ABSTRACT
Estimates by the World Health Organization indicates that 80 % of the global population use herbal medicines for prophylaxis and curative purposes. In rural Kenya, Traditional Medicine is the primary source of healthcare, and most often the only source of healthcare service. This is due to its ease of accessibility, affordability and trust by millions of people. The cost factor of most phytomedicines makes them all the more agreeable at a time of spiralling healthcare expenses and nearly widespread austerity. Even though conventional medicine exists concomitantly with Traditional Medicine, phytomedicines have often been popular for cultural and historical reasons. Herbal medicinal products have turned out to be commercially widely available, particularly in industrialised nations. The rationale for the use of Ocimum americanum L. (Lamiaceae) in antimicrobial phytotherapy is largely based on the long-term experience of traditional medicine practitioners. This study aimed to investigate the antimicrobial activity and cytotoxicity of crude extracts and their fractions in a microbial and brine shrimp model. Several solvents were selected, their crude extracts and fractions evaluated for their antimicrobial activity, cytotoxicity and phytochemical composition. These included: aqueous, acetone, 70 % hydroethanolic, chloroform and ethyl acetate. Standard bacterial strains of Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 25925), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603) and one fungal strain, Candida albicans were used to assess the Minimum Inhibitory Concentration and Minimum Bactericidal Concentration of the Ocimum americanum L. sample extracts and their fractions via standard antimicrobial procedures at the microbiology laboratory, Department of Public Health, Pharmacology and Toxicology University of Nairobi. The data was evaluated for susceptibility of bacterial species and considered significant at 95 % confidence interval. Cytotoxicity of the crude samples and fractions were analysed using brine shrimp lethality test with ten-fold dilutions of 1000 µg/mL, 100 µg/mL and l0 µg/mL. Median Lethal Concentration at p < 0.05 confidence intervals was determined using Probit analysis. Established phytochemical screening tests were performed to show the presence or absence of secondary metabolites. Cardiac glycosides, flavonoids, tannins, phenolics and reducing sugars were present in all sample extracts and fractions while polyuronides were absent. Two crude extracts and their fractions exhibited activity against the tested microorganisms. The hydroethanolic extract and its fractions were most active against the tested microbes. There was no significance difference (p > 0.05) in antibacterial activity between the acetonic and hydroethanolic extracts and their fractions. Gram-positive bacteria were more susceptible to the extracts/fractions than Gram-negative microbes. Bacillus cereus was most susceptible while Escherichia coli exhibited the highest resistance. All the sample extracts had statistically significant (p < 0.05) cytotoxicity at LC50 < 1000 µg/mL. Amongst all the samples the fractions of aqueous alcohol crude samples were more prospective thus good candidate for further research. Chloroform fraction of hydroethanolic sample extract was highly toxic with LC50 value of 0.59 µg/mL. The ethyl acetate sample fractions of aqueous alcohol crude sample have demonstrated promising antimicrobic effects against the Gram-positive microorganism Bacillus cereus. The fractions of hydroethanolic crude samples have potential bioactive molecules which are accountable for antimicrobial and cytotoxicity properties respectively. Results from the present research will provide a groundwork for finding an innovative natural phytomedicine. However extensive study is needed to quantify, isolate and characterise the phytocompounds.
