade that is slightly grooved (Lemmens, 2009).  

It grows up to 7 m tall in the western parts but can grow as tall as 20 to 25 m as recorded in 

some parts of Southern Nigeria and Guinea. The fruit is normally black in colour, ovoid to 

globose drupe which is about 2-6 mm long (Harris & Harris, 2002).  It has greyish brown 

wood with alternate lighter and darker bands (Conti et al., 1976). 

The main types of compounds identified in its volatile oils are fatty acids, sesquiterpenes, 

monoterpenes, , alcohols, phenylpropanoids and esters, (Jeruto, Mutai, Lukhoba, & Ouma, 

2011).  Its volatile oils have been demonstrated to have biological properties like cytotoxicity, 

insecticidal and antimicrobial activities (Lawal et al., 2014; Lawal et al., 2015). 

2.6.2 TRADITIONAL USES 

The leaf of Ehretia cymosa is used in Ghana to treat fractures and to also enhance bone 

modelling. The leaves of the plant are used for treating headaches and fevers and also used for 

its mild laxative effects (Dalziel, 1937; Lewis & Avioli, 1991). Chewing sticks are made out 

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of the branches for maintaining good gum and teeth hygiene (H. M.  Burkill, 1985; Lewis & 

Avioli, 1991). An infusion of the leaf is used as a wash to treat convulsions, muscle spasms 

and fever (Borokini & Omotayo, 2012). The sap of the leaf is reported to be a mild laxative 

and can also be used as a hemostatic. Decoctions from the leaves are used to treat muscle 

stiffness, fevers and toothache. The twigs of the leaves are used in combination with other 

parts of plants to treat gastric ulcers whiles the decoctions of the leaf are used to cure 

dysentery and tetanus. The roots and leaves have been reported to be used as aphrodisiacs 

(Jeruto et al., 2011). Decoctions of the roots and bark are used to manage menstrual problems 

while a decoction of the bark alone is externally used to cure skin conditions. The people from 

Maasai treat brucellosis with the roots which are crushed in water and used against stomach 

diseases. Ehretia cymosa also serves locally as an important feed for livestock and in 

agroforestry in Ethiopia, it is sometimes planted as ornamental trees (Lemmens, 2009). 

The tree has several uses, providing food, wood and medicines for the local population of 

South Western Nigeria (Mulley et al., 2005). There is reported use of the leaves for the 

management of viral conditions like measles among the Southern Western people of Nigeria 

(Mulley et al., 2005). The decoction of the bark is used to regularize menstrual cycle and also 

treat pneumonia (Bankole et al., 2016; Ohemu et al., 2014). There is also reported use of the 

plant for the management of mental problems, venereal diseases, dry cough,  malaria and 

tonsillitis,  (Jeruto, Tooa, Mwamburia, & Amuka, 2015). 

 

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Figure 2.6 An image of Ehretia cymosa Thonn (Boraginaceae) http://tropical.theferns.info 

 

 

  

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CHAPTER 3 MATERIALS AND METHODS 

1.9 DRUGS AND CHEMICALS 

This study employed the use of various chemicals including pentylenetetrazole, picrotoxin, 

phenobarbitone sodium, carbamazepine (Sigma-Aldrich Inc., St. Louis, MO, USA);  

 

1.10 PLANT COLLECTION 

The leaves of Ehretia cymosa were harvested from Kwahu Asakraka 

(6.62942N6˚37'45.9048'', -0.68647W0˚41'11.30253'') in the Eastern Region of Ghana, and 

authenticated at the Pharmacognosy Department, KNUST and a specimen with voucher 

number, KNUST/2020/MN1/L009, was stored at the herbarium of the Faculty of Pharmacy, 

KNUST.  

 

1.11 PREPARATION OF EXTRACT 

The leaves of the plant were powdered and serially extracted with 70% ethanol over 48 hours 

using a Soxhlet apparatus and concentrated under reduced pressure at 40-60 
o
C to a dark 

brown syrupy mass in a rotary evaporator. A water bath was used to dry resulting syrup mass 

and stored in a desiccator. The resulting extract was named Ehretia cymosa extract or ECE.  

 

1.12 QUALITATIVE PHYTOCHEMICAL ANALYSIS ON CRUDE EXTRACT 

Screening of the extract to detect the presence of phytochemical constituents such as 

glycosides, tannins, sterols, alkaloids, terpenoids, saponins and flavonoids was done using 

procedures described by Evans and Trease (2009).  

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1.12.1 TEST FOR TANNINS 

This was done by taking a quantity of 0.2 g of the extract which was dissolved in 25 mL of 

water and the resulting volume was topped up to 25 mL. A 1 mL aliquot of the extract was 

then added to 10 mL of water and 2 drops of 1% ferric chloride which produced the 

appearance of blue-black or green precipitate (ppt). 

