UNIVERSITY OF GHANA 
COLLEGE OF HEALTH SCIENCES 
 
ANTINOCICEPTIVE ACTIVITY OF THE HYDRO-ETHANOLIC EXTRACT OF THE 
LEAVES OF MALLOTUS OPPOSITIFOLIUS (GEISELER) MULL. ARG. IN ACUTE 
AND CHRONIC PAIN MODELS 
BY 
ADDAI, EKOW OKWAN 
(10637568) 
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, 2019 
  
i 
 
DECLARATION 
 
DECLARATION BY THE CANDIDATE 
I hereby declare that this is the product of my own research undertaken under supervision and 
has neither been presented in whole nor in part for another degree elsewhere. I am solely 
responsible for any residual flaws in the work. 
Signature…………………………..                                                 Date: 27/05/2020 
EKOW OKWAN ADDAI (10637568) 
 
DECLARATION BY SUPERVISORS 
We hereby declare that the principal work and presentation of the thesis were supervised by us 
in accordance with guidelines on supervision of thesis laid down by the University of Ghana. 
Principal supervisor 
Signature……………………                                                            Date: 29/05/2020 
(Dr. Patrick Amoateng) 
 
Co-supervisor 
Signature……………………                                                             Date 29/05/2020 
(Dr. Kennedy Kwami Edem Kukuia) 
 
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DEDICATION 
I dedicate this work to the Almighty God. 
  
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ACKNOWLEDGEMENTS 
I want to express my sincere and heartfelt gratitude to the Almighty God for the strength and 
energy to go through this program successfully. 
I would love to express my deep thanks to Dr. Patrick Amoateng and Dr. Kennedy Kwami 
Edem Kukuia of the Department of Pharmacology and Toxicology, School of Pharmacy, 
College of Health Sciences, University of Ghana for their scientific support, guidance, 
encouragement and invaluable supervision. 
I also want to thank the Department of Pharmacology and Toxicology, School of Pharmacy, 
University of Ghana, for the training, exposure and experience they offered me during my 
postgraduate studies. 
My deepest gratitude to all the staff of the Department of Animal Experimentation, Noguchi 
Memorial Institute of Medical Research, University of Ghana, for the training and help they 
offered during the laboratory research period. My sincere gratitude to Dr. Samuel Adjei (Head 
of Department), Mr. Believe Ahedor, Dr. Emmanuel Kodua, Mr. Richard Obeng-Kyeremeh, 
Dr. David Barnes, Ms Naa Adei Kotey and Mr. Benjamin Addo for their immense help and 
technical advice. 
My gratitude goes to Prof. Dorcas Osei-Sarfo, Department of Chemistry, University of Ghana 
for her immense contribution, support and technical advice. I want to thank Emmanuella 
Twumasi, John Obeng and Eric Coffie, all from the Chemistry Department for their technical 
help and advice. 
Special thanks to my parents, Mr and Mrs Nartey for all the financial support and advice, and 
to my siblings for standing by me. May the blessings of the Almighty manifest in your lives 
wherever you find yourselves.   
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TABLE OF CONTENTS 
DEDICATION ....................................................................................................................................... iii 
ACKNOWLEDGEMENTS ................................................................................................................... iv 
LIST OF FIGURES ............................................................................................................................. viii 
LIST OF TABLES .................................................................................................................................. x 
ABSTRACT .......................................................................................................................................... xii 
CHAPTER ONE ................................................................................................................................... 1 
1 INTRODUCTION ............................................................................................................................... 1 
1.1. Background ..................................................................................................................... 1 
1.2. Problem Statement .......................................................................................................... 2 
1.3. Justification ..................................................................................................................... 3 
1.4. Hypothesis ....................................................................................................................... 5 
1.5. Aim of Study ................................................................................................................... 5 
1.6. Specific Objectives ......................................................................................................... 5 
CHAPTER TWO .................................................................................................................................. 6 
2 LITERATURE REVIEW .................................................................................................................... 6 
2.1. Pain: General Overview .................................................................................................. 6 
2.1.1. The Nociceptive System, Pain Pathways and Mechanisms ..................................... 8 
2.1.2. Phases of Nociceptive pain .................................................................................... 10 
2.1.3. Classification of Pain ............................................................................................. 13 
2.2. Types of Analgesics for the pharmacological management of pain ............................. 18 
2.2.1. Non-Steroidal Anti-Inflammatory Drugs (NSAIDS)............................................. 18 
2.2.2. Opioids ................................................................................................................... 19 
2.2.3. Adjuvant analgesics ............................................................................................... 19 
2.3. Traditional Medicine ..................................................................................................... 20 
2.4. Mallotus oppositifolius .................................................................................................. 21 
2.4.1. Ethnopharmacological Uses of M. oppositifolius .................................................. 22 
2.4.2. Non-Medicinal Uses of M. oppositifolius .............................................................. 23 
2.4.3. Previous studies on M. oppositifolius .................................................................... 23 
CHAPTER THREE ............................................................................................................................ 26 
3 MATERIALS AND METHODS ....................................................................................................... 26 
3.1. Plant Material Collection .............................................................................................. 26 
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3.2. Preparation of Plant Extract .......................................................................................... 26 
3.3. Preliminary High-Performance Liquid Chromatography (HPLC) of MOE ................. 26 
3.4. Preliminary Phytochemical Screening .......................................................................... 27 
3.4.1. Test for Tannins ..................................................................................................... 27 
3.4.2. Test for Alkaloids .................................................................................................. 27 
3.4.3. Test for Saponins ................................................................................................... 28 
3.4.4. Test for Reducing Sugars ....................................................................................... 28 
3.4.5. Test for Sterols ....................................................................................................... 28 
3.4.6. Test for Terpenoids ................................................................................................ 28 
3.4.7. Salkowski’s Test for Glycosides ............................................................................ 28 
3.4.8. Test for Anthraquinones and Anthracene Derivatives ........................................... 29 
3.4.9. Test for Flavonoids (Shinoda’s test) ...................................................................... 29 
3.5. Experimental Animals, Housing and Ethical Considerations. ...................................... 29 
3.6. Drugs and Chemicals Used ........................................................................................... 30 
3.7. Experimental Design ..................................................................................................... 30 
3.8. Acute models of pain .................................................................................................... 31 
3.8.1. Formalin Test ......................................................................................................... 31 
3.8.2. Acetic Acid-Induced Writhing Assay .................................................................... 32 
3.8.3. Hot Plate Test ......................................................................................................... 33 
3.8.4. Possible Mechanism(s) of Antinociception ........................................................... 34 
3.9. Assessing the Effect of MOE in Chronic Pain Models ................................................. 36 
3.9.1. Induction of Peripheral Neuropathy ...................................................................... 36 
3.9.2. Thermal Hyperalgesia (Hot Plate test) ................................................................... 37 
3.9.3. Mechanical Hyperalgesia (Randall-Selitto test) .................................................... 38 
3.9.4. Cold Allodynia ....................................................................................................... 38 
3.9.5. Sub-chronic Oral Toxicity (90-Day) ...................................................................... 39 
3.10. Statistical Analysis ...................................................................................................... 41 
CHAPTER FOUR ............................................................................................................................... 42 
4 RESULTS .......................................................................................................................................... 42 
4.1. HPLC Analysis of MOE ............................................................................................... 42 
4.2. Phytochemical Screening .............................................................................................. 44 
4.3. The Effect of MOE in Acute Models of Pain ............................................................... 45 
4.3.1. Formalin Test ......................................................................................................... 45 
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4.3.2. Acetic Acid Induced Writhing Test ....................................................................... 48 
4.3.3. Hot Plate Test ......................................................................................................... 51 
4.3.4. The Possible Mechanism(s) of Antinociception .................................................... 53 
4.4. The Effect of MOE in Paclitaxel-induced Peripheral Neuropathy ............................... 58 
4.4.1. Induction of Peripheral Neuropathy ...................................................................... 58 
4.4.2. Thermal Hyperalgesia (Hot Plate test) ................................................................... 60 
4.4.3. Mechanical Hyperalgesia (Randall-Selitto test) .................................................... 63 
4.4.4. Cold Allodynia ....................................................................................................... 65 
4.5. Sub-chronic Oral Toxicity (90-Day) ............................................................................. 68 
4.5.1. Necropsy and clinical signs ................................................................................... 68 
4.5.2. Effect of daily MOE administration on weight change ......................................... 69 
4.5.3. Effect of daily MOE administration on relative organ weights ............................. 69 
4.5.4. Effect of daily MOE administration on RBC Indices ............................................ 69 
4.5.5. Effect of daily MOE administration on WBC Indices ........................................... 70 
4.5.6. Effect of daily MOE administration on Platelet Indices ........................................ 70 
4.5.7. Effect of daily MOE administration on serum biochemistry parameters .............. 71 
4.5.8. Histopathology ....................................................................................................... 73 
CHAPTER 5 ........................................................................................................................................ 75 
5 DISCUSSION, CONCLUSION AND RECOMMENDATIONS ..................................................... 75 
5.1. DISCUSSION ............................................................................................................... 75 
5.2. CONCLUSION ............................................................................................................. 84 
5.3. RECOMMENDATIONS .............................................................................................. 84 
REFERENCES .................................................................................................................................... 86 
 
 
  
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LIST OF FIGURES 
Figure 2.1. Overview of the pain pathway (dreamstime.com, 2019) ........................................ 9 
Figure 2.3. Illustration showing the substantia gelatinosa and the chemicals involved in 
transmission and modulation. .................................................................................................. 13 
Figure 2.4. The four primary types of pain (Woolf, 2004) ...................................................... 15 
Figure 2.5. Photograph showing the leaves and inflorescence of M. oppositifolius ................ 21 
Figure 2.6. Chemical structures of mallotojapinin C (1), tetramethylmallotojapinin C (2), 
mallotophenone (3) and mallotojaponin B (4). ........................................................................ 25 
Figure 2.7. Chemical structure of Mallotoxin (Rottlerin) ........................................................ 25 
Figure 4.1. HPLC chromatogram of MOE monitored at 272, 278nm .................................... 42 
Figure 4.3.1: Effect of MOE (30, 100 and 300 mg/kg), diclofenac (3, 10 and 30 mg/kg) and 
morphine (0.3, 1 and 3 mg/kg) on the time-course events (A, C, E respectively) and the total 
nociceptive score (calculated as AUCs) (B, D, F respectively) on formalin-induced 
nocifensive behaviour in ICR mice.......................................................................................... 47 
Figure 4.3.2. Effect of MOE (30, 100 and 300 mg/kg) and diclofenac (3, 10 and 30 mg/kg) 
on the time-course events (A and C respectively) and the total nociceptive score (calculated 
as AUCs) (B and D respectively) on acetic acid induced writhing behaviour in ICR mice. ... 50 
Figure 4.3.3. Effect of MOE (30, 100 and 300 mg/kg) and morphine (0.3, 1 and 3 mg/kg) 
on the time-course events (A and C respectively) and the total antinociceptive score 
(calculated as AUCs) (B and D respectively) on thermally induced jumping and paw licking 
behaviour in ICR mice. ............................................................................................................ 52 
Figure 4.4.1. The effect of the pre-treatment of ICR mice with (B) naloxone (2 mg/kg), (C) 
glibenclamide (8 mg/kg), (D) theophylline (10 mg/kg), (E) ondansetron (0.5 mg/kg), (F) 
yohimbine (3 mg/kg), (G) nifedipine (10 mg/kg) and (H) atropine (5 mg/kg) on the 
antinociceptive activity of MOE (100 mg/kg p.o) in the first and second phases of formalin-
induced nociception. ................................................................................................................ 55 
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Figure 4.4.2. The effect of the pre-treatment of ICR mice with (B) naloxone (2 mg/kg), (C) 
glibenclamide (8 mg/kg), (D) theophylline (10 mg/kg), (E) ondansetron (0.5 mg/kg), (F) 
yohimbine (3 mg/kg), (G) nifedipine (10 mg/kg) and (H) atropine (5 mg/kg) on the 
antinociceptive activity of morphine (1 mg/kg i.p) in the first and second phases of formalin-
induced nociception. ................................................................................................................ 56 
Figure 4.4.3. The effect of the pre-treatment of ICR mice with naloxone (2 mg/kg), 
glibenclamide (8 mg/kg), theophylline (10 mg/kg), ondansetron (0.5 mg/kg), yohimbine (3 
mg/kg), nifedipine (10 mg/kg) and atropine (5 mg/kg) on the antinociceptive activity of (A) 
MOE (100 mg/kg p.o) and (B) morphine (1 mg/kg i.p) in the first and second phases of 
formalin-induced nociception. ................................................................................................. 57 
Figure 4.5.1: Effect of the daily intraperitoneal administration of paclitaxel (2 mg/kg) on 
thermal hyperalgesia (A-B), mechanical hyperalgesia (C-D) and cold allodynia (E-F) tests 
in ICR mice for 5 days. ............................................................................................................ 59 
Figure 4.5.2: Effect of MOE (30, 100 and 300 mg/kg) and Pregabalin (10, 30 100 mg/kg) 
on thermal hyperalgesia in paclitaxel induced neuropathy in ICR mice. The left panels 
represent time-course effects of MOE (A) and pregabalin (C) from day 0 to day 5 after 
neuropathy was confirmed. ...................................................................................................... 61 
Figure 4.5.3: Effect of MOE (30, 100 and 300 mg/kg) and Pregabalin (10, 30 100 mg/kg) 
on mechanical hyperalgesia in paclitaxel induced neuropathy in ICR mice. The left panels 
represent time-course effects of MOE (A) and pregabalin (C) from day 0 to day 5 after 
neuropathy was confirmed. ...................................................................................................... 64 
Figure 4.5.4. Effect of MOE (30, 100 and 300 mg/kg) and Pregabalin (10, 30 100 mg/kg) 
on cold allodynia in paclitaxel induced neuropathy in ICR mice. ........................................... 66 
Figure 4.6.1. Mean weights of Sprague-Dawley rats for all groups(n=5) from day 1 to day 
91.............................................................................................................................................. 68 
Plate 4.5.8. Photomicrographs of the brain, kidney, liver, heart and spleen in distilled water 
treated rats and rats treated with MOE (30, 100 and 300 mg/kg) daily over the 90-day sub-
chronic toxicity study period (H & E, ×400). .......................................................................... 74 
 
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LIST OF TABLES 
Table 4.1. Report on the HPLC analysis of MOE ................................................................... 43 
Table 4.2. Preliminary phytochemical screening of MOE ...................................................... 44 
Table 4.3.1.1. ED50s for MOE, diclofenac and morphine in the formalin test ........................ 48 
Table 4.4. ED50s for MOE and diclofenac in the writhing test ................................................ 49 
Table 4.5. ED50s for MOE and morphine in the hot plate test ................................................. 53 
Table 4.5.2. ED50s for MOE and pregabalin in the thermal hyperalgesia test ......................... 62 
Table 4.5.3. ED50s for MOE and pregabalin in the mechanical hyperalgesia test ................... 65 
Table 4.5.4. ED50s for MOE and pregabalin in the cold allodynia test ................................... 67 
Table 4.6.1. The relative organ weights of rats treated with MOE over the 90-day period .... 69 
Table 4.6.2. The haematological indices of rats treated with MOE over the 90-day period ... 71 
Table 4.6.3. Serum biochemistry parameters of rats treated with MOE over the 90-day 
period ....................................................................................................................................... 72 
  
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LIST OF ABBREVIATIONS 
 
MOE Mallotus oppositifolius extract 
5-HT3 5-Hydroxytryptamine 
ATP Adenosine Triphosphate 
CNS Central Nervous System 
COX Cyclooxygenase 
DPX Dibutyl phthalate Polystyrene Xylene 
ED50 Effective Dose at 50% 
EDTA Ethylenediamine Tetraacetic Acid 
GABA Gama Amino Butyric Acid 
IASP International Association for the Study of Pain 
ICR Institute of Cancer Research 
NMDA N-methyl-D-aspartate 
NPC National Pharmaceutical Council 
NSAIDs Non -steroidal Anti- inflammatory drugs 
PAG Periaqueductal Gray 
SNRI Serotonin-Norepinephrine Reuptake Inhibitor 
SSRI Selective Serotonin Reuptake Inhibitor 
TCA Tricyclic Antidepressants 
TNF Tumour necrosis factor 
 
