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     DEPARTMENT OF PHYSIOLOGY 

SCHOOL OF MEDICINE AND DENTISTRY 

COLLEGE OF HEALTH SCIENCES 

UNIVERSITY OF GHANA 

 

 

 

 

 

 

 

 

 

 

MECHANISM OF ACTION OF ETHANOLIC EXTRACT OF Synedrella nodiflora 

(ASTERACEAE) IN MODULATING SEIZURE INTENSITIES. 

 

BY 

 

VINCENT ABOAGYE 

 

(10803760) 

This thesis / dissertation is submitted to the University of Ghana, Legon in partial fulfillment 

of the requirement for the award of MPHIL IN PHYSIOLOGY Degree. 

 

 

 

September, 2023 

 

 

 

 

 

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DECLARATION 
 

I, VINCENT ABOAGYE, hereby declare that with the exception of references made to other 
 

people’s work which I have duly acknowledged, this dissertation which is my original work has 
 

neither in whole nor in part been presented to the University or elsewhere for another degree. 

 

 

Signature:  

                        

Date: 25th September, 2023 

Vincent Aboagye 

 

(Investigator – MPhil Student) 

 

 

 

Signature:  

 

25th September, 2023 

Dr Thomas Amartey Tagoe 

 

(Supervisor) 

 

Signature: 

 

25th September, 2023 

Professor Patrick Amoateng 

 

(Supervisor) 

 

 

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DEDICATION 

I dedicate this work to God Almighty and my Father, Mr. James Yeboah Asuamah. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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ACKNOWLEDGEMENT 

I wish to express my sincere gratitude to my academic supervisors Dr Thomas Amartey Tagoe and 

Dr Patrick Amoateng for their guidance, sponsorship and supervision of this study. My sincere 

thanks also go to Mr Richard Obeng-kyeremeh, Daniel Amoah, and the entire Noguchi Animal 

Experimentation Department for their assistance. Lastly to Mr Addai Phandoh, Mr Safo Kwadwo 

and the entire staff of St Mary’s Hospital Laboratory, Drobo – Bono Region for their assistance in 

the Biochemical Analysis. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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ABSTRACT 

Introduction: Seizure is a neurological disorder which manifests as abnormal cortical nerve cell 

activity. This results from synchronous neuronal activity in the brain with some effects manifesting 

as uncontrolled jerky movements involving much of the body with loss of consciousness. 

Treatment of this neurological condition has been complicated by drug resistance and side effects. 

This has fueled the need to identify new compounds with properties which can be developed into 

drugs. Compounds from plant extracts such as Synedrella nodiflora (SNE) have previously been 

shown to exhibit anti-convulsive properties, making them worthy of further investigation. The aim 

of this study therefore is to explore the potential pathways with which the extract decreases seizure. 

Methods: The anti-convulsant effect of ethanolic extract of Synedrella nodiflora was investigated 

using two animal seizure models; Acute Pentylenetetrazol (PTZ) and Chronic PTZ induced 

seizures. Liver and Renal function tests were assessed after Chronic PTZ induced seizures. Brain 

tissues were resected for histological studies. Flumazenil, a GABA receptor blocker, was used to 

determine whether or not the extract reduced seizures via the GABAergic pathway. 

Results: SNE (1000 mg/kg/body weight) reduced PTZ induced kindling significantly (P<0.05). 

Also, SNE (100, 300, 1000 mg/kg/body weight) reduced the latency and frequency of acute PTZ 

induced seizures. Histology of the hippocampus of SNE- treated mice showed normal cell count 

when compared with control mice. Results of Renal and Hepatic Function were within normal 

limits. SNE had no significant effect after the mice were pretreated with Flumazenil (GABA 

receptor Blocker). 

Conclusion: The result of this study provides evidence that the ethanolic extract of the whole plant 

of Synedrella nodiflora possesses anti-seizure activity probably mediated through GABAergic 

pathway in murine experimental models. 

 

 

 

 

 

 

 

 

 

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LIST OF ABBREVIATIONS 
 

SNE 

GABA 

PTZ 

PHE 

ILAE 

AUC 

E/I 

ICH 

SAH 

AMPA 

NMDA 

IPSP 

CSF 

ALT 

AST 

GGT 

LDH 

NADH 

GFR 

 

Synedrella nodiflora 

Gama Amino Butyric Acid. 

Pentylenetetrazol 

Phenobarbitone Sodium 

International League Against Epilepsy 

Area under Curve 

Excitation/Inhibition 

Intracerebral Hemorrhage 

Subarachnoid Hemorrhage 

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid 

N-Methyl-D-Aspartate Receptor 

Inhibitory Postsynaptic Potential 

Cerebrospinal Fluid 

Alanine Aminotransferase 

Aspartate Aminotransferase 

Gamma-Glutamyl Transferase 

Lactate Dehydrogenase 

Nicotinamide Adenine Diphosphate 

Glomerular Filtration Rate

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Table of Contents 

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

DEDICATION .............................................................................................................................. iii 

ACKNOWLEDGEMENT ............................................................................................................ iv 

ABSTRACT .................................................................................................................................... v 

LIST OF ABBREVIATIONS ....................................................................................................... vi 

LIST OF FIGURES ....................................................................................................................... x 

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

CHAPTER ONE ............................................................................................................................. 1 

1.0 Introduction ............................................................................................................................. 1 

1.1 Background of Study ........................................................................................................... 1 

1.2 Problem Statement ............................................................................................................... 2 

1.4. Aim ..................................................................................................................................... 4 

1.5 Specific Objectives .............................................................................................................. 4 

1.6 Hypothesis ........................................................................................................................... 4 

CHAPTER TWO............................................................................................................................ 5 

2.0 LITERATURE REVIEW ....................................................................................................... 5 

2.1 Neuronal and synaptic Physiology ...................................................................................... 5 

2.2 Cerebral and cortical functions of neurons .......................................................................... 7 

2.3 The hippocampus of the brain ............................................................................................. 9 

2.4 Seizure Mechanisms .......................................................................................................... 10 

2.4.1 Generalized Seizures ...................................................................................................... 11 

2.4.1.1 Absence Seizures ......................................................................................................... 11 

2.4.1.2 Atonic Seizures ............................................................................................................ 11 

2.4.1.3 Tonic - Clonic Seizures ............................................................................................... 12 

2.4.2 Focal Seizures ................................................................................................................. 13 

2.5 Neurochemical mechanisms underlying epilepsy ............................................................. 13 

2.5.1 GABA ............................................................................................................................. 13 

2.5.2 Glutamate ....................................................................................................................... 14 

2.6 Epileptic seizures and Human Genetics ............................................................................ 14 

2.7 Electrophysiology of Neurons During Seizure States ....................................................... 15 

2.8. Biochemical Tests ............................................................................................................ 17 

2.8.1 Liver Function Test ........................................................................................................ 17 

2.8.1.1 Alanine transaminases ................................................................................................. 17 

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2.8.1.2 Aspartate transaminases .............................................................................................. 17 

2.8.1.3 Gamma-glutamyl transferase ....................................................................................... 18 

2.8.1.4 Bilirubin ....................................................................................................................... 19 

2.8.1.5 Albumin ....................................................................................................................... 19 

2.8.1.6 Lactate dehydrogenase ................................................................................................ 20 

2.8.2 Kidney Function Test ..................................................................................................... 20 

2.8.2.1 Creatinine .................................................................................................................... 20 

2.8.2.2 Urea ............................................................................................................................. 21 

3.0 RESEARCH METHODOLOGY ......................................................................................... 22 

3.1 Plant Collection ..................................................................................................................... 22 

3.2 Preparation of Extract of Synedrella nodiflora. .................................................................... 22 

3.3 Animals ................................................................................................................................. 22 

3.4 Determining the Sample Size for an Animal Study .............................................................. 23 

3.5 Chemicals and Drugs ............................................................................................................ 23 

3.6 Pentylenetetrazol (PTZ) seizure Induction. .......................................................................... 23 

3.6.1 Acute PTZ Seizure Induction ......................................................................................... 23 

3.6.2 PTZ kindling. .................................................................................................................. 25 

3.7 Histology of the Hippocampus ............................................................................................. 26 

3.8 Biochemical Analysis ........................................................................................................... 27 

3.9 Statistical Analysis ................................................................................................................ 27 

CHAPTER FOUR ........................................................................................................................ 28 

