ABSTRACT
Leishmaniasis is a disease complex instigated by a protozoan parasite of the genus Leishmania. Contemporary chemotherapies for leishmaniasis employ pentamidine (1), amphotericin B (2) and pentavalent antimonials (3). The efficacy of these drugs has deteriorated due to drug resistance. The drugs also pose unbearable side effects owing to their toxicity. Some metabolites from the genus Pentas (family Rubiaceae) have been reported to show antiprotozoal activity against Plasmodium species, but no studies on antileishmanial activity have been done. The study was focused on investigating five Pentas species for antileishmanial principles. The CH2Cl2/CH3OH (1:1) extracts of the roots and/or stems of Pentas bussei, P. longiflora, P. micrantha, P. parvifolia and P. zanzibarica were subjected to a combination of chromatographic separations resulting in the isolation of 14 compounds. The pure compounds were characterized by utilizing 1H NMR, 13C NMR, 1H-1H COSY, HMBC, HSQC and MS. The crude extract from the roots of Pentas parvifolia yielded busseihydroquinone B (51). The stem bark of Pentas parvifolia yielded β-stigmasterol (50) and β-amyrin (95). The aerial parts of P. parvifolia yielded vanillic acid (96), p-hydroxybenzoic acid (97) and protocatechuic acid (98). The aerial part of P. bussei yielded β-stigmasterol (50), a homoprenylated naphthoquinone (55), busseihydroquinone A (7), busseihydroquinone B (51), busseihydroquinone C (52) and methyl-8-hydroxy-1,4,6,7-tetramethoxy-2-naphthoate (47), which is a new compound. The aerial parts of P. micrantha yielded 2-methoxy-3-methyl-anthracene- 9,10-dione (72). The stem bark of P. zanzibarica yielded rubiadin-1-methyl ether (65) and rubiadin (64). The roots of Pentas longiflora yielded pentalongin (74). Pentalongin (74) showed antileishmanial activity (IC50 = 11 µ M) against the antimony sensitive strain of Leishmania donavani (MHOM/IN/83/AG83). It also generated a substantial amount of nitric oxide in the cell culture (IC50 = 1.08 µM) relative to the positive control, miltefosine (4), (IC50 = 1.11 µ M). Busseihydroquinone A (7) was oxidized with silver (I) oxide to yield 1-hydroxy-4,6-dimethoxy- 7,8-dioxo-7,8-dihydro-naphthalene-2-carboxylic acid methyl ester (99). Through computational modelling, the inhibitory potential of phytochemicals from the genus Pentas for Leishmania infantum trypanothione reductase was studied using UCSF Chimera 1.15. Among the studied compounds, schimperiquinone A (92) exhibited the highest affinity for the binding site of the receptor; with a binding energy of -10.9 kcal/mol. Anthraquinones generally showed superior inhibitory potency for Leishmania infantum trypanothione reductase than naphthoquinones. Overall, the phytochemicals from the genus Pentas showed sustained hydrogen bonds with Thr335, Lys60 and Cys52; these amino acid residues assist FAD to achieve a proper orientation towards the catalytic site of the enzyme. Therefore, the quinones from the genus Pentas have the potential to guide the development of antileishmanial drug agents. Given the distinctive binding mode of some of the anthraquinones and naphthoquinones observed here, the compounds should be subjected to in vitro and in vivo studies.