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF APPENDICES x
ABBREVIATIONS AND ACRONYMS xi
ABSTRACT xii
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background Information 1
1.2 Statement of the problem 3
1.3 Justification 3
1.4 Study hypothesis 4
1.5 Objectives 4
1.5.1 General objective 4
1.5.2 Specific Objectives: 4
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Introduction 5
2.2 Infectious diseases 5
2.2.1 Bacterial pathogens 6
2.2.1.1 Bacillus cereus 6
2.2.1.2 Staphylococcus aureus 7
2.2.1.3 Escherichia coli 8
2.2.1.4 Klebsiella Pneumoniae 8
2.2.2 Fungal pathogens 9
2.2.2.1 Candida albicans 9
2.3 Ethnomedicine 10
2.3.1 Ethnopharmacology 11
2.3.2 Description of the herbal plant Ocimum americanum L. (Lamiaceae) 11
2.3.3 Traditional uses of Ocimum americanum L. (Lamiaceae) 13
2.4 Phytochemicals from O. americanum L. and their pharmacological properties 13
2.4.1 Alkaloids 14
2.4.2 Anthraquinones 16
2.4.3 Cardiac glycosides 17
2.4.4 Cyanogenetic glycosides 18
2.4.5 Flavonoids 19
2.4.6 Phenols 20
2.4.7 Phytosterols/triterpenes 21
2.4.8 Tannins 22
2.4.9 Terpenoids 23
2.4.10 Saponins 24
2.4.11 Reducing sugars 25
2.5 Antimicrobial assays 25
2.5.1 Agar well diffusion technique 25
2.5.2 Broth dilution technique 26
2.6 Cytotoxicity 27
2.6.1 Brine shrimp lethality bioassay 28
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Plant material collection and identification 30
3.2 Preparation and extraction of plant material 30
3.2.1 Aqueous extracts 30
3.2.2 Hydroethanolic extracts 30
3.2.3 Acetonic extracts 31
3.3 Liquid-liquid fractionation (LLF) 31
3.4 Antimicrobial assays 31
3.4.1 Test microorganisms 31
3.4.2 Microbial cultures 32
3.4.3 Preparation of test extracts 32
3.4.4 Agar well diffusion method 32
3.4.5 Broth dilution technique 33
3.5 Brine shrimp cytotoxicity 34
3.5.1 Preparation of artificial marine solution 34
3.5.2 Incubating and harvesting of Artemia salina nauplii 34
3.5.3 Preparation of crude extracts and fractions 34
3.5.4 Brine shrimps bioassay 35
3.6 Phytochemical screening 35
3.6.1 Alkaloids screening (Dragendorff’s/Mayer’s test) 35
3.6.2 Anthraquinones screening (Borntrager’s test) 36
3.6.3 Cardiac glycosides screening (Keller-Killiani test) 36
3.6.4 Cyanogenetic glycosides 36
3.6.5 Flavonoids screening (Ammonia test) 36
3.6.6 Phenolics screening (Ferric chloride test) 37
3.6.7 Terpenoids screening (Salkowski test) 37
3.6.8 Saponins screening (Frothing test) 37
3.6.9 Reducing sugars screening (Benedicts’ test) 37
3.6.10 Polyuronides (gums, mucilage, pectin) 38
3.6.11 Tannins screening (Ferric chloride test) 38
3.6.12 Phytosterols screening (Lieberman-Burchard’s test) 38
CHAPTER FOUR
4.0 RESULTS
4.1 Extraction yield of different solvents 39
4.2 Antimicrobial sensitivity testing of Ocimum americanum L. 39
4.2.1 Agar well diffusion bioassay 39
4.2.1.1 Acetone crude extract… 39
4.2.1.2 Chloroform fraction of the acetonic crude extract 41
4.2.1.3 Ethyl acetate fraction of the acetonic crude extract… 42
4.2.1.4 Hydroethanol crude extract… 44
4.2.1.5 Chloroform fraction of hydroethanolic crude extract… 45
4.2.1.6 Ethyl acetate fraction of hydroethanolic crude extract 47
4.2.1.7 Summarised results of ANOVA, TUKEY and Dunnett test… 48
4.2.2 Broth dilution bioassay 52
4.2.2.1 Summarised results of agar well diffusion and broth microdilution… 53
4.2.3 Brine shrimp bioassay 55
4.2.4 Phytochemical profile 57
CHAPTER FIVE
5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
5.1 Discussion 59
5.2 Conclusions and recommendations 67
REFERENCES 68
APPENDICES 87
LIST OF TABLES
Table 2.1: Major alkaloids classes with examples and pharmacological uses, adapted from Pengelly (2021) 15
Table 4.1: Percentage yield of the O. americanum L. crude extracts with different solvents…39
Table 4.2: Inhibition of microbial growth by acetone crude extract of O. americanum L 40
Table 4.3: Inhibition of microbial growth by chloroform fraction of the acetonic extract 41
Table 4.4: Inhibition of microbial growth by ethyl acetate fraction of the acetonic extract 42
