ABSTRACT
African trypanosomiases (AT) are a group of hemoparasitic diseases caused by multiple flagellated organisms of the genus Trypanosoma. The disease affects humans and livestock animals, and it is lethal if untreated, therefore, AT is of economic and global health importance. Since there is no vaccine for prevention and available drugs produce unsatisfactory outcomes, this work was aimed at discovery of new compounds with potentials for future development of new drugs for AT. Coloured microorganisms of the phylum Cyanobacteria are known to produce therapeutic secondary metabolites, which are yet to be explored for anti-Trypanosoma potentials. Therefore, in search of new anti-Trypanosoma compounds, crude methanolic extracts of four cyanobacteria (Microcystis aeruginosa EAWAG198, Microcystis flos-aquae UTEX 2677, Microcystis wesenbergii and Oscillatoria sp) were prepared and tested for trypanosomes-killing activity. The most active crude extract (M. flos-aquae) was fractionated by gel filtration chromatography with Silica gel 60-120G as the stationary phase and combination of Methanol: Ethyl acetate: Hexane solvent system as mobile phase, while purity was assessed by thin layer chromatography using Hexane: Ethyl acetate (7:3 v/v). Antitrypanosomal fractions were identified by incubating each fraction with Trypanosoma brucei brucei cells and monitoring parasite death for 2 hrs by wet mount under 400× microscopic magnifications. Wistar rats infected with T. b. brucei were treated with 30, 60 and 120 mg/kg body weight dosages of M. flos-aquae crude extract. The most active fraction (E) of M. flos-aquae was characterized by GC/MS.. The crude extract of M. flos-aquae exhibited the highest in vitro trypanocidal activity with a percentage inhibition of 98.44 and 42.18% at 2.5 and 0.3125 mg/mL concentration, respectively, and displaying IC50 value of 0.4140 mg/mL. Fraction E exhibited the highest activity against the parasite with percentage inhibition of 74.21 % at 0.625 mg/mL with IC50 of 0.2991 mg/mL, and its subfraction 76 % inhibition (24 % survival) at the same concentration. Interestingly, the extract of M. flos-aquae suppressed parasite proliferation, and improved weight and PCV in rats at dosages above 60 mg/kg body weight. GC/MS.In conclusion, the screened cyanobacteria are high potential sources of promising bioactive compounds that could be explored for treatment of trypanosomiasis.
TABLE OF CONTENTS
Title page i
Declaration ii
Certification iii
Acknowledgement iv
Dedication v
Abstract vi
Table of contents viii
List of Figures xii
List of Plates xv
List of Appendices xvi
List of Abbreviations and Symbols xvii
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of the study 1
1.2 Statement of Research Problem 4
1.3 Justification 5
1.4 Aim 5
1.5 Objectives 6
1.6 Hypotheses 6
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 African Trypanosomiasis 7
2.1.1 Epidemiology of the African Trypanosomiasis 7
2.1.2 The Biology/Life Cycle of the African Trypanosomes 8
2.1.3 Pathogenesis of African Trypanosomiasis 13
2.1.4 Prevention and Control of African Trypanosomiasis 13
2.2 Ethnobotanical Treatment of Trypanosomiasis 14
2.3 Cyanobacteria 15
2.3.1 Taxonomy of Cyanobacteria 16
2.3.2 Growth Pattern of Cyanobacteria 17
2.4 The Genus Microcystis 21
2.5 The Genus Oscillatoria 21
2.6 Antiprotozoal Activity of Some Algal and Cyanobacterial Extracts 21
2.7 Bioactive Metabolites from Cyanobacteria 22
2.8 Metabolites from Cyanobacteria with Antiprotozoal Activity 26
2.8.1 Metabolites with Antitrypanosomal Activity 26
2.8.2 Metabolites with Antiplasmodial Activity 27
2.8.3 Metabolites with Anti-leishmanial Activity 32
2.10 Cyanobacteria as Source of Nutritional Supplements and Pigments 32