4.0 RESULTS ............................................................................................................................. 28 

Stages of PTZ kindling procedure scored on the Racine scale. .............................................. 28 

Fig 1.0 Seizure Intensity during PTZ-Induced Kindling ......................................................... 29 

Latency, frequency and duration of convulsions following Acute PTZ induced seizures. ........ 30 

Figure 2 .................................................................................................................................... 30 

4.4 Biochemical Analysis after PTZ Kindling Procedure ....................................................... 32 

Table 2: Biochemical analysis after PTZ kindling Procedure. ................................................ 32 

4.5 Histology of the Hippocampus following PTZ kindling. .................................................. 33 

Fig 3. Photomicrograph of the hippocampus of the brain following PTZ induced kindling. . 33 

4.6. Average Mice Body weight After PTZ kindling. ............................................................. 34 

Fig.5 Effects of PTZ kindling on Body weight ....................................................................... 35 

CHAPTER FIVE .......................................................................................................................... 36 

5.0 DISCUSSION ....................................................................................................................... 36 

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5.1 SNE Inhibited Both Acute and Chronic PTZ induced Seizures Suggestively via 

GABAergic Pathway. .............................................................................................................. 36 

5.2 SNE significantly reduced PTZ induced seizures ............................................................. 36 

5.3 Effect of SNE on Biochemical Indices. ............................................................................. 37 

CHAPTER SIX ............................................................................................................................. 39 

6.0 Conclusion and Recommendation ........................................................................................ 39 

6.1 Conclusion ......................................................................................................................... 39 

6.2 Recommendations ............................................................................................................. 39 

REFERENCES ............................................................................................................................. 40 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

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LIST OF FIGURES 

Fig 1.0 Seizure Intensity during PTZ-Induced Kindling ........................................................................... 30 

Figure 2. Effects of SNE on Latency, Frequency and Duration of convulsions ................................ 31 

Fig 3. Photomicrograph of the hippocampus of the brain following PTZ induced kindling. ......... 34 

Fig.4 Effects of PTZ kindling on body weight ............................................................................................. 36 

 

 

 

 

 

 

 

 

 

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LIST OF TABLES 

Table 1. Effect of Flumazenil (GABA receptor blocker) on SNE and Diazepam following acute 

PTZ injection .......................................................................................................................................................... 32 

Table 2: Biochemical analysis after PTZ kindling procedure………………………………………….33 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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CHAPTER ONE 

1.0 Introduction 

1.1 Background of Study 

A seizure is an overwhelming electrical disturbance in the brain that results in changes in behavior, 

movement, sensation, and awareness (Lawal et al., 2018; Spray, 2015). This results from 

synchronous neuronal activity in the brain with some effects manifesting as uncontrolled jerky 

movements involving muscles of the body with loss of consciousness (tonic-clonic seizure), jerky 

movements involving only part of the body with variable levels of consciousness (focal seizures), 

and a subtle momentary loss of awareness (absence seizures)( Leibetseder et al., 2020;Fisher et 

al., 2017). These episodes usually last less than two minutes. During this process, loss of bladder 

control may occur (Brown, 2021). 

Seizures may be either provoked or unprovoked. Provoked seizures are due to a temporary event 

such as low blood sugar, alcohol withdrawal, drug abuse, low blood sodium, fever, brain infection, 

or concussion (Delanty, 2001). Unprovoked seizures occur without a recognized cause, and they 

are likely to persist. Stress or sleep deprivation may also cause unprovoked seizures (Kotagal, 

2001). 

The underlying neurological cause of seizures is a disruption in the brain's normal pattern of 

excitation (E) and inhibition (I) (Bonansco & Fuenzalida, 2016; Stafstrom & Carmant, 2015; 

Ramamoorthi & Lin, 2011). The factors that alter E/I balance can be either genetic or acquired 

(Kang, 2021; Casillas‐Espinosa et al., 2020). 

The developing brain is more susceptible to seizure activity because excitatory synaptic activity 

develops before inhibitory synaptic function, which thereby promotes excitation rather than 

inhibition (Nardou et al., 2013; Owens & Kriegstein, 2002). Furthermore, the neurotransmitter 

GABA produces stimulation rather than inhibition in the cerebral cortex early in life. These       

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findings help to understand why the brain of a child is particularly vulnerable to seizures (Nardou 

et al., 2013; Bowery & Smart, 2006). 

Treatment of this neurological disorder has mostly focused on the use of drugs (Landmark, 2008; 

Löscher, 2002). Drugs such as Phenytoin, carbamazepine, oxcarbazepine block repetitive 

activation of sodium channels (Rogawski & Löscher, 2004). Also, Phenobarbital, 

benzodiazepines, clobazam enhances activity of GABAA receptors and drugs such as Tiagabine 

inhibit GABA reuptake (Greenfield Jr, 2013; Porter & Meldrum, 2001). In summary, these 

medications prevent neuronal depolarization by blocking sodium or calcium channels, enhancing 

potassium channel function, decreasing glutamate-mediated excitation, or promoting GABA-

mediated inhibition. However, this approach to the treatment of seizure disorders is complicated 

by drug resistance and side effects (Chen et al., 2017; Lagae, 2006). Reactions to some of these 

traditional seizure medications have accelerated the search for new compounds, including herbal 

extracts that are effective against recurrent seizure episodes. 

Synedrella nodiflora (SNE) has been demonstrated to have analgesic, antioxidant, and 

anticonvulsant properties (Amoateng et al., 2017; Amoateng et al., 2012). Its analgesic effect has 

been shown to be mediated in part by adenosinergic pathways (Woode et al., 2009), however, the 

mechanism behind its anticonvulsant activity is unknown. The aim of this study therefore is to 

explore the potential pathways through which the extract decreases seizure activity. 

 1.2 Problem Statement 

Seizures are more common in children under the age of one and the elderly, usually after the age 

of 65, as a result of various health issues such as stroke and heart disease (Van Win et al., 2020; 

Liu et al., 2016). According to a prospective multicenter study, the total incidence of seizures after 

intracerebral hemorrhage (ICH) and ischemic strokes are about 10.6% and 8.6%, respectively. 

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Seizures were present in about 10% –25% of individuals after an ICH, and 15.2% after a 

subarachnoid hemorrhage (SAH), (Constant Dit Beaufils et al., 2021).  

 A meta-analysis point to a higher prevalence of seizures and epilepsy in children ranging from 

3.2-5.5/1,000 in developed countries to 3.6-44/1,000 in underdeveloped countries (Camfield & 

Camfield, 2015). Prolonged seizures usually lead to active epilepsy. In a recent study, the 

prevalence rate of epilepsy was about 6.38 per 1,000 people, and a lifetime prevalence of 7.60 per 

1,000 people (Beghi, 2020). This has been found more prevalent in in low- and middle-income 

nations (Kotsopoulos et al., 2002). A major neurological defect has to do with the shrinking of the 

hippocampus of the brain. The hippocampus is reported to be liable to atrophy following repeated 

status epilepticus which may in learning defects (Hattiangady et al., 2008; Marques et al., 2007; 

Silvia et al., 2003). A lot of drugs have been developed in attempting to resolve this neurological 

issue, However, many of the drugs have failed due to their side effects. This therefore has 

influenced the need to research into herbal extracts with their mechanism of action known in 

decreasing seizures. 

 

 

 

1.3 Justification 

Previous studies have looked into Property of Synedrella nodiflora to abort seizures, but there 

has been no study of the mechanism of action of the extract. Research is therefore necessary to 

establish the mechanism(s) of action of the extract. Also, given the high cost of anti-epileptic 

drugs and their numerous adverse effects, there is a pressing need to investigate this plant 

extract and the mechanisms involved in reducing seizure intensity. This study could lead to the 

development of a new anti-seizure drug. 

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1.4. Aim 

The goal of the study is to investigate further the anticonvulsive effects, as well as the mechanism 

of action and safety profile of extracts of Synedrella nodiflora. 

1.5 Specific Objectives 

The specific objectives of the study include: 

1. To determine how SNE affects the latency, frequency, and duration of both acute and chronic 

chemically induced seizures. 

2. To determine the possible receptor pathway by which SNE mediates decreasing seizures. 

3. To determine how SNE affects the viability of hippocampal neurons following a prolonged PTZ 

kindling. 

4. To determine how prolonged SNE administration affects the body's organs (liver and kidney 

functions). 