Table 4.5: Inhibition of microbial growth by hydroethanol extract of O. americanum L 44
Table 4.6: Inhibition of microbial growth by chloroform fraction of hydroethanolic extract 45
Table 4.7: Inhibition of microbial growth by ethyl acetate fraction of hydroethanol extract 47
Table 4.8: Summary of microbial growth inhibition by acetone and hydroethanolic crude extracts and their fractions against test pathogens 49
Table 4.9: The MBC/MFC of Ocimum americanum L. crude extracts and fractions against test microbes… 52
Table 4.10: Summary of MIC, MBC, MFC of Ocimum americanum L. crude extract and their fractions of various solvents against test microbials 54
Table 4.11: Summary of the cytotoxicity of crude extracts and fractions of O. americanum L. against Artemia salina larvae (brine shrimp nauplii) 55
Table 4.12: Phytochemical profile of Ocimum americanum L. crude extracts and their fractions… 57
LIST OF FIGURES
Figure 2.1: Ocimum americanum L. (Lamiaceae) aerial parts sourced from Pate Island, Lamu County 12
Figure 4.1: Microbial growth inhibition against various concentrations of acetone crude extract of O. americanum L 40
Figure 4.2: Microbial growth inhibition against various concentrations of chloroform fraction of the acetonic crude extract of O. americanum L 42
Figure 4.3: Microbial growth inhibition against various concentrations of ethyl acetate fraction of the acetonic crude extract of O. americanum L 43
Figure 4.4: Microbial growth inhibition against various concentrations of the hydroethanol crude extract of O. americanum L 45
Figure 4.5: Microbial growth inhibition against various concentrations of chloroform fraction of the hydroethanolic crude extract of O. americanum L 46
Figure 4.6: Microbial growth inhibition against various concentrations of ethyl acetate fraction of the hydroethanolic crude extract of O. americanum L 48
Figure 4.7: Brine shrimp % mortality against various concentrations of O. americanum L. crude extract and their fractions 56
Figure 4.8: Positive frothing test for saponins, showing aqueous and hydroethanol persistence frothing 58
LIST OF APPENDICES
Appendix 1: Ethical approval of the faculty of biosafety, animal use and ethics committee...87 Appendix 2: Letter of plant identification 88
Appendix 3: Probit analysis of acetone crude extract of Ocimum americanum L 89
Appendix 4: Probit analysis of chloroform fraction of acetonic extract 90
Appendix 5: Probit analysis of ethyl acetate fraction of acetonic extract 91
Appendix 6: Probit analysis of aqueous crude extract of Ocimum americanum L 92
Appendix 7: Probit analysis of chloroform fraction of aqueous crude extract 93
Appendix 8: Probit analysis ethyl acetate fraction of aqueous crude extract 94
Appendix 9: Probit analysis of hydroethanolic crude extract of Ocimum americanum L 95
Appendix 10: Probit analysis of chloroform fraction of hydroethanolic extract 96
Appendix 11: Probit analysis of ethyl acetate fraction of hydroethanolic extract 97
Appendix 12: Probit analysis of positive control drug – Vincristine 98
Appendix 13: Analysis of variance comparing growth inhibition of acetone extract 99
Appendix 14: Analysis of variance comparing growth inhibition of hydroethanolic crude extract and fractions. 99
Appendix 15: Photo for plant specimen collection Pate Island Lamu County 100
Appendix 16: Photos for crude extract extraction process 101
Appendix 17: Photos for Agar well diffusion 103
Appendix 18: Photo for Cytotoxicity 104
Appendix 19: Photos for phytochemicals 104
Appendix 20: Published paper associated with the thesis 106
ABBREVIATIONS AND ACRONYMS
AMDR Antimicrobial Drugs Resistance
AMR Antimicrobial Resistance
ANOVA Analysis of Variance
ATM African Traditional Medicine
ATCC American Type Culture Collection
BACUC Biosafety, Animal Care and Use Committee
DMSO Dimethyl Sulfoxide
EDTA Ethylenediaminetetraacetic Acid
FVM Faculty of Veterinary Medicine,
GBD Global Burden of Disease
GIT Gastrointestinal Tract