TABLE OF CONTENTS
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
LIST OF TABLES ix
LIST OF FIGURES x
LIST OF SCHEMES xi
LIST OF APPENDICES xii
LIST OF ACRONYMS AND SYMBOLS xiv
CHAPTER ONE
1.1 Background Information 1
1.2 Statement of the Problem 2
1.3 Objectives of the Study 3
1.3.1 General Objective 3
1.3.2 Specific Objectives 4
1.4 Justification of the Study 4
CHAPTER TWO
2.1 Leishmaniasis 5
2.1.1 Visceral Leishmaniasis 6
2.1.2 Cutaneous Leishmaniasis 7
2.1.3 Mucocutaneous Leishmaniasis 7
2.2 Prevention and Treatment of Leishmaniasis 8
2.2.1 Use of Antiprotozoal Agents, Antifungal Agents and Antibiotics 8
2.2.2 Use of Pentavalent Antimonials 9
2.2.3 Use of Systemic Antifungal Agents 9
2.2.4 Combination Therapy 10
2.3 Emerging Trends in Leishmaniasis Therapy 10
2.3.1 Immunization 10
2.3.2 Immunotherapy 11
2.3.4 Nanotechnology 11
2.4 Natural Products as Leads for Leishmaniasis Treatment 12
2.4.1 Alkaloids 12
2.4.2 Terpenoids 14
2.4.3 Flavonoids 16
2.4.4 Quinones 17
2.5 The Rubiaceae Family 19
2.5.1 The Genus Pentas 20
2.5.2 Application of the Genus Pentas in Ethnomedicine 20
2.5.3 Phytochemistry of the Genus Pentas 22
2.5.3.1 Chemical Composition of Pentas bussei 22
2.5.3.2 Chemical Composition of Pentas parvifolia 25
2.5.3.3 Anthraquinones of Pentas zanzibarica 28
2.5.3.4 Chemical Composition of Pentas longiflora 28
2.5.3.5 Chemical Composition of Pentas lanceolata 31
2.5.3.6 Anthraquinones of Pentas micrantha 32
2.5.3.7 Chemical Composition of Pentas schimperi 33
2.6 Biosynthesis of Phytochemicals in the Genus Pentas 33
2.7 Biological Activities of Compounds Isolated from Pentas Species 36
2.8 Application of CADD in the Development of Antileishmanial Drugs 37
2.8.1 Leishmania Infantum Trypanothione Reductase (LiTR) 39
CHAPTER THREE
3.1 Materials 41
3.1.1 Plant Materials 41
3.2 Methods 42
3.2.2 General Extraction Method 42
3.2.3 Extraction and Isolation of Compounds from Roots of Pentas parvifolia 43
3.2.3 Extraction and Isolation of Compounds from the Stem Bark of Pentas parvifolia 43
3.2.4 Extraction and Isolation of Compounds from the Aerial Parts of Pentas parvifolia 44
3.2.5 Extraction and Isolation of Compounds from the Aerial Parts of Pentas bussei 44
3.2.6 Extraction and Isolation of Compounds from the Aerial Parts of Pentas micrantha 45
3.2.7 Extraction and Isolation of Compounds from the Stem Bark of Pentas zanzibarica 45
3.2.8 Extraction and isolation of Pentalongin from the Roots of Pentas longiflora 46
3.6 Derivatization 46
3.6.1 Synthesis of Compound 99 46
3.7 Antileishmanial Activity 46
3.7.1 Computational Modelling 46
3.7.2 Ligand Identification 47
3.7.3 Ligand Preparation and Optimisation 47
3.7.4 Receptor Preparation 47
3.7.5 Docking of the Compound Library 48
3.8 In vitro Antileishmanial Assay 48
CHAPTER FOUR
4.1 Introduction 50
4.2 Characterisation of Compounds Isolated from the Roots of Pentas parvifolia 50
4.3 Characterisation of Compounds Isolated from the Stem of Pentas parvifolia 52
4.4 Characterisation of Compounds Isolated from the Aerial Parts of Pentas parvifolia 55
4.5 Characterisation of Compounds Isolated from the Aerial Parts of Pentas bussei 57
