i 
 

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 

  



ii 
 

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) 

 



iii 
 

 

 

 

 

DEDICATION 

I dedicate this work to the Almighty God. 

  



iv 
 

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.   



v 
 

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 



vi 
 

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 



vii 
 

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 

 

 

  



viii 
 

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 



ix 
 

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 

 



x 
 

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 

  



xi 
 

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 

 

  



xii 
 

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. 



xiii 
 

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. 



1 
 

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 



2 
 

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-



3 
 

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 



4 
 

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. 



5 
 

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. 

  



6 
 

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).  



7 
 

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 



8 
 

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).  



9 
 

 

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). 



10 
 

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-



11 
 

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 



12 
 

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). 



13 
 

 

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. 



14 
 

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). 



15 
 

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 



16 
 

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). 



17 
 

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). 



18 
 

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 



19 
 

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 



20 
 

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 



21 
 

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 



22 
 

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 



23 
 

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 



24 
 

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). 



25 
 

 

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) 



26 
 

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 



27 
 

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. 



28 
 

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. 



29 
 

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). 



30 
 

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. 



31 
 

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 



32 
 

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 



33 
 

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: 



34 
 

 

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).  



35 
 

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. 



36 
 

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 



37 
 

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. 



38 
 

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, 



39 
 

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 



40 
 

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 



41 
 

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).  



42 
 

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 

 



43 
 

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   

 

 

  



44 
 

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 

 

  



45 
 

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 neuroge