  
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ABSTRACT 
BACKGROUND: Mallotus oppositifolius is a medicinal plant commonly used in folk 
medicine to treat disorders involving pain and inflammation, among other ailments. However, 
the potential of this herb for the treatment of pain has only rudimentary scientific validation. 
The study sought to investigate the analgesic activity of the hydro-ethanolic extract of the 
leaves of M. oppositifolius (MOE) in order to provide detailed scientific justification for its 
traditional folkloric use.  
METHODS: Preliminary phytochemical screening was conducted on the MOE. 
Antinociceptive activity of MOE (30, 100 and 300 mg/kg) in acute pain models was evaluated 
using the formalin test, the acetic acid-induced writhing assay and the hot plate test. The 
mechanism of antinociception was evaluated by employing various antagonists in the formalin 
test including naloxone (2 mg/kg), glibenclamide (8 mg/kg), theophylline (10 mg/kg), 
ondansetron (0.5 mg/kg), yohimbine (3 mg/kg), nifedipine (10 mg/kg) and atropine (5 mg/kg). 
For the chronic pain model, peripheral neuropathy was induced in ICR mice using paclitaxel 
(2 mg/kg, i.p.) for 5 days and the effect of the extract on paclitaxel-induced peripheral 
neuropathy was evaluated daily for 5 days post induction using the Randall-Selitto test for 
mechanical hyperalgesia, hot plate test for thermal hyperalgesia and cold water at 4℃ for cold 
allodynia with pregabalin (10, 30 and 100 mg/kg) as the positive control. Sub-chronic toxicity 
study was carried out with male Sprague-Dawley rats divided into four groups; group 1 
received vehicle p.o. while groups 2-4 were orally treated with MOE daily at 30, 100 and 300 
mg/kg for 90 days consecutively. At the end of the test mice were sacrificed for their organs 
for histopathological analysis and their whole blood and serum for haematological and 
biochemical analysis respectively. 
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RESULTS: Phytochemical screening confirmed the presence of secondary metabolites 
including saponins, alkaloids, flavonoids, glycosides, sterols, tannins and reducing sugars. 
MOE together with morphine and diclofenac (positive controls) showed significant (P<0.05) 
and dose-dependent decrease in the nociceptive scores in the formalin test, the acetic acid 
induced writhing assay and an increase in antinociceptive scores in the hot plate test. Naloxone, 
theophylline, ondansetron, glibenclamide, atropine, yohimbine and nifedipine failed to block 
the antinociceptive effects of MOE (100 mg/kg) in the formalin test. The extract significantly 
(P<0.05) and dose-dependently increased the paw withdrawal latencies to mechanical and 
thermal hyperalgesia after the 5–day induction of neuropathy with paclitaxel in mice. In the 
90-day sub-chronic toxicity study, data analysis of mortality, body weight gain, clinical 
observations, haematology, biochemistry, organ weights and histopathological findings did not 
show significant differences between control and treated groups.  
CONCLUSION: The oral administration of a hydro-ethanolic leaf extract of Mallotus 
oppositifolius produces dose-dependent antinociceptive effects in formalin, acetic acid-induced 
and thermal acute pain tests, and also significantly inhibited mechanical and thermal 
hyperalgesia in paclitaxel-induced peripheral neuropathy in mice. There was no extract-
treatment related toxicity in rats following a 90-day daily oral administration. The findings 
support the traditional uses of the plant in alleviating pain. 
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Chapter One 
1 INTRODUCTION 
1.1. Background 
Pain is defined by the International Association for the Study of Pain (IASP) as an unpleasant 
sensory and emotional experience associated with actual or potential tissue damage (Merskey 
et al., 1994). In fact, a study by Baigi and Stewart (2015) indicated that pain is a major reason 
for underemployment, absenteeism from work and unemployment, therefore, causing financial 
loss to individuals and countries. Pain is a consequence of many diseases and is among the 
main reasons why several people seek medical care (American Pain Society, 2000). There have 
been substantial advances in the management of pain with the introduction of new drugs 
through research in the last decade. However, many people continue to suffer from pain due to 
the fact that current analgesics are used inappropriately (Chen et al., 2012; Moore, 2013) and 
they are fraught with many side effect and low therapeutic efficacy (Boakye-Gyasi et al., 2017; 
Rajdev et al., 2018). 
In many countries, the use of medicinal plants for the management of pain is common (Kukuia 
et al., 2016). In Ghana, the traditional use of plants for the treatment and management of pain 
and other diseases such as epilepsy, malaria, fever, and depression is widespread (Agyare et 
al., 2014; Bremer Christensen et al., 2015; Kukuia et al., 2016). About 25 percent of drugs 
currently in use are derived from plants (Wachtel-Galor & Benzie, 2011). Among the 
extensively used plants for medicinal purposes is Mallotus oppositifolius, a dioecious shrub 
that is extensively distributed across Africa in countries including Cameroon, Senegal, Angola, 
Ethiopia, Mozambique, Madagascar, Ghana and Nigeria (Farombi et al., 2001; Farombi, 2003). 
In Ghana, it is called ‘sroti’ by the Ewes and ‘sratadua’ by the Akans (Burkill et al., 1985). In 
West Africa, the leaves are chewed or crushed and placed on cuts and sores for antibacterial 
1 
 
effect and wound healing. Crushed leaves are also commonly applied to burns for analgesic 
effect (Adekunle & Ikumapayi, 2006; Agyare et al., 2014). The leaves are used for many 
conditions including worm infestation, diarrhoea, malaria, epilepsy and pain among others 
(Farombi, 2003; Adekunle & Ikumapayi, 2006). Very little work has been done on the 
analgesic effect of M. oppositifolius. Documenting the traditional knowledge and scientifically 
authenticating traditional medicinal plants is a very important path (Uprety et al., 2010). In the 
work done by Farombi et al. (2001), phytochemicals such as the alkaloids, anthraquinones, 
phenols, cardenolides and flavonoids were observed to be present through phytochemical 
screening. Gbedema et al. (2010) also reported that the leaf extract of M. oppositifolius 
exhibited significant antimicrobial activity against Baccilus subtilis, Pseudomanas aeruginosa 
and Staphylococcus aureus.  
Despite the many traditional indications of M. oppositifolius, this research will focus on 
scientifically validating its folkloric use as an analgesic in traditional medicinal practice in 
Ghana. 
1.2. Problem Statement 
Pain can have very detrimental effects on the quality of life of an individual. It can disrupt 
normal living, normal sleep, functional capability and can degrade or deteriorate an 
individual’s health (Elliott et al., 1999). In creating more efficacious medications for pain 
management, the complexity of the disease condition (e.g. diabetes, cancer) from which the 
pain arises can be a challenge (Jarvis & Boyce-Rustay, 2009). Even though pain medication, 
which includes opioids and non-opioids, is widespread, pain management in patients with mild 
to severe pain in many countries is still ineffective (Boakye-Gyasi et al., 2017). This is 
frequently attributed to both an improper use of analgesics and the unavailability of effective 
and efficacious analgesics contributing to an increase in the cost of pain management (Boakye-
2 
 
Gyasi et al., 2017). It has been shown that current analgesics are ineffective in treating 
chemotherapy-induced peripheral neuropathy which develops in about 30 to 40% of cancer 
patients (Deng et al., 2012; Amoateng et al., 2015). This often leads to the discontinuation of 
their cancer chemotherapy (Deng et al., 2012).  
Current optimized pain management strategies in individuals/patients, especially those having 
complicated neuropathic pain, include combining analgesics from several drug classes such as 
anticonvulsants (pregabalin, gabapentin and carbamazepine), tricyclic antidepressants 
(amitriptyline) and analgesics (fentanyl patches and tramadol) to address multiple pain 
mechanisms (Amoateng et al., 2015; Amoateng et al., 2017). This presents a number of severe 
side effects (emesis, gastrointestinal problems, renal damage, addiction) and limited efficacy 
in pain management which in the end impedes effective symptom control (van den Bent, 2005). 
Only about 40 to 60% of patients being given current conventional treatments, which includes 
opioid analgesics, tricyclic antidepressants, and anticonvulsants, achieve partial pain relief 
(Dworkin et al., 2007; Dworkin et al., 2010; Amoateng et al., 2017). Therefore, analgesics that 
are cost-effective, efficacious, easy to access and have fewer side effects are needed. Natural 
medicinal plants could be very important sources of novel analgesics. 
1.3. Justification 
Although pharmacological management of pain remains an important therapeutic option, 
current pain treatment is far from perfect and is riddled with considerable side effects (Woode 
& Abotsi, 2011; Amoateng et al., 2015). In addition, many patients battling with pain are not 
satisfied, prompting the need for research into finding new and promising therapeutic agents 
with minimum adverse effects and an improved efficacy to treat/manage pain. Plants used in 
traditional medicine are alternative sources of drug discovery (Kukuia et al., 2018), from which 
we can obtain lead compounds and commercial drugs (Iliya et al., 2016). Many breakthrough 
3 
 
drugs currently in use were discovered from plant sources. Examples are morphine, which was 
isolated from Papaver somniferum and is used clinically for the management of pain, and 
vincristine, which was isolated from Vinca roseus and is used as an anticancer agent (Staerk et 
al., 2002; Norn et al., 2005). 
Also, the use of plant sources by traditional healers as drugs in disease management brandishes 
these sources as a strong potential for drug discovery and pharmacological management 
(Balunas & Kinghorn, 2005; Abotsi et al., 2017). This makes the assessment of medicinal 
plants as alternative or complementary therapies an important strategy. 
Mallotus oppositifolius (family Euphorbiaceae) is an example of such plants used traditionally 
in managing pain and a spectrum of other diseases including epilepsy and malaria (Woode et 
al., 2012). The genus Mallotus is a widely known rich source of natural bioactive compounds 
(Kabran et al., 2015). Previous preliminary screening of central effects of the hydroethanolic 
leaf extract of Mallotus oppositifolius revealed that it possessed significant antinociceptive 
effect in a tail immersion test model in mice (Woode et al. (2012). Literature search reveals 
that aside the aforementioned study, there are no extensive scientific reports that support either 
the short term or long-term use of the leaves of M. oppositifolius traditionally in the 
management of pain as an effective therapy. Against this backdrop, this study seeks to 
scientifically validate the pharmacological activity of Mallotus oppositifolius leaf extract in the 
management of pain, investigate to provide information on its long-term-usage effects and 
elucidate its possible mechanism(s) of action. This will go a long way to aid in our pursuit of 
discovering alternative drugs that are more effective and safer for the management of pain. 
4 
 
1.4. Hypothesis 
Being traditionally used in the management of pain, the hydro-ethanolic extract of M. 
oppositifolius will demonstrate an analgesic effect in the experiments to be conducted. 
1.5. Aim of Study 
The study seeks to investigate an antinociceptive effect of the hydroethanolic leaf extract of M. 
oppositifolius in acute and chronic models of pain, elucidate the possible mechanism(s) of 
action and conduct a sub-chronic oral toxicity of the leaf extract. 
1.6. Specific Objectives 
1. To qualitatively assess the phytochemicals, present in M. oppositifolius extract. 
2. To assess the antinociceptive properties of the leaf extract using animal models for 
nociceptive pain (acute) and a peripheral neuropathy model (chronic). 
3. To elucidate the possible mechanisms through which the leaf extract exerts its analgesic 
effect. 
4. To evaluate the 90-day sub-chronic oral toxicity of the leaf extract. 
  
5 
 
Chapter Two 
2 LITERATURE REVIEW 
2.1. Pain: General Overview 
Pain is a sensory experience associated with hurting and soreness, and is intrinsically 
unpleasant (Cimmino et al., 2011). The sensation of pain is mostly evoked by a potential 
noxious stimulus or an actual noxious stimulus applied to the body (Schaible, 2007). Pain has 
also been reported in patients suffering from nerve injury or even brain injury (Rang et al., 
2016). Under normal circumstances, the degree of pain felt is directly linked to the noxious 
stimuli and this causes the sufferer to evade situations and behaviours that are known to evoke 
pain. Pain is essentially a sensation but is often described in terms of suffering, associated with 
alterations in autonomic output and avoidance motor reflexes (Woolf, 2004). This means that 
the perception of pain and its thresholds result from complex interactions between emotional, 
behavioural and sensory factors (Cole, 2002). Also, the pain experienced by a person is 
subjective and is modulated by genetic factors as well as other factors such as mood, beliefs 
and cognition (e.g., fear, distraction, anxiety and catastrophizing) making it expedient to tailor 
the management of pain to a particular patient (Marchand, 2008; Nix, 2017; Zortea et al., 2018). 
Pain that is experienced as a result of a disease is dissimilar to “normal pain”, since this usually 
occurs without an external noxious stimulus (Schaible, 2007). This is why it is defined by the 
IASP as an unpleasant sensory and emotional experience associated with actual or potential 
tissue damage. Their definition does not link pain exclusively to a tissue damage, noxious 
stimulus or a pathophysiological cause, as pain is a subjective experience that can occur without 
a noxious stimulus (Treede, 2018).  
6 
 
Pain is not always a bad thing. When a person experiences pain as a direct consequence of any 
activity they are performing, they usually halt the activity because they recognize the pain felt 
as a warning sign that harm is currently occurring. On the other hand, if the pain persists, the 
person’s productivity plummets. Here, pain has ceased to provide the person with a useful 
signal since the pain-causing activity has long ceased. Based on these, pain can be described 
as either adaptive or maladaptive (Tan et al., 2011). Adaptive pain, also referred to as helpful 
pain, contributes to the survival of an organism by producing behaviour or mechanisms that 
protect from damaging stimuli or promotes healing and recovery. It triggers the withdrawal 
reflex and heightens sensitivity after tissue damage to decrease the risk of further damage. 
Maladaptive pain has often been referred to as unhelpful pain as it can persist and remain a 
problem even without the noxious stimulus or after the site of injury is healed (Woolf, 2004; 
Marchand, 2008). It may develop in cases where changes in the nervous system causes it to 
operate abnormally and it may be accompanied by hyperalgesia or allodynia. Hyperalgesia is 
an increase in the response to noxious stimuli, whereas allodynia is when a person perceives 
pain from normally innocuous stimulus. These changes have been referred to central or 
peripheral sensitization, respectively (Mense & Gerwin, 2010). 
Pain cannot easily be measured as it is a subjective experience (first-person perspective) that 
requires consciousness. This description of pain as an experience differentiates pain from 
nociception (Treede, 2018). Nociception can be measured with a number of methodologies 
including electrophysiological techniques (Fabrizi et al., 2011; Johnson, 2016). It is a neural 
process that involves the transduction and processing of noxious stimulus to higher centres via 
certain pathways. Pain therefore results from an intricate interplay between certain signalling 
pathways, modulation of perception from the CNS, as well as the perception of the sufferer 
(Steeds, 2016). Nociception differs in many ways from pain (Cohen et al., 2018). As pointed 
out earlier, pain is subjective and only exists in the individual. However, nociception is an 
7 
 
observable event that occurs in response to stimuli (Treede, 2018). There can be pain without 
nociception, and nociception without pain (NPC, 2001). 
2.1.1. The Nociceptive System, Pain Pathways and Mechanisms 
The skin, muscles, organs, ligaments, bones and joints are interlaced with a dense network of 
nerve fibres that register nociceptive events. Together, neurons that are involved in nociception 
collectively form the nociceptive system. These are the C-fibres and the Aδ fibres, and 
sometimes the Aβ fibres which play a dynamic inhibitory role when recruited (Marchand, 
2008; Golan et al., 2016). The C-fibres have a slow conduction velocity (0.5m–2m per second) 
and represent three-quarters of the sensory afferent input. They are unmyelinated and small. 
Their slow conduction velocity usually results in a more diffuse deep pain sensation when they 
are activated. Comparatively, Aδ fibres are large and myelinated with faster conduction 
velocity (5m–30m per second) and are implicated in pain sensations that is described as a rapid 
pinprick, sharp, transient and well localized sensation (Golan et al., 2016).  
8 
 
 
Figure 2.1. Overview of the pain pathway (dreamstime.com, 2019) 
As the illustration in Figure 2.1 depicts, nociceptors or primary afferent neurons from the 
periphery synapse with certain 2nd order neurons and interneurons inside the dorsal horn. These 
second order neurons project into the thalamus, and by implication the brain, which is known 
to produce conscious pain responses upon stimulation by noxa. The secondary afferents travel 
to superior centres via two major routes: the spino-thalamic tract, that sends afferents to the 
thalamus, and the spino-reticular tract, which transmits stimuli to the medial thalamus, the 
nucleus raphe magnus and the periaqueductal grey area which are part of the descending pain 
modulation pathway. From the thalamus, the nociceptive signal is passed on to the 
somatosensory cortex among other CNS centres to produce the sensation of pain (Woolf, 2004; 
Marchand, 2008; Rang et al., 2016). 
9 
 
Multiple mechanisms that produce pain exist and clinical pain syndromes could involve a 
combination of these mechanisms. The ideal way to treat pain would be to identify and target 
the precise pain mechanisms that are operative in a particular patient and device a treatment 
plan aimed toward either reducing excitatory activity or enhancing inhibitory mechanisms 
(Woolf, 2004; Marchand, 2008). Some of these pain mechanisms include nociception, 
decreased inhibition, central and/or peripheral sensitization, phenotypic switches, ectopic 
excitability, etc. (Marchand, 2008; Rang et al., 2016).  
2.1.2. Phases of Nociceptive pain 
The processes involved can be broken down into five different phases. These are transduction, 
conduction, transmission, modulation and perception (Benzon & Raj, 2008) (Figure 2.2). 
Transduction is defined as the conversion of noxious stimuli into electrical activity at the 
peripheral terminals of nociceptors via specialized proteins and voltage-gated receptors on the 
membranes of these primary afferents (NPC, 2001; Benzon & Raj, 2008). Chemical noxious 
stimuli may refer to those released by damaged cells including cholecystokinin, bradykinin and 
prostaglandins, which when released sensitize other nociceptors that are nearby (Schaible, 
2007; Nix, 2017). This step can be inhibited by opioids, NSAIDS and local anaesthetics 
(Benzon & Raj, 2008). 
Conduction involves the movement of an action potential from the peripheral terminals of a 
nociceptor down to its presynaptic terminal within the dorsal horn in the central nervous system 
at specific laminae (Boos & Aebi, 2008). This process depolarizes the presynaptic terminal. 
Transmission encompasses transfer and modulation at the synapses from one neuronal terminal 
to another (Boos & Aebi, 2008; Marchand, 2008; Nix, 2017). It begins when an action potential 
arrives at the presynaptic terminal of the nociceptor. C-fibres release glutamate, substance P 
and calcitonin at the terminal which activate the postsynaptic α-amino-3-hydroxy-5-methyl-4-
10 
 
isoxazole propionic acid (AMPA) receptors,  NK1 receptors and CGRP receptors respectively 
(Brooks & Tracey, 2005; Krebs et al., 2017) (Figure 2.3). The result is the influx of ions which 
depolarize the interneurons and 2nd order neurons they synapse with. Depolarization results in 
the generation of an action potential which is then relayed through certain tracts to the brain 
stem, medulla or the hypothalamus (Brooks & Tracey, 2005). Both transmission and 
conduction can me modulated by the use of local anaesthetics, opioids and alpha-2-agonists. 
 
