ABSTRACT
Non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and opioids are used in the management of inflammation and pain. However, the use of these drugs is limited by cost, adverse effects, and the reappearance of symptoms after discontinuation. Given these limitations, the search for alternatives may be necessary. The roots of Maerua triphylla are used by Maasai and Kikuyu communities for the management of headaches, stomachaches, migraines, and rheumatism. However, data on the safety and efficacy of this plant is not available to support its use. The aim of this study was to investigate the safety (LD50), phytochemical constituents, analgesic, and anti-inflammatory properties of root extracts of M. triphylla. Cold maceration was used to prepare methanol and aqueous root extracts of M. triphylla.
The safety of these extracts was evaluated in Wistar rats using the Organization for Economic Co- operation and Development (OECD 425) guidelines. Phytochemical composition of the extracts was determined by standard qualitative methods. The acetic acid-induced writhing procedure was used to evaluate the analgesic activity of the extracts in Swiss albino mice. The anti-inflammatory activity of the extracts was determined in Wistar rats using the acetic acid-induced paw oedema method. The percentage yield from the aqueous extraction was 12.4% whereas the percentage yield from the methanol extraction was 6.2%. All the studied plant extracts had LD50 > 2000mg/kg bw and were classified as nontoxic according to the OECD 425 guidelines. Qualitative phytochemical screening revealed the presence of flavonoids, phenols, cardiac glycosides and alkaloids in both extracts. However, saponins were only present in the methanol extract. In the analgesic study, mice that received 100 mg/kg bw and 500 mg/kg bw of aqueous root extract of M. triphylla had significantly lower acetic acid-induced writhing in comparison to mice that received 75 mg/kg bw acetylsalicylic acid (reference drug) (p< 0.05). Additionally, mice that received 500 mg/kg bw of methanol root extract of M. triphylla had significantly lower acetic acid-induced writhing in comparison to mice that received 75 mg/kg bw acetylsalicylic acid (p< 0.05). In the anti-inflammatory study, there was no significant difference (p>0.05) between the inhibitory activity of different doses of the aqueous root extract of M. triphylla and a 50 mg/kg dose of diclofenac sodium (reference drug) on acetic acid-induced paw edema in rats. Moreover, there was no significant difference in the inhibitory activity of 100 mg/kg bw and 500 mg/kg bw doses of the methanol root extract of M. triphylla and a 50 mg/kg dose of diclofenac sodium (p>0.05). These findings suggest that the roots of M. triphylla may be useful in the mitigation of pain and inflammation and therefore support their ethnomedicinal use in the management of inflammation and pain. Further isolation, characterization and quantification of the specific phytochemical constituents in the root extracts of M. triphylla with anti-inflammatory and analgesic activity is recommended. Furthermore, the specific mode(s) through which these extracts exert their reported pharmacological activities should be established. Further toxicological studies on the plant extracts are recommended to fully determine their safety.
Keywords; Maerua triphylla, analgesic, anti-inflammatory
TABLE OF CONTENTS
DECLARATION ii
PLAGIARISM DECLARATION iii
DEDICATION iv
ACKNOWLEDGEMENT v
LIST OF FIGURES ix
LIST OF TABLES x
LIST OF APPENDICES xi
LIST OF ACRONYMS xiii
ABSTRACT xiv
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background information 1
1.2 Problem statement 2
1.3 Justification 2
1.4 Objectives 2
1.4.1 Overall objective 2
1.4.2 Specific objectives 3
1.5 Hypothesis 3
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Pain and Inflammation 4
2.2 Conventional management of pain and inflammation 5
2.3 Herbal medicine used as analgesics and anti-inflammatory agents 6
2.4 Capparidaceae 7
2.4.1 Classification and distribution 7
2.4.2 Biological activities 7
2.4.3 Phytochemistry 8
2.4.4 Ethnomedicinal uses 8
2.4.5 The profile of M. triphylla 9
2.4.5.1 Description 9
2.4.5.2 Classification and distribution 10
2.4.5.3 Ethnomedicinal and economic uses 10
2.4.5.4 Phytochemistry 12
2.4.5.5 Toxicity 12
2.5 Analgesic tests 12
2.6 Anti-inflammatory test 14
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Ethical approval 16
3.2 Study area 16
3.3 Collection and authentication of plant specimen 17
3.4 Preparation of plant material 17
3.5 Preparation of plant extracts 18
3.5.1 Aqueous extract 18
3.5.2 Methanol Extract 19
3.6 Experimental animals 19
3.7 Preparation of doses 20
3.8 Acute oral toxicity study of aqueous and methanol root extracts of M. triphylla 21
3.9 Phytochemical screening of aqueous and methanol root extracts of M. triphylla 21
3.9.1 Saponins (Froth test) 21
3.9.2 Alkaloids (Dragendroff’s test) 21
3.9.3 Terpenoids (Salkowski test) 22
3.9.4 Flavonoids (Alkaline reagent test) 22
