ABSTRACT
Pain and inflammation are the commonest manifestations of various pathologies, and are associated with high morbidities, debility, and economic strife globally, especially in underdeveloped regions of sub-Saharan Africa. The currently available conventional analgesic and anti-inflammatory drugs cause serious side effects, some of which are life threatening, are unaffordable, and unavailable to all patients, especially in low-income countries, hence the need for better alternatives. In the current study, the in vivo anti-inflammatory, analgesic, and in vitro cytotoxic activities of the Phytexponent preparation comprising the ethanolic extracts of Viola tricolor, Echinacea purpurea, Allium sativum, Matricaria chamomilla, and Triticum repens were investigated. The carrageenan- induced paw oedema technique was adopted to investigate the anti-inflammatory activity of the Phytexponent in experimental mice, at doses of 15.625 mg/Kg BW, 31.25 mg/Kg BW, 62.5 mg/Kg BW, 125 mg/Kg BW, 250 mg/Kg BW and 500 mg/Kg BW, with Indomethacin (10 mg/Kg BW) as positive control drug. The paw sizes of respective animals were measured using a plethysmographic technique, and the values used to calculate the percentage reduction in oedematous paw size, as an indicator of anti-inflammatory activity of the Phytexponent. The acetic acid-induced writhing technique was used to determine the analgesic activity of the Phytexponent in experimental Swiss albino mice at similar doses as those used for anti-inflammatory assay and indomethacin (4 mg/Kg BW) as the reference drug. Then, the number of wriths were recorded and expressed as the percentage inhibition of writhing. The standard 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay technique was used to investigate the in vitro cytotoxic effects of the Phytexponent in Vero E6 cell line with cyclophosphamide as a positive cytotoxic agent. The percentage inhibitions of cell proliferation (percentage cytotoxicity) were determined according to a standard procedure. The study findings revealed that the Phytexponent preparation exerted significant anti-inflammatory effects in carrageenan-induced paw oedema mouse model, which ranged from 1.117±0.193% at the first hour to 11.162±0.091% at the fourth hour, at a dose of 31.25 mg/Kg BW, 6.240±0.242 % at the first hour to 17.407±0.186% at the fourth hour at a dose of 62.60 mg/Kg BW, 9.645±0.020% at the first hour to 31.795±0.090% at the fourth hourat a dose of 125,g/Kg BW, and 14.000±0.102% at the first hour to 37.931±0.133% in the fourth hour, at a dose of 250 mg/Kg BW (p<0.05). Notably, the Phytexponent significantly inhibited inflammation in a dose- and time-dependent manner (p<0.05). The Phytexponent preparation exhibited significant analgesic activity (p<0.05) in experimental mice as depicted by reduced writhing frequencies (high percentage inhibitions of acetic acid-induced writhing), which increased from 55.054±0.174% at a dose of 31.25 mg/Kg BW to 94.982±0.098% at a dose of 250 mg/Kg BW, in a dose-dependent manner (p<0.05). The Phytexponent exhibited significantly higher analgesic activity at doses of 125 mg/Kg BW (75.924±0.253%) and 250 mg/Kg BW (94.982±0.098%) than indomethacin (64.786±0.098%), indicating higher analgesic efficacy. The Phytexponent preparation was not cytotoxic to Vero E6 cells as indicated by high CC50 value (>1000 µg/ml) compared to cyclophosphamide (CC50= 2.48µg/ml). The present study indicated that the Phytexponent formulation has significant in vivo anti-inflammatory and analgesic activities in mice models and is not cytotoxic to Vero E6 cell line. Therefore, based on the study findings, the Phytexponent formulation is a potential source of safe analgesic and anti-inflammatory associated phytocompounds. Further empirical studies, determination of mode(s) of anti-inflammatory and analgesic efficacy, and safety of the Phytexponent and its bioactive phytochemicals should be undertaken.