 

1.12.2 TEST FOR REDUCING SUGARS 

Twenty milligrams of the extract were warmed with 5 mL dilute H2SO4 on a water bath for 2 

minutes and allowed to cool and filtered.  Four drops of 20 % NaOH were added to the 

resulting filtrate after which 1mL volume of Fehling`s A and B solutions were added to the 

filtrate. It was then warmed and a red-brown ppt was then observed. 

 

1.12.3 TEST FOR SAPONINS 

About 0.2 g of each extract was taken and shaken vigorously with about 10 mL of water for 

about one minute in a stoppered test tube and later the presence of a persistent froth was then 

observed. 

1.12.4 TEST FOR ALKALOIDS 

Ten milliliter of dilute HCl was boiled with 0.2 g of the extract for 5 minutes. The resulting 

supernatant was then filtered into a separate test tube. About 3 drops of Dragendorff`s reagent 

was added to one milliliter of the filtrate and shaken together the appearance an orange spot 

ppt, was then observed. 

 

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1.12.5 TEST FOR FLAVONOIDS 

A volume of 10 mL of 98% ethanol was added to 0.2 g of the extract. A small amount of zinc 

metal was then added followed by the drop-wise addition of concentrated HCl. The filtrate 

was then examined for the appearance of colors ranging from orange to red representing the 

presence of flavones, orange to crimson indicating flavonols, and crimson to magenta 

indicating presence of flavanones. 

 

1.12.6 TEST FOR STEROLS 

Two milliliter of chloroform was added to 0.2 g and filtered.  An amount of 0.2 g of the 

extract was added to 2 mL of chloroform and filtered. A volume of 2 mL of acetic anhydride 

was added to 1 mL of the filtrate after which few drops of concentrated H2SO4 was carefully 

added along the sides of the test tube. Violet to blue coloration which was produced indicated 

the presence of sterols. 

 

1.12.7 TEST FOR TERPENOIDS 

An amount of 0.2 g of the extract was added to 2 mL of chloroform in a test tube followed by 

the addition of 1 mL of concentrated H2SO4. A reddish-brown coloration at interface 

indicated the presence of terpenoids. 

 

1.13 HANDLING OF ANIMALS 

Sprague-Dawley rats (150-200 g) and Institute of Cancer Research (ICR) mice (20 – 25 g) 

were obtained from the Animal Department of Noguchi Memorial Institute for Medical 

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33 

 

Research, University of Ghana, Legon and kept in their Animal Experimentation labs. The 

animals were housed in cages with wood shavings as bedding and fed with standard mice 

chow, given water ad libitum. All animal procedures and techniques used in these studies 

were done under the Noguchi Institute of Animal Care and Use Committee (NIACUC) 

guidelines with a reference number 2017-05-2R. 

 

1.14 IRWIN OBSERVATION TEST 

Irwin observation test is usually employed to evaluate the effects of a new substance on the 

physiological and behavioural function of experimental animals. The results obtained from the 

this test gives an insight into potential toxicity and enables doses to be selected for specific 

neuropharmacological investigation (Vogel et al., 2015). In this study, mice were grouped 

(n=5) and made to acclimatize for 24 hours before the start of the experiment. The laboratory 

mice were given the extract and compared with a control group given distilled water 10 mL 

kg
-1

, p.o. The effect of the extract was evaluated at 6 doses; 10, 30, 100, 300, 1000, and 3000 

mg kg
-1

 administered p.o. immediately before the test. Observations were performed 15, 30, 

60, 120, and 180 minutes after administration of the extract and 24 and 48 hours later. 

Changes in physiological, behavioural and neurotoxicity symptoms,  pupil diameter and rectal 

temperature were observed and recorded according to a standardized observation grid derived 

from that of Irwin (Biney, Mante, Boakye-Gyasi, Kukuia, & Woode, 2014; Irwin, 1968).  

 

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1.15 HOT PLATE TEST 

The mice were weighed individually and made to acclimatize for 24 hours before the 

commencement of the experiment during which they were not given any food except for 

water. This is due to the fact that some food substances might have analgesic effect. The 

animals were put into 4 groups (n=5). These mice were pre-treated with the extract of Ehretia 

cymosa at 3 doses 30, 100 and 300 mg kg
-1

p.o., and control of normal saline 10 mL kg
-1

. After 

60 minutes of pre-treatment, the animals were placed on a hot plate and the latency time 

which is the time for which mouse remains on the hot plate at (55˚C ± 0.1˚C) without licking 

or flicking of the hind limb or jumping was then recorded. A cut-off time of 30 seconds was 

the time limit for all the animals and this was done to prevent damage to tissues. The readings 

were taken after 0, 30, 60, 90, and 120 min post-administration of the test drug.  The 

percentage analgesia was calculated using the following formula (Naveed, 2014).;  

            
              –                 

     –          –                 
 * 100 

1.16 SKELETAL MUSCLE EFFECTS OF ECE 

1.16.1 ROTAROD TEST 

Some test substances with anticonvulsant effect may impair motor coordination. This can be 

an important factor in the testing for anticonvulsant activity.  The experiment was done to 

ascertain the effect of ECE motor coordination. Mice were randomly put into four groups 

(n=5) and trained to remain on a rotating rod (Ugo-Basile model 7600, Comerio, VA, Italy), 

rotating at 25 revolutions per min for 180 s over three days. This is referred to as habituation. . 