INT p-iodonitrotetrazolium violet
LD50 Median Lethal Dose
MBC Minimum Bactericidal Concentration
MFC Minimum Fungicidal Concentration
MIC Minimum Inhibitory Concentration
NACOSTI National Council for Science and Technology
UNESCO United Nations Educational, Scientific and Cultural Organization
UTI Urinary Tract Infections
RTIs Respiratory Tract Infections
rDNA Recombinant Deoxyribonucleic Acid
SEM Standard Error of the Means
TM Traditional Medicine
WHO World Health Organization
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background Information
Traditional Medicinal Practice (TMP) originated since time immemorial in different parts of the world (Tyler, 2000). African herbal medicinal practice have proceeded via descendants by verbal transmission with little written records or no records at all (Okigbo & Mmeka, 2006). Phytomedicines and folklore practices of established safety and effectiveness endow the primary health care objectives and assures accessibility to the whole society. According to the estimates by World Health Organization for millions of people worldwide, TMP is the primary provenance of healthcare, and most commonly the solitary fount of primary care provision. This is owing to its affordability, ease of accessibility and confidence by millions of individuals. The financial aspect of most herbal medicines render them all the more acceptable at a time of soaring medical care expenditures and virtually rife austerity (WHO, 2013). Although orthodox medicine coexists concurrently with TMP, traditional herbal medicines have commonly been prevalent for historical and traditional motives. Herbal medicinal products have turned out to be commercially widely available, particularly in industrialised nations (Schulz et al., 2001). Estimates by the WHO indicates that 80 % of the worldwide populace consume phytomedicines for prophylaxis and curative purposes, and in Africa the practice is much higher (WHO, 2013).
The benefit of using phytomedicines over xenobiotic alternative is that they are comparatively safer, conferring effective prophylactic or curative therapy as well as affordability. The use of herbal medicines in emerging nations as a cultural basis for healthcare has been extensively observed (UNESCO, 1996). Additionally, the increasing dependence on the use of herbal medicines in the developed countries has been as a result of the progress of several crude extracts from medicinal herbs as well as from folkloric use of countryside therapies (UNESCO, 1998).
Continental Africa is inherently gifted with cornucopia of vegetation, approximated to thousands of types. Phytologists estimate that about 10% of flora in Africa is of therapeutic importance and some of the medicinal herbs have been systematically evaluated and their traditional role established (Gurib-Fakim & Mahomoodally, 2013). The medicinal herb Ocimum americanum L. (Lamiaceae) is native to subtropics and tropical parts of the globe including Indian subcontinent and continental Africa (Ali et al., 2022). The species belong to the genus Ocimum which contains more than 60 species of aromatic herbs and shrubs (Simon et al., 1999; Zengin et al., 2019). The Lamiaceae species are example of herbal plants that are problematic to differentiate on the basis of just leaf morphology, due to the varied shapes of leaf within the species (Vieira et al., 2003). Ocimum americanum L. is a small erect branched, annual perennial aromatic herb which grows up to 1 m high. Stems are somewhat rounded or quadrangular, woody close to the base, hirsute and adpressed. Leaves are scarcely elliptical, typically hairless up to 25 mm in length (Sarma & Babu, 2011). The aromatic medicinal shrub has a widespread topographical dispersal in East Africa, making it the most common phyto- botanical flora in the area (Kokwaro, 2009). In Kenya, it is extensively dispersed in the jungle boundaries, secondary woodland and prairie, riparian spots and in arid zones, mostly in the mounds (Beentje et al., 1994).