2.11 Other Metabolites of Marine Origin with Anti-Trypanosoma Activity 33
2.12 Extraction of Metabolites from Cyanobacteria 34
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Materials 37
3.1.1 Reagents 37
3.1.2 Cyanobacteria 37
3.1.3 Trypanosoma brucei brcei 37
3.1.4 Experimental Animals 38
3.2 Methods 38
3.2.1 Propagation of Trypanosomes in Rats 38
3.2.4 Culturing of Cyanobacteria 38
3.2.5 Extraction of T. b. brucei and Oscillatoria sp. DNA 39
3.2.6 Molecular Identification of Trypanosoma brucei brucei 41
3.2.7 Molecular Identification of the Oscillatoria spp. 42
3.2.8 Preparation of Crude Extract 43
3.5 In vitro Assay for anti-Trypanosoma Activity with Crude Extracts 43
3.6 Fractionation of the Crude Extract 44
3.6.1 Thin Layer Chromatography (TLC) 45
3.7 In Vitro Assay for anti-Trypanosoma Activity with M. flos-aquae fractions 46
3.7.1 Sub-fractionation of Active Fractions 46
3.8 Characterization of the Most Active Fractions 47
3.9 In vivo Antitrypanosomal Activity of Most Active Crude Extract 48
3.9.1 Determination of Acute Toxicity 48
3.9.2 In vivo Antitrypanosomal Assay 48
3.9.2 Determination of Parasitemia and Weight of rats 49
3.9.3 Determination of Packed Cell Volume (PCV) 49
3.10 STATISTICAL ANALYSIS 50
CHAPTER FOUR
4.0 RESULTS
4.1. Molecular Identification of the T. b. brucei 51
4.2 Microscopic and Molecular Characterization of the Isolated Cyanobacteria 51
4.3. Growth Pattern of the Colony Forming Cyanobacteria 55
4.4. Percentage Yields of the Crude Methanol Extract 55
4.5. In vitro Antitrypanosomal Activity of the Cyanobacterial Crude Extracts on T.b brucei 58
4.6. Thin Layer Chromatography of the Pooled Microcystis flos-aquae Fractions 61
4.6.1 In vitro Antitrypanosomal Activity of Microcystis flos-aquae Fractions on T. b. brucei 63
4.7 Thin Layer Chromatography of the Microcystis flos-aquae Sub-fractions 67
4.7.1. In vitro Antitrypanosomal Activity of Sub-fraction from M. flos-aquae 67
4.8. Detection of Compounds in Microcystis flos-aquae Most Active Fractions by GC-MS 70
4.9 In vivo Antitrypanosomal Activity of the Most Active Crude Extract (M. flos-aquae) 73
4.9.1 Acute Toxicity of the Extract 73
4.9.2 Effect of Treatment with Extract on Parasitemia 73
4.9.3 Effect of Treatment with Extract on the Weight of Rats 73
4.9.4 Effect of Treatment with Extract on the PCV of Rats 74
CHAPTER FIVE
5.0 DISCUSSION 79
CHAPTER SIX
6.0 CONCLUSION AND RECOMMENDATIONS
6.1 CONCLUSION 86
6.2 RECOMMENDATIONS 87
6.3 CONTRIBUTION TO KNOWLEDGE 88
REFERENCES 89
APPENDICES 105
LIST OF FIGURES
Figure 2.1: Map of African regions affected by African Trypanosomiasis… 10
Figure 2.2: Diagram of Trypanosoma brucei 11
Figure 2.3: Life cycle of T. brucei 12
Figure 2.4: A typical bacterial growth curve 20
Figure 2.5: Number of isolated compounds from cyanobacteria and percentage of various activities reported… 25
Figure 2.6: Structures of metabolites from cyanobacteria with antiprotozoal activity 29
Figure 2.7: Structures of metabolites from cyanobacteria with antitrypanosomal and antiplasmodial activity 30
Figure 2.8: Structures of metabolites of other marine organisms with antiprotozoal activity 31
Figure 2.8: Structures of metabolites from other marine organisms with antitrypanosomal activity 36
Figure 4.1: Growth curve of the unicellular cyanobacteria cultured for a period of 18 days. 56
Figure 4.2: Effect of crude methanol cyanobacterial extracts on T. b. brucei in vitro 59
Figure 4.3: Plots for IC50 determination of all the cyanobacterial crude extracts at 2 hrs incubation with T.b. brucei 60
Figure 4.4: In vitro antitrypanosomal activity of Microcystis flos-aquae pooled fractions on T. b. brucei 64
Figure 4.5: Plots for IC50 Determination at 2 hrs Incubation of T.b. brucei with the active fractions. 65