3.9.5 Cardiac glycosides (Keller-Kiliani test) 22
3.9.6 Steroids (Salkowski test) 22
3.9.7 Phenols (Ferric chloride test) 22
3.9.8 Tannins (Ferric chloride test) 23
3.10 Determination of the analgesic activity of the aqueous and methanol root extracts of Maerua triphylla 23
3.11 Evaluation of the anti-inflammatory activity of the aqueous and methanol root extracts of Maerua triphylla 24
3.12 Data Analysis 25
CHAPTER FOUR
4.0 RESULTS
4.1 The appearance and percentage yield of M. triphylla root extracts 27
4.2 Evaluation of the safety of the methanol and aqueous root extracts of M. triphylla on oral administration in
Wistar rats 27
4.3 Phytochemical screening 29
4.4 The effect of the methanol and aqueous root extracts of Maerua triphylla on acetic acid-induced writhing in
Swiss albino mice 30
4.5 The effect of the methanol and aqueous root extracts of Maerua triphylla on acetic acid-induced paw edema in Wistar rats 32
4.6 The effect of duration of treatment on acetic acid-induced paw edema in Wistar rats 33
4.7 The effect of treatment and duration on the acetic acid-induced paw edema in Wistar rats 34
CHAPTER FIVE
5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1 DISCUSSION 35
5.2 CONCLUSION 40
5.3 RECOMMENDATIONS 41
REFERENCES 42
LIST OF FIGURES
Figure 2.1: Parts of M. triphylla plant… 10
Figure 3.1: Kajiado County location in Kenya 17
Figure 4.1: A comparison of the analgesic activities of the aqueous and methanol extract of M. triphylla
in Swiss albino mice 31
Figure 4.2: Effects of the duration of treatment on the acetic acid-induced paw edema in Wistar rats…33
LIST OF TABLES
Table 2.1: Adverse effects of some of the commonly used analgesic and anti-inflammatory drugs…….5 Table 2.2: Ethnomedicinal uses of M. triphylla 11
Table 3.1: The treatment procedure used for the evaluation of the analgesic activities of methanol and aqueous root extracts of M. triphylla in Swiss albino mice 23
Table 3.2: The treatment procedure for the evaluation of the anti-inflammatory activities of aqueous and methanol extracts of M. triphylla in Wistar rats 24
Table 4.1: Appearance and percentage yield of M. triphylla root extracts 27
Table 4.2: Observations in Wistar rats treated with 2000mg/kg bw dose of methanol and aqueous M. triphylla root extracts 27
Table 4.3: Effect of a 2000 mg/kg dose of methanol and aqueous root extracts of M. triphylla on the weight of Wistar rats 28
Table 4.4: Phytochemical composition of M. triphylla root extract 29
Table 4.5: Summary of the effect of methanol and aqueous extracts of M. triphylla on acetic acid- induced writhing in Swiss albino mice 30
Table 4.6: Summary of the effect of the methanol and aqueous extracts of M. triphylla on acetic acid- induced paw edema in Wistar rats… 32
Table 4.7: Effects of the duration of treatment on acetic acid-induced paw edema in Wistar rats… 33
LIST OF APPENDICES
Appendix I: Ethical approval document… 56
Appendix II: Research permit… 58
Appendix III: Data output from the analysis of the effect of the aqueous root extract of M. triphylla on acetic acid-induced writhing in Swiss albino mice 60
Appendix IV: Data output from the analysis of the effect of the methanol root extract of M. triphylla on acetic acid-induced writhing in Swiss albino mice 62
Appendix V: Data output from the comparison of the inhibition of acetic acid-induced writhing by aqueous and methanol root extracts of M. triphylla 64
Appendix VI: Data output from the analysis of the effect of duration on the anti-inflammatory activity of the aqueous root extract of M. triphylla on acetic acid-induced paw edema in Wistar rats 64
Appendix VII: Data output from the analysis of the effect of treatment on the anti-inflammatory activity of the aqueous root extract of M. triphylla on acetic acid-induced paw edema in Wistar rats 66
Appendix VIII: Data output of the analysis of the effect of treatment and duration on the anti-inflammatory activity the aqueous root extract of M. triphylla on acetic acid-induced paw edema in Wistar rats 67
Appendix IX: Data output from the analysis of the effect of duration on the anti-inflammatory activity of the methanol root extract of M. triphylla on acetic acid-induced paw edema in Wistar rats 76
Appendix X: Data output from the analysis of the effect of treatment on the anti-inflammatory activity of the methanol root extract of M. triphylla on acetic acid-induced paw edema in Wistar rats 77
Appendix XI: Data output from the analysis of the effect of treatment and duration on the anti- inflammatory activity of the methanol root extract of M. triphylla on acetic acid-induced paw edema in Wistar rats 78
Appendix XII: Data output from the analysis of the acute oral toxicity of the aqueous and methanol root extracts of M. triphylla in Wistar rats 85
LIST OF ACRONYMS
ANOVA Analysis of variance
COX Cyclooxygenase
EDTA Ethylenediaminetetraacetic acid
FDA Food and Drug Administration
GI Gastrointestinal
IL Interleukin
iNOS Inducible nitric oxide synthase
LC50 Lethal concentration of chemical in air responsible for the death of 50% of a population of animals