TABLE OF CONTENTS
DECLARATION i
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
TABLE OF FIGURES vii
LIST OF APPENDICES viii
ACRONYMS AND ABBREVIATIONS ix
ABSTRACT x
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background Information 1
1.2 Statement of the problem and Justification of the study 7
1.3 Study objectives 8
1.3.1 General Objective 8
1.3.2 Specific objectives 8
1.4 Research Questions 9
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Biochemical Basis of Pain 10
2.2 Biochemical and molecular basis of inflammation 11
2.3 Neural Transmitters and Nociceptive Systems 13
2.4 Analgesic assays 14
2.5 Non-steroidal anti-inflammatory drugs 15
2.6 The role of medicinal plants in the management of pain and inflammation 21
2.7 Preparation and composition of the Phytexponent: A polyherbal 23
formulation used in this study 23
2.8 Toxicity of Herbal Products 26
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 The source of the Phytexponent polyherbal formulation 27
3.2 Experimental animals 27
3.3 Determination of in vivo anti-inflammatory activity using Carrageenan 27
induced paw oedema in mice technique 27
3.4 Determination of potental analgesic effects of the Phytexponent 29
3.5 Cell culture technique 31
3.5.1 Vero E6 cell line culture 31
3.5.2 Passaging technique 32
3.5.3 Trypsinisation and resuspension procedures 32
3.5.4 Determination of the in vitro cytotoxic effects of the Phytexponent 32
preparation 32
3.6 Data management and statistical analysis 34
3.7 Ethical Consideration 34
CHAPTER FOUR
4.0 RESULTS
4.1 Anti-inflammatory activity of the Phytexponent preparation in Swiss 36
albino mice 36
4.2 Analgesic activity of the phytexponent preparation 41
4.3 In vitro cytotoxic effects of the Phytexponent preparation 42
CHAPTER FIVE
5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
5.1 Discussion 43
5.2 Conclusions 53
5.3 Recommendations for further studies 53
REFERENCES 54
APPENDICES 80
LIST OF TABLES
Table 3.1: Experimental design for the determination of anti-inflammatory activity of the Phytexponent 29
Table 3.2: Experimental design for the determination of analgesic activity of the Phytexponent 30
Table 4.1:Anti-inflammatory activity of the Phytexponent preparation in Swiss albino mice 40
Table 4.2: In vitro cytotoxic effects of the Phytexponent on Vero cell line 43
TABLE OF FIGURES
Figure 2.1: Structure of diclofenac 17
Figure 2.2: Structure of indomethacin 18
Figure 2.3: Structure of acetylsalicylic acid (aspirin) 19
Figure 4.1: Analgesic effects of the Phytexponent preparation of selected medicinal plants in acetic acid-induced writhing in mice 41
LIST OF APPENDICES
Appendix 1: Ethical Approval Letter 80
Appendix 3: Research Permit granted by the National Commision for Science, Technology, and Innovation 81
Appendix 4: Monograph of the Phytexponent formulation 82
Appendix 5: The researcher carrying out experiments in the laboratory 83
Appendix 6: In vivo anti-inflammatory activity data 84
Appendix 7: Analgesic activity data 87
Appendix 8: In vitro cytotoxicity data (MTT-Assay) 88
ACRONYMS AND ABBREVIATIONS
ANOVA Analysis of Variance
COX Cyclooxygenase
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic Acid
ELISA Enzyme-linked Immunosorbent Assay
EMEM Eagle’s Minimum Essential Medium
FBS Fetal Bovine Serum
GABA Gamma Aminobutyric Acid
KEMRI Kenya Medical Research Institute
LD50 Median Lethal Dose
LPS Lipopolysaccharide
MTT 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
NACOSTI National Commission for Science, Technology, and Innovation
NADH Nicotinamide Adenine Dinucleotide Hydrogen
NCI National Cancer Institute
NF Nuclear Factor
NMDA N-Methyl-D-Aspartate
NSAIDs Non-Steroidal Anti-inflammatory Drugs
OECD Organization for Economic Co-operation and Development
PBS Phosphate-Buffered Saline
PGHS Prostaglandin Endoperoxide H Synthase
PH Potential of Hydrogen
ROS Reactive Oxygen Species
SDH Succinate Dehydrogenase
SEM Standard Error of the Mean
SLE Systemic Lupus Erythematosus
WHO World Health Organization
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background Information
Inflammation is a response of a tissue to a noxious stimulus, such as physical injury, irritant agents and pathogens (Chen et al., 2018). It causes increased vascular permeability, changes in blood flow, and migration of leucocytes to the affected sites (Chen et al., 2018). Pain refers to an unpleasant emotional and sensory experience that results from tissue damage and acts as a signal to warn against further disturbances (Raja et al., 2020). The focus in pain management is to eliminate or remove its cause.
Pain, fever, and inflammation are associated with a myriad of pathological processes in the body (Cross, 1994; Ogoina, 2011; Walter et al., 2016; Woessner, 2006). There are two forms of pain nociceptive that result from tissue injury: due to activation of specific nociceptors and the neuropathic pain that is caused by structural damage to the nerves (Marchand, 2008). Pain is a major health problem with profound debility in the afflicted subjects and persistent inflammation causes chronic diseases and promote tumour development (Olela et al., 2020). On the other hand, fever is a sign of disease colonisation, which signals an inflammatory response aimed at limiting the spread of the microbes (Chen et al., 2018; Pearlman, 1999).