4.6 Characterisation of Compounds Isolated from the Aerial Parts of Pentas micrantha 65
4.7 Characterisation of Compounds Isolated from the Stem Bark of Pentas zanzibarica 66
4.8 Characterisation of Compounds Isolated from the Roots of Pentas longiflora 68
4.9 Characterization of Compound 99 69
4.10 Computational Modelling 72
4.10.1 Docking Protocol Validation 72
4.10.2 Docking of the Compound Library 73
4.10.2.1 Structure-Activity Relationships 76
4.10.2.2 Ligand Interactions for Busseihydroquinone A and its Synthetic Derivative 79
4.11 Predictive Pharmacokinetic Analysis 80
4.12 Antileishmanial Activity 84
4.13 Mechanism of Action of Pentalongin 84
CHAPTER FIVE
5.1 CONCLUSIONS 86
5.2 RECOMMENDATIONS 87
REFERENCES 88
APPENDIX 105
LIST OF TABLES
Table 2.1: Clinical forms of leishmaniasis and their causative agents 6
Table 2.2: Alkaloids with antileishmanial activity 13
Table 2.3: Terpenoids with antileishmanial activity 15
Table 2.4: Flavonoids with antileishmanial activity 16
Table 2.5: Quinones with antileishmanial activity 18
Table 2.6: Geographical distribution of Pentas species in East Africa 20
Table 2.7: Ethnomedical applications of plants from the genus Pentas 21
Table 2.8: Previously studied Pentas species 22
Table 2.9: Phytochemistry of Pentas bussei 23
Table 2.10: Phytochemistry of Pentas parvifolia 25
Table 2.11: Phytochemistry of Pentas zanzibarica 28
Table 2.12: Phytochemistry of Pentas longiflora 28
Table 2.13: A sample of reported targets in Leishmania parasite 38
Table 4.1: NMR data for busseihydroquinone B in CDCl3 (500 MHz) 51
Table 4.2: NMR data for β-stigmasterol in CDCl3 (500 MHz) 52
Table 4.3: NMR data for β-amyrin in CDCl3 (500 MHz) 54
Table 4.4: NMR data for vanillic acid in CDCl3 (500 MHz) 55
Table 4.5: NMR data for p-hydroxybenzoic acid in CD3CN (500 MHz) 56
Table 4.6: NMR data for Protocatechuic acid in DMSO-d6 (500 MHz) 57
Table 4.7: NMR data for busseihydroquinone A in acetone-d6 (500 MHz) 58
Table 4.8: NMR data for compound 48 in CDCl3 (500 MHz) 60
Table 4.9: NMR data for Busseihydroquinone C in CDCl3 (500 MHz) 61
Table 4.10: NMR data for compound 47 in CDCl3 (500 MHz) 63
Table 4.11: NMR data for protocatechuic acid in DMSO-d6 (500 MHz) 64
Table 4.12: NMR data for compound 72 in acetone-d6 66
Table 4.13: NMR data for rubiadin-1-methyl ether (65) in DMSO-d6 (500 MHz) 67
Table 4.14: NMR data for Rubiadin in acetode-d6(500 MHz) 68
Table 4.15: NMR data for Pentalongin in CDCl3 (500 MHz) 69
Table 4.16: NMR data for compound 99 in CDCl3 (500 MHz) 71
Table 4.17: Binding energies for phytochemicals from the genus Pentas 74
Table 4.18: Comparison of binding affinities for the compound classes 76
Table 4.19: Predictive pharmacokinetic analysis 81
Table 4.20: Antileishmanial activity of pentalongin against Leishmania donovani 84
LIST OF FIGURES
Figure 2.1: Alkaloids with antileishmanial activity 14
Figure 2.2: Terpenoids with antileishmanial activity 16
Figure 2.3: Flavonoids with antileishmanial activity 17
Figure 2.4: Quinones with antileishmanial activity 19
Figure 2.5: Phytochemicals from Pentas lanceolata 32
Figure 2.6: Phytochemicals from Pentas micrantha 32
Figure 2.7: Phytochemicals from Pentas schimperi 33