Figure 2.2. An illustration showing the various phases of the pain pathway 
Modulation of nociceptive transmission has been classified as an adaptive process and involves 
both excitatory and inhibitory mechanisms (Schaible, 2007). When the brainstem is activated 
by nociceptive impulses, descending modulation occurs. In a physiologically normal state, 
responses of 2nd order neurons can naturally be facilitated or suppressed. This might be 
impaired in a diseased state. Modulation has been divided into peripheral and central based on 
11 
 
where it occurs along the pain pathway. Three of the ways peripheral modulation can be 
achieved are outlined below:  
 Inhibiting the terminals of nociceptors from being sensitized by using certain drugs (eg. 
COX inhibitors) (Rang et al., 2016).  
 Inhibiting the production and transfer of action potentials in the axons. Local anaesthetics 
like lidocaine are commonly used (Benzon & Raj, 2008; Mense & Gerwin, 2010; Nix, 
2017). 
 Stimulating the Aβ fibres can recruit inhibitory interneurons in substantia gelatinosa 
(Figure 2.3) to release glycine and GABA which prevent glutamate release from the 
afferent terminals. This prevents the depolarization of the 2nd order neuron. This 
phenomenon is known by many as the gate control theory (Marchand, 2008). 
Two of the ways by which central modulation can be achieved are outlined below: 
 The use of opioids like morphine produce their analgesic effect by mimicking endogenous 
opioids and can activate the endorphin receptors: µ, κ and δ. The analgesia produced can 
be blocked by naloxone (Kieffer, 1999; Crain & Shen, 2007; Nix, 2017). 
 Antidepressants enhance the pain relief effect of the descending pain pathway when they 
increase the availability of the synaptic monoamines (Scholz & Woolf, 2002; Costigan et 
al., 2009; Nix, 2017). 
12 
 
 
Figure 2.3. Illustration showing the substantia gelatinosa and the chemicals involved in 
transmission and modulation. 
The perception phase is the stage where the event is recognised as pain by an individual (NPC, 
2001). Both the cortical and limbic system structures are involved. The somatosensory cortex 
is somatotopically mapped to enable us identify the location and intensity of the noxious event 
occurring (NPC, 2001; Brooks & Tracey, 2005; Krebs et al., 2017). Again, perception can be 
modulated with the use of opioids, alpha-2-agonists, TCAs, SNRIs and SSRIs. 
2.1.3. Classification of Pain 
Multiple classification systems for pain have been used. This includes intensity (severe, 
moderate and mild), duration (acute and chronic), physiology (nociceptive, neuropathic, 
inflammatory, (dys)functional), syndromes (cancer, migraine) and also the specific tissue 
involved (heart, tendons etc.), (Schaible, 2007; Marchand, 2008; Costigan et al., 2009; 
Amoateng et al., 2015; Treede et al., 2015; Rang et al., 2016; Nix, 2017). However, the most 
common classifications are based on duration and pain physiology. 
13 
 
2.1.3.1. Classification by Duration 
Acute pain is known to arise from trauma to soft tissue or even inflammation. It has a sudden 
onset and a foreseeable end lasting less than 3 to 6 months. It persists as long as the triggering 
noxa persists. When the noxa is removed, acute pain disappears and the event ends on its own 
(Nix, 2017). It facilitates tissue repair/healing (Macintyre et al., 2010) which is achieved 
through hypersensitizing the injury area (called primary hyperalgesia), and the tissues in the 
area of injury (secondary hyperalgesia) to all stimuli (both damaging/noxious and non-
damaging) in a way that causes us to avoid contact with any external stimulus thereby allowing 
reparative processes to proceed unperturbed (Macintyre et al., 2010). Acute pain does not 
always have a short duration as is always classified. Some sources of acute pain are surgery, 
labour, acute disease states, medical procedures etc. (NPC, 2001; Costigan et al., 2009).  
Chronic pain persists beyond the expected time frame (longer than 6 months) for an injury or 
disease process or expected period of healing (Russo & Brose, 1998; NPC, 2001). It disrupts 
normal living and sleep, and, unlike acute pain, serves no protective or adaptive function. 
Instead, it degrades the health and functional capability of the sufferer and contribute to patient 
morbidity. Here, the nervous system becomes the primary focus of the ongoing pathology 
(Flor, 2000; NPC, 2001). Chronic pain can arise from any sustained noxious stimulus like an 
on-going inflammation and injury, or may exist de novo, with no temporal relation to an 
inciting cause (Flor, 2000). Environmental factors, as well as other affective factors can also 
make chronic pain worse, and lead to disability (Golan et al., 2016). Chronic pain may be either 
neuropathic or nociceptive or both (Golan et al., 2016). 
2.1.3.2. Classification by Pain Physiology 
Pain can be classified based on the underlying pathophysiology. There are four main classes in 
this category distinguishable by their underlying pain mechanisms or pathways (Figure 2.4). 
14 
 
These include nociceptive, inflammatory, neuropathic and dysfunctional pain (Russo & Brose, 
1998; Woolf, 2004; Costigan et al., 2009; Golan et al., 2016; Nix, 2017).  
Figure 2.4. The four primary types of pain (Woolf, 2004) 
2.1.3.3. Nociceptive Pain 
This type is mediated by primary afferents that project into certain nociceptive pathways of the 
CNS. They are sensitive to intense mechanical and thermal stimuli and certain chemical 
15 
 
mediators (Marchand, 2008). The specific pathway is depicted in Figure 2.4. Nociceptive pain 
is basically an acute or chronic pain event in which both the central and peripheral neuronal 
structures are intact (Nix, 2017). However, when inflammation, autoimmune, or metabolic 
aetiology affects these structures causing lesions in the somatosensory system, the pain 
becomes neuropathic. Nociceptive pain is very important to survival. It serves as a protective 
measure against self-mutilation/hurt and additionally sparks/encourages the healing process 
after an encounter with noxa. It is generally transitory and is known to only occur in the 
presence of the noxa and persists as long as that stimulus remains (Costigan et al., 2009). 
However, there are some cases where the pain persists even after the injury/stimulus is removed 
hence advancing to chronic pain. Certain diseases (like osteoarthritis) may generate recurring 
or persistent noxious stimuli that can result in the production of chronic nociceptive pain 
(Schaible, 2007; Costigan et al., 2009). Clinicians are not always able to predict which case 
will devolve into chronic pain following acute nociceptive pain, hence the need to treat this 
type of pain as early as possible. When this system is chronically disabled, its protective 
function is inevitably lost leading to tissue damage including self-induced mutilation of the lips 
and tongue, loss of the tips of fingers, destruction of joints and pressure ulcers (Woolf, 2004).  
2.1.3.4. Inflammatory Pain 
Inflammatory pain is often grouped under nociceptive pain but according to Benzon and Raj 
(2008), it qualifies to stand alone as a pain category as there is evidence of peripheral and 
central reorganization in such pain states. This pain type occurs because of tissue injury and 
the linked inflammatory response that follows (Figure 2.4). This pain stimulus is aimed at 
addressing the consequences of tissue damage as opposed to protecting the organism against 
potentially damaging noxious stimulus (Costigan et al., 2009). In an effort to aid/accelerate 
healing and repair, the CNS is forced to undergo certain changes that can result in hyperalgesia 
and allodynia (Juhl et al., 2008). 
16 
 
2.1.3.5. Neuropathic Pain 
It occurs when signals are aberrantly processed in the nervous system and is an indicator of 
damage to the nervous system (Figure 2.4). It is usually generated by a disease-inducing 
activity within the nociceptive system without an adequate stimulation of the peripheral 
sensory endings of nociceptors (Costigan et al., 2009; Nix, 2017). This can continue beyond 
the disease-inducing activity and the pain thereof can become an independent disease. Even 
though central sensitization/stimulation does not last when the noxa is removed, the resulting 
injury to the nerve that was left causes long lasting changes in the CNS (Costigan et al., 2009). 
From this, many believe that central sensitization can be used to explain why neuropathic pain 
is not proportional to the applied stimuli or why it persists even when no identifiable stimuli 
exist (NPC, 2001; Benzon & Raj, 2008; Nix, 2017). Neuropathic pain is broadly categorized 
as either central or peripheral in origin. Lesions in the nervous system, as found in peripheral 
neuropathic pain, is usually caused by neurotoxic chemicals, trauma arising from mechanical 
sources, infection and tumours. Several of these pathophysiological changes in the nervous 
system are implicated (Woolf, 2004; Nix, 2017). Central neuropathic pain is commonly 
associated with stroke, spinal cord injury etc. (Ducreux et al., 2006).  
According to Benzon and Raj (2008), the pharmacological management of neuropathic pain in 
patients is quite disappointing as no single therapy exists that can properly alleviate the pain in 
such individuals. Some of the drugs prescribed to patients for the management of this type of 
pain are NSAIDs, acetaminophen, tricyclic antidepressants (which include amitriptyline and 
doxepin), anticonvulsants (which include pregabalin and carbamazepine), some local 
anaesthetics (which include lidocaine) and opioids (such as morphine and methadone) (Rang 
et al., 2016; Nix, 2017). 
17 
 
Non-pharmacological treatment options given by some psychologists include hypnosis, 
physical therapy, occupational therapy, individual and family therapy (Benzon & Raj, 2008).  
2.2. Types of Analgesics for the pharmacological management of pain 
An analgesic can be defined as any compound that selectively relieves pain without the loss of 
consciousness (Medical Subject Heading, 2018) and may block nerve impulse conduction 
which is known to alter sensory perception (Moore, 2013). This distinguishes analgesics from 
anaesthetics in that, anaesthetics induce a general or a local loss of sensation or sensory 
perception. The human body intrinsically produces certain endogenous compounds that 
influence pain impulse generation and perception and how the brain responds to these impulses 
(Lesniak & Lipkowski, 2011; Corder et al., 2018). Some of these are endogenous peptides like 
Substance P and chemical messengers such as 5HT3 (serotonin) and GABA (Koneru et al., 
2009). Medications are therefore mostly used to ameliorate the body’s intrinsic pain 
management mechanisms. 
2.2.1. Non-Steroidal Anti-Inflammatory Drugs (NSAIDS) 
NSAIDs exert their therapeutic effect by inhibiting a group of endogenous enzymes known as 
cyclooxygenases (Zarghi & Arfaei, 2011; Nix, 2017). These enzymes mediate the production 
of prostaglandins from arachidonic acid (Zarghi & Arfaei, 2011; Rang et al., 2016). They are 
usually prescribed for mild to moderate pain. The therapeutic activity of NSAIDs stems from 
the inhibition of the COX-2 enzyme. Inhibiting the activity of the COX-1 enzyme generates a 
slew of undesirable effects which include gastric bleeding, stomach ulcers, nausea, etc (Benzon 
& Raj, 2008; Dinarello, 2010; Nix, 2017). Selective COX-2 inhibitors are known to have a 
better gastric safety profile than NSAIDs that inhibit both COX-1 and COX-2. However, their 
usage also comes with certain side effects and may increase the risk of stroke or heart attack in 
certain patients (Benzon & Raj, 2008; Zarghi & Arfaei, 2011). All the side effects limit the 
18 
 
usage of NSAIDs over an extended period. Examples of selective COX-2 inhibitors include 
celecoxib, lumiracoxib etc. Other examples of commonly used NSAIDs include ibuprofen, 
aspirin and indomethacin (Zarghi & Arfaei, 2011). 
2.2.2. Opioids 
Opioids are effective analgesics and are widely used in clinical management of pain (Koneru 
et al., 2009; Corder et al., 2018). The opioid system is one of the main systems engaged in pain 
perception and modulation, reward, addiction and fear behaviours (Lesniak & Lipkowski, 
2011). Some of the opioid peptides synthesised by the human body for these processes include 
endorphins, enkephalins, dynorphins and endomorphins (Przewlocki & Przewlocka, 2005). 
Opioids exert their effect by acting on the pre-synaptic terminal of primary nociceptive 
afferents thereby decreasing the release of excitatory neurotransmitters from the nociceptive 
neurons. They are very effective in treating acute nociceptive pain and to some extent acute 
neuropathic pain. However, their usage in the management of persistent pain is discouraged 
due to concerns about ineffectiveness, the high potential for the development of tolerance, the 
risk of addiction, and a slew of other side effects (Przewlocki & Przewlocka, 2005; Corder et 
al., 2018). Some of these side effects include nausea, vomiting, constipation, sedation, 
confusion and myoclonus. Some commonly used opioid analgesics in clinical practice include 
morphine, codeine, fentanyl, oxycodone, hydromorphone, methadone and buprenorphine 
(Brain & Cox, 2006). 
2.2.3. Adjuvant analgesics 
These are drugs that are prescribed for conditions other than pain but display significant 
analgesic effect in some painful conditions (Lui & Ng, 2011; Mitra & Jones, 2011). They are 
usually co-administered with analgesics such as the NSAIDs, opioids and acetaminophen. 
Major classes include antidepressants (tricyclic antidepressants, selective serotonin reuptake 
19 
 
inhibitors), anticonvulsants (carbamazepine, pregabalin), neuroleptics (olanzapine), NMDA 
receptor antagonists (amantadine, ketamine), corticosteroids (dexamethasone, prednisone), 
local anaesthetics (lidocaine), muscle relaxants (cyclobenzaprine) etc. Some are described as 
multipurpose adjuvant analgesics (antidepressants, corticosteroids etc) as they are prescribed 
for many painful conditions (Lussier et al., 2004; Nix, 2017). Others are prescribed specifically 
for neuropathic pain (anticonvulsants, NMDA receptor antagonists), bone pain (calcitonin, 
bisphosphonates), musculoskeletal pain (muscle relaxants) or pain from bowel obstruction 
(anticholinergics) (Lui & Ng, 2011; Mitra & Jones, 2011; Nix, 2017). These too are riddled 
with side effects such as orthostatic hypotension, erectile dysfunction, nausea, vomiting etc 
(Lussier et al., 2004). 
2.3. Traditional Medicine 
Traditional medicine has been practiced for centuries and still remains common in the 
developing world (Chukwujekwu et al., 2005). In Ghana, it is common to see herbal products 
advertised to treat a plethora of diseases. This is scientifically possible because a plant contains 
a slew of different compounds that can act synergistically on targeted elements of certain 
complex cellular pathways to produce varying effects (Farombi, 2003; Rajdev et al., 2018). 
The use of herbal preparations was popularized by traditionalists as the best alternative to 
allopathic drugs which are riddled with toxicity and mild to severe side effects. It has been 
estimated that about 80 percent of the world’s population rely primarily on medicinal plants 
for the management of various medical conditions (Mahomoodally, 2013; Ekor, 2014). Most 
of these drugs are prepared as extracts of different parts of the medicinal plants or their active 
constituents. Thus, natural products is seen by researchers as a good source of lead compounds 
based on its traditional use in treating and managing ailments  (Hasan et al., 2014; Kukuia et 
al., 2016). Purified natural products from plant sources can be used as lead compounds and 
20 
 
structurally improved upon to produce new generations of therapeutic compounds that have a 
higher therapeutic value and low toxicity. 
2.4. Mallotus oppositifolius 
Mallotus oppositifolius is a dioecious shrub that is widely distributed in Africa across many 
parts of Ghana, Nigeria, Angola, Madagascar, Cameroon, Senegal, Ethiopia etc (Burkill et al., 
1985). It bears many local names in Ghana including “sroti” (Ewe), “sratadua” (Fanti) and 
“osratadua” (Twi). In Ivory Coast, it is referred to as Tomida (Abron) in the Zanzan district 
where it is also abundant and widely used (Farombi, 2003; Okpekon et al., 2004). It has been 
ascribed several names in different parts of Nigeria and these include “Kafar Mutuwaa” by 
Hausas (Northern Nigeria), “Ija” by Yorubas (Western Nigeria) and “Nne Okpo Kirinya” by 
Igbos (Eastern, Nigeria) (Nwaehujor et al., 2014). 
Figure 2.5. Photograph showing the leaves and inflorescence of M. oppositifolius 
21 
 
Mallotus oppositifolius is part of the plant family Euphorbiaceae under the tropical genus 
Mallotus which constitutes about 150 species of shrubs and trees (Woode et al., 2012; 
Harinantenaina et al., 2013) distributed in tropical and subtropical areas which also include 
Australia, Asia and the Pacific (Kulju et al., 2007). Euphorbiaceae is a plant family that has 
afforded several very promising bioactive compounds and is the subject of numerous biological 
investigations (Beutler et al., 1998; Yoder et al., 2007; Harinantenaina et al., 2013). From 
research work done by Burkill et al. (1985), Mallotus oppositifolius sometimes grows to a total 
height of 13 metres. It harbours a raceme inflorescence which may grow to different lengths 
for either the male (10-15) or female (10-18) inflorescence. Its pale yellow-green flowers are 
unisexual with slight differences in the length and shape of the sepals. Its fruits are deeply 3-
lobed and seeded. M. oppositifolius is shown in Figure 2.3.1. 
2.4.1. Ethnopharmacological Uses of M. oppositifolius 
In folklore traditional use, the leaves, stem bark, fruits and roots of M. oppositifolius are 
prepared as either a tincture or a decoction to treat several conditions (Chukwujekwu et al., 
2005). Leaf and/or stem bark infusions are taken as an anthelmintic to treat intestinal 
helminthiasis and as treatment for diarrhoea (Okpekon et al., 2004). The leaves are chewed by 
some traditional healers and placed on sores, skin eruptions, burns and rashes as an 
antibacterial, haemostatic or analgesic for fast healing and relief (Adekunle & Ikumapayi, 
2006). It is also used in folklore for the treatment of epilepsy, headache or mental illness as a 
steam bath. For inflamed eyes and gums in the mouth, the leaves are crushed and applied. The 
leaves are ground with salt and applied to snakebites to nullify poisons (Farombi et al., 2001). 
The root and leaf decoction is taken by some to treat malaria, anaemia, vomiting, pneumonia, 
urinary infections, leprosy, venereal diseases and chickenpox (Noumi & Yomi, 2001; Farombi, 
2003; Okpekon et al., 2004). A paste is also made from the roots and leaves and is applied to 
treat chest pains, convulsions and stomach-ache. In certain parts of East Africa, the root 
22 
 
decoction is drunk as an aphrodisiac (Burkill, 1994). Certain traditional medicine practitioners 
in the south-eastern part of Nigeria hold the claim that M. oppositifolius possess antidiabetic 
activity (Nwaehujor et al., 2013). The stick is also chewed by some for teeth cleaning and oral 
hygiene (Burkill et al., 1985; Nwaehujor et al., 2014). 
2.4.2. Non-Medicinal Uses of M. oppositifolius 
M. oppositifolius leaves are commonly pastured on by cattle (Aschfalk et al., 2000). The dried 
wood is commonly used as firewood and as supplies in making tool handles. The stem is 
sometimes used as support for climbing vines like yam. Farmers use the thin stems from some 
M. oppositifolius plans as rope for binding and the twigs as chewing sticks (Burkill et al., 1985). 
2.4.3. Previous studies on M. oppositifolius 
Phytochemical screening of M. oppositifolius by Farombi et al. (2001), Adekunle and 
Ikumapayi (2006) and Woode et al. (2012) revealed that it constituted certain secondary 
metabolites such as anthocyanins, butacyanins, flavonoids, saponins, steroids, tannins, 
alkaloids, phenols, cardenolides and anthraquinones in both the ethanolic and aqueous leaf 
extracts. Heavy metal analysis by Adekunle and Ikumapayi (2006) revealed the presence of 
insignificant amounts (below the recommended limit of 0.95% in plants) of zinc, copper, iron 
and manganese. Their study also confirmed that the plant extract (aqueous and ethanolic) 
possessed antifungal properties that were very likely attributable to the phytochemical 
compounds present in the extract and not due to the presence of any heavy metals. Significant 
antifungal activity was exhibited against Aspergillus flavus, Microsporum audouinii, Candida 
albicans, Penicillium sp., Trichoderma mentagrophytes and another Trichoderma spps. 
In a study conducted by Gbedema et al. (2010), the leaf extract of M. oppositifolius displayed 
a significant antimicrobial activity against Pseudomanas aeruginosa and Staphylococcus 
aureus and Baccilus subtilis. It also enhanced the antibacterial activity of amoxicillin against 
23 
 