The actual study took place 24 hours after the last habituation during which the animals were 

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given ECE 30, 100 and 300 mg kg
-1 

and distilled water 10 mL kg
-1 

p.o. after which they were 

put on the rotating rod and the latency of the mice to fall off the rotating rod within the 

stipulated time of 180 s was determined one-hour post-treatment. 

 

1.17 ANTICONVULSANT EFFECT OF EHRETIA CYMOSA 

1.17.1 PENTYLENETETRAZOLE (PTZ)-INDUCED SEIZURES 

Clonic-tonic seizures were induced in drug/vehicle pre-treated male Institute of Cancer 

Research (ICR) mice (20-30 g) by subcutaneous injection of 75 mg kg
-1

 pentylenetetrazole 

(PTZ) into the loose skin fold on the back of the neck of the mice. The animals were pre-

treated with ECE (30, 100, and 300 mg kg
-1

) or phenobarbitone sodium (3, 10, 30 mg kg
-1

) 

thirty minutes before the injection of PTZ. The control animals received 0.9 % saline solution 

(10 mL kg
-1

). After the PTZ injection, the animals were placed in a testing chamber (made of 

perspex of dimensions 15×15×15 cm). A mirror angled at 45˚ below the floor of the chamber 

allowed a complete view of the convulsive event of PTZ. The behavior of the animals was 

captured with a camcorder (EverioTM model GZ-MG 130U, JVC, Tokyo, Japan) placed 

directly opposite to the mirror. The video recordings were later analyzed by tracking 

parameters including latencies to myoclonic jerks and clonic-tonic seizures and the duration 

of clonic-tonic seizures using Behavior Tracker Version 4.0 for Windows. The ED50 (a 

measure of anticonvulsant potency) was calculated by plotting the percent seizure inhibition 

of the drug to the vehicle-treated group. The ability of the drug or extract to prevent the 

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seizures or prolong the latency of onset of the hind-limb tonic extensions was used as an 

indication for anticonvulsant activity‖.  

 

1.17.2 PICROTOXIN-INDUCED SEIZURES 

Clonic-tonic seizures were induced in drug or vehicle pre-treated male ICR mice (20-30 g) by 

an intraperitoneal injection of 3 mg kg
-1 

picrotoxin (PIC). The mice were pre-treated with 

ECE (30, 100, 300 mg kg
-1

), or phenobarbitone sodium (3, 10, and 30 mg kg
-1

) thirty minutes 

before the injection of PIC. The control animals received distilled water (10 mL kg
-1

, p.o). 

After the PIC injection, the animals were put in a testing chamber and a video recording of the 

event made. The video recordings were also later analyzed by tracking parameters including 

latency to myoclonic jerks and clonic-tonic seizures and the duration of clonic-tonic seizures 

using Behavior Tracker version 4.0 for Windows. The ED50 was calculated as indicated in the 

PTZ test. The mortality rate was also determined for each drug treatment group. The ability of 

a drug/extract to prevent the seizures or delay/prolong the latency of onset of the hind-limb 

tonic extensions was considered as an indication of anticonvulsant activity. 

 

1.17.3 PENTYLENETETRAZOLE-INDUCED KINDLING 

To kindle the mice to spontaneous seizures, 35 mg kg
-1 

of PTZ was injected i.p. every 48 h 

into saline-, ECE- or phenobarbitone-treated male ICR mice (20-30 g). After the PTZ 

injection, the rats were placed in a testing chamber (made of perspex of dimensions 15×15×15 

cm
3
). A mirror angled at 45˚ below the floor of the chamber allowed a complete view of the 

convulsive event of PTZ. The behavior of the animals was captured with a camcorder (JVC 

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37 

 

Hard Disk Camcorder, GZ-MG 130U) placed directly opposite to the mirror. Seizure 

intensities were classified according to the Racine score (Racine, 1972) as follows:  

Stage 0: no response 

Stage 1: ear and facial twitching  

Stage 2: convulsive waves throughout the body  

Stage 3: myoclonic jerks, rearing  

Stage 4: turning over onto one side  

Stage 5: turning over onto the back, generalized tonic-clonic seizures  

Also, the latency to the onset of myoclonic jerks was measured and analyzed. Each rat was 

considered fully kindled after showing stage 4 or 5 after two consecutive PTZ administrations. 