In Eastern Africa depending with the local tongue variation, the Swahili people refer to Ocimum (basil) as Kivumbani/Mvumbani/Mrihani (Hiltunen & Holm, 1999). Some genus from the family Lamiaceae has important medicinal properties that have high bioprospecting potential. Numerous species indigenous in East Africa are utilised in traditional therapeutic practices and some of their biophysiological effects have been appraised. African (hoary) basil is utilised for non-medicinal and medicinal objectives in diverse local traditions in East Africa (Hiltunen & Holm, 1999; Paton et al., 1999). The main important property of the Lamiaceae (mint) family is associated with its constituent of essential oils. Also known to as volatile oils due to their characteristic high volatility, they are a rich mix with wide spectrum of biophysiological properties. The essential oils obtained from the sweet-smelling herbal plants are a natural blend of phytocompounds with strong fragrance; they are by-products of secondary metabolism (Morsy & Hammad, 2021; Shadia et al., 2007; Sutili et al., 2016). Ocimum americanum L. has a wide range of bioactive compounds in the form of volatile oils. These include camphor, eugenol, methyl eugenol, methyl chavicol, farnesene, linalool, limonene and terpineol (Matasyoh et al., 2006; Paton et al., 1999; Shadia et al., 2007; Sutili et al., 2016).
1.2 Statement of the problem
All antimicrobials drugs that are launched into the marketplace have restricted therapeutic use owing to their intrinsic or acquired mechanism of microbial resistance (Walsh, 2003). Antimicrobial Resistance (AMR) has developed due to the undiscerning use of antibiotic feed additives in animal farming. Conversely humans have intensified the AMR by misuse and noncompliance of their prescribed antimicrobial drugs regimens (Runyoro et al., 2006; Walsh, 2003). Moreover re-emergence of infectious diseases and the inflated rate of antimicrobics have been a key factor exacerbating to the vain control of infectious diseases in the developing nations such as Kenya (Runyoro et al., 2006). Consequently, TM has been an alternative and affordable primary healthcare substitute. Ocimum americanum L. aerial parts are among some of purportedly effective ethnobotanical remedies currently available at a relatively low cost (Malik et al., 2018). The plant materials are mostly sold in raw form and literature on the most efficient standard method of extraction is scanty.
1.3 Justification
Natural products of medicinal plants may provide a potential source of antimicrobial bioactive molecule conceivably with a novel mechanism of action. Ocimum americanum L. (Lamiaceae) is traditionally utilised against diarrhoea and dysentery (Runyoro et al., 2006; Vidhya et al., 2020). Although literature on antimicrobial activity of Ocimum americanum L. plant extracts has been reported, data on the antimicrobial activity and safety of derived fractions is scanty.
This study seeks to bridge these gaps in order to guarantee the safety and efficacy of traditional botanical used Ocimum americanum L. The research thus evaluated the antimicrobial activity, cytotoxicity and phytochemical composition of Ocimum americanum L. crude extracts and fractions. Findings from the present research would provide a potential foundation for finding of a new natural product derived molecule. Cytotoxicity studies are central in hazard assessment and safety evaluation phase of plant extracts. Therefore, this study is highly appropriate because it comes at a time when the use of herbal medicines in the treatment of many bacterial infections has taken a centre stage.
1.4 Study hypothesis
It was hypothesised that Ocimum americanum L. (Lamiaceae) crude extracts and fractions had antimicrobial activity, were cytotoxic and contained significant phytocompounds.
1.5 Objectives
1.5.1 General objective:
To investigate the antimicrobial activity, cytotoxicity and phytochemical composition of Ocimum americanum L. (Lamiaceae).
1.5.2 Specific Objectives:
1. Investigate the antimicrobial activity of Ocimum americanum L. (Lamiaceae) crude extracts and fractions using microbial organisms.
2. Evaluate the cytotoxicity of Ocimum americanum L. (Lamiaceae) crude extracts and fractions using Artemia salina nauplii.
3. Determine the phytochemical composition of Ocimum americanum L. (Lamiaceae) crude extracts and fractions.
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