There are various anti-inflammatory and antinociceptive drugs for the treatment of inflammation and pain (Giorno et al., 2019; Herrero et al., 2003; Newman and Agyare, 2017). However, there is an unending search for new therapeutic compounds to serve as alternatives because of the inaccessibility, unaffordability, adverse effects, and low efficacy of existing conventional medications (Herrero et al., 2003; Olela et al., 2020). In this regard, focus has shifted to investigating natural products, especially medicinal plants, as one of the most promising therapeutic agents for inflammatory diseases (Moriasi et al., 2021b; Raisa et al., 2018; Shojaii et al., 2015; Wambugu et al., 2011).
The most widely used anti-inflammatory agents are the non-steroidal anti- inflammatory drugs (NSAIDs) which act by inhibiting the cyclooxygenase (COX) enzymes, thereby prohibiting the production of prostaglandins (Monteiro and Steagall, 2019; Newman and Agyare, 2017; Ricciotti and Fitzgerald, 2011). However, they have been shown to cause serious side effects, such as liver damage, aseptic meningitis, and bone fractures (Felson, 2016).
In many African communities, especially in the rural areas, herbs are still used to manage various diseases because they are readily available and relatively less expensive compared to conventional medicines (Moriasi et al., 2020a; Waiganjo et al., 2020; World Health Organization (WHO), 2013). According to the World Health Organization, more than 85% of traditional medicine comes from plant extracts (Ighodaro and Omole, 2012). In Kenya, there are various remedies for pain, fever, and inflammation, including some herbs, that are used in traditional medicine (Mukungu et al., 2016; Nankaya et al., 2020; {Ochwang} et al., 2014). Traditionally, analgesic substances have been obtained from plants, with modes of action of some of them already extensively documented (Gwinnutt, 2007; Kumar et al., 2010). Research data shows that plant-derived natural products are a bulwark of future drug discovery, especially for treatment of inflammation and pain (Calixto et al., 2001; Fürst and Zündorf, 2014; Nunes et al., 2020). This is encouraging, considering that more than 80% of the population in third world countries, especially in Africa, do not have access to modern medicine and entirely depend on traditional medicine for healthcare needs { World Health Organization (WHO), 2013].
In the last few years, ethnobotanical research has revisited traditional literature in the search for novel remedies for various ailments (Abreu et al., 2012; Andrade- Cetto et al., 2019; Moriasi et al., 2020b). Plants hold assurance for discovery of new and effective drugs against pain and inflammation (Nunes et al., 2020). Various inflammatory diseases such as ankylosing spondylitis, rheumatoid arthritis, systematic lupus erythematosus, rheumatic fever, and osteoarthritis are currently being managed by an array of synthetic drugs (Monteiro and Steagall, 2019). However, most of them are associated with adverse side effects, high costs, inaccessibility, which limit their usage (Felson, 2016).
Worldwide, drugs derived from plants offer a stable market and they serve as a source of novel drugs (Nunes et al., 2020). In general, natural products, more especially plants, are novel sources of chemical substances with therapeutic capabilities (phytochemicals) (Abreu et al., 2012; Moriasi, et al., 2020c). Most of the anti-inflammatory, anti-malarial, analgesic, and antipyretic drugs have their origin in plants, including chloroquine, morphine, and aspirin (Patridge et al., 2016; Veeresham, 2012). Therefore, there is a need to conduct more studies on plants to discover potent, accessible, affordable and safe products for the alleviation of pain and inflammation, and associated disorders.
Even though medicinal plants have extensive and longstanding utilization in alternative and complementary therapy, various concerns regarding their safety have been raised (George, 2011). For instance, there are no clear guidelines which govern traditional medicine, thus allowing unscrupulous practisoners to thrive (Arora, 2015). Additionally, there is scanty data on herb-herb and herb-drug interactions and associated effects to effectively guide prescriptions (Kaur et al., 2013).
Moreover, in traditional medicine practice, there are no clearly outlined dosage forms for specific diseases and expected side effects (Kaur et al., 2013). Furthermore, the lack of safety and toxicity profiles of many medicinal plants further cripples the confidence accorded to herbal medicine. As a result, it is imperative to evaluate toxicity and safety of herbal preparations used to manage various diseases to avert the development of undesirable effects and fatalities (Arora, 2015; George, 2011; Kaur et al., 2013).