On day seven after kindling had been achieved, the rats were challenged with 35 mg kg
-1

 of 

PTZ and the entire event was also recorded. The ED50 was then calculated.  

 

1.17.4 MAXIMAL ELECTROSHOCK TEST 

The experiment employed the use of male ICR mice put into seven groups (n=10). Out of 

these, three of the groups were treated with the extract (10, 30, and 100 mg/kg, p.o.). Three 

other groups were treated with carbamazepine (10, 30, and 100 mg/kg, p.o.). The last group 

served as the control and they were administered distilled water (10 mL/kg, p.o.). The 

convulsions were then induced in the mice after 1 hour of drug pre-treatment. This was done 

by passing alternating electrical current (50 Hz, 60 mA, and 0.2 s) through the electrodes 

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attached to the earlobe of the mice. After this, the duration of tonic hind limb extension 

seizures was determined in each dose group (K. E. Kukuia et al., 2012). 

 

1.18 TOXICITY STUDIES 

1.18.1 ANIMAL GROUPINGS AND EXTRACT ADMINISTRATION 

The Sprague-Dawley rats were randomly put into four groups (n=10); namely; control group 

(normal saline 10 mL kg
-1

), ECE 30, 100, and ECE 300 mg kg
-1

 doses and made to 

acclimatize for 5 days. The animals were subsequently given a daily dose of the test 

substances for 72 days by oral gavage. This was done to mimic the traditional folkloric route 

of administration. All drug administrations were given at 8:00 GMT each day and the blood 

collection and post-mortem examinations were done before 15:00 GMT. 

 

1.18.2 HISTOPATHOLOGY AND BIOCHEMICAL ANALYSIS 

1.18.2.1 PREPARATION OF SPECIMEN 

All animals were sacrificed and their organs (liver, kidney, lungs, spleen, heart, and brain) 

were harvested and placed in labelled containers containing 10% neutral buffered formalin. 

The organs were transported to the pathology laboratory of School of Dentistry, Korle-bu for 

processing and examination.  

Blood was collected from the jugular vein into serum separating gel tubes (BD Vacutainer® 

blood collection Tube Product, USA) and ethylene diamine-tetracetate (EDTA) tubes 

(Mediplus vacutainer K3, Sunphoria Co. Ltd., Taiwan) for serum preparation and 

haematological analysis respectively. Values for the level liver transaminases (AST, ALT, 

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39 

 

ALP), total protein (TP), direct bilirubin (D. Bilirubin) indirect bilirubin (Indi. Bilirubin), total 

bilirubin (T. Bilirubin), triglycerides and albumin in the serum were measure. Also, the levels 

of kidney urea (Ur), creatinine (Cr), lipid profile (HDL, LDL, total cholesterol) were 

calculated. These were done using an automated clinical chemistry analyzer (ABX Pentra 

C200, Horiba Medical, USA). 

 

1.18.2.2 PROCESSING OF TISSUES 

Portions of the organs were selected into labelled tissue processing cassettes and processed 

into paraffin blocks. Each was passed through ascending grades of alcohol (70%, 80%, 90%, 

and absolute) and further two changes of absolute alcohol for dehydration, cleared in three 

changes of xylene, and finally infiltrated and embedded in paraffin wax. Five-micron sections 

were cut from each block, mounted on microscope slides and stained using the haematoxylin 

and eosin. 

  

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CHAPTER 4 RESULTS                              

1.19 PHYTOCHEMISTRY SCREENING 

Phytochemical analysis of the leaf extract of Ehretia cymosa (ECE) (Table 4.1) revealed the 

presence of these plant secondary metabolites: tannins, saponins, terpenes, triterpenes, phenolic 

compounds, flavanoids, volatile oils, steroids, and glycosides. Alkaloids were however absent. 

Table 4.1: Primary phytochemical screening of ECE 

PHYTOCHEMICAL REMARK 

Tannins + 

Glycosides + 

Alkaloids - 

Flavonoids + 

Terpenoids + 

Saponins + 

Volatile Oils 

Phenolic compounds 

+ 

+ 

(+) Present   (-) Absent 

1.20 IRWIN TEST 

No death was recorded after 48 h, thus LD50 was estimated to be greater than 3000 mg kg
-1

 

(Table 4.2). This test provided the dose ranges (30, 100, and 300 mg kg
-1

) for the actual work.  