Figure 4.6: Effect of M. flos-aquae sub-fractions all on T. b. brucei in vitro 69
Figure 4.7: GC/MS spectrum of fraction E of Microcystis flos-aquae crude extract 72
Figure 4.9: Effect of treatment with M. flos-aquae extract on parasitemia in rats infected with T. b. brucei 76
Figure 4.10: Effect of treatment with M. flos-aquae extract on the weight of rats infected with T. b. brucei 77
Figure 4.11: Effect of treatment with M. flos-aquae extract on packed cell volume (PCV) of rats infected with T.b. brucei 78
LIST OF TABLES
Table 4.1: Biomass and crude cyanobacterial extracts yield of the cyanobacteria used for in vitro assays 57
Table 4.2: List of compounds identified in the most active fractions by GC/MS 71
Table 4.3: Acute toxicity of M. flos-aquae crude extract in Wistar rats… 77
LIST OF PLATES
Plate I: A 1.5 % Agarose gel electrophoregram of the amplified ITS-1 region of T. b. brucei 51
Plate II: Photomicrographs of the screened cyanobacteria 53
Plate III: A 1.5 % Agarose gel electrophoregram of the amplified 16S rDNA region of the Oscillatoria sp… 54
Plate IV: TLC chromatogram of the M. flos-aquae pooled fractions 62
Plate V: Photomicrographs of trypanosomes (400 ×) in blood smears after in vitro treatments; (A) without extract and (B) with 1.25 mg/mL of fraction E 66
Plate VI: TLC chromatogram of the M.flos-aquae sub-fractions 68
LIST OF APPENDICES
APPENDIX I: Composition and Preparation of BG-11 media 105
APPENDIX II: ANOVA Table for the effect of treatments with crude extracts on the Parasitemia in vitro 106
APPENDIX I1I: ANOVA table for the effect of treatments with M. flos-aquae fractions on the parasitemia in vitro 107
APPENDIX IV: ANOVA table for effect of treatments on the parasitaemia in rats 108
APPENDIX V: ANOVA table for effect of treatment on the weight of rats 109
APPENDIX VI: Table showing IC50 values of the crude cyanobacterial extract and the active fractions of M. flos-aquae after 2hrs exposure 110
LIST OF ABBREVIATIONS AND SYMBOLS
AAT =Animal African Trypanosomiasis
DNA = Deoxyribonucleic acid
RNA =Ribonucleic acid
BG-11 = Blue-green-11
ºC =Degree Centigrade
GC-MS = Gas chromatography- Mas spectroscopy
FTIR = Fourier-Transformed Infrared spectroscopy
PBS =Phosphate Buffer Saline
PCR = Polymerase Chain Reaction
DMSO = Dimethyl Sulfoxide
HAT =Human African Trypanosomiasis
AAT= Animal African Trypanosomiasis
PCV =Packed Cell Volume
ANOVA = Analysis of Variance
TLC = Thin Layer Chromatography
Rf = Retention factor
IC50= Inhibitory concentration 50
LD50 = Lethal dosage 50
rRNA = ribosomal Ribonucleic acid (ribosomal RNA)
rDNA = ribosomal Deoxyribonucleic acid (ribosomal DNA)
CDC = Center for Disease Control
CAS = Compound Abstract Service.
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of the study
African trypanosomiasis, commonly called sleeping sickness in humans and nagana in animals is a disease caused by protozoan parasites of the genus Trypanosoma, and transmitted to the mammalian hosts mainly by tsetse flies (Glossina spp) (Steverding, 2008). Sleeping sickness is caused by two subspecies of Trypanosoma brucei which are morphologically indistinguishable. The Trypanosoma bruceei gambiense causes West African sleeping sickness and Trypanosoma brucei rhodesiense causes East African sleeping sickness (CDC, 2015). Trypanosoma brucei brucei is the parental subspecies and does not infect humans, but just as T. congolense, T. vivax, and other animal trypanosomes, is responsible for nagana. According to the World Health Organization (WHO), sustained control efforts have reduced the number of new cases to less than 10,000 people per year but about 65 million people are still at risk in the 36 sub-Saharan countries that are endemic to sleeping sickness (WHO, 2017).