Staph. aureus and B. subtilis. Other studies on the antimicrobial activity of M. oppositifolius 
extract in-vivo and in-vitro have yielded significantly positive results (Chukwujekwu et al., 
2005; Gangoue-Pieboji et al., 2009). 
Extensive work has been done to provide scientific evidence on the anticonvulsant, 
antidepressant and anxiolytic activity of M. oppositifolius (Kukuia, 2012; Woode et al., 2012; 
Kukuia et al., 2014; Kukuia et al., 2016, 2016) as a potent alternative to mainstream drugs for 
the management of these conditions. Mallotoxin (Rottlerin, pictured in figure 2.7) is a 
compound isolated from the M. oppositifolius plant and has been cited to display a spectrum 
of pharmacology (Harinantenaina et al., 2013; Kukuia et al., 2016; Manville & Abbott, 2018). 
In past studies conducted, researchers demonstrated that the leaf extract possesses significant 
antioxidant effect (Nwaehujor et al., 2013; Nwaehujor et al., 2014), anti-inflammatory activity 
(Chukwujekwu et al., 2005), antimalarial effect and anti-parasitic activity (Okpekon et al., 
2004; Harinantenaina et al., 2013; Kabran et al., 2015; Eaton et al., 2016) and significant anti-
blastocystic activity (Bremer Christensen et al., 2015). 
In a study by Harinantenaina et al. (2013), three bioactive compounds, mallotojaponin B and 
C (methylated form of mallotojaponin C, tetramethylmallatojapinin C, was also obtained) and 
mallotophenone were isolated from the ethanol extract of the leaves and inflorescence of M. 
oppositifolius by bioassay-guided fractionation (Figure 2.6). Mallotojaponins B and C showed 
potent antimalarial activity against chloroquine-resistant Plasmodium falciparum. Again, in the 
same study, all three isolated compounds displayed strong antiproliferative activity against the 
A2780 human ovarian cancer cell line. Similarly, Okpekon et al. (2004) tested M. oppositifolius 
methanolic extract against chloroquine-resistant FCB1 strain of Plasmodium falciparum with 
promising results. Following this, several other bioactive compounds have been isolated from 
M. oppositifolius (Kabran et al., 2015). 
24 
 
 
Figure 2.6. Chemical structures of mallotojapinin C (1), tetramethylmallotojapinin C (2), 
mallotophenone (3) and mallotojaponin B (4). 
 
Figure 2.7. Chemical structure of Mallotoxin (Rottlerin) 
25 
 
Chapter Three 
3 MATERIALS AND METHODS 
3.1. Plant Material Collection 
Mallotus oppositifolius leaves were procured from Mampong Centre for Plant Medicine 
Research, Eastern Region, Ghana and were authenticated by a botanist at the Department of 
Botany, University of Ghana, and a voucher specimen (CPMR 314/17) kept. 
3.2. Preparation of Plant Extract 
The leaves were air-dried under shade for seven days and the dried leaves pulverized into fine 
powder with a hammer mill. About 6 kg of the powder was cold macerated with ethanol (70% 
v/v) in a stoppered container and allowed to stand at room temperature for a period of 72 hours. 
The mixture obtained after the 72 hours was strained, the damp solid material left behind was 
also pressed and the liquids combined. The resulting solution was concentrated to a syrupy 
mass using a rotary evaporator (Heidolph Elektro, GmbH, KG, Kcl Hcim, Germany) by 
removing the alcohol. This was done under reduced temperature (60°C) and pressure. The 
syrupy mass was then dried on a water bath to a solid mass and kept in a desiccator till it was 
ready to be used. The percentage yield was 5.18%. The final product yielded was called the 
extract or Mallotus oppositifolius extract (MOE) in this work. 
3.3. Preliminary High-Performance Liquid Chromatography (HPLC) of MOE 
The above procedure was carried out using a Perkin Elmer Flexar LC binary pump coupled to 
a PDA detector. The constituents of MOE were separated on a μBondapak C18 Column (3.9 × 
300 mm, 5μm). The mobile phase used was distilled water (A) and methanol (B). Gradient 
elution started with 99% distilled water for 5 minutes and gradually moved to 0% within 35 
26 
 
minutes. It stayed at 0% distilled water for 5 more minutes and then returned to 99% distilled 
water in 5.1 minutes, bringing the total runtime to 45.1 minutes. The flow rate of 1mL/min and 
a sample injection volume of 20 μL was used. The wavelength was set at 272 nm and 278 nm. 
An initial blank reading using the solvent was taken before the analysis on MOE. 
3.4. Preliminary Phytochemical Screening 
This was performed on the freshly prepared Mallotus oppositifolius extract using methods 
described by Evans et al. (2009) and Kukuia (2012) aimed at the detection of tannins, 
flavonoids, alkaloids, reducing sugars, sterols, saponins and terpenoids. 
3.4.1. Test for Tannins 
About 0.5g of MOE was added to 25ml of water and boiled 5 minutes after which it was cooled 
and then filtered. The filtrate obtained was again topped up to 25 ml with water. To 1 ml of the 
filtrate, 10ml of distilled water followed by 2 drops of 1% ferric chloride was added and 
observed for the appearance of a blue-black or green precipitate. 
3.4.2. Test for Alkaloids 
About 0.1g of the extract was measured and transferred into a test tube. About 5.0 ml of 2.0M 
hydrochloric acid was added to the test tube containing the extract to giving acidified solution. 
This acidified extract solution was swirled vigorously whilst warming over a water bath for 
about 5 minutes and thereafter filtered. The filtrate was divided into three portions designated 
A, B and C. Dragendorff’s reagent was added to portion A of the solution, to portion B, Mayer's 
reagent and to portion C, Wagner's reagent. The presence of a reddish-brown precipitate in 
tubes A and C and a yellowish precipitate in test tube B was an indication that alkaloids were 
present. 
27 
 
3.4.3. Test for Saponins 
About 4 ml of distilled water was added to a small amount of the extract (0.2g). This was then 
shaken for a few seconds. The resulting mixture was observed for the presence of a froth which 
did not break readily when left to stand. 
3.4.4. Test for Reducing Sugars 
An amount of the extract (0.2 g) was boiled in 5 ml of water and the resulting mixture cooled 
and filtered. An equal quantity (5 ml) of Fehling’s A and B solution was added. This mixture 
was then heated and observed for the formation of any red-brown precipitate. 
3.4.5. Test for Sterols 
About 5 ml of acetic anhydride was added to and mixed with about 0.1g of MOE and filtered. 
The filtrate was treated with Libermann-Burchard reagent. The appearance of a dark green ring 
at the interphase was indicative of the presence of sterols and steroids int the extract (Zohra et 
al., 2012). 
3.4.6. Test for Terpenoids 
Thin-layer chromatography (TLC) was carried out on the extract using acetone/petroleum in 
the ratio of 8:2 as the solvent system. The dried plates were sprayed on with Liebermann-
Buchard reagent and placed in an oven for 10 minutes set at a temperature of 110℃. The plate 
was then examined under UV light for any pink spots. The presence of pink spots is indicative 
of the presence of terpenoids in the extract. 
3.4.7. Salkowski’s Test for Glycosides 
About 0.5g of MOE was dissolved with 2 cm3 of chloroform. 2M sulphuric acid was carefully 
poured into the solution down the glassware’s walls to form a lower layer. Glycosides were 
present when a reddish-brown colouration was formed at the interface. 
28 
 
3.4.8. Test for Anthraquinones and Anthracene Derivatives 
Exactly 0.5g of the extract was dissolved in 30 cm3 of distilled water and filtered. The filtrate 
obtained was vigorously mixed with about 10 cm3 of benzene in a separating funnel. The 
benzene layer was transferred into a test tube and shaken with 5 cm3 of 2M ammonia solution. 
The presence of red colour in the ammonia layer was indicative of the presence of 
anthraquinones in the extract.  
3.4.9. Test for Flavonoids (Shinoda’s test) 
About 0.1g of the extract was dissolved in 10.0 ml methanol in a test tube and filtered. Ten 
pieces of Magnesium turnings were added to the filtrate. Few drops of concentrated 
hydrochloric acid were added to the resulting solution. The presence of pink colorations in the 
test solution indicated the presence of flavonoids in the extract 
3.5. Experimental Animals, Housing and Ethical Considerations. 
Male ICR mice, weighing 20–40 g, and male Sprague-Dawley rats, weighing 150–350 g, were 
purchased from and kept at the Department of Animal Experimentation, Noguchi Memorial 
Institute of Medical Research, University of Ghana, where all the experimental procedures 
were undertaken. Experimental animals used were kept in stainless steel cages (dimensions: 47 
× 34 × 18 cm) which had wood shavings as bedding and were fed with normal commercial feed 
(AGRIMAT, Madina), given water ad libitum and maintained under the required laboratory 
conditions of relative humidity 60-70%, temperature 23±2 ˚C and a 12-hour light-dark cycle. 
All animals used in this study were handled according to the Guide for the Care and Use of 
Laboratory Animals (National Research Council (U.S.A.) et al., 2011). All animal 
experimentation was performed during the day from 9 am to 4 pm. Ethical clearance was 
received from the University of Ghana Institutional Animal Care and Use Committee, Noguchi 
Memorial Institute for Medical Research, University of Ghana (NIACUC-2018-02-2V). 
29 
 
3.6. Drugs and Chemicals Used 
These are the drugs and chemicals used in this study: formalin [prepared from 40% 
formaldehyde solution (Research-Lab Fine Chem Industries, Mumbai, India)]; morphine 
(Fresenius Kabi Manufacturing SA (pty) Ltd, SA); diclofenac (KRKA, Slovenia); acetic acid 
(BDH, poole, UK); ondansetron (Novartis Pharmaceuticals Corp, UK); glibenclamide (Ernest 
Chemists Limited, Ghana); nifedipine (Lek Pharmaceuticals, Slovenia); yohimbine (Walter 
Ritter GmbH, Germany); atropine (Yanzhou Xierkangtai Pharma. Co. Ltd., China); naloxone 
(Duopharma Biotech Berhad, Malaysia); theophylline (Adcock Ingram Ltd, SA). All the drugs 
used were of high standard grade and solutions from these were freshly prepared daily before 
use. 
3.7. Experimental Design 
The models used in this study have been divided into acute models, chronic models and the 90-
day sub-chronic oral toxicity study. The first acute model employed in this study was the 
formalin test which can demonstrate whether an agent has peripheral or central analgesic 
activity. To confirm the results from this experiment, the acetic acid-induced writhing test, a 
peripheral pain model was used. Again, to confirm the central activity of MOE, the hot plate 
test was employed. The formalin test was then employed to elucidate the possible 
mechanism(s) by which MOE exerts its antinociceptive effect.  
In another experiment, effect of MOE in chronic models of pain was evaluated. Paclitaxel-
induced peripheral neuropathy was the chronic model used. For peripheral neuropathy, two 
basic symptomologies exist; hyperalgesia and allodynia. For hyperalgesia, mechanical 
hyperalgesia was measured using the Randall-Selitto analgesiometer, whereas thermal 
hyperalgesia was evaluated using the hot plate. For allodynia, cold allodynia test using cold 
water at 4℃ was employed. 
30 
 
Finally, the safety of MOE was assessed for extended use in the 90-day sub-chronic toxicity 
study. 
3.8. Acute models of pain 
3.8.1. Formalin Test 
The formalin test was carried out as described by Hunskaar and Hole (1987) and Woode et al. 
(2009). ICR mice were divided into ten (10) groups (n=5); one negative control group, three 
(3) treatment groups and six (6) positive control groups. Every test animal was assigned one 
formalin test chamber (Perspex chamber of dimensions 15 x 15 x 15 cm) and then acclimatized 
for 1 hour before pre-treatments. Mice were then pre-treated with MOE, morphine, diclofenac 
and distilled water. Mice in the negative control group received distilled water p.o. (vehicle) 
by oral gavage. Those in the three (3) treatment groups were given the Mallotus oppositifolius 
extract at doses of 30, 100 or 300 mg/kg p.o. respectively, all by oral gavage. Finally, mice in 
the positive control groups were given morphine (0.3, 1, 3 mg/kg; i.p) and diclofenac (3, 10 
and 30 mg/kg; i.p). 
The right hind paws of the mice were injected subcutaneously with 10 μL of 5% formalin, 30 
minutes after pre-treatment with morphine or diclofenac (i.p route) and 1 hour after pre-
treatment with MOE (oral route) and immediately returned to individual testing chambers 
where their nociceptive behaviour from time zero (0) was captured for one (1) hour using a 
tripod-mounted video recorder (OnePlus™ A600 camera set at 1080p) placed opposite a mirror 
at the base of the test chamber positioned at an angle of 45°. 
The nociceptive behaviour exhibited by the experimental animals was tracked using the 
BehaviorTracker™ Personal Edition software [version 1.6 (www.BehaviorTracker.com)]. The 
nociceptive scores were calculated in 5-minute time blocks as the product of the duration and 
31 
 
frequency of biting/licking of the right hind paw. The results were designated as 
first/neurogenic phase (0−10 minutes) and second/inflammatory phase (10−60 minutes). A 
time-course curve was plotted from nociceptive scores for each of the three drugs used as well 
as the vehicle. The area under the curve (AUCs) from both phases of the formalin test were 
calculated and plotted as well. ED50 values for MOE, diclofenac and morphine were calculated 
and compared. 
3.8.2. Acetic Acid-Induced Writhing Assay 
This assay was carried out as earlier described by Woode et al. (2009); Woode et al. (2012). 
Thirty-five mice was divided randomly into seven (7) groups of five (n=5); one negative 
control group, three (3) treatment groups and three (3) positive control groups. Each animal 
was assigned and acclimatized to one test chamber for 1 hour prior to 0.6% acetic acid 
injections. After this, the ICR mice were pre-treated with MOE, diclofenac and distilled water. 
Mice in the negative control group received distilled water (vehicle) by oral gavage. Those in 
the three (3) treatment groups were given the Mallotus oppositifolius extract at doses of either 
30, 100 or 300 mg/kg respectively, all by oral gavage. Finally, mice in the positive control 
group were given diclofenac (3, 10 and 30 mg/kg; i.p). 
Each animal was given an intraperitoneal administration of acetic acid (0.6%, 10 ml/kg), 30 
minutes after pre-treatment with diclofenac (i.p route) and 1 hour after pre-treatment with MOE 
(oral route) and immediately returned to individual test chambers. The intraperitoneal 
administration of acetic acid induces a nociceptive behaviour known as writhing (abdominal 
constrictions, pelvic rotation, and hind limb stretching) (Cheng et al., 2016). 
The total time and frequency of writhes were captured for 30 minutes using a tripod-mounted 
video recorder (OnePlus™ A600 camera set at 1080p) placed opposite a mirror at the base of 
the test chamber positioned at an angle of 45°. The nociceptive behaviour exhibited by the 
32 
 
experimental animals was tracked using the BehaviorTracker™ Personal Edition software 
(www.BehaviorTracker.com). The nociceptive score was determined for each 5-minute time 
block and a time-course curve plotted. The total nociceptive scores calculated as AUCs from 
each treatment was determined and plotted as well. ED50 values for both MOE and diclofenac 
were calculated and compared. 
3.8.3. Hot Plate Test  
The hot-plate test is used to evaluate the central analgesic activity of MOE and was conducted 
as previously described (Woode et al., 2012; Simões et al., 2018) with some modifications. 
ICR mice were sorted randomly into seven (7) groups of five; one negative control group, three 
(3) treatment groups and three (3) positive control groups. Before drug administrations, 
baseline measurements on the hot plate apparatus (Model DS37; Ugo Basile, Italy) were 
recorded for all the animals being used. After that, mice in the negative control group received 
distilled water (vehicle) by oral gavage. Those in the three (3) treatment groups were given the 
Mallotus oppositifolius extract at doses of 30, 100 or 300 mg/kg respectively, all by oral 
gavage, 1 hour before being placed on the hot plate apparatus. Finally, mice in the positive 
control group were given morphine (0.3, 1 and 3 mg/kg i.p) 30 minutes before being placed in 
the hot plate apparatus heated to 54±1˚C.  
Latency to withdraw paw (in seconds) was recorded using a stopwatch as the time between 
placing the animal on the hot plate and the appearance of symptoms of discomfort as licking 
or shaking of the hind paws or jumping off from the surface of the hot plate apparatus. A 
significant increase in the latency is considered as indicative of antinociceptive activity. This 
is evaluated at time points 0.5, 1, 1.5, 2 and 4 hours after the administration of MOE, vehicle 
or morphine. A cut-off of 30 seconds was used to prevent damage to mouse paw tissue. The 
percentage maximum effect (%MPE) was calculated from the formula below: 
33 
 