 

 

 

 

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Table 4.2: IRWIN TEST RESULTS 

Dose (mg/kg) Mortality Latency (min) Observed drug effects 

10 0 15-120 Sedation, urination, defecation,  

30 0 15-120 Sedation, urination, defecation, 

analgesia 

100 0 15-180 Sedation, urination, defecation, 

analgesia 

300 0 15-180 Sedation, urination, defecation, 

analgesia 

1000 0 15-120 Sedation, urination, defecation, 

analgesia, altered respiration, tremors 

3000 0 15-120 Sedation, urination, defecation, 

analgesia, altered respiration 

Saline 0 15-30 urination, defecation 

 

1.21 EFFECT OF EXTRACT ON NEUROMUSCULAR ACTIVITY 

1.21.1 ROTAROD 

The extract, Ehretia cymosa (30, 100, 300 mg kg
-1

), did not have any significant effect (F3, 

16=0.3460 P=0.7925) on time spent on the rotating rod when compared to saline group (Figure 

4.1). 

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Figure 4.1: Effect of Ehretia cymosa extract ECE 30, 100, and 300 mg kg-1 on neuromuscular coordination in mice in 

the rotarod test.  Data are Mean ± SEM (n=5) 

 

1.21.2 HOT PLATE TEST 

The latency time (time for which mouse remained on the hot plate (55˚C ± 0.1˚C) without 

licking or flicking of the hind limb or jumping) was recorded. The ECE extract could not 

significantly increase the latency (F 3, 8 =2.907 P=0.1011) as compared to the control (Figure 

4.2). 

Vehicle 30 100 300
0

500

1000

1500

2000

                       ECE (mg/kg)

R
o

ta
ro

d
 A

U
C

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Figure 4.2: Effect of ECE (30, 100, 300 mg kg
-1

) on latency [time for which mouse remained on the hot plate 

(55˚C ± 0.1˚C) without licking or flicking of the hind limb or jumping in seconds. 

 Each column represents the mean ± SEM (n = 5).  

 

1.22 ANTICONVULSANT THRESHOLD TESTS 

1.22.1 PTZ-INDUCED SEIZURE TEST 

The test employed pentylenetetrazole (75 mg kg
-1

s.c) which induced myoclonic convulsions in 

all animals pre-treated with normal saline (10 mL kg
-1

). Ehretia cymosa 30, 100 and 300 mg kg
-

1
 reduced the duration of tonic convulsions significantly; fig 4.3C (P= 0.0024; F 3, 16 = 7.491). In 

the ECE treated animals, the frequency of convulsions was progressively and significantly 

reduced at all three doses 30, 100 and 300 mg kg
-1

 fig. 4.3B (P= 0.0031; F 3, 16 = 7.069). There 

was an observed significant (P<0.0001, F3, 16 = 2.797) increase in latency to the first myoclonic 

jerk fig. 4.3A, with statistical significance at doses 30 mg kg
-1

(P= 0.0031; F 3, 16 = 2.797). 

Phenobarbitone on the other hand which was used as a reference anticonvulsant produced a 

dose-dependent increase in latency to the first myoclonic jerk for the 3 and 30. It significantly 

Veh 30 100 300-30

-20

-10

0

10

20

30

                            ECE ( mg/kg)

L
at

en
cy

 (
s)

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delayed the onset (F3, 16=5.285, P=0.0100) and reduced the frequency (F3, 16=7.069, P=0.0031) 

and duration (F3, 23=4.177, P=0.0231) of PTZ-induced convulsions (Figure 4.3).  

  

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Figure 4.3: Effect of ECE 30, 100, 300 mg kg
-1

 and phenobarbitone 3, 10, 30 mg kg
-1

 on the latency to first 

myoclonic jerks, the total frequency of the seizures, and total frequency of the seizures induced 

by PTZ. Each column represents the mean ± SEM (n = 5). *P < 0.05, **P < 0.01compared with 

vehicle-treated group (one-way analysis of variance followed by Newman–Keuls post hoc test). 

 

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1.22.2 PICROTOXIN INDUCED SEIZURE TEST 

The picrotoxin (3 mg kg
-1

) experiment induced generalized tonic-clonic convulsions in all 

animals treated. ECE 30, 100, 300 significantly; fig. 4.4C (F3, 16=12.57, P=0. 0. 0.0002) reduced 

the duration of tonic-clonic convulsions in the test animals. It (30 mg kg
-1

 100 mg kg
-1

 and 300 

mg kg
-1

)
  
also significantly (P=0.0.0004: F3, 24 =8.422) increased the latency fig. 4.4A to the first 

myoclonic jerk . For the frequency of convulsions, only 100 mg kg
-1

 reduced the frequency but 

not significantly (P=0.0779: F3, 16 =2.735); fig 4.4B. The standard control phenobarbitone, 

reduced the frequency but not significantly (F3, 16=0.0779, P=0. 2.735) and significantly reduced 

both the duration (P=0.0.0403: F3, 16 =3.491) of picrotoxin-induced convulsions and also 

increased significantly (P=0.0007: F3, 16 =49.811) the latency to convulsions in the 3, 10, and 30 

mg kg
-1

 groups (Figure 4.4).  