Chemotherapy has remained the only control measure, with only four approved drugs which have been developed more than three decades ago. Some of the chemotherapeutic drugs used for the treatment of African Animal Trypanosomiasis (AAT) are: diminazene aceturate (Berenil), suramin, melarsoprol (Arsobal) and pentamidine (Kuzeo, 1993; Fairlamb, 2003). These drugs are few and already limited by problems such as resistance by the parasites and toxic side effects (Fairlamb, 2003; Anene et al., 2011; Baker et al., 2013). In addition, there are no approved vaccines for prevention of the disease despite several efforts that have been made so far. Hence, the continuous search for new and better drug candidates.
Trypanosomes have a complicated life cycle that involves alternating between insect-adapted forms such as the procyclic forms (PCFs) in the midgut of the vector and bloodstream forms (BSFs) in the human host (Simpson et al., 2006). Blood Stream Forms of T. brucei rely exclusively on glycolysis for energy production (Verner et al., 2016). The glycolytic pathway of these parasites is compartmentalized within the organelles called glycosomes. Glycerol kinase is one of the glycosomal enzymes and plays a key role in the parasite energy metabolism (Minagawa et al., 1997; Balogun et al., 2010). The rudimentary mitochondrion of the parasites also houses an indispensable cytochrome-independent Trypanosome Alternative Oxidase (TAO) (Chaudhuri et al., 2006). TAO is the essential terminal oxidase for re-oxidization of NADH produced during glycolysis by the trypanosomes, and in addition to glycerol-3-phosphate dehydrogenase, it is also the primary mitochondrial electron transport protein (Kido et al., 2010). The importance of these enzymes to the parasites and their absence in the mammalian hosts has made them a good target for anti-trypanosomal drugs search (Minagawa et al., 1997; Verlinde et al., 2001; Yabu et al., 2003; Balogun et al., 2010).
Cyanobacteria (blue-green algae) are a group of photosynthetic prokaryotic organism found in fresh and marine waters, soil, rock, wall, tree trunks and sewage. They are morphologically diverse and flourish in static and eutrophic water bodies, dominating the aquatic ecosystem through formation of blooms (Ghadouani et al., 2004). During cyanobacteria bloom formation in aquatic environment, there is production of secondary metabolites which are mostly targeted at inhibiting the growth of other competitors, a behavior referred to as allelopathy. Some of these metabolites inhibit the growth of other algal species (algicidal effect), while some are secreted to deter grazers which are mostly zooplankton (Pflungmacher, 2002).Bloom–forming cyanobacteria include Microcystis spp. Oscillatoria spp. Anabaena spp. Nostoc spp. and many others. These species have been known for production of toxins such as microcystins, anatoxins and nodulalins, alongside several metabolites into the aquatic environment (Chia et al 2009; Singh et al., 2017). Cyanobacteria are acknowledged producers of diverse biologically active and structurally diverse secondary metabolites (Demay et al., 2019). Although, most of the isolated metabolites from cyanobacteria eventually exhibit cytotoxic effect, non-toxic metabolites from them exhibits potentials to serve as lead compounds in pharmaceutical, agricultural, or industrial applications (Tan, 2007; Burja et al., 2001; Demay et al., 2019). The activity of these compounds against viruses, bacteria, fungi, protozoa, cancer cells and other algal species have been reported (Mundt et al., 2001; Portman et al., 2008; Mazard et al., 2016). A few of the secondary metabolites have been reported to possess some biological effects on trypanosomes. For instance, aerucyclamide isolated from a cyanobacterium Microcystis aeruginosa displayed anti-trypanosomal activity on T.b. rhodesiense (Portmann et al., 2008); Almiramides isolated from the cyanobacteria Lyngbya majuscula were also found to play a role in disruption of glycosome function in T. b. brucei (Sanchez et al, 2013).
Oscillatoria is a genus of filamentous cyanobacteria which is named after its oscillation movement. Studies on Oscillatoria spp isolated from various water bodies have reported the presence of butylated hydroxyl toluene (BHT), vitamins, minerals, viridamides, antibacterial, antifungal, anticancer and antiprotozoal compounds by the screened species (Shanab, 2007; Linington et al., 2007; Simmons et al., 2008a; Nair and Bhimba, 2013).