 
Time-course curves were plotted from the results. The total antinociceptive scores calculated 
as the area under the curve (AUCs) from each treatment was determined and plotted as well. 
ED50 values for MOE and morphine were calculated and compared. 
3.8.4. Possible Mechanism(s) of Antinociception 
Formalin test (Tjølsen et al., 1992; Woode & Abotsi, 2011) was employed to assess the 
possible involvement of certain pathways in the observed antinociceptive effect of MOE. Here, 
nineteen groups (19) of mice (n=5) were randomly selected and grouped (n=5) and then pre-
treated with seven (7) different inhibitors/antagonists; naloxone (a nonselective opioid receptor 
antagonist), glibenclamide (an ATP sensitive potassium channel inhibitor), theophylline (a 
nonselective adenosine receptor antagonist), ondansetron (a 5-HT3 receptor antagonist), 
yohimbine (a selective adrenoceptor antagonist), nifedipine (L-type voltage-gated calcium 
channel blocker) and atropine (a nonselective muscarinic receptor antagonist).  
Briefly, mice were pre-treated with these antagonists 15 minutes (i.p) and 30 minutes (p.o) 
before the administration of either MOE (100 mg/kg p.o) or morphine (1 mg/kg i.p). Mice then 
received an intraplantar administration of 10 μL of 5% formalin to the right hind limb, 30 
minutes (after morphine) and 1 hour (after MOE) and immediately returned to individual 
testing chambers where their nociceptive behaviour from time zero (0) was captured for one 
(1) hour using a tripod-mounted video recorder (OnePlus™ A600 camera set at 1080p) placed 
opposite a mirror at the base of the test chamber positioned at an angle of 45°. The doses of the 
various antagonists used were chosen based on literature (Woode & Abotsi, 2011; Asante et 
al., 2019).  
34 
 
The nociceptive behaviour exhibited by the experimental animals was tracked using the 
BehaviorTracker™ Personal Edition software [version 1.6 (www.BehaviorTracker.com)]. The 
nociceptive scores were calculated in 5-minute time blocks as the product of the duration and 
frequency of biting/licking of the right hind paw. The results were designated as 
first/neurogenic phase (0−10 minutes) and second/inflammatory phase (10−60 minutes). A 
time-course curve was plotted from nociceptive scores for each of the three drugs used as well 
as the vehicle. The area under the curve (AUCs) from both phases of the formalin test were 
calculated and plotted as well. 
3.8.4.1. Involvement of the Opioid System 
Experimental animals were pre-treated with naloxone (2 mg/kg, i.p.) 15 minutes before the 
administration of MOE (100 mg/kg, p.o.) or morphine (1 mg/kg, i.p.). As described in section 
3.8.4, their nociceptive responses were recorded for 1 hour after the pre-treatments. 
3.8.4.2. Involvement of ATP-sensitive K+ Channels 
Experimental animals were pre-treated with glibenclamide (8 mg/kg, i.p.) 15 minutes before 
the administration of MOE (100 mg/kg, p.o.) or morphine (1 mg/kg, i.p.). As described in 
section 3.8.4, their nociceptive responses were recorded for 1 hour after the pre-treatments. 
3.8.4.3. Involvement of the Adenosinergic System 
Experimental animals were pre-treated with theophylline (10 mg/kg, i.p.) 15 minutes before 
the administration of MOE (100 mg/kg, p.o.) or morphine (1 mg/kg, i.p.). As described in 
section 3.8.4, their nociceptive responses were recorded for 1 hour after the pre-treatments. 
3.8.4.4. Involvement of 5-HT3 Receptors 
Experimental animals were pre-treated with ondansetron (0.5 mg/kg, i.p.) 15 minutes before 
the administration of MOE (100 mg/kg, p.o.) or morphine (1 mg/kg, i.p.). As described in 
section 3.8.4, their nociceptive responses were recorded for 1 hour after the pre-treatments. 
35 
 
3.8.4.5. Involvement of α2-adrenoceptors 
Experimental animals were pre-treated with yohimbine (3 mg/kg, i.p.) 15 minutes before the 
administration of MOE (100 mg/kg, p.o.) or morphine (1 mg/kg, i.p.). As described in section 
3.8.4, their nociceptive responses were recorded for 1 hour after the pre-treatments. 
3.8.4.6. Involvement of Voltage-gated Calcium Channels 
Experimental animals were pre-treated with nifedipine (10 mg/kg, i.p.) 15 minutes before the 
administration of MOE (100 mg/kg, p.o.) or morphine (1 mg/kg, i.p.). As described in section 
3.8.4, their nociceptive responses were recorded for 1 hour after the pre-treatments. 
3.8.4.7. Involvement of the Muscarinic Cholinergic System 
Experimental animals were pre-treated with atropine (5 mg/kg, i.p.) 15 minutes before the 
administration of MOE (100 mg/kg, p.o.) or morphine (1 mg/kg, i.p.). As described in section 
3.8.4, their nociceptive responses were recorded for 1 hour after the pre-treatments. 
3.9. Assessing the Effect of MOE in Chronic Pain Models 
Paclitaxel-induced peripheral neuropathy in ICR mice was performed as previously described 
(Ameyaw et al., 2014; Amoateng et al., 2017) with some modifications. ICR mice were divided 
randomly into seven (7) groups of five (n=5) and allowed to acclimatize to the test environment 
on every testing day, 1 hour or more before drug administrations. Baseline measurements of 
thermal, mechanical and cold stimuli were performed on day zero (0), before paclitaxel 
injections. 
3.9.1. Induction of Peripheral Neuropathy 
ICR mice were injected intraperitoneally with paclitaxel (2 mg/kg) dissolved in distilled water 
on 5 consecutive days (days 1–5) as described by Amoateng et al. (2017). The cumulative 
paclitaxel dose was 10 mg/kg. Behavioural testing for thermal, mechanical and cold stimuli 
36 
 
were performed on day 1, 3 and 5, starting one hour after paclitaxel injections for those days. 
After confirmation of paclitaxel-induced peripheral neuropathy from the behavioural tests, 
mice were assigned to groups as follows: one negative control group, three (3) treatment groups 
and three (3) positive control groups. Mice in the negative control group received distilled 
water (vehicle) by oral gavage. Those in the three (3) treatment groups were given the Mallotus 
oppositifolius extract at doses of 30, 100 or 300 mg/kg respectively, all by oral gavage. Finally, 
mice in the positive control group were given pregabalin (10, 30 and 100 mg/kg; p.o). The 
effect of MOE and pregabalin on paclitaxel-induced peripheral neuropathy was evaluated in 
the hot plate test, the Randall-Selitto test and cold allodynia test for 5 consecutive days starting 
from day 7 post paclitaxel injections. 
3.9.2. Thermal Hyperalgesia (Hot Plate test) 
To assess the effect of MOE (30, 100 and 300 mg/kg), pregabalin (10, 30 and 100 mg/kg) and 
vehicle on thermal hyperalgesia the hot-plate test was employed. The test was conducted daily 
for 5 days as has been described in previous studies (Ameyaw et al., 2014; Amoateng et al., 
2017). ICR mice were divided into seven (7) groups (n=5); one negative control group, three 
(3) treatment groups and three (3) positive control groups. Before drug administrations, 
baseline measurements on the hot plate were recorded for all the animals being used. After that, 
mice in the negative control group received distilled water (vehicle) by oral gavage. Those in 
the three (3) treatment groups were given the Mallotus oppositifolius extract at doses of 30, 
100 or 300mg/kg respectively, all by oral gavage. Finally, mice in the positive control group 
were given pregabalin (10, 30 and 100 mg/kg p.o) 1 hour (p.o.) before being placed in the hot 
plate apparatus. Test was carried out as described in section 3.8.3. 
37 
 
3.9.3. Mechanical Hyperalgesia (Randall-Selitto test) 
To assess the effect of MOE (30, 100 and 300 mg/kg), pregabalin (10, 30 and 100 mg/kg) and 
vehicle on mechanical hyperalgesia the Randall-Selitto paw pressure test was employed. The 
test was conducted daily for 5 days as has been described in previous studies (Ameyaw et al., 
2014; Amoateng et al., 2017) with a few modifications. Mice were divided randomly into seven 
(7) groups of five (n=5); one negative control group, three (3) treatment groups and three (3) 
positive control groups. Before drug administrations, baseline measurements were recorded for 
all the animals being used. After that, mice in the negative control group received distilled 
water (vehicle) by oral gavage. Those in the three (3) treatment groups were given the Mallotus 
oppositifolius extract at doses of 30, 100 or 300 mg/kg respectively, all by oral gavage. Finally, 
mice in the positive control group were given pregabalin (10, 30 and 100 mg/kg p.o).  
In this test, the hind paw of mice was placed in the paw pressure analgesimeter (model 7200, 
Ugo Basile, Varese, Italy) and a steadily increasing pressure stimulus was applied (cut-off of 
250g) to the dorsal surface of the hind paws until there was withdrawal or vocalization. The 
force (g) reading was then recorded for each animal as the nociceptive threshold value. The 
test was performed on both hind paws to obtain two readings. The final value is presented as 
the mean of both hind paw values. The %MPE was calculated and plotted as described in 
section 3.8.3. 
3.9.4. Cold Allodynia 
The antinociceptive effect of Mallotus oppositifolius extract (30-300 mg/kg), pregabalin (10, 
30, 100 mg/kg) and vehicle on cold allodynia was investigated daily for 5 days as has been 
described in previous studies but with minor modifications (Ameyaw et al., 2014; Amoateng 
et al., 2015). ICR Mice were divided into seven (7) groups (n=5); one negative control group, 
three (3) treatment groups and three (3) positive control groups. Before drug administrations, 
38 
 
baseline measurements were recorded for all the animals being used. After that, mice in the 
negative control group received distilled water (vehicle) by oral gavage. Those in the three (3) 
treatment groups were given the Mallotus oppositifolius extract at doses of 30, 100 or 
300mg/kg respectively, all by oral gavage. Finally, mice in the positive control group were 
given pregabalin (10, 30 and 100 mg/kg p.o). 
For this experiment, cold allodynia was investigated by immersing mice tail into cold water 
maintained at 4˚C±0.5 1 hour after drug administrations. The time it takes for a mouse to 
withdraw its tail from the second it was immersed in cold water was measured with a timer. 
The cut-off point was set to 20 seconds. The %MPE was calculated and plotted as described in 
section 3.8.3. 
3.9.5. Sub-chronic Oral Toxicity (90-Day) 
Sprague-Dawley rats were divided randomly into four groups (n=5); one control group and 
three (3) treatment groups. Rats in the control group received distilled water (vehicle) by oral 
gavage. Those in the three (3) treatment groups were given the Mallotus oppositifolius extract 
at doses of 30, 100, or 300 mg/kg p.o. respectively, all by oral gavage. MOE was freshly 
prepared daily with distilled water for the 90-day period. Administration was repeated daily for 
90 days. On each day, the rats are weighed and observed for mortality and clinical signs of 
toxicity or abnormality. 
On day 91, blood samples were collected from all animals via abdominal aorta puncture into 
Eppendorf tubes containing EDTA for haematological analyses to evaluate levels of 
haemoglobin (HGB), red blood cells (RBC), white blood cells (WBC), mean corpuscular 
volume (MCV), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular 
haemoglobin (MCH) and platelets (PLT), granulocytes (Gran), and monocytes (MONO) with 
an automated haematology analyser (KX-2IN, Sysmex Corporation, Japan). Alongside, blood 
39 
 
samples for biochemical analyses were collected into BD-serum separator tubes and 
centrifuged at 3000 rpm for 10 minutes. The serum obtained was analysed for aspartate 
aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), total protein (TP), 
alkaline phosphatase (ALP), triacylglycerol (TG), urea, creatinine (CREA), cholesterol (CHO).  
The rats were euthanized in a chloroform chamber on the 91st day. The organs of interest were 
excised, weighed and visually examined. Principal vital organs (liver, kidney, brain, heart and 
spleen) were preserved in sterile containers with 10% buffered formalin and transported to a 
pathology laboratory for processing and examination. 
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 two further 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 sides and stained by the haematoxylin and eosin 
method and observed under a microscope. The method is outlined below. 
1. Sections on slides were put in two changes of xylene for 15 minute each 
2. Section through three changes of absolute ethanol for 5 minutes each 
3. Sections to 70% ethanol for 15 minutes 
4. Sections to 50% ethanol for 15 minutes 
5. Sections to tap water for 30 minutes 
6. Sections to distilled water for 2 minutes 
7. Sections to Mayer’s haematoxylin stain for 15 minutes 
8. Sections to tap water for 30 minutes 
9. Sections to working eosin solution for 5 minutes 
10. Sections to 80% ethanol for 5 minutes 
40 
 
11. Sections to two changes of absolute ethanol for 5 minutes 
12. Sections to three changes of hemode for 5 minutes each 
13. Sections mounted in DPX 
3.10. Statistical Analysis 
GraphPad Prism 8.0.2 (San Diego, CA) for Windows was the software used to perform all 
statistical analyses and ED50 calculations, and a p-value of ≤ 0.05 was considered statistically 
significant for all analyses. Mean ± standard error of mean (SEM) was used in presenting all 
data. The time-course curves in the study were analysed using two-way (treatment x time) 
analysis of variance (ANOVA) followed by a Bonferroni’s post hoc test. The total (anti-
)nociceptive score for each treatment was calculated as the area under the curve (AUC). One-
way ANOVA followed by Dunnett's multiple comparisons test was used to determine 
differences between treatment groups (areas under the curves). Percentage inhibition was 
calculated using the following formula: 
 
The ED50s for each drug was calculated by using an iterative computer least squares method, 
with the following non-linear regression (three-parameter logistic) equation.  
 
Where X is the logarithm of dose and Y is the response. Y starts at a (the bottom) and goes to b 
(the top) with a sigmoid shape. The fitted midpoints (ED50s) of the curves were compared 
statistically using the F-test. All graphs in this study were plotted using SigmaPlot Version 14.0 
(Systat Software, Germany).  
41 
 
Chapter Four 
4 RESULTS 
4.1. HPLC Analysis of MOE 
A total of 12 constituents were observed beginning at the elution time of 4.78 min to 22.14 min 
with varied peaks (Table 4.1). No constituents were detected after this time period. Figure 4.1 
shows the HPLC chromatogram of the hydroethanolic extract of M. oppositifolius. Three major 
constituents were observed at retention times 4.78, 13.25 and 20.88 min (Table 4.1). The 
percentage composition of these constituents was calculated to be 33.61%, 15.63% and 11.96% 
respectively, using the area under the curves (AUCs). These three constituents make up about 
62% of the extract. 
 
 
Figure 4.1. HPLC chromatogram of MOE monitored at 272, 278nm 
 
42 
 
Table 4.1. Report on the HPLC analysis of MOE 
Peak No. Retention Time Area Height Percentage (%) 
1 4.779 3,017,600.60 115,731.90 33.61 
2 13.228 1,402,988.10 94,766.10 15.63 
3 14.335 77,362.80 5,172.70 0.86 
4 14.739 669,320.60 55,659.90 7.46 
5 15.324 137,361.80 8,034.10 1.53 
6 16.138 510,770.20 31,901.60 5.69 
7 17.86 773,004.80 45,825.80 8.61 
8 18.934 290,487.30 9,878.60 3.24 
9 19.573 467,553.90 22,967.80 5.21 
10 20.875 1,073,238.10 46,666.00 11.96 
11 21.375 364,867.40 22,232.80 4.06 
12 22.137 192,440.70 7,702.40 2.14 
TOTAL  8,976,996.30   
 
 
  
43 
 
4.2. Phytochemical Screening 
Phytochemical screening of the hydroethanolic (70%) extract of M. oppositifolius revealed the 
presence of secondary metabolites including saponins, alkaloids, flavonoids, glycosides, 
sterols, tannins and reducing sugars (Table 4.2). 
 