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Figure 4.4:Effect of ECE 30, 100, 300 mg kg-1 and phenobarbitone 3, 10, 30 mg kg-1 on the latencies to first 

myoclonic jerks, the total frequency of the seizures, and total frequency of the seizures induced 

by picrotoxin. Each column represents the mean ± SEM (n = 5). *P < 0.05, **P < 0.01compared 

with vehicle-treated group (one-way analysis of variance followed by Newman–Keuls post hoc 

test). 

  

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1.22.3 MAXIMAL ELECTROSHOCK TEST 

The maximal electroshock model administered electric current (50 Hz, 60 mA, and 0.2 s) which 

induced hind limb tonic extensions in all animals pre-treated.  For Ehretia cymosa, only the 

highest dose 300 mg kg
-1

 significantly (P<=0.0167: F3, 16 = 4.151) reduced the duration of the 

first hind limb tonic extensions (HLTE); fig. 4.5A. The extract also delayed the total duration of 

the HLTE but was not statistically significant (F3, 16= 0.3300, P=0. 0.8037); fig. 4.6C. The 

standard drug carbamazepine did not reduce the duration of first hind limb tonic extensions 

(HLTE) (P=0.0791: F3, 16 =2.554) nor the total duration of the tonic hind limb convulsions (Fig. 

4.5B and Fig. 4.6D). 

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Figure 4.5: Effect of ECE 30, 100 and 300 mg kg
-1

 and carbamazepine 3, 10 and 30 mg kg
-1

 on the duration 

of First HLE induced by Maximal Electroshock Test (MEST). 

Each column represents the mean ± SEM (n = 7). *P < 0.05, **P < 0.01 ***P < 0.001 compared 

with vehicle-treated group (one-way analysis of variance followed by Newman–Keuls post hoc 

test). 

 

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Figure 4.6: Effect of ECE 30, 100 and 300 mg kg
-1

 and carbamazepine 3, 10 and 30 mg kg
-1

 on the total 

duration of Hind Limb Extensions (HLE) induced by Maximal Electroshock Test (MEST). 

 Each column represents the mean ± SEM (n = 5). *P < 0.05, **P < 0.01 ***P < 0.001 compared 

with vehicle-treated group (one-way analysis of variance followed by Newman–Keuls post hoc 

test). 

 

1.22.4 PTZ KINDLING TEST 

The experiment involved giving the vehicle-treated group, a repeated administration of 35 mg 

kg
-1

 of PTZ on alternate days which caused a gradual increase in the convulsant‘s responses as 

scored using the Racine scale. The score had increased from 0 to 3 by the 9
th

 day and 

maintained a Racine score of 4 from the 27
th

 to the 39
th

 day and reached a peak severity of 

Racine score of 5 by the 41
st
 day which was maintained for 2 weeks (Fig. 4.7A). ECE 

significantly depressed the kindled seizures at all the dose levels tested (F3, 84 = 24.97, P< 

0.0001); fig. 4.7B. None of the animals in the extract-treated groups achieved seizure score 5, 

even after 22 injections of PTZ 35 mg kg
-1

. The percentage severity of seizures (calculated 

from the AUC) shows that ECE attenuated PTZ kindled seizure activity by reducing the 

severity of seizures by 50%--70%. Phenobarbitone also produced a significant dose-dependent 

Veh 30 100 300
0

5

10

15

20
(C)

                          ECE (mg/kg)

T
o

ta
l 

D
u

ra
ti

o
n

 o
f 

H
L
E

Vehicle 3 10 30
0

5

10

15

20

25
(D)

                  Carbamazepine (mg/kg)

T
o

ta
l 

D
u

ra
ti

o
n

 o
f 

H
L
E

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depression of the kindled seizure activity (F3, 84 = 38.09, P < 0.0001); fig. 4.7D, and the 

percent severity of seizures was significantly reduced by 70% (Figure 4.7). 

 

 

 

 

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Figure 4.7:The dose-response effects of ECE 30, 100, and 300 mg kg
-1

 (A and B) and phenobarbitone 3, 10, 

and 30 mg kg
-1

 (C and D) on the PTZ-kindled mice. 

The left panels show the time course of effects over the 32 days and the right panels show the 

percent severity of seizures calculated from the AUCs for the test duration. Values are means ± 

SEM (n=10). *P < 0.05, ** P < 0.01, ***P < 0.001 compared with vehicle-treated group (two-

way analysis of variance followed by Bonferroni’spost hoc test). †P<0.05, †††P<0.001 compared 

with the vehicle-treated group (one-way analysis of variance followed by Newman–Keul’spost 

hoc test). 