1.6 Hypothesis 

Synedrella nodiflora extract will decrease seizures via the GABAergic pathway. 

 

 

 

 

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CHAPTER TWO 

2.0 LITERATURE REVIEW 

2.1 Neuronal and synaptic Physiology 

In the mammalian brain, excitatory and inhibitory neurotransmission are mediated by the 

transmitter’s glutamate and GABA, respectively (Wen et al., 2022; Obata, 2013). Fast signaling 

between neurons occurs at specialized synaptic contacts formed between the axons of presynaptic 

cells and the soma or dendrites of postsynaptic target neurons (Jan & Jan, 2001; Isaacson, 2000). 

The narrow width of the synaptic cleft ensures that transmitters reach receptors quickly and at high 

concentrations. The lifetime of transmitter action at excitatory and inhibitory synapses is governed 

by a combination of diffusion of transmitter molecules away from receptors in the postsynaptic 

region, and uptake of transmitter molecules by specific transporters in neuronal and glial membranes 

(Farrant & Kaila, 2007; Cherubini & Conti, 2001). 

Neurons form a complex biological neural network through which action potentials travel. Neurons do 

not touch each other except in the case of an electrical synapse through a gap junction; instead, neurons 

interact at close contact points called synapses (Hormuzdi et al., 2004). A neuron transports its 

information by way of an action potential. When the nerve impulse arrives at the synapse, it may cause 

the release of neurotransmitters, which influence another postsynaptic neuron (Hormuzdi et al., 2004). 

The postsynaptic neuron may receive inputs from many additional neurons, both excitatory and 

inhibitory. The excitatory and inhibitory influences are summed, and if the net effect is inhibitory, the 

neuron will be less likely to generate an action potential, and if the net effect is excitatory, the neuron 

will be more likely to generate action potential (Huang et al., 2012; Yoshimura et al., 2005).  How likely 

a neuron is to fire depends on how far its membrane potential is from the threshold potential, the voltage 

at which an action potential is triggered because enough voltage-dependent sodium channels are 

activated so that the net inward sodium current exceeds all outward currents (Khaliq & Bean, 2010; 

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Denac et al., 2000).  Excitatory inputs bring a neuron closer to threshold, while inhibitory inputs bring 

the neuron farther from threshold. 

Neurotransmission is the process by which signaling molecules known as neurotransmitters are released 

by the axon terminal of a neuron (the presynaptic neuron), and bind to and react with the receptors on 

the dendrites of another neuron (the postsynaptic neuron) (Togashi et al., 2009; Süudhof, 2008; Stahl, 

2000).  

Glutamate is a divalent anion of glutamic acid and it acts as a neurotransmitter (Heath & Shaw, 2002). 

It is by a wide margin the most abundant excitatory neurotransmitter in the vertebrate nervous system 

(Omote et al., 2011). It is involved in every major excitatory function in the vertebrate brain and accounts   

for over 90% of the synaptic connections in the human brain (Heath & Shaw, 2002). It also serves as the 

primary neurotransmitter for some localized brain regions, such as granule cells in the cerebellum 

(Hisano, 2003). 

Biochemical receptors for glutamate fall into three major classes, known as α-amino-3-hydroxy-5-

methyl-4-isoxazolepropionic acid (AMPA) receptors, N-methyl-D-aspartate (NMDA) receptors, and 

metabotropic glutamate receptors (Fu et al., 2018; Sherman, 2014). Many synapses use multiple types 

of glutamate receptors. AMPA receptors are ionotropic receptors specialized for fast excitation. In many 

synapses they produce excitatory electrical responses in their targets a fraction of a millisecond after 

being stimulated (Reiner & Levitz, 2018; Kullmann & Lamsa, 2007). NMDA receptors are also 

ionotropic, but they differ from AMPA receptors in being permeable, when activated, to calcium (Cull- 

Candy et al., 2006). Their properties make them particularly important for learning and memory (Riedel 

et al., 2003). Metabotropic receptors act through second messenger systems to create slow, sustained 

effects on their targets (Reiner & Levitz, 2018). 

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GABA on the other hand mediates fast inhibitory transmission via the activation of ionotropic 

receptors. Its actions are thought to be primarily excitatory in the developing brain (Ben-Ari, 2014). 

Activation of a GABAA receptor thus leads to efflux of Cl
−
 ions from the cell and this manifest as 

depolarizing current (Nawafleh et al., 2022). GABAergic interneurons mature faster in the 

hippocampus and the GABA machinery appears earlier than glutamatergic transmission. Thus, 

GABA is considered the major excitatory neurotransmitter in many regions of the brain before the 

maturation of glutamatergic synapses (Ben-Ari et al., 2007; Luján et al., 2005; Herlenius & 

Lagercrantz, 2004). 

One approach to understanding the physiological role of GABAergic inhibition is to record from 

synaptically-coupled pairs of neurons. In the hippocampus, dual electrophysiological recordings of 

GABAergic interneurons and their postsynaptic target cells, pyramidal neurons, have shown that 

single interneurons exert powerful inhibitory actions on the excitability of individual pyramidal cells 

(Isaacson, 2000). 

2.2 Cerebral cortical functions of neurons 

Cerebral and cortical functions of neurons are integral components of the brain's overall function 

and are responsible for various cognitive and sensory processes (Maldonado, 2018). These functions 

involve complex interactions among different types of neurons, neurotransmitters, and neural 

networks within the cerebral cortex, which is the outermost layer of the brain responsible for higher-

order cognitive functions (Molnár et al., 2019). Some of these complex interactions include 

information processing, sensory perception, motor control, memory formation among others. 

Neurons in the cerebral cortex are primarily responsible for processing and integrating information 

from various sensory modalities, such as sight, sound, touch, and taste (Molnár et al., 2019). They 

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also play a crucial role in higher cognitive processes like thinking, reasoning, and decision-making. 

The cerebral cortex is divided into different regions, each with specialized functions, such as the 

visual cortex for processing visual information and the prefrontal cortex for executive functions like 

planning and problem-solving (Carpenter et al., 2000). 

Also, the cerebral cortex contains regions dedicated to each of the senses. For example, the 

somatosensory cortex is responsible for processing tactile sensations like touch and pressure, while 

the auditory cortex processes sound, and the olfactory cortex is involved in the sense of smell (Rupini 

& Nandagopal, 2015). Neurons in these areas receive and process sensory input, allowing us to 

perceive and interpret the world around us. 

Neurons in the cerebral cortex also control voluntary movements (Isomura et al., 2009). The primary 

motor cortex, for instance, is responsible for planning and executing motor actions. When you decide 

to move a limb or perform a specific action, neurons in this region send signals to the muscles to 

carry out those movements (Koch & Rothwell, 2009). 

Adding up, the cerebral cortex is vital for memory processing (Mizuno-Matsumoto et al., 2020). 

Different regions, such as the hippocampus and various parts of the temporal and frontal lobes, are 

involved in encoding, storing, and retrieving memories (Simons & Spiers, 2003). Neurons play a 

crucial role in forming and strengthening synaptic connections associated with memory traces 

(Lisman et al., 2018). 

In summary, cerebral and cortical functions of neurons are diverse and intricate, underpinning the 

brain's ability to process sensory information, control movements, form memories, regulate 

emotions, communicate, and engage in complex cognitive processes. These functions result from 

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the coordinated activity of billions of neurons working together within the cerebral cortex, making 

it the most sophisticated part of the human brain.  

2.3 The hippocampus of the brain 

The hippocampus is a crucial region in the brain associated with various functions related to learning 

and memory, especially spatial memory and the formation of new long-term memories (Deng et al., 

2010). Neural connections in the hippocampus play a significant role in these processes. There are 

several key regions and connections within the hippocampus, and each has its specific function. 

The dentate gyrus is the first region of the hippocampus that receives input from the entorhinal cortex 

(Jonas & Lisman, 2014). It serves as a gateway to the hippocampal circuitry. One of its primary 

functions is pattern separation, which helps in distinguishing similar inputs and forming distinct 

representations of memories (Vivar et al., 2012). 

The CA3 (Cornu Ammonis 3) is a region of the hippocampus that receives input from the dentate 

gyrus (Chauhan et al., 2021). It is known for its recurrent connections, which are thought to be 

crucial for memory consolidation and the rapid encoding of new information. CA3 is also involved 

in associating different elements of a memory, which can contribute to the formation of complex 

memories (Cherubini & Miles, 2015). 