Table 4.2. Preliminary phytochemical screening of MOE 
PHYTOCHEMICALS INFERENCE 
Saponins Present 
Alkaloids Present 
Flavonoids Present 
Terpenoids Absent 
Glycosides Present 
Sterols Present 
Tannins Present 
Reducing sugars Present 
 
  
44 
 
4.3. The Effect of MOE in Acute Models of Pain 
4.3.1. Formalin Test 
The intraplantar injection of 10µL of 5% formalin of the right hind paw of mice induced a 
nociceptive response characterized by the biting and licking of the injected paw. Figure 4.3.1 
shows the effect of the pre-treatment of mice with MOE, diclofenac and morphine in the 
formalin test. As depicted by the time course curves (Figure 4.3.1A,C,E) all the drug-treated 
groups displayed a significant reduction in nociceptive scores when compared to the vehicle 
(VEH) treated group [(MOE: F3,16=32.52, P<0.0001; diclofenac: F3,16=41.81, P<0.0001 
morphine: F3,16=34.46, P<0.0001 (two-way ANOVA)]. Two-way ANOVA revealed that the 
oral administration of MOE (30, 100, 300 mg/kg) one hour before the intraplantar injection of 
formalin significantly suppressed the formalin-induced nociceptive behaviour in mice in both 
the neurogenic (F3,16=7.91, P=0.002) and inflammatory (F3,16=24.28, P<0.0001) phases (Figure 
4.3.1B). Analysis of the AUCs (Figure 4.3.1B) for the doses used revealed that MOE attenuated 
formalin-induced nociceptive behaviour by 58.30 to 73.74% in the early/neurogenic phase and 
53.60 to 93.17% in the late/inflammatory phase. Also, one-way ANOVA followed by Dunnett's 
multiple comparisons test revealed a significant decrease in total nociceptive scores in both 
phases in the presence of MOE (F3,16=7.87, P=0.001; and F3,16=23.45, P<0.0001; respectively) 
(Figure 4.3.1 B). 
Similarly, the intraperitoneal administration of the positive analgesic control used, morphine 
(0.3, 1, 3 mg/kg), a centrally acting opioid, 30 minutes before formalin injection resulted in a 
significant and dose-dependent reduction in nociceptive scores (Figure 4.3.1F) in both the early 
(F3,16=6.67, P=0.004) and late (F3,16=27.50, P<0.0001) phases (two-way ANOVA). Analysis 
of AUCs (Figure 4.3.1F) show that morphine produced a maximum inhibition of 66.43% in 
the neurogenic phase (F3,16=6.77, P=0.004) and 91.69% in the inflammatory phase 
(F3,16=26.65, P<0.001).  
45 
 
Also, an intraperitoneal administration of the reference drug, diclofenac (3-30 mg/kg) a 
peripherally acting analgesic, given 30 minutes before formalin administration dose-
dependently inhibited the inflammatory phase (F3,16=36.90, P<0.0001 two-way ANOVA 
followed by Bonferroni's multiple comparisons test) but was not active in the neurogenic phase 
(F3,16=0.7226, P=0.55) as shown in Figure 4.3.1D. The percentage inhibitions calculated from 
the AUCs in Figure 4.3.1.1D were 84.54 to 97.24% in the late phase. 
The ED50 values of the three drugs used were calculated using non-linear regression (Table 
4.3.1) for both phases of the formalin test. ED50s for Diclofenac and morphine were 
significantly different for that of MOE in both phases (phase 1: F2,42=22.13, P<0.0001; phase 
2: F2,42=47.20, P<0.0001). Comparison of ED50s by the F-test revealed that MOE was 1.5 times 
more potent in the second phase than in the first phase (F1,14=6.159, P=0.03). Also, MOE was 
four (4) times more potent than diclofenac in the neurogenic phase and about 40 times less 
potent than diclofenac in the inflammatory phase. 
 
46 
 
 
Figure 4.3.1: Effect of MOE (30, 100 and 300 mg/kg), diclofenac (3, 10 and 30 mg/kg) and morphine (0.3, 1 and 
3 mg/kg) on the time-course events (A, C, E respectively) and the total nociceptive score (calculated as AUCs) 
(B, D, F respectively) on formalin-induced nocifensive behaviour in ICR mice.  
Nociceptive scores are shown in 5-minute time blocks up to the 60th minute post formalin injection. Plotted data 
are mean ± SEM (n = 5). *P<0.05, **P<0.01 and ***P<0.001 compared to the vehicle-treated group (two-way 
ANOVA followed by Bonferroni’s post hoc test). †P<0.05, ††P<0.01, †††P<0.001 compared to the vehicle-treated 
group (one-way ANOVA followed by Dunnett's multiple comparisons test).  
47 
 
Table 4.3.1.1. ED50s for MOE, diclofenac and morphine in the formalin test 
DRUG ED50 (mg/kg) 
Phase 1 Phase 2 
MOE 34.33 ± 13.77 22.91 ± 4.01 
Diclofenac 137.42 ± 91.04 0.58 ± 0.10 
Morphine 0.49 ± 0.17 0.02 ± 0.03 
 
4.3.2. Acetic Acid Induced Writhing Test 
The intraperitoneal injection of mice in this experiment with acetic acid (0.6%, 10 mL/kg) 
induced a characteristic nociceptive behaviour known as writhing (abdominal constrictions, 
pelvic rotation, and hind limb stretching). Figure 4.3.2 shows the effect (nociceptive score) of 
the pre-treatment of mice with MOE and diclofenac on mice in the acetic acid induced writhing 
test over the 30-minute observation period. The oral administration of MOE (30-300 mg/kg) 
one hour before the administration of 0.6% acetic acid significantly inhibited abdominal 
writhes over the 30-minute period as compared to the control group [F3,15=13.92, P<0.001 
(two-way ANOVA)] (Figure 4.3.2A). Analysis of the AUCs shows that MOE at 30, 100 and 
300 mg/kg significantly (F3,15=14.16, P<0.001) and dose-dependently attenuated acetic acid 
induced writhes by 73.14% (P=0.003), 92.92% (P<0.0001) and 96.49% (P<0.0001). 
In a similar way, the intraperitoneal administration of the peripherally acting non-steroidal anti-
inflammatory drug (NSAID), diclofenac (3, 10 and 30 mg/kg), 30 minutes before in a 0.6% 
acetic acid administration significantly reduced acetic acid induced writhes [F3,16=17.54, 
P<0.0001 (two way ANOVA)] (Figure 4.3.2C) in comparison to the negative control. Analysis 
of the AUCs shows that diclofenac at 3, 10 and 30 mg/kg significantly reduced acetic acid 
induced writhes by 95.13% (P<0.0001), 97.01% (P<0.0001) and 99.69% (P<0.0001). 
48 
 
ED50s were calculated by non-linear regression method (table 4.3.2). Comparison with the F-
test revealed that diclofenac was more potent than MOE in the writhing test (P<0.001).  
Table 4.4. ED50s for MOE and diclofenac in the writhing test 
DRUG ED50 (mg/kg) 
MOE 10.41 ± 1.14 
Diclofenac 0.17 ± 0.06 
 
 
 
49 
 
 
Figure 4.3.2. Effect of MOE (30, 100 and 300 mg/kg) and diclofenac (3, 10 and 30 mg/kg) on the time-course 
events (A and C respectively) and the total nociceptive score (calculated as AUCs) (B and D respectively) on 
acetic acid induced writhing behaviour in ICR mice.  
Nociceptive scores are shown in 5-minute time blocks up to 30 minutes post acetic acid injection. Plotted data are 
mean ± SEM (n=5) *P<0.05, **P<0.01 and ***P<0.001 compared to the vehicle-treated group (two-way ANOVA 
followed by Bonferroni’s post hoc test). †P<0.05, ††P<0.01, †††P<0.001 compared to the vehicle-treated group 
(one-way ANOVA followed by Dunnett's multiple comparisons test). 
 
  
50 
 
4.3.3. Hot Plate Test 
Figure 4.3.3 shows the effect of MOE (30-300 mg/kg) and morphine (0.3-3 mg/kg) on paw 
withdrawal latencies calculated as %MPE in the hot plate test. As depicted by the time course 
curve in Figure 4.3.3A, the oral administration of MOE significantly increased paw withdrawal 
latencies (F3,16=6.23, P=0.005 [two-way ANOVA]) compared to the distilled water treated 
group over the 3-hour test period. Analysis of the AUCs (Figure 4.3.3B) shows that MOE 
significantly (F3,16=6.48, P=0.004 [one-way ANOVA]) increased total paw withdrawal 
thresholds with a maximum effect (P=0.002) [one-way ANOVA]) at the highest dose (300 
mg/kg). However, MOE at 30 mg/kg (Figure 4.3.3B) was not statistically significant (P=0.21 
[one-way ANOVA]). 
In a very similar way, the intraperitoneal injection of the positive control drug used, morphine 
(Figure 4.3.3C) the centrally acting opioid analgesic, 30 minutes before the hot plate testing 
displayed a significant and dose-dependent increase in paw withdrawal latencies (F3,16=6.23, 
P=0.005 [two-way ANOVA]), compared to the distilled water treated group over the 3 hour 
test period. AUC analysis (Figure 4.3.3D) revealed that overall, morphine significantly 
(F3,16=14.92, P<0.001 [one-way ANOVA]) and dose-dependently increased total paw 
withdrawal thresholds with a maximum effect at the highest dose (3 mg/kg; P<0.001; [one-
way ANOVA]).  
ED50s were calculated by non-linear regression method (table 4.3.3). Comparison with the F-
test revealed that diclofenac was more potent than MOE in the writhing test (P<0.001). 
 
51 
 
 
Figure 4.3.3. Effect of MOE (30, 100 and 300 mg/kg) and morphine (0.3, 1 and 3 mg/kg) on the time-course 
events (A and C respectively) and the total antinociceptive score (calculated as AUCs) (B and D respectively) on 
thermally induced jumping and paw licking behaviour in ICR mice.  
Nociceptive scores are shown from 0 to 180 minutes post MOE administration (p.o.). Plotted data are mean ± 
SEM (n = 5). *P<0.05, **P<0.01 and ***P<0.001 compared to the vehicle-treated group (two-way ANOVA followed 
by Bonferroni’s post hoc test). †P<0.05, ††P<0.01, †††P<0.001 compared to the vehicle-treated group (one-way 
ANOVA followed by Dunnett's multiple comparisons test). 
 
52 
 
Table 4.5. ED50s for MOE and morphine in the hot plate test 
DRUG ED50 (mg/kg) 
MOE 380.41 ± 91.28 
Morphine 1.33 ± 0.35 
 
4.3.4. The Possible Mechanism(s) of Antinociception 
Figure 4.4.1-4.4.3 shows the effect of the pre-treatment of mice with 7 different antagonists on 
the antinociceptive activity of MOE (100 mg/kg p.o) and morphine (1 mg/kg i.p) in the 
formalin test.  
Systemic pre-treatment of ICR mice with naloxone (2 mg/kg), glibenclamide (8 mg/kg), 
theophylline (10 mg/kg), ondansetron (0.5 mg/kg), yohimbine (3 mg/kg), nifedipine (10 
mg/kg) and atropine (5 mg/kg) could not significantly block the antinociceptive activity of 
MOE in the first phase of the formalin test (Figure 4.4.3) [naloxone (P=0.17), glibenclamide 
(P=0.99), theophylline (P=0.19), ondansetron (P<0.24), yohimbine (P>0.99), nifedipine 
(P=0.16) and atropine (P=0.39) (One-way ANOVA followed by Dunnett's multiple 
comparisons test)]. Similarly, naloxone, glibenclamide, theophylline, ondansetron, yohimbine 
nifedipine and atropine could not significantly reverse the antinociceptive effects of MOE in 
the second phase of the formalin test (P=0.37; P=0.95; P=095; P=0.64; P=0.92; P=0.15 and 
P=0.73 respectively [Figure 4.4.3]).  
In the positive control tests, morphine (1 mg/kg) was used as the reference drug (Figure 4.4.2). 
Pre-treatment of ICR mice with naloxone and theophylline significantly blocked the 
antinociceptive activity of morphine in both the first phase (P<0.001 for both) and the second 
phase (P<0.01 and P<0.03 respectively) of the formalin test exhibited as an increase in the 
53 
 
frequency and time of formalin-induced paw licking in mice (Figure 4.4.3). Similarly, pre-
treatment of mice with ondansetron, a 5-HT3 receptor antagonist, significantly reversed the 
antinociception caused by morphine in both phase one (P<0.001) and phase two (P<0.03) of 
the formalin test. In contrast, pre-treatment with glibenclamide, yohimbine, nifedipine and 
atropine did not significantly reverse the antinociceptive activity of morphine in the first phase 
(P>0.99, P>0.99, P=0.43, P>0.99 respectively) and second phase (P=0.97, P>0.99, P=0.93, 
P=0.72 respectively) of the formalin test in mice (Figure 4.4.3). 
54 
 
 
Figure 4.4.1. The effect of the pre-treatment of ICR mice with (B) naloxone (2 mg/kg), (C) glibenclamide (8 
mg/kg), (D) theophylline (10 mg/kg), (E) ondansetron (0.5 mg/kg), (F) yohimbine (3 mg/kg), (G) nifedipine (10 
mg/kg) and (H) atropine (5 mg/kg) on the antinociceptive activity of MOE (100 mg/kg p.o) in the first and second 
phases of formalin-induced nociception.  
Nociceptive scores are shown in 5-minute time blocks up to the 60th minute post formalin injection. Plotted data 
are mean ± SEM (n = 5). 
55 
 
 
Figure 4.4.2. The effect of the pre-treatment of ICR mice with (B) naloxone (2 mg/kg), (C) glibenclamide (8 
mg/kg), (D) theophylline (10 mg/kg), (E) ondansetron (0.5 mg/kg), (F) yohimbine (3 mg/kg), (G) nifedipine (10 
mg/kg) and (H) atropine (5 mg/kg) on the antinociceptive activity of morphine (1 mg/kg i.p) in the first and second 
phases of formalin-induced nociception.  
Nociceptive scores are shown in 5-minute time blocks up to the 60th minute post formalin injection. Plotted data 
are mean ± SEM (n = 5). 
56 
 
 
Figure 4.4.3. The effect of the pre-treatment of ICR mice with naloxone (2 mg/kg), glibenclamide (8 mg/kg), 
theophylline (10 mg/kg), ondansetron (0.5 mg/kg), yohimbine (3 mg/kg), nifedipine (10 mg/kg) and atropine (5 
mg/kg) on the antinociceptive activity of (A) MOE (100 mg/kg p.o) and (B) morphine (1 mg/kg i.p) in the first 
and second phases of formalin-induced nociception.  
Each column represents mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 compared to the vehicle-treated group 
(two-way ANOVA followed by Bonferroni’s post hoc test). †P<0.05, ††P<0.01, †††P<0.001 compared to the either 
MOE- or morphine-treated group (one-way ANOVA followed by Dunnett's multiple comparisons test). 
57 
 
4.4. The Effect of MOE in Paclitaxel-induced Peripheral Neuropathy 
4.4.1. Induction of Peripheral Neuropathy 
Assessment of thermal hyperalgesia, cold allodynia and mechanical hyperalgesia during the 5-
day induction (day 1 to 5) of peripheral neuropathy with a cumulative paclitaxel dose of 10 
mg/kg yielded a gradual decline in latencies and nociceptive thresholds within the groups used, 
from day 0 to day 5 as depicted by the time course curves (Figure 4.5.1 A,C,E). 
Two-way ANOVA followed by Bonferroni’s post hoc test showed a significant decline in 
latencies and paw withdrawal thresholds in the thermal hyperalgesia test (P<0.0001) and in the 
mechanical hyperalgesia test (P<0.0001) respectively, comparing day 0 (baseline) to day 5 (day 
of final paclitaxel administration) (Figure 4.5.1A-D). This suggests a reduced pain threshold 
and confirms the induction of peripheral neuropathy. Analysis of the responses between day 0 
and day 5 in the cold allodynia test (Figure 4.5.1E-F) revealed a significant reduction in tail 
withdrawal latencies (group 1: P=0.003; group 2: P=0.04; group 3: P=0.04; group 6: P=0.001; 
group 7: P=0.03). However, decline in latencies for group 4 (P=0.051) and group 5 (P=0.23) 
did not reach statistical significance. 
Two deaths were recorded on day 0, pre-paclitaxel administration. No deaths were recorded 
during the induction period. However, two deaths were recorded on day 2, two on day 3, two 
on day 4 and one on day 5 due to cannibalism by cage members.  
 
 
58 
 
 
Figure 4.5.1: Effect of the daily intraperitoneal administration of paclitaxel (2 mg/kg) on thermal hyperalgesia 
(A-B), mechanical hyperalgesia (C-D) and cold allodynia (E-F) tests in ICR mice for 5 days.  
A, C, E: Time-course event from day 0 to 5. B, D, F: A comparison of reaction latency on day 0 and day 5. Plotted 
data are mean ± SEM (n = 5). *P<0.05, **P<0.01 and ***P<0.001compared to the vehicle-treated group (two-
way ANOVA followed by Bonferroni’s post hoc test).  
59 
 
4.4.2. Thermal Hyperalgesia (Hot Plate test) 
As depicted in Figure 4.5.2, sustained thermal hyperalgesia was observed in the mice in the 
vehicle treated group from day 1 through to day 5. MOE (30-300 mg/kg) and pregabalin (10-
100 mg/kg) produced significant and dose dependent reversal of thermal hyperalgesia (MOE: 
F3,65=82.6, P<0.0001; pregabalin: F3,66=33.38, P<0.0001) displayed as an increase in latency 
to nocifensive behaviour on the hot plate apparatus. Treatment of mice with MOE at the doses 
of 100 mg/kg and 300 mg/kg (Figure 4.5.2A, B) produced significant reversal of thermal 
hyperalgesia each day from test day one to day five. However, results obtained when mice were 
treated daily with MOE at 30 mg/kg produced significant reversal of thermal hyperalgesia on 
day 5 (P=0.009). Analysis of AUCs revealed that overall, MOE significantly (F3,15=4.86; 
P=0.01; [one-way ANOVA]) and dose-dependently reversed thermal hyperalgesia with a 
maximum effect at the highest dose (300 mg/kg; P<0.001; [one-way ANOVA]). 
Similarly, treatment of mice with pregabalin at the dose of 100 mg/kg (Figure 4.5.2C-D) 
produced a significant reversal of thermal hyperalgesia in the hot plate test, daily, from test day 
one to day five. The pregabalin dose at dose 30 mg/kg yielded significant analgesic effect from 
day 2 to day 5. Treatment of mice with the lowest dose of pregabalin yielded no statistically 
significant results except on day 5 (P=0.03). AUC analysis revealed that pregabalin 
significantly inhibited thermal hyperalgesia at the doses of 100 mg/kg (P<0.001) and 30 mg/kg 
(P<0.001).             
The ED50s (mg/kg) obtained by non-linear regression for MOE and pregabalin are 150.1 and 
42.43 respectively. Thus, MOE was about 3.5 times less potent than pregabalin in the thermal 
hyperalgesia test. 
 