 

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1.23 TOXICITY STUDIES 

1.23.1 BIOCHEMICAL PARAMETERS 

The study evaluated the lipid profile, kidney and liver functions of the animals. There was no 

significant difference (P=0.08-0.99) between the vehicle-treated and the ECE (30, 100, and 

300 mg kg
-1

)-treated rats regarding the serum concentrations of urea or creatinine (kidney 

function) of the rat subjects. There was also no significant difference between the treatment 

groups with regards to HDL (P=0.99), LDL (P=0.92), total cholesterol (P=0.51), and 

triglycerides (P=0.25), when compared to the control. The other parameters measured for the 

lipid profile were not significantly different (P=0.25-0.99) for the ECE-treated animals and 

the vehicle. However, ECE-treated rats (30 mg kg
-1

) showed significant difference in direct 

bilirubin (P=0.02) when compared to vehicle-treated rats (Table 4.3). 

 

 

 

 

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Table 4.3: A table showing the biochemical analysis of a single administration of ECE (30, 100 and 300 mg kg
-1

) after a 72-day study period in 

Sprague-Dawley male rats 

Parameters Vehicle ECE 30 ECE 100 ECE 300 P-value  

Renal function test (mmol 

L
-1

) 

     

Urea 8.41 ± 1.46  8.14 ± 0.78  8.34 ± 0.92  8.34 ± 0.36  0.99 

Creatinine  67.04± 12.06  38.38 ± 2.37  42.72 ± 6.24  46.66 ± 4.30  0.08 

Lipid Profile (mmol L
-1

)      

Total Cholesterol  2.04 ± 0.10 1.85± 0.14 2.0 ± 0.16 2.25 ± 0.11  0.51  

Triglycerides  1.41 ± 0.21 1.30 ± 0.06  1.21 ± 0.28  1.77 ± 0.16  0.25  

HDL  0.92 ± 0.11 0.86 ± 0.058 1.13 ± 0.09  1.19 ± 0.11  0.99  

LDL  0.48 ± 0.12 0.48 ± 00.11 0.43 ± 0.072 0.51 ± 0.11  0.92 

Liver function test      

Total Protein (g L-1)  88.98± 2.21 81.13±1.713 81.64±4.647 85.46±1.617 0.24 

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Albumin (gL-1)  39.44± 1.247 35.70±1.369 37.64±2.157 37.56± 0.96 0.44 

D.Bilirubin (μmolL-1)  1.33 ± 0.024 1.003±0.059 1.22 ± 0.089 1.23± 0.05 0.02* 

Ind.Bilirubin (μmolL-1)  0.54 ± 0.44 0.43 ± 0.13 0.52 ± 0.17 1.30±0.1897 0.13 

T.Bilirubin (μmolL-1)  1.86 ± 0.44 1.45± 0.15 1.76± 0.20 2.53 ± 0.16 0.09 

ALT (UL-1)  91.50 ± 27.92  67.73±5.663 88.76±19.23 87.90±6.624 0.82 

AST (IUL-1)  269.6 ± 42.09 183.4±18.09 276.5±55.94 205.5±27.65 0.32 

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1.23.2 HISTOLOGICAL EXAMINATION OF ISOLATED TISSUES 

. The liver of extract-treated rats showed normal liver sections with a characteristic 

hexagonal arrangement of hepatocytes in lobules surrounding a central vein (Figure 4.9). 

The examination of the heart sections showed a characteristic branching arrangement of 

myocardial fibres with centrally placed nuclei (Figure 4.11). The kidney sections revealed 

normal tubules, glomeruli, and renal capsules (Figure 4.10). The histology of the lungs 

showed normal alveolar sacs, alveoli as well as alveolar ducts, bronchus, and bronchioles. 

Lastly, the sectioned brain revealed normal pia mater, molecular layer, granular layer, and 

white matter as well as dura mater and with normal Purkinje and pyramidal cells (Figure 

4.8). There were no distortions of the normal architecture of tissues of any of the organs 

with immigrant cell types which may have constituted a form of pathology in all the doses 

levels as compared to that of the control.  

 

 

 

 

 

 

Figure 4.8: The Photomicrographs of Brain isolated from rats after 72-day continuous administration 

of (A) Vehicle, (B) ECE (30, 100 and 300 mg kg
-1

), (H&E staining, 40×). 

 

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Figure 4.9:The Photomicrographs of livers isolated from rats after 72-day continuous administration of 

(A) Vehicle, (B) 30 (C) 100 and (D) 300 mg kg
-1

) (H&E staining, 40×). 

The arrow shows the evenly distributed hepatocytes. 

 

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Figure 4.10:Photomicrographs of kidneys isolated from rats after 72-day continuous administration of 

(A) Vehicle, (B) ECE 30, (C) 100 and (D) 300 mg kg
-1

 showing normal renal tubule and 

glomeruli (H&E staining, 40×). 

 

 

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Figure 4.11:Photomicrographs of hearts isolated from rats after 72-day continuous administration of 

(A) Vehicle, (B) 30, (C) 100, (D) ECE 300 mg kg
-1

) showing normal myocardial fibers with 

characteristic central nuclei and branching arrangement as indicated by the arrows. 