Another region within the hippocampus is the CA1 (Cornu Ammonis 1), and it receives input from 

CA3. CA1 plays a critical role in the retrieval of stored memories and their integration into existing 

knowledge (Schlichting & Preston, 2015). It also sends information back to the entorhinal cortex 

and other brain regions, allowing for the transfer of information between the hippocampus and the 

neocortex (Schlichting et al., 2014). 

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Also, the subiculum which is another region of the hippocampus is located at the output end of the 

hippocampus and is responsible for transmitting information from the hippocampus to other brain 

regions, including the cortex (O’Mara, 2006). It plays a role in spatial memory and navigation 

(Buzsáki, 2015). 

Finally, the entorhinal cortex is a key interface between the hippocampus and the neocortex (Rozov 

et al., 2020). It provides input to the hippocampus, conveying information about spatial and 

contextual aspects of the environment. It is also involved in the formation of cognitive maps, which 

are essential for spatial memory (Steffenach et al., 2005). 

These neural connections and their functions within the hippocampus are essential for the formation, 

storage, and retrieval of memories, as well as for spatial orientation and navigation in the 

environment (Vismer et al., 2015). Damage to the hippocampus, as seen in conditions like Epileptic 

states and Alzheimer's disease, can severely impair these functions, leading to memory deficits and 

spatial disorientation (Van Hoesen et al., 2000). 

2.4 Seizure Mechanisms 

An electrical disturbance in the brain known as a seizure is caused by brain cells that fire very 

rhythmically. It is defined by the temporary development of symptoms and signs brought on by 

abnormally synchronized neuronal activity in the brain (Bowman et al., 2001; Nye & Thadani, 

2015). 

Physiologically, excitatory neurons are subjected to refractoriness after it has fired. Some of the 

factors contributing to this refractoriness includes; inhibitory neurons' impact, electrical changes 

inside excitatory neurons, and the detrimental effects of adenosine (Neske et al., 2015). 

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Over 1,600 ion-channel mutations and 41 ion-channel genes have been linked to the onset of 

epileptic seizures (Chen et al., 2017; Wei et al., 2017). An influx of Ca
2+

 can lead to a prolonged 

depolarization in individual neurons, which results in prolonged activation of Na
+
 channels and 

recurrent action potentials.  

2.4.1 Generalized Seizures 

A generalized seizure happens when the aberrant electrical activity that causes a seizure starts at 

synchronously in both hemispheres of the brain (Englot & Blumenfeld, 2009). This usually shoots 

from a localized region and quickly fires through the two hemispheres of the brain. Different types 

of Generalized Seizures include; Absence seizures, atonic, myoclonic, tonic and clonic seizures. 

2.4.1.1 Absence Seizures 

Absence seizure is also known as Petit mal seizure. It is characterized by abrupt onset of staring 

spells. A person experiencing an absence seizure will often stop moving or may blink rapidly or stare 

into space than 15 seconds in one place (Chen et al., 2005; Tononi & Koch, 2008). Even though the 

individual may not recall what occurred during the seizure, their normal level of attentiveness returns 

right afterward. 

2.4.1.2 Atonic Seizures 

An Atonic seizure An Atonic seizure is characterized by loss of muscle tone (atonia) which leads to 

limping and slumping and may cause to injury to the individual (Thijs et al., 2009). Certain epilepsy 

syndromes, including Lennox-Gastaut syndrome, are characterized by atonic seizures (Crumrine, 

2002). 

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2.4.1.3 Tonic - Clonic Seizures 

A person experiences a tonic seizure, when the muscles undergo spasm and the individual loses 

consciousness (Tononi & Koch, 2008). It presents with arching of the trunk as a result of spasm of 

muscles of the spine, chest and limb and rolling of the eyes (Jankovic & Lang, 2004). Breathing is 

interrupted as a result of spasm of the muscles of the chest, and the person's lips and face may turn gray 

or blue (Devinsky, 2007). 

Clonic seizures cause a person’s muscles to contract and relax leading to jerky movements of the 

body. Elbow, leg, and neck muscles all quickly flex and then relax (Rudzinski & Shih, 2010). As 

the seizure passes, the jerking motion becomes less severe until it eventually stops. The individual 

usually lets out a long sigh as the jerking ceases before starting to breathe normally again. When 

tonic and clonic movements occur synchronously, tonic-clonic seizures, also known as "grand mal" 

or "convulsive" seizures, take place (Brodie et al., 2018). Depending on the severity of the seizure, 

a seizure usually lasts a few minutes or less, after which the person is likely to stay unconscious for 

another few minutes. 

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2.4.2 Focal Seizures 

Focal seizures which could be olfactory, motor, sensory, cognitive, autonomic, or autonomic-

autonomic phenomena can make a person confused or dazed during a complicated partial seizure 

and be unable to reply to questions or instructions (Wang et al., 2017). This is usually coupled 

with a Jacksonian march, which occurs when jerking activity spreads from one muscle group to 

another (Unterberger et al., 2018). 

Focal seizures develop from networks that are confined to one hemisphere (Burman & Parrish, 

2018) with Subcortical structures being the source. Seizure semiology can be used to pinpoint the 

specific region of the brain, lobe, or hemisphere that is responsible for the development and spread 

of the condition (McGonigal & Chauvel, 2004). 

2.5 Neurochemical mechanisms underlying epilepsy 

2.5.1 GABA 

Several studies have shown that GABA is involved in the pathophysiology of epilepsy in both 

animal models and patients suffering from epilepsy (Bozzi et al., 2018; Engelborghs et al., 2000; 

Yin et al., 2013). The GABA hypothesis of epilepsy implies that a reduction of GABAergic 

inhibition results in epilepsy whereas an enhancement of GABAergic inhibition results in an anti-

epileptic effect (Catterall et al., 2010). Inhibitory postsynaptic potentials (IPSPs) gradually 

decrease in amplitude during repetitive activation of cortical circuits. This phenomenon might be 

caused by decreases in GABA release from terminals, desensitization of GABA receptors that are 

coupled to increases in Cl conductance or alterations in the ionic gradient because of intracellular 

accumulation of Cl
-
 (Engelborghs et al., 2000). Moreover, Cl

-
-K

+
 co-transport becomes less 

effective during seizures as it depends on the K
+
 gradient. As Cl

-
-K

+
 co-transport depends on 

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metabolic processes, its effectiveness may be affected by hypoxia or ischemia as well (Pedersen 

et al., 2006). 

GABA levels and glutamic acid decarboxylase (GAD) activity were shown to be reduced in 

epileptic foci surgically excised from patients with intractable epilepsy and in CSF of patients with 

certain types of epilepsy (Badawy et al., 2009). Several endogenous (guanidino compounds) and 

exogenous (e.g., bicuculline, picrotoxin, penicillin, pilocarpine, pentylenetetrazol) convulsant 

inhibit GABAergic transmission through inhibition of GABA synthesis or through interaction with 

distinct sites at the postsynaptic GABA(A) receptor (De Risio & Platt, 2014).  

2.5.2 Glutamate 

Glutamatergic synapses play a critical role in all epileptic phenomena (Engelborghs et al., 2000). 

Activation of both ionotropic and metabotropic postsynaptic glutamate receptors serve as 

proconvulsant (Alexander & Godwin, 2006). 

An increased sensitivity to the action of glutamate at NMDA receptors is seen in hippocampal 

slices from kindled rats and in cortical slices from cortical foci in human epilepsy (Alexander & 

Godwin, 2006). This results in an enhanced entry of Ca2+ into neurons during synaptic activity. 

Changes in metabotropic glutamate receptor function may also play a key role in onset of seizures 

(Chapman, 2000). 

2.6 Epileptic seizures and Human Genetics 

An estimated 40% of patients who experience epileptic seizures have genetic predispositions that 

play a role in the etiology of seizures (Anwar et al., 2020). The majority of familial epilepsies, 

including juvenile myoclonic epilepsy, childhood absence epilepsy, and benign childhood 

epilepsy, have a complex pattern of inheritance caused by the interplay of several loci with 

environmental factors (Berkovic & Scheffer, 2001; Helbig et al., 2008). Biochemical alterations 

(such as elevated plasma glutamate levels) that can be connected to a widespread increase in 

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cortical excitability have been seen in absence seizure sufferers (and their first-degree relatives) 

(Brambilla et al., 2003; Palmieri et al., 2010). 