60 
 
 
Figure 4.5.2: Effect of MOE (30, 100 and 300 mg/kg) and Pregabalin (10, 30 100 mg/kg) on thermal hyperalgesia 
in paclitaxel induced neuropathy in ICR mice. The left panels represent time-course effects of MOE (A) and 
pregabalin (C) from day 0 to day 5 after neuropathy was confirmed.  
The right panels represent the total antinociceptive effects (calculated as AUCs) of MOE (B) and pregabalin (D). 
Plotted data are mean ± SEM (n = 5). **P<0.01 and ***P<0.001 compared to the vehicle-treated group (two-way 
ANOVA followed by Bonferroni’s post hoc test). †P<0.05, ††P<0.01, †††P<0.001 compared to the vehicle-treated 
group (one-way ANOVA followed by Dunnett's multiple comparisons test). 
 
61 
 
Table 4.5.2. ED50s for MOE and pregabalin in the thermal hyperalgesia test 
DRUG ED50 (mg/kg) 
MOE 150.1 ± 58.03 
Pregabalin 42.43 ± 9.94 
  
62 
 
4.4.3. Mechanical Hyperalgesia (Randall-Selitto test) 
Both mice hind paws demonstrated distinct mechanical hyperalgesia in baseline measurements 
taken on day 0 in this experiment. From figure 4.5.3, sustained mechanical hyperalgesia was 
observed in the vehicle-treated mice.  MOE (30-300 mg/kg) and pregabalin (10-100 mg/kg) 
produced significant and dose dependent reversal of mechanical hyperalgesia (MOE: 
F3,65=53.75, P<0.0001; pregabalin: F3,65=81.56, P<0.0001; two-way ANOVA). Treatment of 
mice with MOE at the doses of 100 mg/kg and 300 mg/kg (Figure 4.5.3A) significantly 
reversed mechanical hyperalgesia from day one to five (MOE 300 mg/kg) and day two to five 
(MOE 100 mg/kg). Treatment with MOE at the dose of 30 mg/kg significantly reversed 
mechanical hyperalgesia on day 5 (P=0.05). One-way ANOVA followed by Dunnett's multiple 
comparisons test of the AUCs (Figure 4.5.3B) shows that MOE significantly and dose-
dependently reversed mechanical hyperalgesia at the doses of 100 mg/kg (P=0.03) and 300 
mg/kg (P=0.01).  
As depicted by figure 4.5.3C, daily treatment of mice with pregabalin at the doses of 30 mg/kg 
and 100 mg/kg significantly (P<0.0001) reversed mechanical hyperalgesia from day one to 
five. However, inhibition of mechanical hyperalgesia by pregabalin at 10 mg/kg was not 
statistically significant on any of the test days as revealed by two-way ANOVA analysis 
followed by Bonferroni's multiple comparisons test. Analysis of the AUCs (figure 4.5.3D) 
shows that overall, pregabalin dose-dependently and significantly (F3,13=25.54, P<0.001 [one-
way ANOVA]) reversed mechanical hyperalgesia in both hind paws of the mice from day 1 to 
5 with maximum inhibitions (P<0.001) occurring at 30-100 mg/kg dose levels. 
The ED50s (mg/kg) obtained by non-linear regression for MOE and pregabalin in this test are 
112.4 and 25.11 respectively (Table 4.5.3). Thus, MOE was about 4.5 times less potent than 
pregabalin in the mechanical hyperalgesia test. 
63 
 
 
Figure 4.5.3: Effect of MOE (30, 100 and 300 mg/kg) and Pregabalin (10, 30 100 mg/kg) on mechanical 
hyperalgesia in paclitaxel induced neuropathy in ICR mice. The left panels represent time-course effects of MOE 
(A) and pregabalin (C) from day 0 to day 5 after neuropathy was confirmed.  
The right panels represent the total antinociceptive effects (calculated as AUCs) of MOE (B) and pregabalin (D). 
Plotted data are mean ± SEM (n = 5). *P<0.05, **P<0.01 and ***P<0.001 compared to the vehicle-treated group 
(two-way ANOVA followed by Bonferroni’s post hoc test). †P<0.05, ††P<0.01, †††P<0.001 compared to the 
vehicle-treated group (one-way ANOVA followed by Dunnett's multiple comparisons test). 
 
 
64 
 
Table 4.5.3. ED50s for MOE and pregabalin in the mechanical hyperalgesia test 
DRUG ED50 (mg/kg) 
MOE 112.4 ± 45.72 
Pregabalin 25.11 ± 6.05 
 
4.4.4. Cold Allodynia 
The daily administration of MOE to mice from treatment day 1 to 5 (figure 4.5.4A) yielded 
significant anti-cold allodynic effects at the dose of 300 mg/kg on the fourth (P=0.01) and fifth 
(P=0.005) days (two-way ANOVA) exhibited in the test as an increased latency to tail 
withdrawal from cold water at 4˚C. Reversal of cold allodynia when mice were treated daily 
with MOE at 30 mg/kg and 100 mg/kg was not statistically significant on any of the 5 test days. 
Analysing the AUCs with one-way ANOVA followed by Dunnett's multiple comparisons test 
(figure 4.5.4B) reveals that overall inhibition of cold allodynia was not significant (F3,13=0.93, 
P=0.45) when compared to the AUC of the vehicle treated group [(MOE 30: P>0.99; MOE 
100: P=0.89; MOE 300: P=0.34). 
From figure 4.5.4C pregabalin at the dose of 100 mg/kg produced significant analgesic effect 
in the cold allodynia test on the 4th (P<0.001) and 5th (P<0.001) days of administration and 
testing. Analgesic effect of pregabalin at 30 mg/kg produced significant antiallodynic effect on 
the 4th day (P<0.03) alone. Daily treatment with the dose of 10 mg/kg did not yield any 
significant anti-allodynic effects on any of the test days. Analysis of AUCs in figure 4.5.4D 
reveals that overall, pregabalin at 100 mg/kg produced significant antiallodynic effect (P=0.03) 
in the test conducted. Overall, analgesic effects of the doses of pregabalin at 10 mg/kg (P=0.42) 
and 30 mg/kg (P=0.12) did not reach statistical significance in the test. 
65 
 
The ED50s (mg/kg) obtained by non-linear regression for MOE and pregabalin in this test are 
584.8 and 58.07 respectively (Table 4.5.4). Thus, MOE was about 10 times less potent than 
pregabalin in this cold allodynia test. 
 
Figure 4.5.4. Effect of MOE (30, 100 and 300 mg/kg) and Pregabalin (10, 30 100 mg/kg) on cold allodynia in 
paclitaxel induced neuropathy in ICR mice.  
The left panels represent of MOE (A) and pregabalin (C) from day 0 to day 5 after neuropathy was confirmed. 
The right panels represent the total antinociceptive effects (calculated as AUCs) of MOE (B) and pregabalin (D). 
Plotted data are mean ± SEM (n = 5). *P<0.05, **P<0.01 and ***P<0.001 compared to the vehicle-treated group 
(two-way ANOVA followed by Bonferroni’s post hoc test). †P<0.05, ††P<0.01, †††P<0.001 compared to the 
vehicle-treated group (one-way ANOVA followed by Dunnett's multiple comparisons test). 
66 
 
Table 4.5.4. ED50s for MOE and pregabalin in the cold allodynia test 
DRUG ED50 (mg/kg) 
MOE 584.8 ± 395.41 
Pregabalin 58.07 ± 23.86 
  
67 
 
4.5. Sub-chronic Oral Toxicity (90-Day) 
Two Sprague-Dawley rats from the control group, the low dose group and the median dose 
group died. Two more died from the high dose group. This was due to oral administration 
problems and affected rats were isolated.  
4.5.1. Necropsy and clinical signs 
For the rats that were not isolated, no death or serious clinical signs were found in any of the 
groups all through the 90-day experimental period. Daily visual observation of the MOE (30, 
100 and 300 mg/kg; group 1-3) treated rats reported no signs of toxicity on their skin, fur or 
eyes. They didn’t exhibit any behavioural changes, coma, tremors or salivation. Rats in the 
vehicle treated groups produced similar results on routine checks. LD50 is estimated above 3000 
mg/kg. 
 
Figure 4.6.1. Mean weights of Sprague-Dawley rats for all groups(n=5) from day 1 to day 91.  
68 
 
4.5.2. Effect of daily MOE administration on weight change 
Figure 4.6.1 is a graphical representation of the change in mean weights for all groups from 
day 1 to day 91. There was consistent weight gain over time in all the groups (P<0.001). Rats 
in all groups demonstrated rapid weight gain during the first 5 weeks and a slower rate 
thereafter. However, compared with the vehicle treated group, there were no dose-specific 
patterns in body weight related to drug treatment (F3,160=0.47, P=0.70). 
4.5.3. Effect of daily MOE administration on relative organ weights 
The relative organ weights of the rats are shown in Tables 4.6.1. The relative organ weight for 
each organ (liver, kidney, heart, spleen, brain) recorded at necropsy for rats in the extract 
treated groups did not show any significant difference compared to the control (P>0.5).  
Table 4.6.1. The relative organ weights of rats treated with MOE over the 90-day period 
Organs Control  MOE (mg/kg)  
  30 100 300 
Liver 3.93±0.06 4.43±0.08 3.13±0.79 3.70±0.44 
Kidneys 0.32±0.02 0.43±0.02 0.37±0.01 0.37±0.09 
Heart 0.39±0.01 0.44±0.05 0.40±0.02 0.48±0.03 
Spleen 0.30±0.05 0.30±0.01 0.27±0.04 0.31±0.04 
Brain 0.80±0.19 0.90±0.16 1.02±0.14 1.27±0.50 
The values are presented as Means ± SEM 
 
4.5.4. Effect of daily MOE administration on RBC Indices 
The influence of the daily administration of MOE on haematological indices for the male 
Sprague-Dawley rats after the 90-day sub-chronic toxicity study are displayed on Table 4.6.2. 
Analysis of the RBC indices for all groups revealed that the MOE treated groups showed no 
69 
 
significant changes/differences in any of the RBC indices (F3,48=1.545, P=0.22) compared to 
the vehicle treated group.  
4.5.5. Effect of daily MOE administration on WBC Indices 
Table 4.6.2 shows the effect of MOE on the levels of lymphocytes, neutrophils, eosinophils, 
basophils, monocytes and the total WBC in the 90-day toxicity study. No significant 
differences were observed for the levels of monocytes, eosinophils and basophils when 
compared to the vehicle treated group. However, the levels of neutrophils for the group treated 
with the median dose (100 mg/kg) was significantly higher (P=0.01) compared to the vehicle 
treated group. Overall, differences in neutrophil levels was not dose-dependent. Percent 
lymphocyte levels were also significantly lower (P=0.03) for the median dose group. Overall, 
treatment with MOE had no significant dose dependent effect on the WBC indices 
(F3,36=0.1205, P=0.95). 
4.5.6. Effect of daily MOE administration on Platelet Indices 
Analysis of the platelet indices displayed on Table 4.6.2 revealed a significant decrease in 
platelet count (PLT) for the 30 mg/kg dose P=0.01 and the 100 mg/kg dose (P=0.02) compared 
to the control group. However, there were no significant difference in the platelet ratio (PCT) 
and size (PDW and MPV) for any of the 3 MOE treated groups compared to the vehicle treated 
group. 
 
 
 
70 
 
Table 4.6.2. The haematological indices of rats treated with MOE over the 90-day period    
Parameter Unit Control  MOE (mg/kg) 
   30 100 300 
RBC indices      
RBC 1012/L 9.63±0.34 9.59±0.21 10.54±0.99 9.41±0.16 
HGB g/dL 16.23±0.49 15.4±0.30 16.77±1.05 15.10±0.50 
HCT % 40.97±1.53 38.7±0.90 43.00±3.44 38.45±0.75 
MCV fL 42.6±1.22 40.45±0.05 40.97±0.60 40.90±0.10 
MCH g/dL 16.83±0.50 16.00±0.00 15.93±0.47 16.00±0.30 
MCHC g/dL 39.33±0.33 40.00±0.00 39.33±0.67 39.50±0.50 
RDW-SD fL 35.23±1.03 33.3±1.20 34.47±1.36 35.05±0.55 
RDW-CV % 12.6±0.46 12.9±0.00 12.73±0.70 12.75±0.05 
WBC indices      
WBC 109/L 12.69±3.55 4.18±0.15 16.29±3.51 15.68±7.92 
LYM % 63.91±3.44 73.2±0.29 42.29±14.04* 52.41±8.25 
MON % 8.65±1.60 9.59±0.73 9.61±1.62 8.72±1.80 
NEU % 25.7±4.72 15.32±1.57 46.38±14.65* 37.03±10.37 
EOS % 1.59±0.36 1.78±0.64 1.51±0.34 1.67±0.23 
BASO % 0.16±0.10 0.13±0.08 0.22±0.03 0.17±0.09 
Platelet indices     
PLT 109/L 1272.67±70.05 818.5±6.5* 882±297.76* 1021.5±125.5 
MPV fL 3.83±0.13 4.15±0.15 3.87±0.09 4.05±0.05 
PDW fL 6.30±0.32 5.40±0.00 5.47±0.62 6.05±0.35 
PCT % 0.48±0.03 0.34±0.01 0.34±0.12 0.41±0.06 
The values are presented as Means ± SEM              *P < 0.05 
4.5.7. Effect of daily MOE administration on serum biochemistry parameters 
Displayed on Table 4.6.3 are the results for the biochemical parameters at the end of the 90-
day treatment period. The AST levels for the group treated with the median dose (100 mg/kg) 
71 
 
were significantly lower (P=0.03) compared to the vehicle treated group. Significantly lower 
values (P<0.001) were also recorded for ALP at the dose of 30 mg/kg. Differences were also 
recorded for other parameters such as the blood urea levels but these were not significant when 
compared to the vehicle treated group (P>0.05). 
Table 4.6.3. Serum biochemistry parameters of rats treated with MOE over the 90-day period 
Parameter Unit Control MOE (mg/kg) 
   30 100 300 
Liver      
TBil µmol/L 5.10±1.01 9.55±2.34 5.95±0.88 7.63±3.15 
DBil µmol/L 1.79±0.33 2.16±1.85 3.00±1.70 2.86±0.63 
IBil µmol/L 3.31±0.94 7.40±0.50 2.95±0.84 4.77±2.52 
ALT U/L 71.63±6.08 72.45±4.45 58.45±3.53 77.1±12.4 
AST U/L 148.53±27.96 116.85±6.85 94.9±10.42* 116.75±15.95 
ALP U/L 265.13±7.00 129.6±74.6* 303.85±64.87 238.11±4.11 
GGT U/L 4.00±2.31 5.50±3.50 9.33±0.88 12.50±6.50 
Total Protein g/L 70.47±1.43 64.70±1.30 65.7±2.43 59.20±3.80 
ALB g/L 27.37±1.62 27.50±1.10 23.93±1.36 23.55±3.35 
GLB g/L 43.10±3.04 37.20±0.20 41.77±1.34 35.65±0.45 
A/G  0.65±0.08 0.74±0.03 0.57±0.03 0.66±0.09 
      
Renal      
UREA mmol/L 14.80±0.80 7.30±1.30 19.87±8.88 7.25±4.15 
CREAT µmol/L 71.05±7.64 74.72±18.09 68.9±12.54 70.32±14.04 
The values are presented as Means ± SEM               *P < 0.05 
 
72 
 
4.5.8. Histopathology 
The photomicrographs obtained from sections of the brain, kidney, liver, heart and spleen from 
Sprague-Dawley rats treated with vehicle and MOE (30, 100 and 300 mg/kg) for the 90-day 
sub-chronic toxicity study period are shown in Plate 4.5.8 Histopathological evaluation of the 
organs revealed no significant extract-related changes when compared to the control animals 
except for some changes in the kidney for only one animal in the group receiving the low dose 
of 30 mg/kg which showed acute mononuclear cell inflammation and glomerulopathy. 
Comparison of liver morphological structure for the MOE treated groups to the distilled water 
treated group revealed no remarkable abnormalities (Plate 4.5.8 I, J, K, L). There was no tissue 
necrosis or cellular degeneration. The morphology of the renal corpuscles and hepatic lobules 
were all normal. The histomorphology of the spleen was not affected by the 90-day oral 
administration of MOE (Plate 4.5.8 Q, R, S, T). Splenic congestion or haemorrhage considered 
important features of toxicity in the spleen were absent. Cardiac myocytes (Plate 4.5.8 M, N, 
O,P) did not suffer any injury after the 90-day treatment. 
73 
 
 
Plate 4.5.8. Photomicrographs of the brain, kidney, liver, heart and spleen in distilled water 
treated rats and rats treated with MOE (30, 100 and 300 mg/kg) daily over the 90-day sub-
chronic toxicity study period (H & E, ×400).  
74 
 