Herbal remedies, such as the Phytexponent preparation containing ethanolic extracts of Viola tricolor Echinacea purpurea, Allium sativum, Matricaria chamomilla, and Triticum repens have been used in complementary and alternative medicine to manage inflammation and pain, and associated syndromes, and has demonstrated appreciable level of efficacy (Moriasi et al., 2021). Polyherbal preparations, such as the Phytexponent are relatively cheap, readily available, cause fewer side effects, and are easy to administer (Atawodi, 2001; Girish et al., 2004; Jangle, 2012). The plants used to formulate the Phytexponent are used in traditional medicine since they possess various pharmacologic activities against a variety of disease conditions. For instance, Viola tricolor has been traditionally used for treatment of inflammatory lung and skin ailments, such as ulcers, itching, scabs, psoriasis, and eczema (Hellinger et al., 2014). Besides, Echinacea purpurea, which is indigenous to North America, is the most widely cultivated medicinal plant for use in chemotherapy, and is commonly used to alleviate cold symptoms. Manayi et al. (2015) noted that the herb has anti- inflammatory and immunostimulatory properties.
Additionally, Allium sativum (Garlic) is widely used as a food ingredient, and as an aphrodisiac to cause sexual arousal,pleasure and performance (Jayanthi and Dhar, 2011). Garlic extracts have more than 200 chemicals that have been identified to date, and determonstrated to be effective in treating various conditions, including some types of cancer (Martins et al., 2016). (Arreola et al. (2015) reported that garlic products can be prepared in liquid or solid forms. The plant has many anti-inflammatory effects, including anticancer, antingiogenic, and free radical-mediated anti-inflammatory effects, antiobesity, among others (Yang et al., 2018; Moriasi, et al., 2021a).
Recently Moriasi et al. (2021a) investigated the in vitro anti-inflammatory, antioxidant activities of the Phytexponent and observed significant efficacy. Furthermore, qualitative phytochemistry of the Phytexponent revealed the presence of bioactive phytochemicals with diverse pharmacologic effects, including anti-inflammation (Moriasi et al., 2021a) . However, there is a scarcity of documented studies on the in vivo efficacy of this polyherbal product, its mode(s) of action in various disease states, its toxicity, and safety. Therefore, this study was designed to investigate the in vivo anti-inflammatory, analgesic, and cytotoxic effects of the Phytexponent preparation of Viola tricolor, Echinacea purpurea, Allium sativum, Matricaria chamomilla, and Triticum repens, as a potential alternative source of affordable, accessible, potent, and safe analgesic and anti-inflammatory lead compounds for drug discovery and development.
1.2 Statement of the problem and Justification of the study
Fever, inflammation, and pain are critical signs manifesting in many diseases affecting humans and other animals, and lead to poor quality of life, disability, depression, mortality, and financial loss (Ricciotti and Fitzgerald, 2011; Taylor et al., 2011; Khandaker et al., 2015; Réus et al., 2015; Walter et al., 2016; Sahlmann and Ströbel, 2016; Sommer et al., 2018).
Unfortunately, the management of pain, and inflammation is expensive, and it typically entails the administration of different classes of drugs which are associated with various insufficiencies (Felson, 2016). Most of these drugs have serious side effects, such as gastric ulcers, hepatotoxicity, nephrotoxicity, cardiotoxicity, among others, caused by non-steroidal anti-inflammatory drugs like aspirin, diclofenac, among others (Fokunang, 2018; Harirforoosh et al., 2013; Sylvester, 2019). Research has established that herbal remedies are cheap, easily available, effective, and elicit fewer side effects (Azab et al., 2016; Nasri and Shirzad, 2013; Olela et al., 2020). However, many of the plant-based remedies have not been scrutinised with scientific precision to determine their efficacy, composition, mode of action, toxicity profile and safety.
Herbal remedies, such as the Phytexponent preparation composed of ethanolic extracts of Viola tricolor Echinacea purpurea, Allium sativum, Matricaria chamomilla, and Triticum repens, have been used to manage pain and inflammatory conditions with demonstrable degree of efficacy (Moriasi et al., 2021a). Additionally, herbal preparations are cost effective, readily available, with fewer side effects, and easy to administer. Therefore, scientific studies on their pharmacologic efficacy, toxicity, safety is a worthy undertaking as they present a viable alternative source of potent therapies for various maladies, including pain and inflammation.
1.3 Study objectives
1.3.1 General Objective
The main objective of the study was to investigate the in vivo anti-inflammatory, analgesic, and cytotoxic effects of the Phytexponent preparation: A polyherbal formulation.
1.3.2 Specific objectives
i. To determine the in vivo anti-inflammatory activity of the Phytexponent preparation in Swiss albino mice.
ii. To investigate the analgesic effects of the Phytexponent preparation in Swiss albino mice.
iii. To evaluate the cytotoxic effects of the Phytexponent preparation in Vero E6 cell line from the green monkey kidney cells.
1.4 Research Questions
This study was guided by the following research questions:
i. Does the Phytexponent preparation have anti-inflammatory activity in Swiss albino mice?
ii. Does the Phytexponent preparation have analgesic activity in Swiss albino mice?
iii. What are the cytotoxic effects of the Phytexponent preparation in Vero E6 cell line?
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