Genetic data from research on absence epilepsy in animals reveals a very straightforward 

inheritance factor of one gene that decides whether an individual is epileptic or not, while other 

genes regulate the frequency and length of epileptic fits (Grone & Baraban, 2015; Winawer, 2002). 

A rare autosomal dominant condition called episodic ataxia type 1 is characterized by transient 

ataxic episodes accompanied by myokymia. Partial epileptic episodes are also present in patients 

with this disease (Jen et al., 2007; Rajakulendran et al., 2007). Point mutations in the human 

voltage-gated potassium channel gene on chromosome 12p13 are related to the syndrome (Eunson 

et al., 2000).  

2.7 Electrophysiology of Neurons During Seizure States 

Neurons communicate through electrical impulses, which are generated and propagated by 

changes in membrane potential (Fields, 2008). During a seizure, there is a significant increase in 

neuronal excitability, making neurons more prone to firing action potentials (Misonou, 2010). This 

heightened excitability is often a result of an imbalance between excitatory and inhibitory 

neurotransmitters, such as glutamate and GABA (Uzunova et al., 2016). 

In a healthy brain, neurons fire in a coordinated but asynchronous manner. During a seizure, 

however, groups of neurons become excessively synchronized, leading to the generation of 

hypersynchronous electrical activity (Margineanu, 2010). This synchronization can occur in 

specific brain regions or even propagate throughout the entire brain, depending on the seizure type 

(Schwaller et al., 2004). 

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One other effect of neurons during seizure states includes ionic imbalance which can cause 

abnormal neuronal firing (Scharfman, 2007). Neurons maintain their resting membrane potential 

through the selective movement of ions, primarily sodium (Na+), potassium (K+), and chloride (Cl-

) (Clausen & Poulsen, 2013). During seizures, there is often an imbalance in these ions. For 

example, an excessive influx of sodium or calcium ions can lead to depolarization and 

hyperexcitability, triggering seizure activity (Johnkennedy, 2021). 

Also, neurotransmitter Alterations plays a key role in abnormal firing of neurons during seizure 

states. Dysregulation of neurotransmitters is a key factor in seizure generation. Glutamate, the 

brain's primary excitatory neurotransmitter, can be excessively released during seizures, further 

promoting excitatory signaling (Alcoreza et al., 2021). Conversely, the inhibitory neurotransmitter 

gamma-aminobutyric acid (GABA) may be impaired, reducing its ability to counterbalance 

excitatory activity. 

All these abnormal activities of neurons during seizure states can be monitored using 

electrophysiological tests (Englot et al., 2008; Klaassen et al., 2006). 

Electrophysiological tests like electroencephalography (EEG) are commonly used to diagnose 

seizures and identify their location in the brain (Bowyer, 2016). EEG records the electrical activity 

of the brain through electrodes placed on the scalp, and abnormal electrical patterns detected 

during seizures help doctors diagnose and classify different types of seizures (Siuly et al., 2016). 

Also, Electrophysiology is used to investigate the mechanisms underlying seizures (Staba et al., 

2014). Techniques such as patch-clamp recordings or extracellular field potential recordings to 

study the electrical properties of neurons and how they contribute to the generation and 

propagation of seizures (Jung et al., 2014). In a nutshell, seizures are complex events that involve 

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alterations in the electrophysiology of neurons. Understanding these changes is essential for both 

the diagnosis and treatment of epilepsy 

2.8. Biochemical Tests 

2.8.1 Liver Function Test 

2.8.1.1 Alanine transaminases 

Serum alanine transaminases (ALT) is not only measured to determine a possible liver damage, 

but also useful in monitoring the general health status. This is an enzyme that is mainly found in 

the cytosol of the hepatocytes, and plays a role in gluconeogenesis. Typically, it catalyzes the 

transfer of amino acid from L-alanine to alpha ketoglutarate to produce L-glutamate and pyruvate 

in the liver. ALT is released as a result of liver injury from the injured liver cells into the serum, 

which causes the levels to increase significantly in the serum. While serum levels of ALT have 

been used almost extensively as a marker for liver dysfunction. Gender related ALT levels studied 

showed that, men have slightly higher serum levels of ALT compared with their female 

counterparts (Parti et al., 2002). Gender however did not affect the levels of serum ALT in another 

study (Kotila et al., 2005). 

2.8.1.2 Aspartate transaminases 

Elevated levels of Aspartate transaminases may be as a result of several conditions such as increased 

hemolysis, and not hepatic damage (Nsiah et al., 2011). Nonetheless, serum levels of aspartate can be 

used in combination with other specific enzymes that assess liver damage to make a very sound 

conclusion regarding its (liver) integrity. Aspartate transaminases and alanine transaminases are 

however, considered as the two common tests when measuring liver enzymes. Thus, it plays a very 

important role in the assessment of normal function of the liver. Aspartate transaminases has been 

known as an enzyme that catalyzes the formation of glucose from other substrates. Thus, they play a 

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role in gluconeogenesis, which is essential for the body’s metabolic activities. Patients whose 

biochemical test for AST showed marked elevation are usually present with acute liver injury (Giannini 

et al., 2005). 

2.8.1.3 Gamma-glutamyl transferase 

Gamma-glutamyl transferase (GGT) is a cell-surface protein, which is mainly produced in the liver 

and present in serum. It contributes to the extracellular catabolism of glutathione (an antioxidant that 

protects the cell against oxidative stress) (Emdin et al., 2005; Whitfield, 2001). Thus, the higher the 

levels of GGT in the serum, the more the oxidative stress. It is important to bring to light that, 

significant increase in GGT levels may reflect a possible increased exposure to certain xenobiotics of 

organic sources that are metabolized in the liver through the process of glutathionylation. GGT serves 

as a serum biomarker of hepatobiliary damage. The serum activity of GGT has been used as early 

marker of inflammation and oxidative stress (due to its catabolic activity on the antioxidant 

glutathione) (Nothlings et al., 2012; Schmelzer et al., 2011; Menke et al., 2000). In most cases, GGT 

levels in the serum has been used as a biomarker of alcohol-related liver disease (Whitfield, 2001).

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2.8.1.4 Bilirubin 

Bilirubin is formed as a result of the breakdown of a component that is mostly derived from the 

haemoglobin of erythrocytes, called haeme. It could also be produced from the breakdown of 

haeme derived from other haemoproteins including catalase, myoglobin as well as cytochrome. 

The haeme is broken down to form biliverdin, by haeme oxygenase (HO), a rate-limiting enzymes 

in haeme catabolism. The biliverdin is consequently converted to bilirubin by biliverdin reductase 

(Rochette et al., 2018; O’Brien et al., 2015). The formed bilirubin then binds to albumin in the 

circulation, where it is transported to the liver. In the liver, it is conjugated by uridine diphosphate-

glucuronyl transferase 1A1 with glucuronic acid. (Keppler et al., 2014). 

2.8.1.5 Albumin 

Although studies have documented the presence of albumin mRNA in extra-hepatic places of the body 

including the brain, pancreas and kidney, albumin is predominantly synthesized in the liver (Yoshida 

et al., 1997). Among the several functions of albumin are its anti-inflammatory and anti-oxidant 

properties, through a number of mechanisms, as well as a binding capacity. Albumin can bind to a 

number of substances including drugs and bilirubin. In conditions such as liver cirrhosis, the ongoing 

systemic inflammation leads to imbalanced redox state. Thus, there is the formation of more oxidized 

forms of albumin. The increase in the percentages of the oxidized forms of the albumin has been 

associated with the severity of the liver cirrhosis and mortality (Domenicali et al., 2014: Oetti et al., 

2013). Albumin has been documented in a large study as a major prognostic factor that can significantly 

predict death in patients with cirrhosis (D’Amico et al., 2013). Albumin synthesis is invariably reduced 

in conditions such as liver dysfunction, as a result of at least in part, decreased synthetic capacity. Thus, 

hypoalbuminemia could reflect a destruction in liver synthetic function, and the possibility of 

developing hepatic deterioration (Carvalho and Machado, 2018). 

 

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Reduced levels of albumin lead to decreased oncotic pressure, which will allow the leakage of 

fluids from the interstitial spaces into the peritoneal cavity, leading to conditions such as ascites. 

A longitudinal study previously conducted has documented a strong association of albumin with 

liver fibrosis (Pinto et al., 2003). 