Figure 4.1: Crystal Structure of Leishmania infantum trypanothione reductase 72
Figure 4.2: Ligand interactions for FAD (100) 73
Figure 4.3: Ligand interactions for rubiadin-3-O-β-primeveroside (62) 77
Figure 4.4: Ligand interactions for rubiadin (64) 77
Figure 4.5: Ligand interactions for schimperiquinone A (92) 78
Figure 4.6: Ligand interactions for schimperiquinone B (93) 78
Figure 4.7: Ligand interactions for isagarin (77, A) and busseihydroquinone C (52, B) 79
Figure 4.8: Ligand interactions for busseihydroquinone A (7) and compound 99 80
Figure 4.9: Ligand interaction for pentalongin (74) 85
LIST OF SCHEMES
Scheme 2.1: Biosynthesis of naphthoquinones in the genus Pentas 34
Scheme 2.2: Biosynthesis of quinones in the genus Pentas 35
Scheme 2.3: Trypanothione reductase-catalysed reduction of trypanothione 40
Scheme 4.1: Proposed fragmentation pattern of busseihydroquinone A 58
Scheme 4.2: Oxidation of busseihydroquinone A (7) using silver (I) oxide 69
Scheme 4.3: Proposed fragmentation pattern of compound 99 70
LIST OF APPENDICES
APPENDIX A: Physical and Spectroscopic Data 105
APPENDIX 1.0: 1H NMR Spectrum of Busseihydroquinone B (CD3CN, 500 MHz) 109
APPENDIX 1.1: 13C NMR Spectrum of Busseihydroquinone B (CD3CN, 500 MHz) 110
APPENDIX 1.2: ESI-MS Spectrum for Busseihydroquinone A 111
APPENDIX 2.0: 1H NMR Spectrum of β-Stigmasterol (CD3CN, 500 MHz) 112
APPENDIX 2.1: 13C NMR Spectrum of β-Stigmasterol (CD3CN, 500 MHz) 113
APPENDIX 3.0: 1H NMR Spectrum of β-Amyrin (CDCl3, 500 MHz) 114
APPENDIX 3.1: 13C NMR Spectrum of β-Amyrin (CDCl3, 500 MHz) 115
APPENDIX 4.0: 1H NMR Spectrum of Busseihydroquinone A (CDCl3, 500 MHz) 116
APPENDIX 4.1: 13C NMR Spectrum of Busseihydroquinone A (Acetone-d6, 500 MHz) 117
APPENDIX 4.1: ESI-MS Spectrum for Busseihydroquinone A 118
APPENDIX 5.0: 1H NMR Spectrum of Vanillic acid (CD3CN, 500 MHz) 119
APPENDIX 5.1: 13C NMR Spectrum of Vanillic acid (CD3CN, 500 MHz) 120
APPENDIX 5.2: ESI-MS Spectrum for Vanillic acid 121
APPENDIX 6.0: 1H NMR Spectrum of p-Hydroxybenzoic acid (CD3CN, 500 MHz) 122
APPENDIX 6.1: 13C NMR Spectrum of p-Hydroxybenzoic acid (CD3CN, 500 MHz) 123
APPENDIX 6.2: ESI-MS Spectrum for p-Hydroxybenzoic acid 124
APPENDIX 7.0: 1H NMR Spectrum of Protocatechuic acid (DMSO-d6, 500 MHz) 125
APPENDIX 7.1: 13C NMR Spectrum of Protocatechuic acid (DMSO-d6, 500 MHz) 126
APPENDIX 7.2: ESI-MS Spectrum for Protocatechuic acid 127
APPENDIX 8.0: 1H NMR Spectrum of Compound 48 (CDCl3, 500 MHz) 128
APPENDIX 8.1: 13C NMR Spectrum of Compound 48 (CDCl3, 500 MHz) 129
APPENDIX 8.2: H-H COSY Spectrum of Compound 48 (CDCl3, 500 MHz) 130
APPENDIX 8.3: HMBC Spectrum of Compound 48 (CDCl3, 500 MHz) 131
APPENDIX 8.4: NOESY Spectrum of Compound 48 (CDCl3, 500 MHz) 132
APPENDIX 8.5: ESI-MS Spectrum for Compound 48 133
APPENDIX 9.0: 1H NMR Spectrum of Busseihydroquinone C (CDCl3, 500 MHz) 134
APPENDIX 9.1: 13C NMR Spectrum of Busseihydroquinone C (CD3CN, 500 MHz) 135
APPENDIX 9.2: ESI-MS Spectrum for Busseihydroquinone C 136
APPENDIX 10.0: 1H NMR Spectrum of Compound 47 (CDCl3, 500 MHz) 137
APPENDIX 10.1: 13C NMR Spectrum of Compound 47 (CDCl3, 500 MHz) 138
APPENDIX 11.0: 1H NMR Spectrum of Compound 72 ((CD3)2CO, 500 MHz) 139