Microcystis is a genus of cyanobacteria characterized by colony of spherical shape cells (about 2- 8µm) containing gas vesicles and constitutes the most common bloom-forming cyanobacteria in many water bodies worldwide (Mazur-Marzec et al., 2010). Although, Microcystis species have been known to be synthesizers of hepatotoxins such as microcystin, bioactive compounds of pharmacological importance have also been isolated from them (Portmann et al., 2008).
Previous reports on cyanobacteria as rich sources of bioactive compounds with therapeutic potential inspired the design of this work. Research into biological activity of these compounds reported in the literature mostly focused on screening of laboratory cultures, most of which are from the marine environment; rarely species from the local environment are assessed for their pharmacological properties (Mundt et al., 2001). This study was therefore designed to assess the biological activity of extracts from an indigenous freshwater cyanobacterial strain alongside three other exotics strains on Trypanosoma brucei brucei.
1.2 Statement of Research Problem
African human and animal Trypanosomiasis have been acknowledged as the cause of morbidity and mortality to humans and livestock throughout sub-Saharan Africa, and a major constraint to agricultural activity. An estimated 65 million people are at risk in the 36 sub-Saharan African countries. Methods of vector control through tsetse traps and spraying with insecticides such as Dichloro-Diphenyl-Trichloroethane (DDT) have a lot of limitations and have not been effective (Adamu et al, 2011). Chemotherapy, which is the control measure, exacerbates the situation in that it relies on a few drugs that have negative side effects, and must be administered intramuscularly or intravenously by qualified medical personnel (Fairlamb 2003; Barret et al., 2007). The few available drugs are already facing the problem of emerging resistance because drugs currently in use have been employed continuously for a long time (Baker et al., 2013). Hence, the need for continuous search of better alternative drugs to tackle this majorly Africa’s health problem.
1.3 Justification
Studies into natural products for discovery of novel therapeutics have been directed more towards screening of higher plants. There is less focus on marine and freshwater cyanobacteria and microalgae as promising sources of new compounds of interest in pharmacology and biotechnology. Due to structural diversity in their metabolites, the probability of rediscovery of compounds already identified in other organisms is very low. Cyanobacteria (blue-green algae) are a promising yet underexplored source for novel natural products with potent biological activities.
Although, most of the compounds isolated from cyanobacteria in the past are cytotoxic, they also produce a significant number of compounds that possess anti-infective activities (Niedermeyer, 2015). Cyclic hexapeptides isolated from Oscillatoria species and Microcystis species from marine environment have shown activity against T. cruzi and T. b. rhodesiense (Linington et al., 2007; Portmann et al., 2008). Quite a number of works have reported antimicrobial activities of marine cyanobacteria, sponges and other microalgae, but there is scarcity of information on the anti-infective potential of freshwater cyanobacteria. As a result, this study investigated the anti- Trypanosoma potential of methanol extracts from exotic strains and an indigenous isolate.
1.4 Aim
The aim of this work is to evaluate the extracts of Microcystis flos-aquae, Microcystis aeruginosa, Microcystis wesenbergii and Oscillatoria sp. for alternative chemotherapeutic principles against trypanosomiasis.
1.5 Objectives
The specific objectives are to:
i. Carry out molecular characterization of the isolated indigenous cyanobacterium.
ii. Determine the anti-Trypanosoma activity of crude extracts of the four cyanobacteria isolates on T. b. brucei in vitro
iii. Determine the anti-Trypanosoma activity of fractions from most active extract on T. b. brucei in vitro
iv. Detect bioactive metabolites from the most active fraction of the cyanobacterial extract
v. Carry out in vivo screening of the most active crude extract on T. b. brucei in rats.
1.6 Hypotheses
i. The isolated cyanobacterium is not Oscillatoria sp.
ii. Crude extracts of the four cyanobacteria has no anti-Trypanosoma activity.
iii. Fractions most active cyanobacterial extract has no anti-Trypanosoma activity.
iv. There are no bioactive metabolites in the fractions of the cyanobacteria
v. The most active cyanobacteria extract does no possess in vivo antitrypanosomal activity.
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