2.8.1.6 Lactate dehydrogenase 

The major cytoplasmic enzyme in the glycolytic anaerobic pathway that catalyzes the reduction of 

pyruvate to L-lactate is lactate hydrogenase (LDH) with oxidation of NADH is to NAD+. LDH is 

present in all tissues, with its isoenzymes in specific tissues. LDH 1 is mostly found in heart, 

erythrocytes, and the kidney. While LDH 3 is found predominantly in the lungs, LDH 4 is mainly 

present in the kidneys, placenta, and the pancreas. The predominant isozyme found in the liver and 

the skeletal muscle is LDH 5 (Panteginini et al., 2014). Lactate dehydrogenase (LDH) has been 

measured in haemoglobinopathies, and used as a surrogate marker for intravascular hemolysis. 

Although LDH levels in the serum has been used invariably as a marker of hemolysis, the levels 

in combination with other blood tests may also reflect tissue damage in conditions such as liver 

disease. 

2.8.2 Kidney Function Test 

2.8.2.1 Creatinine 

Creatinine is the waste product of creatine, which the muscles use to produce energy, (Kumar & Gill, 

2018). The amino acids arginine, glycine, and methionine are trans-aminated to form creatine in the 

liver, pancreas, and kidneys. Then, when creatine circulates throughout the body, it is phosphorylated 

in the brain and skeletal muscle to become phosphocreatine (Heim, 2010). It is measured in milligrams 

per deciliter (mg/dL) or millimoles per liter (mmol/L) (Schwartz & Furth, 2007). Normal ranges are 

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0.6 to 1.2 mg/dL or 60 -120 µmol/L. Creatinine levels greater than 2.0 or more in babies and 5.0 or 

more in adults   indicate severe kidney impairment. 

Kidney obstruction caused by an enlarged prostate or kidney stones, is one disorder that might affect 

the kidneys' ability to remove creatinine from the body (Gulmi & Felsen, 2012).  

Increased protein intake can potentially be a deciding factor, however studies over a longer time 

frame have indicated that high-protein diets have no appreciable effect on blood creatinine levels 

after two years (Ikizler et al., 2013).   Reduced or loss of muscle mass, rapid weight loss, and 

pregnancy all lower creatinine levels (Cartin-Ceba et al., 2007; Naylor et al., 2000). 

2.8.2.2 Urea 

Urea is a byproduct of protein metabolism and is often referred to as blood urea nitrogen (Salazar, 

2014). Protein-derived amino acids are converted to ammonia via the process of deamination. The 

liver's enzymes then change ammonia into urea (Agnelli, 2016). As a result, the amount of urea in the 

blood depends on the amount of protein consumed, how well the body can break down protein, and 

how well the kidneys can remove urea from the body. Urea is transported by the blood to the kidneys, 

where it is filtered and expelled from the body through the urine (Rawitch & Baynes, 2022, Rawitch 

et al., 2014). About 90% of the body's urea production is eliminated if the kidneys are operating 

normally (Amin et al., 2014; Dobre et al., 2013). Thus, the blood urea levels can indicate how 

effectively the kidneys are functioning. Since it is almost entirely eliminated from the body by the 

kidneys, measuring it in the blood and urine can help detect kidney illness (Yakubu et al., 2003). 

The quantity of urea in the blood can be impacted by a variety of liver and renal illnesses (Makris & 

Spanou, 2016). Urea concentrations will be high if the liver produces more of it or the kidneys are less 

effective at removing it.  

 

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3.0 RESEARCH METHODOLOGY 

3.1 Plant Collection 

The Ghana Herbarium, Department of Plant and Environmental Biology, University of Ghana, 

Legon, Accra was the place where whole plant of Synedrella nodiflora was gathered, identified, 

and validated. 

3.2 Preparation of Extract of Synedrella nodiflora. 

The stem and the leaves were air-dried for seven days, pulverized into powder, and cold-macerated 

in water containing 70% v/v ethanol. To remove traces of ethanol, the hydro-ethanolic content 

was evaporated under decreased pressure using a rotary evaporator (Buchi rotavapor® r-300, 

Flawil, Switzerland). The aqueous fraction was lyophilized after being frozen at -200°C (bench-

top freeze dryer, Labfreez Instruments Co. Ltd. Beijing, China). The extract was labeled (as SNE) 

and stored in a refrigerator at 4-8°c after calculating the percentage yield of dried SNE (10% w/w) 

(Amoateng et al., 2012). 

3.3 Animals 

The Animal Experimentation Department of the Noguchi Memorial Institute for Medical 

Research, University of Ghana, Legon, was where ICR mice (4-5 weeks old) weighing 20-30g 

were procured and kept. The animals were housed in stainless steel cages (34 cm long, 47cm wide and 

18 cm high), fed a normal commercial pellet diet (Gafco, Tema), water ad libitum, and kept under 

laboratory temperature of 24–28°C, relative humidity 60–70%. 

All of the procedures and techniques utilized in these studies followed the Standards of the 

National Institute for the care and use of laboratory animals (NIH, Department of Health Services 

publication no. 83-23, revised 1985). The Ethical and Protocol Review Committee of the 

University of Ghana's College of Health Sciences approved the study. 

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3.4 Determining the Sample Size for an Animal Study 

The resource equation approach (E= Total number of animals – Total Number of groups) was used 

to estimate sample size for the various tests (Charan & Biswas, 2013). Based on the sample size, 

a value "E" is calculated in this approach. “E” equals the total number of animals minus the total 

number of groups. The value of “E” should be between 10 and 20 for an optimum sample size. If 

“E” is less than 10, more animals should be added; if it is greater than 20, the sample size should 

be reduced. A sample size for the following tests was determined as listed below:  

a. PTZ kindling (n = 10) in three groups 

b. Acute PTZ (8 groups, n=5) 

Where “n” represents number of mice in each group. 

3.5 Chemicals and Drugs 

PTZ, Carbamazipine, Diazepam, Phenobarbitone sodium, and Flumazenil were all purchased at 

different periods of the study.  

3.6 Pentylenetetrazol (PTZ) seizure Induction. 

3.6.1 Acute PTZ Seizure Induction 

The anticonvulsant testing method described by (Amoateng et al., 2012) was used. For the acute 

PTZ behavioral experiments, the following groups were used; 

Group 1: vehicle (0.9% saline, 0.01 ml/kg) + PTZ (75 mg/kg) – Control group 

Group 2: SNE (100 mg/kg) + PTZ (75 mg/kg) 

Group 3: SNE (300 mg/kg) + PTZ (75 mg/kg) 

Group 4: SNE (1000 mg/kg) + PTZ (75 mg/kg) 

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Group 5: Phenobarbitone sodium (30 mg/kg) + PTZ (75 mg/kg) 

Group 6: Flumazinil (2 mg/kg) + PTZ (75 mg/kg) 

Group 7: Flumazinil (2 mg/kg) + SNE (1000 mg/kg) + PTZ (75 mg/kg) 

Group 8: Flumazinil (2 mg/kg) + Phenobarbitone sodium (30 mg/kg) + PTZ (75 mg/kg) 

Acute seizures were generated in drug/vehicle-pretreated male ICR mice by administering 75 

mg/kg PTZ intraperitoneally. SNE (100–1000 mg/kg, orally) or Phenobarbitone sodium (30 

mg/kg) intraperitoneally 30 minutes before PTZ administration. 

The control animals received 0.9% saline solution orally (0.01 ml/kg). After the intraperitoneal 

PTZ injection, the animals were placed in a testing chamber (made of Perspex of dimensions 15 

cm×15 cm× 15 cm). The episodes of convulsion induced by PTZ could be observed in a mirror 

angled at 45 degrees below the floor of the chamber. A camcorder (EverioTM model GZ-mg 130u, 

JVC, Tokyo, Japan) was set directly opposite the mirror to record the animals. The latencies as 

well as the duration of the seizures were determined from the video recordings using the public 

domain software JwatcherTM version 1.0 (University of California, Los Angeles, USA, and 

Macquarie University, Sydney, Australia; available at http://www.jwatcher.ulca.edu. 

Anticonvulsant action was defined as the ability of a drug/extract to prevent seizures or to prolong 

the latency or onset of tonic hind-limb extensions

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3.6.2 PTZ kindling. 

ICR Male mice (4 to 5 weeks old) weighing 25-30g were used for this procedure. Animals were 

observed in transparent Plexiglas containers that were open on the top for proper aeration. 16 

Plexiglas enclosures with enough space for mice to move freely were used. The containers were 

thoroughly rinsed using water followed by disinfection with 70% (v/v) alcohol after each group. 