Chapter 5 
5 DISCUSSION, CONCLUSION AND RECOMMENDATIONS 
5.1. DISCUSSION 
This study was aimed at assessing the possible antinociceptive activity of the hydroethanolic 
leaf extract of Mallotus oppositifolius (Geisler) Mull. Arg. using acute and chronic pain models 
in rodents. The findings obtained clearly suggest that the Mallotus oppositifolius extract has 
potent anti-nociceptive properties. They also clearly suggest that MOE possesses anti-
hyperalgesia activity against thermally and mechanically induced hyperalgesia in paclitaxel-
induced peripheral neuropathy mice. Findings clearly confirm an earlier report of the analgesic 
activity of the hydroethanolic leaf extract of Mallotus oppositifolius by Woode et al. (2012). 
The secondary metabolites detected in preliminary phytochemical analysis was consistent with 
previous findings from work done on the hydroethanolic leaf extract by Woode et al. (2012), 
Adekunle and Ikumapayi (2006) and Farombi (2003). Several alkaloids and their derivatives 
have been isolated from plant sources and have been used for their analgesic effect. Examples 
are morphine, which was isolated from Papaver somniferum and is used clinically for the 
management of pain, and vincristine, which was isolated from Vinca roseus and is used as an 
anticancer agent (Staerk et al., 2002; Norn et al., 2005). Sterols are known to lower blood 
cholesterol levels and boost the immune system (Kukuia, 2012; Harcombe & Baker, 2014). 
Saponins have been cited to have antinociceptive properties, anti-inflammatory properties and 
anticancer properties (Waller & Yamasaki, 1996; Kukuia, 2012; Dong et al., 2019; Jega et al., 
2019). Flavonoids are compounds known to possess anti-inflammatory activity (Liang et al., 
1999). It has been reported that some flavonoids block both the lipoxygenase and 
cyclooxygenase pathways of the arachidonate cascade at relatively high concentrations, and 
block only the lipoxygenase pathway at lower concentrations (Di Carlo et al., 1999). Certain 
75 
 
flavonoids are known to exert their antinociception via the activation of opioid receptors 
(Rajendran et al., 2000; Otuki et al., 2005). According to Middleton et al. (2002) flavonoids 
also exhibit inhibitory effects against phospholipase A2 and phospholipase C pathways. The 
pharmacological activity of the extract may be due to the presence of one or more of these 
secondary metabolites. 
MOE was evaluated in three murine models of pain to get a clearer picture of its analgesic 
activity. In all the tests conducted, MOE showed significant activity against all noxious stimuli. 
It significantly and dose-dependently reduced nociceptive scores in both phases of the formalin 
test suggesting that it possesses both central and peripheral analgesic activity. This was 
confirmed with follow-up tests in the acetic acid induced writhing test which showed that MOE 
possessed peripheral analgesic effect, and in the hot plate test which confirmed that MOE has 
central analgesic activity. 
The formalin test is widely known as a highly predictive pain model and mimics acute clinical 
pain due to tissue injury (Abotsi et al., 2017). In this experiment, we measured nociception 
continuously over a specific time period. This pain model makes it possible to the assess the 
onset and duration of action of analgesics (Dubuisson & Dennis, 1977; Abotsi et al., 2017). In 
this chemically induced pain model, the antinociceptive effect demonstrated by MOE was 
significant and comparable to that of diclofenac and morphine. However, both morphine and 
diclofenac were more potent. Intraplantar injection of formalin after pre-treatment with MOE, 
diclofenac or morphine produced a biphasic response, the result of two different nociceptive 
pathways. The first/neurogenic phase occurs almost immediately after formalin injections and 
is elicited by the direct chemical activation of central nociceptive afferent terminals of the Aδ 
fibres. The second/inflammatory phase is an inflammatory response due to central sensitization 
in the dorsal horn and the direct stimulation of chemical nociceptors which results in an 
76 
 
increase in the input from C fibres (Hunskaar & Hole, 1987; Ameyaw et al., 2016; Abotsi et 
al., 2017; Afify et al., 2017). It is known that centrally acting analgesics like the reference drug 
used, morphine, inhibit both the neurogenic and inflammatory phases of the formalin test. The 
extract significantly inhibited both phases of the formalin test as well, which infers a central 
analgesic activity. Also, peripherally acting analgesics which includes some NSAIDS only 
effectively inhibit the late or inflammatory phase. Inhibition of the second phase by MOE 
suggests that it possesses peripheral and anti-inflammatory activity. The later has been 
confirmed in previous studies (Farombi, 2003; Chukwujekwu et al., 2005; Nwaehujor et al., 
2014). 
The acetic acid-induced writhing test was conducted in this study to confirm findings from the 
formalin test. This model has been used frequently to screen compounds for peripheral 
antinociceptive activity (Dhouibi et al., 2018). It is a sensitive peritoneovisceral pain model, 
and is a convenient test for assessing the antinociceptive activity of compounds (Abotsi et al., 
2017). The visceral pain induced by the intraperitoneal injection of 0.6% acetic acid is 
perceived via C fibres (Koster et al., 1959). The writhing response results from the activation 
of certain acid-sensitive ion channels and the transient receptor potential vanilloid 1 (TRPV1) 
located in the afferent primary fibres (Cavalcante-Silva et al., 2014; Abotsi et al., 2017). 
Additionally, the local irritation caused by acetic acid injection causes the release of pro-
inflammatory cytokines such as interleukin 1β, interleukin 8 and TNF-α by resident peritoneal 
macrophages and mast cells as well as prostaglandins (PGE2 and PGF2α), bradykinin, substance 
P, serotonin, histamine and sympathomimetic amines (Pavao-de-Souza et al., 2012; Abotsi et 
al., 2017; Dhouibi et al., 2018) which stimulate nociceptive neurons that are sensitive to 
NSAIDs and narcotics (Dhouibi et al., 2018). The endogenous chemicals released stimulate 
nerve endings and peritoneal receptors and incite abdominal constriction responses attributable 
to receptor sensitization to prostaglandins (Hassan et al., 2015; Dhouibi et al., 2018). MOE 
77 
 
significantly and dose-dependently inhibited the abdominal writhes induced by acetic acid 
suggesting peripheral mediated analgesic activity which may result from the inhibition of the 
synthesis of arachidonic acid metabolites. This confirms the activity of the extract seen in the 
inflammatory phase of the formalin test. The effect of MOE seen in this model could be 
attributed to an interference with the stimulation of nociceptive fibres by the released 
endogenous nociceptive mediators or the suppression of the sensitization effect of the pro-
inflammatory cytokines (Abotsi et al., 2017). The extract probably exerts its effect by 
inhibiting the biosynthesis, release and action of prostaglandins. According to Nwafor et al. 
(2007) acetic acid also causes inflammatory pain by inducing capillary permeability and in part 
by sensitisation of peritoneal chemosensitive nociceptors by prostaglandins, particularly PGE2 
and PGF2α as well as lipoxygenase products (Bentley et al., 1983). These results then suggest 
that in part, MOE may exert its effect through the cyclooxygenase and/or the lipoxygenase 
system. 
The hot plate test employs the use of a thermal noxa to induce pain by the stimulation of the 
Aδ fibers (Dhouibi et al., 2018). This test is specific for centrally mediated activity therefore 
selective for centrally acting analgesics (Afify et al., 2017). In this model, the hot plate is 
maintained at a constant temperature and this is known to produce two nociceptive behavioural 
responses, paw licking and jumping, which are measured as how long it takes for these 
behavioural responses to appear. Both of these behaviours are considered as supra-spinally 
mediated responses (Milind & Monu, 2013). According to Le Bars et al. (2001), the paw 
licking behaviour is affected by only opioids whereas the latency to jumping response is 
equally increased by less powerful analgesics such as paracetamol or aspirin. The pre-treatment 
of the mice with MOE and morphine significantly increased latency to paw licking and 
jumping. However, morphine was far more potent than MOE. M. oppositifolius demonstrated 
some central antinociceptive activity by increasing the latency to reaction to thermal stimulus 
78 
 
and may in part act like centrally active drugs. Some of these centrally active drugs exert their 
effect by stimulating the periaqueductal grey matter to release endogenous peptides (i.e., 
endorphins, enkephalins) (Pathan & Williams, 2012). The endogenous peptides travel to the 
synapse in the dorsal horn and inhibit the transmission of the thermal pain impulses (Woolf, 
2004; Benzon & Raj, 2008). 
With MOE demonstrating antinociceptive effects in the three different pain models, further 
studies to assess the possible mechanism(s) of antinociception were carried out. The formalin 
test was used as it is more specific for site of action and mechanism of action. In all the tests, 
the antinociceptive effect of MOE was not significantly reversed by any of the antagonists 
used. It is therefore necessary that further tests be carried out on the possible mechanism of 
antinociception of MOE and the doses of antagonists used in this study be modified. 
This study also demonstrated that MOE possesses potent analgesic activity in paclitaxel-
induced peripheral neuropathy in mice further confirming the results from the nociceptive pain 
models in this research work. Daily administration of paclitaxel produced significant peripheral 
neuropathy demonstrated by a lowering in the mechanical and thermal pain thresholds as well 
as a reduction in the latency to tail withdrawal from cold water at 4℃ from day 0 to day 5.  
Peripheral neuropathy (a chronic condition) is a common side effect of paclitaxel 
administration and is manifested as allodynia or hyperalgesia with direct action on the 
processing and secretion of proinflammatory mediators, which include TBARS, IL-1b and 
glutathione (Miranda et al., 2018). Neuropathic pain is characterized by spontaneous pain, 
hyperalgesia and allodynia. These remain without adequate treatment and compromise the 
quality of life (Woolf, 2004; Costa et al., 2011). The onset of hyperalgesia is known to occur 
within a few hours to 7 days after paclitaxel administration and can last several weeks (Dina et 
al., 2001; Polomano et al., 2001; Cata et al., 2006). Similarly, thermal and mechanical 
79 
 
hyperalgesia markedly increased from day 1 to day 5 during the induction period and lasted 
throughout the test period for mice that were not treated with MOE or pregabalin.  
Though the exact mechanism of pathogenesis by paclitaxel is yet to be fully elucidated, many 
findings have come up to suggest the involvement of several molecules and systems. Some 
findings suggest that paclitaxel may induce nerve damage by disrupting the action of 
microtubules which are needed for transport in axons (Pourmohammadi et al., 2012), and cause 
the swelling of mitochondria in axons (Fidanboylu et al., 2011; Ameyaw et al., 2014) leading 
to the loss of certain intracellular functions. Consequently, the calcium levels in the axons are 
altered as well as an initiation of apoptosis (Flatters & Bennett, 2006; Amoateng et al., 2017). 
The allodynia that develops from the administration of paclitaxel has been attributed to 
apoptosis in the dorsal root ganglion neurons (Ameyaw et al., 2014). In a study by Zhang et al. 
(2012) they demonstrated that paclitaxel treatments induced the activation of spinal astrocytes 
and not microglia. They also showed that the involvement of spinal astrocytes in the 
pathogenesis of paclitaxel-induced neuropathy was possibly by downregulating glial glutamate 
transporters GLAST and GLT-1 but not by releasing proinflammatory cytokines such as TNFα, 
IL-1b, and IL-6 in spinal dorsal horn. GLAST and GLT-1 are two types of glial glutamate 
transporters that are predominantly expressed on spinal astrocytes for the uptake of glutamate 
(Liaw et al., 2005; Yaster et al., 2011). Blocking spinal GLAST and GLT-1 induces 
spontaneous nociceptive behaviours and hypersensitivity to both peripheral mechanical and 
thermal stimuli (Liaw et al., 2005). Blockage leads to enhanced neuronal activity and excessive 
activation of post-synaptic AMPA and NMDA receptors in the dorsal horn (Weng et al., 2007; 
Nie & Weng, 2009). The downregulation of GLAST and GLT-1 in the dorsal horn caused by 
damage to peripheral nerves is known to facilitate the development of neuropathic pain which 
has been reversed when the activity of these transporters were restored (Sung et al., 2003; 
Zhang et al., 2012). 
80 
 
The neuropathic mice were treated with MOE and pregabalin for 5 consecutive days. Much 
like pregabalin, the extract significantly and dose-dependently inhibited mechanical and 
thermal hyperalgesia in the mice. However, pregabalin was more potent. Pregabalin is effective 
both experimentally and clinically in the management of chemotherapy induced neuropathic 
pain (Ameyaw et al., 2014; Mangaiarkkarasi et al., 2015). It is known that its action is as a 
result of antagonist effects on α2-δ1 Ca2+ channel subunit of N-type voltage dependent calcium 
channels (Mangaiarkkarasi et al., 2015) which consequently leads to a reduction in neuronal 
excitability and inhibition of other cellular enzymatic reactions that lead to pain sensation 
(Kumar et al., 2010; Ameyaw et al., 2014; Amoateng et al., 2017).  
The exact mechanism by which MOE attenuates mechanical and thermal hyperalgesia in this 
model of peripheral neuropathy is yet to be established and will be investigated in future 
studies. However, earlier reports (Kukuia et al., 2014; Kukuia et al., 2016) suggest that the 
analgesic effect of MOE in this model may be associated with an inhibitory effect on 
glutamatergic neurotransmission and inhibition of the excessive activation of NMDA 
receptors, as this neurotransmitter system and its associated NMDA receptors are implicated 
in paclitaxel-induced neuropathic pain (Liaw et al., 2005; Nie & Weng, 2009; Yaster et al., 
2011). 
Sub-chronic studies are designed to assess the undesirable effects of the continuous or repeated 
exposure to drugs, compounds and plant extracts over an extended time period in experimental 
animals (Zhang et al., 2016). It provides very useful information on the adverse effects 
associated with these molecules and how this affect specific target organs (Yuet Ping et al., 
2013). In this study, the sub-chronic toxicity of MOE was evaluated in Sprague-Dawley rats at 
doses of 30, 100 and 300 mg/kg for 90 days. 
81 
 
No significant and dose-dependent changes in the weights of the harvested organs were 
observed, suggesting that the extended oral administration of MOE has no effect on normal 
growth. In general, body weight change is an index of adverse effects after exposure to toxic 
substances and animals that survive this exposure should not lose any more than 10% of their 
initial body weight (Hemalatha & Hari, 2014). A high increase in body weight may be 
attributable to appetite stimulating drugs or agents which affect the lipid profile of the animals 
causing fat deposition. On the other hand, decrease in body weight could be attributed to the 
anorexic effect of certain drugs (Gura & Ciccone, 2010; Koumba Madingou et al., 2016). 
Organ weights are very sensitive to toxicity, enzyme induction, acute injury and so on making 
them useful in toxicological studies (Zhang et al., 2016). As such, they are frequently targeted 
in toxicity studies (Michael et al., 2007). The Society of Toxicologic Pathology has 
recommended that organ weights be included in toxicity studies especially when multiple dose 
levels are given in durations from one week to one year (Sellers et al., 2007). Results from this 
study reveals that there was no significant and treatment related differences in relative organ 
weights between the distilled water groups and any of the MOE treated groups, suggesting that 
MOE had no effect on organ weights. 
Analysis of blood parameters/indices is very important when assessing the toxicity of a drug 
as they have a high predictive value for toxicity in humans (Olson et al., 2000; Gautam & Goel, 
2014). Blood is a transport medium and as such carries many drugs and xenobiotics that enter 
the body. Inadvertently, blood components are forced to interact with high amounts of 
xenobiotics which may be toxic. As such, evaluating haematological indices can be useful in 
pointing out the extent of toxic effect of a given plant extract (Olson et al., 2000). In the study, 
there were no significant differences in RBC indices between the vehicle treated groups and 
the extract treated groups indicating that the extract has no significant dose-dependent effects 
82 
 
on erythropoiesis, morphology or osmotic fragility of the RBCs. Total WBC count for the drug 
treated groups was not significantly different when compared to the vehicle group. However, 
the group treated with the median dose showed a significantly high neutrophil count suggestive 
of an increase in their production in the bone marrow which may be attributed to stress or an 
ongoing infection or inflammation. This observation was however not dose dependent.  
Serum biochemistry analyses were conducted to assess possible extract related effects on 
hepatic and renal functions which are very important in the evaluation of toxicity as it has been 
demonstrated in certain studies that phytotherapeutic products may cause liver or kidney 
damage (Féres et al., 2006).  
Among the parameters measured, ALT, ALP, AST, GGT, bilirubin and albumin are considered 
liver function markers. Drug metabolism and elimination occur majorly in the liver (Zhang et 
al., 2016). Elevated ALP, AST and ALT levels are usually reported in liver diseases or 
hepatotoxicity and are often used to differentiate between hepatocellular and cholestatic 
diseases (Féres et al., 2006). Another typical function of the liver is the production and 
maintenance of TP and ALB levels. A decrease in their levels is indicative of reduced synthesis 
function of the liver attributable to an hepatocellular impairment or injury (Yuet Ping et al., 
2013). There were no significant differences in biochemical parameters for treatment groups 
and vehicle groups when compared suggesting that liver function was not impaired. However, 
the median dose treated group (MOE at 100 mg/kg) had significantly low AST levels. Low 
AST levels are expected in such tests and are normal. On the other hand, high AST levels may 
suggest an ongoing inflammatory event in the liver or even a blockage of bile ducts (Zhang et 
al., 2016).  The group treated with the MOE at 30 mg/kg recorded significantly low ALP levels. 
This can be attributed to low levels of serum magnesium and zinc as well as vitamin C and B12 
deficiency (Lum, 1995; Ray et al., 2017). From the histopathological analysis, acute 
83 
 
inflammation was observed from two different liver sections from only two rats from the 
median dose group with one showing high mononuclear cell infiltration (Plate 4.5.8 K). This 
was not apparent from the biochemistry parameters measured. This phenomenon will be 
investigated in subsequent studies. 
Comparison of kidney parameters for the treated group to the control did not show any 
significant difference. However, histopathological analysis revealed acute mononuclear cell 
inflammation and glomerulopathy in only one of the test subjects treated with MOE at 30 
mg/kg. Histological analysis of isolated organs did not show any dose dependent toxicity 
pattern. 
5.2. CONCLUSION 
The oral administration of a hydro-ethanolic leaf extract of Mallotus oppositifolius produces 
dose-dependent antinociceptive effects in formalin, acetic acid-induced and thermal acute pain 
tests, and also significantly inhibited mechanical and thermal hyperalgesia in paclitaxel 
induced peripheral neuropathy in mice. There was no MOE dose-dependent toxicity in rats 
following a 90-day daily oral administration. The findings support the traditional uses of the 
plant in alleviating pain. 
5.3. RECOMMENDATIONS 
 The extract should be fractionated to isolate the compounds responsible for the observed 
antinociceptive effects.  
 This study should be repeated in non-human primates in order to assess and/or confirm the 
scientific information gathered or discover those that were not apparent in this study. 
 Other pain models should be used to elucidate the possible mechanism(s) of action. 
84 
 
 Budget should be increased in further chronic studies to cover the histopathological 
investigation of more organs in the gastrointestinal tract. 
 
 
 
 
 
  
85 
 
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