APPENDIX 11.1: 13C NMR Spectrum of Compound 72 ((CD3)2CO, 500 MHz) 140
APPENDIX 11.2: HMBC Spectrum of Compound 72 ((CD3)2CO, 500 MHz) 141
APPENDIX 12.0: 1H NMR Spectrum of Rubiadin-1-methylether (DMSO-d6, 500 MHz) 142
APPENDIX 12.1: 13 C NMR Spectrum of Rubiadin-1-methylether (DMSO-d6, 500 MHz) 143
APPENDIX 12.2: HMBC Spectrum of Rubiadin-1-methylether (DMSO-d6, 500 MHz) 144
APPENDIX 12.3: ESI-MS Spectrum for Rubiadin-1-methyl ether 145
APPENDIX 13.0: 1H NMR Spectrum of Rubiadin ((CD3)2CO, 500 MHz) 146
APPENDIX 13.1: 13C NMR Spectrum of Rubiadin ((CD3)2CO, 500 MHz) 147
APPENDIX 13.2: COSY Spectrum of Rubiadin ((CD3)2CO, 500 MHz) 148
APPENDIX 13.3: HMBC Spectrum of Rubiadin ((CD3)2CO, 500 MHz) 149
APPENDIX 13.4: ES-MS Spectrum for Rubiadin 150
APPENDIX 14.0: 1H NMR Spectrum of Compound 99 (CDCl3, 500 MHz) 151
APPENDIX 14.1: 13C NMR Spectrum of Compound 99 (CDCl3, 500 MHz) 152
APPENDIX 14.2: HSQC Spectrum of Compound 99 (CDCl3, 500 MHz) 153
APPENDIX 14.3: HMBC Spectrum of Compound 99 (CDCl3, 500 MHz) 154
APPENDIX 14.4: HMBC Spectrum of Compound 99 for Aromatic Region 155
APPENDIX 14.5: HMBC Spectrum of Compound 99 for Methoxy Groups 156
APPENDIX 14.6: ESI-MS Spectrum for Compound 99 157
LIST OF ACRONYMS AND SYMBOLS
CADD Computer Aided Drug Design
CFR Case Fatality Rate
ESI-MS Electron Spray Ionisation Mass Spectrometry
FAD Flavin-adenine dinucleotide
GCG Bis(gamma-glutamyl-cysteinyl glycinyl)spermidine
HMG-CoA
3-Hydroxy-3-methylglutaryl coenzyme A
HMBC Heteronuclear Multiple Bond Correlation
HSQC Heteronuclear Single Quantum Coherence
IC50 50% Inhibition Concentration
LiTR Leishmania infantum trypanothione reductase
MMFF Merck Molecular Force Field
NMR Nuclear Magnetic Resonance
NOESY Nuclear Overhauser Effect Spectroscopy
PTLC Preparative Thin Layer Chromatography
QSAR Quantitative Structural Activity Relationship
UV Ultraviolet
WHO World Health Organization
CHAPTER ONE
INTRODUCTION
1.1 Background Information
Leishmaniasis is a set of protozoan-based infections with numerous clinical indicators: destructive mucosal inflammation and ulcerative skin lesions; it is caused by over 20 species of Leishmania parasite (Kaye et al., 2020). Worldwide, two (2) million cases of leishmaniasis occur annually, and three hundred fifty million people are at risk of getting infected (Jawed and Majumdar, 2018). According to WHO (2017), over 95% of the new cases of visceral Leishmaniasis were reported to occur in four Asian and five African countries. In July 2019, a total of 1,564 leishmaniasis cases were reported by WHO (2019) in Marsabit and Wajir, Counties of Kenya. WHO (2021) reported 873 cases of visceral leishmaniasis in Marsabit, Garissa, Kitui, Baringo, West Pokot, Mandera and Wajir since January 2020 which accounted for 9 deaths (CFR 1.0%).
Leishmaniasis is one of the neglected tropical diseases; it affects mainly the marginalised communities that cannot afford to pay for the medication even when it is made available. Consequently, pharmaceutical companies, which are primarily profit-oriented do not find it cost effective to invest in the manufacture of drugs for such diseases (de Menezes et al., 2015).