Observations on determining the onset of different phases of convulsions were made using a 

tracker software. 

Different groups of mice were pretreated with Synedrella nodiflora Extract (SNE), Phenobarbitone 

sodium (PHE) and Normal Saline orally and was allowed a time lapse of 30 minutes before 

Pentylenetetrazol (PTZ) was injected at a threshold convulsant dose (35 mg/kg, I.P.). Animals 

demonstrated different phases of convulsions as follows:  

Stage 0: no response 

Stage 1: twitching of the ears and face 

Stage 2: Neck jerks 

Stage 3: myoclonic jerks  

Stage 4: rolling onto the side 

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

The mice were allowed to acclimatize for 2 hours in the testing experimental room at the Animal 

Experimentation Department at the Noguchi Memorial Institute for Medical Research, University 

of Ghana, Legon before initiating the kindling procedures. Animals were marked and weighed and 

kept in a cage for proper identification by randomization. 

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For the PTZ kindling behavioral investigations, the following groups were used. (n = 10 where 

“n” represents the number of mice in each group). 

Group 1: vehicle (0.9% saline) + PTZ (35 mg/kg) – Control group 

Group 2: SNE (1000 mg/kg) + PTZ (35 mg/kg) 

Group 3: Phenobarbitone sodium (30 mg/kg) + PTZ (35 mg/kg). 

The dose volume for all the injections were kept constant (10 ml/kg). On day one, each mouse 

received either Normal Saline, SNE or PHE, the volume of which was based on their body weight, 

30 minutes before PTZ injection. Mice were then observed for the stages of the PTZ kindling for 

30 minutes. When all three groups had been treated, animals were placed   in cages for overnight 

recovery. The kindling procedure repeated every other day over a period of 35 days and the mice 

were considered fully kindled if the mice   demonstrated stage 4 or 5 on two consecutive PTZ 

doses. On every kindling day, mice were weighed and also observed for abnormalities. A week 

after the last PTZ injection, animals were challenged with same sub-convulsant dose of 

pentylenetetrazol (35 mg/kg, i.p.). After completion of the experiment, mice were euthanized using 

a carbon dioxide euthanasia chamber. Blood samples were immediately taken for biochemical 

analysis. The hippocampus of the brain was resected for histological studies as described below. 

3.7 Histology of the Hippocampus 

Brain tissue was taken and placed in a formaldehyde solution after the mice were quickly 

decapitated before being dried with ethanol (70 percent for 24 h, 90 percent for 1 h and 100 percent 

for 1 h). After that, it was cleaned in xylene before being embedded in paraffin. Coronal slices of 

the temporal lobe of the brain were cut at 5 µm thickness exposing the hippocampus with a 

microtome (Leica RM 2025, Germany). The slices were mounted on glass slides, and stained with 

hematoxylin and eosin. Hematoxylin is used to illustrate nuclear detail in cells whilst Eosin is the 

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most commonly used counterstain that distinguishes between the cytoplasm and nuclei of cells 

(Blumcke et al., 2017). The hippocampus neuronal cell count was subsequently determined using 

light microscopy. 

3.8 Biochemical Analysis 

Intraperitoneal blood sample was collected and stored in plain glass tubes. Serum was obtained 

after centrifugation of the clotted blood. The serum was for evaluation of liver and kidney 

functions. Albumin, Globulins, Alkaline Phosphate, ALT, AST, Direct Bilirubin, Indirect 

Bilirubin, Total Bilirubin, Total Protein, Creatinine, Urea, and Blood Urea Nitrogen were all 

measured using an automated multi-parameter blood analyzer (Selectra pro and Cobas 311). 

(Thapa & Walia, 2007). 5 milliliters of serum were pipetted into the micro titer, which was then 

placed in the blood analyzer and analyzed automatically. 

3.9 Statistical Analysis 

GraphPad Prism version 5.0 for Windows, (GraphPad software, La Jolla, California, USA, 

www.graphpad.com), was used to analyze quantitative data. The means of Racine scores, as well 

as the weight of mice during the PTZ induced kindling procedure, were compared using a two-

way ANOVA followed by Bonferroni post-tests. One-way ANOVA was used to examine the area 

under the curve, latency, frequency, and duration of convulsions during acute PTZ. P<0.05 was 

considered statistically significant. The mean ± SEM was used to express all of these variables. 

The frequencies were used to present all category data. 

 

 

 

 

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CHAPTER FOUR 

4.0 RESULTS 

4.1 Stages of PTZ kindling procedure scored on the Racine scale. 

Repeated administration of 35 mg/kg of PTZ every other day caused a gradual increase in the 

seizure intensity as scored by the Racine scale in the Control group as compared to SNE and PHE 

group. By the 11th day, the score had increased from 0 to 2, and attained a severity on the Racine 

score of 3 by day 35 It showed a steady rise to 4 by the 35th day through to the 42nd day (Figure 

1A). SNE significantly depressed the kindled seizures (F2, 20 =12.55, P = 0.0003; Figure 1A) and 

none of the animals in these treatment group attained seizure score 4 or 5. The percentage severity 

of seizures (calculated from the AUC) shows that SNE reduced PTZ kindled seizure activity by 

reducing the Score 4 on the Racine scale by 35%, Figure 1B). Phenobarbitone (30 mg/kg) also 

produced a significant depression of the kindled seizure activity (P < 0.0003; Figure 1A) and the 

percent severity of seizures was significantly reduced by 25%, p value of 0.001Figure 1B). 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Fig 1.0 Seizure Intensity during PTZ-Induced Kindling 

SNE (1000 mg/kg) and PHE (30 mg/kg) dose–response effects on PTZ-kindled mice. 

(A) The time course of effects for all three groups (CTL- Red, SNE- Green, and PHE-blue) 

over a 42-day period was plotted using mean SEM (n = 10) and the values were displayed using 

Mean ± SEM. (n=10), P<0.05 when compared to the control group (2-way ANOVA followed by 

bonferroni post-tests) 

(B) For the course of the test, the AUCS was used to compute the % severity of seizures. The 

Mean ± SEM (n = 10) was used to plot the data. (+++p=0.0003, One way ANOVA followed by 

Newman-Keuls Post Hoc test). 

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4.2 Latency, frequency and duration of convulsions following Acute PTZ induced seizures. 

PTZ induced a sequence of events starting with myoclonic jerks which was then followed by an 

intense clonic convulsive phase. The SNE extract, showed significant anticonvulsant effect against 

seizures induced by PTZ. SNE increased the latency of the myoclonic jerks (F3, 20 = 4.111, P 

=0.0250; Figure 2A) and these effects were significant at dose levels of SNE 100, 300 and 1000 

mg/kg. It equally reduced the frequency of convulsions significantly (F4, 20 =5.18, P=0.0051, 

Figure 3B). However, SNE produced no significant effect on the duration of the seizures (F 4, 18 = 

2.396, P = 0.0885; Figure 2C). 

 

Figure 2: Effects of SNE (100, 300 1000 mg/kg) and PHE (30 mg/kg) on PTZ-induced seizure 

latencies, frequencies, and duration. The mean ± SEM (n = 5) is represented in each column 

compared to the Control group (one-way analysis of variance followed by Newman-Keuls post 

hoc test) (P=0.0250; figure 3a, p=0.0051, figure 3b, p = 0.0885; figure 3c). 

 

 

 

 

 

 

 

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Table 1. Effect of Flumazenil (GABA receptor blocker) on SNE and Phenobarbitone Sodium 

following PTZ convulsions. 

 

Drug (Dose) Latency Frequency Duration 

      

Flumazenil (10 ml) + CTL (0.9w/v saline) 155.8 ± 25.90 2.4 ± 0.7 29.25 ± 10.05 

Flumazenil (10 ml) + SNE (1000 mg/kg) 96.00 ± 7.66 1.8 ± 0.2 29.25 ± 8.03 

Flumazenil (10 ml) + PHE (30mg/kg) 242.2 ± 25.21* 2.8 ± 1.1 28.50 ± 5.07 

Values are expressed as Mean ± SEM (n=5). *P<0.05 compared to vehicle treated control group. 

(One-way ANOVA followed by Newman-Keuls Post hoc test). 