 

 

 

 

 

 

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CHAPTER 5 DISCUSSION 

The present work demonstrated that ECE has anticonvulsant potential. The extract reduced 

seizure activity in mice in various models at doses that were considered non-toxic. Animal 

models used for screening the pharmacological effect of ECE include Irwin‘s test, PTZ-

kindling, picrotoxin induced seizure test, rotarod test, maximal electroshock test, the 

hotplate test and acute and sub-chronic toxicity studies. 

Irwin observation test is employed to assess the effects of an investigative drug on 

physiological and behavioural activity. The results obtained from this test are used to 

assess the  potential toxicity and to help in the selection of doses for a particular 

neuropharmacological investigation (Vogel et al., 2015). It provides an understanding of 

the possible toxicity or otherwise of the test substance.  From this, novel therapeutic agents 

may be discovered through a systematic way of assessing behavioural and physiological 

functions of the test substance qualitatively (Biney et al., 2014; Irwin, 1968). After the 

observation test employing an oral gavage of 6 different doses of the extract and control, 

mortality was zero after 48 hours and the LD50 was found to be greater than 3000 mg kg
-1

. 

Irwin test revealed reduced sensitivity to tail pinch touch indicating a potential analgesic 

effect, and reduced activity which also indicated potential sedative and neuromuscular 

effects. There was however altered respiration and slight tremors elicited at higher doses of 

1000 and 3000 mg kg
-1

 but mortality was zero in these groups too. Most of the CNS effect 

was elicited at the 30, 100 and 300 mg kg
-1

 dose groups and no tremors or altered 

respiration was also seen in those groups. The CNS effects were mostly elicited between 

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30 minutes to one hour after the oral administration. Based on these findings, the 30, 100, 

and 300 mg kg
-1 

dose groups were selected for this work, and a one hour waiting time was 

also selected as pre-treatment time for this work. 

From Irwin test, a potential neuromuscular effect of the extract was indicated so the 

rotarod apparatus test was used to validate that effect. The rotarod test is used to assess the 

behavioural effect of drugs on motor coordination  (Vogel et al., 2015). The present study 

showed that ECE could cause a reduction in time spent on the rotating rod but this was not 

statistically significant at all doses tested i.e. 30, 100, 300 mg kg
-1

. This means that the 

doses that can cause a potential reduction in neuromuscular activity with regards to the 

effects seen or elicited during Irwin's test can be said to be above the doses 30, 100, and 

300 mg kg
-1

 that were used for this work. 

The hot plate test is a simple behavioural test used for assessing the consequence of 

investigative drugs on the pain threshold sensitivity. The test is based on the logic that 

rodents will lick their paws or attempt escaping by jumping when they are put on a hot 

surface,. Investigative drugs that can alter the nociception threshold will either increase the 

latency to jumping or licking indicating an analgesic effect while those with hyperalgesic 

effect decrease the latency to licking/jumping (Vogel et al., 2015). The test, however, 

could not significantly increase the latency (F 3, 8 =2.907 P=0.1011 fig 4.1) as compared to 

the control. It increased the latency to paw licking of the 100 and 300 mg kg
-1

 but not 

significantly when compared to the control. But again, drugs that have a low analgesic 

effects, specifically, those that are essentially anti-inflammatory agents like ibuprofen, 

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aspirin and paracetamol, exhibit less pronounced outcome than well-known analgesics 

such as the opioids (Vogel et al., 2015). This means that the extract‘s possible analgesic 

effect seen during Irwin test could be due to the extract's anti-inflammatory effects and not 

necessarily an analgesic effect. 

This experimental work employed various models of seizures and provided evidence that 

the ethanolic extract of the leaves of Ehretia cymosa has anticonvulsant activity and that 

the extract is also safe in murine models. The anticonvulsant activity was evident in both 

the acute and chronic models of seizures and no toxicity was recorded in both models of 

toxicity that were used.  

The study employed convulsive threshold tests that are used in estimating the probability 

of an investigative drug to induce alterations in the propensity to the occurrence of seizures 

either spontaneously or, more importantly, in the company with other treatments (Vogel et 

al., 2015). In the acute models, PTZ, picrotoxin, and the maximal electroshock tests were 

employed whereas the PTZ kindling model was used for the chronic model.  

The PTZ acute model is a model that is thought to have an antagonistic action on 

GABAA receptors (Auvin & Nehlig, 2017) and represents a well-founded model for 

human absence and generalized seizures (Patrick Amoateng, Eric Woode, & Samuel B 

Kombian, 2012; Löscher & Schmidt, 1988). The extract elicited anticonvulsant activity 

against seizures induced by PTZ by increasing the latency to the first myoclonic jerks and 

clonic seizures in mice as well as reducingthe duration and frequency of the clonic 

seizures. Thus, the ability of the extract to inhibit seizures induc