To date, an effective vaccine against leishmaniasis has not been found. Leishmaniasis is largely treated using chemotherapeutic agents, usually entailing the use of antimony-based compounds administered by injection. In addition to the antimony-based drugs, pentamidine (1), amphotericin B (2) and Pentostam (sodium stibogluconate, 3) are used (Jawed and Majumdar, 2018).
Pentamidine and pentostam are employed in East Africa to treat visceral leishmaniasis (Marlet et al., 2003). However, these drugs are highly toxic and relatively expensive. The current therapies for leishmaniasis are also faced with drug resistance, which significantly compromises their efficacy in combating the disease (Tiuman et al., 2011).
Nature-derived therapies have for millennia been irrefutably fundamental in the fight against protozoan based infections such as malaria, amoebiasis and leishmaniasis. Phytochemicals belonging to the classes of alkaloids, terpenoids, saponins, phenolics and quinones were reported to exhibit antileishmanial activity (Manuel and Luis, 2001). Some natural products from the Rubiaceae family, most notably quinine, have been reported to exhibit significant antiprotozoal activity. Endale (2012) reported substantial antiplasmodial activities for some phytochemicals in the genus Pentas.
1.2 Statement of the Problem
The available knowledge on leishmania has not yet translated into successful antileishmanial drug agents (de Menezes et al., 2015). Many of the therapies used for treating leishmaniasis such as pentamidine (1), miltefosine (4) and paromomycin (5) exhibit species-specific activity; they are only effective against particular strains of the pathogen (Arevalo et al., 2001 ; Alvar et al., 2006 ; Reithinger et al., 2007 ; Miranda- Verastegui et al., 2009). Furthermore, the conventional therapies for leishmaniasis widely involve the application of pentavalent antimony compounds which are associated with agonizing secondary side effects such as muscular-skeletal pains, renal failure, hepatotoxicity and cardiotoxicity (Reithinger et al., 2007; de Menezes et al., 2015; Jawed and Majumdar, 2018). Consequently, this has dramatically lessened drug tolerability and resulted in treatment noncompliance and abandonment. Ultimately, the emergence of drug- resistant strains of leishmania is prevalent, this accounts for the remarkable decline in the efficacy of the conventional therapies. There is, therefore, an urgent need for interventions that include exploration of alternative drug molecules. The study was focused on the investigation of natural products for antileishmanial compounds. Particularly, the study was anchored on the phytochemical investigation of Pentas species; P. parvifolia, P. bussei, P. micrantha, P. longiflora and P. zanzibarica in the pursuit of safer and affordable leishmaniasis therapies.
1.3 Objectives of the Study
1.3.1 General Objective
The general objective of this study was to identify antileishmanial principles from five
Pentas species.
1.3.2 Specific Objectives
The specific objectives of this study were:
i. To isolate and characterize secondary metabolites from P. parvifolia, P. bussei, P. micrantha, P. longiflora and P. zanzibarica.
ii. To determine the antileishmanial activity of secondary metabolites isolated from P. parvifolia, P. bussei, P. micrantha, P. longiflora and P. zanzibarica.
iii. To predict the inhibitory potency of phytochemicals in the genus Pentas for Leishmania infantum trypanothione reductase through computational modelling.
iv. To enhance the antileishmanial activities of promising phytochemicals through structural modification.
1.4 Justification of the Study
Naphthalene derivatives such as plumbagin (6), a naphthoquinone isolated from Plumbago rosea (Kapadia et al., 2005), were reported to show antileishmanial activity with IC50 of 0.42 and 1.10 μgmL-1 against the amastigotes of Leishmania donovani and Leishmania amazonensis, respectively (Manuel and Luis, 2001). Endale (2012) reported naphthalene derivatives such as busseihydroquinone A (7), from the genus Pentas with antiprotozoal activity against Plasmodium falciparum; D6 clone (IC50 11.10 μgmL-1) and W2 clone (IC50 44.50μgmL-1). Leishmaniasis, like malaria, is caused by a protozoan parasite. In addition, quinone derivatives were reported to bind effectively in the FAD binding cavity of Leishmania infantum trypanothione reductase (Venkatesan et al., 2010). Therefore, compounds from Pentas species are attractive candidates for designing antileishmanial drugs.
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