 

 

4.3 Effect of Flumazenil (GABA receptor Blocker) on SNE and Phenobarbitone sodium 

following induced convulsions 

SNE (1000 mg/kg) administered after Flumazenil (10ml, i.p) injection did not significantly 

affect latency, frequency and duration compared to control group. Diazepam, on the other 

hand, increased the latency of onset of seizures significantly (p <0.01), but had no significant 

effect on the frequency and duration of seizures (p<0.6 and p<0.065 respectively) compared 

to control (table 1). 

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4.4 Biochemical Analysis after PTZ Kindling Procedure 

The liver and the kidney parameters were examined after the PTZ procedure. Parameters such as 

albumin and Alkaline Phosphate (ALP) showed a significant difference comparing SNE (1000 

mg/kg) to the control group (CTL) (p<0.3). Generally, there was a slight insignificant rise in liver 

enzymes ALT and AST across the three groups however. Creatinine levels were generally reported 

to be low across all the groups with Urea and Blood Urea nitrogen (BUN) been reported to be in 

normal ranges.  

Table 2: Biochemical analysis after PTZ kindling Procedure. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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4.5 Histology of the Hippocampus following PTZ kindling. 

Fig 4 shows the stained sections of the hippocampus as well as the neuronal cell density count (in 

thousands of cells per mm
3
) after chronic PTZ induced kindling procedure. There was a slight 

hippocampal neuronal cell increment of SNE (1000 mg/kg) and PHE (30mg/kg) compared to the 

control group, however they were not statistically significant (P= 0.0631). 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig 3. Photomicrograph of the hippocampus of the brain following PTZ induced kindling. 

A- Control group, B- SNE and C-PHE). A graph of the neuronal cells count has been plotted (D). 

Values were plotted using Mean ± SEM (One-way ANOVA followed by Newman-Keuls post hoc 

test). 

 

 

 

 

 

 

 

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4.6. Average Mice Body weight After PTZ kindling. 

Fig. 5 shows the weight of the mice following the PTZ kindling procedure. In the control group, 

there was a steady rise in weight reaching a peak weight of 35g on day14. This was followed by a 

fall in weight till day 17 and started to rise again. This is slightly different in the case of SNE which 

achieved a peak weight of 34g on day 12 followed by a steady decline till day 19. It was steady 

after day 21. There were no statistical differences observed comparing the weight of SNE (1000 

mg/kg) with the control group. Phenobarbitone Sodium (30 mg/kg), a reference drug did reach a 

peak weight of 32g on day 13 after which it was characterized with steady decline till the last day. 

 

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Fig.4 Effects of PTZ kindling on Body weight 

The effect of SNE (1000 mg/kg) and PHE (30 mg/kg) on body weight following PTZ induced 

convulsions. Values were plotted using mean ± SEM (n = 10). (A- 2-way ANOVA followed by 

Bonferroni post-tests). (B- One-way ANOVA followed by Newman-Keuls Post Hoc Test). 

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CHAPTER FIVE 

5.0 DISCUSSION 

5.1 SNE Inhibited Both Acute and Chronic PTZ induced Seizures Suggestively via 

GABAergic Pathway. 

The study revealed that SNE inhibited PTZ-induced seizures in chronic phases. The PTZ test 

represents a valid model for human generalized and absence seizures. PTZ has been used 

experimentally to study seizure phenomenon and to identify drugs that may control seizure 

susceptibility (Amoateng et al., 2012; Hamed, 2019). 

The neuronal basis underlining the mechanism of seizure action of PTZ still remains unclear.  

In this study, pretreating the mice with flumazenil (GABAA receptor blocker) followed by SNE 

administration showed no significant difference after PTZ induced seizures compared to the 

control group. This somewhat suggest that the extract reduce epileptic seizures via the 

GABAergic Pathway. 

Reference anticonvulsant drugs such as diazepam and phenobarbitone, inhibit PTZ-induced seizure 

by enhancing the action of GABAA receptors, possibly by facilitating the GABA-mediated opening 

of chloride channels (Mohler et al., 2002). Postsynaptic GABAA receptors are multiunit complexes 

with binding sites for the endogenous ligand GABA, benzodiazepines, barbiturates, and other 

ligands with a central chloride ion channel (Ralvenius et al., 2015; Rudolph & Knoflach, 2011). 

Hence for SNE to reduce both latency, frequency of convulsions   when applied alone but not when 

administered after flumazenil during acute PTZ induced seizures supports the assertion that SNE 

acts via the GABAergic pathway. 

5.2 SNE significantly reduced PTZ induced seizures 

The outcome of this study also points to a significant progressive decrement of seizure induced 

by PTZ kindling measured using the Racine score. PTZ-induced kindling is an acknowledged 

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experimental model of human seizures and useful for the study of seizure mechanisms 

(Samokhina & Samokhin 2018; Dhir 2012). It is characterized by cerebral deficit, changes in 

emotional behavior, and neuronal cell loss in hippocampal CA1, CA3, CA4 structures and 

dentate gyrus (Zhu et al., 2016; Dhir 2012). This in contrast with the result of this study wherein 

there was some neuronal cell lost in the control group compared to the extract group even 

though it was not statistically significant. 

SNE's ability to prevent full kindling by PTZ in mice may be as a result of its ability to inhibit 

lipid peroxidation and/or scavenge free radicals. This is supported by the fact that free radical 

generation due to increased activity of the glutamate plays a fundamental role in neuronal cell 

death associated with PTZ kindling in mice (Kutluhan et al., 2009; Bashkatova et al., 2003). 

When the production of free radicals increases or the defense mechanism of the body decreases, 

lipid peroxidation at polyunsaturated sites on biological membranes occurs leading to cell 

dysfunction (Akbas et al., 2004). 

5.3 Effect of SNE on Biochemical Indices. 

The study as well investigated the effects of chronic PTZ induced kindling on Renal and Liver 

indices of the ICR mice. Although over all, there was a general increment in the serum 

concentrations for the liver enzymes, however, it was not significant comparing treatment 

groups to the control. The observed increment in the enzyme levels associated with liver 

function (ALT, AST & ALP) following the PTZ induce kindling procedure suggests organ 

damage especially the liver. This may not be extensive and as such does not raise cause for 

concern. However, further studies will be recommended to ensure the full safety profile of SNE 

should these studies proceed to clinical stages. 

Serum alanine transaminases (ALT) is not only measured to determine a possible liver damage, 

but also useful in monitoring the general health status (Vazquez et al., 2020; Joni et al., 2020). 

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This enzyme is mainly found in the cytosol of the hepatocytes, and plays a role in 

gluconeogenesis (Moriles and Azer 2020). ALT as well as the other enzymes are released as a 

result of liver injury from the injured liver cells into the serum, which causes the levels to 

increase significantly in the serum (Ghosh et al., 2020; Smith et al., 2020).  

Also, low creatinine levels observed and it can be associated with less muscle metabolism in 

the mice. This correlates with a study by Rodrigues et al., (2014) whose study sought to 

establish standards for renal functions in mice through body size adjusted creatinine and urea 

levels. In their study they found out body size and muscle mass had influence on creatinine 

levels. As there was treatment induced weight changes in this study, changes in the creatinine 

levels would not have been strange. Nonetheless, one can only conclude that the weight 

changes were not drastic enough to be reflected in the creatinine levels. 

Overall, the biochemical parameters (Kidney and Liver functions) assessed did not show any 

deteriorating effect following the application of the extract SNE.  

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

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CHAPTER SIX 

6.0 Conclusion and Recommendation 

6.1 Conclusion 

The result of this study provides evidence that the ethanolic extract of the whole plant of S. 

nodiflora possesses antiseizure activity and suggested to be via the GABAergic pathway in 

murine experimental models. Also, the biochemical analysis which assesses the overall health 

of the kidney and the liver was normal in the dose dependent SNE group. Therefore, the 

effectiveness of the plant's extract used suggests that the herb can be used in treating seizures.  

6.2 Recommendations 

Future studies that will seek to explore the other pathways (eg. Glutamate pathway) is highly 

recommended. Also, an electrophysiological study to measure electrical changes in the entire 

brain specifically the hippocampus of the brain following the extract application would yield 

a better understanding of the mechanism of action of the extract in the brain. And lastly future 

studies focusing on Calcium Imaging Technique to check the impact of the extract on calcium 

ions at the synapse is greatly recommended. 

 

 

 

 

 

 

 

 

 

 

 

 

 

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