LD50 Lethal dose responsible for the death of 50% of a population of animals
mm Millimetres
M. triphylla Maerua triphylla
NF-B Nuclear factor kappa-light-chain-enhancer of activated B cells
NSAIDS Nonsteroidal anti-inflammatory drugs
OECD Organization for Economic Co-operation and Development
STIs Sexually Transmitted Infections
TNF Tumor Necrosis Factor
V/V Volume per volume
W/W Weight by weight
WHO World Health Organization
CHAPTER ONE
INTRODUCTION
1.1 Background information
Traditional medicine is described by the World Health Organization (WHO) as the expertise, understanding, as well as practises founded on models, experiences and beliefs indigenous of various traditions used in diagnosis, treatment and prevention of mental and physical ailments (WHO, 2022). Medicinal plants naturally contain numerous phytochemicals and hence are used worldwide in traditional medicine (Singh and Geetanjali, 2013). In poor rural areas, most Kenyan communities depend heavily on herbal treatments (Kigen et al., 2013). Some of these therapeutic plants apply in the treatment and management of inflammation and pain in diseases associated with these symptoms such as rheumatism. The International Association for the Study of Pain (IASP) describes pain as unwanted emotional or receptive sensation brought about by potential or actual damage to the tissue (Raja et al., 2020). Inflammation, on the other hand is the body’s immune system’s response to an irritant and this can be part of various pathological conditions like arthritis, rheumatism, and atherosclerosis (Vogl et al., 2013).
For these illnesses, treatment generally relies on a large number of commercial preparations such as Nonsteroidal Anti-inflammatory Drugs (NSAIDs) like acetylsalicylic acid and steroidal drugs such as prednisone. They do this by inhibiting cyclooxygenase enzymes (COX) that act on arachidonic acid and cause the synthesis of prostaglandins (Katzung, 2018). However, most of these drugs have adverse effects like peptic ulcer, dyspepsia, and gastrointestinal bleeding (McGettigan and Henry, 2011). Furthermore, conventional drugs have low efficacy and are costly (Amaral et al., 2007). Therefore, a large number of medicinal plants need to be investigated for their potential analgesic and anti-inflammatory activity to avoid these adverse effects. One such plant is Maerua triphylla that has been used traditionally for the management of rheumatism, headache, migraine and tooth aches (Alfred, 2020).
Maerua triphylla is an evergreen shrub belonging to the family Capparidaceae. In terms of ethnomedicinal use, M. triphylla has been used to suppress conditions like rheumatism, headache, migraine, diarrhoea and stomach ache by the Maasai and Kikuyu communities (Alfred, 2020). Maerua triphylla has medicinal properties of the two conditions being investigated above but insufficient information is present on the safety, phytochemistry, and pharmacological properties of crude M. triphylla extracts. The study therefore aimed at assessing the acute oral toxicity (LD50), phytochemical composition, analgesic, as well as anti-inflammatory properties of M. triphylla root extracts.
1.2 Problem statement
Many communities use M. triphylla to treat pain and inflammation because it is regarded as cheap, easily accessible and believed to be more effective than modern-day drugs. However, prior to this study there was no scientific evidence of the anti-inflammatory and analgesic properties of M. triphylla to confirm these ethnomedicinal uses.
1.3 Justification
Modern-day chemotherapy against autoimmune diseases like rheumatism has faced a huge blow due to the associated adverse effects of existing drugs and costly treatment (Maina et al., 2015). There was therefore a need for efficacious and cheaper alternatives such as medicinal plants to manage these conditions. Given the above facts, studies of the plants having potential analgesic and anti-inflammatory properties such as M. triphylla are important. Phytochemicals such as proline betaine isolated from M. triphylla have been shown to suppress inflammation (McLean et al., 1996). Maerua triphylla roots are also known to be toxic (Alfred, 2020). Scarce scientific data existed to support the safe ethnomedicinal use of M. triphylla to mitigate conditions associated with pain and inflammation hence making this study necessary.
1.4 Objectives
1.4.1 Overall objective
To assess the acute oral toxicity, phytochemical composition, analgesic, and anti-inflammatory properties of M. triphylla root extracts.
1.4.2 Specific objectives
i. To determine acute oral toxicity (LD50) of M. triphylla root extracts in Wistar rats.
ii. To find out the phytochemical constituents of M. triphylla root extracts.
iii. To evaluate analgesic properties of M. triphylla root extracts.
iv. To examine the anti-inflammatory activities of M. triphylla root extracts.
1.5 Hypothesis
Maerua triphylla root extracts did not have analgesic or anti-inflammatory properties
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